Biochemical Journal Immediate Publications

Mechanism and specificity of the human paracaspase MALT1

2012-02-06T15:29:47Z

The paracaspase domain of MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of a gene translocation fused to the N-terminal domains of the cellular inhibitor of apoptosis protein 2. The paracaspase itself, commonly known as MALT1, participates in the NF-κB pathway, likely by driving survival signals downstream of the B-cell antigen receptor through MALT1 proteolytic activity. We have developed methods for the expression and purification of recombinant full-length MALT1 and its constituent catalytic domain alone. Both are activated by dimerization without cleavage, with a similar dimerization barrier to the distantly related cousins, the apical caspases. By using positional scanning peptidyl substrate libraries we demonstrate that the activity and specificity of full-length MALT1 is recapitulated by the catalytic domain alone, showing a stringent requirement for cleaving after Arg, and with striking peptide length constraints for efficient hydrolysis. Rates of cleavage (k_cat/K_m values) of optimal peptidyl substrates are in the same order (10³ – 10⁴ M^-1 s^-1) as for a putative target protein CYLD. Thus, MALT1 has many similarities to caspase 8, even cleaving the putative target protein CYLD with comparable efficiencies, but with diametrically opposite primary substrate specificity.

Readthrough of long-QT syndrome type 1 nonsense mutations rescues function but alters the biophysical properties of the channel

S C Harmer, J S Mohal, D Kemp, A Tinker — 2012-02-06T15:18:15Z

The nonsense mutations R518X-KCNQ1 and Q530X-KCNQ1 cause long-QT syndrome type 1 (LQT1) and result in a complete loss of I_Ks channel function. In this study we attempted to rescue the function of these mutants, in human embryonic kidney-293 cells, by promoting readthrough of their premature termination codons (PTCs) using the pharmacological agents G-418, gentamicin and PTC124. Gentamicin and G-418, acted to promote full-length channel protein expression from R518X at 100 µM and from Q530X at 1 mM. In contrast, PTC124 did not, at any dose tested, induce readthrough of either mutant. G-418 (1 mM) treatment also acted to significantly (P<0.05) increase current density and peak-tail current density, at +80 mV, for R518X, but not Q530X, to 58±11% and 82±17% of the wild-type level respectively. However, the biophysical properties of the currents produced, from R518X, while similar were not identical to wild-type as the voltage dependence of activation was significantly (P<0.05) shifted by +25 mV. Overall, these findings indicate that although functional rescue of LQT1 nonsense mutations is possible it is dependent on the degree of readthrough achieved and the effect on channel function of the amino acid substituted for the PTC. Such considerations will determine the success of future therapies.

Identification of a molecular component of the mitochondrial acetyl transferase program; a novel role for GCN5L1

I Scott, B R. Webster, J H. Li, M N. Sack — 2012-02-06T15:07:51Z

SIRT3 modulates respiration via the deacetylation of lysine residues in electron transport chain proteins. Whether mitochondrial protein acetylation is controlled by a counter-regulatory program has remained elusive. Here we identify an essential component of this previously undefined mitochondrial acetyltransferase system. We show that GCN5L1/Bloc1s1 counters the acetylation and respiratory effects of SIRT3. GCN5L1 is mitochondrial-enriched and displays significant homology to a prokaryotic acetyltransferase. Genetic knockdown of GCN5L1 blunts mitochondrial protein acetylation, and its reconstitution in intact mitochondria restores protein acetylation. GCN5L1 interacts with and promotes acetylation of SIRT3 respiratory chain targets and reverses global SIRT3 effects on mitochondrial protein acetylation, respiration and bioenergetics. These data identify GCN5L1 as a critical, prokaryote-derived component of the mitochondrial acetyltransferase program.

Oxygen Activation in Neuronal NO Synthase: Resolving the Consecutive Monooxygenation Steps

D Papale, C Bruckmann, B Gazur, C S Miles, C G Mowat, S Daff — 2012-02-03T11:49:02Z

The vital signalling molecule nitric oxide is produced by mammalian NOS enzymes in two steps. L-arginine is converted to N-hydroxy-L-arginine NOHA, which is converted to NO and citrulline. Both steps are thought to proceed via similar mechanisms in which the cofactor tetrahydrobiopterin (H₄B) activates dioxygen at the heme site by electron transfer. The subsequent events are poorly understood due to the lack of stable intermediates. By analogy with cytochromes P450, a heme-iron oxo species may be formed, or direct reaction between a heme-peroxy intermediate and substrate may occur. The two steps may also occur via different mechanisms. Here we analyse the two reaction steps using the G586S mutant of nNOS, which introduces an additional H-bond in the active site and provides an additional proton source. In the mutant, H₄B activates dioxygen as in the wild-type enzyme, but an interesting intermediate heme species is then observed. This may be a stabilized form of the active oxygenating species. The mutant is able to perform step 2 (reaction with NOHA), but not step 1 (with L-Arg) indicating that the extra H-bond enables it to discriminate between the two monooxygenation steps. This implies that the two steps follow different chemical mechanisms.

Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex

L Troeberg, B Mulloy, P Ghosh, M Lee, G Murphy, H Nagase — 2012-02-03T11:43:36Z

The semi-synthetic sulfated polysaccharide pentosan polysulfate (PPS) increases affinity between the aggrecan-degrading adamalysins with thrombospondin motifs (ADAMTSs) and their endogenous inhibitor, tissue inhibitor of metalloproteinases (TIMP)-3. Here we demonstrate that PPS mediates the formation of a high affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular matrix binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Spacer domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of 8 saccharide units, indicating the involvement of extended or multiple protein interaction sites. The formation of a high affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.

Cytosolic Ca2{+} regulates the energisation of isolated brain mitochondria by formation of pyruvate through the malate{-}aspartate shuttle

2012-02-01T16:25:47Z

The glutamate-dependent respiration of isolated brain mitochondria (BM) is regulated by cytosolic Ca²⁺ (Ca²⁺_cyt) (S_0.5= 225 ± 22 nM) through its effects on aralar. We now also demonstrate that the a-glycerophosphate-dependent respiration is controlled by Ca²⁺_cyt (S_0.5 = 60 ± 10 nM). At higher Ca²⁺_cyt(< 600 nM), BM accumulate Ca²⁺ which enhances the rate of action of intramitochondrial dehydrogenases. The highest Ca²⁺-induced increments of state 3 respiration decrease with substrate in the order glutamate < a-ketoglutarate < isocitrate < a-glycerophosphate < pyruvate. Whereas the oxidation of pyruvate is only slightly influenced by Ca²⁺_cyt, we show that the formation of pyruvate is tightly controlled by Ca²⁺_cyt. Through its common substrate couple NADH/NAD⁺, the formation of pyruvate by lactate dehydrogenase (LDH) is linked to the malate–aspartate shuttle (MAS) with aralar as a central component. A rise of Ca²⁺_cyt in a reconstituted system consisting of BM, cytosolic enzymes of MAS and LDH causes an up to five-fold enhancement of OXPHOS rates that is due to an increased substrate supply, acting in a manner similar to a “gas pedal”. In contrast, mitochondrial Ca²⁺ (Ca²⁺_mit) regulates the oxidation rates of substrates which are present within mitochondrial matrix. We postulate that Ca²⁺_cytis a key factor in adjusting the mitochondrial energisation to the requirements of intact neurons.

A naturally occurring nonapeptide functionally compensates the CP1 domain of leucyl-tRNA synthetase to modulate aminoacylation activity

M Tan, W Yan, R Liu, M Wang, X Chen, X Zhou, E Wang — 2012-02-01T11:52:02Z

Aminoacyl-tRNA synthetases (aaRSs) establish the rules of the genetic code by catalyzing the formation of aminoacyl-tRNA. The quality control for aminoacylation reaction is achieved by editing activity, which is usually carried out by a discrete editing domain. For leucyl-tRNA synthetase (LeuRS), the connective peptide 1 (CP1) domain is the editing domain responsible for hydrolyzing mis-charged tRNA. The CP1 domain is universally present in LeuRSs except LeuRS from Mycoplasma mobile (MmLeuRS). The substitute of CP1 in MmLeuRS is a nonapeptide (MmLinker). We show here that the MmLinker, which is critical for aminoacylation activity of MmLeuRS, could confer remarkable tRNA charging activity to the inactive CP1-deleted LeuRS from Escherichia coli (EcLeuRS) and Aquifex aeolicus (AaLeuRS). Furthermore, CP1 from EcLeuRS could functionally compensate the MmLinker and endow MmLeuRS with post-transfer editing capability. These investigations provide a mechanistic framework for the modular construction of aaRSs and their coordination to achieve catalytic efficiency and fidelity. These results also show that the pre-transfer editing function of LeuRS originates from its conserved synthetic domain, and shed light on future mechanism study.

Cross-talk between TGF{beta}1 and EGFR signaling pathways induces TM4SF5 expression and Epithelial-Mesenchymal Transition

2012-01-31T14:13:54Z

The epithelial-mesenchymal transition (EMT) is involved in fibrosis and cancer, and regulated by different signaling pathways mediated through soluble factors, actin reorganization, and transcription factor actions. Because tetraspan(in) transmembrane 4 L6 family member 5 (TM4SF5) is highly expressed in hepatocellular carcinoma and induces EMT, understanding how TM4SF5 expression in hepatocytes is regulated is important. We explored the mechanisms that induce TM4SF5 expression and whether impaired signaling pathways for TM4SF5 expression inhibit acquisition of mesenchymal cell features, using human and mouse normal hepatocytes. We found that transforming growth factor beta 1 (TGFβ1)-mediated Smad activation caused TM4SF5 expression and EMT, and activation of the epidermal growth factor receptor (EGFR) pathway. Inhibition of EGFR activity following TGFβ1 treatment abolished acquisition of EMT, suggesting a link from Smads to EGFR for TM4SF5 expression. Further, TGFβ1-mediated EGFR activation and TM4SF5 expression were abolished by EGFR suppression or extracellular EGF depletion. Smad overexpression mediated EGFR activation and TM4SF5 expression in the absence of serum, and EGFR kinase inactivation or EGF depletion abolished Smad overexpression-induced TM4SF5 and mesenchymal cell marker expression. Inhibition of Smad, EGFR, or TM4SF5 using Smad7 or small compounds also blocked TM4SF5 expression and/or EMT. These results indicate that TGFβ1- and growth factor-mediated signaling activities mediate TM4SF5 expression leading to acquisition of mesenchymal cell features, suggesting that TM4SF5 induction may be involved in development of liver pathologies.

The novel function of HINFP as a co-activator in sterol-regulated transcription of PCSK9 in HepG2 cells

H Li, J Liu — 2012-01-31T12:11:41Z

PCSK9 (proprotein convertase subtilisin/kexin type 9 ) plays an important role in control of plasma LDL cholesterol metabolism by modulating the degradation of hepatic LDL receptor. Previous studies demonstrated that PCSK9 is a target gene of the sterol regulatory element (SRE) binding protein 2 (SREBP2) that activates PCSK9 gene transcription through an SRE motif of the promoter. In addition to SREBP2, hepatic nuclear factor 1a (HNF1a) positively regulates PCSK9 gene transcription in hepatic cells through a binding site located 28 bp upstream from SRE. In this study, we have identified a novel histone nuclear factor P (HINFP) recognition motif residing between HNF1 motif and SRE that is essential for basal and sterol-regulated transcriptions of the PCSK9 promoter. Mutation of this motif lowers the basal promoter activity and abolishes the sterol-mediated repression as well as the SREBP2-induced activation of the PCSK9 promoter. We further show that the activity of SREBP2 in stimulating PCSK9 promoter activity is greatly enhanced by HINFP. Additional experiments suggest that HINFP and its cofactor NPAT form a functional complex, and NPAT may subsequently recruit HAT cofactor TRRAP to facilitate the histone H4 acetylation of the PCSK9 promoter. Knockdown of HINFP, NPAT or TRRAP each markedly reduces the amount of acetylated histone H4 on the PCSK9 promoter region and lowers PCSK9 protein levels. Importantly, by utilizing co-immunoprecipitation assays, we have demonstrated a direct interaction between SREBP2 and HINFP and its cofactor NPAT/TRRAP. These new findings, altogether, identify HINFP as a co-activator in SREBP-mediated transactivation of PCSK9 gene expression.

p53Ser15 Phosphorylation disrupts p53-RPA70 complex and induces RPA70-mediated DNA repair in hypoxia

E Madan, R Gogna, U Pati — 2012-01-30T13:47:24Z

Cellular stressors are known to inhibit p53-RPA70 complex and RPA70 increases cellular DNA repair in cancer cells. We hypothesized that regulation of RPA70-mediated DNA repair might be responsible for inhibition of apoptosis in hypoxic tumors. We have shown that, in cancer cells, hypoxia disrupts p53-RPA70 complex thereby enhancing RPA70-mediated NER/NHEJ-repair. In normal cells, RPA70 binds to p53 N-terminus (NTD) whereas this binding is disrupted in hypoxia. Phosphorylation of p53-NTD is a crucial event in dissociating both NTD-RPA70 and p53-RPA70 complexes. Serial mutations at serine and threonine residues in NTD confirm that p53^Ser15phosphorylation induces dissociation of p53-RPA70 complex in hypoxia. DNA-PK is shown to induce p53^Ser15phosphorylation thus enhancing RPA70-mediated NER/NHEJ-repair. Further, RPA70 gene silencing induces significant increase in the cellular apoptosis in the resistant hypoxic cancer cells. We have thus elucidated a novel pathway showing how DNA-PK-mediated p53^Ser15 phosphorylation dissociates p53-RPA70 complex in enhancing NER/NHEJ-repair that causes resistance to apoptosis, in hypoxic cancer cells. This novel finding may open new strategies in developing cancer therapeutics based upon regulation of RPA70-mediated NER/NHEJ-repair.

Ribonucleoprotein Y-box binding protein-1 regulates mitochondrial oxidative phosphorylation (OXPHOS) protein expression after serum stimulation through binding to OXPHOS mRNA

2012-01-27T11:58:49Z

Mitochondria play key roles in essential cellular functions such as energy production, metabolic pathways and aging. Growth factor-mediated expression of the mitochondrial oxidative phosphorylation (OXPHOS) complex proteins has been proposed to play a fundamental role in metabolic homeostasis. Although protein translation is affected by general RNA-binding proteins, very little is known about the mechanism involved in mitochondrial OXPHOS protein translation. In the present study, serum stimulation induced the nuclear-encoded OXPHOS protein expression such as NDUFA9, NDUFB8, SDHB and UQCRFS1 and mitochondrial ATP production in translation-dependent manners. We also observed that the major ribonucleoprotein Y-box binding protein-1 (YB-1) preferentially bound to these OXPHOS mRNA and regulated the recruitment of mRNAs from inactive messenger ribonucleoprotein particles (mRNPs) to active polysomes. YB-1 depletion led to upregulation of mitochondrial function through induction of OXPHOS protein translation from inactive mRNP release. In contrast, YB-1 overexpression suppressed the translation of these OXPHOS mRNAs through reduced polysome formation, suggesting that YB-1 regulated the translation of mitochondrial OXPHOS mRNAs through mRNA binding. Taken together, our findings suggest that YB-1 is a critical factor for translation that may control OXPHOS activity.

Fibulin-5 binds urokinase type plasminogen activator and mediates urokinase-stimulated {beta}1-integrin-dependent cell migration

2012-01-26T15:50:10Z

Urokinase-type plasminogen activator (uPA) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPA receptor (uPAR/CD87), integrins and lipoprotein receptor-related protein 1 (LRP1) that activate intracellular signaling pathways. We report uPA-mediated cell migration requires its interaction with fibulin-5. uPA stimulates migration of wild-type mouse embryonic fibroblasts (FBLN5^+/+ MEFs), but has no effect on fibulin-5-deficient FBLN5^-/- MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (FBLN5^RGE/RGEMEFs) do not migrate in response to uPA. Moreover, a blocking anti-human β1 antibody inhibited the migration of pulmonary arterial smooth muscle cells (PASMCs) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal calcium-binding epidermal growth factor-like (cbEGF) domain, leading to loss of b1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from b1 integrin and thereby unblocks integrin’s capacity to facilitate cell motility.

Regulation of the inter subunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD{+} synthetase

W Chuenchor, T I. Doukov, M Resto, A Chang, B Gerratana — 2012-01-26T14:20:27Z

Glutamine-dependent NAD⁺ synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyzes the ATP-dependent formation of NAD⁺from NaAD⁺ at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 Å long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism, however, its regulatory mechanism is unclear. We report the structures of the inactive-glutaminase C176A variant in an apo form and in three synthetase ligand complexes with substrates (NaAD⁺/ATP), substrate analog (NaAD⁺/AMPCPP) and intermediate analogs (NaAD⁺/AMP/PP_i), as well as the structure of wild type mtuNadE in a products complex (NAD⁺/AMP/PPi/Glu). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: 1) at the entrance near the glutaminase active site, 2) in the middle of the tunnel and 3) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.

