Evolution of antioxidant mechanisms: Thiol-Dependent peroxidases and thioltransferase among procaryotes

Summary Glutathione peroxidase and glutathione S-transferase both utilize glutathione (GSH) to destroy organic hydroperoxides, and these enzymes are thought to serve an antioxidant function in mammalian cells by catalyzing the destruction of lipid hydroperoxides. Only two groups of procaryotes, the purple bacteria and the cyanobacteria, produce GSH, and we show in the present work that representatives from these two groups (Escherichia coli, Beneckea alginolytica, Rhodospirillum rubrum, Chromatium vinosum, andAnabaena sp. strain 7119) lack significant glutathione peroxidase and glutathione S-transferase activities. This finding, coupled with the general absence of polyunsaturated fatty acids in procaryotes, suggests that GSH-dependent peroxidases evolved in eucaryotes in response to the need to protect against polyunsaturated fatty acid oxidation. A second antioxidant function of GSH is mediated by glutathione thiol-transferase, which catalyzes the reduction of various cellular disulfides by GSH. Two of the five GSH-producing bacteria studied (E. coli andB. alginolytica) produced higher levels of glutathione thiol-transferase than found in rat liver, whereas the activity was absent in the other three species studied. The halobacteria produced γ-glutamylcysteine rather than GSH, and assays for γ-glutamylcysteine-dependent enzymes demonstrated an absence of peroxidase and S-transferase activities but the presence of significant thioltransferase activity. Based upon these results it appears that GSH and γ-glutamylcysteine do not function in bactera as antioxidants directed against organic hydroperoxides but do play a significant, although not universal, role in main-taining disulfides in a reduced state. The function of GSH in the photosynthetic bacteria, aside from providing a form of cysteine resistant toward autoxidation, remains a puzzle, as none of the GSH-dependent enzymes tested other than glutathione reductase were present in these organisms.     

Crystal structure of cyclophilin A complexed with a binding site peptide from the HIV-1 capsid protein.

The cellular protein, cyclophilin A (CypA), is incorporated into the virion of the type 1 human immunodeficiency virus (HIV-1) via a direct interaction with the capsid domain of the viral Gag polyprotein. We demonstrate that the capsid sequence 87His-Ala-Gly-Pro-Ile-Ala92 (87HAGPIA92) encompasses the primary cyclophilin A binding site and present an X-ray crystal structure of the CypA/HAGPIA complex. In contrast to the cis prolines observed in all previously reported structures of CypA complexed with model peptides, the proline in this peptide, Pro 90, binds the cyclophilin A active site in a trans conformation. We also report the crystal structure of a complex between CypA and the hexapeptide HVGPIA, which also maintains the trans conformation. Comparison with the recently determined structures of CypA in complexes with larger fragments of the HIV-1 capsid protein demonstrates that CypA recognition of these hexapeptides involves contacts with peptide residues Ala(Val) 88, Gly 89, and Pro 90, and is independent of the context of longer sequences.     

Effect of homopolyribonucleotides on messenger ribonucleoprotein particles.

A discrete set of polypeptides copurify with and appear to be specifically attached to mRNA from polysomes of eukaryotic cells. This report describes the effect of homopolyribonucleotides on mRNA-protein complexes separated from ribosome subunits by oligo(dT)-cellulose chromatography. It is shown that poly (U) and poly (A) can release mRNA-protein complexes adsorbed to oligo(dT)-cellulose, whereas poly (C) and poly(I) are much less effective in this process. Analysis of polyribonucleotide released material showed that poly(U) effectively dissociated the mRNA-protein complexes while poly(A) caused no or only partial derangement of these particles. The specificities seen in the polyribonucleotide effects in turn suggest a high degree of specificity in the interaction between the proteins and mRNA.     

