ClpP2蛋白失活对集胞藻PCC6803生长、光合作用和蛋白质组的影响

Effects of ClpP2 protein inactivation on growth, photosynthesis, and proteome of Synechocystis sp. PCC6803

摘要:

【背景】蛋白酶能够降解细胞中错误折叠或是无功能的蛋白，Clp家族蛋白就是一类重要的蛋白酶复合物。Clp蛋白酶复合物的水解核心是ClpP，集胞藻PCC6803中存在4种不同的ClpP蛋白，分别为ClpP1-ClpP4。作为重要的蛋白水解复合物的功能组分，目前对集胞藻ClpP的研究十分有限，对其生理功能与调控底物的研究甚少。【目的】选择集胞藻为研究对象探究ClpP2蛋白的功能，鉴定其潜在底物，为集胞藻ClpP2作用机制提供实验支撑。【方法】构建集胞藻ClpP2突变株(ΔClpP2)，进行其生长实验和光合生理功能研究。通过标记定量蛋白质组学技术(isobaric tag for relative absolute quantitation, iTRAQ)鉴定ClpP2调控的靶标蛋白，生物信息学分析底物蛋白参与的代谢通路，最后利用平行反应监测(parallel reaction monitoring, PRM)技术对部分定量数据进行验证。【结果】ΔClpP2可以在自然条件下光合自养生长至对数生长期，但高光或高温胁迫下则无法正常生长。相较于野生型，ΔClpP2有着显著降低的PSⅡ电子传递效率及PSⅠ环式电子传递活性。通过iTRAQ定量蛋白质组学手段，ΔClpP2相对于WT共鉴定到206个差异表达蛋白，其中131个上调、75个下调，为ClpP2蛋白酶提供了丰富的潜在底物库。基因本体论(gene ontology, GO)分析发现ClpP2主要参与各种物质的转运，其中ABC蛋白转运途径显著被富集。利用PRM技术对34个差异表达的蛋白进行了验证。【结论】ClpP2蛋白不是集胞藻生长所必需，但在高温或者高光胁迫下是必不可少的，其失活会降低集胞藻光合系统活性。ClpP2可能通过调控离子转运进而影响光合系统。ClpP2很可能与ClpX结合形成蛋白酶复合物。

关键词:ClpP2;iTRAQ;集胞藻PCC6803;蛋白质组学

Abstract:

[Background] Proteases can degrade the misfolded or nonfunctional proteins in cells. Clp family protein is one of the important protease complexes. ClpP is the core of proteolysis of Clp protease complex. According to genomic data, there are four different ClpP proteins in Synechocystis sp. PCC6803, namely ClpP1–ClpP4. As a vital functional component of proteolytic complex, ClpP in Synechocystis is currently poorly studied. The study on its physiological function and substrate regulation is limited. [Objective] To explore the functions of ClpP2 in Synechocystis and identify potential substrate clusters, thereby providing experimental support for the mechanism research of ClpP2. [Methods] The ClpP2 mutant strain (ΔClpP2) was constructed, and the growth experiment and photosynthetic system characteristic experiment were carried out. Target proteins regulated by ΔClpP2 were identified by isobaric tag for relative absolute quantitation (iTRAQ), and the metabolic pathways involved in the substrate proteins were analyzed via bioinformatics. Finally, parallel reaction monitoring (PRM) was used to verify part of the quantitative data. [Results]ΔClpP2 grew into the logarithmic phase through photoautotrophy under natural conditions, but did not grow normally when met high-light or high-temperature stress. Compared with the wild-type (WT), ΔClpP2 showed significantly reduced photosystem Ⅱ (PSⅡ) electron transport efficiency and circle electron transport activity of photosystemⅠ(PSⅠ). A total of 206 differentially expressed proteins in ΔClpP2 were identified by iTRAQ quantitative proteomics. Among them, 131 were up-regulated and 74 were down-regulated, which provided a rich substrate library. Gene Ontology (GO) analysis showed that ClpP2 was mainly involved in the transport of various substances, and ABC transporter pathway was enriched notably. Thirty-four differentially expressed proteins were successfully verified by PRM technology. [Conclusion] ClpP2 is not necessary for the growth of Synechocystis, but is essential when Synechocystis meets high-temperature or high-light stress. ClpP2 inactivation reduces the activity of the photosynthetic system in Synechocystis. ClpP2 might affect the photosynthetic system by regulating ion transport. ClpP2 is likely to bind with ClpX to form a protease complex.

Key words:ClpP2;iTRAQ;Synechocystis sp. PCC6803;proteomics