Endothelial Cell Protein Targeting by Myeloperoxidase-Derived 2-Chlorofatty Aldehyde.

Neutrophils are important cellular mediators of injury and repair in diseases including ischemic heart disease, atherosclerosis, and sepsis. Myeloperoxidase-derived (MPO)-oxidants released from neutrophils are potential mediators of endothelial injury in disease. MPO-derived HOCl attacks plasmalogen phospholipid to liberate 2-chlorofatty aldehyde (2-ClFALD). Both 2-ClFALD and its oxidation product, 2-chlorofatty acid (2-ClFA), are electrophilic lipids, and both probably react with proteins through several mechanisms. In the present study, we investigate protein modification specifically by 2-ClFALD under non-reducing conditions (e.g., without stabilizing Schiff base bonds), which likely reflects nucleophilic targeting of the electrophilic chlorinated carbon. Protein modification by the ω-alkyne analog of 2-chlorohexadecanal (2-ClHDA), 2-ClHDyA, was compared to that with the ω-alkyne analog of 2-chlorohexadecanoic acid (2-ClHA), 2-ClHyA, in multiple cell lines, which demonstrated 2-ClFALD preferentially modifies proteins compared to 2-ClFA. The 2-ClHDyA modified proteins from EA.hy926 cells and human lung microvascular endothelial cells analyzed by shotgun proteomics and over-representation analysis included adherens junction, cell adhesion molecule binding, and cell substrate junction enrichment categories. It is possible that proteins in these groups may have roles in previously described 2-ClFALD-elicited endothelial barrier dysfunction.

The role of environmental factors on the evolution of phenotypic diversity in vesicular stomatitis virus populations.

Virus adaptation to an ever-changing environment requires the availability of variants with phenotypes that can fulfil new requirements for replication. High mutation rates result in the generation of these variants. The factors that contribute to the maintenance or elimination of this diversity, however, are not fully understood. This study used a collection of vesicular stomatitis virus strains generated under different conditions to measure the extent of variation within each population, and tested the effects of several environmental factors on diversity. It was found that the host-cell type used for selection sometimes had an effect on the extent of variation and that there may be different levels of variation over time. Persistent infections promoted higher levels of diversity than acute infections, presumably due to complementation. In contrast, environmental heterogeneity, host breadth and the cell type used for testing (as opposed to the cell type used for selection) did not seem to have an effect on the amount of phenotypic diversity observed.

Newly discovered viral E3 ligase pK3 induces endoplasmic reticulum-associated degradation of class I major histocompatibility proteins and their membrane-bound chaperones.

Viral immune invasion proteins are highly effective probes for studying physiological pathways. We report here the characterization of a new viral ubiquitin ligase pK3 expressed by rodent herpesvirus Peru (RHVP) that establishes acute and latent infection in laboratory mice. Our findings show that pK3 binds directly and specifically to class I major histocompatibility proteins (MHCI) in a transmembrane-dependent manner. This binding results in the rapid degradation of the pK3/MHCI complex by a mechanism dependent upon catalytically active pK3. Subsequently, the rapid degradation of pK3/MHCI secondarily causes the slow degradation of membrane bound components of the MHCI peptide loading complex, tapasin, and transporter associated with antigen processing (TAP). Interestingly, this secondary event occurs by cellular endoplasmic reticulum-associated degradation. Cumulatively, our findings show pK3 uses a unique mechanism of substrate detection and degradation compared with other viral or cellular E3 ligases. More importantly, our findings reveal that in the absence of nascent MHCI proteins in the endoplasmic reticulum, the transmembrane proteins TAP and tapasin that facilitate peptide binding to MHCI proteins are degraded by cellular quality control mechanisms.

Ubiquitination of substrates by esterification.

Post-translational modification by ubiquitination determines intracellular location and fate of numerous proteins, thus impacting a diverse array of physiologic functions. Past dogma has been that ubiquitin was only coupled to substrates by isopeptide bonds to internal lysine residues or less frequently peptide bonds to the N-terminus. Enigmatically, however, several proteins lacking lysines had been reported to retain ubiquitin-dependent fates. Resolution of this paradox was afforded by recent observations that ubiquitination of substrates can also occur on cysteine or serine and threonine residues by thio- or oxy-ester bond formation, respectively (collectively called esterification). Although chemically possible, these bonds were considered too labile to be of physiological relevance. In this review we discuss recent evidence for the ubiquitination of protein substrates by esterification and speculate on its mechanism and its physiological importance.

