甲羟戊酸激酶突变导致光敏性汗孔角化症的致病机制研究

发布时间：2018-04-29 01:27

本文选题：DSAP + 甲羟戊酸激酶　；参考：《中南大学》2013年硕士论文

【摘要】：目的：甲羟戊酸激酶(mevalonate kinase, MVK)可将甲羟戊酸磷酸化为5-磷酸甲羟戊酸。前期研究中在多个光敏性汗孔角化症(disseminated superficial actinic porokeratosis, DSAP)家系及散发患者中发现MVK基因突变,但是其具体的致病机制不明。本研究拟探讨MVK相关突变导致DSAP的机制,加深我们对这一疾病的认识。方法：采集正常人和患者外周血,抽提淋巴细胞总RNA, real time PCR和RT-PCR研究mRNA表达水平。构建相关真核表达载体,在HEK293中表达MVK野生型和DSAP相关突变,免疫印迹观察蛋白表达。建立MVK野生型和DSAP相关突变的HEK293稳定表达细胞系,使用放线菌酮(Chcloheximide CHX)抑制蛋白合成,在不同时间点收集蛋白,免疫印迹检测MVK野生型和DSAP相关突变型的半衰期。使用蛋白酶体抑制剂(MG132),溶酶体抑制剂(CQ)阻断MVK的降解,免疫印迹观察MVK野生型和DSAP相关突变型的降解途径。在HEK293细胞中瞬时表达GFP标签或FLAG标签的MVK野生型和DSAP相关突变型,对过氧化物酶体、线粒体、内质网等细胞器的标志蛋白进行免疫荧光染色,研究MVK野生型和DSAP相关突变型进行亚细胞定位。构建pGEX-4T-2骨架的MVK野生型及DSAP相关突变质粒,在BL21感受态中通过IPTG诱导原核表达GST融合蛋白,GST pull down研究MVK野生型和DSAP相关突变形成同源二聚体的能力。在HEK293细胞中共转GFP标签和FLAG标签的MVK野生型和/或DSAP相关突变型,免疫共沉淀研究MVK野生型和DSAP相关突变型形成同源二聚体的能力。结果： 1.A189V突变患者mRNA表达低于正常人；DSAP相关突变蛋白表达低于野生型； 2. DSAP目关突变蛋白的降解较野生型蛋白明显加快；DSAP野生型和相关相关突变蛋白主要通过蛋白酶体途径降解； 3.MVK野生型和DSAP相关突变型为细胞质分布；MVK野生型和DSAP相关突变与过氧化物酶体、线粒体、内质网没有共定位； 4.MVK野生型和A189V可以形成同源二聚体,MVK野生型不能和H312R形成同源二聚体,H312R自身不能形成同源二聚体。结论： 1.MVK DSAP相关突变的细胞亚定位没有改变。 2.MVK DSAP相关突变蛋白半衰期缩短,通过蛋白酶体途径降解。 3.MVKH312R形成同源二聚体的能力有缺陷。
[Abstract]:Objective: Mevalonate kinase (MVK) phosphorylated mevalate to 5-phosphate. In previous studies, MVK gene mutations were found in many families and sporadic patients with Guang Min porokeratosis, but the specific pathogenesis of MVK gene mutation was unknown. This study aims to explore the mechanism of MVK related mutations leading to DSAP and to deepen our understanding of the disease. Methods: Peripheral blood samples were collected from normal subjects and patients. Total RNAs of lymphocytes were extracted, real time PCR and RT-PCR were used to study the expression of mRNA. The related eukaryotic expression vector was constructed and MVK wild-type and DSAP related mutations were expressed in HEK293. Western blot was used to observe the expression of proteins. MVK wild-type and DSAP related mutant HEK293 stable expression cell line was established. The protein synthesis was inhibited by actinomycin Chcloheximide CHX. The half-life of MVK wild-type and DSAP related mutant was detected by Western blotting at different time points. Proteasome inhibitor (MG132) and lysosome inhibitor (CQ) were used to block the degradation of MVK. Western blot was used to observe the degradation pathway of MVK wild type and DSAP related mutant type. The transient expression of MVK wild type and DSAP related mutant type of GFP tag or FLAG tag in HEK293 cells was detected by immunofluorescence staining of peroxisome, mitochondria, endoplasmic reticulum, and other marker proteins of the organelle, such as peroxisome, mitochondria and endoplasmic reticulum. To study the subcellular localization of MVK wild type and DSAP associated mutant. The wild-type and DSAP related mutant plasmids of pGEX-4T-2 skeleton were constructed, and the expression of GST fusion protein GST-pull down in BL21 was induced by IPTG. The ability of MVK wild-type and DSAP related mutations to form homologous dimer was studied. The wild and / or DSAP related mutants of MVK cotransduced with GFP and FLAG tags were co-transformed into HEK293 cells. Immunoprecipitation was used to study the ability of MVK wild type and DSAP related mutant to form homologous dimer. Results: The expression of mRNA in patients with 1.A189V mutation was lower than that in wild type. 2. The degradation of DSAP mutants was significantly faster than that of wild type proteins. The degradation of wild type and related mutant proteins was mainly through proteasome pathway. The wild type and DSAP related mutation of 3.MVK were cytoplasmic distribution and DSAP related mutation and peroxisome, mitochondria and endoplasmic reticulum were not colocated. 4.MVK wild-type and A189V can form homologous dimer, MVK wild type can not form homologous dimer with H312R and H312R itself can not form homodimer. Conclusion: The sublocalization of 1.MVK DSAP related mutations was not changed. The half-life of 2.MVK DSAP associated mutant protein was shortened and degraded by proteasome pathway. The ability of 3.MVKH312R to form homologous dimers is deficient.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R751

【共引文献】