Essential role of GEXP15, a specific Protein Phosphatase type 1 partner, in Plasmodium berghei in asexual erythrocytic proliferation and transmission.

The essential and distinct functions of Protein Phosphatase type 1 (PP1) catalytic subunit in eukaryotes are exclusively achieved through its interaction with a myriad of regulatory partners. In this work, we report the molecular and functional characterization of Gametocyte EXported Protein 15 (GEXP15), a Plasmodium specific protein, as a regulator of PP1. In vitro interaction studies demonstrated that GEXP15 physically interacts with PP1 through the RVxF binding motif in P. berghei. Functional assays showed that GEXP15 was able to increase PP1 activity and the mutation of the RVxF motif completely abolished this regulation. Immunoprecipitation assays of tagged GEXP15 or PP1 in P. berghei followed by immunoblot or mass spectrometry analyses confirmed their interaction and showed that they are present both in schizont and gametocyte stages in shared protein complexes involved in the spliceosome and proteasome pathways and known to play essential role in parasite development. Phenotypic analysis of viable GEXP15 deficient P. berghei blood parasites showed that they were unable to develop lethal infection in BALB/c mice or to establish experimental cerebral malaria in C57BL/6 mice. Further, although deficient parasites produced gametocytes they did not produce any oocysts/sporozoites indicating a high fitness cost in the mosquito. Global proteomic and phosphoproteomic analyses of GEXP15 deficient schizonts revealed a profound defect with a significant decrease in the abundance and an impact on phosphorylation status of proteins involved in regulation of gene expression or invasion. Moreover, depletion of GEXP15 seemed to impact mainly the abundance of some specific proteins of female gametocytes. Our study provides the first insight into the contribution of a PP1 regulator to Plasmodium virulence and suggests that GEXP15 affects both the asexual and sexual life cycle

Étude du syndrome néphrotique idiopathique par des approches protéomiques

Idiopathic nephrotic syndrome (INS) is a podocytopathy of presumable immune origin and poorly understood pathogenesis. Our team has identified a gene (CMIP) whose expression is associated with certain forms of INS. The main objective of my thesis is to better understand the pathophysiology of INS by using proteomic approaches. After optimizing the FASP method to improve sensitivity, we studied the proteome of T-cells overexpressing CMIP. The results show, among other effects, the link between CMIP expression and T-cell cytoskeleton disorganization. As CMIP can locate in membrane rafts, we developed a very sensitive method for raft protein analysis by mass spectrometry. We observed the impact of CMIP on raft-mediated signaling in T-cells via altered recruitment of proteins in these microdomains. We also explored the proximal signaling in podocytes exposed to plasma from SNI patients. By studying the raftome and phosphoproteome of plasma-stimulated podocytes, we highlighted a deregulation of mTORC1, of autophagy, and a disorganization of the podocyte cytoskeleton. Finally, we analyzed plasma exchanges (both the soluble fraction and extracellular vesicles -EVs-) from post-transplant INS patients to identify proteins potentially involved in INS recurrence. We found in the EV fraction from SNI patients the differential presence of amino acid transporters, known to be involved in the mTOR pathway. We also observed a high presence of neutrophil degranulation proteins in both the soluble fraction and EV from these patients. In conclusion, my PhD work has highlighted the dysregulation of cytoskeleton organization in both T-cells and podocytes, as well as the implication of the mTOR pathway in INS pathogenesis in podocytes. The results of the study on post-transplant patient plasma open new perspectives on the potential involvement of neutrophils in the recurrence of the diseaseLe syndrome néphrotique idiopathique (SNI) est une podocytopathie d’origine immunitaire dont la pathogenèse n’est pas bien connue. Notre équipe a identifié un gène (CMIP) dont l’expression est associée à certaines formes de SNI. Le principal objectif de cette étude est de mieux comprendre la physiopathologie du SNI par des approches protéomiques. Après avoir optimisé la méthode FASP pour l’analyse des lymphocytes T (LT) afin d’améliorer la sensibilité de l’analyse protéomique, nous avons étudié le protéome total du LT en fonction de la présence de CMIP. Nous avons ainsi montré l’implication de CMIP dans la désorganisation du cytosquelette du LT. CMIP pouvant être localisée dans les rafts, nous avons alors développé une méthode très sensible permettant l’analyse de ces microdomaines par spectrométrie de masse afin d’évaluer l’impact de CMIP sur le raftome de LT obtenus à partir d’une souris. Nos résultats suggèrent un recrutement différent des protéines de signalisation des LT stimulés en fonction de la présence de CMIP. Nous avons également exploré la signalisation proximale podocytaire pour comprendre l’effet sur ces cellules du plasma des patients SNI. En étudiant le raftome et le phosphoprotéome des podocytes, nous avons observé une dérégulation de mTORC1, de l’autophagie et une désorganisation du cytosquelette podocytaire. Enfin, les échanges plasmatiques (la fraction soluble et les vésicules extracellulaires -EVs-) des patients SNI en récidive post-greffe ont été analysés, afin de mettre en évidence des biomarqueurs de la récidive. Nos expériences ont montré que les EVs des patients SNI présentent une augmentation des transporteurs d’acides aminés, impliqués dans la voie mTOR. De plus, nous observons une plus forte présence des protéines de la dégranulation du neutrophile chez ces patients. En conclusion, mes travaux de thèse ont permis de mettre en évidence la désorganisation du cytosquelette au niveau des LT exprimant CMIP et des podocytes incubés en présence de plasma SNI, ainsi que l’implication de la voie mTOR dans les podocytes dans ce contexte. Les résultats issus de l'analyse du plasma des patients a permis d’ouvrir la voie à de nouvelles hypothèses sur l’implication des neutrophiles dans la récidive de la maladi

