PeptideShaker - PeptideShaker is a user friendly tool for shotgun proteomic data interpretation and reprocessing.
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Permanent Cell Line Cell, Hela Cell
SUBMITTER: 
Marc Vaudel
LAB HEAD: Lennart Martens
PROVIDER: PXD000674 | Pride | 2014-01-27
REPOSITORIES: Pride
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Project description:Caveolins are structural and functional proteins in plasma membrane invaginations called caveolae. Mutations in Caveolin-3 cause myopathies of variable severity. The pathogenicity of the Caveolin-3 variant G55S is still unclear. Here, we report on three patients suffering from mild to moderate myopathy. In all three patients, but also in two seemingly unaffected family members of patient one the G55S variant was found. Histology revealed moderate chronic myopathic changes and reduced sarcolemmal Caveolin-3 immunoreactivity in all three cases. Immunoblots for Caveolin-3 were abnormal in all cases. By electron microscopy, enlarged caveolae were detected in case one and three and vacuolar myopathy in case two. EM studies of RCMH myoblasts transfected with G55S Caveolin-3 revealed autophagic vacuoles. The alterations in Golgi morphology were in line with pathological Caveolin-3 deposits in this organelle detected by immunofluorescence and indicative for activation of autophagy. Phospho-blotting demonstrated that G55S affects EGFR signaling. Proteomic profiling of transfected RCMH myoblasts demonstrated alterations in levels of physiologically relevant proteins which are indicative for antagonization of G55S Caveolin-3 expression. Some proteomic alterations were enhanced by osmotic/mechanical stress. In conclusion, our results suggest that the G55S Caveolin-3 sequence variant can be compensated by cellular defense mechanisms and that additional stress may lead to vulnerability of G55S Caveolin-3 expressing muscle cells.
2018-07-05 | PXD003719 | Pride
Project description:Marinesco-Sjögren syndrome (MSS) is a neurodegenerative disorder caused by autosomal recessive SIL1 mutations. SIL1 acts as a nucleotide exchange factor for the endoplasmic reticulum (ER) resident chaperone BiP. As BiP controls many ER-related processes, it is likely to contribute to MSS pathology. Owing to the absence of appropriate in vitro models, the precise pathophysiological mechanisms leading to neurodegeneration in MSS are still elusive. Here, we demonstrate for the first time that SIL1-depleted HEK293 cells can be used to reveal these mechanisms using ultra-structural, cell biological, and biochemical approaches.
2017-02-28 | PXD001197 | Pride
Project description:SIL1 acts as a co-chaperone for the major ER-resident chaperone BiP and thus plays a role in many BiP-dependent cellular functions such as protein folding control and unfolded protein response. Whereas increase of BiP upon cellular stress conditions is a well-known phenomenon, elevation of SIL1 under stress conditions was thus far solely studied in yeast and different studies indicated an adverse effect of SIL1 increase. This is seemingly in contrast with the beneficial effect of SIL1 increase in surviving neurons in neurodegenerative disorders such as Amyotrophic Lateral Sclerosis and Alzheimer disease. In the present study, we systematically addressed these controversial findings. Using cell biological, morphological and biochemical methods, we could demonstrate that that SIL1 is increased in various mammalian cells and neuronal tissues upon induction of cellular stress. Investigation of heterozygous SIL1/Sil1 mutant cells and tissues supported this finding. Moreover, SIL1 protein was found to be stabilized during ER stress. Increased SIL1 initiates ER stress in a concentration-dependent manner which is in accordance with the described adverse effect of increased SIL1. However, our results also suggest that protective levels are achieved by the secretion of excessive SIL1 and GRP170 (no longer perturbing ER homeostasis) and that moderately increased SIL1 also ameliorates cellular fitness under stress conditions. Results of our immunoprecipitation studies indicate that under cellular stress conditions SIL1 might act in a BiP-independent manner. Unbiased proteomic studies revealed that elevated SIL1 levels alter the expression of several proteins including crucial players in neurodegeneration, especially in Alzheimer disease. This finding is in accordance with our observation of increased SIL1-immunoreactivity in surviving neurons of Alzheimer disease autopsy cases and supports the assumption that SIL1 plays a protective role in neurodegenerative disorders.
