ABSTRACT: This data set collection is of time-resolved TMT data from HRASG12V expressing fibroblasts and from top down data under 30kDa from the secretome of senescent cells
Project description:To understand how cells communicate with each other, it is essential to define the cellular secretome, a collection of proteins including soluble secreted, unconventionally secreted and proteolytically-shed proteins. Quantitative methodologies to decipher the secretome are challenging, due to the requirement of large cell numbers and abundant serum proteins that interfere with the detection of low-abundant cellular secretome proteins. Here, we miniaturized secretome analysis by developing the improved secretome-protein-enrichment-with-click-sugars method (iSPECS), which identifies the glyco-secretome. We applied this method to provide a cell type-resolved mouse brain glyco-secretome resource. Our data show that a surprisingly high number of secreted proteins are generated by ectodomain shedding in a cell type-specific manner. Two examples are neuronally secreted ADAM22 and CD200, which we identified as new substrates of the Alzheimer-linked protease BACE1. Taken together, iSPECS and the brain glyco-secretome resource can be exploited for a wide range of applications to study protein secretion and shedding.
Project description:Cardiac fibroblasts (CF) are key players after myocardial infarction (MI), but their signaling is only incompletely understood. Here we report a first secretome atlas of CF in control (cCF) and post-MI mouse hearts (miCF), combining a rapid cell isolation technique with SILAC and click chemistry. In CF, numerous paracrine factors involved in immune homeostasis are identified. Comparing secretome, transcriptome (SLAMseq), and cellular proteome disclose protein turnover. In miCF at day 5 post-MI, significantly upregulated proteins include SLIT2, FN1, and CRLF1 in mouse and human samples. Comparing the miCF secretome at days 3 and 5 post-MI reveals the dynamic nature of protein secretion. Specific in-vivo labeling of miCF proteins via biotin ligase TurboID using the POSTN promotor mirrors the in-vitro data. In summary, we identify numerous paracrine factors specifically secreted from CF in mice and humans. This secretome atlas may lead to new biomarkers and/or therapeutic targets for the activated CF.
Project description:Senescent cells are one source of the chronic inflammation that contributes to the diseases and debilities of aging. Whereas cellular senescence in fibroblasts is well documented, how this process is orchestrated in epithelial cells, the origin of human carcinomas, is much less understood. We used normal primary human oral keratinocytes (NOKs) to elucidate senescence programs in a prototype mucosal epithelial cell that undergoes senescence spontaneously. We employed widely-accepted assays to characterize senescence phenotypes in these cells and found that p21WAF1/CIP1 is not a reliable marker of senescence for NOKs, as it is for fibroblasts. We performed transcriptome analysis by RNA-seq and proteomic analysis of secreted proteins, both non-vesicular and those associated with extracellular vesicles (EVs). NOKs, while sharing with fibroblasts certain important aspects of inflammation, such as upregulation of the NF-κB, interferon, and p38MAPK pathways, also exhibit novel senescence associated characteristics. In two of the donors, we observed an expected repression of DNA repair genes, correlating with downregulation of E2F1 mRNA and protein, but saw a divergent result for the third donor. We were able to highlight potential senolytic targets. Our secretome analysis led us to propose additions to the senescence associated secretory phenotype, including HSP60, which we found on the surface of EVs. Moreover, EVs from senescent NOKs can create inflammation by stimulating interferon pathway signaling in THP-1 monocytes in culture. Our results highlight important senescence changes in epithelial cells in terms of how these cells contribute to chronic inflammation, aging, and age-related diseases.
Project description:Senescent cells are one source of the chronic inflammation that contributes to the diseases and debilities of aging. Whereas cellular senescence in fibroblasts is well documented, how this process is orchestrated in epithelial cells, the origin of human carcinomas, is much less understood. We used normal primary human oral keratinocytes (NOKs) to elucidate senescence programs in a prototype mucosal epithelial cell that undergoes senescence spontaneously. We employed widely-accepted assays to characterize senescence phenotypes in these cells and found that p21WAF1/CIP1 is not a reliable marker of senescence for NOKs, as it is for fibroblasts. We performed transcriptome analysis by RNA-seq and proteomic analysis of secreted proteins, both non-vesicular and those associated with extracellular vesicles (EVs). NOKs, while sharing with fibroblasts certain important aspects of inflammation, such as upregulation of the NF-κB, interferon, and p38MAPK pathways, also exhibit novel senescence associated characteristics. In two of the donors, we observed an expected repression of DNA repair genes, correlating with downregulation of E2F1 mRNA and protein, but saw a divergent result for the third donor. We were able to highlight potential senolytic targets. Our secretome analysis led us to propose additions to the senescence associated secretory phenotype, including HSP60, which we found on the surface of EVs. Moreover, EVs from senescent NOKs can create inflammation by stimulating interferon pathway signaling in THP-1 monocytes in culture. Our results highlight important senescence changes in epithelial cells in terms of how these cells contribute to chronic inflammation, aging, and age-related diseases.
