| 1. |
- Andersson, Rebecca, et al.
(författare)
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Differential role of cytosolic Hsp70s in longevity assurance and protein quality control
- 2021
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Ingår i: PLoS Genetics. - : Public Library of Science (PLoS). - 1553-7404 .- 1553-7390. ; 17:1
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Tidskriftsartikel (refereegranskat)abstract
- 70 kDa heat shock proteins (Hsp70) are essential chaperones of the protein quality control network; vital for cellular fitness and longevity. The four cytosolic Hsp70's in yeast, Ssa1-4, are thought to be functionally redundant but the absence of Ssa1 and Ssa2 causes a severe reduction in cellular reproduction and accelerates replicative aging. In our efforts to identify which Hsp70 activities are most important for longevity assurance, we systematically investigated the capacity of Ssa4 to carry out the different activities performed by Ssa1/2 by overproducing Ssa4 in cells lacking these Hsp70 chaperones. We found that Ssa4, when overproduced in cells lacking Ssa1/2, rescued growth, mitigated aggregate formation, restored spatial deposition of aggregates into protein inclusions, and promoted protein degradation. In contrast, Ssa4 overproduction in the Hsp70 deficient cells failed to restore the recruitment of the disaggregase Hsp104 to misfolded/aggregated proteins, to fully restore clearance of protein aggregates, and to bring back the formation of the nucleolus-associated aggregation compartment. Exchanging the nucleotide-binding domain of Ssa4 with that of Ssa1 suppressed this 'defect' of Ssa4. Interestingly, Ssa4 overproduction extended the short lifespan of ssa1 Delta ssa2 Delta mutant cells to a lifespan comparable to, or even longer than, wild type cells, demonstrating that Hsp104-dependent aggregate clearance is not a prerequisite for longevity assurance in yeast. Author summary All organisms have proteins that network together to stabilize and protect the cell throughout its lifetime. One of these types of proteins are the Hsp70s (heat shock protein 70). Hsp70 proteins take part in folding other proteins to their functional form, untangling proteins from aggregates, organize aggregates inside the cell and ensure that damaged proteins are destroyed. In this study, we investigated three closely related Hsp70 proteins in yeast; Ssa1, 2 and 4, in an effort to describe the functional difference of Ssa4 compared to Ssa1 and 2 and to answer the question: What types of cellular stress protection are necessary to reach a normal lifespan? We show that Ssa4 can perform many of the same tasks as Ssa1 and 2, but Ssa4 doesn't interact in the same manner as Ssa1 and 2 with other types of proteins. This leads to a delay in removing protein aggregates created after heat stress. Ssa4 also cannot ensure that misfolded proteins aggregate correctly inside the nucleus of the cell. However, this turns out not to be necessary for yeast cells to achieve a full lifespan, which shows us that as long as cells can prevent aggregates from forming in the first place, they can reach a full lifespan.
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| 2. |
- Eisele-Bürger, Anna Maria, et al.
(författare)
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Calmodulin regulates protease versus co-chaperone activity of a metacaspase
- 2023
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Ingår i: Cell Reports. - 2211-1247. ; 42:11
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Tidskriftsartikel (refereegranskat)abstract
- Metacaspases are ancestral homologs of caspases that can either promote cell death or confer cytoprotection. Furthermore, yeast (Saccharomyces cerevisiae) metacaspase Mca1 possesses dual biochemical activity: proteolytic activity causing cell death and cytoprotective, co-chaperone-like activity retarding replicative aging. The molecular mechanism favoring one activity of Mca1 over another remains elusive. Here, we show that this mechanism involves calmodulin binding to the N-terminal pro-domain of Mca1, which prevents its proteolytic activation and promotes co-chaperone-like activity, thus switching from pro-cell death to anti-aging function. The longevity-promoting effect of Mca1 requires the Hsp40 co-chaperone Sis1, which is necessary for Mca1 recruitment to protein aggregates and their clearance. In contrast, proteolytically active Mca1 cleaves Sis1 both in vitro and in vivo, further clarifying molecular mechanism behind a dual role of Mca1 as a cell-death protease versus gerontogene.
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| 3. |
- Eisele, Frederik, et al.
(författare)
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An Hsp90 co-chaperone links protein folding and degradation and is part of a conserved protein quality control
- 2021
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 35:13
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Tidskriftsartikel (refereegranskat)abstract
- In this paper, we show that the essential Hsp90 co-chaperone Sgt1 is a member of a general protein quality control network that links folding and degradation through its participation in the degradation of misfolded proteins both in the cytosol and the endoplasmic reticulum (ER). Sgt1-dependent protein degradation acts in a parallel pathway to the ubiquitin ligase (E3) and ubiquitin chain elongase (E4), Hul5, and overproduction of Hul5 partly suppresses defects in cells with reduced Sgt1 activity. Upon proteostatic stress, Sgt1 accumulates transiently, in an Hsp90- and proteasome-dependent manner, with quality control sites (Q-bodies) of both yeast and human cells that co-localize with Vps13, a protein that creates organelle contact sites. Misfolding disease proteins, such as synphilin-1 involved in Parkinson's disease, are also sequestered to these compartments and require Sgt1 for their clearance. © 2021 The Author(s)
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| 4. |
- Ruiz-Solani, N., et al.
