Histone deacetylases (HDACs) are a family of enzymes involved in the regulation of gene expression, DNA repair, and stress response. These processes often are altered in tumors, and HDAC inhibitors have had pronounced antitumor activity with promising results in clinical trials. Here, we report the crystal structure of human HDAC8 in complex with a hydroxamic acid inhibitor. Such a structure of a eukaryotic zinc-dependent HDAC has not be described previously. Similar to bacterial HDAC-like protein, HDAC8 folds in a single ␣͞␤ domain. The inhibitor and the zinc-binding sites are similar in both proteins. However, significant differences are observed in the length and structure of the loops surrounding the active site, including the presence of two potassium ions in HDAC8 structure, one of which interacts with key catalytic residues. CD data suggest a direct role of potassium in the fold stabilization of HDAC8. Knockdown of HDAC8 by RNA interference inhibits growth of human lung, colon, and cervical cancer cell lines, highlighting the importance of this HDAC subtype for tumor cell proliferation. Our findings open the way for the design and development of selective inhibitors of HDAC8 as possible antitumor agents.T he epigenetic control of gene expression is operated through a series of posttranslational modifications of chromatin that influence the electrostatics of DNA-protein interactions and generate docking sites for a large number of chromatininteracting proteins (1, 2). The acetylation status of lysine residues found in the accessible N termini of core histones is one of the posttranslational chromatin modifications that impinge on gene expression. Acetylation and deacetylation of histones are controlled by the enzymatic activity of histone acetyltransferases and histone deacetylases (HDACs) (3, 4). Alterations of gene expression are a hallmark of cancer, and mounting evidence suggests that at least a part of these alterations is mediated by epigenetic mechanisms (5, 6). Importantly, the aberrant recruitment of HDACs has been mechanistically linked to malignancy in leukemias and lymphomas (7,8), and small-molecule HDAC inhibitors show antitumor activity in preclinical models and in clinical trials and have the promise to become effective, new antineoplastic therapeutics (9).At least 18 HDAC subtypes exist, and they are subdivided into three classes (10): class I (HDACs 1-3 and 8), homologous to the yeast Rpd3 deacetylase; class II (HDACs 4-7, 9, and 10), related to the yeast Hda1 deacetylase; and class III proteins (Sirtuins 1-7), which are yeast Sir2 homologs. HDAC11 has homology to both class I and II enzymes but cannot unambiguously be assigned to either class. Class I and II HDACs, as well as HDAC11, are all zinc-dependent hydrolases. The therapeutically relevant HDAC inhibitors are thought to be nonselective or poorly selective inhibitors of all or most of class I and II enzymes but do not inhibit class III HDACs (9). It is not clear whether the antitumor properties of HDAC inhibitors are due to their l...

The nonstructural protein NS3 of the hepatitis C virus (HCV) harbors a serine protease domain that is responsible for most of the processing events of the nonstructural region of the polyprotein. Its inhibition is presently regarded as a promising strategy for coping with the disease caused by HCV. In this work, we show that the NS3 protease undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B cleavage sites, whereas no inhibition is observed with a cleavage product of the intramolecular NS3-NS4A junction. The Ki values of the hexamer inhibitory products [Ki(NS4A) = 0.6 microM, Ki(NS5A) = 1.4 microM, and Ki(NS4B) = 180 microM] are lower than the Km values of the respective substrate peptides [Km(NS4A-NS4B) = 10 microM, Km(NS5A-NS5B) = 3.8 microM, and Km(NS4B-NS5A) > 1000 microM]. Mutagenesis experiments have identified Lys136 as an important determinant for product binding. The phenomenon of product inhibition can be exploited to optimize peptide inhibitors of NS3 protease activity that may be useful in drug development.

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a serine protease activity resides in the N-terminal one-third of the protein, whereas RNA helicase activity and RNA-stimulated nucleoside triphosphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full-length NS3 polypeptide of 67 kDa was expressed as a nonfusion protein in Escherichia coli, purified to homogeneity, and shown to retain all three enzymatic activities. The protease activity of the full-length NS3 was strongly dependent on the activation by a synthetic peptide spanning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the isolated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isolated protease domain, the protease activity of full-length NS3 undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characterized and quantified the NS3 ATPase, RNA helicase, and RNA-binding activities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyzed in independent in vitro assays. We have further explored the possible interdependence of the NS3 N-terminal and C-terminal domains by analyzing the effect of polynucleotides on the modulation of all NS3 enzymatic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the helicase RNA-binding domain.

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The aim of this study was to investigate the mechanism of activation of human heparanase, a key player in heparan sulfate degradation, thought to be involved in normal and pathologic cell migration processes. Active heparanase arises as a product of a series of proteolytic processing events. Upon removal of the signal peptide, the resulting, poorly active 65 kDa species undergoes the excision of an intervening 6 kDa fragment generating an 8 kDa polypeptide and a 50 kDa polypeptide, forming the fully active heterodimer. By engineering of tobacco etch virus protease cleavage sites at the N- and C-terminal junctions of the 6 kDa fragment, we were able to reproduce the proteolytic activation of heparanase in vitro using purified components, showing that cleavage at both sites leads to activation in the absence of additional factors. On the basis of multiple-sequence alignment of the N-terminal fragment, we conclude that the first beta/alpha/beta element of the postulated TIM barrel fold is contributed by the 8 kDa subunit and that the excised 6 kDa fragment connects the second beta-strand and the second alpha-helix of the barrel. Substituting the 6 kDa fragment with the topologically equivalent loop from Hirudinaria manillensis hyaluronidase or connecting the 8 and 50 kDa fragments with a spacer of three glycine-serine pairs resulted in constitutively active, single-chain heparanases which were comparable to the processed, heterodimeric enzyme with regard to specific activity, chromatographic profile of hydrolysis products, complete inhibition at NaCl concentrations above 600 mM, a pH optimum of pH approximately 5, and inhibition by heparin with IC(50)s of 0.9-1.5 ng/microL. We conclude that (1) the heparanase heterodimer (alpha/beta)(8)-TIM barrel fold is contributed by both 8 and 50 kDa subunits with the 6 kDa connecting fragment leading to inhibition of heparanase by possibly obstructing access to the active site, (2) proteolytic excision of the 6 kDa fragment is necessary and sufficient for heparanase activation, and (3) our findings open the way to the production of recombinant, constitutively active single-chain heparanase for structural studies and for the identification of inhibitors.