Étude du syndrome néphrotique idiopathique par des approches protéomiques

Idiopathic nephrotic syndrome (INS) is a podocytopathy of presumable immune origin and poorly understood pathogenesis. Our team has identified a gene (CMIP) whose expression is associated with certain forms of INS. The main objective of my thesis is to better understand the pathophysiology of INS by using proteomic approaches. After optimizing the FASP method to improve sensitivity, we studied the proteome of T-cells overexpressing CMIP. The results show, among other effects, the link between CMIP expression and T-cell cytoskeleton disorganization. As CMIP can locate in membrane rafts, we developed a very sensitive method for raft protein analysis by mass spectrometry. We observed the impact of CMIP on raft-mediated signaling in T-cells via altered recruitment of proteins in these microdomains. We also explored the proximal signaling in podocytes exposed to plasma from SNI patients. By studying the raftome and phosphoproteome of plasma-stimulated podocytes, we highlighted a deregulation of mTORC1, of autophagy, and a disorganization of the podocyte cytoskeleton. Finally, we analyzed plasma exchanges (both the soluble fraction and extracellular vesicles -EVs-) from post-transplant INS patients to identify proteins potentially involved in INS recurrence. We found in the EV fraction from SNI patients the differential presence of amino acid transporters, known to be involved in the mTOR pathway. We also observed a high presence of neutrophil degranulation proteins in both the soluble fraction and EV from these patients. In conclusion, my PhD work has highlighted the dysregulation of cytoskeleton organization in both T-cells and podocytes, as well as the implication of the mTOR pathway in INS pathogenesis in podocytes. The results of the study on post-transplant patient plasma open new perspectives on the potential involvement of neutrophils in the recurrence of the diseaseLe syndrome néphrotique idiopathique (SNI) est une podocytopathie d’origine immunitaire dont la pathogenèse n’est pas bien connue. Notre équipe a identifié un gène (CMIP) dont l’expression est associée à certaines formes de SNI. Le principal objectif de cette étude est de mieux comprendre la physiopathologie du SNI par des approches protéomiques. Après avoir optimisé la méthode FASP pour l’analyse des lymphocytes T (LT) afin d’améliorer la sensibilité de l’analyse protéomique, nous avons étudié le protéome total du LT en fonction de la présence de CMIP. Nous avons ainsi montré l’implication de CMIP dans la désorganisation du cytosquelette du LT. CMIP pouvant être localisée dans les rafts, nous avons alors développé une méthode très sensible permettant l’analyse de ces microdomaines par spectrométrie de masse afin d’évaluer l’impact de CMIP sur le raftome de LT obtenus à partir d’une souris. Nos résultats suggèrent un recrutement différent des protéines de signalisation des LT stimulés en fonction de la présence de CMIP. Nous avons également exploré la signalisation proximale podocytaire pour comprendre l’effet sur ces cellules du plasma des patients SNI. En étudiant le raftome et le phosphoprotéome des podocytes, nous avons observé une dérégulation de mTORC1, de l’autophagie et une désorganisation du cytosquelette podocytaire. Enfin, les échanges plasmatiques (la fraction soluble et les vésicules extracellulaires -EVs-) des patients SNI en récidive post-greffe ont été analysés, afin de mettre en évidence des biomarqueurs de la récidive. Nos expériences ont montré que les EVs des patients SNI présentent une augmentation des transporteurs d’acides aminés, impliqués dans la voie mTOR. De plus, nous observons une plus forte présence des protéines de la dégranulation du neutrophile chez ces patients. En conclusion, mes travaux de thèse ont permis de mettre en évidence la désorganisation du cytosquelette au niveau des LT exprimant CMIP et des podocytes incubés en présence de plasma SNI, ainsi que l’implication de la voie mTOR dans les podocytes dans ce contexte. Les résultats issus de l'analyse du plasma des patients a permis d’ouvrir la voie à de nouvelles hypothèses sur l’implication des neutrophiles dans la récidive de la maladi

