家蚕血淋巴营养储藏类蛋白乙酰化修饰组鉴定及其提高蛋白稳定性的分子机制研究

Acetylation is an important post-translational modification, which can compete for the same ubiquitination sites of target protein, resulting in an improvement of protein stabilization in the cells and thus regulating the protein functions. The previous work showed that extremely highly acetylated sites were found in nutrient-storage proteins from hemolymph in bombyx mori. We further confirmed lysine acetylation could improve the protein stabilization of SP2 and 30K-3 through competing for the ubiquitination sites, thus up-regulate the protein level in the cells. Based on the above results, we will systemically identify the lysine acetylation profiling of nutrient-storage proteins from hemolymph in Bombyx mori and the molecular mechanism about protein stabilization by acetylation. Firstly, the acetylation profiling and expression profiling of nutrient-storage proteins in hemolymph and their relationship in different developmental stages of Bombyx mori will be identified using many methods such as Western blot and nano-HPLC/MS/MS. Furthermore, the nutrient-storage proteins SPs and 30K were selected as the research target. Using many methods and technologies such as acetylated sites mutation and eukaryotic over-expression, blocking protein expression and proteasomal degradation pathway, IP and WB, we will further validate the acetylation sites of SPs/30K proteins at a molecular level and identify their function and molecular mechanism on the protein stabilization, confirming that acetylation modification of nutrient-storage proteins could inhibit ubiquitin-mediated proteasomal degradation pathway through competing for the same ubiquitination sites, thus stabilize the protein in the cells. Therefore, a new mechanism for nutrient storage, release and utilization will be expected to find in silkworm and even in insects.

乙酰化是一类重要的蛋白翻译后修饰，非组蛋白乙酰化能通过竞争泛素化修饰来提高蛋白稳定性。我们前期研究发现家蚕营养储藏类蛋白存在罕见的高度乙酰化，并证实乙酰化能提高其中SP2和30K-3蛋白的稳定性，竞争其泛素化修饰。基于前期研究，本项目系统开展家蚕血淋巴营养储藏类蛋白的乙酰化修饰组鉴定及其提高蛋白稳定性的分子机制研究。采用WB和nano-HPLC/MS/MS技术鉴定家蚕各时期血淋巴营养储藏类蛋白乙酰化组和蛋白表达谱，分析两者相关性；选择其中SP和30K蛋白为研究对象，采用乙酰化位点突变过表达、蛋白合成和蛋白酶体降解途径阻断、IP和WB等方法进一步开展SP/30K蛋白乙酰化修饰的分子验证、对蛋白稳定性的调控及其调控的分子机制研究。确定乙酰化通过竞争营养储藏类蛋白的泛素化，抑制泛素介导的蛋白酶体降解途径，从而提高蛋白稳定性的分子机制，有望发现一种乙酰化调控家蚕乃至昆虫营养储藏和水解利用的新机制。

家蚕拥有分布全身的血淋巴循环系统，可将营养和氧传递到体内各部位。血淋巴是营养和能量的储藏仓库，其含有大量营养储藏类蛋白。营养储藏类蛋白能够将家蚕唯一摄食期幼虫期摄入的多余营养和能量储藏起来，以供后续非摄食期发育和丝蛋白合成所需营养和能量的供应，因此营养的储藏与利用调控对研究家蚕生长发育和产丝性能提高显得尤为重要，但其调控的机制还有待研究。. 本项目前期研究首次发现家蚕血淋巴中主要营养储藏类蛋白存在高度乙酰化，暗示了一种乙酰化调控的家蚕营养储藏与利用的新机制。在前期研究基础上，本项目系统开展家蚕血淋巴营养储藏类蛋白乙酰化修饰鉴定与功能以及分子机制的研究工作。通过对家蚕主要营养储藏类蛋白SP1、SP3、BmApoLp-II、BmApoLp-III、30K-3、30K-15和30K-24等蛋白的乙酰化鉴定与功能研究，发现乙酰化是家蚕血淋巴营养储藏类蛋白中的一种普遍的修饰现象，证实乙酰化修饰可以提高家蚕主要的营养储藏类蛋白的稳定性、细胞含量与抗细胞凋亡活性，并进一步证实乙酰化可通过竞争泛素化修饰，抑制泛素介导的蛋白酶体降解途径，从而提高家蚕营养储藏类蛋白的稳定性和细胞含量的分子机制；进一步研究成功筛选得到家蚕乙酰转移酶BmCBP的特异抑制剂C646和A485，并发现两者可降低上述多个营养储藏类蛋白的表达和乙酰化水平，同时发现BmCBP可以和多种营养储藏类蛋白结合，催化其乙酰化修饰进而调控其蛋白表达，并影响家蚕细胞的TCA循环，表明BmCBP参与了家蚕营养储藏类蛋白的乙酰化修饰和营养储藏的调控，还有待进一步研究。. 上述结果揭示了一种乙酰化修饰调控家蚕营养储藏和水解利用的新机制，为家蚕中BmCBP调控的营养储藏和利用的新机制提供进一步的理论依据，为家蚕产丝性能提高提供新的理论参考，同时对研究其他鳞翅目昆虫发育过程中营养储藏和水解利用的调控机制以及鳞翅目农业害虫的生物防治具有较好的借鉴意义。