(författare)
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Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates, and delays senescence
- 2023
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Ingår i: Plant Cell. - 1040-4651. ; 35:9, s. 3325-3344
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Tidskriftsartikel (refereegranskat)abstract
- The Arabidopsis metacaspase MC1 is recruited to stress granules upon proteotoxic stress, participates in clearance of pathological protein aggregates, and delays senescence. Stress granules (SGs) are highly conserved cytoplasmic condensates that assemble in response to stress and contribute to maintaining protein homeostasis. These membraneless organelles are dynamic, disassembling once the stress is no longer present. Persistence of SGs due to mutations or chronic stress has been often related to age-dependent protein-misfolding diseases in animals. Here, we find that the metacaspase MC1 is dynamically recruited into SGs upon proteotoxic stress in Arabidopsis (Arabidopsis thaliana). Two predicted disordered regions, the prodomain and the 360 loop, mediate MC1 recruitment to and release from SGs. Importantly, we show that MC1 has the capacity to clear toxic protein aggregates in vivo and in vitro, acting as a disaggregase. Finally, we demonstrate that overexpressing MC1 delays senescence and this phenotype is dependent on the presence of the 360 loop and an intact catalytic domain. Together, our data indicate that MC1 regulates senescence through its recruitment into SGs and this function could potentially be linked to its remarkable protein aggregate-clearing activity.
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| 5. |
- Schneider, Kara, et al.
(författare)
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Using reporters of different misfolded proteins reveals differential strategies in processing protein aggregates
- 2022
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 298:11
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Tidskriftsartikel (refereegranskat)abstract
- The accumulation of misfolded proteins is a hallmark of aging and many neurodegenerative diseases, making it important to understand how the cellular machinery recognizes and processes such proteins. A key question in this respect is whether misfolded proteins are handled in a similar way regardless of their genetic origin. To approach this question, we compared how three different misfolded proteins, guk1-7, gus1-3, and pro3-1, are handled by the cell. We show that all three are nontoxic, even though highly overexpressed, highlighting their usefulness in analyzing the cellular response to misfolding in the absence of severe stress. We found significant differences between the aggregation and disaggregation behavior of the misfolded proteins. Specifically, gus1-3 formed some aggregates that did not efficiently recruit the protein disaggregase Hsp104 and did not colocalize with the other misfolded reporter proteins. Strikingly, while all three misfolded proteins generally coaggregated and colocalized to specific sites in the cell, disaggregation was notably different; the rate of aggregate clearance of pro3-1 was faster than that of the other misfolded proteins, and its clearance rate was not hindered when pro3-1 colocalized with a slowly resolved misfolded protein. Finally, we observed using super-resolution light microscopy as well as immunogold labeling EM in which both showed an even distribution of the different misfolded proteins within an inclusion, suggesting that misfolding characteristics and remodeling, rather than spatial compartmentalization, allows for differential clearance of these misfolding reporters residing in the same inclusion. Taken together, our results highlight how properties of misfolded proteins can significantly affect processing.
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| 6. |
- Babazadeh, Roja, et al.
(författare)
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Syntaxin 5 Is Required for the Formation and Clearance of Protein Inclusions during Proteostatic Stress
- 2019
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 28:8
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Tidskriftsartikel (refereegranskat)abstract
- Spatial sorting to discrete quality control sites in the cell is a process harnessing the toxicity of aberrant proteins. We show that the yeast t-snare phosphoprotein syntaxin5 (Sed5) acts as a key factor in mitigating proteotoxicity and the spatial deposition and clearance of IPOD (insoluble protein deposit) inclusions associates with the disaggregase Hsp104. Sed5 phosphorylation promotes dynamic movement of COPII-associated Hsp104 and boosts disaggregation by favoring anterograde ER-to-Golgi trafficking. Hsp104-associated aggregates co-localize with Sed5 as well as components of the ER, trans Golgi network, and endocytic vesicles, transiently during proteostatic stress, explaining mechanistically how misfolded and aggregated proteins formed at the vicinity of the ER can hitchhike toward vacuolar IPOD sites. Many inclusions become associated with mitochondria in a HOPS/vCLAMP-dependent manner and co-localize with Vps39 (HOPS/vCLAMP) and Vps13, which are proteins providing contacts between vacuole and mitochondria. Both Vps39 and Vps13 are required also for efficient Sed5-dependent clearance of aggregates.
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| 7. |
- Chen, Xin, 1980, et al.