ROCKII serine 1366 phosphorylation reflects the activation status

H Chuang, C Yang, Y Tsay, C Hsu, L Tseng, Z Chang, H Lee — 2012-01-24T14:38:48Z

Rho-associated protein kinase (ROCK), a downstream effector of RhoA, plays an important role in many cellular processes. Accumulating evidence has shown the involvement of ROCK activation in the pathogenesis of many diseases. However, a reagent capable of directly detecting ROCK activation is lacking. In this study, we show autophosphorylation of ROCKII in an in vitro kinase reaction. The phosphorylation sites were identified by mass spectrometry and the major phosphorylation site was found to be at the highly conserved S1366 residue. A phospho-specific antibody was generated that can specifically recognize ROCKII S1366 phosphorylation. We found that the extent of S1366 phosphorylation of endogenous ROCKII is correlated with that of myosin light chain phosphorylation in cells in response to RhoA stimulation, showing that S1366 phosphorylation reflects its kinase activity. In addition, ROCKII S1366 phosphorylation could be detected in human breast tumors by immunohistochemical staining. Our study provides a new approach for revealing ROCKII activation status by directly probing ROCKII S1366 phosphorylation in cells or tissues.

First identification of small molecule inhibitors of Pontin by combining virtual screening and enzymatic assay

2012-01-24T14:13:58Z

The human protein Pontin, which belongs to the AAA+ family, is overexpressed in several cancers and its silencing in vitro leads to tumor cell growth arrest and apoptosis, making it a good target for cancer therapy. In particular, high levels of expression were found in hepatic tumors for which the therapeutic arsenal is rather limited. The 3D structure of Pontin had previously been resolved, revealing an hexameric assembly with one ADP molecule co-crystallized in each subunit. Using Vina, Drugscore and Xscore, structure-based virtual screening of 2,200 commercial molecules was conducted into the ATP binding site formed by a dimer of Pontin in order to prioritize the best candidates. Complementary to the in silico screening, a versatile and sensitive colorimetric assay was set up to measure the disruption of the ATPase activity of Pontin. This assay allowed the determination of inhibition curves for more than twenty top scoring compounds, resulting in the identification of four ligands presenting an inhibition constant in the micromolar concentration range. Three of them inhibited tumor cell proliferation. The association of virtual screening and experimental assay thus proved successful for the discovery of the first small molecule inhibitors of Pontin.

A NOVEL LECTIN FROM AGROCYBE AEGERITA SHOWS HIGH BINDING SELECTIVITY FOR TERMINAL N-ACETYLGLUCOSAMINE

S Jiang, Y Chen, M Wang, Y Yin, Y Pan, B Gu, G Yu, Y Li, B Wong, Y Liang, h sun — 2012-01-23T15:09:25Z

A novel lectin was isolated from the mushroom Agrocybe aegerita (designated AAL-2) by affinity chromatography with N-acetylglucosamine (GlcNAc) coupled Sepharose 6B after (NH₄)₂SO₄precipitation. The AAL-2 coding sequence (1224 bp) was identified by performing a homologous search of the five tryptic peptides identified by mass spectrometry against the translated transcriptome of A. aegerita. The molecular weight of AAL-2 was calculated to be 43.175 kDa from mass spectrometry (MS), which was consistent with the data calculated from the amino acid sequence. To analyze the sugar binding properties of AAL-2, a glycan array composed of 465 glycan candidates was employed and the result showed that AAL-2 bound with high selectivity to terminal, nonreducing GlcNAc residues, and further analysis revealed that AAL-2 bound to terminal, nonreducing GlcNAc residues with higher affinity than previously well-known GlcNAc-binding lectins such as wheat germ agglutinin (WGA) and Griffonia simplicifolia lectin-II (GSL-II). Isothermal titration calorimetry (ITC) further showed that GlcNAc bound to AAL-2 in a sequential manner with moderate affinity. In the current study, we also evaluated the antitumor activity of AAL-2. The results showed that AAL-2 could bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro. Furthermore, AAL-2 exerted an anti-hepatoma effect via inhibition of tumor growth and prolongation of survival time of tumor bearing mice in vivo.

The nonsteroidal anti-inflammatory drug indomethacin activates the eIF2alpha kinase PKR, causing a translational block in human colorectal cancer cells

C Brunelli, C Amici, M Angelini, C Fracassi, G Belardo, M Santoro — 2012-01-23T14:57:01Z

The non-steroidal anti-inflammatory drug (NSAID) indomethacin, a cyclooxygenase-1 and -2 inhibitor with anti-inflammatory and analgesic properties, is known to possess anticancer activity against colorectal cancer (CRC) and other malignancies in humans; however, the mechanism underlying the anticancer action remains elusive. Herein we show that indomethacin selectively activates the double-stranded RNA (dsRNA)-dependent protein kinase PKR in a cyclooxygenase-independent manner, causing rapid phosphorylation of the alpha-subunit of eukaryotic translation initiation-factor 2 (eIF2alpha) and inhibiting protein synthesis in colorectal carcinoma and other types of cancer cells. The PKR-mediated translational block was followed by inhibition of CRC cell proliferation and apoptosis induction. Indomethacin did not affect the activity of eIF2alpha-kinases PERK, GCN2 and HRI, and induced eIF2alpha phosphorylation in PERK-knockout and GCN2-knockout cells, but not in PKR-knockout cells or in human PKR-silenced CRC cells, identifying PKR as a selective target for indomethacin-induced translational inhibition. The fact that indomethacin induced PKR activity in-vitro, an effect reversed by PKR-inhibitor 2-aminopurine, suggests a direct effect of the drug in the kinase activation. The results identify PKR as a novel target of indomethacin, opening new scenarios on the molecular mechanisms underlying the pleiotropic activity of this traditional NSAID.

Involvement of PGE2 and cyclic AMP signaling pathway in the up-regulation of COX-2 and mPGES-1 expression in LPS -activated macrophages

2012-01-23T14:40:59Z

Prostaglandin (PG) E₂ plays an important role in the modulation of the immune response and the inflammatory process. In this study, we describe a PGE₂positive feedback for Cyclooxygenase (COX) -2 and microsomal PGE Synthase (mPGES) -1 expression in the macrophage cell line RAW 264.7. Our results show that PGE₂induces COX-2 and mPGES-1 expression, an effect mimicked by dibutyryl-cAMP (dbcAMP) or Forskolin. Furthermore, cAMP signaling pathway cooperates with LPS in the induction of COX-2 and mPGES-1 transcriptional activation. Analysis of the involvement of EP receptors showed that incubation with EP2 agonists up-regulated both COX-2 and mPGES-1 mRNA levels. Moreover, EP2 receptor over expression enhanced the transcriptional activation of COX-2 and mPGES-1 promoters, being this induction abolished by the PKA inhibitor, H89. Activation of PGE₂/EP2/PKA signaling pathway induced the phosphorylation of the cAMP response element-binding protein (CREB) in macrophages and stimulated the specific binding of this transcription factor to COX-2 and mPGES-1 promoters. Deletion or mutation of potential CRE sites in both promoters diminished their transcriptional activity. In summary, our data demonstrate that activation of PKA/CREB signaling through the EP2 receptor by PGE₂ plays a key role in the expression of COX-2 and mPGES-1 in activated macrophages.

Transforming Growth Factor Beta-1 represses proximal tubular cell microRNA-192 expression via decreased Hepatocyte Nuclear Factor DNA binding

R H. Jenkins, J Martin, A O. Phillips, T Bowen, D J. Fraser — 2012-01-23T12:05:33Z

MicroRNA-192 (miR-192) plays key roles in renal pathological and physiological responses, by repressing targets including Zeb1, Zeb2, and Wnk1. Here, we have studied regulation of miR-192 expression. We found that Transforming Growth Factor Beta-1 (TGF-b1) down-regulates miR-192 and miR-194, co-transcribed in the shared precursor pri-miR-192/194. Luciferase reporter analysis showed constitutive promoter activity within nucleotides +21 to -223. We identified Hepatocyte Nuclear Factor and p53 binding sites within this region that were required for constitutive promoter activity, which was decreased by TGF-b1 via an Alk5-dependent mechanism. TGF-b1-treatment decreased HNF binding to the miR-194-2/192 promoter, while knockdown of HNF-1 inhibited mature miR-192 and -194 expression. miR-192, -194 and HNF expression were restricted to a defined subset of human tissues including kidney, small intestine, colon, and liver. Our data identify coordinated regulation of miR-192 and -194, with binding of HNF and p53 transcription factors necessary for activation of transcription, and TGF-b1 mediated repression via decreased HNF binding to its cognate promoter element.

Regulation of Human Microsomal Prostaglandin E Synthase-1 by IL-1{beta} requires a Distal Enhancer Element with a Unique Role for C/EBP{beta}

2012-01-20T12:25:53Z

The studies of prostaglandin E2 (PGE₂) biosynthesis have primarily focused on the role of cyclooxygenases. Efforts have shifted towards the specific PGE₂ terminal synthases, particularly microsomal PGE synthase (mPGES-1), which has emerged as the crucial inducible synthase with roles in pain, cancer and inflammation. mPGES-1 is induced by proinflammatory cytokines with studies focusing on the proximal promoter, mediated specifically through Egr-1. Numerous studies demonstrate that the mPGES-1 promoter alone cannot account for the level of IL-1β induction. We identified two DNase I hypersensitive sites within the proximal promoter near the Egr-1 element and a novel distal site near -8.6kb. Functional analysis of the distal site revealed two elements that cooperate with basal promoter expression and a stimulus-dependent enhancer. A specific binding site for CCAAT/enhancer binding protein beta (C/EBPβ) in the enhancer was directly responsible for inducible enhancer activity. ChIP analysis demonstrated constitutive Egr-1 binding to the promoter and induced RNA polymerase II and C/EBPβ binding to the promoter and enhancer, respectively. Knockout/knockdown studies established a functional role for C/EBPβ in mPGES-1 gene regulation and documented interaction between Egr-1 and C/EBPβ highlights the proximal promoter cooperation with a novel distal enhancer element in regulating inducible mPGES-1 expression.

Green tea polyphenol EGCG induces lipid raft clustering and apoptotic cell death by activating protein kinase C{delta} and acid sphingomyelinase through 67-kDa laminin receptor in multiple myeloma cells

2012-01-19T11:59:48Z

(－)-Epigallocatechin-3-O-gallate (EGCG), the major polyphenol of green tea, has cancer chemopreventive and chemotherapeutic activities. EGCG selectively inhibits cell growth and induces apoptosis in cancer cells without adversely affecting normal cells; however, the underlying molecular mechanism in vivo is unclear. In this study, we show that EGCG-induced apoptotic activity is attributed to a lipid raft clustering mediated through 67-kDa laminin receptor (67LR) that is significantly elevated in multiple myeloma (MM) cells relative to normal peripheral blood mononuclear cells, and that acid sphingomyelinase (aSMase) is critical for the lipid raft clustering and the apoptotic cell death induced by EGCG. We also found that EGCG induces aSMase translocation to the plasma membrane and protein kinase C delta (PKCδ) phosphorylation at Ser⁶⁶⁴, which was necessary for aSMase/ceramide signaling, via 67LR. Additionally, orally administered EGCG activated PKCδ and aSMase in a murine MM xenograft model. These results elucidate a novel cell death pathway triggered by EGCG for the specific killing of MM cells.

{gamma}-Enolase C-terminal peptide promotes cell survival and neurite outgrowth by activation of PI 3-K/Akt and MAPK/ERK signaling pathways

A Hafner, N Obermajer, J Kos — 2012-01-19T11:42:24Z

γ-Enolase, a glycolytic enzyme, is expressed specifically in neurons. It exerts neurotrophic activity and has been suggested to regulate growth, differentiation, survival and regeneration of neurons. In this study, we investigated the involvement of γ-enolase in PI 3-K/Akt (phosphatidylinositol 3-kinase/Akt) and MAPK/ERK (mitogen-activated protein kinase/ extracellular-signal-regulated kinase) signaling, the two pathways triggered predominantly by neurotrophic factors. While the PI 3-K/Akt pathway, rather than the MAPK/ERK pathway, is involved in γ-enolase-enhanced cell survival, γ-enolase-stimulated neurite outgrowth requires both pathways, i.e. the activation of both PI 3-K and ERK1/2, leading to subsequent expression of growth cone-specific GAP-43 protein. MEK (mitogen-activated protein kinase kinase) and PI 3-K inhibition blocked or attenuated the neurite outgrowth associated with dynamic remodeling of the actin-based cytoskeleton. We show that γ-enolase–mediated PI 3-K activation regulates RhoA kinase, a key regulator of actin cytoskeleton organization. Moreover, the inhibition of RhoA down-stream effector ROCK (Rho-associated kinase) results in enhanced γ-enolase induced neurite outgrowth, accompanied by actin polymerization and its redistribution to growth cones. Our results show that γ-enolase controls neuronal survival, differentiation and neurite regeneration, by activating PI 3-k/Akt and MAPK/ERK signaling pathways, resulting in down-stream regulation of the molecular and cellular processes of cytoskeleton reorganization and cell remodeling, activation of transcriptional factors and regulation of the cell cycle.

Proteoglycans of uterine fibroids and keloid scars: similarity in their proteoglycan composition

D A. Carrino, S Mesiano, N M. Barker, W W. Hurd, A I. Caplan — 2012-01-19T11:22:29Z

Fibrosis is the formation of excess and abnormal fibrous connective tissue as a result of either a reparative or reactive process. A defining feature of connective tissue is its extracellular matrix, which provides structural support and also influences cellular activity. Two common human conditions that result from fibrosis are uterine fibroids (leiomyomas) and keloid scars. Because these conditions share a number of similarities and because their growth is due primarily to excessive extracellular matrix deposition, we compared the proteoglycans of uterine fibroids and keloid scars to corresponding normal tissues. Our analysis indicates that uterine fibroids and keloid scars contain higher amounts of proteoglycans relative to normal myometrium and normal adult skin, respectively. Proteoglycan composition is also different in the fibrotic tissues. Compared to unaffected tissues, uterine fibroids and keloid scars contain higher relative amounts of versican and lower relative amounts of decorin. There is also evidence for a higher level of versican catabolism in the fibrotic tissues compared to unaffected tissues. These qualitative and quantitative proteoglycan differences may play a role in the expansion of these fibrotic conditions and in their excessive matrix deposition and matrix disorganization due to effects on cell proliferation, TGF-β signaling, and/or collagen fibril formation.

The Golgi-Associated Long Coiled-Coil Protein NECC1 Participates in the Control of the Regulated Secretory Pathway in PC12 Cells

2012-01-18T12:34:00Z

Golgi-associated long coiled-coil proteins, often referred to as golgins, are involved in the maintenance of the structural organization of the Golgi apparatus and the regulation of membrane traffic events occurring in this organelle. Little information is available on the contribution of golgins to Golgi function in cells specialized in secretion such as endocrine cells or neurons. Here, we characterize the intracellular distribution as well as the biochemical and functional properties of a novel long coiled-coil protein present in neuroendocrine tissues, neuroendocrine long coiled-coil protein 1 (NECC1). Our studies show that NECC1 is a peripheral membrane protein displaying high stability to detergent extraction, which distributes across the Golgi apparatus in neuroendocrine cells. In addition, NECC1 partially localizes to post-Golgi carriers containing secretory cargo in PC12 cells. Overexpression of NECC1 resulted in the formation of juxtanuclear aggregates together with a slight fragmentation of the Golgi and a decrease in K⁺-stimulated hormone release. In contrast, NECC1 silencing did not alter Golgi architecture but enhanced K⁺-stimulated hormone secretion in PC12 cells. In all, our results identify NECC1 as a novel component of the Golgi matrix and support a role for this protein as a negative modulator of the regulated trafficking of secretory cargo in neuroendocrine cells.

n-3 polyunsaturated fatty acids suppress phosphatidylinositol-(4,5)-bisphosphate dependent actin remodeling during CD4{+} T cell activation

2012-01-18T11:24:15Z

n-3 polyunsaturated fatty acids (PUFA), i.e. docosahexaenoic acid (DHA), found in fish oil, exhibit anti-inflammatory properties; however, the molecular mechanisms remain unclear. Since phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P₂) resides in raft domains and DHA can alter the size of rafts, we hypothesized that PI(4,5)P₂ and downstream actin remodeling are perturbed by the incorporation of n-3 PUFA into membranes, resulting in suppressed T cell activation. CD4⁺ T cells isolated from Fat-1 transgenic mice (membranes enriched in n-3 PUFA) exhibited a 50% decrease in PI(4,5)P₂. Upon activation by plate bound anti-CD3/anti-CD28 or PMA/ionomycin, Fat-1 CD4⁺ T cells failed to metabolize PI(4,5)P₂. Furthermore, actin remodeling, failed to initiate in Fat-1 CD4⁺ T cells upon stimulation; however, the defect was reversed by incubation with exogenous PI(4,5)P₂. When Fat-1 CD4⁺ T cells were stimulated with anti-CD3/anti-CD28 coated beads, the Wiskott-Aldrich syndrome protein (WASP) failed to translocate to the immunological synapse. The suppressive phenotype, consisting of defects in PI(4,5)P₂ metabolism and actin remodeling, were recapitulated in CD4⁺ T cells isolated from mice fed a 4% DHA triglyceride-enriched diet. Collectively, these data demonstrate that DHA alters PI(4,5)P₂ in CD4⁺ T cells, thereby suppressing the recruitment of WASP to the IS, and impairing actin remodeling in CD4⁺ T cells.

