Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Controlling protein homeostasis through regulation of Heat shock factor 1
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2019. , p. 60
Keywords [en]
stress, protein homeostasis, molecular chaperones, heat shock response, Hsf1, Hsp70, Saccharomyces cerevisiae
National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-167081ISBN: 978-91-7797-688-2 (print)ISBN: 978-91-7797-708-7 (electronic)OAI: oai:DiVA.org:su-167081DiVA, id: diva2:1298604
Public defence
2019-05-21, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 4: Manuscript.

Available from: 2019-04-25 Created: 2019-03-25 Last updated: 2019-04-11Bibliographically approved
List of papers
1. Cytoplasmic protein misfolding titrates nuclear Hsp70 to unleash active Hsf1
Open this publication in new window or tab >>Cytoplasmic protein misfolding titrates nuclear Hsp70 to unleash active Hsf1
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-167079 (URN)
Available from: 2019-03-15 Created: 2019-03-15 Last updated: 2019-03-25Bibliographically approved
2. Cytosolic splice isoform of Hsp70 nucleotide exchange factor Fes1 is required for the degradation of misfolded proteins in yeast
Open this publication in new window or tab >>Cytosolic splice isoform of Hsp70 nucleotide exchange factor Fes1 is required for the degradation of misfolded proteins in yeast
Show others...
2016 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 27, no 8, p. 1210-1219Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Cell Biology
Research subject
Cell Biology; Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-129116 (URN)10.1091/mbc.E15-10-0697 (DOI)000375753600006 ()26912797 (PubMedID)
Available from: 2016-04-14 Created: 2016-04-14 Last updated: 2019-03-25Bibliographically approved
3. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae
Open this publication in new window or tab >>Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae
2016 (English)In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 33, no 5, p. 191-200Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
bioluminescence, reporter, luciferase, gene expression, protein stability
National Category
Biological Sciences Environmental Biotechnology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-131221 (URN)10.1002/yea.3155 (DOI)000375782300003 ()
Available from: 2016-06-14 Created: 2016-06-14 Last updated: 2019-03-25Bibliographically approved
4. A co-translational folding reporter to monitor proteostasis
Open this publication in new window or tab >>A co-translational folding reporter to monitor proteostasis
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-167080 (URN)
Available from: 2019-03-15 Created: 2019-03-15 Last updated: 2019-03-25Bibliographically approved
5. Mitochondrial Translation Efficiency Controls Cytoplasmic Protein Homeostasis
Open this publication in new window or tab >>Mitochondrial Translation Efficiency Controls Cytoplasmic Protein Homeostasis
Show others...
2018 (English)In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 27, no 6, p. 1309-1322Article in journal (Refereed) Published
Abstract [en]

Cellular proteostasis ismaintained via the coordinated synthesis, maintenance, and breakdown of proteins in the cytosol and organelles. While biogenesis of the mitochondrial membrane complexes that execute oxidative phosphorylation depends on cytoplasmic translation, it is unknown how translation within mitochondria impacts cytoplasmic proteostasis and nuclear gene expression. Here we have analyzed the effects of mutations in the highly conserved accuracy center of the yeast mitoribosome. Decreased accuracy of mitochondrial translation shortened chronological lifespan, impaired management of cytosolic protein aggregates, and elicited a general transcriptional stress response. In striking contrast, increased accuracy extended lifespan, improved cytosolic aggregate clearance, and suppressed a normally stress-induced, Msn2/4-dependent interor-ganellar proteostasis transcription program (IPTP) that regulates genes important for mitochondrial proteostasis. Collectively, the data demonstrate that cytosolic protein homeostasis and nuclear stress signaling are controlled by mitochondrial translation efficiency in an inter-connected organelle quality control network that determines cellular lifespan.

National Category
Biological Sciences
Research subject
Biochemistry; Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-157770 (URN)10.1016/j.cmet.2018.04.011 (DOI)000434480000016 ()29754951 (PubMedID)
Available from: 2018-06-25 Created: 2018-06-25 Last updated: 2019-03-29Bibliographically approved

Open Access in DiVA

Controlling protein homeostasis through regulation of Heat shock factor 1(1280 kB)26 downloads
File information
File name FULLTEXT01.pdfFile size 1280 kBChecksum SHA-512
dd9621267eccd1f3149dcd7441f47bcb311a684b94a0c7b981a60199719cdca4e876cdad191c4ae0d0c8e112edc8dcb5872ad4ca91a5c989cda6735b3d4de34d
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Masser, Anna E.
By organisation
Department of Molecular Biosciences, The Wenner-Gren Institute
Biochemistry and Molecular BiologyCell Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 26 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 264 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf