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
Regulation of cellular Hsp70: Proteostasis and aggregate management
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins have to be folded to their native structures to be functionally expressed. Misfolded proteins are proteotoxic and negatively impact on cellular fitness. To maintain the proteome functional proteins are under the constant surveillance of dedicated molecular chaperones that perform protein quality control (PQC). Using the model organism yeast Saccharomyces cerevisiae this thesis investigates the molecular mechanisms that cells employ to maintain protein homeostasis (proteostasis). In Study I the role of the molecular chaperone Hsp110 in the disentanglement and reactivation of aggregated proteins was investigated. We found that Hsp110 is essential for cellular protein disaggregation driven by the molecular chaperones Hsp40, Hsp70 and Hsp104 and characterized its involvement via regulation of Hsp70 ATPase activity as a nucleotide exchange factor. In Study II we found out that Hsp110 undergoes translational frameshifting during its expression resulting in a nuclear targeting. Nuclear Hsp110 interacts with Hsp70 and reprograms the proteostasis system to better deal with stress and to confer longevity. Study III describes regulation of Hsp70 function in PQC by the nucleotide exchange factor Fes1. We found that rare alternative splicing regulates Fes1 subcellular localization in the cytosol and nucleus and that the cytosolic isoform has a key role in PQC. In Study IV we have revealed the molecular mechanism that Fes1 employ in PQC. We show that Fes1 carries a specialized release domain (RD) that ensures the efficient release of protein substrates from Hsp70, explaining how Fes1 maintains the Hsp70-chaperone system clear of persistent misfolded proteins. In Study V we report on the use of a novel bioluminescent reporter (Nanoluc) for use in yeast to measure the gene expression and protein levels. In summary, this thesis contributes to the molecular understanding of chaperone-dependent PQC mechanisms both at the level of individual components as well as how they interact to ensure proteostasis.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2017.
National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-148410ISBN: 978-91-7649-998-6 (print)ISBN: 978-91-7649-999-3 (electronic)OAI: oai:DiVA.org:su-148410DiVA, id: diva2:1152364
Public defence
2017-12-08, E306, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, 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 2: Manuscript. Paper 4: Manuscript.

Available from: 2017-11-15 Created: 2017-10-24 Last updated: 2017-11-09Bibliographically approved
List of papers
1. Coordinated Hsp110 and Hsp104 Activities Power Protein Disaggregation in Saccharomyces cerevisiae
Open this publication in new window or tab >>Coordinated Hsp110 and Hsp104 Activities Power Protein Disaggregation in Saccharomyces cerevisiae
2017 (English)In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 37, no 11, article id e00027-17Article in journal (Refereed) Published
Abstract [en]

Protein aggregation is intimately associated with cellular stress and is accelerated during aging, disease, and cellular dysfunction. Yeast cells rely on the ATP-consuming chaperone Hsp104 to disaggregate proteins together with Hsp70. Hsp110s are ancient and abundant chaperones that form complexes with Hsp70. Here we provide in vivo data showing that the Saccharomyces cerevisiae Hsp110s Sse1 and Sse2 are essential for Hsp104-dependent protein disaggregation. Following heat shock, complexes of Hsp110 and Hsp70 are recruited to protein aggregates and function together with Hsp104 in the disaggregation process. In the absence of Hsp110, targeting of Hsp70 and Hsp104 to the aggregates is impaired, and the residual Hsp104 that still reaches the aggregates fails to disaggregate. Thus, coordinated activities of both Hsp104 and Hsp110 are required to reactivate aggregated proteins. These findings have important implications for the understanding of how eukaryotic cells manage misfolded and amyloid proteins.

Keywords
Hsp110, Sse1, Sse2, Hsp104, Ssa1, protein disaggregation, chaperone, protein misfolding, heat shock, protein folding, protein quality control, stress proteins
National Category
Biological Sciences
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-144802 (URN)10.1128/MCB.00027-17 (DOI)000401345800001 ()
Available from: 2017-07-12 Created: 2017-07-12 Last updated: 2017-10-24Bibliographically approved
2. Nuclear targeting of Hsp110 modifies the proteostasis system by mobilizing latent Hsp70 chaperones
Open this publication in new window or tab >>Nuclear targeting of Hsp110 modifies the proteostasis system by mobilizing latent Hsp70 chaperones
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-148368 (URN)
Available from: 2017-10-23 Created: 2017-10-23 Last updated: 2017-10-30Bibliographically approved
3. 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: 2017-10-24Bibliographically approved
4. Substrate-mimicking domain of nucleotide-exchange factor Fes1/HspBP1 ensures efficient release of persistent substrates from Hsp70
Open this publication in new window or tab >>Substrate-mimicking domain of nucleotide-exchange factor Fes1/HspBP1 ensures efficient release of persistent substrates from Hsp70
Show others...
(English)Manuscript (preprint) (Other academic)
Keywords
Hsp70, Fes1, protein quality control
National Category
Cell Biology
Research subject
Cell Biology; Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-129327 (URN)
Available from: 2016-04-20 Created: 2016-04-20 Last updated: 2017-10-30Bibliographically approved
5. 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: 2017-10-24Bibliographically approved

Open Access in DiVA

Regulation of cellular Hsp70: Proteostasis and aggregate management(711 kB)31 downloads
File information
File name FULLTEXT01.pdfFile size 711 kBChecksum SHA-512
8cbeaf05b8b342ca137ea5cd05fd72577119023c0f6207b7097f3aee283842957819f70ce7f982e18daa8748159d7ef01b3af810471454f1c8b957f2a3891fb4
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Kaimal, Jayasankar Mohanakrishnan
By organisation
Department of Molecular Biosciences, The Wenner-Gren Institute
Biochemistry and Molecular BiologyCell Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 31 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: 304 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