Project description:Oligosaccharyl transferase (OST) protein complex mediates the N-linked glycosylation of substrate proteins in the endoplasmic reticulum (ER), which regulates stability, activity and localization of its substrates. Although many OST substrate proteins have been identified, the physiological role of OST complex remains incompletely understood. Here, we show that OST complex in C. elegans is crucial for ER protein homeostasis and enhances resistance to pathogenic bacteria, Pseudomonas aeruginosa (PA14), via immune-regulatory PMK-1/p38 MAP kinase.
Project description:To better understand how OMA1 affects mitochondrial proteostasis and stress responses, we performed a proteomic survey of OMA1-deficient cells for proteolytic substrates. We demonstrate that OMA1 cleaves the mitochondrial chaperone DNAJC15 facilitating its degradation by the mitochondrial m-AAA protease. The loss of DNAJC15 alters protein import by TIM23 protein translocases in the IM and limits the accumulation of OXPHOS-related mitochondrial matrix and IM proteins. Non-imported mitochondrial preproteins accumulate at the endoplasmic reticulum (ER) and trigger an ATF6-related unfolded protein response. These results demonstrate that OMA1 allows to adapt mitochondrial protein biogenesis to stress and reveal an intricate network of cellular stress responses to proteostasis disturbances.
Project description:To understand how OMA1 affects mitochondrial proteostasis and stress responses, we performed a proteomic survey of OMA1-deficient cells for proteolytic substrates. We demonstrate that OMA1 cleaves the mitochondrial chaperone DNAJC15 facilitating its degradation by the mitochondrial m-AAA protease. The loss of DNAJC15 alters protein import by TIM23 protein translocases in the IM and limits the accumulation of OXPHOS-related mitochondrial matrix and IM proteins. Non-imported mitochondrial preproteins accumulate at the endoplasmic reticulum (ER) and trigger an ATF6-related unfolded protein response. These results demonstrate that OMA1 allows to adapt mitochondrial protein biogenesis to stress and reveal an intricate network of cellular stress responses to proteostasis disturbances.
Project description:The majority of secretory proteins undergo N-glycosylation, a process catalyzed by oligosaccharyltransferase (OST), a membrane-bound protein complex that associates with the translocation channels within the endoplasmic reticulum (ER). Proteins failing quality control undergo ER-associated degradation (ERAD) by retrotranslocation to cytosolic proteasomes. Using a misfolded protein bait and SILAC proteomics, we unexpectedly identified several OST subunits among top hits. For further details, Please refer to the publication "Oligosaccharyltransferase is involved in targeting to ER-associated degradation" by Marina Shenkman et al..
Project description:Multiple myeloma (MM) is an incurable malignancy characterized by mutated plasma cell clonal expansion in the bone marrow, leading to severe clinical symptoms. Thus, identifying new therapeutic targets for MM is crucial. We identified the oligosaccharyltransferase (OST) complex as a novel vulnerability in MM cells. Elevated expression of this complex is associated with relapsed, high-risk MM, and poor prognosis. Disrupting the OST complex suppressed MM cell growth, inducing cell cycle arrest and apoptosis. Combined inhibition with bortezomib synergistically eliminated MM cells in vitro and in vivo, via suppressing genes related to bortezomib-resistant phenotypes. Mechanistically, OST complex disruption downregulated pathological gene signatures (NF-kB signaling, mTORC1 pathway, glycolysis, MYC targets, and cell cycle), induced TRAIL-mediated apoptosis and inflammatory pathways. MYC translation was robustly suppressed upon inhibiting the OST complex. Collectively, the OST complex presents a novel target for MM treatment, and combining its inhibition with bortezomib offers a promising approach for relapsed MM patients.
Project description:Investigating gene expression patterns in mice with conditional expression of the most common RVCL mutation, V235fs, and another mice expressing a conditional C-terminal mutation, D272fs, associated with a case of human SLE. Mutations at the N-terminus affecting TREX1 DNase activity are associated with autoimmune and inflammatory conditions such as Aicardi-Goutières syndrome (AGS). Mutations in the C-terminus of TREX1 cause loss of localization to the ER and dysregulation of oligosacchryltransferase (OST) activity, and are associated with retinal vasculopathy with cerebral leukodystrophy (RVCL) and in some cases with systemic lupus erythematosus (SLE).