Familial risks for common diseases: etiologic clues and guidance to gene identification

Familial clustering of a disease is a direct indicator of a possible heritable cause, provided that environmental sharing can be excluded. If the familial clustering is lacking, the likelihood of a heritable influence is also small. In the era of genome scans, the consideration of data on heritability should be important in the assessment of the likely success of the genome scan. The availability of a Multigeneration Register in Sweden provides a reliable access to families throughout the last century. This Register has been extensively used to study a number of different diseases through linkage to the Hospital Discharge Register. In the present article we review the obtained and some unpublished results for nine main disease classes. For each of these, familial risks are given for four disease subtypes. As measures of familial clustering we use risks between siblings, twins and spouses. Disease correlation between spouses suggests environmental sharing and a higher correlation between siblings and particularly twins shows heritable effects. We will also comment on the established susceptibility genes and the risks conferred by them. The data suggest high heritabilities for chronic obstructive pulmonary disease, asthma, noninfective enteritis and colitis, cerebral palsy and endocrine and metabolic diseases. Among the performed first-generation genome scans on various diseases, the success appears to be related to the a priori heritability estimates. To our knowledge this is a first attempt to summarize familial risks for a large number of diseases using data from a single population on which reasonable uniform diagnostic criteria have been applied.     

MyD88 and IFN-alphabeta differentially control maturation of bystander but not Salmonella-associated dendritic cells or CD11cintCD11b+ cells during infection

The interface between dendritic cells (DCs) and T cells is critical to elicit effective immunity against pathogens. The maturation state of DCs determines the quality of the interaction and governs the type of response. DCs can be matured directly through activating Toll-like receptors (TLRs) or indirectly by cytokines. We explore the role of the TLR adaptor MyD88 on DC maturation during Salmonella infection. Using Salmonella expressing GFP, we also examine the phenotype and function of bacteria-associated DCs matured in the absence of bacteria-mediated TLR signalling. MyD88 was required for upregulation of CD80 on DCs during infection, whereas CD86 and CD40 were upregulated independently of MyD88, although requiring a higher bacterial burden in the MLN. MyD88-independent upregulation was mediated by IFN-αβ produced during infection. In infected MyD88^−/−IFN-αβR^−/− mice, which lack most bacteria-driven TLR signalling, indirect DC maturation was abolished. In contrast, DCs containing Salmonella upregulated co-stimulatory molecules independently of MyD88 and IFN-αβ, revealing a pathway of phenotypic maturation active in infected DCs. However, despite high co-stimulatory molecule expression, Salmonella -containing DCs from MyD88^−/− or MyD88^−/−IFN-αβR^−/− mice had a compromised capacity to activate T cells. Thus, bacterial stimulation of TLRs influences DC function at multiple levels that modulates their capacity to direct antibacterial immunity.     

The role of LIP5 and CHMP5 in multivesicular body formation and HIV-1 budding in mammalian cells

We examined the function of LIP5 in mammalian cells, because the yeast homologue Vta1p was recently identified as a protein required for multivesicular body (MVB) formation. LIP5 is predominantly a cytosolic protein. Depletion of LIP5 by small inhibitory RNA (siRNA) does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does reduce the degradation of internalized epidermal growth factor receptor (EGFR), with EGFR accumulating in intracellular vesicles. Depletion of LIP5 by siRNA also decreases human immunodeficiency virus type 1 (HIV-1) budding by 70%. We identify CHMP5 as a LIP5-binding protein and show that CHMP5 is primarily cytosolic. Depletion of CHMP5 by siRNA does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does result in reduced degradation of the EGFR similar to silencing of LIP5. Surprisingly, CHMP5 depletion results in an increase in the release of infectious HIV-1 particles. Overexpression of CHMP5 with a large carboxyl-terminal epitope affects the distribution of both early and late endocytic compartments, whereas overexpression of LIP5 does not alter the endocytic pathway. Comparison of overexpression and siRNA phenotypes suggests that the roles of these proteins in MVB formation may be more specifically addressed using RNA interference and that both LIP5 and CHMP5 function in MVB sorting, whereas only LIP5 is required for HIV release.     

Homologue of macrophage-activating lipoprotein in Mycoplasma gallisepticum is not essential for growth and pathogenicity in tracheal organ cultures