2020-07-24 | PXD005218 | Pride
Project description:Mass spectrometry-based proteomics data, obtained experimentally or via public repositories, remains challenging to analyze. This challenge is particularly felt when re-analysing or re-purposing public data sets, endeavours that otherwise hold great potential. PeptideShaker is an open source system that greatly simplifies the interpretation and dissemination of proteomics data, and that automates the re-analysis of public data, thus completing the proteomics data cycle (http://peptide-shaker.googlecode.com).
2014-06-24 | PXD001077 | Pride
Project description:In this study, we describe new patients suffering from INPP5K mutations and hereby expand the mutational and clinical spectrum of the underlying disease. Pathogenicity of a new INPP5K missense mutation has been functionally confirmed. In addition, we systematically addressed the need to identify common molecular key players to link phenotypically similar rare diseases. For this purpose, utilizing cells derived from INPP5K and SIL1 patients, we performed proteomic profiling and identified PHGDH, as a protein significantly altered in abundance in the in vitro systems of both diseases. Prompted by the striking molecular link between MSS, INPP5K- and PHGDH-phenotypes and the fact that PHGDH-patients notoriously respond to L-serine treatment, respective zebrafish models have been generated and response to L-serine treatment has been addressed in vivo. Apart from the biochemical link between MSS, INPP5K-and PHGDH-phenotypes, affection of CDK9, a protein modulating the activity of CTDP1 biochemically links the INPP5K-phenotype to CCFDN. Thus, our study not only allowed to build molecular bridges between four phenotypically overlapping rare diseases but more importantly allowed to transfer the acquired molecular knowledge to the pre-clinical testing of a therapeutic intervention concept.
2021-04-06 | PXD009272 | Pride
Project description:Recessive mutations in DNAJC3, an endoplasmic reticulum (ER)-resident BiP co-chaperone, have been identified in patients with multisystemic neurodegeneration and diabetes mellitus. To further unravel these pathomechanisms we employed a non-biased proteomic approach and identified dysregulation of several key cellular pathways, suggesting a pathophysiological interplay of perturbed lipid metabolism, mitochondrial bioenergetics, ER-Golgi function, and amyloid-beta processing. Further functional investigations in fibroblasts of patients with DNAJC3 mutations detected cellular accumulation of lipids, and an increased sensitivity to cholesterol-stress, which led to activation of the unfolded protein response (UPR), alterations of the ER-Golgi machinery, a defect of APP. In line with the results of previous studies, we describe here alterations in mitochondrial morphology and function, as a major contributor to the DNAJC3 pathophysiology. Hence, we propose that the loss of DNAJC3 affects lipid/cholesterol homeostasis, leading to UPR activation, Aβ accumulation and impairment of mitochondrial oxidative phosphorylation.
2022-02-15 | PXD028168 | Pride
Project description:Filamin C (encoded by the FLNC gene) is a large actin-cross-linking protein involved in shaping the actin cytoskeleton in response to signaling events both at the sarcolemma and at myofibrillar Z-discs of cross-striated muscle cells. Multiple mutations in FLNC are associated with myofibrillar myopathies of autosomal dominant inheritance. Here, we describe a boy with congenital onset of generalized muscular hypotonia and muscular weakness, delayed motor development but no cardiac involvement associated with a homozygous FLNC mutation affecting the rod domain of the protein. To demonstrate pathogenicity of this homozygous FLNC-mutation described, ultra-morphological, proteomic and functional investigations were performed in addition to immunological studies of known marker proteins for dominant filaminopathies. Our results showed that the mutant protein is expressed to similar quantities as the wildtype variant in control skeletal muscle fibres, alters the proteomic signature of quadriceps muscle, and results in the presence of ultrastructural perturbations. Moreover, comparable findings for filaminopathy marker proteins were found in both, our homozygous and a dominant case. The mutant protein is less stable and more prone to degradation by proteolytic enzymes than the wildtype variant. These combined findings extend the currently recognized clinical, genetic and biochemical spectrum of filaminopathies. The unusual congenital presentation of the disease indicates that homozygosity for a mutation in filamin C severely aggravates the phenotype.