Ube2j2 ubiquitinates hydroxylated amino acids on ER-associated degradation substrates.

Ubiquitin (Ub) modification of proteins plays a prominent role in the regulation of multiple cell processes, including endoplasmic reticulum-associated degradation (ERAD). Until recently, ubiquitination of substrates was thought to occur only via isopeptide bonds, typically to lysine residues. Several recent studies suggest that Ub can also be coupled to nonlysine residues by ester/thiolester bonds; however, the molecular basis for these novel modifications remains elusive. To probe the mechanism and importance of nonlysine ubiquitination, we have studied the viral ligase murine K3 (mK3), which facilitates the polyubiquitination of hydroxylated amino acids serine/threonine on its ERAD substrate. In this paper, we identify Ube2j2 as the primary cellular E2 recruited by the mK3 ligase, and this E2-E3 pair is capable of conjugating Ub on lysine or serine residues of substrates. However, surprisingly, Ube2j2-mK3 preferentially promotes ubiquitination of hydroxylated amino acids via ester bonds even when lysine residues are present on wild-type substrates, thus establishing physiological relevance of this novel ubiquitination strategy.

Role of the RING-CH domain of viral ligase mK3 in ubiquitination of non-lysine and lysine MHC I residues.

A plethora of ubiquitin ligases determine the intracellular location and fate of numerous proteins in a substrate-specific manner. However, the mechanisms for these functions are incompletely understood. Most ligases have structurally related RING domains that are critical for ligase activity including the recruitment of ubiquitin conjugating enzymes. Here we probe the function of the RING-CH domain of murine gamma-herpesvirus-68 ligase mK3 that functions as an immune evasin by targeting major histocompatibility complex (MHC) class I heavy chains for endoplasmic reticulum-associated degradation (ERAD). Interestingly, mK3 mediates ubiquitin conjugation via ester bonds to S or T residues in addition to conventional isopeptide linkages to K residues. To determine the mechanism of non-K ubiquitination of substrates, we introduced into an mK3 background the RING-CH domains of related viral and cellular MARCH (membrane associated RING-CH) ligases. We found that although a conserved W present in all viral RING-CH domains is critical for mK3 function, sequences outside the RING-CH domain determine whether and which non-lysine substrate residues can be ubiquitinated by mK3. Our findings support the model that viral ligases have evolved a highly effective strategy to optimally orient their RING domain with substrate allowing them to ubiquitinate non-K residues.

Viral and cellular MARCH ubiquitin ligases and cancer.

Covalent conjugation of proteins with ubiquitin is one the most important post-translational modifications because it controls intracellular protein trafficking typically resulting in protein degradation. Frequently ubiquitinated proteins are targeted to the proteasome for degradation in the cytosol. However, ubiquitinated membrane bound proteins can also be targeted for endocytosis and degradation in the lysosome. Ubiquitin-dependent degradation pathways have clear cancer relevance due to their integral involvement in protein quality control, regulation of immune responses, signal transduction, and cell cycle regulation. In spite of its fundamental importance, little is known regarding how proteins are specifically identified for ubiquitin-dependent degradation. In this article we review a newly discovered family of viral and cellular ubiquitin ligases called MARCH proteins. Recent studies of MARCH proteins define new paradigms showing how ubiquitin E3 ligases determine the intracellular location and fate of proteins.

Evaluation of two homologous proline-rich proteins of Coccidioides posadasii as candidate vaccines against coccidioidomycosis.

