Differential proteomics argues against a general role for CD9, CD81 or CD63 in the sorting of proteins into extracellular vesicles.

The tetraspanins CD9, CD81 and CD63 are major components of extracellular vesicles (EVs). Yet, their impact on EV composition remains under-investigated. In the MCF7 breast cancer cell line CD63 was as expected predominantly intracellular. In contrast CD9 and CD81 strongly colocalized at the plasma membrane, albeit with different ratios at different sites, which may explain a higher enrichment of CD81 in EVs. Absence of these tetraspanins had little impact on the EV protein composition as analysed by quantitative mass spectrometry. We also analysed the effect of concomitant knock-out of CD9 and CD81 because these two tetraspanins play similar roles in several cellular processes and associate directly with two Ig domain proteins, CD9P-1/EWI-F/PTGFRN and EWI-2/IGSF8. These were the sole proteins significantly decreased in the EVs of double CD9- and CD81-deficient cells. In the case of EWI-2, this is primarily a consequence of a decreased cell expression level. In conclusion, this study shows that CD9, CD81 and CD63, commonly used as EV protein markers, play a marginal role in determining the protein composition of EVs released by MCF7 cells and highlights a regulation of the expression level and/or trafficking of CD9P-1 and EWI-2 by CD9 and CD81.

Human OTULIN haploinsufficiency impairs cell-intrinsic immunity to staphylococcal α-toxin.

The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor κB signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor α-toxin. Naturally elicited antibodies against α-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to α-toxin in nonleukocytic cells.

The 10q26 Risk Haplotype of Age-Related Macular Degeneration Aggravates Subretinal Inflammation by Impairing Monocyte Elimination.

A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age-related macular degeneration (AMD). Here, we examined the mechanisms underlying this susceptibility. We found that monocytes from homozygous carriers of the 10q26 AMD-risk haplotype expressed high amounts of the serine peptidase HTRA1, and HTRA1 located to mononuclear phagocytes (MPs) in eyes of non-carriers with AMD. HTRA1 induced the persistence of monocytes in the subretinal space and exacerbated pathogenic inflammation by hydrolyzing thrombospondin 1 (TSP1), which separated the two CD47-binding sites within TSP1 that are necessary for efficient CD47 activation. This HTRA1-induced inhibition of CD47 signaling induced the expression of pro-inflammatory osteopontin (OPN). OPN expression increased in early monocyte-derived macrophages in 10q26 risk carriers. In models of subretinal inflammation and AMD, OPN deletion or pharmacological inhibition reversed HTRA1-induced pathogenic MP persistence. Our findings argue for the therapeutic potential of CD47 agonists and OPN inhibitors for the treatment of AMD.

Reverse Immunology Approach to Define a New HIV-gp41-Neutralizing Epitope.

The design of immunogens susceptible to elicit potent and broadly neutralizing antibodies against the human immunodeficiency virus type 1 (HIV-1) remains a veritable challenge in the course of vaccine development. Viral envelope proteins adopt different conformational states during the entry process, allowing the presentation of transient neutralizing epitopes. We focused on the highly conserved 3S motif of gp41, which is exposed to the surface envelope in its trimeric prefusion state. Vaccination with a W614A-modified 3S peptide induces in animals neutralizing anti-HIV-1 antibodies among which we selected clone F8. We used F8 as bait to select for W614A-3S phage-peptide mimics. Binding and molecular docking studies revealed that F8 interacts similarly with W614A-3S and a Mim_F8-1 mimotope, despite their lack of sequence homology, suggesting structural mimicry. Finally, vaccination of mice with the purified Mim_F8-1 phage elicited HIV-1-neutralizing antibodies that bound to the cognate W614A-3S motif. Collectively, our findings provide new insights into the molecular design of immunogens to elicit antibodies with neutralizing properties.

The Tumor Suppressor SASH1 Interacts With the Signal Adaptor CRKL to Inhibit Epithelial-Mesenchymal Transition and Metastasis in Colorectal Cancer.

