Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
Vertebrate genomes contain around 20,000 protein-encoding genes, of which a large fraction is still not associated with specific functions. A major task in future genomics will thus be to assign physiological roles to all open reading frames revealed by genome sequencing. Here we show that C2orf62, a highly conserved protein with little homology to characterized proteins, is strongly expressed in testis in zebrafish and mammals, and in various types of ciliated cells during zebrafish development. By yeast two hybrid and GST pull-down, C2orf62 was shown to interact with TTC17, another uncharacterized protein. Depletion of either C2orf62 or TTC17 in human ciliated cells interferes with actin polymerization and reduces the number of primary cilia without changing their length. Zebrafish embryos injected with morpholinos against C2orf62 or TTC17, or with mRNA coding for the C2orf62 C-terminal part containing a RII dimerization/docking (R2D2) - like domain show morphological defects consistent with imperfect ciliogenesis. We provide here the first evidence for a C2orf62-TTC17 axis that would regulate actin polymerization and ciliogenesis.
ISSN: 1932-6203

hal-00955242
https://hal-univ-rennes1.archives-ouvertes.fr/hal-00955242
https://hal-univ-rennes1.archives-ouvertes.fr/hal-00955242/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-00955242/file/C2orf62_and_TTC17-accepted.pdf
DOI : 10.1371/journal.pone.0086476

Éditeur(s) : HAL CCSD Wiley-VCH Verlag
Résumé : International audience
Proteomic profiling plays a decisive role in the elucidation of molecular signatures representative of a specific clinical context. MuStem cell-based therapy represents a promising approach for clinical applications to cure Duchenne Muscular Dystrophy (DMD). To expand our previous studies collected in the clinically relevant DMD animal model, we decided to investigate the skeletal muscle proteome four months after systemic delivery of allogenic MuStem cells. Quantitative proteomics with isotope-coded protein labelling (ICPL) was used to compile quantitative changes in the protein expression profiles of muscle in transplanted Golden Retriever Muscular Dystrophy (GRMD) dogs as compared to GRMD dogs. A total of 492 proteins were quantified, including 25 that were overrepresented and 46 that were underrepresented after MuStem cell transplantation. Interestingly, this study demonstrates that somatic stem cell therapy impacts on the structural integrity of the muscle fascicle by acting on fibres and its connections with the extracellular matrix. We also show that cell infusion promotes protective mechanisms against oxidative stress and favours the initial phase of muscle repair. This study allows us to identify putative candidates for tissue markers that might be of great value in objectively exploring the clinical benefits resulting from our cell-based therapy for DMD.
ISSN: 1615-9853

Droits : info:eu-repo/semantics/OpenAccess
hal-01332030
https://hal.archives-ouvertes.fr/hal-01332030
https://hal.archives-ouvertes.fr/hal-01332030/document
https://hal.archives-ouvertes.fr/hal-01332030/file/Quantitative%20proteome%20profiling%20of%20dystrophic%20dog%20skeletal%20muscle_accepted.pdf
DOI : 10.1002/pmic.201600002

Éditeur(s) : HAL CCSD
Résumé : International audience
Among infertile couples, approximately half have to face with male infertility. In men with non-obstructive azoospermia, surgical retrieval testicular sperm extraction (TESE) of spermatozoa can be attempted, but with low success rates. A specific biomarker that could predict residual spermatogenesis would obviously be of interest before performing TESE. Thus, our aim was to identify biomarkers of residual spermatogenesis in seminal plasma of patients with non-obstructive azoospermia (NOA) using an isotope-coded protein label (ICPL)-based proteomic strategy coupled with conventional protein assay. This retrospective study was conducted in 40 men with NOA at the University Hospital of Nantes and Proteomics Core facility Biogenouest - Inserm U1085 - IRSET, Rennes, France. Comparative ICPL proteomic screening of frozen seminal plasma and correlation with TESE outcome allowed the identification of some differentially expressed proteins. Among them, lectin galactoside-binding, soluble 3 binding protein (LGALS3BP) expression was further confirmed using conventional protein assay, and its interest as a predictor of TESE outcome was then evaluated and compared with conventional clinical and hormonal markers of residual spermatogenesis. Among the 12 differentially expressed proteins identified with comparative ICPL proteomic strategy, seminal LGALS3BP expression was found to be significantly higher in men with successful TESE. Finally, comparative ICPL proteomic screening of seminal plasma appears to be a promising approach for the identification of biomarkers of residual spermatogenesis. LGALS3BP, associated with clinical and hormonal markers, could potentially be used as a predictive marker of successful TESE outcome in patients with NOA.
Andrology

inserm-00818514
http://www.hal.inserm.fr/inserm-00818514
DOI : 10.1111/j.2047-2927.2012.00059.x
PUBMED : 23427166

