去棕榈酰化修饰酶APT1和APT2的生理功能研究

Protein palmitoylation is the most commonly detected lipid modification in the brain, which might manipulate the modified cytosolic proteins to insert into membrane compartments, signaling transduction and protein-protein interaction. The only reversible lipid modification, also referred as S-palmitoylation, is composed of two processes: palmitoylation and depalmitoylation, which are catalyzed by two different groups of protein enzymes. Hence, the study on these protein enzymes has been becoming an essential step to understand the physiological functions of protein palmitoylation. It is known that PPT1 and -2, APT1 and -2 are involved in the depalmitoylation process, of which the PPT1 and -2 and APT1 and -2 are not only alike in protein sequence but share a conserved functional domain i.e. the abhydrolase domain. The natural mutation of PPT1 in human and PPT1 or -2 knockout mouse presented a neurodegenerative disease phenotype, yet the physiological function and potentially, the pathological mechanism resulted from the mutation of APT1 or -2 genes in mouse or human remained unknown. Our previous study showed that APTs regulated the membrane localizations and signaling of the proto oncogene H-Ras and GAP-43. Here, by taking the advantage of the APT1 and -2 knockout mice, we set out to investigate the downstream target proteins regulated by APTs, aiming to understand the physiological functions and potentially, the pathological mechanism of APT1 and -2, as well as the mutual-interactions between APT1 and -2.

蛋白棕榈酰化是大脑中蛋白翻译后脂肪酸化修饰的最常见方式，它调控了蛋白在细胞膜上的定位、信号传导以及蛋白之间的相互作用。可逆的棕榈酰化修饰是棕榈酰化和去棕榈酰化交替的动态循环过程，由不同蛋白酶催化调节，对这些蛋白酶的研究是理解棕榈酰化生理功能的关键。去棕榈酰化蛋白酶包括PPT1、PPT2、 APT1、APT2等。人类PPT1基因自然突变以及PPT1、PPT2基因敲除小鼠均呈现神经退行性病变，但APT1、APT2的生理功能和潜在病理机制研究尚未见报道。申请人前期研究证实APT1、APT2参与调控重要蛋白H-Ras 和GAP-43在细胞膜上的定位和信号传导。籍建立APT1、APT2基因敲除小鼠模型，申请人拟在前期工作的基础上对受APT1、APT2调控的下游靶蛋白做进一步深入研究，探讨APT1、APT2在机体水平上的生理功能和潜在的病理机制，以及APT1和APT2之间的相互作用关系。

蛋白棕榈酰化修饰通过硫酯键连接到被修饰蛋白的特定半胱氨酸残基上完成修饰过程。棕榈酰化修饰提高了被修饰蛋白的疏水性进而影响了蛋白的定位，蛋白-蛋白间相互作用和信号通路的开关等。由于缺乏特异棕榈酰化蛋白抗体等客观原因，本领域的研究进展较为缓慢。申请人通过建立去棕榈酰化修饰催化酶APT1/2缺失的细胞学模型和小鼠模型，阐明APT1/2的细胞学功能和其潜在的催化底物。进一步的研究发现APT1/2单基因敲除小鼠未出现明显的表型异常。在此技术平台上，我们利用Crispr-Cas9构建DHHC2敲除小鼠，发现DHHC2通过影响IRF7的磷酸化和IFN-a的表达调控了机体的炎症水平从而改善了银屑病的病理进程。此外，我们利用棕榈酰化蛋白组学挖掘神经系统和恶性肿瘤中特异变化的棕榈酰化修饰蛋白，其中发现GFAP能被棕榈酰化修饰且其过高的棕榈酰化修饰水平促进了神经退行性疾病的病理进程，抑制该修饰能有效缓解神经细胞的死亡和延长小鼠的寿命。综上所述，申请人实验室以蛋白棕榈酰化修饰为聚焦，致力于阐明该修饰在多种人类疾病小鼠模型中的潜在分子病理机制和发现有价值的药物靶点。主要研究结果已整理并以通讯作者身份发表在包括PNAS等高水平科研期刊上。