Proteomics approaches to study idiopathic nephrotic syndrome

Le syndrome néphrotique idiopathique (SNI) est une podocytopathie d’origine immunitaire dont la pathogenèse n’est pas bien connue. Notre équipe a identifié un gène (CMIP) dont l’expression est associée à certaines formes de SNI. Le principal objectif de cette étude est de mieux comprendre la physiopathologie du SNI par des approches protéomiques. Après avoir optimisé la méthode FASP pour l’analyse des lymphocytes T (LT) afin d’améliorer la sensibilité de l’analyse protéomique, nous avons étudié le protéome total du LT en fonction de la présence de CMIP. Nous avons ainsi montré l’implication de CMIP dans la désorganisation du cytosquelette du LT. CMIP pouvant être localisée dans les rafts, nous avons alors développé une méthode très sensible permettant l’analyse de ces microdomaines par spectrométrie de masse afin d’évaluer l’impact de CMIP sur le raftome de LT obtenus à partir d’une souris. Nos résultats suggèrent un recrutement différent des protéines de signalisation des LT stimulés en fonction de la présence de CMIP. Nous avons également exploré la signalisation proximale podocytaire pour comprendre l’effet sur ces cellules du plasma des patients SNI. En étudiant le raftome et le phosphoprotéome des podocytes, nous avons observé une dérégulation de mTORC1, de l’autophagie et une désorganisation du cytosquelette podocytaire. Enfin, les échanges plasmatiques (la fraction soluble et les vésicules extracellulaires -EVs-) des patients SNI en récidive post-greffe ont été analysés, afin de mettre en évidence des biomarqueurs de la récidive. Nos expériences ont montré que les EVs des patients SNI présentent une augmentation des transporteurs d’acides aminés, impliqués dans la voie mTOR. De plus, nous observons une plus forte présence des protéines de la dégranulation du neutrophile chez ces patients. En conclusion, mes travaux de thèse ont permis de mettre en évidence la désorganisation du cytosquelette au niveau des LT exprimant CMIP et des podocytes incubés en présence de plasma SNI, ainsi que l’implication de la voie mTOR dans les podocytes dans ce contexte. Les résultats issus de l'analyse du plasma des patients a permis d’ouvrir la voie à de nouvelles hypothèses sur l’implication des neutrophiles dans la récidive de la maladieIdiopathic nephrotic syndrome (INS) is a podocytopathy of presumable immune origin and poorly understood pathogenesis. Our team has identified a gene (CMIP) whose expression is associated with certain forms of INS. The main objective of my thesis is to better understand the pathophysiology of INS by using proteomic approaches. After optimizing the FASP method to improve sensitivity, we studied the proteome of T-cells overexpressing CMIP. The results show, among other effects, the link between CMIP expression and T-cell cytoskeleton disorganization. As CMIP can locate in membrane rafts, we developed a very sensitive method for raft protein analysis by mass spectrometry. We observed the impact of CMIP on raft-mediated signaling in T-cells via altered recruitment of proteins in these microdomains. We also explored the proximal signaling in podocytes exposed to plasma from SNI patients. By studying the raftome and phosphoproteome of plasma-stimulated podocytes, we highlighted a deregulation of mTORC1, of autophagy, and a disorganization of the podocyte cytoskeleton. Finally, we analyzed plasma exchanges (both the soluble fraction and extracellular vesicles -EVs-) from post-transplant INS patients to identify proteins potentially involved in INS recurrence. We found in the EV fraction from SNI patients the differential presence of amino acid transporters, known to be involved in the mTOR pathway. We also observed a high presence of neutrophil degranulation proteins in both the soluble fraction and EV from these patients. In conclusion, my PhD work has highlighted the dysregulation of cytoskeleton organization in both T-cells and podocytes, as well as the implication of the mTOR pathway in INS pathogenesis in podocytes. The results of the study on post-transplant patient plasma open new perspectives on the potential involvement of neutrophils in the recurrence of the diseas