CNGA3 is expressed in inner ear hair cells and binds to an intracellular carboxy terminus domain of EMILIN1

2012-01-17T12:23:32Z

The molecular characteristics of cyclic nucleotide-gated (CNG) channels in auditory/vestibular hair cells are largely unknown, unlike those of CNG channels mediating sensory transduction in vision and olfaction. Here, we report full-length sequence for three CNGA3 variants in a hair cell preparation from the trout saccule with high identity to CNGA3 in olfactory receptor neurons/cone photoreceptors. A custom antibody targeting amino terminus sequence immunolocalized CNGA3 to the stereocilia and subcuticular plate region of saccular hair cells. The cytoplasmic carboxyl terminus of CNGA3 was found by yeast two-hybrid analysis to bind the carboxy terminus of elastin microfibril interface-located protein 1 (EMILIN1) in both the vestibular hair cell model and rat organ of Corti (OC). Specific binding between CNGA3 and EMILIN1 was confirmed with surface plasmon resonance analysis, predicting dependence on Ca²⁺ with a K_d = 1.6 x 10^-6 M for trout hair cell proteins and a K_d = 2.7 x 10^-7 M for OC proteins at 68 µM Ca²⁺. Pull-down assays indicated that the binding to OC CNGA3 was attributable to EMILIN1 intracellular sequence that follows a predicted transmembrane domain in the carboxy terminus. Saccular hair cells also express transcript for phosphodiesterase 6C (PDE6C), which in cone photoreceptors regulates degradation of cGMP used to gate CNGA3 in phototransduction. Taken together, the evidence supports the existence in saccular hair cells of a molecular pathway linking CNGA3, its binding partner EMILIN1 (and beta1-integrin) and cGMP-specific PDE6C, which is potentially replicated in cochlear outer hair cells, given stereociliary immunolocalizations of CNGA3, EMILIN1 and PDE6C.

The antineurodegenerative agent clioquinol regulates the transcription factor FOXO1a

A R Cameron, K Wallace, L Logie, A R Prescott, T G Unterman, J Harthill, G Rena — 2012-01-16T14:43:21Z

Many diseases of ageing including Alzheimer’s Disease (AD) and type 2 diabetes (T2D) are strongly associated with common risk factors, such as hypertension, hyperglycaemia and hyperinsulinaemia, suggesting that there may be shared ageing mechanisms underlying these diseases, with scope to identify common cellular targets for therapy. Here we have studied insulin-like signalling properties of an experimental AD 8-hydroxyquinoline drug known as clioquinol. The insulin/IGF-1 signal transduction (IIS) kinase Akt/PKB inhibits the transcription factor FOXO1a by phosphorylating it on residues that trigger its exit from the nucleus and in 293 cells we found that clioquinol treatment induces similar effects. A key transcriptional response to IIS is inhibition of hepatic gluconeogenic gene expression and in rat liver cells, clioquinol represses expression of the key gluconeogenic regulatory enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase). The effects on FOXO1a and gluconeogenic gene expression require the presence of zinc ions, reminiscent of much earlier literature examining diabetogenic properties of 8-hydroxyquinolines. Comparative investigation of the signalling properties of a panel of these compounds demonstrates that CQ alone exhibits FOXO1a regulation without diabetogenicity. Our results suggest that zinc-dependent regulation of FOXOs and gluconeogenesis may contribute to the therapeutic properties of this drug. Further investigation of this signalling response might illuminate novel pharmacological strategies for the treatment of age-related diseases.

Biochemical identification of the OsMKK6-OsMPK3 signaling pathway for chilling stress tolerance in rice

G Xie, H Kato, R Imai — 2012-01-16T14:27:38Z

Mitogen-activated protein kinase (MAPK) pathways have been implicated in stress signaling in plants. In this study, we performed yeast two-hybrid screening to identify partner MAPKs for OsMKK6, a rice MAPK kinase, and revealed specific interactions of OsMKK6 with OsMPK3 and OsMPK6. OsMPK3 and OsMPK6 each co-immunoprecipitated OsMKK6, and both were directly phosphorylated by OsMKK6 in vitro. A MBP kinase assay of the immunoprecipitation complex indicated that OsMPK3 and OsMPK6 were activated in response to a moderately low temperature (12°C) but a severely low temperature (4°C) in rice seedlings. A constitutively active form of OsMKK6, OsMKK6DD, showed elevated phosphorylation activity against OsMPK3 and OsMPK6 in vitro. OsMPK3, but not OsMPK6, was constitutively activated in transgenic plants overexpressing OsMKK6DD, indicating that OsMPK3 is an in vivo target of OsMKK6. Enhanced chilling tolerance was observed in the transgenic plants overexpressing OsMKK6DD. Taken together, our data suggest that OsMKK6 and OsMPK3 constitute a moderately low-temperature signaling pathway and regulate cold stress tolerance in rice.

Lipocalin-type Prostaglandin D Synthase Protects Against Oxidative Stress-induced Neuronal Cell Death

A Fukuhara, M Yamada, K Fujimori, Y Miyamoto, T Kusumoto, H Nakajima, T Inui — 2012-01-16T14:00:57Z

Lipocalin-type prostaglandin D synthase (L-PGDS) is a dual functional protein, acting as a PGD₂-producing enzyme and a lipid-transporter. L-PGDS is a member of the lipocalin superfamily and can bind a wide variety of lipophilic molecules. Here we show the protective effect of L-PGDS on H₂O₂-induced apoptosis in neuroblastoma cell line SH-SY5Y. L-PGDS expression was increased in H₂O₂-treated neuronal cells, and the L-PGDS level was highly associated with H₂O₂-induced apoptosis, indicating that L-PGDS protected the neuronal cells against H₂O₂-mediated cell death. Cell viability assay revealed that L-PGDS protected against H₂O₂-induced cell death in a concentration-dependent manner. Further, the titration of free-thiols in H₂O₂-treated L-PGDS revealed that H₂O₂ reacted with the thiol of Cys65 of L-PGDS. The MALDI-TOF MS spectrum of H₂O₂-treated L-PGDS showed a 32-Da increase in the mass relative to that of the untreated protein, showing that the thiol was oxidized to sulfinic acid. The binding affinities of oxidized L-PGDS for lipophilic molecules were comparable to those of untreated L-PGDS. Taken together, these results demonstrate that L-PGDS protected against neuronal cell death by scavenging reactive oxygen species without losing its ligand-binding function. The novel function of L-PGDS could be useful for the suppression of oxidative stress-mediated neurodegenerative diseases.

The Guanine-Nucleotide Exchange Factor (GEF) P-Rex1 is Activated by Protein Phosphatase 1{alpha} (PP1{alpha})

2012-01-16T12:34:24Z

P‑Rex1 is a guanine-nucleotide exchange factor (GEF) for the small G protein Rac that is activated by PIP₃ and Gbg subunits and inhibited by PKA. Here, we show that Protein Phosphatase 1a (PP1a) binds P‑Rex1 through an RVxF-type docking motif. PP1a activates P‑Rex1 directly in vitro, both independently of and additively to PIP₃ and Gbg. PP1a also substantially activates P‑Rex1 in vivo, both in basal and PDGF- or LPA-stimulated cells. The phosphatase activity of PP1a is required for P‑Rex1 activation. PP1b, a close homologue of PP1a, is also able to activate P‑Rex1, but less effectively. PP1a stimulates P-Rex1-mediated, Rac-dependent changes in endothelial cell morphology. Mass spectrometric analysis of wild-type P‑Rex1 and a PP1a-binding deficient mutant revealed that endogenous PP1a dephosphorylates P‑Rex1 on at least three residues, S834, S1001 and S1165. Site-directed mutagenesis of S1165 to alanine caused activation of P-Rex1 to a similar degree as did PP1a, confirming S1165 as a dephosphorylation site important in regulating P‑Rex1 Rac-GEF activity. In summary, we have identified a novel mechanism for direct activation of P‑Rex1 through PP1a-dependent dephosphorylation.

The antioxidative effect of de novo generated vitamin B6 in Plasmodium falciparum validated by protein interference

2012-01-16T12:05:38Z

The malaria parasite Plasmodium falciparum is able to synthesize de novo pyridoxal-5-phosphate (PLP), the active form of vitamin B6. We show here that the de novo synthesized PLP is used by the parasite to detoxify singlet molecular oxygen (¹O₂), a highly destructive reactive oxygen species arising from hemoglobin digestion. The formation of singlet oxygen and the response of the parasite were monitored by life cell fluorescence microscopy, by transcription analysis and by determination of PLP levels in the parasite, respectively. Pull-down experiments of transgenic parasites overexpressing the vitamin B6 biosynthetic enzymes PfPdx1 and PfPdx2 clearly demonstrated an interaction of the two proteins in vivo which results in an elevated PLP level from 12.5 microM in wild-type parasites to 36.6 microM in the Pdx1/Pdx2-overexpressing cells and thus to a higher tolerance towards ¹O₂. In contrast, by applying cellular protein interference using inactive Pdx1 and Pdx2 mutants P. falciparum became susceptible to singlet oxygen. Our results clearly demonstrate the crucial role of vitamin B6 biosynthesis in the detoxification of singlet oxygen in P. falciparum. Besides the known role of PLP as a cofactor of many essential enzymes this second important task of the vitamin B6 de novo synthesis as antioxidant emphasizes the high potential of this pathway as a target of new anti-malarial drugs.

Novel structural arrangement of nematode cystathionine beta-synthases: characterization of Caenorhabditis elegans CBS-1

R Vozdek, A Hnizda, J Krijt, M Kostrouchova, V Kozich — 2012-01-13T12:14:15Z

Cystathionine beta-synthases are eukaryotic pyridoxal 5'-phosphate (PLP)-dependent proteins that maintain cellular homocysteine homeostasis and produce cystathionine and hydrogen sulfide. In this study, we describe a novel structural arrangement of the cystathionine beta-synthase (CBS) enzyme encoded by the cbs-1 gene of the nematode Caenorhabditis elegans. The CBS-1 protein contains a unique tandem repeat of two evolutionarily conserved catalytic regions in a single polypeptide chain. These repeats include a catalytically active C-terminal module containing a PLP-binding site and a less conserved N-terminal module that is unable to bind the PLP cofactor and cannot catalyze CBS reactions, as demonstrated by analysis of truncated variants and active site-mutant proteins. In contrast to other metazoan enzymes, CBS-1 lacks the heme and the regulatory Bateman domain essential for activation by S-adenosylmethionine and only forms monomers. We determined the tissue and subcellular distribution of CBS-1 and showed that cbs-1 knockdown by RNA interference leads to delayed development and to an approximately 10-fold elevation of homocysteine concentrations in nematode extracts. This paper provides the first insight into the metabolism of sulfur amino acids and hydrogen sulfide in C. elegans and shows that nematode cystathionine beta-synthases possess a structural feature that is unique among CBS protein.

Transcriptional regulation mechanisms of Hypoxia-induced neuroglobin gene expression

N liu, Z Yu, S Xiang, S Zhao, A Tjärnlund Wolf, C Xing, J Zhang, X Wang — 2012-01-13T11:45:00Z

Neuroglobin (Ngb) has been identified as a novel endogenous neuroprotectant. However, little is known about the regulation mechanisms of Ngb expression, especially under hypoxia conditions. In this study, we located the core proximal promoter of mouse Ngb gene to a 554 bp segment, which harbors putative conserved NFκB, Egr1 binding sites. Over-expression and knock-down of transcription factors p65, p50, Egr1 or Sp1 increased and decreased Ngb expression, respectively. Experimental assessments with transfections of mutational Ngb gene promoter constructs, as well as EMSA and ChIP assays demonstrated that NFκB family members (p65, p50, cRel), Egr1, and Sp1 bound in vitro and in vivo to the proximal promoter region of Ngb gene. Moreover, a κB3 site was found as a pivotal cis-element responsible for hypoxia-induced Ngb promoter activity. NFκB (p65) and Sp1 were also responsible for hypoxia-induced upregulation of Ngb expression. Although there are no conserved HREs (hypoxia-response elements) in the promoter of mouse Ngb gene, our results suggested that HIF1α is also involved in hypoxia-induced Ngb upregulation. In conclusion, we identified that NFκB, Egr1, and Sp1 played important roles in regulation of basal Ngb expression via specific interactions with the mouse Ngb promoter. NFκB, Sp1 and HIF1α contributed to the upregulation of mouse Ngb gene expression under hypoxic conditions.

3{'}-5{'}phosphoadenosine phosphate is an inhibitor of Poly(ADP-ribose) Polymerase 1 and a potential mediator of the lithium-dependent inhibition of PARP-1 in vivo

E Toledano, V Ogryzko, A Danchin, D Ladant, U Mechold — 2012-01-12T14:34:48Z

3’-5’phosphoadenosine phosphate (pAp) is a by-product of sulfur and lipid metabolism and has been shown to have strong inhibitory properties on RNA catabolism. We report here a new target of pAp, Poly(ADP-ribose) Polymerase 1 (PARP-1), a key enzyme in the detection of DNA single strand breaks. We show that pAp can interact with PARP-1 and inhibit its poly(ADP-ribosyl)ation activity. In vitro, inhibition of PARP-1 was detectable at micromolar concentrations of pAp and altered both PARP-1 automodification and heteromodification of histones. Analysis of the kinetic parameters revealed that pAp acted as a mixed inhibitor that modulates both the K_M and the V_M of PARP-1. In addition, we showed that upon treatment by lithium, a very potent inhibitor of the enzyme responsible of pAp recycling, HeLa cells exhibited a reduced level of poly(ADP-ribosyl)ation in response to oxidative stress. From these results, we propose that pAp might be a physiological regulator of PARP-1 activity.

Distinct roles in folding, CD81 receptor binding and viral entry for conserved histidines of HCV glycoprotein E1 and E2.

I Boo, K teWierek, F Douam, D LAVILLETTE, P Poumbourios, H Edelgard Drummer — 2012-01-12T14:09:38Z

The protonation of histidine in acidic environments underpins its role in regulating the function of pH-sensitive proteins. For pH-sensitive viral fusion proteins, His protonation in the endosome leads to the activation of their membrane fusion function. The hepatitis C virus (HCV) glycoprotein E1/E2 heterodimer mediates membrane fusion within the endosome but the roles of conserved histidines in the formation of a functional heterodimer and in sensing pH changes is unknown. We examined the functional roles of conserved histidines located within E1 and E2. The E1 mutations, H222A/R, H298R, and H352A, disrupted E1-E2 heterodimerization and reduced virus entry. Five of 6 histidines located within the E2 receptor-binding domain (RBD) were important for the E2 fold, their substitution with Arg or Ala causing aberrant heterodimerization and/or CD81 binding. Distinct roles in E1/E2 heterodimerization and in virus entry were identified for His-691 and His-693, respectively, within the membrane-proximal stem region. Viral entry and cell-cell fusion at neutral and low pH were enhanced with H445R, indicating that the protonation state of His-445 is a key regulator of HCV fusion. However, H445R did not overcome the block to virus entry induced by bafilomycin A1 indicating a requirement for an endosomal activation trigger in addition to acidic pH.

Caveolae Optimize Tissue Factor-Factor VIIa Inhibitory Activity of Cell Surface Associated Tissue Factor Pathway Inhibitor

S A. Maroney, P E. Ellery, J P. Wood, J P. Ferrel, C E. Bonesho, A E. Mast — 2012-01-12T13:53:45Z

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that prevents intravascular coagulation through inhibition of factor Xa (fXa) and the tissue factor (TF)-factor VIIa complex (TF-fVIIa). Localization of TFPI within caveolae enhances its anticoagulant activity. To further define how caveolae contribute to TFPI anticoagulant activity, CHO cells were co-transfected with TF and membrane associated TFPI targeted to either caveolae (TFPI-GPI) or to bulk plasma membrane (TFPI-TM). Stable clones had equal expression of surface TF and TFPI. TX-114 cellular lysis confirmed localization of TFPI-GPI to detergent insoluble membrane fractions, while TFPI-TM localized to the aqueous phase. TFPI-GPI and TFPI-TM were equally effective direct inhibitors of fXa in amidolytic assays. However, TFPI-GPI was a significantly better inhibitor of TF-FVIIa than TFPI-TM, as measured in both amidolytic and plasma clotting assays. Disrupting caveolae by removing membrane cholesterol from EA.hy926 cells, which make TFPIa, CHO cells transfected with TFPIb, and HUVECs, did not affect their fXa inhibition but significantly decreased their inhibition of TF-fVIIa. These studies confirm and quantify the enhanced anticoagulant activity of TFPI localized within caveolae, demonstrate that caveolae enhance the inhibitory activity of both TFPI isoforms, and define the effect of caveolae as specifically enhancing the anti-TF activity of TFPI.