2020-07-24 | PXD016657 | Pride
Project description:Diabetes is associated with platelet hyperactivation and enhanced risk of thrombosis development. We have previously shown that calpain inhibition stabilizes the platelet proteome in diabetic patients. However, a global proteome analysis comparing platelets from healthy donors and from diabetic patients is missing. Therefore, we performed comparative proteomic studies to identify differentially expressed proteins and their possible function in platelet activation. Mass spectrometry analysis identified cyclin Y in platelets and levels were found to be reduced in platelets from diabetic patients. In the latter, cyclin Y was degraded by the protease calpain. In order to assess the function of cylcin Y in platelets, mice globally lacking cyclin Y were generated using CRISPR/Cas9 technology by inserting a premature Stop codon in exon 1 of Ccny gene. Cyclin Y deletion resulted in a reduction in the circulating platelet number but the agonist-induced platelet aggregation was comparable in platelets from wild-type and Ccny-/- mice, as was agonist-induced α and dense granule secretion. However, cyclin Y-deficient platelets demonstrated enhanced adhesion to fibronectin and collagen as well as an attenuated spreading and clot retraction, indicating an alteration in “outside in” integrin signalling. The phenotype was accompanied by a significant reduction in the agonist-induced tyrosine phosphorylation of the β3 integrin in Ccny-/- platelets. Stimulation of human platelets or platelets from wildtype mice with thrombin led to the membrane translocation of cyclin Y where it associates with the tyrosine kinase Fyn suggesting that cyclin Y might play a role as an adaptor protein for the tyrosine kinase to phosphorylate the cytoplasmic tail of the β3 integrin. Thus, cyclin Y is present in anucleated platelets where it is involved in the regulation of integrin-mediated outside in signalling.
2021-05-31 | PXD021271 | Pride
Project description:Schwann cells (SCs) are not only decisive to produce the axon-wrapping myelin sheath thus ensuring a proper nerve conduction but also exhibit with trophic function and can direct repair mechanisms of the peripheral nervous system. Consequently, suitable and well-characterized SC in vitro models are needed to perform appropriate pre-clinical studies including the investigation of the complex biochemical adaptations occurring in the peripheral nervous system (PNS) under different (patho)physiological conditions. MSC80 cells represent a SC line derived from mouse used in laboratories as a suitable in vitro system for neuropathological studies. As a comprehensive MSC80 protein catalogue remained elusive, here we introduce the most abundant 9532 proteins identified via mass spectrometry based protein analytics and thus provide the most comprehensive SC protein catalogue published thus far. Hereby, not only proteins causative for inherited neuropathies were covered but it has also been demonstrated that apart from cytoplasmic and nuclear proteins, mitochondrial proteins and such belonging to the protein processing machinery are very well covered. Moreover, we addressed the suitability of MSC80 to examine molecular effect of a drug-treatment by analyzing the proteomic signature of Vitamin C-treated MSC80 cells. Proteomic findings along with further immunological and functional experiments support the concept of a beneficial role influence of Vitamin C on oxidative stress and identified TMX1 as an oxidative stress protective factor in SC which might represent a promising target for therapeutic intervention of PNS disorders with marked oxidative stress burden such as diabetic neuropathy.
2018-08-13 | PXD006910 | Pride
Project description:The study aimed to comprehensively characterize human myoblastic cell line RCMH using using electron microscopic and proteomic approaches. Myoblastic cell lines can be useful to investigate the complex biochemical changes occuring under different conditions that reflect the physiological and pathophysiological mechanisms of muscle. So far, there are no suitable in vitro models of human muscle origin to study a variety of muscle related processes including responses to mechanical stress, EC-coupling and (ER-associated) myopathic disorders. Therefore, we characterized the human immortal myoblastic cell line RCMH and the results suggest RCMH as a suitable in vitro model for investigating human muscle related processes and disorders.
2016-01-26 | PXD001974 | Pride