Plasma Membrane Profiling Defines an Expanded Class of Cell Surface Proteins Selectively Targeted for Degradation by HCMV US2 in Cooperation with UL141

Human cytomegalovirus (HCMV) US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I). Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin β1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2’s impact on HCMV pathogenesis.     

Antigen presentation: TAP dances with ATP.

Assembly of antigen-presenting complexes between class I MHC molecules and peptide requires formation of a complex between the 'ABC' peptide transporter, TAP, and newly synthesized class I molecules. Recent studies have provided new insights into the role of ATP in peptide binding, transport and release.     

Predicting risk of decompression sickness in humans from outcomes in sheep.

In animals, the response to decompression scales as a power of species body mass. Consequently, decompression sickness (DCS) risk in humans should be well predicted from an animal model with a body mass comparable to humans. No-stop decompression outcomes in compressed air and nitrogen-oxygen dives with sheep (n = 394 dives, 14.5% DCS) and humans (n = 463 dives, 4.5% DCS) were used with linear-exponential, probabilistic modeling to test this hypothesis. Scaling the response parameters of this model between species (without accounting for body mass), while estimating tissue-compartment kinetic parameters from combined human and sheep data, predicts combined risk better, based on log likelihood, than do separate sheep and human models, a combined model without scaling, and a kinetic-scaled model. These findings provide a practical tool for estimating DCS risk in humans from outcomes in sheep, especially in decompression profiles too risky to test with humans. This model supports the hypothesis that species of similar body mass have similar DCS risk.     

The ubiquitin E3 ligase MARCH7 is differentially regulated by the deubiquitylating enzymes USP7 and USP9X

Protein modification by one or more ubiquitin chains serves a critical signalling function across a wide range of cellular processes. Specificity within this system is conferred by ubiquitin E3 ligases, which target the substrates. Their activity is balanced by deubiquitylating enzymes (DUBs), which remove ubiquitin from both substrates and ligases. The RING-CH ligases were initially identified as viral immunoevasins involved in the downregulation of immunoreceptors. Their cellular orthologues, the Membrane-Associated RING-CH (MARCH) family represent a subgroup of the classical RING genes. Unlike their viral counterparts, the cellular RING-CH proteins appear highly regulated, and one of these in particular, MARCH7, was of interest because of a potential role in neuronal development and lymphocyte proliferation. Difficulties in detection and expression of this orphan ligase lead us to search for cellular cofactors involved in MARCH7 stability. In this study, we show that MARCH7 readily undergoes autoubiquitylation and associates with two deubiquitylating enzymes – ubiquitin-specific protease (USP)9X in the cytosol and USP7 in the nucleus. Exogenous expression and short interfering RNA depletion experiments demonstrate that MARCH7 can be stabilized by both USP9X and USP7, which deubiquitylate MARCH7 in the cytosol and nucleus, respectively. We therefore demonstrate compartment-specific regulation of this E3 ligase through recruitment of site-specific DUBs.     

Comparative genomic assessment of Multi-Locus Sequence Typing: rapid accumulation of genomic heterogeneity among clonal isolates of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

Background  

Multi-Locus Sequence Typing (MLST) has emerged as a leading molecular typing method owing to its high ability to discriminate among bacterial isolates, the relative ease with which data acquisition and analysis can be standardized, and the high portability of the resulting sequence data. While MLST has been successfully applied to the study of the population structure for a number of different bacterial species, it has also provided compelling evidence for high rates of recombination in some species. We have analyzed a set of Campylobacter jejuni strains using MLST and Comparative Genomic Hybridization (CGH) on a full-genome microarray in order to determine whether recombination and high levels of genomic mosaicism adversely affect the inference of strain relationships based on the analysis of a restricted number of genetic loci.     

Perivascular cells of the supraspinatus tendon express both tendon- and stem cell-related markers

Tendons and ligaments are often affected by mechanical injuries or chronic impairment but other than muscle or bone they possess a low healing capacity. So far, little is known about regeneration of tendons and the role of tendon precursor cells in that process. We hypothesize that perivascular cells of tendon capillaries are progenitors for functional tendon cells and are characterized by expression of marker genes and proteins typical for mesenchymal stem cells and functional tendon cells. Immunohistochemical characterization of biopsies derived from intact human supraspinatus tendons was performed. From these biopsies perivascular cells were isolated, cultured, and characterized using RT-PCR and Western blotting. We have shown for the first time that perivascular cells within tendon tissue express both tendon- and stem/precursor cell-like characteristics. These findings were confirmed by results from in vitro studies focusing on cultured perivascular cells isolated from human supraspinatus tendon biopsies. The results suggest that the perivascular niche may be considered a source for tendon precursor cells. This study provides further information about the molecular nature and localization of tendon precursor cells, which is the basis for developing novel strategies towards tendon healing and facilitated regeneration. H. Tempfer and A. Wagner have contributed equally to this paper.     

Es-x/Ces1 prevents triacylglycerol accumulation in McArdle-RH7777 hepatocytes

Mouse esterase-x/carboxylesterase 1 (Es-x/Ces1) is a close homolog of triacylglycerol hydrolase/carboxylesterase 3 (TGH/Ces3). Es-x possesses a conserved esterase/lipase active site motif, suggesting that like TGH it could play a role in hepatic triacylglycerol (TG) metabolism. McArdle-RH7777 cells stably transfected with Es-x cDNA accumulated significantly less TG and had increased production of acid-soluble metabolites (an indicator of β-oxidation) during incubations with 0.4 mM oleic acid when compared to empty vector or TGH cDNA transfected cells. Reduction of cellular TG persisted in the presence of esterase/lipase inhibitor E600 indicating that Es-x-mediated TG lowering can be largely explained by reduced partitioning of exogenous fatty acids to TG and increased redirection to β-oxidation, rather than by increased TG turnover. Glycerol supplementation increased TG synthesis in both control and Es-x expressing cells to similar extent suggesting that Es-x expression did not reduce flux of metabolic intermediates through the glycerol-3-phosphate pathway. While Es-x expression reduced cellular TG levels, secretion of TG and apolipoprotein B remained unchanged when compared to control cells. Overall, these results suggest that Es-x limits hepatic TG accumulation by promoting β-oxidation.