| 1. |
- Andreasson, Claes, et al.
(författare)
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Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:9, s. e74207-
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Tidskriftsartikel (refereegranskat)abstract
- Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors.
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| 2. |
- Ciccarelli, Michela, et al.
(författare)
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Genetic inactivation of essential HSF1 reveals an isolated transcriptional stress response selectively induced by protein misfolding
- 2023
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 34:11
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Tidskriftsartikel (refereegranskat)abstract
- Heat Shock Factor 1 (Hsf1) in yeast drives the basal transcription of key proteostasis factors and its activity is induced as part of the core heat shock response. Exploring Hsf1 specific functions has been challenging due to the essential nature of the HSF1 gene and the extensive overlap of target promoters with environmental stress response (ESR) transcription factors Msn2 and Msn4 (Msn2/4). In this study, we constructed a viable hsf1 increment strain by replacing the HSF1 open reading frame with genes that constitutively express Hsp40, Hsp70, and Hsp90 from Hsf1-independent promoters. Phenotypic analysis showed that the hsf1 increment strain grows slowly, is sensitive to heat as well as protein misfolding and accumulates protein aggregates. Transcriptome analysis revealed that the transcriptional response to protein misfolding induced by azetidine-2-carboxylic acid is fully dependent on Hsf1. In contrast, the hsf1 increment strain responded to heat shock through the ESR. Following HS, Hsf1 and Msn2/4 showed functional compensatory induction with stronger activation of the remaining stress pathway when the other branch was inactivated. Thus, we provide a long-overdue genetic test of the function of Hsf1 in yeast using the novel hsf1 increment construct. Our data highlight that the accumulation of misfolded proteins is uniquely sensed by Hsf1-Hsp70 chaperone titration inducing a highly selective transcriptional stress response.
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| 3. |
- Gowda, Naveen Kumar Chandappa, et al.
(författare)
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Cytosolic splice isoform of Hsp70 nucleotide exchange factor Fes1 is required for the degradation of misfolded proteins in yeast
- 2016
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 27:8, s. 1210-1219
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Tidskriftsartikel (refereegranskat)abstract
- Cells maintain proteostasis by selectively recognizing and targeting misfolded proteins for degradation. In Saccharomyces cerevisiae, the Hsp70 nucleotide exchange factor Fes1 is essential for the degradation of chaperone-associated misfolded proteins by the ubiquitin-proteasome system. Here we show that the FES1 transcript undergoes unique 3' alternative splicing that results in two equally active isoforms with alternative C-termini, Fes1L and Fes1S. Fes1L is actively targeted to the nucleus and represents the first identified nuclear Hsp70 nucleotide exchange factor. In contrast, Fes1S localizes to the cytosol and is essential to maintain proteostasis. In the absence of Fes1S, the heat-shock response is constitutively induced at normally non-stressful conditions. Moreover, cells display severe growth defects when elevated temperatures, amino acid analogues or the ectopic expression of misfolded proteins, induce protein misfolding. Importantly, misfolded proteins are not targeted for degradation by the ubiquitin-proteasome system. These observations support the notion that cytosolic Fes1S maintains proteostasis by supporting the removal of toxic misfolded proteins by proteasomal degradation. This study provides key findings for the understanding of the organization of protein quality control mechanisms in the cytosol and nucleus.
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| 4. |
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| 5. |
- Masser, Anna E., 1987-
(författare)
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Controlling protein homeostasis through regulation of Heat shock factor 1
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In order to thrive in a changing environment all organisms need to ensure protein homeostasis (proteostasis). Proteostasis is ensured by the proteostasis system that monitors the folding status of the proteome and regulates cell physiology and gene expression to counteract any perturbations. An increased burden on the proteostasis system activates Heat shock factor 1 (Hsf1) to induce transcription of the heat shock response (HSR), a transiently induced transcriptional program including core proteostasis genes, importantly those encoding the Hsp70 class of molecular chaperones. The HSR assists cells in counteracting the harmful effects of protein folding stress and restoring proteostasis. The work presented in this thesis is based on experiments with the Saccharomyces cerevisiae (yeast) model with the overall goal of deciphering how Hsp70 detects and impacts on perturbations of cellular proteostasis and controls Hsf1 activity.In Study I we describe the fundamental mechanism by which Hsp70 maintains Hsf1 in its latent state by controlling its ability to bind DNA. We found that Hsf1 and unfolded proteins directly compete for binding to the Hsp70 substrate-binding domain. During heat shock the pool of unfolded proteins mainly consist of misfolded, newly synthesized proteins. Severe out-titration of Hsp70 by misfolded substrates resulted in unrestrained Hsf1 activity inducing a previously uncharacterized genetic hyper-stress program. More insight into regulation of Hsp70 availability was gained in Study II where the two splice isoforms of the Hsp70 nucleotide exchange factor Fes1 were characterized. We found that the cytosolic splice isoform Fes1S is crucial to release unfolded proteins from Hsp70 and that impaired release results in strong Hsf1 activation.In Study III we developed methodology to easily measure the rapid changes in Hsf1 activity upon proteostatic perturbations and to monitor protein turnover using the novel bioluminescent reporter NanoLuc optimized for yeast expression (yNluc). In Study IV we report that yNluc also functions as an in vivo reporter that detects severe perturbations of de novo protein folding by its failure to fold to an active conformation under such conditions.Finally, in Study V we investigated how organellar proteostasis impacts on the availability of cytosolic Hsp70. We found that a lowered mitochondrial proteostatic load as a result of high translation accuracy extended lifespan and improved cytosolic proteostasis capacity, evidenced by more rapid stress recovery and less sensitivity to toxic misfolded proteins. In contrast, lowered mitochondrial translation accuracy decreased lifespan and impaired management of cytosolic protein aggregates as well as elicited a general transcriptional stress response.Taken together, the findings presented in this thesis advance our understanding of how the regulatory mechanisms of the proteostasis system function. Furthermore, they provide novel methodology that will facilitate future studies to improve our understanding how cells integrate internal and external stress cues to control proteostasis.