Evaluation of the protective efficacy of recombinant T-cell-reactive proteins of Coccidioides posadasii in a murine model of coccidioidomycosis has led to the discovery of potential vaccines against this respiratory disease. A recombinant proline-rich antigen (rAg2/Pra) has been reported to be a leading vaccine candidate. However, contradictory results exist on the protection afforded by this antigen. Subcutaneous vaccination of either C57BL/6 or BALB/c mice with rAg2/Pra plus adjuvant followed by intraperitoneal challenge with C. posadasii resulted in a significant reduction of the fungal burden at 12 to 14 days postchallenge compared to that in nonvaccinated animals. Use of the same vaccination protocol followed by intranasal (i.n.) challenge of C57BL/6 mice with an equal number of organisms culminated in chronic pulmonary infection or death over a 90-day period. Early studies of Ag2/Pra suggested that it is a component of an immunogenic complex. We reveal in this study that C. posadasii produces a homolog of the reported proline-rich antigen, designated Prp2, which shows 69% protein sequence identity and 86% similarity to Ag2/Pra. Protection against i.n. challenge of C57BL/6 mice was evaluated by vaccination with the single bacterially expressed homolog, rAg2/Pra, or rPrp2 in combination with rAg2/Pra, each in the presence of the same adjuvant. The combined vaccine provided significantly better protection than either of the single recombinant protein vaccines. Results of enzyme-linked immunospot assays of the immunized mice revealed that the two proline-rich homologs contain unique T-cell epitopes. In combination, the recombinant proteins stimulate a more heterogeneous and protective T-cell repertoire than the monovalent vaccines.

Ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce ERAD of MHC-I by viral E3 ligase mK3.

The mechanism by which substrates for endoplasmic reticulum-associated degradation are retrotranslocated to the cytosol remains largely unknown, although ubiquitination is known to play a key role. The mouse gamma-herpesvirus protein mK3 is a viral RING-CH-type E3 ligase that specifically targets nascent major histocompatibility complex I heavy chain (HC) for degradation, thus blocking the immune detection of virus-infected cells. To address the question of how HC is retrotranslocated and what role mK3 ligase plays in this action, we investigated ubiquitin conjugation sites on HC using mutagenesis and biochemistry approaches. In total, our data demonstrate that mK3-mediated ubiquitination can occur via serine, threonine, or lysine residues on the HC tail, each of which is sufficient to induce the rapid degradation of HC. Given that mK3 has numerous cellular and viral homologues, it will be of considerable interest to determine the pervasiveness of this novel mechanism of ubiquitination.

Improved protection of mice against lethal respiratory infection with Coccidioides posadasii using two recombinant antigens expressed as a single protein.

Two recombinant antigens which individually protect mice from lethal intranasal infection were studied in combination, either as a mixture of two separately expressed proteins or as a single chimeric expression product. Mice vaccinated with either combination survived longer than mice given single antigens. Immunized mice also exhibited specific IgG immunoglobulins and yielded splenocytes which produced interferon-gamma in response to either antigen. The chimeric antigen has the practical advantage of offering enhanced protection from multiple components without increasing production costs.

Positive directional selection in the proline-rich antigen (PRA) gene among the human pathogenic fungi Coccidioides immitis, C. posadasii and their closest relatives.

In this study, we investigate the possibility of selection acting on the proline-rich antigen (PRA) gene in natural populations of the two human pathogens, Coccidioides immitis and Coccidioides posadasii, and three of their close relatives, Chrysosporium lucknowense, Chrysosporium queenslandicum, and Uncinocarpus reesii. We addressed the following questions: Is diversifying selection acting on PRA in the pathogenic species as a result of avoidance of the host's immune system, and has adaptation to a pathogenic life style lead to positive directional selection and increased rate of evolution in PRA between the species? For these purposes, we amplified and sequenced from 40 individuals belonging to the five species, the entire coding region of the PRA gene, as well as partial sequences from the coding region of each of the three housekeeping genes glyderaldehyde-3-phosphate dehydrogenase, glutamine synthetase A, and hexokinase A. We used likelihood-based methods to compare models of different types of selective pressure among codons to analyze the mode of evolution of the genes and found that the PRA gene evolves under positive selection, but the investigated parts of the housekeeping genes evolve primarily under purifying selection. We found a very low level of intraspecific variability and no evidence of diversifying selection, suggesting that the increased rate of evolution in the PRA gene is not a result of avoidance of the host's immune system. Neither did likelihood-based analyses suggest that selection was stronger on the branch separating pathogenic and nonpathogenic species. Instead, we suggest that positive selection act on PRA as a consequence of spore cell-wall morphogenesis unique to each species.