叶绿体分子伴侣Cpn60亚基协作与功能分化调控

Chaperones are essential for maintaining cellular protein homeostasis and exist in all kingdoms of life. Chaperonins are a subfamily of chaperones, folding and refolding of a wide variety of newly synthesised or stress-denatured polypeptides, which have diverged into two distinct groups. Group I chaperonins, found in prokaryotes or in organelles of prokaryotic origin, include GroEL/ES in E.coli, Hsp60/10 in mitochondria, and Cpn60/20 in chloroplasts.Cpn60 was discovered as Rubisco large subunit binding protein which is distinguished from other group I chaperonin by its complex composition. In contrast to homologues GroEL or Hsp60, Cpn60 consists of multiple subunits divided into two distinct α and β types. Our previous studies investigated that CPN60 complexes contain all three subunits in Chlamydomonas, and each protomer has specific function. Though CPN60β1 and CPN60β2 share very high sequence similarity (more than 90%), CPN60β oligomers were differentially affected by ATP, with ATP causing no observable effect on CPN60β1 oligomers and complete dissassembly of CPN60β2 oligomers. The project aims to investigate the cooperation and functional partition of chloroplast chaperonin, and study the mechanism of their different behaviors. The chaperonin subunit contains three structural domains: an apical domain, an intermediate domain and an equatorial domain, which were divided into five fragments in the primary sequences. We plan to swap the five fragments from three CPN60 subunits and GroEL subunit, and construct over 30 CPN60/GroEL chimeras. The structure and function of chimeras will be investigated in vivo and in vitro to determine the elements influencing the cooperation and functional partition of subunits. The underlying mechanism will be investigated by crystal structure analysis.

分子伴侣蛋白Hsp60参与折叠组装新合成或胁迫变性的蛋白质。叶绿体Cpn60复合体折叠光合作用限速酶Rubisco大亚基，是Rubisco全酶生物合成的基础。Cpn60是由多个不同亚基组成的双环结构，并根据序列特异性分为α或β型。我们前期工作已经证实莱茵衣藻叶绿体内三种CPN60亚基都参与复合体的形成，且每个亚基在形成复合体中功能不同。尽管两个CPN60β亚基高度同源，但在响应ATP水解作用时表现完全不同。本项目拟利用遗传学、生物化学以及结构生物学手段，探讨CPN60复合体内三个亚基的协作与功能分化，并解析其分子机理。Chaperonin亚基含有三个结构域，体现在氨基酸序列上为五个片段。我们拟将三个CPN60亚基与GroEL亚基的五个片段重新组合，构建各种嵌合体，体内遗传互补GroEL的功能，体外生化研究其功能的分子基础。并利用结构生物学手段，解析各亚基功能分化的元件与分子机理。

1,5-二磷酸核酮糖羧化酶／加氧酶（Rubisco）在光合作用中将大气中游离的二氧化碳转化为生物体内储能分子，是无机碳进入生物圈的主要途径。Rubisco的折叠组装依赖于叶绿体分子伴侣素系统CPN10/60。所有进行光合作用的真核生物基因组均编码多个分子伴侣素基因，这些伴侣蛋白可分为α和β亚型，而分子伴侣素蛋白需要在ATP和Mg2 +存在下与可拆卸的盖子状的辅伴侣素蛋白进行适当的相互作用，以进行底物封装和折叠。 叶绿体分子伴侣素系统如何辅助Rubisco的生物合成，是非常重要的科学问题。本项目关注此科学问题，研究工作按计划完成，取得了重要的研究进展和阶段性成果。莱茵衣藻叶绿体内三种CPN60 （CPN60α、CPN60β1，CPN60β2）亚基都参与复合体的形成，且每个亚基在形成复合体中功能不同。尽管两个CPN60β亚基高度同源，但在响应ATP水解作用时表现完全不同。为了解析三个CPN60亚基功能分化的分子基础， 我们构建系列CPN60嵌合体共表达载体。在三个CPN60顶端区对GroEL该区的替换嵌合体中，只有CPN60α所提供的顶端区无法互补GroEL的功能。表明CPN60α顶端区是不同于CPN60β的，两大类型的亚基在该区存在明显的分化。进一步分析发现CPN60α顶端结构域结合底物的能力更强，而CPN60β与辅分子伴侣素相互作用。CPN60α与CPN60β顶端结构域的晶体结构分析以及突变分析解析了Q203、T241两个氨基酸决定了CPN60α与CPN60β 亚基的功能分化。在莱茵衣藻中有三个辅分子伴侣素：CPN20，CPN23，CPN11。我们发现，尽管单独的辅分子伴侣CPN20复合体可以在体外行驶功能，但在体内，辅分子伴侣复合体含有三个亚基。利用大肠杆菌分子伴侣素缺陷型菌株，我们证明了叶绿体分子伴侣素CPN60α/β1/β2与辅分子伴侣CPN11/20/23的相互作用复合体具有完善的功能，能够在极端热处理条件下互补GroEL/ES的功能。同时，我们解析了同源十四聚体CPN60β1的晶体结构。并建立了从莱茵衣藻中直接纯化內源叶绿体分子伴侣素复合体的方法，分析其生化特性、亚基摩尔比等以及解析了其冷冻电镜结构。我们的研究结果解析了Cpn60调控叶绿体内蛋白质稳态，维持叶绿体功能的分子机理，为体外改造Rubisco奠定理论基础。

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