Project description:<p>Perturbations in lysosome integrity are tightly linked to neurological disorders and ageing, but the underlying pathogenic mechanisms are incompletely understood. Using an unbiased proteomic approach, we here identified the bridge-like lipid transport protein VPS13C/PARK23 as a key component of a global early response pathway to lysosome damage. VPS13C readily binds lysosomes under mechanical or osmotic tension in anticipation of membrane lesions. The latter trigger a conformational change in the protein’s <em>C</em>-terminus, involving its ATG2C domain acting as sensor of damage-induced lipid packing defects. We show that ER-lysosome contacts formed by VPS13C provide critical binding platforms for OSBP/ORPs to enable efficient ER wrapping of damaged lysosomes. A chemical approach to assess directional ER-to-lysosome lipid transport revealed that VPS13C is essential for large-scale lipid delivery to acutely damaged lysosomes to facilitate their repair. Our findings offer new mechanistic insights into how loss-of-function mutations in <em>VPS13C</em> may enhance the risk of Parkinson’s disease.</p><p><br></p><p>This study contains shotgun lipidomics data from HeLa cells treated with L-leucyl-L-leucine methyl ester (LLOMe), a lysosomotropic agent that induces lysosomal membrane damage. Lipid profiles were obtained from both whole-cell lysates and isolated lysosomal fractions. Lipids were extracted and quantified by mass spectrometry-based shotgun lipidomics.</p>
Project description:The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) results in the condition called âER stressâ which induces the unfolded protein response (UPR) which is a complex cellular process that includes changes in expression of many genes. Failure to restore homeostasis in the ER is associated with human diseases. To identify the underlying changes in gene expression in response to ER stress, we induced ER stress in human B-cells and then measured gene expression at 10 time-points. We followed up those results by studying cells from 60 unrelated people. We rediscovered genes that were known to play a role in ER stress response and uncovered several thousand genes that are not known to be involved. Two of these are VLDLR and INHBE which showed significant increase in expression following ER stress in B-cells and in primary fibroblasts. To study the links between unfolded protein response and disease susceptibility, we identified ER stress responsive genes that are associated with human diseases and assessed individual differences in ER stress response. Many of the UPR genes are associated with Mendelian disorders such as Wolfram syndrome and complex human diseases including amyotrophic lateral sclerosis and diabetes. Data from two independent samples showed extensive individual variability in ER stress response. Additional analyses with monozygotic twins revealed significant correlations within twin pairs in their responses to ER stress thus showing evidence for heritable variation among individuals. These results have implications for basic understanding of ER function and its role in disease susceptibility. Keywords: array-based gene expression We measured gene expression levels in immortalized B cells from members of 60 unrelated CEPH-Utah grandparents. Each individual was treated for 8 hours with either DMSO or with 4 ug/ml of tunicamycin. Gene expression was measured to identify tunicamycin-responsive genes. To assess whether there is a genetic component to the individual variation in gene expression response to ER stress, we used microarrays to measure expression of genes in 26 monozygotic twin pairs treated with either DMSO or 500 nM thapsigargin for 4 hours.
Project description:The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) results in the condition called “ER stress” which induces the unfolded protein response (UPR) which is a complex cellular process that includes changes in expression of many genes. Failure to restore homeostasis in the ER is associated with human diseases. To identify the underlying changes in gene expression in response to ER stress, we induced ER stress in human B-cells and then measured gene expression at 10 time-points. We followed up those results by studying cells from 60 unrelated people. We rediscovered genes that were known to play a role in ER stress response and uncovered several thousand genes that are not known to be involved. Two of these are VLDLR and INHBE which showed significant increase in expression following ER stress in B-cells and in primary fibroblasts. To study the links between unfolded protein response and disease susceptibility, we identified ER stress responsive genes that are associated with human diseases and assessed individual differences in ER stress response. Many of the UPR genes are associated with Mendelian disorders such as Wolfram syndrome and complex human diseases including amyotrophic lateral sclerosis and diabetes. Data from two independent samples showed extensive individual variability in ER stress response. Additional analyses with monozygotic twins revealed significant correlations within twin pairs in their responses to ER stress thus showing evidence for heritable variation among individuals. These results have implications for basic understanding of ER function and its role in disease susceptibility. Keywords: array-based gene expression