Project description:Proteomics data from gel band excision in support of "The proteasome as a drug target in the metazoan pathogen, Schistosoma mansoni"
Project description:We compared the expression changes in Ewing sarcoma cell lines following treatment with 2 known 20S proteasome inhibitors versus 2 novel compounds
Project description:Alzheimer's disease (AD) is a relentlessly progressive, fatal neurodegenerative disorder that results in widespread protein dysfunctions. However, the full extent of aberrant proteomic changes in AD and their impact on cellular physiology remains unknown. This is, in part, because of the challenges of comprehensively measuring the proteome. Here, we used plexDIA, an approach that provides deep proteomic coverage and high throughput by parallelizing the acquisition of peptides and samples, to characterize proteomic changes in AD. Using human dorsolateral prefrontal cortex tissue, we identified 281 differentially abundant proteins in AD. By systematically analyzing compartment and protein complex-specific shifts in protein abundance, we identified an AD-specific decrease in levels of the 20S proteasome, the catalytic core of the cell's primary protein degradation pathway. This alteration was accompanied by widespread decreases in proteasome subunit stoichiometries. Many proteasome substrate proteins were negatively correlated with 20S levels and increased in AD, suggesting that reduced 20S levels leads to abnormal protein accumulation. By analyzing proteins increased in AD, we identify key properties of such proteins: They have fast degradation rates, they contain signal sequences that allow them to be targeted for proteasomal degradation, and they are targeted by quality control pathways that recognize mislocalized proteins. Changes in these gene products at the protein and mRNA levels were highly discordant, providing additional evidence for increased protein abundance driven by impaired clearance. We also identified coherent sets of ubiquitin system enzymes, proteins that target substrates for proteasomal degradation, whose levels robustly discriminate AD from non-AD samples. One subset exhibited consistent increases in AD, while another, which contained the tau E3 ligase Cul5, exhibited consistent decreases, revealing complex changes in the ubiquitin system in AD. Taken together, our results suggest that decreased ubiquitin-proteasome system capacity and impaired clearance of short-lived and mislocalized proteins contribute substantially to proteopathic burden in AD.
Project description:Cell lines have been used for drug discovery as useful models of cancers; however, they do not recapitulate cancers faithfully, especially in the points of rapid growth rate and microenvironment independency. Consequently, the majority of conventional anti-cancer drugs are less sensitive to slow growing cells and do not target microenvironmental support, although most primary cancer cells grow slower than cell lines and depend on microenvironmental support. Here, we developed a novel high throughput drug screening system using patient-derived xenograft (PDX) cells of lymphoma that maintained primary cancer cell phenotype more than cell lines. The library containing 2613 known pharmacologically active substance and off-patent drugs were screened by this system. We could find many compounds showing higher cytotoxicity than conventional anti-tumor drugs. Especially, pyruvinium pamoate showed the highest activity, and its strong anti-tumor effect was confirmed also in vivo. We extensively investigated its mechanism of action and found that it inhibited glutathione supply from stromal cells to lymphoma cells, implying the importance of the stromal protection from ox 1 idative stress for lymphoma cell survival and a new therapeutic strategy for lymphoma. Our system introduces a primary cancer cell phenotype into cell-based phenotype screening and sheds new light on anti-cancer drug development. Global gene expression profiles of PDX cells showed high similarity to those of original primary cells. The correlation coefficient of gene expression profiles between PDX cells and the originalprimary cells was 0.814-0.890.
Project description:The 20S proteasome is responsible for the catalytic activity of all proteasome complexes. Structural constraints mean that only unfolded, extended polypeptide chains may enter the catalytic core of the 20S proteasome. Here we conducted a comprehensive analysis of the 20S CP substrates in-vitro. We revealed that the 20S CP substrates are highly structually disordered. The 20S proteasome substrate group, termed 20S-IDPome are characterized by having significantly more protein binding partners, more post-translational modification sites and are highly enriched for RNA binding proteins. Remarkably, we found that low complexity proteins with prion-like domain which interact with GR or PR di-peptide repeats are the most preferential 20S proteasome substrates. Our finding suggests roles of the 20S proteasome in gene transcription and formation of phase-separated granules.
