ALS Associated Mutations In Matrin 3 Alter Protein-Protein Interactions And Impede Mrna Nuclear Export

Mutations in Matrin 3 have recently been linked to ALS, though the mechanism that induces disease in these patients is unknown. To define the protein interactome of wild-type and ALS-linked MATR3 mutations, we performed immunoprecipitation followed by mass spectrometry using NSC-34 cells expressing human wild-type or mutant Matrin 3. Gene ontology analysis identified a novel role for Matrin 3 in mRNA transport centered on proteins in the TRanscription and EXport (TREX) complex, known to function in mRNA biogenesis and nuclear export. ALS-linked mutations in Matrin 3 led to its re-distribution within the nucleus, decreased co-localization with endogenous Matrin 3 and increased co-localization with specific TREX components. Expression of disease-causing Matrin 3 mutations led to nuclear mRNA export defects of both global mRNA and more specifically the mRNA of TDP-43 and FUS. Our findings identify a potential pathogenic mechanism attributable to MATR3 mutations and further link cellular transport defects to ALS

Identification of a Blood-Based Protein Biomarker Panel for Lung Cancer Detection

Lung cancer is the deadliest cancer worldwide, mainly due to its advanced stage at the time of diagnosis. A non-invasive method for its early detection remains mandatory to improve patients’ survival. Plasma levels of 351 proteins were quantified by Liquid Chromatography-Parallel Reaction Monitoring (LC-PRM)-based mass spectrometry in 128 lung cancer patients and 93 healthy donors. Bootstrap sampling and least absolute shrinkage and selection operator (LASSO) penalization were used to find the best protein combination for outcome prediction. The PanelomiX platform was used to select the optimal biomarker thresholds. The panel was validated in 48 patients and 49 healthy volunteers. A 6-protein panel clearly distinguished lung cancer from healthy individuals. The panel displayed excellent performance: area under the receiver operating characteristic curve (AUC) = 0.999, positive predictive value (PPV) = 0.992, negative predictive value (NPV) = 0.989, specificity = 0.989 and sensitivity = 0.992. The panel detected lung cancer independently of the disease stage. The 6-protein panel and other sub-combinations displayed excellent results in the validation dataset. In conclusion, we identified a blood-based 6-protein panel as a diagnostic tool in lung cancer. Used as a routine test for high- and average-risk individuals, it may complement currently adopted techniques in lung cancer screening.publishedVersio

Arginine Depletion Therapy with ADI-PEG20 Limits Tumor Growth in Argininosuccinate Synthase–Deficient Ovarian Cancer, Including Small-Cell Carcinoma of the Ovary, Hypercalcemic Type

PurposeMany rare ovarian cancer subtypes, such as small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), have poor prognosis due to their aggressive nature and resistance to standard platinum- and taxane-based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes.Experimental designWe compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype, high-grade serous ovarian cancer (HGSC), to identify potential therapeutic targets. IHC of tissue microarrays was used as validation of arginosuccinate synthase (ASS1) deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed in vitro using cell lines and patient-derived xenograft mouse models representing SCCOHT.ResultsGlobal proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared with HGSC. Low ASS1 levels were validated through IHC in large patient cohorts. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 12 of 31 cases, and expressed in less than 5% of the tumor cells in 9 of 31 cases. ASS1-deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype in vitro. Furthermore, in two cell line mouse xenograft models and one patient-derived mouse xenograft model of SCCOHT, once-a-week treatment with ADI-PEG20 (30 mg/kg and 15 mg/kg) inhibited tumor growth in vivo.ConclusionsPreclinical in vitro and in vivo studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers, including SCCOHT

Re-expression of SMARCA4/BRG1 in Small Cell Carcinoma of Ovary, Hypercalcemic Type (SCCOHT) promotes an epithelial-like gene signature through an AP-1-dependent mechanism

