O‐linked N‐acetylglucosamine (O‐GlcNAc) modification of specific serines/threonines on intracellular proteins in higher eukaryotes has been shown to directly regulate important processes such as the cell cycle, insulin sensitivity and transcription. The structure, molecular mechanisms of catalysis, protein substrate recognition/specificity of the eukaryotic O‐GlcNAc transferase and hydrolase are largely unknown. Here we describe the crystal structure, enzymology and in vitro activity on human substrates of Clostridium perfringens NagJ, a close homologue of human O‐GlcNAcase (OGA), representing the first family 84 glycoside hydrolase structure. The structure reveals a deep active site pocket highly conserved with the human enzyme, compatible with binding of O‐GlcNAcylated peptides. Together with mutagenesis data, the structure supports a variant of the substrate‐assisted catalytic mechanism, involving two aspartic acids and an unusually positioned tyrosine. Insights into recognition of substrate come from a complex with the transition state mimic O‐(2‐acetamido‐2‐deoxy‐D‐glucopyranosylidene)amino‐N‐phenylcarbamate (Ki=5.4 nM). Strikingly, the enzyme is inhibited by the pseudosubstrate peptide Ala‐Cys(‐S‐GlcNAc)‐Ala, and has OGA activity against O‐GlcNAcylated human proteins, suggesting that the enzyme is a suitable model for further studies into the function of human OGA.

This article reviews the mechanisms by which glucosinolate breakdown products are thought to inhibit carcinogenesis. It describes how isothiocyanates, thiocyanates, nitriles, cyano-epithioalkanes and indoles are produced from glucosinolates through the actions of myrosinase, epithiospecifier protein and epithiospecifier modifier protein released from cruciferous vegetables during injury to the plant. The various biological activities displayed by these phytochemicals are described. In particular, their abilities to induce cytoprotective genes, mediated by the Nrf2 (NF-E2 related factor 2) and AhR (arylhydrocarbon receptor) transcription factors, and their abilities to repress NF-kappaB (nuclear factor-kappaB) activity, inhibit histone deacetylase, and inhibit cytochrome P450 are outlined. Isothiocyanates appear to alter gene expression through modification of critical thiols in regulatory proteins such as Keap1 (Kelch-like ECH-associated protein 1) or IKK (IkappaB kinase), causing activation of Nrf2 and inactivation of NF-kappaB, respectively. Certain indoles act as ligands for AhR. Isothiocyanates and indoles are also capable of affecting cell cycle arrest and stimulating apoptosis. The mechanisms responsible for these anti-proliferative responses are discussed.

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The paralogous multifunctional adaptor proteins YAP and TAZ are the nuclear effectors of the Hippo pathway, a central mechanism of organ size control and stem cell self-renewal. WW domains, mediators of protein-protein interactions, are essential for YAP and TAZ function, enabling interactions with PPxY motifs of numerous partner proteins. YAP has single and double WW domain isoforms (YAP1 and YAP2) whereas only a single WW domain isoform of TAZ has been described to date. Here we identify the first example of a double WW domain isoform of TAZ. Using NMR, we have characterized conformational features and peptide binding of YAP and TAZ tandem WW domains (WW1-WW2). The solution structure of YAP WW2 confirms that it has a canonical three-stranded antiparallel β-sheet WW domain fold. While chemical shift-based analysis indicates that the WW domains in the tandem WW pairs retain the characteristic WW domain fold, 15N relaxation data show that, within the respective WW pairs, YAP WW1 and both WW1 and WW2 of TAZ undergo conformational exchange. 15N relaxation data also indicate that the linker between the WW domains is flexible in both YAP and TAZ. Within both YAP and TAZ tandem WW pairs, WW1 and WW2 bind single PPxY-containing peptide ligand concurrently and noncooperatively with sub-mM affinity. YAP and TAZ WW1-WW2 bind a dual PPxY-containing peptide with approximately 6-fold higher affinity. Our results indicate that both WW domains in YAP and TAZ are functional and capable of enhanced affinity binding to multi-PPxY partner proteins such as LATS1, ErbB4, and AMOT.

Trypanothione reductase (TryR) is a key enzyme involved in the oxidative stress management of the Trypanosoma and Leishmania parasites, which helps to maintain an intracellular reducing environment by reduction of the small-molecular-mass disulphide trypanothione (T[S](2)) to its di-thiol derivative dihydrotrypanothione (T[SH](2)). TryR inhibition studies are currently impaired by the prohibitive costs of the native enzyme substrate T[S](2). Such costs are particularly notable in time-dependent and high-throughput inhibition assays. In the present study we report a protocol that greatly decreases the substrate quantities needed for such assays. This is achieved by coupling the assay with the chemical oxidant 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), which can rapidly re-oxidize the T[SH](2) product back into the disulphide substrate T[S](2), thereby maintaining constant substrate concentrations and avoiding deviations from rate linearity due to substrate depletion. This has enabled the development of a continuous microplate assay for both classical and time-dependent TryR inhibition in which linear reaction rates can be maintained for 60 min or more using minimal substrate concentrations (<1 microM, compared with a substrate K (m) value of 30 microM) that would normally be completely consumed within seconds. In this manner, substrate requirements are decreased by orders of magnitude. The characterization of a novel time-dependent inhibitor, cis -3-oxo-8,9b-bis-(N(1)-acrylamidospermidyl)-1,2,3,4,4a,9b-hexahydrobenzofuran (PK43), is also described using these procedures.

SummaryChitinase inhibitors have chemotherapeutic potential as fungicides, pesticides, and antiasthmatics. Argifin, a natural product cyclopentapeptide, competitively inhibits family 18 chitinases in the nanomolar to micromolar range and shows extensive substrate mimicry. In an attempt to map the active fragments of this large natural product, the cyclopentapeptide was progressively dissected down to four linear peptides and dimethylguanylurea, synthesized using a combination of solution and solid phase peptide synthesis. The peptide fragments inhibit chitinase B1 from Aspergillus fumigatus (AfChiB1), the human chitotriosidase, and chitinase activity in lung homogenates from a murine model of chronic asthma, with potencies ranging from high nanomolar to high micromolar inhibition. X-ray crystallographic analysis of the chitinase-inhibitor complexes revealed that the conformations of the linear peptides were remarkably similar to that of the natural product. Strikingly, the dimethylguanylurea fragment, representing only a quarter of the natural product mass, was found to harbor all significant interactions with the protein and binds with unusually high efficiency. The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.