Phosphate is Not an Absolute Requirement for The Inhibitory Effects of Cyclosporine-A or Cyclophilin-D Deletion on Mitochondrial Permeability Transition

A M McGee, C P Baines — 2012-01-11T16:38:05Z

Cyclophilin-D (CypD) has been established as a critical regulator of the mitochondrial permeability transition (MPT) pore, and pharmacological or genetic inhibition of CypD attenuates MPT in numerous systems. However, it has recently been suggested that the inhibitory effects of CypD inhibition are only manifest when phosphate (P_i) is present, and that inhibition is lost when P_i is substituted with arsenate (As_i) or vanadate (V_i). To test this, liver mitochondria were isolated from wildtype and CypD-deficient (Ppif^-/-) mice and then incubated in buffer containing P_i, As_i, or V_i. MPT was induced under both energized and de-energized conditions by addition of Ca²⁺, and the resultant mitochondrial swelling measured spectrophotometrically. For pharmacological inhibition of CypD, wildtype mitochondria were pre-incubated with cyclosporine-A (CsA) prior to the addition of Ca²⁺. In energized and de-energized mitochondria, Ca²⁺ induced MPT regardless of the anion present, although the magnitude differed between P_i, As_i, and V_i. However, in all cases, pretreatment with CsA significantly inhibited MPT. Moreover, these effects were independent of mouse strain, organ type, and rodent species. Similarly, attenuation of Ca²⁺-induced MPT in the Ppif^-/- mitochondria was still observed irrespective of whether P_i, As_i, or V_i was present. We conclude that the pharmacological and genetic inhibition of CypD is still able to attenuate MPT even in the absence of P_i.

A CRITICAL TYROSINE RESIDUE OF THE MITOCHONDRIAL OXALOACETATE CARRIER DETERMINES ITS UNCOUPLING PROTEIN (UCP)-LIKE FUNCTION IN YEAST.

2012-01-11T14:10:00Z

The mitochondrial oxaloacetate carrier (Oac) that can be found in fungi and plants catalyzes the uptake of oxaloacetate, malonate and sulfate. Despite their sequence similarity, transport specificity varies considerably between Oac’s. Indeed, while in Saccharomyces cerevisiae the Oac (ScOac) is a specific anion-proton symporter, the Yarrowia lipolytica Oac (YlOac) has the added ability to transport protons, thereby mimicking an uncoupling protein (UCP). Significantly, we identified two amino acid changes at the matrix gate of YlOac and ScOac, Tyr/Phe and Met/Leu, and we studied their role by expressing them in an Oac-null S. cerevisiae strain following site-directed mutagenesis. No phenotype could be associated to the Met/Leu substitution, whereas UCP-like activity was dependent on the presence of the Tyr normally expressed in the YlOac, i.e., Tyr-ScOac also mediated proton transport, while Phe-YlOac lost its protonophoric activity. These findings indicate that, the UCP-like activity of YlOac is determined by a single tyrosine residue.

AMP-activated protein kinase phosphorylates and inactivates liver glycogen synthase

2012-01-11T12:12:00Z

Recombinant muscle glycogen synthase-1 (GYS1) and recombinant liver glycogen synthase-2 (GYS2) were phosphorylated by recombinant AMP-activated protein kinase (AMPK) in a time-dependent manner and to a similar stoichiometry. The phosphorylation site in GYS2 was identified as Ser7, which lies in a favorable consensus for phosphorylation by AMPK. Phosphorylation of GYS1 or GYS2 by AMPK led to enzyme inactivation by decreasing the affinity for both UDP-Glc (assayed in the absence of Glc-6-P) and Glc-6-P (assayed at low UDP-Glc concentrations). Incubation of freshly isolated rat hepatocytes with pharmacological AMPK activators 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICA riboside) or A769662 led to persistent GYS inactivation and Ser7 phosphorylation, whereas inactivation by glucagon treatment was transient. In hepatocytes from mice harboring a liver-specific deletion of the AMPK catalytic a1/a2 subunits, GYS2 inactivation by AICA riboside and A769662 was blunted whereas inactivation by glucagon was unaffected. The results suggest that GYS inactivation by AMPK activators in hepatocytes is due to GYS2 Ser7 phosphorylation.

Activating transcription factor 4-dependent induction of FGF21 during amino acid deprivation.

A De Sousa-Coelho, P F. Marrero, D Haro — 2012-01-11T12:05:00Z

Nutrient deprivation or starvation frequently correlates with amino acid limitation. Amino acid starvation initiates a signal transduction cascade starting with the activation of the kinase GCN2 phosphorylation of eukaryotic initiation factor 2, global protein synthesis reduction and increased activating transcription factor (ATF) 4. ATF4 modulates a wide spectrum of genes involved in the adaptation to dietary stress. The hormone FGF21 is induced during fasting in liver, and its expression induces a metabolic state that mimics long-term fasting. Thus, FGF21 is critical for the induction of hepatic fat oxidation, ketogenesis and gluconeogenesis, metabolic processes which are essential for the adaptive metabolic response to starvation. In this article, we show that FGF21 is induced by amino acid deprivation in both mouse liver and HepG2 cultured cells. We have identified the human FGF21 gene as a target gene for ATF4 and we have localized two conserved ATF4 binding sequences in the 5’ regulatory region of the human FGF21 gene, which are responsible for the ATF4-dependent transcriptional activation of this gene. These results add FGF21 gene induction to the transcriptional program initiated by increased levels of ATF4 and offer a new mechanism for the induction of the FGF21 gene expression under nutrient deprivation.

Protein kinase D2 has a restricted but critical role in T cell antigen receptor signaling in mature T cells

2012-01-10T16:35:00Z

Protein Kinase D2 (PKD2) is a serine/threonine kinase activated by diacylglycerol in response to engagement of antigen receptors in lymphocytes. To explore PKD2 regulation and function in T cell antigen receptor (TCR) signal transduction we expressed TCR complexes with fixed affinity for self antigens in T cells of PKD2 null mice or mice deficient in PKD2 catalytic activity. We also developed a single cell assay to quantify PKD2 activation as T cells respond to developmental stimuli or engagement of a/b TCR complexes in vivo. Strikingly, PKD2 loss caused increases in thymic output, lymphadenopathy and splenomegaly in TCR transgenic mice. The precise magnitude and timing of PKD2 activation during T cell development is thus critical to regulate thymic homeostasis. PKD2 null T cells that exit the thymus have a normal transcriptome but show a limited and abnormal transcriptional response to antigen. Transcriptional profiling reveals the full consequences of PKD2 loss and maps in detail the selective but critical function for PKD2 in signaling by a/b mature TCR complexes in peripheral T cells.

A novel, non-canonical mechanism of regulation of mammalian Ste20-related kinase 3 (MST3)

2012-01-09T16:15:00Z

The canonical pathway of regulation of the germinal centre kinase (GCK) III subgroup member, mammalian Sterile20-related kinase 3 (MST3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr¹⁷⁸-), induction of Ser-/Thr-protein kinase activity and nuclear localisation. We identified an alternative ‘non-canonical’ pathway of MST3 activation (regulated primarily through dephosphorylation) which may also be applicable to other GCKIII (and GCKVI) subgroup members. In the basal state, inactive MST3 co-immunoprecipitated with the Golgi protein, GOLGA2/gm130. Activation of MST3 by calyculin A (a protein Ser-/Thr- phosphatase 1/2A inhibitor) stimulated (auto)phosphorylation of MST3(Thr¹⁷⁸-) in the catalytic domain with essentially simultaneous cis-autophosphorylation of MST3(Thr³²⁸-) in the regulatory domain, an event also requiring the MST3(341-376) sequence which acts as a putative docking domain. MST3(Thr¹⁷⁸-) phosphorylation increased MST3 kinase activity but this activity was independent of MST3(Thr³²⁸-) phosphorylation. Interestingly, MST3(Thr³²⁸-) lies immediately C-terminal to a STRAD pseudokinase-like site recently identified as being involved in binding of GCKIII/GCKVI members to MO25 scaffolding proteins. MST3(Thr¹⁷⁸- /Thr³²⁸-) phosphorylation was concurrent with dissociation of MST3 from GOLGA2/gm130 and association of MST3 with MO25, and MST3(Thr³²⁸-) phosphorylation was necessary for formation of the activated MST3-MO25 holocomplex.

The importance of an asymmetric distribution of acidic lipids for Synaptotagmin 1 function as a Ca2+ sensor

Y Lai, Y Shin — 2012-01-09T15:31:00Z

Synaptotagmin 1 (Syt1) is a major Ca²⁺ sensor for synaptic vesicle fusion. Although Syt1 is known to bind to SNARE complexes and to the membrane, the mechanism by which Syt1 regulates vesicle fusion is controversial. Here we used in vitro lipid mixing assays to investigate the Ca²⁺-dependent Syt1 function in proteoliposome fusion. To study the role of acidic lipids, the concentration of negatively charged DOPS in the vesicle was varied. Syt1 stimulated lipid mixing by factors of 3-10 without Ca²⁺. However, with Ca²⁺ there was additional factors of 2-5 enhancement. But the Ca²⁺-dependent stimulation was observed only when there was excess PS on the t-SNARE side, and if there was equal or more PS on v-SNARE side, the Ca²⁺-dependent stimulation was not observed. We found that Ca²⁺ in a few ten µM level was sufficient to give rise to the maximal enhancement. The single vesicle fusion assay indicates that the Ca²⁺-dependent enhancement was mainly on docking while its effect on lipid mixing was small. Thus, for Syt1 to function as a Ca²⁺ sensor, a charge asymmetry appears to be important and it may play a role in steering Syt1 to make productive trans binding to the plasma membrane.

Serum Copper as a Novel Biomarker for Resistance to Thyroid Hormone

2012-01-06T11:16:00Z

Thyroid hormone action is mediated by the thyroid hormone receptors TRa1 and TRb. Defects in TRb lead to “Resistance to Thyroid Hormone” (RTHb), a syndrome characterized by high levels of thyroid hormone and non-suppressed thyroid-stimulating hormone (TSH). However, a correct diagnosis of RTHb patients is difficult as the clinical picture varies. A biochemical serum marker indicative of defects in TRb signaling is needed and could simplify the diagnosis of RTHb, in particular the differentiation to TSH-secreting pituitary adenomas, which present with clinically similar symptoms. Here we show that serum copper levels are regulated by thyroid hormone, which stimulates the synthesis and the export of the hepatic Cu-transport protein ceruloplasmin into the serum. This is accompanied by a concerted reduction of the mRNA levels of other Cu-containing proteins such as metallothioneins 1 and 2 or superoxide dismutase 1. The induction of serum Cu was abolished in genetically hyperthyroid mice lacking TRb and human RTHb patients, demonstrating an important role of TRb for this process. Together with a previously reported TRa1 specific regulation of serum selenium, we furthermore show that the ratio of serum Cu and Se – which is largely independent of thyroid hormone levels, volume changes or sample degradation – can constitute a valuable novel biomarker for RTHb. Moreover, it could also provide a suitable large-scale screening parameter to identify RTHa patients, which have not been identified to date.

Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol monooxygenase

2012-01-06T10:57:00Z

Alkylglycerol monooxygenase (E.C. 1.14.16.5) forms a third, distinct class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a diiron centre, which comprises eight conserved histidines. Membrane enzymes containing this motif, including alkylglycerol monooxygenase, are especially labile and could not be purified to homogeneity in active form so far. To get a first insight on structure-function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild type and mutant proteins containing a C-terminal myc tag together with fatty aldehyde dehydrogenase in Chinese hamster ovary cells. Among all acidic residues within the eight-histidine motif, only mutation of glutamate 137 to alanine led to an 18-fold increase in the Michaelis Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine essential for activity was identified. Nine membrane domains were predicted by 4 programs ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using Rosetta-Membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol monooxygenase.

The Q43L Mutant of Neuregulin 2beta Is A Pan-ErbB Receptor Antagonist

2012-01-05T10:32:00Z

The ErbB4 receptor tyrosine kinase possesses both tumor suppressor and oncogenic activities. Thus, pharmacologic agents are needed to help elucidate ErbB4 functions. However, limitations of existing ErbB4 agonists and antagonists have led us to seek novel ErbB4 antagonists. The Q43L mutant of the ErbB4 agonist NRG2beta stimulates ErbB4 tyrosine phosphorylation, yet fails to stimulate ErbB4 coupling to cell proliferation. Thus, here we hypothesize that NRG2beta/Q43L may be an ErbB4 antagonist. NRG2beta/Q43L competitively antagonizes agonist stimulation of ErbB4 coupling to cell proliferation. NRG2beta/Q43L stimulates less ErbB4 tyrosine phosphorylation than does NRG2beta. In addition, NRG2beta stimulation of cell proliferation requires PI3K activity and NRG2beta stimulates greater Akt phosphorylation than does NRG2beta/Q43L. Moreover, EGFR kinase activity (but not that of ErbB4) is critical for coupling ErbB4 to proliferation. Experiments utilizing ErbB4 splicing isoforms and mutants suggest that NRG2beta and NRG2beta/Q43L may differentially stimulate ErbB4 coupling to the transcriptional coregulator YAP. Finally, NRG2beta/Q43L competitively antagonizes agonist stimulation of EGFR and ErbB2/ErbB3, indicating that NRG2beta/Q43L is a pan-ErbB antagonist. Thus, we postulate that NRG2beta/Q43L and other antagonistic ligands stimulate ErbB tyrosine phosphorylation on a set of residues distinct from that stimulated by agonists, thus suggesting a novel mechanism of ErbB receptor regulation. Moreover, NRG2beta/Q43L and related ligand-based antagonists establish a paradigm for the discovery of anti-ErbB therapeutics.

Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase

2012-01-04T15:17:00Z

Eukaryotic elongation factor 2 kinase (eEF2K) phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily but instead belongs to a small group of so-called a-kinases. The activity of eEF2K is normally dependent upon Ca²⁺ and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. Here we identified several autophosphorylation sites including Thr-348, Thr-353, Ser-366 and Ser-445, all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser-78, a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser-78, Thr-348 and Ser-366 to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr-348 was detected by immunoblotting after transfecting wild-type eEF2K into HEK 293 cells, but not after transfection with a kinase-inactive construct confirming that it is indeed a site of autophosphorylation. Thr-348 appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other a-kinases is also autophosphorylated and contributes to the activation of these enzymes (Crawley et al., J. Biol. Chem. 2011, 286, 2607-2616). Ser-366 phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is not only phosphorylated autocatalytically but also in trans by other kinases.

Autoinhibition Mechanism of the Plasma Membrane Calcium Pump Isoforms 2 and 4 Studied Through Lipid-Protein Interaction

2012-01-04T12:05:00Z

The autoinhibition/activation of the plasma membrane calcium pump (PMCA) involves conformational changes in the membrane region of the protein that affect the amount of lipids directly associated with the transmembrane domain. The lipid-protein dependence of PMCA isoforms 2 and 4 expressed and obtained in purified form from Saccharomyces cerevisiae, was investigated using the phosphatidylcholine analogue [¹²⁵I]TID-PC/16, which was incorporated into mixtures of dimyristoyl-phosphatidylcholine and the nonionic detergent C₁₂E₁₀. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca²⁺-calmodulin of PMCA2 showed 30 times higher affinity than PMCA4. PMCA2 exhibited a lower level labeling in the autoinhibited conformation relative to PMCA4 indicating that the lower autoinhibition was correlated with a lower exposure to lipids in the autoinhibited state. Analysis of the lipid:protein stoichiometry show that the lipid annulus of PMCA varies: (i) in accordance to the conformational state of the enzyme and (ii) depending on the different isoforms of PMCA. PMCA2 during Ca²⁺transport changes its conformation to a lesser extent than PMCA4, an isoform more sensitive to modulation by calmodulin and acidic phospholipids. This is the first demonstration of a dynamic behavior of annular lipids and PMCA.

Structure-function studies of a plant tyrosyl-DNA phosphodiesterase provide novel insights into DNA repair mechanisms of Arabidopsis thaliana

H Kim, S Na, S Lee, Y Jeong, H Hwang, J Hur, S Park, J Woo, S Kim — 2012-01-03T16:40:00Z

Tyrosyl-DNA phosphodiesterase 1 (TDP1), a member of the phospholipase D (PLD) superfamily, catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and the 3′-phosphate of DNA. We previously identified and characterized the AtTDP gene in Arabidopsis thaliana, an orthologue of yeast and human TDP1 genes (Lee et al (2010) Plant Physiology 154; 1460-1469). Sequence alignment of TDP1 orthologues revealed that AtTDP has both a conserved C-terminal TDP domain and, uniquely, an N-terminal SMAD/forkhead-associated (FHA) domain. To help understand the function of this novel enzyme, we analyzed the substrate saturation kinetics of fulllength AtTDP versus a truncated AtTDP mutant lacking the N–terminal FHA domain. The recombinant AtTDP protein hydrolyzed a single-stranded DNA substrate with K_m and k_cat / K_m values of 703 ± 137 nM and 1.5 x 10⁹ ± 0.04 x 10⁹ M^-1 min^-1, respectively. The AtTDP D1–122 protein (TDP domain) showed kinetic parameters that were equivalent to those of the fulllength AtTDP protein. A basic amino acid sequence (RKKVKP) within the AtTDP D123–605 protein (FHA domain) was necessary for nuclear localization of AtTDP. Analysis of active site mutations showed that a histidine and a lysine residue in each of the HKD motifs were critical for enzyme activity. Vanadates, inhibitors of phosphoryl transfer reactions, inhibited AtTDP enzymatic activity and retarded the growth of an Arabidopsis tdp mutant. Finally, we showed that expression of the AtTDP gene could complement a yeast tdp1Drad1D mutant, rescuing the growth inhibitory effects of vanadate analogs and camptothecin (CPT). Taken together, our data demonstrate the structure-based function of AtTDP through which AtTDP can repair DNA strand breaks in plants.

