Metadata-driven comparative analysis tool for sequences (meta-CATS): an automated process for identifying significant sequence variations that correlate with virus attributes.

The Virus Pathogen Resource (ViPR; www.viprbrc.org) and Influenza Research Database (IRD; www.fludb.org) have developed a metadata-driven Comparative Analysis Tool for Sequences (meta-CATS), which performs statistical comparative analyses of nucleotide and amino acid sequence data to identify correlations between sequence variations and virus attributes (metadata). Meta-CATS guides users through: selecting a set of nucleotide or protein sequences; dividing them into multiple groups based on any associated metadata attribute (e.g. isolation location, host species); performing a statistical test at each aligned position; and identifying all residues that significantly differ between the groups. As proofs of concept, we have used meta-CATS to identify sequence biomarkers associated with dengue viruses isolated from different hemispheres, and to identify variations in the NS1 protein that are unique to each of the 4 dengue serotypes. Meta-CATS is made freely available to virology researchers to identify genotype-phenotype correlations for development of improved vaccines, diagnostics, and therapeutics.

Randomized clinical trial of effect of synbiotics, neomycin and mechanical bowel preparation on intestinal barrier function in patients undergoing colectomy.

BACKGROUND: The aim of this study was to investigate whether it is possible to modulate gut microflora and preserve intestinal barrier function during elective colorectal surgery by using combinations of oral antibiotics, synbiotics and mechanical bowel preparation (MBP). METHODS: Ninety-two patients were randomly assigned to one of four groups. Group 1 had MBP only, group 2 had neomycin + MBP, group 3 had synbiotics + neomycin + MBP, and group 4 had synbiotics + neomycin but no MBP. Changes in gut microflora were assessed by culturing nasogastric aspirates and polymerase chain reaction-denaturing gradient gel electrophoresis of faecal samples. Intestinal barrier function was determined by microbiological confirmation of bacterial translocation and measurement of intestinal permeability. The inflammatory response was monitored by measurement of serum C-reactive protein and interleukin 6, and septic morbidity was recorded prospectively. RESULTS: Four patients were excluded owing to protocol violation, leaving 24 patients in group 1, 22 in group 2, 20 in group 3 and 22 in group 4 for analysis. There was a significant decrease in Enterobacteriaceae in group 3 compared with the other groups. Group 3 had a significantly lower incidence of translocation after bowel mobilization (P < 0.001). There was no significant difference between the groups in intestinal permeability, inflammatory response or septic morbidity. CONCLUSION: The combination of MBP, neomycin and synbiotics reduces the prevalence of faecal Enterobacteriaceae and bacterial translocation; however, this was not associated with a reduction in inflammatory response or septic morbidity in this study. Larger trials are needed before a change in practice can be recommended.

Dose-response study of probiotic bacteria Bifidobacterium animalis subsp lactis BB-12 and Lactobacillus paracasei subsp paracasei CRL-341 in healthy young adults.

OBJECTIVE: This study was performed to investigate the dose-response effects of supplementation with Bifidobacterium animalis subsp lactis (BB-12) and Lactobacillus paracasei subsp paracasei (CRL-431) on blood lipids, recovery from feces and bowel habits. Changes of the fecal microflora was analyzed in the 10(10) CFU/day probiotic and placebo group. DESIGN: The study was designed as a randomized, placebo-controlled, double-blinded, parallel dose-response study. SUBJECTS: Healthy young adults (18-40 years) were recruited by advertising in local newspapers. Of the 75 persons enrolled, 71 (46 women, 25 men, mean age 25.6 years (range 18-40 years)) completed the study. INTERVENTION: The volunteers were randomly assigned into five groups receiving either placebo or a mixture of the two probiotics in the concentration of 10(8), 10(9), 10(10) or 10(11) CFU/day in 2 weeks run-in period, 3 weeks intervention and 2 weeks wash-out. Diary reporting bowel habits and well being (abdominal bloating, flatulence and headache) was kept for all 7 weeks and blood lipids, fecal recovery of BB-12 and CRL-431, as well as fecal microflora was tested before, immediately and 2 weeks after intervention. RESULTS: The fecal recovery of BB-12 increased significantly (P < 0.001) with increasing dose. In the group receiving 10(11) CFU/day BB-12 was recovered from 13 out of 15 volunteers. CRL-431 was not recovered in any of the fecal samples. Supplementation with probiotics did not change the fecal bacterial composition. A significant linear increase in fecal consistency (looser stool) with increasing probiotic dose (P = 0.018) was observed. No overall dose-response effect was found on the blood lipids. High doses of probiotics were well tolerated. CONCLUSION: A dose-related recovery of BB-12 from feces was observed.

The binding site for UCH-L3 on ubiquitin: mutagenesis and NMR studies on the complex between ubiquitin and UCH-L3.