Éditeur(s) : HAL CCSD American Chemical Society
Résumé : International audience
The present study is a contribution to the "neXt50 challenge", a coordinated effort across C-HPP teams to identify the SO most tractable missing proteins (MPs) on each chromosome. We report the targeted search of 38 theoretically detectable MPs from chromosomes 2 and 14 in Triton X-100 soluble and insoluble sperm fractions from a total of 15 healthy donors. A targeted mass spectrometry-based strategy consisting of the development of LC-PRM assays (with heavy labeled synthetic peptides) targeting 92 proteotypic peptides of the 38 selected MPs was used. Out of the 38 selected MPs, 12 were identified with two or more peptides and 3 with one peptide after extensive SDS-PAGE fractionation of the two samples and with overall low-intensity signals. The PRM data are available via ProteomeXchange in PASSEL (PASS01013). Further validation by immunohistochemistry on human testes sections and cytochemistry on sperm smears was performed for eight MPs with antibodies available from the Human Protein Atlas. Deep analysis of human sperm still allows the validation of MPs and therefore contributes to the C-HPP worldwide effort. We anticipate that our results will be of interest to the reproductive biology community because an in-depth analysis of these MPs may identify potential new candidates in the context of human idiopathic infertilities.
ISSN: 1535-3893

hal-01681656
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01681656
DOI : 10.1021/acs.jproteome.7b00374

Éditeur(s) : HAL CCSD Wiley-VCH Verlag
Résumé : International audience
Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer-membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer-membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB-dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT-PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment.
ISSN: 1615-9853

hal-00864093
https://hal.archives-ouvertes.fr/hal-00864093
DOI : 10.1002/pmic.201100644

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
Mammalian spermatogenesis, which takes place in complex testicular structures called seminiferous tubules, is a highly specialized process controlled by the integration of juxtacrine, paracrine and endocrine information. Within the seminiferous tubules, the germ cells and Sertoli cells are surrounded by testicular fluid (TF), which probably contains most of the secreted proteins involved in crosstalk between these cells. It has already been established that germ cells can modulate somatic Sertoli cell function through the secretion of diffusible factors. We studied the germ cell secretome, which was previously considered inaccessible, by analyzing the TF collected by microsurgery in an "integrative omics" strategy combining proteomics, transcriptomics, genomics and interactomics data. This approach identified a set of proteins preferentially secreted by Sertoli cells or germ cells. An interaction network analysis revealed complex, interlaced cell-cell dialog between the secretome and membranome of seminiferous cells, mediated via the TF. We then focused on germ cell-secreted candidate proteins, and we identified several potential interacting partners located on the surface of Sertoli cells. Two interactions, APOH/CDC42 and APP/NGFR, were validated in situ, in a proximity ligation assay (PLA). Our results provide new insight into the crosstalk between germ cells and Sertoli cells occurring during spermatogenesis. Our findings also demonstrate that this "integrative omics" strategy is powerful enough for data mining and highlighting meaningful cell-cell communication events between different types of cells in a complex tissue, via a biological fluid. This integrative strategy could be applied more widely, to gain access to secretomes that have proved difficult to study whilst avoiding the limitations of in vitro culture.
PLoS ONE

hal-01063901
https://hal.archives-ouvertes.fr/hal-01063901
DOI : 10.1371/journal.pone.0104418
PUBMED : 25111155