Fructose 2,6-bisphosphate is essential for glucose-regulated gene transcription of glucose 6-phosphatase and other ChREBP target genes in hepatocytes

2012-01-03T16:08:00Z

Glucose metabolism in the liver activates transcription of various genes encoding enzymes of glycolysis and lipogenesis and also glucose 6-phosphatase (G6pc). Allosteric mechanisms involving glucose 6-P or xylulose 5-P and covalent modification of ChREBP have been implicated in this mechanism. However evidence supporting an essential role for a specific metabolite or pathway in hepatocytes remains equivocal. By using diverse substrates and inhibitors and a kinase-deficient bisphosphatase-active variant of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/FBP2), we demonstrate an essential role for fructose 2,6-bisphosphate in the induction of G6pc and other ChREBP target genes by glucose. Selective depletion of fructose 2,6-bisphosphate inhibits glucose-induced recruitment of ChREBP to the G6pc promoter and also induction of G6pc by xylitol and gluconeogenic precursors. The requirement for fructose 2,6-bisphosphate for ChREBP recruitment to the promoter does not exclude involvement of additional metabolites acting either co-ordinately or at downstream sites. Glucose raises fructose 2,6-bisphosphate in hepatocytes by reversing the phosphorylation of PFK2/FBP2 at ser32 but also independently of ser32 dephosphorylation. This supports a role for the bifunctional enzyme as the phosphometabolite sensor and for its product, fructose 2,6-bisphosphate, as the metabolic signal for substrate-regulated ChREBP-mediated expression of G6pc and other ChREBP target genes.

Transcriptional control of glyoxalase 1 by Nrf2 provides a stress responsive defence against dicarbonyl glycation

2011-12-22T10:55:00Z

Abnormal cellular accumulation of the dicarbonyl metabolite methylglyoxal occurs on exposure to high glucose concentration, inflammation, cell ageing and senescence. It is associated with increased methylglyoxal-adduct content of protein and DNA linked to increased DNA strand breaks and mutagenesis, mitochondrial dysfunction and reactive oxygen species formation and cell detachment from the extracellular matrix. Methylglyoxal–mediated damage is countered by glutathione-dependent metabolism by glyoxalase-1. It is not known, however, if glyoxalase-1 has stress responsive up-regulation to counter periods of high methylglyoxal concentration or dicarbonyl stress. We identified a functional antioxidant response element in the 5’-untranslated region of exon-1 of the mammalian glyoxalase-1 gene. Transcription factor Nrf2 binds to this antioxidant response element increasing basal and inducible expression of glyoxalase 1. Activators of Nrf2 induced increased glyoxalase-1 mRNA, protein and activity. Increased expression of glyoxalase-1 decreased cellular and extracellular concentrations of methylglyoxal, methylglyoxal -derived protein adducts, mutagenesis and cell detachment. Hepatic, brain, heart, kidney and lung glyoxalase-1 mRNA and protein were decreased in Nrf2 (-/-) mice and urinary excretion of methylglyoxal protein and nucleotide adducts were increased ca. 2-fold. We conclude that dicarbonyl stress is countered by up-regulation of glyoxalase-1 in the Nrf2 stress responsive system, protecting protein and DNA from increased damage and preserving cell function.

The human gene SLC25A17 encodes a peroxisomal transporter of coenzyme A, FAD and NAD+

G Agrimi, A Russo, P Scarcia, F Palmieri — 2011-12-21T12:59:00Z

The essential cofactors coenzyme A (CoA), FAD and NAD⁺ are synthesized outside the peroxisomes and therefore must be transported into the peroxisomal matrix where they are required for important processes. In this work we have functionally identified and characterized SLC25A17, which is the only member of the mitochondrial carrier family that has previously been shown to be localized in the peroxisomal membrane. Herein, recombinant and purified SLC25A17 was reconstituted into liposomes. Its transport properties and kinetic parameters demonstrate that SLC25A17 is a transporter of CoA, FAD, FMN, AMP and to a lesser extent of NAD⁺, adenosine 3’,5’-diphosphate (PAP) and ADP. SLC25A17 functioned almost exclusively by a counter-exchange mechanism, was saturable and inhibited by pyridoxal-5’-phosphate and other mitochondrial carrier inhibitors. It was expressed to various degrees in all the human tissues examined. Its main function is probably to transport free CoA, FAD and NAD⁺ into peroxisomes in exchange for intraperoxisomally generated PAP, FMN and AMP. This is the first report describing the identification and characterization of a transporter for multiple free cofactors in peroxisomes.

A proximal pair of positive charges provides the dominant ligand binding contribution to complement-like domains from the low density lipoprotein receptor-related protein (LRP)

P GW Gettins, K Dolmer — 2011-12-20T12:00:00Z

The low density lipoprotein receptor-related protein (LRP) can bind a wide range of structurally diverse ligands to regions composed of clusters of ~40 residue Ca²⁺-dependent, disulfide-rich, complement-like repeats (CR). While lysine residues from the ligands have been implicated in binding, there has been no quantitation of the energetic contributions of such interactions and hence of their relative importance in overall affinity, or of the ability of arginine or histidine to bind. We have used four representative CR domains from the principal ligand cluster of LRP to determine the energetics of interaction with well defined small ligands, that include methyl esters of lysine, arginine, histidine, and aspartic acid, as well as N-terminally blocked lysine methyl ester. We found that, not only lysine, but also arginine and histidine bound well, and when present with an additional proximal positive charge, accounted for about half the total binding energy of a protein ligand such as PAI-1. Two such sets of interactions, one to each of two CR domains could thus account for almost all the necessary binding energy of a real ligand such as PAI-1. For the CR domains, a central aspartate in the sequence DxDxD tightens Kd by ~20-fold, while DxDDD is no more effective. Together these findings establish the rules for determining binding specificity for protein ligands to LRP and to other LDL receptor family members.

Crystal structure of the Sox4 HMG/DNA complex suggests a mechanism for the positional interdependence in DNA recognition

R Jauch, C Keow Leng Ng, K Narasimhan, P R Kolatkar — 2011-12-19T14:40:00Z

It has recently been proposed that the sequence preferences of DNA-binding transcription factors can be well described by models that include the positional interdependence of the nucleotides of the target sites. Such binding models allow for multiple motifs to be invoked, such as principal and secondary motifs differing at two or more nucleotide positions. However, the structural mechanisms underlying the accommodation of such variant motifs by TFs remain elusive. Here we present the crystal structure of the high-mobility group (HMG) domain of Sox4 bound to DNA. By comparing this structure with previously solved structures of Sox17 and Sox2 we observed subtle conformational differences at the DNA binding interface. Furthermore, using quantitative electrophoretic mobility shift assays (EMSAs) we validated the positional interdependence of two nucleotides and the presence of a secondary Sox motif in the affinity landscape of Sox4. These data suggest that a concerted rearrangement of two interface amino acids enables Sox4 to accommodate primary and secondary motifs. The structural adoptions lead to altered dinucleotide preferences that mutually reinforce each other. These analyses underline the complexity of the DNA recognition by TFs and provide an experimental validation for the conceptual framework of positional interdependence and secondary binding motifs.

Novel Sterol Metabolic Network of Trypanosoma brucei Procyclic and Bloodstream Forms

2011-12-19T12:20:00Z

Trypanosoma brucei is the protozoan parasite that causes African trypanosomiasis, a neglected disease of people and animals. Co-metabolite analysis, labeling studies using [methyl-²H₃]methionine and substrate/product-specificities of the cloned sterol C24-methyl transferase (24-SMT) and sterol C14-demethylase (14-SDM) from T. brucei afforded an uncommon sterol metabolic network that proceeds from lanosterol and 31-norlanosterol to ergosta-5,7,25(27)-trien-3β-ol (ETO), 24-dimethyl ergosta-5,7,25(27)-trienol (DTO) and ergosta-5,7,22(23)-trienol (ergosterol). To assess the possible carbon sources of ergosterol biosynthesis, specifically ¹³C-labeled specimens of lanosterol, acetate, leucine and glucose were administered to T. brucei and the ¹³C distributions found were in accord with the operation of the acetate-mevalonate pathway, with leucine as an alternate precursor, to ergostenols in either the insect or bloodstream form. In searching for metabolic signatures of procyclic cells, we observed that the ¹³C-labeling treatments induce fluctuations between the acetyl-CoA (mitochondrial) and sterol (cytosolic) synthetic pathways detected in the progressive increase in ¹³C-ergosterol production (control < [2-¹³C]leucine < [2-¹³C]acetate < [1-¹³C]glucose) and corresponding depletion of cholesta-5,7,24-trienol. We conclude that anabolic fluxes originating in mitochondrial metabolism constitute a flexible part of sterol synthesis that is further fluctuated in the cytosol yielding distinct sterol profiles in relation to cell demands on growth.

The yeast vacuolar Rab GTPase Ypt7p has an activity beyond membrane recruitment of the HOPS/Class C Vps complex

C Stroupe — 2011-12-16T11:28:00Z

A previous report described lipid mixing of reconstituted proteoliposomes made using lipid mixtures that mimic the composition of yeast vacuoles. This lipid mixing required SNARE proteins, Sec18p and Sec17p (yeast NSF[1] and a-SNAP), and the HOPS/Class C Vps complex, but not the vacuolar Rab GTPase Ypt7p. The present study investigates the activity of Ypt7p in proteoliposome lipid mixing. Ypt7p is required for lipid mixing of proteoliposomes lacking cardiolipin. Omission of other lipids with negatively charged and/or small headgroups does not cause Ypt7p dependence for lipid mixing. Yeast vacuoles made from strains disrupted for cardiolipin synthase (CRD1) fuse to the same extent as vacuoles from strains with functional CRD1. Disruption of CRD1 does not alter dependence on Rab GTPases for vacuole fusion. It has been proposed that HOPS complex recruitment to membranes is the main function of Ypt7p. However, Ypt7p is still required for lipid mixing even when the concentration of HOPS complex in lipid-mixing reactions is adjusted such that cardiolipin-free proteoliposomes with or without Ypt7p bind equal amounts of HOPS. Ypt7p therefore must stimulate membrane fusion by a mechanism in addition to HOPS-membrane recruitment. This is the first demonstration of such a stimulatory activity – that is, beyond bulk effector recruitment – for a Rab GTPase.

New mimetic peptides of Kinase Inhibitory Region (KIR) of SOCS1 through focused peptide libraries

2011-12-14T12:38:00Z

Suppressor Of Cytokine Signalling (SOCS) proteins are negative feedback regulators of the Janus Kinase (JAK) and Signal Transducer and Activator of Transcription (STAT) pathway. Their expression levels are low in physiological conditions, but they are up-regulated in response to cytokine stimulation in many immune and inflammatory processes. Overexpression of SOCS1 in keratinocyte clones abrogates the IFN-γ-induced expression of many pro-inflammatory genes and the release of related chemokines by blocking the JAK-STAT pathway. SOCS1 inhibits JAK2 kinase activity by binding the catalytic site of JAK2, with its Kinase Inhibitory Region (KIR) acting as a pseudo-substrate of the enzyme. Here we screened a focused combinatorial peptide library of KIR to identify new peptides able to mimic its function with an improved affinity towards the JAK2 catalytic site. Using an Ala-scanning method, KIR residues that are crucial for the interaction with JAK2 were unveiled. In this way the KIR sequence was restricted to a shorter segment and “non-essential” residues were substituted with different amino acids following a simplified combinatorial approach. We selected a new unnatural sequence able to bind to JAK2 with K_D values in the nanomolar range. This peptide was tested in human keratinocyte cultures and reduced the phosphorylation of STAT1 and the expression levels of the Interferon Regulatory Factor-1 (IRF-1).

I{kappa}B Kinase {beta} (IKK{beta}) does not mediate feedback inhibition of the insulin-signaling cascade

2011-12-14T12:17:00Z

Herein, we have examined whether IκB Kinase β (IKKβ) plays a role in feedback inhibition of the insulin-signaling cascade. Insulin induces the phosphorylation of IKKβ, in vitro and in vivo, and this effect is dependent on intact signaling via phosphatidylinositol 3-kinase (PI3K), but not protein kinase B (PKB). To test the hypothesis that insulin activates IKKβ as a means of negative feedback, we employed a variety of experimental approaches. Firstly, pharmacological inhibition of IKKβ via BMS-345541 did not potentiate insulin-induced IRS1 tyrosine phosphorylation, PKB phosphorylation or 2-deoxyglucose uptake in differentiated 3T3-L1 adipocytes. BMS-345541 did not prevent insulin-induced IRS1 serine phosphorylation on known IKKβ target sites. Secondly, adenoviral-mediated over-expression of wild type (WT) IKKβ in differentiated 3T3-L1 adipocytes did not suppress insulin-stimulated 2-deoxyglucose uptake, insulin receptor substrate 1 (IRS1) tyrosine phosphorylation, IRS1 association with the p85 regulatory subunit of PI3K or PKB phosphorylation. Thirdly, insulin signaling was not potentiated in mouse embryo fibroblasts lacking IKKβ (Ikkβ^-/- MEF). Finally, insulin treatment of 3T3-L1 adipocytes did not promote the recruitment of IKKβ to IRS1, supporting our data that IKKβ, while activated by insulin, does not promote direct serine phosphorylation of IRS1 and does not contribute to the feedback inhibition of the insulin-signaling cascade.

Functional analysis of membraneous Fo-a subunit of F1Fo-ATP synthase by in vitro protein synthesis

Y Kuruma, T Suzuki, S Ono, M Yoshida, T Ueda — 2011-12-14T12:08:00Z

F_o-a subunit of F₁F_o-ATP synthase (F₁F_o) is a highly hydrophobic protein with five putative transmembrane helices and it plays a central role in H⁺-translocation that is coupled with ATP synthesis/hydrolysis. Here, we show that F_o-a subunit produced by the in vitro protease-free protein synthesis system (PURE system) is integrated into a preformed F_o-a-less F₁F_o complex in the Escherichia coli membrane vesicles and in liposomes. The resulting F₁F_o has H⁺-coupled ATP synthesis/hydrolysis activity that is approximately half of that of the native F₁F_o. By using this procedure, we analyzed five mutations of F₁F_o, where the conserved residues in F_o-a subunit (N90, D112, R169, N173, and Q217) were individually replaced with alanine. All of the mutant F_o-a subunits were successfully incorporated into F₁F_o, showing the advantage over conventional expression in E. coli by which three (N90A, D112A, and Q217A) mutant F_o-a subunits were not found in F₁F_o. A mutant N173A retained full activity and mutants D112A and Q217A weak but detectable activity. No activity was observed for mutants of R169A, as reported, and N90A. N90 is located in the middle of putative second transmembrane helix and likely to play an important role in H⁺-translocation. This work exemplifies that the PURE system provides an alternative approach when in vivo expression of membraneous components in protein complexes turns out to be difficult.

Plastoquinone-9 biosynthesis in cyanobacteria differs from that in plants and involves a novel 4-hydroxybenzoate solanesyltransferase

R Sadre, C Pfaff, S Buchkremer — 2011-12-14T11:14:00Z

Plastoquinone-9 (PQ-9) has a central role in the energy transformation processes in cyanobacteria by mediating electron transfer in both, the photosynthetic as well as the respiratory electron transport chain. The present study provides evidence that the PQ-9 biosynthetic pathway in cyanobacteria substantially differs from that in plants. We identified 4-hydroxybenzoate as being the aromatic precursor for PQ-9 in Synechocystis sp. PCC6803 and report here on the role of the membrane-bound 4-hydroxybenzoate solanesyltransferase, Slr0926, in PQ-9 biosynthesis and on the properties of the enzyme. The catalytic activity of Slr0926 was demonstrated by in vivo labelling experiments in Synechocystis sp., complementation studies in an E. coli mutant with a defect in ubiquinone biosynthesis as well as by in vitro assays using the recombinant as well as the native enzyme. While Slr0926 was highly specific for the prenyl acceptor substrate, 4-hydroxybenzoate, it displayed a broad specificity with regard to the prenyl donor substrate and used not only solanesyl diphosphate (SPP) but also a number of shorter-chain prenyl diphosphates. In combination with in silico data, our results indicate that Slr0926 evolved from bacterial 4-hydroxybenzoate prenyltransferase catalysing prenylation in the course of ubiquinone biosynthesis.

