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Deg/HtrA proteases of the cyanobacterium Synechocystis sp. PCC 6803: from biochemical characterization to their physiological functions
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The family of Deg/HtrA proteases is present in a wide range of organisms from bacteria, archaea to eukaryota. These ATP-independent serine endopeptidases play key roles in the cellular protein quality control. The cyanobacterium Synechocystis sp. PCC 6803, a model organism for studies on photosynthesis, metabolism and renewable energy, contains three Deg proteases known as HhoA, HhoB and HtrA. The three proteases are important for survival in stress conditions, such as high light or temperature.

In my work the biochemical characteristics of each protease were revealed in vitro and in vivo. In vitro studies performed using recombinant Synechocystis Deg proteases allowed conclusions about their oligomerization states, proteolytic activities and tertiary structure. The in vivo studies addressed their sub-cellular localization, expression and physiological importance by comparing wild-type Synechocystis cells with the three single mutants lacking one of the Deg proteases.

HhoA seems to be involved in the cytoplasmic protein quality control. This protease is regulated post-transcriptional and post-translational: oligomerization, pH and/or cation-binding are some of the important factors to stimulate its proteolytic activity. Instead HhoB acts on periplasmic proteins and seems to be important for the transportation/secretion of proteins. While it has low proteolytic capacity, it may act as a chaperone. The stress-induced HtrA functions in the cellular tolerance against photosynthetic stress; additionally it might act as a protease partner of HhoB, generating a protease/chaperone complex.

The results presented in this thesis lay the foundation for a better understanding of the dynamic protein quality control in cyanobacteria, which is undoubtedly important for various cellular metabolic pathways.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2015. , 35 p.
Keyword [en]
Deg, HtrA, protease, chaperone, Synechocystis sp. PCC 6903, biochemical characterization, physiological function, proteomics, structure
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-99719ISBN: 978-91-7601-223-9 (print)OAI: oai:DiVA.org:umu-99719DiVA: diva2:787965
Public defence
2015-03-16, KB3A9, KBC building, Umeå University, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2015-02-23 Created: 2015-02-12 Last updated: 2015-03-06Bibliographically approved
List of papers
1. Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism
Open this publication in new window or tab >>Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism
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2011 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 435, no 3, 733-742 p.Article in journal (Refereed) Published
Abstract [en]

Cyanobacteria require efficient protein quality control mechanisms to survive under dynamic, often stressful environmental conditions. It was reported that three serine proteases, HtrA, HhoA and HhoB are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. Here we show that all three proteases can degrade unfolded model substrates, but differ in respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg2+ and Ca2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison to each other and to the well-studied Escherichia coli orthologues DegP and DegS. Deletion of the PDZ domain decreased, but not abolished proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterisation of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant stress resistance functions in Synechocystis sp. PCC 6803.

Identifiers
urn:nbn:se:umu:diva-40402 (URN)10.1042/BJ20102131 (DOI)21332448 (PubMedID)
Note
Published as BJ Immediate Publication 21 February 2011 Available from: 2011-03-10 Created: 2011-02-22 Last updated: 2017-12-11Bibliographically approved
2. Degradation of PsbO by the deg protease HhoA is thioredoxin dependent
Open this publication in new window or tab >>Degradation of PsbO by the deg protease HhoA is thioredoxin dependent
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 9, e45713- p.Article in journal (Refereed) Published
Abstract [en]

The widely distributed members of the Deg/HtrA protease family play an important role in the proteolysis of misfolded and damaged proteins. Here we show that the Deg protease rHhoA is able to degrade PsbO, the extrinsic protein of the Photosystem II (PSII) oxygen-evolving complex in Synechocystis sp. PCC 6803 and in spinach. PsbO is known to be stable in its oxidized form, but after reduction by thioredoxin it became a substrate for recombinant HhoA (rHhoA). rHhoA cleaved reduced eukaryotic (specifically, spinach) PsbO at defined sites and created distinct PsbO fragments that were not further degraded. As for the corresponding prokaryotic substrate (reduced PsbO of Synechocystis sp. PCC 6803), no PsbO fragments were observed. Assembly to PSII protected PsbO from degradation. For Synechocystis sp. PCC 6803, our results show that HhoA, HhoB, and HtrA are localized in the periplasma and/or at the thylakoid membrane. In agreement with the idea that PsbO could be a physiological substrate for Deg proteases, part of the cellular fraction of the three Deg proteases of Synechocystis sp. PCC 6803 (HhoA, HhoB, and HtrA) was detected in the PSII-enriched membrane fraction.  

Place, publisher, year, edition, pages
San Francisco: Public Library of Science, 2012
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-58436 (URN)10.1371/journal.pone.0045713 (DOI)000309388400123 ()23029195 (PubMedID)
Available from: 2012-08-30 Created: 2012-08-30 Last updated: 2017-12-07Bibliographically approved
3. Proteomic approaches to identify substrates of the three Deg/HtrA proteases of the cyanobacterium Synechocystis sp. PCC 6803
Open this publication in new window or tab >>Proteomic approaches to identify substrates of the three Deg/HtrA proteases of the cyanobacterium Synechocystis sp. PCC 6803
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(English)Article in journal (Refereed) Submitted
National Category
Organic Chemistry
Identifiers
urn:nbn:se:umu:diva-99838 (URN)
Available from: 2015-02-13 Created: 2015-02-13 Last updated: 2015-02-16
4. Structure of the HhoA protease from Synechocystis sp. PCC 6803
Open this publication in new window or tab >>Structure of the HhoA protease from Synechocystis sp. PCC 6803
(English)Manuscript (preprint) (Other academic)
National Category
Organic Chemistry
Identifiers
urn:nbn:se:umu:diva-99839 (URN)
Available from: 2015-02-13 Created: 2015-02-13 Last updated: 2015-02-16

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Lâm, Xuân Tâm

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