Human OTULIN haploinsufficiency impairs cell-intrinsic immunity to staphylococcal alpha-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 kappa B 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 alpha-toxin. Naturally elicited antibodies against alpha-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to alpha-toxin in nonleukocytic cells.Peer reviewe

Etude protéomique de la potentialisation à long terme dans l'hippocampe chez le rongeur

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Proteomic analysis of Plasmodium sporozoites limited samples using the timsTOF Pro

International audienc

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 Ca2+ transport ability of SERCA1a, the Ca2+ 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 Ca2+ 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 Lys515 within the nucleotide binding pocket of SERCA1a. A remarkable fact was that both the steady-state ability of SR vesicles to sequester Ca2+ 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 Ca2+, 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 150–160 kDa, 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 Ca2+ leakage pathways might be involved in the general toxic effects of HNE

Profiling tear film enzymes reveals major metabolic pathways involved in the homeostasis of the ocular surface

Abstract The ocular surface (OS) enzymes are of great interest due to their potential for novel ocular drug development. We aimed first to profile and classify the enzymes of the OS to describe major biological processes and pathways that are involved in the maintenance of homeostasis. Second, we aimed to compare the enzymatic profiles between the two most common tear collection methods, capillary tubes (CT) and Schirmer strips (ScS). A comprehensive tear proteomic dataset was generated by pooling all enzymes identified from nine tear proteomic analyses of healthy subjects using mass spectrometry. In these studies, tear fluid was collected using CT (n = 4), ScS (n = 4) or both collection methods (n = 1). Classification and functional analysis of the enzymes was performed using a combination of bioinformatic tools. The dataset generated identified 1010 enzymes. The most representative classes were hydrolases (EC 3) and transferases (EC 2). Phosphotransferases, esterases and peptidases were the most represented subclasses. A large portion of the identified enzymes was common to both collection methods (n = 499). More enzymes were specifically detected in the ScS-extracted proteome. The major pathways in which the identified enzymes participate are related to the immune system and protein, carbohydrate and lipid metabolism. Metabolic processes for nucleosides, cellular amides, sugars and sulfur compounds constituted the most enriched biological processes. Knowledge of these molecules highly susceptible to pharmacological manipulation might help to predict the metabolism of ophthalmic medications and develop novel prodrug strategies as well as new drug delivery systems. Combining such extensive knowledge of the OS enzymes with new analytical approaches and techniques might create new prospects for understanding, predicting and manipulating the metabolism of ocular pharmaceuticals. Our study reports new, essential data on OS enzymes while also comparing the enzyme profiles obtained via the two most popular methods of tear collection, capillary tubes and Schirmer strips

Native presynaptic metabotropic glutamate receptor 4 (mGluR4) interacts with exocytosis proteins in rat cerebellum.

International audienceThe eight pre- or/and post-synaptic metabotropic glutamatergic receptors (mGluRs) modulate rapid excitatory transmission sustained by ionotropic receptors. They are classified in three families according to their percentage of sequence identity and their pharmacological properties. mGluR4 belongs to group III and is mainly localized presynaptically. Activation of group III mGluRs leads to depression of excitatory transmission, a process that is exclusively provided by mGluR4 at parallel fiber-Purkinje cell synapse in rodent cerebellum. This function relies at least partly on an inhibition of presynaptic calcium influx, which controls glutamate release. To improve the understanding of molecular mechanisms of the mGluR4 depressant effect, we decided to identify the proteins interacting with this receptor. Immunoprecipitations using anti-mGluR4 antibodies were performed with cerebellar extracts. 183 putative partners that co-immunoprecipitated with anti-mGluR4 antibodies were identified and classified according to their cellular functions. It appears that native mGluR4 interacts with several exocytosis proteins such as Munc18-1, synapsins, and syntaxin. In addition, native mGluR4 was retained on a Sepharose column covalently grafted with recombinant Munc18-1, and immunohistochemistry experiments showed that Munc18-1 and mGluR4 colocalized at plasma membrane in HEK293 cells, observations in favor of an interaction between the two proteins. Finally, affinity chromatography experiments using peptides corresponding to the cytoplasmic domains of mGluR4 confirmed the interaction observed between mGluR4 and a selection of exocytosis proteins, including Munc18-1. These results could give indications to explain how mGluR4 can modulate glutamate release at parallel fiber-Purkinje cell synapses in the cerebellum in addition to the inhibition of presynaptic calcium influx

New mortalin and histidyl Trna synthetase isoforms point out a pitfall in proteomic analysis of Egrl-gentically modified mince.