Solution structure of Pdp1 PWWP domain reveals its unique binding sites for methylated H4K20 and DNA

2011-12-13T12:32:00Z

Methylation of H4K20 plays an important role in the regulation of diverse cellular processes. In fission yeast, all the three states of H4K20 methylation are catalyzed by Set9. Pdp1 is a PWWP domain-containing protein, which associates with Set9 to regulate its chromatin localization and methyltransferase activity towards H4K20. The structure of Pdp1 PWWP domain, which is the first identified PWWP domain that binds to methyl-lysine at H4K20 site, was determined by solution NMR in the present study. Pdp1 PWWP domain adopts a classical PWWP fold, with a 5-strand antiparallel β-barrel followed by three α-helices. However, it differs significantly from other PWWP domains in some structural aspects that account in part for its molecular recognition. Moreover, we revealed a unique binding pattern of the PWWP domain in that the PWWP domain of Pdp1 bound not only to H4K20me3 but also to dsDNA via an aromatic cage and a positively charged area respectively. Electrophoretic Mobility Shift Assay (EMSA) illustrated the ability of Pdp1 PWWP domain to bind the nucleosome core particle and further mutagenesis experiments indicated the crucial role of this binding activity in histone H4K20 di- and tri- methylation in yeast cells. Our study may shed light on a novel mechanism of histone methylation regulation by the PWWP domain.

Human protein derived peptides for intracellular delivery of biomolecules

A K Haas, D Maisel, J Adelmann, C von Schwerin, I Kahnt, U Brinkmann — 2011-12-13T11:55:00Z

Access of therapeutic biomolecules to cytoplasmic and nuclear targets is hampered by the inability of these molecules to cross biological membranes. Approaches to overcome this hurdle involve cell penetrating peptides (CPPs) or protein transduction domains. Most of these require rather high concentrations to elicit cell penetrating functionality, are non-human, pathogen-derived or synthetic entities and may therefore not be tolerated or even immunogenic. We identified novel human protein derived CPPs by a combination of in-silico and experimental analyses: polycationic CPP candidates were identified in an in-silico library of all 30mer peptides of the human proteome. 60 of these peptides derived from extracellular proteins were evaluated experimentally. Cell viability and siRNA transfection assays revealed that 20 of the 60 peptides were functional. Three of these showed CPP functionality without interfering with cell viability. A peptide derived from human Neurturin (NRTN) that contains an alpha helix performed best in our screen and was uniformly taken up by cultured cells. Examples for payloads that can be delivered to the cytosol by the NRTN peptide include complexed siRNAs and both N- and C-terminally fused pro-apoptotic peptides.

RCD1-DREB2A interaction in leaf senescence and stress responses in Arabidopsis thaliana

2011-12-12T16:52:00Z

Transcriptional regulation of gene expression is one major determinant of developmental control and stress adaptation in virtually all living organisms. In recent years numerous transcription factors controlling various aspects of plant life have been identified. The activity of transcription factors needs to be regulated to prevent unspecific, prolonged or inappropriate responses. The transcription factor DEHYDRATION-RESPONSIVE ELEMENT BINDING 2A (DREB2A) has been identified as one of the main regulators of drought and heat responses, and it is regulated through protein stability. Here we present evidence that the interaction with RADICAL-INDUCED CELL DEATH1 (RCD1) contributes to the control of DREB2A under a range of conditions. The interaction is mediated by a novel protein motif in DREB2A and a splice variant of DREB2A which lacks the interaction domain accumulates during heat stress and senescence. In addition RCD1 is rapidly degraded during heat stress, thus our results suggest that removal of RCD1 protein or the loss of the interaction domain in DREB2A appears to be required for proper DREB2A function under stress conditions.

The chromatin binding protein HMGN3 stimulates histone acetylation and transcription across the Glyt1 gene

2011-12-12T16:17:00Z

HMGNs are nucleosome-binding proteins that alter the pattern of histone modifications and modulate the binding of linker histones to chromatin. The HMGN3 family member exists as two splice forms, HMGN3a which is full length and HMGN3b which lacks the C-terminal regulatory domain. Here, we have used the Glyt1 gene as a model system to investigate where HMGN proteins are bound across the locus in vivo, and to study how the two HMGN3 splice variants affect histone modifications and gene expression. We demonstrate that HMGN1, HMGN2, HMGN3a and HMGN3b are bound across the Glyt1 gene locus and surrounding regions, and are not enriched more highly at the promoter or putative enhancer. We conclude that the peaks of H3K4me3 and H3K9ac at the active Glyt1a promoter do not play a major role in recruiting HMGN proteins. HMGN3a/b binding leads to increased H3K14 acetylation and stimulates Glyt1a expression, and but does not alter the levels of H3K4me3 or H3K9ac enrichment. Acetylation assays show that HMGN3a stimulates the ability of PCAF to acetylate nucleosomal H3 in vitro, whereas HMGN3b does not. We propose a model where HMGN3a/b–stimulated H3K14 acetylation across the bodies of large genes like Glyt1 can lead to more efficient transcription elongation and increased mRNA production.

Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein-D: Implications for lipid antioxidant activity and Alzheimer{'}s disease

S Bhatia, B Knoch, J Wong, W Scott Kim, P L. Else, A J Oakley, B Garner — 2011-12-12T15:07:00Z

Apolipoprotein-D (apoD) is upregulated in Alzheimer’s disease (AD) and upon oxidative stress. ApoD inhibits brain lipid peroxidation in vivo but the mechanism is unknown. Specific Met residues may inhibit lipid peroxidation by reducing radical-propagating lipid hydroperoxides to non-reactive hydroxides via a reaction that generates methionine sulfoxide (MetSO). Since apoD has three conserved Met residues (M49, M93, M157), we generated recombinant proteins with either one or all Met residues replaced by Ala and assessed their capacity to reduce hydroperoxyeicosatetraenoic acids (HpETEs) to their hydroxyeicosatetraenoic acid (HETE) derivatives. ApoD, apoD(M49-A) and apoD(M157-A) all catalysed the reduction of HpETEs to their corresponding HETEs. Amino acid analysis of HpETE-treated apoD revealed a loss of one third of the Met residues accompanied by the formation of MetSO. Additional studies using apoD(M93-A) indicated M93 was required for HpETE reduction. We also assessed the impact that apoD MetSO formation has on protein aggregation by Western blotting of HpETE-treated apoD and human brain samples. ApoD Met oxidation was associated with formation of apoD aggregates that were also detected in AD hippocampus. In conclusion, conversion of HpETE to HETE is mediated by apoD M93, a process that may contribute to apoD antioxidant function.

A Molten Globule-to-Ordered Structure Transition of Drosophila melanogaster Crammer Is Required for its Ability to Inhibit Cathepsin

T Tseng, C Cheng, D Chen, M Shih, Y Liu, S Danny Hsu, P Lyu — 2011-12-09T14:32:00Z

Drosophila melanogaster crammer is a novel cathepsin inhibitor that is involved in long-term memory (LTM) formation. The mechanism by which the inhibitory activity is regulated remains unclear. Here we have shown that the oligomeric state of crammer is pH dependent. At neutral pH, crammer is predominantly dimeric in vitro as a result of disulfide bond formation, and is monomeric at acidic pH. Our inhibition assay shows that monomeric crammer, not disulfide bonded dimer, is a strong competitive inhibitor of cathepsin L. Crammer is a monomeric molten globule in acidic solution, a condition that is similar to the environment in the lysosome where crammer is likely located. Upon binding to cathepsin L, however, crammer undergoes a molten globule-to-ordered structural transition. Using high-resolution NMR spectroscopy, we have shown that a cysteine-to-serine point mutation at position 72 (C72S) renders crammer monomeric at pH 6.0 and that the structure of the C72S variant highly resembles that of wild-type crammer in complex with cathepsin L at pH 4.0. We have determined the first solution structure of propeptide-like protease inhibitor in its active form and examined in detail using a variety of spectroscopic methods the folding properties of crammer in order to delineate its biomolecular recognition of cathepsin.

Macromolecular and small molecule modulation of intracellular A{beta}42 aggregation and associated toxicity

2011-12-09T14:08:00Z

Aβ peptide has a central role in Alzheimer’s disease (AD) where neuronal toxicity is linked to its extracellular and intracellular accumulation as oligomeric species. Searching for molecules that attenuate Aβ aggregation could uncover novel therapies for AD, but most studies in mammalian cells have inferred aggregation indirectly by assessing levels of secreted Aβ peptide. Here we establish a mammalian cell system for the direct visualisation of β-amyloid formation by expression of an Aβ₄₂-EGFP fusion protein in the HEK cell line, T-REx293, and use this to identify both macromolecules and small molecules that reduced aggregation and associated cell toxicity. Thus, a molecular shield protein, AavLEA1, which limits aggregation of proteins with expanded polyglutamine (polyQ) repeats, is also effective against Aβ₄₂-EGFP when co-expressed in T-REx293 cells. A screen of polysaccharide and small organic molecules from medicinal plants and fungi reveals one candidate in each category, PS5 and ganoderic acid DM, respectively, with activity against Aβ peptide. Both PS5 and ganoderic acid DM probably promote Aβ aggregate clearance indirectly through the proteasome. The model is therefore of value to study the effects of intracellular Aβ peptide on cell physiology, and to identify reagents that counteract those effects.

Reprogramming of pancreatic exocrine cells towards a beta cell character using Pdx1, Ngn3 and MafA

E Akinci, A Banga, L V Greder, J R Dutton, J M.W. Slack — 2011-12-09T12:11:00Z

Pdx1, Ngn3 and MafA have been reported to bring about the transdifferentiation of pancreatic exocrine cells to beta cells in vivo (Zhou et al. (2008) Nature 455, 627-630), We have investigated the mechanism of this process using a standard in vitro model of pancreatic exocrine cells, the rat AR42j-B13 cell line. We constructed a new adenoviral vector encoding all three genes, called Ad-PNM. When introduced into AR42j-B13 cells, Ad-PNM causes a rapid change to a flattened morphology, and a cessation of cell division. The expression of exocrine markers is suppressed. Both insulin genes are upregulated as well as a number of transcription factors normally characteristic of beta cells. At the chromatin level, histone tail modifications of the Pdx1, Ins1 and Ins2 gene promoters are shifted in a direction associated with gene activity, and the level of DNA CpG methylation is reduced at the Ins1 promoter. The transformed cells secrete insulin and are capable of relieving diabetes in streptozotocin-treated NOD-SCID mice. However the transformation is not complete. The cells lack expression of several genes important for beta cell function and they do not show glucose-sensitive insulin secretion. We conclude that, for this exocrine cell model, although the transformation is dramatic, the reprogramming is not complete and lacks critical aspects of the beta cell phenotype.

A Golgi-localized MATE transporter mediates iron homoeostasis under osmotic stress in Arabidopsis

P Seo, J Park, M Park, Y Kim, S Kim, J Jung, C Park — 2011-12-08T11:39:00Z

Iron is an essential micronutrient that acts as a cofactor in a wide variety of pivotal metabolic processes, such as electron transport chain of respiration, photosynthesis, and redox reactions, in plants. However, its overload exceeding the cellular capacity of iron binding and storage is potentially toxic to plant cells by causing oxidative stress and cell death. Consequently, plants have developed versatile mechanisms to maintain iron homeostasis. Organismal iron content is tightly regulated at the steps of uptake, translocation, and compartmentalization. Whereas iron uptake is fairly well understood at the cellular and organismal levels, intracellular and intercellular transport is only poorly understood. Here, we show that a multidrug and toxic compound extrusion (MATE) transporter, designated BCD1, contributes to iron homeostasis during stress responses and senescence in Arabidopsis. The BCD1 gene is induced by excessive iron but repressed by iron deficiency. It is also induced by cellular and tissue damages occurring under osmotic stress. The activation-tagged mutant bcd1-1D exhibits leaf chlorosis, typical symptom of iron deficiency. The chlorotic lesion of the mutant was partially recovered by iron feeding. Whereas the bcd1-1D mutant accumulated a lower amount of iron, the iron level was elevated in the knockout mutant bcd1-1. The BCD1 protein is localized to the Golgi complex. We propose that the BCD1 transporter plays a role in sustaining iron homeostasis by reallocating excess iron released from stress-induced cellular damages.

Folded Functional Lipid-Poor Apolipoprotein A-I Obtained by Heating of High-Density Lipoproteins: Relevance to HDL Biogenesis

S Jayaraman, G Cavigiolio, O Gursky — 2011-12-08T11:26:00Z

High-density lipoproteins (HDL) remove cell cholesterol and protect from atherosclerosis. The major HDL protein is apolipoprotein A-I (apoA-I). Most plasma apoA-I circulates in lipoproteins, yet ~5% forms monomeric lipid-poor/free species. This metabolically active species is a primary cholesterol acceptor and is central to HDL biogenesis. Structural properties of lipid-poor apoA-I are unclear due to difficulties in isolating this transient species. We used thermal denaturation of human HDL to produce lipid-poor apoA-I. Analysis of the isolated lipid-poor fraction showed protein:lipid weight ratio 3:1, with apoA-I, phosphatidylcholine and cholesterol ester at approximate molar ratios of 1:8:1. Compared to lipid-free apoA-I, lipid-poor apoA-I showed slightly altered secondary structure and aromatic packing, reduced thermodynamic stability, lower self-associating propensity, increased adsorption to phospholipid surface, and comparable ability to remodel phospholipids and form reconstituted HDL. Lipid-poor apoA-I can be formed by heating of either plasma or reconstituted HDL. We propose the first structural model of lipid-poor apoA-I which corroborates its distinct biophysical properties and postulates the lipid-induced ordering of the labile C-terminal region. In summary, HDL heating produces folded functional monomolecular lipid-poor apoA-I that is distinct from lipid-free apoA-I. Increased adsorption to phospholipid surface and reduced C-terminal disorder may help direct lipid-poor apoA-I towards HDL biogenesis.

A novel glutaredoxin domain-containing peroxiredoxin {'}All1541{'} protects the N2-fixing cyanobacterium Anabaena PCC 7120 from oxidative stress

M Banerjee, A Ballal, S Kumar Apte — 2011-12-08T10:29:00Z

Peroxiredoxins (Prxs) are ubiquitous thiol-based peroxidases that detoxify toxic peroxides. The Anabaena PCC 7120 genome harbours seven genes/ORFs with homology to peroxiredoxins. One of these (all1541) was identified to encode a novel glutaredoxin (Grx) domain-containing peroxiredoxin by bioinformatic analysis. A recombinant N-terminal His-tagged All1541 protein was overexpressed in E. coli and purified. Analysis with protein alkylating agent AMS showed All1541 to form an intra-molecular disulfide bond. The All1541 protein used glutathione (GSH) more efficiently than thioredoxin (Trx) to detoxify H₂O₂. Deletion of Grx domain from All1541 resulted in loss of GSH-dependent peroxidase activity. Employing site directed mutagenesis, the cysteine residues at position 50 and 75 were identified as peroxidatic and resolving cysteine residues respectively, while both the cysteine residues within the Grx domain (position 181 and 184) were shown to be essential for GSH-dependent peroxidase activity. Based on these data, a reaction mechanism has been proposed for All1541. In vitro All1541 protein protected plasmid DNA from oxidative damage. In Anabaena PCC 7120, the all1541 was transcriptionally activated under oxidative stress. Recombinant Anabaena PCC 7120 strain over-expressing All1541 protein showed superior oxidative stress tolerance to H₂O₂ as compared to the wild-type strain. The results suggest that the glutathione dependent peroxidase All1541 plays an important role in protecting Anabaena from oxidative stress.

A Cohesin-RAD21 Interactome

A K Panigrahi, N Zhang, S K Otta, D Pati — 2011-12-07T12:10:00Z

The cohesin complex holds the sister chromatids together from S phase until the metaphase-to-anaphase transition and ensures both their proper cohesin and timely separation. In addition to its canonical function in chromosomal segregation, cohesion has been suggested by several lines of investigation in recent years to play additional roles in apoptosis, DNA damage response, transcriptional regulation, and hematopoiesis. To better understand the basis of the disparate cellular functions of cohesin in these various processes, we have characterized a comprehensive protein interactome of cohesin-RAD21 by using three independent approaches: yeast 2-hybrid (Y2H) screening, immunoprecipitation-coupled-mass spectrometry (IP-M) of cytoplasmic and nuclear extracts from MOLT-4 T lymphocytes in the presence and absence of etoposide induced apoptosis, and affinity-pull down assays of chromatographically purified nuclear extracts from proapoptotic MOLT-4 cells. Our analyses revealed 112 novel protein interactors of cohesin RAD21 that function in different cellular processes including mitosis, regulation of apoptosis, chromosome dynamics, replication, transcription regulation, RNA processing, DNA damage response, protein modification and degradation, and cytoskeleton and cell motility. Identification of cohesin interactors provides a framework for explaining the various non-canonical functions of the cohesin complex.

Targeting of proapoptotic TLR adaptor, SARM, to mitochondria: definition of critical region and residues in the signal sequence

P Panneerselvam, L Pradeepkumar Singh, B Ho, J Chen, J L Ding — 2011-12-07T11:51:00Z

The fifth and the most well conserved member of the TLR adaptor, SARM, has been reported to be an important mediator of apoptosis. However, the exact cellular localization of SARM with respect to its role is unclear. Here we show that SARM specifically co-localizes with the mitochondria. Endogenous SARM is mainly found in the mitochondria. We demonstrated that the N terminal 27 amino acids (S27) of SARM, which is hydrophobic and polybasic, acts as a mitochondria-targeting signal sequence, associating SARM to the mitochondria. The S27 peptide has an inherent ability to bind to lipids and mitochondria. This sequence effectively translocates the soluble EGFP reporter into the mitochondria. Positioning S27 downstream of the EGFP abrogates its mitochondria-targeting ability. Transmission electron microscopy confirms the ability of S27 to import EGFP into the mitochondria. Importantly, by mutagenesis study, we delineated the specificity of the mitochondria-targeting ability to the Arginine residue at the 14^th position. Arg14Ala SARM mutant also showed reduced apoptotic potential when compared to the wild type. Taken together S27, which is a bona fide signal sequence that targets SARM to the mitochondria, explains the proapoptotic activity of SARM.

Angiotensin II-dependent Phosphorylation at Ser-11/Ser-18 and Ser-938 shift the E2 Conformations of Rat Kidney Na,K-ATPase

K J. Massey, Q Li, N F. Rossi, R R. Mattingly, D R. Yingst — 2011-12-07T09:52:00Z

Kidney plasma membranes, which contain a single a-1 isoform of Na,K-ATPase, simultaneously contain two sub-conformations of EP₂, differing in their rate of digoxin release in response to Na and ATP. Treating cells with angiotensin II (Ang II) somehow changes the conformation of both, because it differentially inhibits the rate of digoxin release. We test if Ang II regulates release by increasing phosphorylation at Ser-11/Ser-18 and Ser-938. Opossum kidney cells co-expressing the AT_1a receptor and either alpha-1.wild-type, alpha-1.S11A/S18A or alpha-1.S938A were treated ± 10 nM Ang II for 5 min, increasing phosphorylation at the three sites. Na,K-ATPase was bound to digoxin-affinity columns in the presence of Na, ATP, and Mg. A solution containing 30 mM NaCl and 3 mM ATP eluted ~20% of bound untreated Na,K-ATPase (Population #1). Pre-treating cells with Ang II slowed the elution of Population #1 in alpha-1.wild-type and alpha-1.S938A, but not alpha-1.S11A/S18A cells. Another 50% of bound Na,K-ATPase (Population #2) was subsequently eluted in two phases by a solution containing 150 mM NaCl and 3 mM ATP. Ang II increased the initial rate and slowed the second phase in alpha-1.wild-type, but not a-1.S938A cells. Thus, Ang II changes the conformation of two forms of EP₂via differential phosphorylation.

Inhibition of protein translocation at the endoplasmic reticulum promotes activation of the unfolded protein response

2011-12-06T14:06:00Z

Selective small-molecule inhibitors represent powerful tools for the dissection of complex biological processes. Eeyarestatin I (ES_I) is a novel modulator of endoplasmic reticulum (ER) function. Here, we show that in addition to acutely inhibiting ERAD, ES_I causes production of mislocalised polypeptides that are ubiquitinated and degraded. Unexpectedly, our results suggest these nontranslocated polypeptides promote activation of the unfolded protein response (UPR), and indeed we can recapitulate UPR activation with an alternative and quite distinct inhibitor of ER translocation. These data suggest that the accumulation of nontranslocated proteins in the cytosol may represent a novel mechanism that contributes to UPR activation.

Secreted CXCL12 (SDF-1) forms dimers under physiological conditions

2011-12-06T11:31:00Z

Chemokine CXCL12 signaling through receptors CXCR4 and CXCR7 has essential functions in development and underlies diseases including cancer, atherosclerosis, and autoimmunity. Chemokines may form homodimers that regulate receptor binding and signaling, but previous studies with synthetic CXCL12 have produced conflicting evidence for homodimerization. We used bioluminescence imaging with Gaussia luciferase fusions to investigate dimerization of CXCL12 secreted from mammalian cells. By column chromatography and Gaussia luciferase complementation, we established that CXCL12 was secreted from mammalian cells as both monomers and dimers. Secreted CXCL12 also formed homodimers in the extracellular space. Monomeric CXCL12 preferentially activated CXCR4 signaling through Gαi and AKT, while dimeric CXCL12 more effectively promoted recruitment of β-arrestin 2 to CXCR4 and chemotaxis of CXCR4-expressing breast cancer cells. We also showed that CXCR7 preferentially sequestered monomeric CXCL12 from the extracellular space and had minimal effects on dimeric CXCL12 in cell-based assays and an orthotopic tumor xenograft model of human breast cancer. These studies establish that CXCL12 secreted from mammalian cells forms homodimers under physiologic conditions. Since monomeric and dimeric CXCL12 have distinct effects on cell signaling and function, our results have important implications for ongoing efforts to target CXCL12 pathways for therapy.

Membrane-integration of a mitochondrial signal-anchored protein does not require additional proteinaceous factors

E Merklinger, Y Gofman, A Kedrov, A J.M. Driessen, N Ben-Tal, Y Shai, D Rapaport — 2011-12-05T14:30:00Z

The mitochondrial outer membrane (OM) contains signal-anchored proteins that bear at their N-terminus a single hydrophobic segment that serves as both a mitochondrial targeting signal and an anchor at the membrane. These proteins, like the vast majority of mitochondrial proteins, are encoded in the nucleus and have to be imported into the organelle. Currently, the mechanisms by which they are targeted to and inserted into the OM are unclear. To shed light on these issues, we employed a recombinant version of the signal-anchored protein OM45 and a synthetic peptide corresponding to its signal-anchor segment. Both forms associated with isolated mitochondria independently of cytosolic factors. Interaction with mitochondria was diminished when a mutated form of the signal-anchor was employed. We demonstrate that the signal-anchor peptide acquires an alpha-helical structure in lipid environment and adopted a transmembrane topology within artificial lipid bilayers. Moreover, the peptide´s affinity to artificial membranes with OM-like lipid composition was much higher than that to membranes with ER-like lipid composition. Collectively, our results suggest that signal-anchor proteins are specifically inserted into the mitochondrial OM by a process that is not dependent on additional proteins but is rather facilitated by the distinct lipid composition of this membrane.

Lactococcus lactis HemW (HemN) is a haem-binding protein with a putative role in haem trafficking

H K Abicht, J Martinez, G Layer, D Jahn, M Solioz — 2011-12-05T14:11:00Z

Lactococcus lactis cannot synthesize heme, but when supplied with heme, expresses a cytochrome bd oxidase. Aside of the cydAB structural genes for this oxidase, L. lactis features two additional genes, hemH and hemW (hemN), with conjectured functions in heme metabolism. While it appears clear that hemH encodes a ferrochelatase, no function is know for hemW. HemW-like proteins occur in bacteria, plants, and animals, and are usually annotated as coproporphyrinogen III dehydrogenases. However, such a function has never been demonstrated for a HemW-like protein. We here studied HemW of L. lactis and showed that it is devoid of coproporphyrinogen III dehydrogenase activity in vivo and in vitro. Recombinantly produced, purified HemW contained an iron-sulfur cluster and was dimeric; upon loss of the iron, the protein became monomeric. Both forms of the protein covalently bound heme b in vitro, with a stoichiometry of one heme per monomer and a K_D of 8 µM. In vivo, HemW occurred as a heme free, cytosolic form, as well as a heme-containing, membrane-associated form. Addition of L. lactis membranes to heme-containing HemW triggered the release of heme from HemW in vitro. Based these findings, we propose a role of HemW in heme trafficking. HemW-like proteins form a distinct phylogenetic clade which has not previously been recognized.

Epidermal growth factor induces tumour marker AKR1B10 expression through activator protein-1 signalling in hepatocellular carcinoma cells

2011-12-02T16:19:00Z

Synopsis Aldo-keto reductase1B10 (AKR1B10) is overexpressed in live and lung cancer and plays a critical role in tumor development and progression through promoting lipogenesis and eliminating cytotoxic carbonyls. AKR1B10 is a secretory protein and potential tumor marker. However, little is known about the regulatory mechanism of AKR1B10 expression. This study showed that AKR1B10 is induced by mitogens epidermal growth factor (EGF) and insulin through the activator protein-1 (AP-1) signaling pathway. In human hepatocellular carcinoma cells (HepG2 and Hep3B), EGF (50ng/ml) and insulin (10nM) stimulated endogenous AKR1B10 expression and promoter activity. In the AKR1B10 promoter, a putative AP-1 element was found at -222 to -212bp. Deletion or mutations of this AP-1 element abrogated the basal promoter activity and response to EGF and AP-1 proteins. This AP-1 element bound to nuclear proteins extracted from HepG2 cells, and this binding was stimulated by EGF and insulin in a dose-dependent manner. Chromatin immunoprecipitation showed that AP-1 proteins c-Fos and c-Jun were predominant factors bound to the AP-1 consensus, followed by JunD and then JunB. The same order was followed in the stimulation of endogenous AKR1B10 expression by AP-1 proteins. Furthermore, c-Fos shRNA and AP-1 inhibitors/antagonists (U0126 and Tanshinone IIA) inhibited the endogenous AKR1B10 expression and promoter activity in HepG2 cells cultured in vitro or inoculated subcutaneously in nude mice. U0126 also inhibited AKR1B10 expression induced by EGF. Together these data suggest that AKR1B10 is upregulated by EGF and insulin through the AP-1 mitogenic signaling and may be implicated in hepatocarcinogenesis.

Structural determinants of rCNT2 sorting to the plasma membrane of polarized and non-polarized cells

I Pinilla-Macua, F Casado, M Pastor-Anglada — 2011-12-02T15:33:00Z

rCNT2 (Slc28a2) is the purine-preferring concentrative nucleoside transporter. It is expressed in both non polarized and polarized cells, where it is localized in the brush border membrane. Since no information about the domains implicated in the plasma membrane sorting of rCNT2 is available, this work aims at identifying structural and functional requirements for rCNT2 trafficking. The comprehensive topological mapping of the intracellular N terminal tail revealed two main features: 1) a glutamate enriched region (NPGLELME) between residues 21 and 28 seems to be implicated in the stabilization of rCNT2 in the cell surface, since mutagenesis of these conserved glutamates resulted in enhanced endocytosis. 2) Mutation of a potential Casein Kinase 2 (CK2) domain led to a loss of brush border-specific sorting. Although the shortest proteins assayed (-74AA, -48AA and -37AA) were accumulated intracellularly and lost their brush border membrane preference, they were still functional. A deeper analysis of CK2 implication in CNT2 trafficking, using a CK2 specific inhibitor (DMAT) and other complementary mutations mimicking the negative charge provided by phosphorylation (S46D and S46E), demonstrated an effect of this kinase on CNT2 activity. In summary, rCNT2 N-terminal tail contains dual sorting signals. An acidic region is responsible for its proper stabilization at the plasma membrane whereas the putative CK2 domain (Ser⁴⁶) is implicated in the apical sorting of the transporter.

Allosteric antibody inhibition of human Hepsin protease

2011-12-02T12:25:00Z

Hepsin is a type II transmembrane serine protease that is expressed in several human tissues. Overexpression of hepsin has been found to correlate with tumor progression and metastasis, which is so far best studied for prostate cancer, where more than 90% of such tumors show this characteristic. To enable improved future patient treatment, we developed a monoclonal humanized antibody that selectively inhibits human hepsin and does not inhibit other related proteases. We found that our antibody hH35 potently inhibits hepsin enzymatic activity at nanomolar concentrations. Kinetic characterization revealed non-linear, slow, tight-binding inhibition. This correlates with the crystal structure we obtained for the human hepsin-hH35 antibody Fab fragment complex, which showed that the antibody binds hepsin around a3-helix, located far from the active center. The unique allosteric mode of inhibition of hH35 is distinct from the recently described HGFA (Hepatocyte Growth Factor Activator) allosteric antibody inhibition. We further explain how a small change in the antibody design induces dramatic structural rearrangements in the hepsin antigen upon binding leading to complete enzyme inactivation.

2-Carboxy-D-arabinitol 1-phosphate (CA1P) phosphatase {-} evidence for a wider role in plant Rubisco regulation

2011-12-02T11:13:00Z

The genes for 2-carboxy-D-arabinitol 1-phosphate phosphatase (CA1P phosphatase) from French bean, wheat, Arabidopsis and tobacco were identified and cloned. The deduced protein sequence included an N-terminal motif identical to the phosphogycerate mutase (PGM) active site sequence [LIVM]-x-R-H-G-[EQ]-x-x-[WN]. The corresponding gene from wheat coded for an enzyme with the properties published for CA1P phosphatase. The expressed protein lacked PGM activity but rapidly dephosphorylated 2,3-diphosphoglycerate to 3-phosphoglycerate. DTT activation and GSSG inactivation of this enzyme was pH sensitive, the greatest difference being apparent at pH 8. The presence of the expressed protein during in vitro measurement of Rubisco carboxylase activity prevented a progressive decline in Rubisco turnover. This was due to the removal of an inhibitory bisphosphate which was present in the RuBP preparation, and was found to be D-glycero-2,3-pentodiulose-1,5-bisphosphate (PDBP). The substrate specificity of the expressed protein indicates a role for CA1P phosphatase in the removal of misfire products of Rubisco.

Oligoamine analogues in combination with 2-difluoromethylornithine synergistically induce re-expression of aberrantly silenced tumour-suppressor genes

Y Wu, N Steinbergs, T Murray-Stewart, L J. Marton, R A Casero Jr. — 2011-12-02T10:56:00Z

Epigenetic gene silencing is an important mechanism in the initiation and progression of cancer. Abnormal DNA CpG island hypermethylation and histone modifications are involved in aberrant silencing of tumor suppressor genes. Lysine-specific demethylase 1 (LSD1) was the first enzyme identified to specifically demethylate lysine 4 of histone H3 (H3K4). Methylated H3K4 is an important mark associated with transcriptional activation. The flavin adenine dinucleotide binding, amine oxidase domain of LSD1 is homologous to two polyamine oxidases, spermine oxidase and N¹-acetylpolyamine oxidase. We have demonstrated that long chain polyamine analogues, the oligoamines, are inhibitors of LSD1. Here we report the synergistic effects of specific oligoamines in combination with 2-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, in human colorectal cancer cells. DFMO treatment depletes natural polyamines and increases the uptake of exogenous polyamines. The combination of oligoamines and DFMO results in a synergistic re-expression of aberrantly silenced tumor suppressor genes, including the secreted frizzled-related protein 2 (SFRP2) gene, which encodes a Wnt signaling pathway antagonist and plays an anti-tumorigenic role in colorectal cancer. The treatment-induced re-expression of SFRP2 is associated with increased H3K4me2 in the gene promoter. The combination of LSD1-inhibiting oligoamines and DFMO represents a novel approach to epigenetic therapy of cancer.

Up-regulated miR-17 promotes cell proliferation, tumor growth and cell cycle progression by targeting RND3 tumor suppressor gene in colorectal carcinoma

H Luo, J Zou, Z Dong, Q Zeng, D Wu, L Liu — 2011-12-01T15:35:00Z

Emerging evidence indicates that the miR-17 family may have a causal role in human cancer tumorigenesis, but their specific effects on occurrence of colorectal carcinoma (CRC) are still poorly understood. In this study, we profiled CRC tissue samples by miRNA microarray and found that four members of the miR-17 family had higher expressions in CRC tissues than in normal tissues. This finding was further validated by qRT-PCR. Transfecting CRC cells with an inhibitor of miR-17 lowered their ability to proliferate and induced G₀/G₁ arrest. We also confirmed that miR-17 exerted this function by directly targeting RND3 in vitro and that the expression of miR-17 was negatively correlated with that of RND3 in CRC tissues and CRC cells. Moreover, miR-17 inhibition led to tumor growth suppression and up-regulation of RND3 expression in a nude mouse xenograft model. RND3 expression was found significantly lower in CRC tissues than in normal tissues and adenomas, indicating RND3 may act as a tumor suppressor gene in CRC. In conclusion, our study suggests that miR-17 plays an important role in CRC carcinogenesis by targeting RND3 and may be a therapeutic agent for CRC.

An aspartate residue in the external vestibule of GLYT2 (glycine transporter 2) controls cation access and transport coupling

2011-12-01T15:15:00Z

Synaptic glycine levels are controlled by glycine transporters (GLYTs). GLYT1 is the main regulator of synaptic glycine concentrations, which catalyzes Na⁺/Cl^-/glycine cotransport with a 2:1:1 stoichiometry. By contrast, neuronal GLYT2 supplies glycine to the presynaptic terminal with a 3:1:1 stoichiometry. We subjected homology models of GLYT1 and GLYT2 to molecular dynamics simulations in the presence of Na⁺. Using Molecular Interaction Potential maps and in silico mutagenesis, we identified a conserved region in the GLYT2 external vestibule likely to be involved in Na⁺ interactions. Replacement of D471 in this region reduced Na⁺ affinity and Na⁺ cooperativity of transport, an effect not produced in the homologous position (D295) in GLYT1. Unlike the GLYT1D295 mutation, this D471 mutant increased sodium leakage and non-stoichiometric uncoupled ion movements through GLYT2, as determined by simultaneously measuring current and [³H]glycine accumulation. The homologous D471 and D295 positions exhibited distinct cation-sensitive external accessibility, and they were involved in Na⁺ and Li⁺-induced conformational changes. While these two cations had opposite effects on GLYT1 they had comparable effects on accessibility in GLYT2, explaining the inhibitory and stimulatory responses to lithium exhibited by the two transporters. Based on these findings, we propose a role for D471 in controlling cation access to GLYT2 Na⁺ sites, ion coupling during transport and the subsequent conformational changes.

