Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.
ABSTRACT The relationship between lipid homeostasis and protein homeostasis (proteostasis) is complex and remains incompletely understood. We conducted a screen for genes required for efficient degradation of Deg1 -Sec62, a model aberrant translocon-associated substrate of the endoplasmic reticulum (ER) ubiquitin ligase Hrd1, in Saccharomyces cerevisiae . This screen revealed that INO4 is required for efficient Deg1 -Sec62 degradation. INO4 encodes one subunit of the Ino2/Ino4 heterodimeric transcription factor, which regulates expression of genes required for lipid biosynthesis. Deg1 -Sec62 degradation was also impaired by mutation of genes encoding several enzymes mediating phospholipid and sterol biosynthesis. The degradation defect in ino4 Δ yeast was rescued by supplementation with metabolites whose synthesis and uptake are mediated by Ino2/Ino4 targets. Stabilization of a panel of substrates of the Hrd1 and Doa10 ER ubiquitin ligases by INO4 deletion indicates ER protein quality control is generally sensitive to perturbed lipid homeostasis. Further, loss of INO4 sensitized yeast to proteotoxic stress, suggesting a broad requirement for lipid homeostasis in maintaining proteostasis. Abundance of the ER ubiquitin-conjugating enzyme Ubc7 was reduced in the absence of INO4 , consistent with a model whereby perturbed lipid biosynthesis alters the abundance of critical protein quality control mediators, with broad consequences for ER proteostasis. A better understanding of the dynamic relationship between lipid homeostasis and proteostasis may lead to improved understanding and treatment of several human diseases associated with altered lipid biosynthesis.
Proteostasis
Endoplasmic-reticulum-associated protein degradation
The endoplasmic reticulum (ER) translocon is a molecular channel responsible for moving secreted and endomembrane system proteins across the ER membrane. Proteins that stall within or otherwise aberrantly engage the translocon channel may impair transport of other proteins across the membrane. Proteins that persistently or aberrantly engage the translocon are targeted for degradation via the conserved ER‐resident ubiquitin ligase Hrd1. A better understanding of this system may prove useful in treating elevated cholesterol in humans as, under certain circumstances, the major protein component of low‐density lipoproteins (LDL; bad cholesterol) stalls within the translocon prior to LDL synthesis. Protein co‐factors that assist Hrd1 in targeting other substrates for degradation are dispensable for Hrd1‐dependent degradation of translocon‐obstructing proteins. We hypothesize that Hrd1 requires the function of a distinct cohort of protein co‐factors to recognize and ubiquitylate proteins that aberrantly engage the translocon. We have developed a growth‐based screen to identify genes in Saccharomyces cerevisiae that mediate the degradation of proteins that aberrantly engage the translocon. We fused the His3 enzyme, which is required for histidine biosynthesis, to a model protein that aberrantly engages with the translocon. In this way, histidine prototrophy is an indicator of impaired protein degradation. We introduced this fusion protein into the non‐essential yeast gene knockout collection using Synthetic Genetic Array (SGA) technology. Genes found to be required for degradation of the model translocon‐associated protein may represent therapeutic targets for patients with cholesterol‐related pathology. Support or Funding Information National Institutes of Health Grant R15 GM111713 (to EMR). Funds from the Ball State University Provost's Office and Department of Biology (to EMR). Ball State University Chapter of Sigma Xi Research Award (to CJI).
Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.
Abstract Background A thorough understanding of a patient’s inflammatory response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is crucial to discerning the associated, underlying immunological processes and to the selection and implementation of treatment strategies. Defining peripheral blood biomarkers relevant to SARS-CoV-2 infection is fundamental to detecting and monitoring this systemic disease. This safety-focused study aims to monitor and characterize the immune response to SARS-CoV-2 infection via analysis of peripheral blood and nasopharyngeal swab samples obtained from patients hospitalized with Coronavirus disease 2019 (COVID-19), in the presence or absence of bamlanivimab treatment. Methods 23 patients hospitalized with COVID-19 were randomized to receive a single dose of the neutralizing monoclonal antibody, bamlanivimab (700 mg, 2800 mg or 7000 mg) or placebo, at study initiation (Clinical Trial; NCT04411628). Serum samples and nasopharyngeal swabs were collected at multiple time points over 1 month. A Proximity Extension Array was used to detect inflammatory profiles from protein biomarkers in the serum of hospitalized COVID-19 patients relative to age/sex-matched healthy controls. RNA sequencing was performed on nasopharyngeal swabs. A Luminex serology assay and Elecsys® Anti-SARS-CoV-2 immunoassay were used to detect endogenous antibody formation and to monitor seroconversion in each cohort over time. A mixed model for repeated measures approach was used to analyze changes in serology and serum proteins over time. Results Levels of IL-6, CXCL10, CXCL11, IFNγ and MCP-3 were > fourfold higher in the serum of patients with COVID-19 versus healthy controls and linked with observations of inflammatory and viral-induced interferon response genes detected in nasopharyngeal swab samples from the same patients. While IgA and IgM titers peaked around 7 days post-dose, IgG titers remained high, even after 28 days. Changes in biomarkers over time were not significantly different between the bamlanivimab and placebo groups. Conclusions Similarities observed between nasopharyngeal gene expression patterns and peripheral blood biomarker profiles reveal a connection between the circulation and processes in the nasopharyngeal cavity, reinforcing the potential utility of systemic blood biomarker profiling for therapeutic monitoring of patient response. Serological antibody responses in patients correlated closely with reductions in the COVID-19 inflammatory protein biomarker signature. Bamlanivimab did not affect the biomarker dynamics in this hospitalized patient population.
Conserved homologues of the Hrd1 ubiquitin ligase target for degradation proteins that persistently or aberrantly engage the endoplasmic reticulum translocon, including mammalian apolipoprotein B (apoB; the major protein component of low-density lipoproteins) and the artificial yeast protein Deg1-Sec62.A complete understanding of the molecular mechanism by which translocon-associated proteins are recognized and degraded may inform the development of therapeutic strategies for cholesterol-related pathologies.Both apoB and Deg1-Sec62 are extensively post-translationally modified.Mass spectrometry of a variant of Deg1-Sec62 revealed that the protein is acetylated at the N-terminal methionine and two internal lysine residues.N-terminal and internal acetylation regulates the degradation of a variety of unstable proteins.However, preventing N-terminal and internal acetylation had no detectable consequence for Hrd1mediated proteolysis of Deg1-Sec62.Our data highlight the importance of empirically validating the role of post-translational modifications and sequence motifs on protein degradation, even when such elements have previously been demonstrated sufficient to destine other proteins for destruction.
Conserved homologues of the Hrd1 ubiquitin ligase target for degradation proteins that persistently or aberrantly engage the endoplasmic reticulum translocon, including mammalian apolipoprotein B (apoB; the major protein component of low-density lipoproteins) and the artificial yeast protein Deg1-Sec62.A complete understanding of the molecular mechanism by which translocon-associated proteins are recognized and degraded may inform the development of therapeutic strategies for cholesterol-related pathologies.Both apoB and Deg1-Sec62 are extensively post-translationally modified.Mass spectrometry of a variant of Deg1-Sec62 revealed that the protein is acetylated at the N-terminal methionine and two internal lysine residues.N-terminal and internal acetylation regulates the degradation of a variety of unstable proteins.However, preventing N-terminal and internal acetylation had no detectable consequence for Hrd1mediated proteolysis of Deg1-Sec62.Our data highlight the importance of empirically validating the role of post-translational modifications and sequence motifs on protein degradation, even when such elements have previously been demonstrated sufficient to destine other proteins for destruction.
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