Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA137605 | ENA |
REPOSITORIES: ENA
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Project description:The protein secretory pathway must maintain homoeostasis while producing a wide assortment of proteins in different conditions. It is also used extensively to produce many useful proteins in biotechnology. As such, secretory pathway dysfunction can be highly detrimental to the cell, resulting in the molecular basis for many human diseases, and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. To better understand some of these dysfunctions, we measured multiple systems-level states of the cell (physiology, transcriptome, metabolism) while secreting a small protein (insulin precursor) or a large protein (?-amylase). This was carried out in the presence and absence of HAC1, a key transcription factor in maintaining secretory homeostasis. Clear trends in cellular stress were apparent across multiple data resulting from our perturbations. In particular, processes involving (1) degradation of protein / recycling amino acids, (2) overall transcription/translation repression, and (3) oxidative stress. Apparent runaway oxidative radical production was explained by a thermodynamic model that we put forward for disulfide formation in the endoplasmic reticulum. This model predicts that balancing the relative rates of protein folding and disulfide bond formation are key to easing oxidative stress. These predictions have direct implications in how to engineer a broad range of recombinant proteins for secretion and provide potential hypotheses for the root causes of several secretory-associated diseases. Yeast strains were constructed that produce and secrete (a) IP or (b) ?-amylase and were compared to yeast strains containing (c) an empty vector in both wild-type and HAC1 deletion backgrounds. These strains are named WN (WT with empty vector), WI (WT secreting IP), WA (WT secreting ?-amylase), dN (?hac1 with empty vector), dI (?hac1 secreting IP), and dA (?hac1 secreting ?-amylase). Strains were characterized in batch fermentation and samples were taken in mid-exponential phase. Triplicate fermentations were carried out for each strain.
2011-12-01 | E-GEOD-27062 | biostudies-arrayexpress
Project description:The protein secretory pathway must maintain homoeostasis while producing a wide assortment of proteins in different conditions. It is also used extensively to produce many useful proteins in biotechnology. As such, secretory pathway dysfunction can be highly detrimental to the cell, resulting in the molecular basis for many human diseases, and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. To better understand some of these dysfunctions, we measured multiple systems-level states of the cell (physiology, transcriptome, metabolism) while secreting a small protein (insulin precursor) or a large protein (α-amylase). This was carried out in the presence and absence of HAC1, a key transcription factor in maintaining secretory homeostasis. Clear trends in cellular stress were apparent across multiple data resulting from our perturbations. In particular, processes involving (1) degradation of protein / recycling amino acids, (2) overall transcription/translation repression, and (3) oxidative stress. Apparent runaway oxidative radical production was explained by a thermodynamic model that we put forward for disulfide formation in the endoplasmic reticulum. This model predicts that balancing the relative rates of protein folding and disulfide bond formation are key to easing oxidative stress. These predictions have direct implications in how to engineer a broad range of recombinant proteins for secretion and provide potential hypotheses for the root causes of several secretory-associated diseases.
2011-12-01 | GSE27062 | GEO
Project description:The endoplasmic reticulum (ER) lumen provides the proper redox environment for disulfide bond formation, which is essential for the correct folding of proteins entering the secretory pathway and forming membranes. However, the precise mechanisms by which disruptions in protein folding within the ER activate proteostatic mechanisms remain to be fully elucidated. In this study, we demonstrate that in Schizosaccaromyces pombe the antineoplastic agent hydroxyurea (HU) induces a transient perinuclear ER expansion, Bip1 accumulation and the clustering of nuclear pore complexes in a specific region of the nuclear envelope. This striking phenotype is mimicked by diamide (DIA), a specific inducer of thiol stress, and can be prevented or rapidly reversed by dithiothreitol, a reducing agent, suggesting that ER expansion results from disulfide stress. Furthermore, HU or DIA treatments resulted in the accumulation of misfolded proteins in cytoplasmic foci containing Hsp104 disaggregase and Hsp70/Ssa1 chaperones. Our data show that HU impacts redox-dependent protein folding, impairs the secretory pathway and activates specific proteostatic mechanisms in both the ER and the cytoplasm.
2025-10-03 | GSE309439 | GEO
Project description:Understanding the conformational sampling of translation-arrested ribosome nascent chain complexes is key to understand co-translational folding. Up to now, coupling of cysteine oxidation, disulfide bond formation and structure formation in nascent chains has remained elusive. Here, we investigate the eye-lens protein γB-crystallin in the ribosomal exit tunnel. Using mass spectrometry, theoretical simulations, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance and cryo-electron microscopy, we show that thiol groups of cysteine residues undergo S-glutathionylation and S-nitrosylation and form non-native disulfide bonds. Thus, covalent modification chemistry occurs already prior to nascent chain release as the ribosome exit tunnel provides sufficient space even for disulfide bond formation which can guide protein folding.
2020-11-09 | PXD021574 | Pride
Project description:The yeast Komagataella phaffii (syn. Pichia pastoris) is a highly effective and well-established host for the production of recombinant proteins. The redox balance of its secretory pathway, which is multi-organelle dependent, is of high importance for producing secretory proteins. Redox imbalance and oxidative stress an significantly influence protein folding and secretion. Glutathione serves as the main redox buffer of the cell and cellular redox conditions can be assessed through the status of the glutathione redox couple (GSH-GSSG). Previous research often focused on the redox potential of the endoplasmic reticulum (ER), where oxidative protein folding and disulfide bond formation occur. In this study, in vivo measurements of the glutathione redox potential were extended to different subcellular compartments by targeting genetically encoded redox sensitive fluorescent proteins (roGFPs) to the cytosol, ER, mitochondria and peroxisomes. Using these biosensors, the impact of oxygen availability on the redox potentials of the different organelles was investigated in non-producing and producing K. phaffii strains in glucose-limited chemostat cultures. It was found that the transition from normoxic to hypoxic conditions affected the redox potentials of all investigated organelles, while the exposure to hyperoxic conditions did not impact them. Also, as reported previously, hypoxic conditions led to increased recombinant protein secretion. Finally, transcriptome and proteome analyses provided novel insights into the short-term adaptation of the cells from normoxic to hypoxic conditions.