(författare)
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FMN reduces Amyloid-beta toxicity in yeast by regulating redox status and cellular metabolism
- 2020
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer's disease (AD) is defined by progressive neurodegeneration, with oligomerization and aggregation of amyloid-beta peptides (A beta) playing a pivotal role in its pathogenesis. In recent years, the yeast Saccharomyces cerevisiae has been successfully used to clarify the roles of different human proteins involved in neurodegeneration. Here, we report a genome-wide synthetic genetic interaction array to identify toxicity modifiers of A beta 42, using yeast as the model organism. We find that FMN1, the gene encoding riboflavin kinase, and its metabolic product flavin mononucleotide (FMN) reduce A beta 42 toxicity. Classic experimental analyses combined with RNAseq show the effects of FMN supplementation to include reducing misfolded protein load, altering cellular metabolism, increasing NADH/(NADH+NAD(+)) and NADPH/(NADPH+NADP(+)) ratios and increasing resistance to oxidative stress. Additionally, FMN supplementation modifies Htt103QP toxicity and alpha-synuclein toxicity in the humanized yeast. Our findings offer insights for reducing cytotoxicity of A beta 42, and potentially other misfolded proteins, via FMN-dependent cellular pathways.Saccharomyces cerevisiae is a model organism to study proteins involved in neurodegeneration. Here, the authors performed a yeast genome-wide synthetic genetic interaction array (SGA) to screen for toxicity modifiers of A beta 42 and identify riboflavin kinase and its metabolic product flavin mononucleotide as modulators that alleviate cellular A beta 42 toxicity, which is supported by further experimental analyses.
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| 8. |
- Guerra, Lina, et al.
(författare)
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Bacterial genotoxin triggers FEN1-dependent RhoA activation, cytoskeleton remodeling and cell survival
- 2011
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 124:16, s. 2735-2742
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Tidskriftsartikel (refereegranskat)abstract
- The DNA damage response triggered by bacterial cytolethal distending toxins (CDTs) is associated with activation of the actin-regulating protein RhoA and phosphorylation of the downstream-regulated mitogen-activated protein kinase (MAPK) p38, which promotes the survival of intoxicated (i.e. cells exposed to a bacterial toxin) cells. To identify the effectors of this CDT-induced survival response, we screened a library of 4492 Saccharomyces cerevisiae mutants that carry deletions in nonessential genes for reduced growth following inducible expression of CdtB. We identified 78 genes whose deletion confers hypersensitivity to toxin. Bioinformatics analysis revealed that DNA repair and endocytosis were the two most overrepresented signaling pathways. Among the human orthologs present in our data set, FEN1 and TSG101 regulate DNA repair and endocytosis, respectively, and also share common interacting partners with RhoA. We further demonstrate that FEN1, but not TSG101, regulates cell survival, MAPK p38 phosphorylation, RhoA activation and actin cytoskeleton reorganization in response to DNA damage. Our data reveal a previously unrecognized crosstalk between DNA damage and cytoskeleton dynamics in the regulation of cell survival, and might provide new insights on the role of chronic bacteria infection in carcinogenesis.
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| 9. |
- Öling, David, et al.
(författare)
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Opposing roles of Ubp3-dependent deubiquitination regulate replicative life span and heat resistance
- 2014
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Ingår i: Embo Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 33:7, s. 747-761
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Tidskriftsartikel (refereegranskat)abstract
- Abstract The interplay between molecular chaperones, ubiquitin/deubiquitinating enzymes, and proteasomes is a critical element in protein homeostasis. Among these factors, the conserved deubiquitinase, Ubp3, has the interesting ability, when overproduced, to suppress the requirement for the major cytosolic Hsp70 chaperones. Here, we show that Ubp3 overproduction counteracts deficiency of Hsp70s by the removal of damaged proteins deposited in inclusion bodies (JUNQ) during both aging and heat stress. Consistent with this, Ubp3 destabilized, deubiquitinated, and diminished the toxicity of the JUNQ-associated misfolded protein Ubc9(ts) in a proteasome-dependent manner. In contrast, another misfolded model protein, increment ssCPY*, was stabilized by Ubp3-dependent deubiquitination demonstrating a dual role for Ubp3, saving or destroying aberrant protein species depending on the stage at which the damaged protein is committed for destruction. We present genetic evidence for the former of these activities being key to Ubp3-dependent suppression of heat sensitivity in Hsp70-deficient cells, whereas protein destruction suppresses accelerated aging. We discuss the data in view of how heat stress and aging might elicit differential damage and challenges on the protein homeostasis network. The conserved deubiquitinase Ubp3 regulates both heat stress resistance and replicative life span in yeast through dual roles in cellular protein homeostasis. Ubp3 aids removal of damaged proteins either by stabilizing them in a rescue pathway or by accelerating their proteasome-mediated degradation. Ubp3 promotes the removal of damaged proteins deposited in JUNQ inclusion bodies. Misfolded model proteins are either stabilized or degraded by Ubp3 overproduction. Ubp3 suppresses heat sensitivity in Hsp70-deficient cells by diverting damaged proteins from destruction. Ubp3-mediated proteasomal degradation of cytotoxic proteins suppresses accelerated replicative aging.
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