International audienceEgr1 (Zif268) is an immediate early gene encoding an inducible transcription factor involved in synaptic plasticity and several forms of memory in rodents. Using 2-DE and MS, we compared proteomes of hippocampal subregions and cortex in Egr1-deficient and wild-type littermates. Two significant differences were identified: a shift in the pI of the molecular chaperone mortalin (mtHsp70/PBP74/Grp75) and the apparent disappearance of histidyl tRNA synthetase (HisRS). We found that the pI shift for mortalin in Egr1-deficient mice was caused by a difference in protein sequence: D626G. Using cDNA sequencing, we demonstrated for both mortalin and HisRS that protein differences were not due to a lack of Egr1 but to DNA polymorphism between the C57Bl/6J and 129/Sv strains used to generate the Egr1-deficient mice. Our results show that mortalin and HisRS genes, which map closely to the Egr1 locus, have conserved the 129/Sv haplotype despite numerous back-crossing of the null mice progeny with C57Bl/6J animals. This demonstrates that allelic differences between mouse strains can introduce variations in differential proteomic analyses of genetically modified organisms. Finally, we report the identification of new isoforms of HisRS and mortalin (mot-3) encoded by the 129/Sv haplotype

Proteomic Analysis of Tears and Conjunctival Cells Collected with Schirmer Strips Using timsTOF Pro: Preanalytical Considerations

International audienceThis study aimed to investigate the human proteome profile of samples collected from whole (W) Schirmer strips (ScS) and their two parts—the bulb (B) and the rest of the strip (R)—with a comprehensive proteomic approach using a trapped ion mobility mass spectrometer, the timsTOF Pro. Eight ScS were collected from two healthy subjects at four different visits to be separated into three batches, i.e., 4W, 4B, and 4R. In total, 1582 proteins were identified in the W, B, and R batches. Among all identified proteins, binding proteins (43.4%) and those with catalytic activity (42.2%) constituted more than 80% of the molecular functions. The most represented biological processes were cellular processes (31.2%), metabolic processes (20.8%), and biological regulation (13.1%). Enzymes were the most represented protein class (41%), consisting mainly of hydrolases (47.5%), oxidoreductases (22.1%), and transferases (16.7%). The bulb (B), which is in contact with the conjunctiva, might collect both tear and cell proteins and therefore promote the identification of more proteins. Processing B and R separately before mass spectrometry (MS) analysis, combined with the high data acquisition speed and the addition of ion-mobility-based separation in the timsTOF Pro, can bring a new dimension to biomarker investigations of a limited sample such as tear fluid

Evaluation of dermal extracellular matrix and epidermal-dermal junction modifications using matrix-assisted laser desorption/ionization mass spectrometric imaging, in vivo reflectance confocal microscopy, echography, and histology: effect of age and peptide applications

International audienceThis study was conducted to establish a new methodology for evaluating elements of dermal extracellular matrix (ECM), of epidermal-dermal junction (EDJ), and effects of molecules which can modulate their synthesis. This methodology is based on matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI). In vivo reflectance confocal microscopy (in vivo RCM) and echography were also used. Using immunohistochemistry methods on explants, age-related modification data were obtained for selected dermal ECM and EDJ proteins (collagen I, collagen IV, collagen VII, collagen XVII, nidogen I, decorin/decorunt) and used as reference for MALDI-MSI studies. A methodology was developed with MALDI-MSI to map epidermis and dermis proteins. Then MALDI-MSI was used to study age modifications. In vivo RCM and high-frequency ultrasounds were used to evaluate ECM and EDJ undulation modifications caused by aging. Anti-aging molecule evaluations were performed with a blend of palmitoyl oligopeptide and palmitoyl tetrapeptide-7. Immunohistochemistry studies demonstrated that the selected proteins were found to be less abundant in aged group explants vs. young group except for decorin. MALDI-MSI studies correlated the results obtained for decorin. In vivo RCM measurements indicated a decrease of EDJ undulation depth with age and ECM modifications in the upper part of dermis. Echography demonstrated that the peptide blend reduced subepidermal low-echogenic band thickness and improved its density. In vivo RCM studies indicated that the peptides improved the ECM structure vs. placebo. This preliminary MALDI-MSI study raised some technical difficulties that were overcome. Further studies will be conducted to identify more proteins and to demonstrate the interest of this method for cosmetic evaluations

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

International audienceIn 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