2025-02-10 | GSE277464 | GEO
Project description:N-glycosylation and disulfide bond formation are two essential steps in protein folding, that both take place in the endoplasmic reticulum (ER). These two modifications can influence each other, but the exact timing, as well as the mediators of this important crosstalk, are still not completely elucidated. In previous work, we demonstrated that cells deficient in ER oxidoreductin 1-alpha (ERO1-alpha), a protein disulfide oxidase, are impaired in their ability to secrete Vascular Endothelial Growth Factor-A (VEGF121), a key regulator of vascular homeostasis in health and of metastasis in cancer. Here, to analyze the crosstalk between N-glycosylation and disulfide bond formation in newly synthesized proteins of the ER, we investigated how ERO1-alpha deficit affects glycosylation of VEGF121. We found that reduced ER redox poise imposed by ERO1 deficiency, while slowing down the intracellular formation of disulfide-bonds in VEGF121, promoted the full utilization of its single N-glycosylation consensus site, which lies close to an intra-polypeptide disulfide bridge. Unexpectedly, this hyperglycosylation impairs the kinetics of VEGF121 secretion. Unbiased mass-spectrometric data of VEGF121 immunoprecipitates followed by pathway analysis indicated a stable interaction between VEGF121 and proteins involved in the N-glycosylation in ERO1-alpha knockout, but not wild-type cells. Notably, MAGT1, a thioredoxin-containing protein and part of the post-translational oligosaccharyltransferase complex (OST), was a major hit exclusive to ERO1-deficient cells. We demonstrate that post-translational N-glycosylation VEGF121 is increased under the altered redox poise caused by ERO1 deficit: on the one hand by a reduced rate of formation of its disulfide bridges, and on the other, by the increased trapping potential of MAGT1's thioredoxin domain. These findings have implications for a variety of pathological processes involving altered redox conditions in the ER.
2022-08-29 | PXD033941 | Pride
Project description:Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively-fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1alpha, ERO1beta and PRDX4, compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly-modest delay in disulfide bond formation in secreted proteins but a profound, five-fold lower procollagen 4 hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content, in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H2O2 generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation and a non-canonical form of scurvy. double and triple mutants and wild type
2012-09-15 | E-GEOD-39492 | biostudies-arrayexpress
Project description:Cancer cells exhibit accelerated protein production to accommodate their high metabolic demands. This elevated protein load creates a dependency on endoplasmic reticulum-resident proteins and chaperones, which are required to maintain proteostasis. In this study, we identify the protein disulfide isomerases (PDIs) PDIA1 and PDIA5, which play a critical role in folding of client proteins in the ER, as important regulators of prostate cancer growth and therapy response. PDIA1 and PDIA5 are upregulated in prostate cancer and induced by the androgen receptor (AR) signalling axis. Genetic or pharmacological targeting of PDIA1/PDIA5 causes mitochondrial dysfunction, growth inhibition and apoptosis of prostate cancer cells in vitro and in vivo. Loss of PDIA1/PDIA5 activity leads to AR ubiquitination and degradation, revealing the existence of a feedback loop between these chaperones and the AR pathway. Mechanistically, PDIA1/PDIA5 regulate AR stability by catalysing disulfide bond formation in AR, an activity that requires cysteines 670 and 845 in AR’s ligand-binding domain. Importantly, targeting PDIAs sensitizes prostate cancer cells to the AR antagonist, enzalutamide. Collectively, this study reveals a novel mechanism governing AR proteostasis in prostate cancer and positions PDIA1/5 as viable therapeutic targets.
2025-10-14 | GSE288596 | GEO
Project description:Mycobacterium, including Mycobacterium tuberculosis, the etiological agent of tuberculosis, have a unique cell envelope critical for their survival and antibacterial resistance. The cell envelope's assembly and maintenance influence permeability, making it a key target against multidrug resistant strains. Disulfide bond (SB) formation is crucial for the folding of cell envelope proteins The DSB pathway in mycobacteria includes two enzymes, DsbA and VKOR, required for survival. Using bioinformatics and cystine prfiling proteomics, we identified cell envelope proteins dependent on DSBs. We validated via in vivo alkylation, that key proteins like LamA (MmpS3, PstP, LpqW, and EmbB rely on DSBs for stability. Their stability is disrupted in the Delta VKOR mutant or by VKOR inhibition. Thus targeting DsbA and VKOR systems could compromise both cell division and mycomembrane integrity. These findings emphasize the potential of DSB inhibition as a novel strategy to combat mycobacterial infections.
2025-01-16 | MSV000096877 | MassIVE
Project description:E. coli CyDisCo strain enables a high yield secretion of disulfide bond-containing proteins to the periplasm via Twin-arginine (Tat) pathway. Introducing two exogenous oxidases: the yeast sulfydryl oxidase (Erv1p) and human protein disulfide isomerase (PDI), the CyDisCo strain changes the cytoplasm into an oxidized environment, where the disulfide bonds can efficiently be formed. In this study, we analyzed the proteome changes upon the expression of disulfide bond-containing scFv and the misfolded scFv in the CyDisCo strain. The correctly folded protein is secreted to the periplasm, while the misfolded protein accumulates exclusively in the inclusion body fraction. We observed a high number of significant changes mostly in proteins associated with protein folding and degradation, oxidative stress, membrane transport and integrity.
2019-02-18 | PXD010078 | Pride