Éditeur(s) : HAL CCSD
Résumé : International audience
Spermatogenesis is a highly sophisticated process involved in the transmission of genetic heritage. It includes halving ploidy, repackaging of the chromatin for transport, and the equipment of developing spermatids and eventually spermatozoa with the advanced apparatus (e.g., tightly packed mitochondrial sheat in the mid piece, elongating of the tail, reduction of cytoplasmic volume) to elicit motility once they reach the epididymis. Mammalian spermatogenesis is divided into three phases. In the first the primitive germ cells or spermatogonia undergo a series of mitotic divisions. In the second the spermatocytes undergo two consecutive divisions in meiosis to produce haploid spermatids. In the third the spermatids differentiate into spermatozoa in a process called spermiogenesis. Paracrine, autocrine, juxtacrine, and endocrine pathways all contribute to the regulation of the process. The array of structural elements and chemical factors modulating somatic and germ cell activity is such that the network linking the various cellular activities during spermatogenesis is unimaginably complex. Over the past two decades, advances in genomics have greatly improved our knowledge of spermatogenesis, by identifying numerous genes essential for the development of functional male gametes. Large-scale analyses of testicular function have deepened our insight into normal and pathological spermatogenesis. Progress in genome sequencing and microarray technology have been exploited for genome-wide expression studies, leading to the identification of hundreds of genes differentially expressed within the testis. However, although proteomics has now come of age, the proteomics-based investigation of spermatogenesis remains in its infancy. Here, we review the state-of-the-art of large-scale proteomic analyses of spermatogenesis, from germ cell development during sex determination to spermatogenesis in the adult. Indeed, a few laboratories have undertaken differential protein profiling expression studies and/or systematic analyses of testicular proteomes in entire organs or isolated cells from various species. We consider the pros and cons of proteomics for studying the testicular germ cell gene expression program. Finally, we address the use of protein datasets, through integrative genomics (i.e., combining genomics, transcriptomics, and proteomics), bioinformatics, and modelling.
Systems Biology in Reproductive Medicine

hal-00877749
https://hal.archives-ouvertes.fr/hal-00877749
DOI : 10.3109/19396368.2012.691943
PUBMED : 22788530

Éditeur(s) : HAL CCSD
Résumé : International audience
9th International Conference on Toxic Cyanobacteria “Living with toxigenic cyanobacteria”
Pilanesberg, South Africa

hal-01159035
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01159035

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
UNLABELLED: Glioblastoma (GB) is the most frequent and aggressive tumor of the central nervous system. There is currently growing interest in proteomic studies of GB, particularly with the aim of identifying new prognostic or therapeutic response markers. However, comparisons between different proteomic analyses of GB have revealed few common differentiated proteins. The types of control samples used to identify such proteins may in part explain the different results obtained. We therefore tried to determine which control samples would be most suitable for GB proteomic studies. We used an isotope-coded protein labeling (ICPL) method followed by mass spectrometry to reveal and compare the protein patterns of two commonly used types of control sample: GB peritumoral brain zone samples (PBZ) from six patients and epilepsy surgery brain samples (EB) pooled from three patients. The data obtained were processed using AMEN software for network analysis. We identified 197 non-redundant proteins and 35 of them were differentially expressed. Among these 35 differentially expressed proteins, six were over-expressed in PBZ and 29 in EB, showing different proteomic patterns between the two samples. Surprisingly, EB appeared to display a tumoral-like expression pattern in comparison to PBZ. In our opinion, PBZ may be more appropriate control sample for GB proteomic analysis. BIOLOGICAL SIGNIFICANCE: This manuscript describes an original study in which we used an isotope-coded protein labeling method followed by mass spectrometry to identify and compare the protein patterns in two types of sample commonly used as control for glioblastoma (GB) proteomic analysis: peritumoral brain zone and brain samples obtained during surgery for epilepsy. The choice of control samples is critical for identifying new prognostic and/or diagnostic markers in GB.
ISSN: 1874-3919

inserm-00871494
http://www.hal.inserm.fr/inserm-00871494
http://www.hal.inserm.fr/inserm-00871494/document
http://www.hal.inserm.fr/inserm-00871494/file/ManuscritProt-accepted.pdf
DOI : 10.1016/j.jprot.2013.04.031

