Previous findings have suggested that class IIa histone deacetylases (HDACs) (HDAC4, -5, -7, and -9) are inactive on acetylated substrates, thus differing from class I and IIb enzymes. Here, we present evidence supporting this view and demonstrate that class IIa HDACs are very inefficient enzymes on standard substrates. We identified HDAC inhibitors unable to bind recombinant human HDAC4 while showing inhibition in a typical HDAC4 enzymatic assay, suggesting that the observed activity rather reflects the involvement of endogenous copurified class I HDACs. Moreover, an HDAC4 catalytic domain purified from bacteria was 1,000-fold less active than class I HDACs on standard substrates. A catalytic Tyr is conserved in all HDACs except for vertebrate class IIa enzymes where it is replaced by His. Given the high structural conservation of HDAC active sites, we predicted the class IIa His-N2 to be too far away to functionally substitute the class I Tyr-OH in catalysis. Consistently, a Tyr-to-His mutation in class I HDACs severely reduced their activity. More importantly, a His-976-Tyr mutation in HDAC4 produced an enzyme with a catalytic efficiency 1,000-fold higher than WT, and this ''gain of function phenotype'' could be extended to HDAC5 and -7. We also identified trifluoroacetyl-lysine as a class IIa-specific substrate in vitro. Hence, vertebrate class IIa HDACs may have evolved to maintain low basal activities on acetyl-lysines and to efficiently process restricted sets of specific, still undiscovered natural substrates.catalytic domain ͉ enzymatic activity ͉ trifluoroacetyl-lysine ͉ gain of function
The solution structure of the hepatitis C virus (BK strain) NS3 protein Nterminal domain (186 residues) has been solved by NMR spectroscopy. The protein is a serine protease with a chymotrypsin-type fold, and is involved in the maturation of the viral polyprotein. Despite the knowledge that its activity is enhanced by the action of a viral protein cofactor, NS4A, the mechanism of activation is not yet clear. The analysis of the folding in solution and the differences from the crystallographic structures allow the formulation of a model in which, in addition to the NS4A cofactor, the substrate plays an important role in the activation of the catalytic mechanism. A unique structural feature is the presence of a zincbinding site exposed on the surface, subject to a slow conformational exchange process.
Azumamide E, a cyclotetrapeptide isolated from the sponge Mycale izuensis, is the most powerful carboxylic acid containing natural histone deacetylase (HDAC) inhibitor known to date. In this paper, we describe design and synthesis of two stereochemical variants of the natural product. These compounds have allowed us to clarify the influence of side chain topology on the HDAC-inhibitory activity. The present contribution also reveals the identity of the recognition pattern between azumamides and the histone deacetylase-like protein (HDLP) model receptor and reports the azumamide E unprecedented isoform selectivity on histone deacetylases class subtypes. From the present studies, a plausible model for the interaction of azumamides with the receptor binding pocket is derived, providing a framework for the rational design of new cyclotetrapeptide-based HDAC inhibitors as antitumor agents.
The NS3 protein of hepatitis C virus contains a chymotrypsin-like serine proteinase domain. We built a homology model of this domain which predicts the presence of a tetradentate metal binding site formed by three cysteines and one histidine. These residues are strictly conserved in all known hepatitis C viral genotypes as well as in other recently discovered related hepatitis viruses. We show that the hepatitis C virus enzyme does indeed contain a Zn2+ ion with S3N ligation and that the metal is required for structural integrity and activity of the enzyme. Strikingly, the residues forming the metal binding site are also conserved in the chymotrypsin-like 2A cysteine proteinases of picornaviruses. Remarkably, in these highly variable viral genomes the metal binding site is more conserved than the catalytic residues and thus allows us to define a novel class of zinc binding chymotrypsin-like proteinases and to identify a new attractive target for antiviral therapy.