The ubiquitin fold is a versatile and widely used targeting signal that is added post-translationally to a variety of proteins. Covalent attachment of one or more ubiquitin domains results in localization of the target protein to the proteasome, the nucleus, the cytoskeleton or the endocytotic machinery. Recognition of the ubiquitin domain by a variety of enzymes and receptors is vital to the targeting function of ubiquitin. Several parallel pathways exist and these must be able to distinguish among ubiquitin, several different types of polymeric ubiquitin, and the various ubiquitin-like domains. Here we report the first molecular description of the binding site on ubiquitin for ubiquitin C-terminal hydrolase L3 (UCH-L3). The site on ubiquitin was experimentally determined using solution NMR, and site-directed mutagenesis. The site on UCH-L3 was modeled based on X-ray crystallography, multiple sequence alignments, and computer-aided docking. Basic residues located on ubiquitin (K6, K11, R72, and R74) are postulated to contact acidic residues on UCH-L3 (E10, E14, D33, E219). These putative interactions are testable and fully explain the selectivity of ubiquitin domain binding to this enzyme.

Functional analysis of the proteasome regulatory particle.

We have developed S. cerevisiae as a model system for mechanistic studies of the 26S proteasome. The subunits of the yeast 19S complex, or regulatory particle (RP), have been defined, and are closely related to those of mammalian proteasomes. The multiubiquitin chain binding subunit (S5a/Mcb1/Rpn10) was found, surprisingly, to be nonessential for the degradation of a variety of ubiquitin-protein conjugates in vivo. Biochemical studies of proteasomes from deltarpn10 mutants revealed the existence of two structural subassemblies within the RP, the lid and the base. The lid and the base are both composed of 8 subunits. By electron microscopy, the base and the lid correspond to the proximal and distal masses of the RP, respectively. The base is sufficient to activate the 20S core particle for degradation of peptides, but the lid is required for ubiquitin-dependent degradation. The lid subunits share sequence motifs with components of the COP9/signalosome complex, suggesting that these functionally diverse particles have a common evolutionary ancestry. Analysis of equivalent point mutations in the six ATPases of the base indicate that they have well-differentiated functions. In particular, mutations in one ATPase gene, RPT2, result in an unexpected defect in peptide hydrolysis by the core particle. One interpretation of this result is that Rpt2 participates in gating of the channel through which substrates enter the core particle.

Active site mutants in the six regulatory particle ATPases reveal multiple roles for ATP in the proteasome.

A family of ATPases resides within the regulatory particle of the proteasome. These proteins (Rpt1-Rpt6) have been proposed to mediate substrate unfolding, which may be required for translocation of substrates through the channel that leads from the regulatory particle into the proteolytic core particle. To analyze the role of ATP hydrolysis in protein breakdown at the level of the individual ATPase, we have introduced equivalent site-directed mutations into the ATPbinding motif of each RPT gene. Non-conservative substitutions of the active-site lysine were lethal in four of six cases, and conferred a strong growth defect in two cases. Thus, the ATPases are not functionally redundant, despite their multiplicity and sequence similarity. Degradation of a specific substrate can be inhibited by ATP-binding-site substitutions in many of the Rpt proteins, indicating that they co-operate in the degradation of individual substrates. The phenotypic defects of the different rpt mutants were strikingly varied. The most divergent phenotype was that of the rpt1 mutant, which was strongly growth defective despite showing no general defect in protein turnover. In addition, rpt1 was unique among the rpt mutants in displaying a G1 cell-cycle defect. Proteasomes purified from an rpt2 mutant showed a dramatic inhibition of peptidase activity, suggesting a defect in gating of the proteasome channel. In summary, ATP promotes protein breakdown by the proteasome through multiple mechanisms, as reflected by the diverse phenotypes of the rpt mutants.

Substrate specificity of deubiquitinating enzymes: ubiquitin C-terminal hydrolases.

Ubiquitin C-terminal hydrolases (UCH) are deubiquitinating enzymes which hydrolyze C-terminal esters and amides of ubiquitin. Here we report the processing of a number of ubiquitin derivatives by two human UCH isozymes (isozymes L1 and L3) and find that these enzymes show little discrimination based on the P1' amino acid, except that proline is cleaved slowly. Ubiquitinyllysine derivatives linked by the alpha- or epsilon-amino group are hydrolyzed at identical rates. Isozyme-specific hydrolytic preferences are only evident when the leaving group is large. The ubiquitin gene products can be cotranslationally processed by one or both of these UCH isozymes, and purified UbCEP52 can be hydrolyzed by UCH isozyme L3. Binding of nucleic acid by UbCEP52 converts it to a form resistant to processing by these enzymes, apparently because of the formation of a larger, more tightly folded substrate. Consistent with this postulate is the observation that these enzymes do not hydrolyze large ubiquitin derivatives such as N epsilon-ubiquitinyl-cytochrome-c, N epsilon-K48polyubiquitinyl-lysozyme, or an N alpha-ubiquitinyl-beta-galactosidase fusion protein. Thus, these enzymes rapidly and preferentially cleave small leaving groups such as amino acids and oligopeptides from the C-terminus of ubiquitin, but not larger leaving groups such as proteins. These data suggest that the physiological role of UCH is to hydrolyze small adducts of ubiquitin and to generate free monomeric ubiquitin from ubiquitin proproteins, but not to deubiquitinate ubiquitin-protein conjugates or disassemble polyubiquitin chains.

Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 A resolution.

Ubiquitin C-terminal hydrolases catalyze the removal of adducts from the C-terminus of ubiquitin. We have determined the crystal structure of the recombinant human Ubiquitin C-terminal Hydrolase (UCH-L3) by X-ray crystallography at 1.8 A resolution. The structure is comprised of a central antiparallel beta-sheet flanked on both sides by alpha-helices. The beta-sheet and one of the helices resemble the well-known papain-like cysteine proteases, with the greatest similarity to cathepsin B. This similarity includes the UCH-L3 active site catalytic triad of Cys95, His169 and Asp184, and the oxyanion hole residue Gln89. Papain and UCH-L3 differ, however, in strand and helix connectivity, which in the UCH-L3 structure includes a disordered 20 residue loop (residues 147-166) that is positioned over the active site and may function in the definition of substrate specificity. Based upon analogy with inhibitor complexes of the papain-like enzymes, we propose a model describing the binding of ubiquitin to UCH-L3. The UCH-L3 active site cleft appears to be masked in the unliganded structure by two different segments of the enzyme (residues 9-12 and 90-94), thus implying a conformational change upon substrate binding and suggesting a mechanism to limit non-specific hydrolysis.

Substrate binding and catalysis by ubiquitin C-terminal hydrolases: identification of two active site residues.

Ubiquitin C-terminal hydrolases (UCH's) are a newly-defined class of thiol proteases implicated in the proteolytic processing of polymeric ubiquitin. They are important for the generation of monomeric ubiquitin, the active component of the eukaryotic ubiquitin-dependent proteolytic system. There are at least three mammalian isozymes which are tissue specific and developmentally regulated. To study the structure and functional roles of these highly homologous enzymes, we have subcloned and overexpressed two of these isozymes, UCH-L1 and UCH-L3. Here, we report their purification, physical characteristics, and the mutagenesis of UCH-L1. Site-directed mutagenesis of UCH-L1 reveals that C90 and H161 are involved in catalytic rate enhancement. Data from circular dichroic and Raman spectroscopy, as well as secondary structure prediction algorithms, indicate that both isozymes have a significant amount of alpha-helix (> 35%), and contain no disulfide bonds. Both enzymes are reasonably stable, undergoing a reversible thermal denaturation at 52 degrees C. These transitions are characterized by thermodynamic parameters typical of single domain globular proteins. Substrate binding affinity to UCH-L3 was directly measured by equilibrium gel filtration (Kd = 0.5 microM), and the results are similar to the kinetically determined Km for ubiquitin ethyl ester (o.6 microM). The binding is primarily electrostatic in nature and indicates the existence of a specific and extensive binding site for ubiquitin on the surface of the enzyme.

Metabolism of the polyubiquitin degradation signal: structure, mechanism, and role of isopeptidase T.

A necessary step in ubiquitin-dependent proteolysis is the addition of a polyubiquitin chain to the target protein. This ubiquitinated protein is degraded by a multisubunit complex known as the 26S proteasome. The polyubiquitin chain is probably not released until a late stage in the proteolysis by the proteasome. It is subsequently disassembled to yield functional ubiquitin monomers. Here we present evidence that a 93 kDa protein, isopeptidase T, has the properties expected for the enzyme which disassembles these branched polyubiquitin chains. Protein and cDNA sequencing revealed that isopeptidase T is a member of the ubiquitin specific protease family (UBP). Isopeptidase T disassembles branched polyubiquitin chains (linked by the G76-K48 isopeptide bond) by a sequential exo mechanism, starting at the proximal end of the chain (the proximal ubiquitin contains a free carboxyl-terminus). Isopeptidase T prefers to disassemble chains in which there is an intact and unblocked RGG sequence at the C-terminus of the proximal subunit. Rates of disassembly are reduced when G76 of the proximal ubiquitin is modified, for example, by ligation to substrate protein, by esterification, by replacement of the proximal glycine with alanine (G76A), or by truncation. Linear proubiquitin is only a poor substrate. Observed rates and specificity are consistent with isopeptidase T playing a major role in disassembly of polyubiquitin chains. The high discrimination against chains that are blocked or modified at the proximal end indicates that the enzyme acts after release of the chains from conjugated proteins or degradation intermediates. Thus, the proteolytic degradation signal is not disassembled by isopeptidase T before the ubiquitinated protein is degraded. These (and earlier) results suggest that UBP isozymes may exhibit significant substrate specificity, consistent with a role in the regulated catabolism of the polymeric ubiquitin, including the polyubiquitin protein degradation signal.