Project description:Senescent cells release a variety of cytokines, proteases and growth factors collectively defined as the Senescent Secretory Associated Phenotype (SASP). Sustained SASP induces a pattern of chronic inflammation associated with aging and implicated in multiple age-related diseases. Here, we investigated the expression and function of the immunomodulatory cytokine BAFF (B-cell activating factor), a SASP factor, in multiple senescence models. First, we characterized BAFF production across different senescence models, including senescent human diploid fibroblasts (WI-38, IMR-90) and monocytic leukemia cells (THP-1), and tissues of mice induced to undergo senescence. We identified IRF1 (interferon response factor 1) as a transcription factor required for promoting BAFF mRNA transcription in senescence, and found that suppressing BAFF production decreased the senescent phenotype in both monocytes and fibroblasts. Importantly, the influence of BAFF on the senescence program was cell type-specific: in monocytes, BAFF promoted the early activation of NF-κB and general SASP secretion, while in fibroblasts, BAFF contributed to the production and function of TP53 (p53). Overall, BAFF silencing affected shared senescence-associated phenotypes including IL6 secretion, SA-beta-Gal staining, and γ-H2AX accumulation. We propose that BAFF is a novel biomarker of senescence and a potential target for senotherapy.
Project description:We found the bone marrow stromal-derived neural progenitor cells secretome have the neural protection effect. Proteomic analysis was performed nn order to analyze the protection factor in the secretome. Keywords: Neural protection, secretome
Project description:With advanced mass spectrometry (MS)-based proteomics, genome-scale proteome coverage can be achieved from bulk tissues. However, such bulk measurement lacks spatial resolution and obscures important tissue heterogeneity, which make it impossible for proteome mapping of tissue microenvironment. Here we report an integrated wet collection of single tissue voxel and Surfactant-assisted One-Pot voxel processing method termed wcSOP for robust label-free single voxel proteomics. wcSOP capitalizes on buffer droplet-assisted wet collection of single tissue voxel dissected by LCM into the PCR tube cap and MS-compatible surfactant-assisted one-pot voxel processing in the collection cap. This convenient method allows reproducible label-free quantification of ~900 and ~4,600 proteins for single voxel from fresh frozen human spleen tissue at 20 µm × 20 µm × 10 µm (close to single cells) and 200 µm × 200 µm × 10 µm (~100 cells), respectively. 100s-1000s of protein signatures were identified to be spatially resolved between spleen red and white pulp regions depending on the voxel size. Region-specific signaling pathways were enriched from single voxel proteomics data. To evaluate its broad applicability, we applied wcSOP-MS to two commonly accessible, OCT-embedded and FFPE, human archived tissues. It enabled to identify spatially resolved proteome changes and enriched pathways between diseased (breast cancer tumor or AD amyloid plaque) and adjacent normal regions. Antibody-based CODEX and IHC imaging validated label-free MS quantitation for single voxel analysis. The wcSOP-MS method paves the way for routine robust single voxel proteomics and spatial proteomics.
Project description:In response to various cellular stresses, the cyclin-dependent kinase inhibitor p21 transiently halts cell cycle progression until the stress is resolved, or indefinitely when permanently damaged cells undergo cellular senescence. p21 does so by repressing the transcription of E2F target genes through hypophosphorylation of Rb, but whether regulation of Rb-mediated gene transcription by p21 controls stress responses beyond cell cycle regulation is unknown. Here we report that in concert with arresting cell growth, a p21-responsive Rb pool interacts with specific Stat and Smad transcription factors at select gene promoters to establish a bioactive secretome, referred to as the p21-activated secretory phenotype (PASP). The PASP consists of several hundred secreted factors that continued to be expressed in a p21-dependent fashion after transiently arrested cells have become senescent. We found that the PASP places stressed cells under immunosurveillance, a property that largely depended on a single factor, the macrophage attractant Cxcl14. In mice, hepatocytes promptly attracted macrophages upon induction of p21 and were eliminated after sequential recruitment of B and T cells several days later. Cells normalizing p21 expression within several days after its induction resumed proliferation and were released from immunosurveillance. Collectively, these findings demonstrate that stressed cells are simultaneously placed under both proliferative arrest and immunosurveillance (to prevent the expansion of stressed, potentially damaged cells), and that p21 coordinates these cell-autonomous and non-autonomous activities at the transcriptional level through regulation of Rb.