While the genomes of a number of Mycoplasma species have been fully determined, there has been limited characterization of which genes are essential. The surface protein (p47) identified by monoclonal antibody B3 is the basis for an enzyme-linked immunosorbent assay for serological detection of Mycoplasma gallisepticum infection and appears to be constitutively expressed. Its gene was cloned, and the DNA sequence was determined. Subsequent analysis of the p47 amino acid sequence and searches of DNA databases found homologous gene sequences in the genomes of M. pneumoniae and M. genitalium and identity with a gene family in Ureaplasma urealyticum and genes in M. agalactiae and M. fermentans. The proteins encoded by these genes were found to belong to a family of basic membrane proteins (BMP) that are found in a wide range of bacteria, including a number of pathogens. Several of the BMP family members, including p47, contain selective lipoprotein-associated motifs that are found in macrophage-activating lipoprotein 404 of M. fermentans and lipoprotein P48 of M. agalactiae. The p47 gene was predicted to encode a 59-kDa peptide, but affinity-purified p47 had a molecular mass of approximately 47 kDa, as determined by polyacrylamide gel analysis. Analysis of native and recombinant p47 by mass peptide fingerprinting revealed the absence of the carboxyl end of the protein encoded by the p47 gene in native p47, which would account for the difference seen in the predicted and measured molecular weights and indicated posttranslational cleavage of the lipoprotein at its carboxyl end. A DNA construct containing the p47 gene interrupted by the gene encoding tetracycline resistance was used to transform M. gallisepticum cells. A tetracycline-resistant mycoplasma clone, P2, contained the construct inserted within the genomic p47 gene, with crossovers occurring between 73 bp upstream and 304 bp downstream of the inserted tetracycline resistance gene. The absence of p47 protein in clone P2 was determined by the lack of reactivity with rabbit anti-p47 sera or monoclonal antibody B3 in Western blots of whole-cell proteins. There was no difference between the p47(-) mutant and wild-type M. gallisepticum in pathogenicity in chicken tracheal organ cultures. Thus, p47, although homologous to genes that occur in many prokaryotes, is not essential for growth in vitro or for attachment and the initial stages of pathogenesis in chickens.     

Structure of the Tsg101 UEV domain in complex with the PTAP motif of the HIV-1 p6 protein

The structural proteins of HIV and Ebola display PTAP peptide motifs (termed 'late domains') that recruit the human protein Tsg101 to facilitate virus budding. Here we present the solution structure of the UEV (ubiquitin E2 variant) binding domain of Tsg101 in complex with a PTAP peptide that spans the late domain of HIV-1 p6(Gag). The UEV domain of Tsg101 resembles E2 ubiquitin-conjugating enzymes, and the PTAP peptide binds in a bifurcated groove above the vestigial enzyme active site. Each PTAP residue makes important contacts, and the Ala 9-Pro 10 dipeptide binds in a deep pocket of the UEV domain that resembles the X-Pro binding pockets of SH3 and WW domains. The structure reveals the molecular basis of HIV PTAP late domain function and represents an attractive starting point for the design of novel inhibitors of virus budding.     

The human endosomal sorting complex required for transport (ESCRT-I) and its role in HIV-1 budding

Efficient human immunodeficiency virus type 1 (HIV-1) budding requires an interaction between the PTAP late domain in the viral p6(Gag) protein and the cellular protein TSG101. In yeast, Vps23p/TSG101 binds both Vps28p and Vps37p to form the soluble ESCRT-I complex, which functions in sorting ubiquitylated protein cargoes into multivesicular bodies. Human cells also contain ESCRT-I, but the VPS37 component(s) have not been identified. Bioinformatics and yeast two-hybrid screening methods were therefore used to identify four novel human proteins (VPS37A-D) that share weak but significant sequence similarity with yeast Vps37p and to demonstrate that VPS37A and VPS37B bind TSG101. Detailed studies produced four lines of evidence that human VPS37B is a Vps37p ortholog. 1) TSG101 bound to several different sites on VPS37B, including a putative coiled-coil region and a PTAP motif. 2) TSG101 and VPS28 co-immunoprecipitated with VPS37B-FLAG, and the three proteins comigrated together in soluble complexes of the correct size for human ESCRT-I ( approximately 350 kDa). 3) Like TGS101, VPS37B became trapped on aberrant endosomal compartments in the presence of VPS4A proteins lacking ATPase activity. 4) Finally, VPS37B could recruit TSG101/ESCRT-I activity and thereby rescue the budding of both mutant Gag particles and HIV-1 viruses lacking native late domains. Further studies of ESCRT-I revealed that TSG101 mutations that inhibited PTAP or VPS28 binding blocked HIV-1 budding. Taken together, these experiments define new components of the human ESCRT-I complex and characterize several TSG101 protein/protein interactions required for HIV-1 budding and infectivity.