BJ ChemBio Immediate Publications

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.

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.

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.

Novel Sterol Metabolic Network of Trypanosoma brucei Procyclic and Bloodstream Forms

2011-12-19T12:18:58Z

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.

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

2011-12-14T12:37:44Z

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).

Human protein derived peptides for intracellular delivery of biomolecules

A K Haas, D Maisel, J Adelmann, C von Schwerin, I Kahnt, U Brinkmann — 2011-12-13T11:54:08Z

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.

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

2011-12-06T14:04:59Z

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.

Allosteric antibody inhibition of human Hepsin protease

2011-12-02T12:24:11Z

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.

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 — 2011-11-08T11:21:40Z

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.