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and aggressive form of ovarian cancer. SCCOHT tumors have inactivating mutations in SMARCA4 (BRG1), one of the two mutually exclusive ATPases of the SWI/SNF chromatin remodeling complex. To address the role that BRG1 loss plays in SCCOHT tumorigenesis, we performed integrative multi-omic analyses in SCCOHT cell lines +/- BRG1 reexpression. BRG1 reexpression induced a gene and protein signature similar to an epithelial cell and gained chromatin accessibility sites correlated with other epithelial originating TCGA tumors. Gained chromatin accessibility and BRG1 recruited sites were strongly enriched for transcription-factor-binding motifs of AP-1 family members. Furthermore, AP-1 motifs were enriched at the promoters of highly upregulated epithelial genes. Using a dominant-negative AP-1 cell line, we found that both AP-1 DNA-binding activity and BRG1 reexpression are necessary for the gene and protein expression of epithelial genes. Our study demonstrates that BRG1 reexpression drives an epithelial-like gene and protein signature in SCCOHT cells that depends upon by AP-1 activity

Applications biologiques du NanoSIMS

La spectrométrie de masse à ions secondaires (SIMS) est une technique de caractérisation chimique de la matière par balayage avec un faisceau d’ions primaires. L’impact des ions primaires provoque une cascade de collisions atomiques causant un déplacement des éléments constitutifs de l’échantillon, émis sous forme d’ions, et séparés selon les critères d’énergie et de masse par un spectromètre. Le NanoSIMS fait partie de la famille des microscopes SIMS dynamiques capable d’imager à haute résolution la distribution d’ions élémentaires dans un échantillon. Les travaux présentés dans cette thèse sont axés sur le développement de techniques de préparations d’échantillons biologiques pour le NanoSIMS, et sur la recherche d’applications biologiques spécifique. Plusieurs outils informatiques ont été développés afin de permettre le traitement d’images provenant de cet instrument. Des suggestions sont proposées afin d’améliorer les conditions d’analyses et d’acquérir des images de meilleure qualité. Grâce à sa résolution en masse, le NanoSIMS a permis de suivre directement des molécules marquées par des isotopes résultant d’un marquage métabolique, démontré lors d’expériences « pulse-chase » et d’immunomarquage par des anticorps marqués à l’15N. Il découle de nos travaux que la sensitivité et la résolution de l’instrument le rendent particulièrement intéressant pour des études de pollution atmosphérique. Cette affirmation est soutenue par une étude sur la distribution de métaux traces dans des lichens, comme bioindicateurs. Lors de ces études, l’utilisation du NanoSIMS a été complémentée par des techniques d’imagerie alternatives telles que la microscopie confocale et électronique.Secondary Ion Mass Spectrometry (SIMS) is based upon the sputtering of a few atomic layers from the surface of a sample, induced by a ’primary ion’ bombardment. An energetic primary ion impact triggers a cascade of atomic collisions resulting in an erosion of atoms and molecules. Some of the ejected particles can be spontaneously ionized and are representative of the target area composition. In a SIMS instrument, these “secondary ions” are accelerated and separated in function of their mass/charge ratio (m/z) before detection. The NanoSIMS is a dynamic SIMS ion microprobe capable of imaging the distribution of elemental ions at high lateral resolution (50 nm) with a high mass resolution. The aim of this thesis was to define the requirements of such instruments, to evaluate their utility in life sciences and to develop potential applications in biology. In order to do so, preparative and analytical methods had to be devised to improve ion imaging by SIMS. The limited knowledge about the behaviour of biological samples under a primary ion beam impeded the identification of tissue and cellular features and required supplementary confirmation by complementary techniques. The ability of SIMS to discriminate isotopes of a same element has encouraged us to develop isotopically labelled biomolecules of varying specificity. Abiding to the experience gained in the course of the thesis, several applications are proposed in the areas of trace metal detection, antigen uptake and protein localization. Finally, NanoSIMS is compared to other techniques of microanalytical imaging and the role of the ion microprobe in life sciences is discussed as well as its prospects