Background & Aims: The tumor-suppressor sterile α motif- and Src-homology 3-domain containing 1 (SASH1) has clinical relevance in colorectal carcinoma and is associated specifically with metachronous metastasis. We sought to identify the molecular mechanisms linking decreased SASH1 expression with distant metastasis formation. Methods: SASH1-deficient, SASH1-depleted, or SASH1-overexpressing HCT116 colon cancer cells were generated by the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9-method, RNA interference, and transient plasmid transfection, respectively. Epithelial-mesenchymal transition (EMT) was analyzed by quantitative reverse-transcription polymerase chain reaction, immunoblotting, immunofluorescence microscopy, migration/invasion assays, and 3-dimensional cell culture. Yeast 2-hybrid assays and co-immunoprecipitation/mass-spectrometry showed V-Crk avian sarcoma virus CT10 oncogene homolog-like (CRKL) as a novel interaction partner of SASH1, further confirmed by domain mapping, site-directed mutagenesis, co-immunoprecipitation, and dynamic mass redistribution assays. CRKL-deficient cells were generated in parental or SASH1-deficient cells. Metastatic capacity was analyzed with an orthotopic mouse model. Expression and significance of SASH1 and CRKL for survival and response to chemotherapy was assessed in patient samples from our department and The Cancer Genome Atlas data set. Results: SASH1 expression is down-regulated during cytokine-induced EMT in cell lines from colorectal, pancreatic, or hepatocellular cancer, mediated by the putative SASH1 promoter. Deficiency or knock-down of SASH1 induces EMT, leading to an aggressive, invasive phenotype with increased chemoresistance. SASH1 counteracts EMT through interaction with the oncoprotein CRKL, inhibiting CRKL-mediated activation of SRC kinase, which is crucially required for EMT. SASH1-deficient cells form significantly more metastases in vivo, depending entirely on CRKL. Patient tumor samples show significantly decreased SASH1 and increased CRKL expression, associated with significantly decreased overall survival. Patients with increased CRKL expression show significantly worse response to adjuvant chemotherapy. Conclusions: We propose SASH1 as an inhibitor of CRKL-mediated SRC signaling, introducing a potentially druggable mechanism counteracting chemoresistance and metastasis formation.

Protein profile of mouse ovarian follicles grown in vitro.

STUDY QUESTION: Could the follicle proteome be mapped by identifying specific proteins that are common or differ between three developmental stages from the secondary follicle (SF) to the antrum-like stage? SUMMARY ANSWER: From a total of 1401 proteins identified in the follicles, 609 were common to the three developmental stages investigated and 444 were found uniquely at one of the stages. WHAT IS KNOWN ALREADY: The importance of the follicle as a functional structure has been recognized; however, up-to-date the proteome of the whole follicle has not been described. A few studies using proteomics have previously reported on either isolated fully-grown oocytes before or after meiosis resumption or cumulus cells. STUDY DESIGN, SIZE, DURATION: The experimental design included a validated mice model for isolation and individual culture of SFs. The system was chosen as it allows continuous evaluation of follicle growth and selection of follicles for analysis at pre-determined developmental stages: SF, complete Slavjanski membrane rupture (SMR) and antrum-like cavity (AF). The experiments were repeated 13 times independently to acquire the material that was analyzed by proteomics. PARTICIPANTS/MATERIALS, SETTING, METHODS: SFs (n = 2166) were isolated from B6CBA/F1 female mice (n = 42), 12 days old, from 15 l. About half of the follicles isolated as SF were analyzed as such (n = 1143) and pooled to obtain 139 µg of extracted protein. Both SMR (n = 359) and AF (n = 124) were obtained after individual culture of 1023 follicles in a microdrop system under oil, selected for analysis and pooled, to obtain 339 µg and 170 µg of protein, respectively. The follicle proteome was analyzed combining isoelectric focusing (IEF) fractionation with 1D and 2D LC-MS/MS analysis to enhance protein identification. The three protein lists were submitted to the 'Compare gene list' tool in the PANTHER website to gain insights on the Gene Ontology Biological processes present and to Ingenuity Pathway Analysis to highlight protein networks. A label-free quantification was performed with 1D LC-MS/MS analyses to emphasize proteins with different expression profiles between the three follicular stages. Supplementary western blot analysis (using new biological replicates) was performed to confirm the expression variations of three proteins during follicle development in vitro. MAIN RESULTS AND THE ROLE OF CHANCE: It was found that 609 out of 1401 identified proteins were common to the three follicle developmental stages investigated. Some proteins were identified uniquely at one stage: 71 of the 775 identified proteins in SF, 181 of 1092 in SMR and 192 of 1100 in AF. Additional qualitative and quantitative analysis highlighted 44 biological processes over-represented in our samples compared to the Mus musculus gene database. In particular, it was possible to identify proteins implicated in the cell cycle, calcium ion binding and glycolysis, with specific expressions and abundance, throughout in vitro follicle development. LARGE SCALE DATA: Data are available via ProteomeXchange with identifier PXD006227. LIMITATIONS, REASONS FOR CAUTION: The proteome analyses described in this study were performed after in vitro development. Despite fractionation of the samples before LC-MS/MS, proteomic approaches are not exhaustive, thus proteins that are not identified in a group are not necessarily absent from that group, although they are likely to be less abundant. WIDER IMPLICATIONS OF THE FINDINGS: This study allowed a general view of proteins implicated in follicle development in vitro and it represents the most complete catalog of the whole follicle proteome available so far. Not only were well known proteins of the oocyte identified but also proteins that are probably expressed only in granulosa cells. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Portuguese Foundation for Science and Technology, FCT (PhD fellowship SFRH/BD/65299/2009 to A.A.), the Swedish Childhood Cancer Foundation (PR 2014-0144 to K.A.R-.W.) and Stockholm County Council to K.A.R-.W. The authors of the study have no conflict of interest to report.