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| 6. |
- Masser, Anna E., et al.
(författare)
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Cytoplasmic protein misfolding titrates Hsp70 to activate nuclear Hsf1
- 2019
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Ingår i: eLIFE. - 2050-084X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Hsf1 is an ancient transcription factor that responds to protein folding stress by inducing the heat-shock response (HSR) that restore perturbed proteostasis. Hsp70 chaperones negatively regulate the activity of Hsf1 via stress-responsive mechanisms that are poorly understood. Here, we have reconstituted budding yeast Hsf1-Hsp70 activation complexes and find that surplus Hsp70 inhibits Hsf1 DNA-binding activity. Hsp70 binds Hsf1 via its canonical substrate binding domain and Hsp70 regulates Hsf1 DNA-binding activity. During heat shock, Hsp70 is out-titrated by misfolded proteins derived from ongoing translation in the cytosol. Pushing the boundaries of the regulatory system unveils a genetic hyperstress program that is triggered by proteostasis collapse and involves an enlarged Hsf1 regulon. The findings demonstrate how an apparently simple chaperone-titration mechanism produces diversified transcriptional output in response to distinct stress loads.
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| 7. |
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| 8. |
- Masser, Anna E., et al.
(författare)
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Hsf1 on a leash - controlling the heat shock response by chaperone titration
- 2020
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 396:1
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Forskningsöversikt (refereegranskat)abstract
- Heat shock factor 1 (Hsf1) is an ancient transcription factor that monitors protein homeostasis (proteostasis) and counteracts disturbances by triggering a transcriptional programme known as the heat shock response (HSR). The HSR is transiently activated and upregulates the expression of core proteostasis genes, including chaperones. Dysregulation of Hsf1 and its target genes are associated with disease; cancer cells rely on a constitutively active Hsf1 to promote rapid growth and malignancy, whereas Hsf1 hypoactivation in neurodegenerative disorders results in formation of toxic aggregates. These central but opposing roles highlight the importance of understanding the underlying molecular mechanisms that control Hsf1 activity. According to current understanding, Hsf1 is maintained latent by chaperone interactions but proteostasis perturbations titrate chaperone availability as a result of chaperone sequestration by misfolded proteins. Liberated and activated Hsf1 triggers a negative feedback loop by inducing the expression of key chaperones. Until recently, Hsp90 has been highlighted as the central negative regulator of Hsf1 activity. In this review, we focus on recent advances regarding how the Hsp70 chaperone controls Hsf1 activity and in addition summarise several additional layers of activity control.
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| 9. |
- Masser, Anna E.
(författare)
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Induction of the heat-shock response by misfolded proteins
- 2016
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cells respond to stress by transcriptional changes intended for stress survival and adaption to further stresses. Heat shock is an intensively studied stress condition that induces in a plethora of physiological changes including increased protein misfolding. The heat-shock response (HSR) is an evolutionary conserved transcriptional response to elevated temperatures. In yeast, the HSR is governed by the transcription factors Hsf1 and Msn2/4. The HSR counteracts protein-folding stress by upregulating the expression of proteins involved in the proteostasis network including molecular chaperones and proteins involved in proteasomal degradation. Msn2/4 activity is controlled through complex signal transduction pathways involving Protein Kinase A (PKA). Hsf1 is thought to be regulated by chaperone titration, a model adapted from bacterial stress-sensing, but the mechanisms underlying its regulation are poorly understood. Here we investigate the influence that accumulated misfolded proteins have on the HSR. In study I, we evaluate a novel bioluminescent reporter system, Nanoluciferase (Nluc), for studies of the HSR in yeast. We find that codon-optimized Nluc faithfully reports on the rapid changes associated with gene induction during stress. In study II, we investigate the Hsp70 nucleotide exchange factor Fes1 and find that its nuclear splice-isoform Fes1S is involved in the degradation of misfolded proteins and a negative regulator of the HSR. In study III, we build on study II and make use of fes1Δ cells as a model for cells that have accumulated misfolded proteins. We find that accumulated misfolded proteins specifically activate Hsf1 and not Msn2/4. Interestingly, accumulated misfolded proteins potently modify both the amplitude and range of the HSR. Our findings lay the groundwork for further studies on the mechanisms that make accumulated proteins rewire cellular stress responses and Hsf1 activation.
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| 10. |
- Masser, Anna E., et al.
(författare)
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Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae
- 2016
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Ingår i: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 33:5, s. 191-200
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Tidskriftsartikel (refereegranskat)abstract
- Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo (R) is compatible with detection of yNluc bioluminescence in < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat-shock promoter (PCYC1-HSE), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate.
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