The crystal structure of human UDP-glucose pyrophosphorylase reveals a latch effect that influence enzymatic activity

Q Yu, X Zheng — 2011-12-01T14:43:00Z

UDP-glucose pyrophosphorylase (UGPase) is highly conserved among eukaryotes. UGPase reversely catalyzes the formation of UDP-glucose and is critical in carbohydrate metabolism. Previous studies have mainly focused on the UGPases from plants, fungi and parasites and indicate that the regulatory mechanisms responsible for the enzyme activity vary among different organisms. Here the crystal structure of human UGPase (hUGPase) was determined and shown to form octamers through end-to-end and side-by-side interactions. The observed latch loop in hUGPase distinctly differs from yeast UGPase (yUGPase), which could explain why hUGPase and yUGPase possess different enzymatic activities. Mutagenesis studies showed that both dissociation of octamers and mutations of the latch loop can significantly affect the UGPase activity. Moreover, this latch effect is also evolutionarily meaningful in UGPase from different species.

Inactivation of ceramide transfer protein during pro-apoptotic stress by Golgi disassembly and caspase cleavage

S Chandran, C E. Machamer — 2011-12-01T11:55:00Z

The mammalian Golgi apparatus is composed of multiple stacks of cisternal membranes organized laterally into a polarized ribbon. Furthermore, trans-Golgi membranes come in close apposition with endoplasmic reticulum (ER) membranes to form ER-trans-Golgi contact sites, which may facilitate transfer of newly synthesized ceramide from the ER to sphingomyelin (SM) synthase at the trans-Golgi via ceramide transfer protein (CERT). CERT interacts with both ER and Golgi membranes, and together with Golgi morphology contributes to efficient SM synthesis. Here, we show that Golgi disassembly during proapoptotic stress induced by tumor necrosis factor (TNFa) and anisomycin results in decreased levels of CERT at the Golgi region. This is accompanied by a caspase-dependent loss of full-length CERT and reduction in de novo SM synthesis. In vitro, CERT is cleaved by caspases-2, -3 and -9. Truncated versions of CERT corresponding to fragments generated by caspase-2 cleavage at Asp²¹³ were mislocalized and did not promote efficient de novo SM synthesis. Thus, it is likely that during cellular stress, disassembly of Golgi structure together with inactivation of CERT by caspases causes a reduction in ceramide trafficking and SM synthesis, and could contribute to the cellular response to proapoptotic stress.

Functional mapping of the disparate activities of the yeast moonlighting protein Hal3

J Abrie, A González, E Strauss, J Ariño — 2011-11-29T15:04:00Z

The Saccharomyces cerevisiae Hal3 protein is a moonlighting protein, able to function both as an inhibitory subunit of the Ppz1 protein phosphatase and as a constituent protomer of an unprecedented heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC), the third enzyme of the CoA biosynthetic pathway. We initiated here the dissection of the structural elements required for both disparate cellular tasks by using a combination of biochemical and genetic approaches. We show that the conserved Hal3 core (the PPCDC domain) is necessary for both functions, as determined by in vitro and in vivo assays. The Hal3 N-terminal domain is not functional by itself, although in vitro experiments indicate that when this domain is combined with the core it has a relevant function in Hal3’s heteromeric PPCDC activity. Both the N-terminal and the acidic C-terminal domain also appear to be important for Hal3’s Ppz1 regulatory function, although our results indicate that the C-terminal domain fulfils the key role in this regard. Finally, we show that the introduction of two key Asn and Cys residues, essential for monofunctional PPCDC activity but absent in Hal3, is not sufficient to convert it to such a homomeric PPCDC, and that additional modifications of Hal3’s PPCDC domain aimed at increasing its resemblance to known PPCDC also fails to introduce this activity. This suggests that Hal3 has undergone significant evolutionary drift from ancestral PPCDC proteins. Taken together, our work highlights specific structural determinants that could be exploited for full understanding of Hal3’s cellular functions.

Hydroperoxide and peroxynitrite reductase activity of poplar thioredoxin-dependent glutathione peroxidase 5 : kinetics, catalytic mechanism and oxidative inactivation

2011-11-29T12:31:00Z

Glutathione peroxidases constitute a family of peroxidases, including selenocysteine- or cysteine-containing isoforms ((SeCys- or Cys-Gpxs) which are regenerated by glutathione or thioredoxins, (Trxs) respectively. We present here new data concerning the substrates of poplar Gpx5 and the residues involved in its catalytic mechanism. This study establishes the capacity of this Cys-Gpx to reduce peroxynitrite with a catalytic efficiency of 10⁶ M^-1s^-1. In PtGpx5, Glu79, which replaces the Gln usually found in Gpx catalytic tetrad, is likely involved in substrate selectivity. Although the redox midpoint potential of the Cys44-Cys92 disulfide and the pKa of Cys44 are not modified in the E79Q variant, it exhibited significantly improved kinetic parameters (K_peroxide and k_cat) with tert-butyl hydroperoxide. The characterization of the monomeric Y151R variant demonstrated that PtGpx5 is not an obligate homodimer. Also, we show that the conserved Phe90 is important for Trx recognition and that Trx-mediated recycling of PtGpx5 occurs via the formation of a transient disulfide between the Trx catalytic cysteine and the Gpx5 resolving cysteine. Finally, we demonstrate that the conformational changes observed during the transition from the reduced to the oxidized form of PtGpx5 are primarily determined by the oxidation of the peroxidatic cysteine into sulfenic acid. Besides, mass spectrometry analysis of in vitro oxidized PtGpx5 demonstrated that the peroxidatic cysteine can be over-oxidized into sulfinic or sulfonic acids. This suggests that some isoforms could have dual functions potentially acting as hydrogen peroxide- and peroxynitrite-scavenging systems and/or as mediators of peroxide signalling as proposed for 2-Cys peroxiredoxins.

MOCA is an integrator of the neuronal death signals that are activated by familial Alzheimer{'}s disease-related mutants of amyloid beta precursor protein and presenilins

N Tachi, Y Hashimoto, M Matsuoka — 2011-11-24T15:31:00Z

Death in cholinergic neurons in cerebral cortex and certain subcortical regions is linked to irreversible dementia relevant to Alzheimer’s disease (AD). Although multiple studies have shown that expression of a familial AD (FAD)-linked amyloid beta precursor protein (APP) or a presenilin (PS) mutant, but not that of wild-type APP or PS, induced neuronal death by activating intracellular death signals, it remains to be addressed how these signals are interrelated and what the key molecule involving this process is. In this study, we show that the PS1-mediated (or possibly the PS2-mediated) signal is essential for the APP-mediated death in a gamma-secretase-independent manner and vice versa. MOCA (modifier of cell adhesion), originally identified as being a PS- and Rac1-binding protein, is a common downstream constituent of these neuronal death signals. The detailed molecular analysis indicates that MOCA is a key molecule of the AD-relevant neuronal death signals that links the PS-mediated death signal with the APP-mediated death signal at a point between Rac1 (or Cdc42) and ASK1.

Interfacial water molecules in SH3 interactions: a revised paradigm for polyproline recognition.

J Martin-Garcia, J Ruiz-Sanz, I Luque — 2011-11-24T14:17:00Z

In spite of its biomedical relevance, polyproline recognition is still not fully understood. The disagreement between the current description of SH3 complexes and their thermodynamic behavior calls for a revision of the SH3 binding paradigm. Recently, Abl-SH3 was demonstrated to recognize its ligands by a dual binding mechanism involving a robust network of water-mediated hydrogen bonds that complements the canonical hydrophobic interactions. The systematic analysis of the SH3 structural database presented here reveals that this dual binding mode is universal to SH3 domains. Tightly bound, buried interfacial water molecules were found in all SH3 complexes studied mediating the interaction between the peptide ligand and the domain. Moreover, structural waters were also identified in a high percentage of free SH3 domains. A detailed analysis of the pattern of water-mediated interactions enabled the identification of conserved hydration sites in the polyproline-recognition region and the establishment of relationships between hydration profiles and the sequence of both, ligands and SH3 domains. Water-mediated interactions were also systematically observed in WW, UEV and EVH-1 structures. These results clearly indicate that the current description of proline-rich sequence recognition by protein-protein interaction modules is incomplete and insufficient for a correct understanding of these systems. A new binding paradigm is required that includes interfacial water molecules as relevant elements in polyproline recognition.

Progestins activate 6-phosphofructo-2-kinase/fructose-2,6-disphosphatase (PFKFB3) in breast cancer cells

2011-11-24T14:09:00Z

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) catalyzes the synthesis and degradation of fructose-2,6-bisphosphate (Fru-2,6-P₂), a key modulator of glycolysis and gluconeogenesis. PFKFB3 gene is extensively involved in cell proliferation due to its key role in carbohydrate metabolism. Here we analyse its mechanism of regulation by progestins in breast cancer cells. We report that exposure of T47D cells to synthetic progestins (ORG2058 or Norgestrel) leads to a rapid increase in Fru-2,6-P₂ concentration. Western blot results are compatible with a short-term activation due to PFKFB3 isoenzyme phosphorylation and a long-term sustained action due to increased PFKFB3 protein levels. Transient transfection of T47D cells with deleted gene promoter constructs allowed us to identify a progesterone regulatory element (PRE) to which progesterone receptor binds and thus transactivates PFKFB3 gene transcription. Progesterone Receptor (PR) expression, in the PR-negative cell line (MDA-MB-231), induces endogen PFKFB3 expression in response to Norgestrel. Direct Binding of PR to the PRE box (-3490 nt) was confirmed by ChIP experiments. A dual mechanism affecting PFKFB3 protein and gene regulation operates in order to assure glycolysis in breast cancer cells. An immediate early response through the ERK-RSK pathway leading to phosphorylation of PFKFB3 on Ser 461 is followed by activation of mRNA transcription via cis-acting sequences on PFKFB3 promoter.

Oxidation of Met1606 in von Willebrand factor is a risk factor for thrombotic and septic complications in chronic renal failure

2011-11-18T11:50:00Z

Chronic Kidney Disease (CKD) is a life-threatening pathology, often requiring haemodialysis (HD) and characterized by high oxidative stress, inflammation and perturbation of vascular endothelium. HD-patients have increased levels of von Willebrand factor (vWF), a large protein (~240kDa) released as ultra large polymers (UL-vWF, M.W.~20000-50000kDa) from vascular endothelial cells and megakaryocytes and responsible for the initiation of primary haemostasis. The pro-haemostatic potential of vWF increases with its length, which is proteolytically regulated by ADAMTS-13, a zinc-protease cleaving vWF at the single Tyr1605-Met1606 bond, and by leukocyte serine proteases, released by activated polymorphonuclear cells during bacterial infections. Previous studies showed that in vitro oxidation of Met1606 hinders vWF cleavage by ADAMTS-13, resulting in the accumulation of UL-vWF, which are not only more prothrombotic than shorter vWF oligomers but also more efficient in binding to bacterial adhesins during sepsis. Notably, HD-patients have greatly increased risk of developing dramatic cardiovascular and septic complications, whose underlying mechanisms are largely unknown. In this study, we first purified vWF from HD-patients and then chemically characterized its oxidative state. Interestingly, HD-vWF contains high carbonyl levels and increased proportion of UL-vWF polymers, which are also more resistant to ADAMTS-13. Using targeted mass spectrometry techniques, we estimated that HD-vWF contains <10% of Met1606 in the sulphoxide form. We conclude that oxidation of Met1606, impairing ADAMTS-13 cleavage, results in the accumulation of UL-vWF polymers, which recruit and activate more efficiently platelets and bind more tightly to bacterial adhesins, thus contributing to the development of thrombotic and septic complications in CKD.

ALAS1 gene expression is down-regulated by Akt-mediated phosphorylation and nuclear exclusion of FOXO1 by vanadate in diabetic mice

L Maria Oliveri, C Davio, A María del Cármen Batlle, E Noemi Gerez — 2011-11-10T09:14:00Z

Porphyrias are diseases caused by partial deficiencies of haem biosynthesis enzymes. Acute porphyrias are characterized by a neuropsychiatric syndrome with intermittent induction of hepatic δ-aminolevulinate synthase 1 (ALAS-1), first and rate-limiting enzyme of haem pathway. Porphyria acute attacks are usually treated with glucose administration, its effect is apparently related to its ability of inhibiting ALAS-1 by modulating insulin plasma levels. It was shown that insulin blunts hepatocytes ALAS-1 induction, by disrupting the interaction of the Forkhead box O1 (FOXO1) and the proliferator-activated receptor γ coactivator 1α (PGC-1α). We evaluated the expression of ALAS-1 in a murine model of diabetes and determined the effects of the insulinomimetic vanadate, on the enzyme regulation to evaluate its potential for the treatment of porphyria acute attacks. Both ALAS-1 mRNA and protein content were induced in diabetic animals, accompanied by decreased Akt phosphorylation and increased nuclear FOXO1, PGC-1α and FOXO1-PGC-1α complex. Vanadate reversed ALAS-1 induction with a concomitant PI3K/Akt pathway activation and subsequent reduction of nuclear FOXO1, PGC-1α and FOXO1-PGC-1α complex levels. These finding support that the FOXO1-PGC-1α complex is involved in the control of ALAS-1 expression and further suggest that a vanadate-based therapy could be beneficial for the treatment of porphyria acute attacks.

Inhibition of glutaminyl cyclase attenuates cell migration modulated by monocyte chemoattractant proteins

Y Chen, K Huang, W Kuo, Y Lo, Y Lee, A H.-J. Wang — 2011-11-08T11:22:00Z

Glutaminyl cyclase (QC) catalyzes the formation of N-terminal pyroglutamate (pGlu) in peptides and proteins. pGlu formation in chemoattractants may participate in the regulation of macrophage activation and migration. However, a clear molecular mechanism for the regulation is lacking. This study examines the role of QC-mediated pGlu formation on monocyte chemoattractant proteins (MCPs) in inflammation. We demonstrated in vitro the pGlu formation on MCPs by QC using mass spectrometry. A potent QC inhibitor, PBD150, significantly reduced the N-terminal uncyclized MCPs precursor (preMCPs)-stimulated monocyte migration, whereas pGlu-containing MCPs (pMCPs)-induced cell migration was unaffected. QC siRNA revealed a similar inhibitory effect. Lastly, we demonstrated that inhibiting QC can attenuate cell migration by LPS. These results strongly suggest that QC-catalyzed N-terminal pGlu formation of MCPs is required for monocyte migration, and provide new insights into the role of QC in the inflammation process. Our results also suggest that QC could be a drug target for some inflammatory disorders.

Mesothelin Enhances Invasion of Ovarian Cancer by Inducing MMP-7 through MAPK/ERK and JNK Pathways

M Chang, C Chen, P Chen, Y Chiang, Y Chen, T Mao, H Lin, W Lin Chiang, W Cheng — 2011-10-14T15:23:00Z

Ovarian cancer has one of the highest mortalities in malignancies in women but little is known of its tumor progression properties and there is still no effective molecule that can monitor its growth or therapeutic responses. Mesothelin, a secreted protein that is over-expressed in ovarian cancer tissues with a poor clinical outcome, has been previously identified to activate PI3K/Akt signaling and inhibit paclitaxel-induced apoptosis. This study investigated the correlation between mesothelin and matrix metalloproteinases-7 (MMP-7) in the progression of ovarian cancer, and mesothelin’s mechanism of enhancing ovarian cancer invasion. The expression of mesothelin correlated well with MMP-7 expression in human ovarian cancer tissues. Mesothelin over-expressed or mesothelin-treated ovarian cancer cells enhanced the migration and invasion of cancer cells through the inducation of MMP-7. Mesothelin regulated the expression of MMP-7 through the ERK (extracellular-signal-regulated kinase) 1/2, Akt (phosphoinositide 3-kinase), and JNK (c-jun-N-terminal kinase) pathways. The expression of MMP-7 and the migrating ability of mesothelin-treated ovarian cancer cells were suppressed by the ERK1/2 or JNK specific inhibitor, or decoy AP-1 oligonucleotide by in vitro experiments, while in vivo animal experiments also demonstrated that mice treated with MAPK/ERK or JNK-specific inhibitors could decrease intra-tumor MMP-7 expression, delay tumor growth, and extend the survival of the mice. In conclusion, mesothelin enhances ovarian cancer invasion by MMP-7 expression through the MAPK/ERK and JNK signal transduction pathways. Blocking the mesothelin-related pathway can be a potential strategy for inhibiting the growth of ovarian cancer.