Applications biologiques du nanoSIMS

La spectrométrie de masse à ions secondaires (SIMS) est une technique de caractérisation chimique de la matière par balayage avec un faisceau d ions primaires. L impact des ions primaires provoque une cascade de collisions atomiques causant un déplacement des éléments constitutifs de l échantillon, émis sous forme d ions, et séparés selon les critères d énergie et de masse par un spectromètre. Le NanoSIMS fait partie de la famille des microscopes SIMS dynamiques capable d imager à haute résolution la distribution d ions élémentaires dans un échantillon.Les travaux présentés dans cette thèse sont axés sur le développement de techniques de préparations d échantillons biologiques pour le NanoSIMS, et sur la recherche d applications biologiques spécifique. Plusieurs outils informatiques ont été développés afin de permettre le traitement d images provenant de cet instrument. Des suggestions sont proposées afin d améliorer les conditions d analyses et d acquérir des images de meilleure qualité. Grâce à sa résolution en masse, le NanoSIMS a permis de suivre directement des molécules marquées par des isotopes résultant d un marquage métabolique, démontré lors d expériences pulse-chase et d immunomarquage par des anticorps marqués à l 15N. Il découle de nos travaux que la sensitivité et la résolution de l instrument le rendent particulièrement intéressant pour des études de pollution atmosphérique. Cette affirmation est soutenue par une étude sur la distribution de métaux traces dans des lichens, comme bioindicateurs. Lors de ces études, l utilisation du NanoSIMS a été complémentée par des techniques d imagerie alternatives telles que la microscopie confocale et électronique.Secondary Ion Mass Spectrometry (SIMS) is based upon the sputtering of a few atomic layers from the surface of a sample, induced by a primary ion bombardment. An energetic primary ion impact triggers a cascade of atomic collisions resulting in an erosion of atoms and molecules. Some of the ejected particles can be spontaneously ionized and are representative of the target area composition. In a SIMS instrument, these secondary ions are accelerated and separated in function of their mass/charge ratio (m/z) before detection. The NanoSIMS is a dynamic SIMS ion microprobe capable of imaging the distribution of elemental ions at high lateral resolution (50 nm) with a high mass resolution.The aim of this thesis was to define the requirements of such instruments, to evaluate their utility in life sciences and to develop potential applications in biology. In order to do so, preparative and analytical methods had to be devised to improve ion imaging by SIMS. The limited knowledge about the behaviour of biological samples under a primary ion beam impeded the identification of tissue and cellular features and required supplementary confirmation by complementary techniques. The ability of SIMS to discriminate isotopes of a same element has encouraged us to develop isotopically labelled biomolecules of varying specificity. Abiding to the experience gained in the course of the thesis, several applications are proposed in the areas of trace metal detection, antigen uptake and protein localization. Finally, NanoSIMS is compared to other techniques of microanalytical imaging and the role of the ion microprobe in life sciences is discussed as well as its prospects.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Immunoprecipitation And Mass Spectrometry Defines An Extensive Rbm45 Protein–Protein Interaction Network

The pathological accumulation of RNA-binding proteins (RBPs) within inclusion bodies is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RBP aggregation results in both toxic gain and loss of normal function. Determining the protein binding partners and normal functions of disease-associated RBPs is necessary to fully understand molecular mechanisms of RBPs in disease. Herein, we characterized the protein–protein interactions (PPIs) of RBM45, a RBP that localizes to inclusions in ALS/FTLD. Using immunoprecipitation coupled to mass spectrometry (IP–MS), we identified 132 proteins that specifically interact with RBM45 within HEK293 cells. Select PPIs were validated by immunoblot and immunocytochemistry, demonstrating that RBM45 associates with a number of other RBPs primarily via RNA-dependent interactions in the nucleus. Analysis of the biological processes and pathways associated with RBM45-interacting proteins indicates enrichment for nuclear RNA processing/splicing via association with hnRNP proteins and cytoplasmic RNA translation via eiF2 and eiF4 pathways. Moreover, several other ALS-linked RBPs, including TDP-43, FUS, Matrin-3, and hnRNP-A1, interact with RBM45, consistent with prior observations of these proteins within intracellular inclusions in ALS/FTLD. Taken together, our results define a PPI network for RBM45, suggest novel functions for this protein, and provide new insights into the contributions of RBM45 to neurodegeneration in ALS/FTLD. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease