Tam Lam X, Aigner Harald, Timmerman Evy, Gevaert Kris, Funk Christiane
Department of Chemistry, Umeå University, S-90187 Umeå, Sweden.
Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
Biochem J. 2015 Jun 15;468(3):373-84. doi: 10.1042/BJ20150097. Epub 2015 Apr 16.
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.
Deg/HtrA蛋白酶家族在多种生物体的细胞蛋白质质量控制中发挥着重要作用。在光合研究和可再生能源产品的模式生物——蓝藻集胞藻PCC 6803的基因组中，编码了三种Deg蛋白酶，分别称为HhoA、HhoB和HtrA。在本研究中，我们将野生型（WT）集胞藻细胞与单插入突变体ΔhhoA、ΔhhoB和ΔhtrA进行了比较。在单插入突变体中，其余Deg/HtrA蛋白酶的蛋白质表达受到强烈影响。使用差异凝胶电泳（DIGE）和N端组合分数对角线色谱法（N-terminal combined fractional diagonal chromatography，N-terminal COFRADIC）进行的详细蛋白质组学研究表明，单个Deg蛋白酶的失活对这三个突变体的蛋白质组具有相似的影响；在参与主要代谢途径的酶中观察到了与野生型的差异。仅在插入突变体ΔhhoB中观察到磷酸通透酶系统Pst-1数量的变化。对ΔhhoB细胞裂解物进行的N端COFRADIC分析证实，与野生型相比，许多细胞膜蛋白的数量发生了变化，包括磷酸通透酶系统。进行了体外COFRADIC研究，以确定添加到集胞藻野生型蛋白质组中的重组蛋白酶rHhoA、rHhoB或rHtrA的特异性谱。体内和体外N端COFRADIC数据集的综合分析表明，RbcS是HhoA的天然底物，PsbO是HhoB和HtrA的天然底物，Pbp8是HtrA的天然底物。因此，我们认为集胞藻的每种Deg蛋白酶都通过不同但相互关联的机制保护细胞。
