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Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism
Department of Biology, University of Konstanz, Germany. (Centre for Blood Research, University of British Columbia, Vancouver, Canada)
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Department of Biology, University of Konstanz, Germany.
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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.

Place, publisher, year, edition, pages
2011. Vol. 435, no 3, 733-742 p.
Identifiers
URN: urn:nbn:se:umu:diva-40402DOI: 10.1042/BJ20102131PubMedID: 21332448OAI: oai:DiVA.org:umu-40402DiVA: diva2:402946
Note
Published as BJ Immediate Publication 21 February 2011 Available from: 2011-03-10 Created: 2011-02-22 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Stress response in the cyanobacterium Synechocystis sp. PCC 6803
Open this publication in new window or tab >>Stress response in the cyanobacterium Synechocystis sp. PCC 6803
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Adaptation to environmental changes is important for the survival of living organisms. Under extreme abiotic conditions, organic molecules (such as lipids, proteins and nucleic acids) are prone to damage. Under these conditions stress response mechanisms are activated, either to prevent the source of damage or to promote the rapid turnover of damaged molecules. Like all photoautotrophic organisms, cyanobacteria are sensitive to high light intensity and oxidative stress, which induces damage to the photosynthetic apparatus. My thesis is divided in two subjects related to particular stress responses in the cyanobacterium Synechocystis sp. PCC 6803: 1) the role of Deg/HtrA proteases and 2) investigations on the small CAB-like proteins.

Deg/HtrA proteases are ATP-independent serine endopeptidases with a characteristic C-terminal PDZ domain. These proteases are largely dispersed among living organisms, with many different functions, mostly involved in protein quality control. The genome of Synechocystis sp. PCC 6803 contains three genes coding for Deg/HtrA proteases: HtrA, HhoA and HhoB. These proteases are essential for survival under high light and heat stress, and may overlap in their functions. During my Ph.D. studies I focused on the identification of the precise localization of the Deg/HtrA proteases in the cyanobacterial cell, analyzed the biochemical properties of recombinant Synechocystis Deg/HtrA proteases in vitro and adopted proteomic and metabolomic approaches to study the physiological importance of these proteases. My data show that Deg/HtrA proteases are not only important in stress response mechanisms under adverse conditions, but are also involved in the stabilization of important physiological processes, such as polysaccharides biosynthesis and peptidoglycan turnover.

The small CAB-like proteins (SCPs) belong to the light harvesting-like family of stress induced proteins and are thought to be involved in the photoprotection of the photosynthetic apparatus. Five small CAB-like proteins where identified in Synechocystis sp. PCC 6803 (ScpA-E). In my studies I identified another relative to the SCPs, LilA, which I found to be co-transcribed with ScpD. I also focused on the subcellular localization and identification of potential interaction partners of the SCPs.

Place, publisher, year, edition, pages
Umeå: Department of Chemistry, Umeå University, 2011. 49 p.
Keyword
Cyanobacteria, High light, heat stress, ROS, Photosynthesis, Photosystem II, Deg/HtrA proteases, small CAB-like proteins, Refraction-2D™, MALDI-TOF, 2D-gel, GC-MS, PCA, OPLS-DA, EPS, S-layer
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-43086 (URN)978-91-7459-201-6 (ISBN)
Public defence
2011-05-13, KBC-huset, KB3B1, Umeå University, Umeå, Sweden, 13:00 (English)
Opponent
Supervisors
Available from: 2011-04-20 Created: 2011-04-18 Last updated: 2011-04-19Bibliographically approved
2. Deg/HtrA proteases of the cyanobacterium Synechocystis sp. PCC 6803: from biochemical characterization to their physiological functions
Open this publication in new window or tab >>Deg/HtrA proteases of the cyanobacterium Synechocystis sp. PCC 6803: from biochemical characterization to their physiological functions
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
Deg, HtrA, protease, chaperone, Synechocystis sp. PCC 6903, biochemical characterization, physiological function, proteomics, structure
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-99719 (URN)978-91-7601-223-9 (ISBN)
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

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