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  • 1. Hall, M.
    et al.
    Lâm, Xuân Tâm
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Persson, K.
    Structure of the HhoA protease from Synechocystis sp. PCC 6803Manuscript (preprint) (Other academic)
  • 2.
    Hall, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wagner, Raik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lam, Xuan Tam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The HhoA protease from Synechocystis sp. PCC 6803: novel insights into structure and activity regulation2017In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 198, no 3, p. 147-153Article in journal (Refereed)
    Abstract [en]

    Proteases play a vital role in the removal of proteins, which become damaged due to temperature or oxidative stress. Important to this process in the cyanobacterium Synechocystis sp. PCC6803 is the family of Deg/HtrA proteases; HhoA (sll1679), HhoB (sll1427) and HtrA (slr1204). While previous studies have elucidated the structures of Deg/HtrA proteases from Escherichia coli and from the chloroplast of the higher plant Arabidopsis thaliana, no structural data have been available for any Deg/HtrA protease from cyanobacteria, the evolutionary ancestor of the chloroplast. To gain a deeper insight into the molecular mechanisms and regulation of these proteins we have solved the structure of the Synechocystis HhoA protease in complex with a co-purified peptide by X-ray crystallography. HhoA assembles into stable trimers, mediated by its protease domain and further into a cage-like hexamer by a novel interaction between the PDZ domains of opposing trimers. Each PDZ domain contains two loops for PDZ-PDZ formation: interaction clamp one and two (IC1, IC2). IC1 interacts with IC2 on the opposing PDZ domain and vice versa. Our structure shows a peptide bound to a conserved groove on the PDZ domain and the properties of this pocket suggest that it binds substrate proteins as well as the neo C-termini of cleaved substrates. In agreement with previous studies showing the proteolytic activity of HhoA to be activated by Ca2+ or Mg2+, binding of divalent metal ions to the central channel of the trimer by the L1 activation loop was observed.

  • 3.
    Hall, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wagner, Raik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Tam, Lam Xuan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The HhoA protease from Synechocystis sp. PCC 6803 – Novel insights into structure and activity regulation2017In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 198, no 3, p. 147-153Article in journal (Refereed)
    Abstract [en]

    Abstract Proteases play a vital role in the removal of proteins, which become damaged due to temperature or oxidative stress. Important to this process in the cyanobacterium Synechocystis sp. PCC6803 is the family of Deg/HtrA proteases; HhoA (sll1679), HhoB (sll1427) and HtrA (slr1204). While previous studies have elucidated the structures of Deg/HtrA proteases from Escherichia coli and from the chloroplast of the higher plant Arabidopsis thaliana, no structural data have been available for any Deg/HtrA protease from cyanobacteria, the evolutionary ancestor of the chloroplast. To gain a deeper insight into the molecular mechanisms and regulation of these proteins we have solved the structure of the Synechocystis HhoA protease in complex with a co-purified peptide by X-ray crystallography. HhoA assembles into stable trimers, mediated by its protease domain and further into a cage-like hexamer by a novel interaction between the PDZ domains of opposing trimers. Each PDZ domain contains two loops for PDZ-PDZ formation: interaction clamp one and two (IC1, IC2). IC1 interacts with IC2 on the opposing PDZ domain and vice versa. Our structure shows a peptide bound to a conserved groove on the PDZ domain and the properties of this pocket suggest that it binds substrate proteins as well as the neo C-termini of cleaved substrates. In agreement with previous studies showing the proteolytic activity of HhoA to be activated by Ca2+ or Mg2+, binding of divalent metal ions to the central channel of the trimer by the L1 activation loop was observed.

  • 4.
    Huesgen, Pitter Florian
    et al.
    Department of Biology, University of Konstanz, Germany.
    Miranda, Helder
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lam, Xuan Tam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Perthold, Manuela
    Department of Biology, University of Konstanz, Germany.
    Schuhmann, Holger
    Department of Biology, University of Konstanz, Germany.
    Adamska, Iwona
    Department of Biology, University of Konstanz, Germany.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 435, no 3, p. 733-742Article in journal (Refereed)
    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.

  • 5.
    Lâm, Xuân Tâm
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Deg/HtrA proteases of the cyanobacterium Synechocystis sp. PCC 6803: from biochemical characterization to their physiological functions2015Doctoral 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.

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    Doctoral thesis _ LXT
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  • 6.
    Lâm, Xuân Tâm
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Aigner, Harald
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Timmerman, Evy
    Gevaert, Kris
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Proteomic approaches to identify substrates of the three Deg/HtrA proteases of the cyanobacterium Synechocystis sp. PCC 68032015In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 468, no 3, p. 373-384Article in journal (Refereed)
    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.

  • 7.
    Roberts, Irma N
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Lam, Xuan Tam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Miranda, Helder
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kieselbach, Thomas
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Degradation of PsbO by the deg protease HhoA is thioredoxin dependent2012In: PLOS ONE, E-ISSN 1932-6203, Vol. 7, no 9, p. e45713-Article in journal (Refereed)
    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.  

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  • 8.
    Roberts, Irma N
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Miranda, Hélder
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Tam, Lam Xuan
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Kieselbach, Thomas
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    PsbO degradation by deg proteases under reducing conditions2013In: Photosynthesis research for food, fuel and future: 15th International Conference on Photosynthesis / [ed] Tingyun Kuang; Congming Lu; Lixin Zhang, Springer Berlin/Heidelberg, 2013, , p. 4p. 599-602Conference paper (Refereed)
    Abstract [en]

    DegP/HtrA proteases are ATP-independent serine endopeptidases widely distributed in nearly all organisms. As yet, their physiological role in oxygenic photosynthetic organisms is unclear, although it has been widely speculated that they participate in the photosystem II repair cycle. Here, we investigated the ability of Deg proteases to degrade PsbO according to its redox state. A sample of purified PsbO or photosystem II complex was incubated together with recombinant Deg proteases of Synechocystis sp. PCC 6803 (HhoA, HhoB or HtrA). The reducing media was conferred by the Escherichia coli thioredoxin/thioredoxin reductase system. The results obtained showed that HhoA is able to hydrolyze reduced PsbO while HhoB and HtrA are not. HhoA was active against free PsbO of spinach as well as PsbO of Synechocystis attached to photosystem II, only under reducing conditions. The finding that all three Deg proteases of Synechocystis co-purify with photosystem II supports the hypothesis of PsbO as a substrate for Deg proteases in vivo.

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