Mapping PP1c and Its Inhibitor 2 Interactomes Reveals Conserved and Specific Networks in Asexual and Sexual Stages of Plasmodium

International audienceMalaria parasites require multiple phosphorylation and dephosphorylation steps to drive signaling pathways for proper differentiation and transformation. Several protein phosphatases, including protein phosphatase 1 (PP1), one of the main dephosphorylation enzymes, have been shown to be indispensable for the Plasmodium life cycle. The catalytic subunit of PP1 (PP1c) participates in cellular processes via dynamic interactions with a vast number of binding partners that contribute to its diversity of action. In this study, we used Plasmodium berghei transgenic parasite strains stably expressing PP1c or its inhibitor 2 (I2) tagged with mCherry, combined with the mCherry affinity pulldown of proteins from asexual and sexual stages, followed by mass spectrometry analyses. Mapped proteins were used to identify interactomes and to cluster functionally related proteins. Our findings confirm previously known physical interactions of PP1c and reveal enrichment of common biological processes linked to cellular component assembly in both schizonts and gametocytes to biosynthetic processes/translation in schizonts and to protein transport exclusively in gametocytes. Further, our analysis of PP1c and I2 interactomes revealed that nuclear export mediator factor and peptidyl-prolyl cis-trans isomerase, suggested to be essential in P. falciparum, could be potential targets of the complex PP1c/I2 in both asexual and sexual stages. Our study emphasizes the adaptability of Plasmodium PP1 and provides a fundamental study of the protein interaction landscapes involved in a myriad of events in Plasmodium, suggesting why it is crucial to the parasite and a source for alternative therapeutic strategies

Dataset of a comparative proteomics experiment in a methylmalonyl-CoA mutase knockout HEK 293 cell model

Methylmalonic acidemia is a rare inborn error of metabolism with severe clinical complications and poor outcome. The present data article is related to a proteomic investigation conducted on a HEK 293 cell line which has been ge- netically modified using CRISPR-CAS9 system to knockout the methylmalonyl-CoA mutase enzyme (MUT-KO). Thus, the generated cell model for methylmalonic acidemia was used for a proteomic comparison with respect to HEK 293 wild type cells performing a label-free quantification (LFQ) ex- periment. A comparison between FASP and S-Trap digestion methods was performed on protein extracts before to pro- ceed with the proteomic analysis of the samples. Four bio- logical replicates were employed for LC-MS/MS analysis and each was run in technical triplicates. MaxQuant and Perseus platforms were used to perform the LFQ of the proteomes and carry out statistical analysis, respectively. Globally, 4341 proteins were identified, and 243 as differentially regulated,of which 150 down-regulated and 93 up-regulated in the MUT-KO condition. MS proteomics data have been deposited to the ProteomeXchange Consortium with the dataset iden- tifier PXD017977. The information provided in this dataset shed new light on the cellular mechanisms altered in this rare metabolic disorder, highlighting quantitative unbalances in proteins acting in cell structure and architecture organiza- tion and response to the stress. This article can be used as a new source of protein actors to be validated and a starting point for the identification of clinically relevant therapeutic targets

A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome

International audienceThe analysis of T cell lipid raft proteome is challenging due to the highly dynamic nature of rafts and the hydrophobic character of raft-resident proteins. We explored an innovative strategy for bottom-up lipid raftomics based on suspension trapping (S-trap) sample preparation. Mouse T cells were prepared from splenocytes by negative immunoselection and rafts were isolated by a detergent-free method and OptiPrep gradient ultracentrifugation. Microdomains enriched in Flotillin-1, LAT and cholesterol were subjected to proteomic analysis through an optimized protocol based on STrap and high pH fractionation, followed by nano-LC-MS/MS. Using this method, we identified 2680 proteins in the raft-rich fraction and established a database of 894 T cell raft proteins. We then performed a differential analysis on the raft-rich fraction from non-stimulated vs. anti-CD3/CD28 TCR-stimulated T cells. Our results revealed 42 proteins present in one condition and absent in the other. For the first time, we performed a proteomic analysis on rafts from ex-vivo T cells obtained from individual mice, before and after TCR activation. This work demonstrates that the proposed method utilizing an Strap based approach for sample preparation increases the specificity and sensitivity of lipid raftomics. Data are available via ProteomeXchange with identifier PXD016476