The NS3 region of the hepatitis C virus encodes for a serine protease activity, which is necessary for the processing of the nonstructural region of the viral polyprotein. The minimal domain with proteolytic activity resides in the N terminus, where a structural tetradentate zinc binding site is located. The ligands being been identified by x-ray crystallography as being three cysteines (Cys 97 has shown the importance of this residue in the metal incorporation pathway and for achieving an active fold. The metal coordination of the protease was also investigated by circular dichroism and electronic absorption spectroscopies using a Co(II)-substituted enzyme. We show evidence for rearrangements of the metal coordination geometry induced by complex formation with an NS4A peptide cofactor. No such changes were observed upon binding to a substrate peptide. Also, CN ؊ and N 3 ؊ induced Co(II) ligand field perturbations, which went along with an 1.5-fold enhancement of protease activity.The hepatitis C Virus (HCV) 1 has been identified as the major etiologic agent of parenterally transmitted non-A non-B hepatitis (1, 2). HCV is an enveloped virus with a positivestranded RNA genome of 9.4 kb, which is translated into a precursor polyprotein of about 3010 amino acids (3). Both cellular and virally encoded proteases are involved in the maturational proteolytic processing of this precursor. Whereas the structural viral proteins arise from signal peptidase-catalyzed cleavages (4), two different proteolytic activities encoded by the HCV NS2 and NS3 proteins are responsible for the processing of the nonstructural region of the polyprotein. The NS2-NS3 precursor is cleaved intramolecularly by an autoprotease, the activity of which was shown to be zinc-dependent (5). The N-terminal part of the NS3 protein, furthermore, contains a 20-kDa serine protease domain that accomplishes all cleavage events downstream of NS3, including the generation of the mature viral polymerase (6). In order to perform its physiological task, the NS3 serine protease has to bind to the viral protein NS4A (7,8). This binding event leads to an enhancement of the protease activity and to a stabilization of NS3. In vitro, activation of NS3 can be achieved by addition of peptides harboring residues 21-34 of NS4A (9 -12).Based on a homology model, we were able to predict the presence, in the NS3 protease domain, of a tetradentate metal binding site formed by three cysteines (Cys 97 , Cys 99 , and Cys 145 ) and one histidine residue (His 149 ) (13). Biochemical characterization has confirmed this prediction and demonstrated the presence of a zinc ion in a tetrahedral environment (13,14). This zinc ion was shown to be essential for the structural integrity of the protein; its removal leads to unfolding and aggregation of the enzyme. Mutagenesis experiments have shown that mutations affecting any of the three cysteine residues resulted in an impaired NS3 protease activity as judged from in vitro translation experiments (14, 15). On the other hand, mutagenesis...
The developmental expression pattern and localization of calreticulin were studied in Nicotiana tabacum L. anthers, pollen and pollen tubes. High transcript and protein levels were detected throughout anther development. Immunolocalization of calreticulin in the anthers showed particular dense label in tapetum and pollen at developmental stage 2, when the tapetum is highly active and the pollen tetrads are formed. Much lower transcript and protein levels were detected in dry and hydrated pollen and in pollen tubes. Immunofluorescence labeling of both chemically fixed and cryo-fixed and freeze-substituted pollen tubes showed the presence of calreticulin in Golgi apparatus and endoplasmic reticulum (ER). Calreticulin was seen throughout the stacks in the Golgi apparatus and in the areas with coated-Golgi vesicles but much less so in the ER. Calreticulin was not found in the secretory vesicles. A relatively intense label was occasionally seen adjacent to the wall of the tube. No significant label was observed in mitochondria, vacuoles, generative cells, cell wall or callose plugs. The present results are consistent with a role of calreticulin in Ca2+-dependent folding of secreted glycoproteins in tapetum, pollen and pollen tubes.
It is highly desirable to design ligand-dependent transcription regulation systems based on transactivators unresponsive to endogenous ligands but induced by synthetic small molecules unable to activate endogenous receptors. Using molecular modeling and yeast selection, we identified an estrogen receptor ligand binding domain double mutant (L384M, M421G) with decreased affinity to estradiol and enhanced binding to compounds inactive on estrogen receptors. Nonresponsiveness to estrogen was achieved by additionally adding the G521R substitution while introducing an "antagonistic-type" side chain in the compound, as in 4-hydroxytamoxifen. The triple-substituted ligand binding domain is insensitive to physiological concentrations of estradiol and has nanomolar affinity for the ligand. In this binary system, both receptor and ligand are, therefore, reciprocally specific. The mutated variant in the context of a chimeric transcription factor provides tight, ligand-dependent regulation of reporter gene expression.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.