2018-07-30 | PXD010132 | Pride
Project description:20S proteasome subunits as prognostic and resistant markers in cancer
Project description:Mature Red Blood Cells (RBCs) lack internal organelles and canonical defense mechanisms, making them both a fascinating host cell, in general, and an intriguing choice for the deadly malaria parasite Plasmodium falciparum (Pf), in particular. While growing inside RBCs, Pf are known to secrete multipurpose extracellular vesicles (EVs), yet their influence on this parasite’s essential host cell, the RBC, remains unknown. Here we demonstrate that Pf parasites export within such EVs assembled and functional 20S proteasome complexes (EV-20S). The EV-20S proteasomes modulate the mechanical properties of naïve host RBCs by remodeling the cytoskeleton network. Furthermore, we identified four novel degradation targets of the exported 20S proteasome, the phosphorylated cytoskelatal proteins β-adducin, ankyrin-1, dematin and Epb4.1. Overall, our findings reveal a previously unknown 20S proteasome export mechanism of Pf-iRBCs, which prime naïve RBC by altering membrane stiffness, to facilitate malaria parasite growth.
Project description:Programmed degradation of maternal proteins is essential for the oocyte-to-embryo transition (OET). While pharmacological inhibition studies have established the importance of proteasomes in ovarian reserve maintenance, oocyte maturation and fertilization, the physiological impact of intrinsic proteasome insufficiency and underlying molecular mechanisms remain poorly understood. In mice, endolysosomal vesicular assemblies (ELVAs), specialized membraneless compartments composed of proteasomes, endolysosomes and autophagosomes, facilitate protein degradation during oocyte maturation and early embryogenesis. In this study, we generated mice with oocyte-specific deletion of the proteasomal core subunit Psma7, to investigate the physiological function of the 20S proteasome and its roles in ELVAs-mediated protein degradation. PSMA7-deficiency destabilized 20S proteasomes and disrupted translocation of ELVAs, leading to pronounced accumulation of ubiquitinated proteins in oocytes and zygotes. Consequently, maternal Psma7 deletion resulted in female infertility, manifested by impaired oocyte maturation and developmental arrest at one- to two-cell stage. Furthermore, we observed reduced proteasome abundance and dysfunction of ELVAs in aged oocytes, providing a mechanistic explanation for the decline in developmental competence associated with oocyte aging. Taken together, our findings elucidate the critical function of proteasome-regulated proteostasis within ELVAs in maintaining oocyte quality during OET and reproductive aging.
Project description:Mature Red Blood Cells (RBCs) lack internal organelles and canonical defense mechanisms, making them both a fascinating host cell, in general, and an intriguing choice for the deadly malaria parasite Plasmodium falciparum (Pf), in particular. Pf, while growing inside its natural host, the human RBC, secretes multipurpose extracellular vesicles (EVs), yet their influence on this essential host cell remains unknown. Here we demonstrate that Pf parasites, cultured in fresh human donor blood, export within such EVs assembled and functional 20S proteasome complexes (EV-20S). The EV-20S proteasomes modulate the mechanical properties of naïve human RBCs by remodeling their cytoskeletal network. Furthermore, we identify four novel degradation targets of the exported 20S proteasome, the phosphorylated cytoskeletal proteins β-adducin, ankyrin-1, dematin and Epb4.1. Overall, our findings reveal a previously unknown 20S proteasome export mechanism employed by the human malaria parasite, which primes RBCs for parasite invasion by altering membrane stiffness, to facilitate malaria parasite growth.
Project description:We used Affymetrix Chicken Genome Array to identify transcripts differentially expressed between cells treated with DMIOA and control cells and between DMIOA and DMIOA+20S treated cells Preadipocytes isolated from laying hen were treated with DMIOA and DMIOA+20S to identify transcripts differentailly expressed between control cells and DMIOA treated and between DMIOA and DMIOA+20S treated cells