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Proteomic approaches to identify substrates of the three Deg/HtrA proteases of the cyanobacterium Synechocystis sp. PCC 6803
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
Vise andre og tillknytning
2015 (engelsk)Inngår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 468, nr 3, s. 373-384Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

The family of Deg/HtrA proteases plays an important role in quality control of cellular proteins in a wide range of organisms. In the genome of the cyanobacterium Synechocystis sp. PCC 6803, a model organism for photosynthetic research and renewable energy products, three Deg proteases are encoded, termed HhoA, HhoB and HtrA. In the present study, we compared wild-type (WT) Synechocystis cells with the single insertion mutants ΔhhoA, ΔhhoB and ΔhtrA. Protein expression of the remaining Deg/HtrA proteases was strongly affected in the single insertion mutants. Detailed proteomic studies using DIGE (difference gel electrophoresis) and N-terminal COFRADIC (N-terminal combined fractional diagonal chromatography) revealed that inactivation of a single Deg protease has similar impact on the proteomes of the three mutants; differences to WT were observed in enzymes involved in the major metabolic pathways. Changes in the amount of phosphate permease system Pst-1 were observed only in the insertion mutant ΔhhoB. N-terminal COFRADIC analyses on cell lysates of ΔhhoB confirmed changed amounts of many cell envelope proteins, including the phosphate permease systems, compared with WT. In vitro COFRADIC studies were performed to identify the specificity profiles of the recombinant proteases rHhoA, rHhoB or rHtrA added to the Synechocystis WT proteome. The combined in vivo and in vitro N-terminal COFRADIC datasets propose RbcS as a natural substrate for HhoA, PsbO for HhoB and HtrA and Pbp8 for HtrA. We therefore suggest that each Synechocystis Deg protease protects the cell through different, but connected mechanisms.

sted, utgiver, år, opplag, sider
Portland Press, 2015. Vol. 468, nr 3, s. 373-384
Emneord [en]
N-terminal combined fractional diagonal chromatography (COFRADIC), Synechocystis 6803, difference gel electrophoresis (DIGE), expression, proteases
HSV kategori
Identifikatorer
URN: urn:nbn:se:umu:diva-99838DOI: 10.1042/BJ20150097ISI: 000360966500003PubMedID: 25877158Scopus ID: 2-s2.0-84935014065OAI: oai:DiVA.org:umu-99838DiVA, id: diva2:788279
Merknad

Originally included in thesis in manuscript form.

Tilgjengelig fra: 2015-02-13 Laget: 2015-02-13 Sist oppdatert: 2023-03-24bibliografisk kontrollert
Inngår i avhandling
1. Deg/HtrA proteases of the cyanobacterium Synechocystis sp. PCC 6803: from biochemical characterization to their physiological functions
Åpne denne publikasjonen i ny fane eller vindu >>Deg/HtrA proteases of the cyanobacterium Synechocystis sp. PCC 6803: from biochemical characterization to their physiological functions
2015 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Umeå: Umeå University, 2015. s. 35
Emneord
Deg, HtrA, protease, chaperone, Synechocystis sp. PCC 6903, biochemical characterization, physiological function, proteomics, structure
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-99719 (URN)978-91-7601-223-9 (ISBN)
Disputas
2015-03-16, KB3A9, KBC building, Umeå University, Umeå, 10:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2015-02-23 Laget: 2015-02-12 Sist oppdatert: 2018-06-07bibliografisk kontrollert

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