Importance of host cell arginine uptake in Francisella phagosomal escape and ribosomal protein amounts

International audienceUpon entry into mammalian host cells, the pathogenic bacterium Francisella must import host cell arginine to multiply actively in the host cytoplasm. We identified and functionally characterized an arginine transporter (hereafter designated ArgP) whose inactivation considerably delayed bacterial phagosomal escape and intracellular multiplication. Intramacrophagic growth of the ΔargP mutant was fully restored upon supplementation of the growth medium with excess arginine, in both F. tularensis subsp. novicida and F. tularensis subsp. holarctica LVS, demonstrating the importance of arginine acquisition in these two subspecies. High-resolution mass spectrometry revealed that arginine limitation reduced the amount of most of the ribosomal proteins in the ΔargP mutant. In response to stresses such as nutritional limitation, repression of ribosomal protein synthesis has been observed in all kingdoms of life. Arginine availability may thus contribute to the sensing of the intracellular stage of the pathogen and to trigger phagosomal egress. All MS data have been deposited in the ProteomeXchange database with identifier PXD001584 (http://proteomecentral.proteomexchange.org/dataset/PXD001584)

Proteomics Reveals that Methylmalonyl-CoA Mutase Modulates Cell Architecture and Increases Susceptibility to Stress

Methylmalonic acidemia (MMA) is a rare inborn error of metabolism caused by deficiency of the methylmalonyl-CoA mutase (MUT) enzyme. Downstream MUT deficiency, methylmalonic acid accumulates together with toxic metabolites from propionyl-CoA and other compounds upstream of the block in the enzyme pathway. The presentation is with life-threatening acidosis, respiratory distress, brain disturbance, hyperammonemia, and ketosis. Survivors develop poorly understood multi-organ damage, notably to the brain and kidneys. The HEK 293 cell line was engineered by CRISPR/Cas9 technology to knock out the MUT gene (MUT-KO). Shotgun label-free quantitative proteomics and bioinformatics analyses revealed potential damaging biological processes in MUT-deficient cells. MUT-KO induced alteration of cellular architecture and morphology, and ROS overproduction. We found the alteration of proteins involved in cytoskeleton and cell adhesion organization, cell trafficking, mitochondrial, and oxidative processes, as validated by the regulation of VIM, EXT2, SDC2, FN1, GLUL, and CHD1. Additionally, a cell model of MUT-rescuing was developed in order to control the specificity of MUT-KO effects. Globally, the proteomic landscape of MUT-KO suggests the cell model to have an increased susceptibility to propionate- and H2O2-induced stress through an impairment of the mitochondrial functionality and unbalances in the oxidation-reduction processes

Identification and Relative Quantification of hFSH Glycoforms in Women’s Sera via MS–PRM-Based Approach

Follicle-stimulating hormone (FSH) is a glycohormone synthesized by adenohypophysis, and it stimulates ovulation in women and spermatogenesis in men by binding to its receptor (FSHR). FSHR is involved in several mechanisms to transduce intracellular signals in response to the FSH stimulus. Exogenous FSH is currently used in the clinic for ovarian hyperstimulation during in vitro fertilization in women, and for treatment of infertility caused by gonadotropin deficiency in men. The glycosylation of FSH strongly affects the binding affinity to its receptor, hence significantly influencing the biological activity of the hormone. Therefore, the accurate measurement and characterization of serum hFSH glycoforms will contribute to elucidating the complex mechanism of action by which different glycoforms elicit distinct biological activity. Nowadays ELISA is the official method with which to monitor serum hFSH, but the test is unable to distinguish between the different FSH glycovariants and is therefore unsuitable to study the biological activity of this hormone. This study presents a preliminary alternative strategy for identifying and quantifying serum hFSH glycoforms based on immunopurification assay and mass spectrometry (MS), and parallel reaction monitoring (PRM) analysis. In this study, we provide an MS–PRM data acquisition method for hFSH glycopeptides identification with high specificity and their quantification by extracting the chromatographic traces of selected fragments of glycopeptides. Once set up for all its features, the proposed method could be transferred to the clinic to improve fertility treatments and follow-ups in men and women