内质网累积PMP22突变蛋白的清除及降解促进因子的研究

Charcot-Marie-Tooth disease (CMT) is a group of the most common hereditary sensorimotor peripheral neuropathies characterized by progressive sensory loss and muscle weakness. The autosomal dominant “demyelinating” form, CMT1A, accounts for about 35% of CMT patients. CMT1A is caused by a duplication on chromosome 17p11.2 that contains the peripheral myelin protein-22 (PMP22) gene or by missense mutations in the PMP22 gene. PMP22 is a peripheral myelin protein and is involved in the regulation of Schwann cell proliferation, differentiation and apoptosis. Of significance, these mutations generally occur in the hydrophobic or transmembrane domains of the PMP22 protein. Similar with the effect of overexpression of duplicated PMP22, the mutant PMP22 proteins are abnormally retained in the endoplasmic reticulum (ER)，resulting in ER stress, apoptosis of myelinating Schwann cells, and consequently, thin myelin sheaths, demyelinating and decreased nerve conduction velocities in CMT patients. Clearance of ER-accumulated mutant PMP22 may achieve a new therapeutic approach. .A spontaneous dominant mutation L16P, which is located in the hydrophobic domain of PMP22, has been proviously identified in both CMT1A patient and Trembler-J (TrJ) mouse. It has been demonstrated that misfolded PMP22TrJ accumulates in ER and is stabilized, while the wild type mainly appear at the plasma membrane of Schwann cells and degrade promptly. In this start-up project, we will use the intracellular location and degradation rate of PMP22TrJ as indicators of pathologic status and establish cell-based system to screen cofactors that enhance the transport of PMP22TrJ from ER to plasma membrane or that promote ER-associated degradation of PMP22TrJ. Initially we will test the effects of ER chaperones or ER-resident proteins, which generally function on ER quality control and degradation pathway, on PMP22 using biochemistry approaches, such as Western-blot, immunoprecipitation or pulse-chase experiments. Through the study, we can optimize our screen system and techniques, and make better preparation in experimental materials for further study in developing new therapeutic strategies to improve the neuropathologic phenotype in CMT-1A patients. .

腓骨肌萎缩症（CMT）是一组最常见的遗传性进行性感觉运动周围神经病变。CMT1A型患者约占35%，以脱髓鞘为特征，由周围髓鞘蛋白22(PMP22)基因区域的大片段重复或基因内点突变所致。PMP22为髓鞘结构蛋白并参与调节雪旺细胞的增殖、分化和凋亡。其跨膜域突变蛋白在内质网累积，引发内质网应激，致雪旺细胞凋亡，患者呈现髓鞘变薄、脱髓鞘及神经传导速率降低。本研究以CMT1A的TrJ小鼠模型的跨膜域突变PMP22L16P为代表，建立以生化检测为基础的细胞筛查系统；以PMP22突变蛋白的亚细胞定位的改善和降解速率的提高为指标，通过检测内质网质控和蛋白降解途径相关分子伴侣对PMP22突变蛋白代谢过程的作用，探索促进PMP22突变蛋白从内质网转运、降解的作用因子，为深入研究促髓鞘形成、延缓或逆转CMT病程的机制奠定基础。

腓骨肌萎缩症1A型（CMT1A）患者以脱髓鞘为特征，由周围髓鞘蛋白22(PMP22)基因区域的大片段重复或基因内点突变所致。PMP22跨膜域突变蛋白在内质网累积，引发内质网应激，致雪旺细胞凋亡，患者呈现髓鞘变薄、脱髓鞘及神经传导速率降低。本研究总体目标是通过检测内质网质控和蛋白降解途径相关分子伴侣对PMP22突变蛋白代谢过程的作用，发现促进PMP22突变蛋白从内质网转运、降解的作用因子，探索促髓鞘形成、延缓或逆转CMT病程的机制。本一年期主任基金项目为实现上述总体目标奠定基础和进行实验材料和方法上的准备。以CMT1A的TrJ小鼠模型的跨膜域突变PMP22L16P为代表，确立了以“PMP22突变蛋白亚细胞定位的改善和降解速率的提高”为指征的筛查指标，建立了针对PMP22突变蛋白分子病理过程细胞筛查体系和相应的检测方法，并研究2种内质网蛋白质控及降解体系关联蛋白ER-mannosidase 1 （ERM1) 和calnexin（CNX）对PMP22突变蛋白的作用。结果显示ERM1可促进PMP22wt和TrJ降解，提示其可作为改善TrJ突变病理状态的候选因子进一步研究。本课题完成既定研究目标，已建立的筛查系统可用于筛查更多作用分子对TrJ突变病理状态的改善作用，也可利用该检测体系预测其他类型PMP22突变蛋白功能。