Éditeur(s) : HAL CCSD Nature Publishing Group
Résumé : International audience
The regulatory mechanisms involved in the acquisition of thermal tolerance are unknown in insects. Reversible phosphorylation is a widespread post-translational modification that can rapidly alter proteins function(s). Here, we conducted a large-scale comparative screening of phosphorylation networks in adult Drosophila flies that were cold-acclimated versus control. Using a modified SIMAC method followed by a multiple MS analysis strategy, we identified a large collection of phosphopeptides (about 1600) and phosphoproteins (about 500) in both groups, with good enrichment efficacy (80%). The saturation curves from the four biological replicates revealed that the phosphoproteome was rather well covered under our experimental conditions. Acclimation evoked a strong phosphoproteomic signal characterized by large sets of unique and differential phosphoproteins. These were involved in several major GO superclusters of which cytoskeleton organization, positive regulation of transport, cell cycle, and RNA processing were particularly enriched. Data suggest that phosphoproteomic changes in response to acclimation were mainly localized within cytoskeletal network, and particularly within microtubule associated complexes. This study opens up novel research avenues for exploring the complex regulatory networks that lead to acquired thermal tolerance.
ISSN: 2045-2322

hal-01532256
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01532256
DOI : 10.1038/s41598-017-01974-z
PUBMED : 28490779

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Risk assessment related to the exposure of humans to chemicals released into the environment is a major concern of our modern societies. In this context, toxicology plays a crucial role to characterize the effects of this exposure on health and identify the targets of these molecules. MALDI imaging mass spectrometry (IMS) is an enabling technology for biodistribution studies of chemicals. Although the majority of published studies are presented in a pharmacological context, the concepts discussed in this review can be applied to the toxicological evaluation of chemicals released into the environment. The major asset of IMS is the simultaneous localization and identification of a parent molecule and its metabolites without labeling and without any prior knowledge. Quantification methods developed in IMS are presented with application to an environmental pollutant. IMS is effective in the localization of chemicals and endogenous species. This opens unique perspectives for the discovery of molecular alterations in metabolites and protein biomarkers that could help for a better understanding of toxicity mechanisms. Distribution studies of agrochemicals in plants by IMS can contribute to a better understanding of their mode of action and to a more effective use of these chemicals, avoiding the current concern of environmental damage.
ISSN: 1874-3919

hal-01367141
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01367141
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01367141/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01367141/file/Potential%20of%20MALDI%20imaging.pdf
DOI : 10.1016/j.jprot.2016.05.008

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
The strikingly complex structural organization of the mammalian testis in vivo creates particular difficulties for studies of its organization, function and regulation. These difficulties are particularly pronounced for investigations of the molecular communication networks within the seminiferous tubules that govern spermatogenesis. The use of classical molecular and cell biology approaches to unravel this complexity has proved problematic, due to difficulties in maintaining differentiated germ cells in vitro, in particular. The lack of a suitable testing ground has led to a greater reliance on high-quality proteomic and genomic analyses as a prelude to the in vitro antx1d in vivo testing of hypotheses. In this study, we highlight the options currently available for research, as used in our laboratory, in which proteomic and integrative genomic strategies are applied to the study of spermatogenesis in mammals. We will comment on results providing insight into the molecular mechanisms underlying normal and pathological spermatogenesis and new perspectives for the treatment of male infertility in humans. Finally, we will discuss the relevance of our strategies and the unexpected potential and perspectives they offer to teams involved in the study of male reproduction, within the framework of the Human Proteome Project. SIGNIFICANCE: Integrative genomics is becoming a powerful strategy for discovering the biological significance hidden in proteomic datasets. This work introduces some of the integrative genomic concepts and works used by our team to gain new insight into mammalian spermatogenesis, a remarkably sophisticated process. We demonstrate the relevance of these integrative approaches to understand the cellular cross talks established between the somatic Sertoli cells and the germ cell lineage, within the seminiferous epithelium. Our work also contributes to new knowledge on the pathophysiology of testicular function, with promising clinical applications. This article is part of a Special Issue entitled: 20years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini, Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez.
ISSN: 1874-3919

hal-01063931
https://hal.archives-ouvertes.fr/hal-01063931
DOI : 10.1016/j.jprot.2014.04.013
PUBMED : 24751586