Dp71Δ78-79 dystrophin mutant stimulates neurite outgrowth in PC12 cells via upregulation and phosphorylation of HspB1.

PC12 cells acquire a neuronal phenotype in response to nerve growth factor (NGF). However, this phenotype is more efficiently achieved when the Dp71Δ78-79 dystrophin mutant is stably expressed in PC12-C11 cells. To investigate the effect of Dp71Δ78-79 overexpression on the protein profile of PC12-C11 cells, we compared the expression profiles of undifferentiated and NGF-differentiated PC12-C11 and PC12 cells by 2DE. In undifferentiated cultures, one protein was downregulated, and five were upregulated. Dp71Δ78-79 overexpression had a greater effect on differentiated cultures, with ten proteins downregulated and seven upregulated. The protein with the highest upregulation was HspB1. Changes in HspB1 expression were validated by Western blot and immunofluorescence analyses. Interestingly, the neurite outgrowth in PC12-C11 cells was affected by a polyclonal antibody against HspB1, and the level of HspB1 and HspB1Ser86 decreased, suggesting an important role for this protein in this cellular process. Our results show that Dp71Δ78-79 affects the expression level of some proteins and that the stimulated neurite outgrowth produced by this mutant is mainly through upregulation and phosphorylation of HspB1.

SASH1, a new potential link between smoking and atherosclerosis.

OBJECTIVE: We have previously reported that SASH1 expression is increased in circulating human monocytes from smokers and was positively correlated with the number of carotid atherosclerotic plaques. The aim of this study was to further validate the link between smoking, SASH1 and atherosclerosis within the vascular wall and to assess the impact of SASH1 expression on endothelial cell functions. METHOD: Human carotids with atherosclerotic plaques were obtained from 58 patients (45 of them with known smoking status: smoker, non-smoker, ex-smokers), and were processed for gene expression analyses and immunostaining. To investigate its function, SASH1 was silenced in human aortic endothelial cells (HAECs) using two different siRNA and subcellular localization of SASH1 was determined by immunostaining and subcellular fractionation. Subsequently the transcriptomic analyses and functional experiments (wound healing, WST-1 proliferation or Matrigel assays) were performed to characterize SASH1 function. RESULTS: SASH1 was expressed in human vascular cells (HAECs, smooth muscle cells) and in monocytes/macrophages. Its tissue expression was significantly higher in the atherosclerotic carotids of smokers compared to non-smokers (p < 0.01). In HAECs, SASH1 was expressed mostly in the cytoplasm and SASH1 knockdown resulted in an increased cell migration, proliferation and angiogenesis. Transcriptomic and pathway analyses showed that SASH1 silencing results in a decreased CYP1A1 expression possibly through the inhibition of TP53 activity. CONCLUSION: We showed that SASH1 expression is increased in atherosclerotic carotids in smokers and its silencing affects endothelial angiogenic functions; therefore we provide a potential link between smoking and atherosclerosis through SASH1 expression.

First proteomic study of S-glutathionylation in cyanobacteria.

Glutathionylation, the reversible post-translational formation of a mixed disulfide between a cysteine residue and glutathione (GSH), is a crucial mechanism for signal transduction and regulation of protein function. Until now this reversible redox modification was studied mainly in eukaryotic cells. Here we report a large-scale proteomic analysis of glutathionylation in a photosynthetic prokaryote, the model cyanobacterium Synechocystis sp. PCC6803. Treatment of acellular extracts with N,N-biotinyl glutathione disulfide (BioGSSG) induced glutathionylation of numerous proteins, which were subsequently isolated by affinity chromatography on streptavidin columns and identified by nano LC-MS/MS analysis. Potential sites of glutathionylation were also determined for 125 proteins following tryptic cleavage, streptavidin-affinity purification, and mass spectrometry analysis. Taken together the two approaches allowed the identification of 383 glutathionylatable proteins that participate in a wide range of cellular processes and metabolic pathways such as carbon and nitrogen metabolisms, cell division, stress responses, and H2 production. In addition, the glutathionylation of two putative targets, namely, peroxiredoxin (Sll1621) involved in oxidative stress tolerance and 3-phosphoglycerate dehydrogenase (Sll1908) acting on amino acids metabolism, was confirmed by biochemical studies on the purified recombinant proteins. These results suggest that glutathionylation constitutes a major mechanism of global regulation of the cyanobacterial metabolism under oxidative stress conditions.

