Unraveling the hidden catalytic activity of vertebrate class IIa histone deacetylases.

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-Nepsilon2 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.

Characterization of the hepatitis C virus NS2/3 processing reaction by using a purified precursor protein.

The NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction. Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction. We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor. Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction. This truncated protein was efficiently expressed and processed in Escherichia coli. The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies. This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor. Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein. However, we were unable to observe trans cleavage activity between cleavage-site mutants and active-site mutants. Furthermore, the cleavage reaction of the wild-type protein was not inhibited by addition of a mutant that was unable to undergo self-processing. Site-directed mutagenesis data and the independence of the processing rate from the nature of the added metal ion argue in favor of NS2/3 being a cysteine protease having Cys993 and His952 as a catalytic dyad. We conclude that a purified protein can efficiently reproduce processing at the NS2/3 site in the absence of additional cofactors.

Role of charged residues in the catalytic mechanism of hepatitis C virus NS3 protease: electrostatic precollision guidance and transition-state stabilization.

Maturational cleavage of the hepatitis C virus polyprotein involves the viral chymotrypsin-like serine protease NS3. The substrate binding site of this enzyme is unusually flat and featureless. We here show that NS3 has a highly asymmetric charge distribution that is characterized by strong positive potentials in the vicinity of its active site and in the S5/S6 region. Using electrostatic potential calculations, we identified determinants of this positive potential, and the role of six different residues was explored by site-directed mutagenesis. Mutation of residues in the vicinity of the active site led to changes in k(cat) values of a peptide substrate indicating that basic amino acids play a role in the stabilization of the transition state. Charge neutralization in the S5/S6 region increased the K(m) values of peptide substrates in a manner that depended on the presence of negatively charged residues in the P5 and P6 positions. K(i) values of hexapeptide acids spanning P6-P1 (product inhibitors) were affected by charge neutralization in both the active site region and the S5/S6 region. Pre-steady-state kinetic data showed that the electrostatic surface potential is used by this enzyme to enhance collision rates between peptidic ligands and the active site. Calculations of the interaction energies of protease-substrate or protease-inhibitor complexes showed that electrostatic interaction energies oppose the formation of a tightly bound complex due to an unfavorable change in the desolvation energy. We propose that desolvation costs are minimized by avoiding the formation of individual ion pair interactions through the use of clusters of positively charged residues in the generation of local electrostatic potentials.

Characterization of human gamma-tryptases, novel members of the chromosome 16p mast cell tryptase and prostasin gene families.

Previously, this laboratory identified clusters of alpha-, beta-, and mast cell protease-7-like tryptase genes on human chromosome 16p13.3. The present work characterizes adjacent genes encoding novel serine proteases, termed gamma-tryptases, and generates a refined map of the multitryptase locus. Each gamma gene lies between an alpha1H Ca2+ channel gene (CACNA1H) and a betaII- or betaIII-tryptase gene and is approximately 30 kb from polymorphic minisatellite MS205. The tryptase locus also contains at least four tryptase-like pseudogenes, including mastin, a gene expressed in dogs but not in humans. Genomic DNA blotting results suggest that gammaI- and gammaII-tryptases are alleles at the same site. betaII- and betaIII-tryptases appear to be alleles at a neighboring site, and alphaII- and betaI-tryptases appear to be alleles at a third site. gamma-Tryptases are transcribed in lung, intestine, and in several other tissues and in a mast cell line (HMC-1) that also expresses gamma-tryptase protein. Immunohistochemical analysis suggests that gamma-tryptase is expressed by airway mast cells. gamma-Tryptase catalytic domains are approximately 48% identical with those of known mast cell tryptases and possess mouse homologues. We predict that gamma-tryptases are glycosylated oligomers with tryptic substrate specificity and a distinct mode of activation. A feature not found in described tryptases is a C-terminal hydrophobic domain, which may be a membrane anchor. Although the catalytic domains contain tryptase-like features, the hydrophobic segment and intron-exon organization are more closely related to another recently described protease, prostasin. In summary, this work describes gamma-tryptases, which are novel members of chromosome 16p tryptase/prostasin gene families. Their unique features suggest possibly novel functions.

Characterization of genes encoding known and novel human mast cell tryptases on chromosome 16p13.3.

Tryptases are serine proteases implicated in asthma and are very highly expressed in human mast cells. They fall into two groups, alpha and beta. Although several related tryptase mRNAs are known, it is unclear which if any are transcripts of separate haploid genes. The studies described here investigated the nature and number of human tryptases and sought possibly novel members of the family. To this end, two human bacterial artificial chromosome (BAC) clones containing tryptase genes were identified and mapped to chromosome 16p13.3, of which approximately 2.2 megabases are syntenic with the part of mouse chromosome 17 containing tryptase genes mouse mast cell protease (mMCP)-6 and -7. Sequencing and restriction mapping suggest that the BACs may partially overlap. Sequenced BAC genes correspond to three known beta-tryptases (betaI, betaII, and betaIII), an alpha-like gene, and a pair of novel hybrid genes related partly to alpha/beta-tryptases and partly to orthologs of mMCP-7. betaII and betaIII, betaI and alphaII, as well as the two mMCP-7-like genes, may be alleles at single loci; in total, there are at least three nonallelic tryptase genes in the isolated BAC clones. DNA blotting and restriction analysis suggest that the BACs include most members of the immediate tryptase family. Thus, chromosome 16p13.3 harbors a cluster of known and previously undescribed members of the tryptase gene family.

Structural and functional properties of Bos taurus tryptase: a search for a possible propeptide processing role.

Some structural features of bovine tryptase were discussed based on spectroscopic analysis. The far UV-CD spectrum of the enzymatically active bovine tryptase is consistent with a structure containing very little, if any alpha-helix, as found for other serine proteases. The analysis of near UV-CD and UV absorption spectra reveals the presence of a high number of Trp residues arranged probably in strong structural motifs. At variance with other tryptases, the bovine enzyme shows an electrophoretic behaviour in native and denaturating conditions compatible with an association state larger than a tetramer (probably a dodecamer). From a biochemical point of view, the bovine tryptase shares with the human counterpart, the preference for cleaving substrates bearing dibasic cleavage sites. Thus, it is hypothesized that tryptase may be involved in some proprotein processing mechanism(s).

cDNA cloning and primary structure of tryptase from bovine mast cells, and evidence for the expression of bovine pancreatic trypsin inhibitor mRNA in the same cells.

A partial cDNA encoding bovine tryptase, an oligomeric serine proteinase previously isolated from bovine mast cells, was obtained by reverse transcription/polymerase chain reaction of mast cell mRNA, using combinations of primers designed on the basis of information obtained from partial sequencing of the purified protein. The complete amino acid sequence of bovine tryptase (245 residues) was deduced from a 711-bp nucleotide sequence and from Edman degradation of the protein. Bovine tryptase primary structure has an identity of about 75% with tryptases from other species and includes all the essential residues of the active-site regions; sequence data in the region of the putative substrate binding pocket suggest a rearrangement capable of maintaining the specificity of trypsin-like proteinases. From the same mast cell mRNA, cDNA encoding bovine trypsin protease inhibitor (BPTI) was obtained and amplified with specific primers, confirming the synthesis of BPTI in these cells. Results are consistent with previous data on the presence of BPTI and bovine tryptase in the same granules of bovine mast cells and with their interaction in vitro.