Abstract：
Protein homeostasis is essential for cyanobacteria to maintain proper cellular function under adverse and fluctuating conditions. The AAA+ superfamily of proteolytic complexes in cyanobacteria plays a critical role in this process, including ClpXP, which comprises a hexameric ATPase ClpX and a tetradecameric peptidase ClpP. Despite the physiological effects of ClpX on growth and photosynthesis, its potential substrates and underlying mechanisms in cyanobacteria remain unknown. In this study, we employed a streptavidin-biotin affinity pull-down assay coupled with label-free proteome quantitation to analyze the interactome of ClpX in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We identified 503 proteins as potential ClpX-binding targets, many of which had novel interactions. These ClpX-binding targets were found to be involved in various biological processes, with particular enrichment in metabolic processes and photosynthesis. Using protein-protein docking, GST pull-down, and biolayer interferometry assays, we confirmed the direct association of ClpX with the photosynthetic proteins, ferredoxin-NADP+ oxidoreductase (FNR) and phycocyanin subunit (CpcA). Subsequent functional investigations revealed that ClpX participates in the maintenance of FNR homeostasis and functionality in Synechocystis grown under different light conditions. Overall, our study provides a comprehensive understanding of the extensive functions regulated by ClpX in cyanobacteria to maintain protein homeostasis and adapt to environmental challenges.
摘要：
蛋白质稳态对于蓝细菌在不利和波动的条件下维持适当的细胞功能至关重要。蓝细菌中蛋白水解复合物的AAA+超家族在这一过程中起着至关重要的作用,包括ClpXP，其包含六聚体ATP酶ClpX和十四聚体肽酶ClpP。尽管ClpX对生长和光合作用有生理作用，其在蓝藻中的潜在底物和潜在机制仍然未知。在这项研究中,我们采用链霉亲和素-生物素亲和下拉测定法结合无标记蛋白质组定量分析，以分析ClpX在蓝细菌集胞藻模型中的相互作用。PCC6803（以下简称集胞藻）。我们确定了503种蛋白质作为潜在的ClpX结合靶标，其中许多都有新颖的互动。发现这些ClpX结合靶标参与各种生物过程，在代谢过程和光合作用中特别富集。利用蛋白质-蛋白质对接，GST下拉式,和生物层干涉测量，我们证实了ClpX与光合蛋白的直接关联，铁氧还蛋白-NADP+氧化还原酶(FNR)和藻蓝蛋白亚基(CpcA)。随后的功能研究表明，ClpX参与了在不同光照条件下生长的集胞藻的FNR稳态和功能的维持。总的来说,我们的研究全面了解了ClpX在蓝藻中调节的广泛功能，以维持蛋白质稳态并适应环境挑战。