The Synechocystis PCC6803 MerA-like enzyme operates in the reduction of both mercury and uranium under the control of the glutaredoxin 1 enzyme.

In a continuing effort to analyze the selectivity/redundancy of the three glutaredoxin (Grx) enzymes of the model cyanobacterium Synechocystis PCC6803, we have characterized an enzyme system that plays a crucial role in protection against two toxic metal pollutants, mercury and uranium. The present data show that Grx1 (Slr1562 in CyanoBase) selectively interacts with the presumptive mercuric reductase protein (Slr1849). This MerA enzyme plays a crucial role in cell defense against both mercuric and uranyl ions, in catalyzing their NADPH-driven reduction. Like MerA, Grx1 operates in cell protection against both mercury and uranium. The Grx1-MerA interaction requires cysteine 86 (C86) of Grx1 and C78 of MerA, which is critical for its reductase activity. MerA can be inhibited by glutathionylation and subsequently reactivated by Grx1, likely through deglutathionylation. The two Grx1 residues C31, which belongs to the redox active site (CX(2)C), and C86, which operates in MerA interactions, are both required for reactivation of MerA. These novel findings emphasize the role of glutaredoxins in tolerance to metal stress as well as the evolutionary conservation of the glutathionylation process, so far described mostly for eukaryotes.

Inhibition by 4-hydroxynonenal (HNE) of Ca2+ transport by SERCA1a: low concentrations of HNE open protein-mediated leaks in the membrane.

Exposure of sarcoplasmic reticulum membranes to 4-hydroxy-2-nonenal (HNE) resulted in inhibition of the maximal ATPase activity and Ca(2+) transport ability of SERCA1a, the Ca(2+) pump in these membranes. The concomitant presence of ATP significantly protected SERCA1a ATPase activity from inhibition. ATP binding and phosphoenzyme formation from ATP were reduced after treatment with HNE, whereas Ca(2+) binding to the high-affinity sites was altered to a lower extent. HNE reacted with SH groups, some of which were identified by MALDI-TOF mass spectrometry, and competition studies with FITC indicated that HNE also reacted with Lys(515) within the nucleotide binding pocket of SERCA1a. A remarkable fact was that both the steady-state ability of SR vesicles to sequester Ca(2+) and the ATPase activity of SR membranes in the absence of added ionophore or detergent were sensitive to concentrations of HNE much smaller than those that affected the maximal ATPase activity of SERCA1a. This was due to an increase in the passive permeability of HNE-treated SR vesicles to Ca(2+), an increase in permeability that did not arise from alteration of the lipid component of these vesicles. Judging from immunodetection with an anti-HNE antibody, this HNE-dependent increase in permeability probably arose from modification of proteins of about 150-160kDa, present in very low abundance in longitudinal SR membranes (and in slightly larger abundance in SR terminal cisternae). HNE-induced promotion, via these proteins, of Ca(2+) leakage pathways might be involved in the general toxic effects of HNE.

Cadmium response and redoxin targets in Chlamydomonas reinhardtii: a proteomic approach.

A proteomic approach including two-dimensional electrophoresis and MALDI-TOF analysis has been developed to identify the soluble proteins of the unicellular photosynthetic algae Chlamydomonas reinhardtii. We first described the partial 2D-picture of soluble proteome obtained from whole cells grown on acetate. Then we studied the effects of the exposure of these cells to 150 muM cadmium (Cd). The most drastic effect was the decrease in abundance of both large and small subunits of the ribulose-1,5-bisphosphate carboxylase/oxygenase, in correlation with several other enzymes involved in photosynthesis, Calvin cycle and chlorophyll biosynthesis. Other down-regulated processes were fatty acid biosynthesis, aminoacid and protein biosynthesis. On the other hand, proteins involved in glutathione synthesis, ATP metabolism, response to oxidative stress and protein folding were up-regulated in the presence of cadmium. In addition, we observed that most of the cadmium-sensitive proteins were also regulated via two major cellular thiol redox systems, thioredoxin and glutaredoxin.