体外重构STUB1介导α-1抗胰蛋白酶突变体Z蛋白泛素化修饰反应体系

Reconstitution of the in vitro STUB1-mediated ubiquitination reaction system for α-1 antitrypsin mutant Z protein

摘要:

α-1抗胰蛋白酶Z型突变体蛋白(α-1 antitrypsin Z-mutant protein, ATZ)是引发α-1抗胰蛋白酶缺陷症(α-1 antitrypsin deficiency, AATD)的主要原因，研究ATZ蛋白的泛素化修饰和降解对于治疗AATD具有重要意义。STUB1是一种重要的E3泛素连接酶，参与调节多种蛋白质的泛素化修饰。然而，STUB1是否参与ATZ的泛素化修饰尚未明确。本研究首先将ATZ和STUB1的编码基因克隆到pET28a质粒，构建了这2个蛋白的表达质粒。随后，将重组质粒转入大肠杆菌表达系统，在优化诱导条件实现了重组蛋白的异源表达。通过金属螯合亲和层析技术纯化得到目的蛋白，并通过蛋白质谱分析验证了其氨基酸序列的准确性。利用纯化的ATZ和STUB1重组蛋白，构建了一个体外泛素化修饰反应体系。实验结果显示，在ATP、E1泛素激活酶和E2泛素结合酶的协同作用下，STUB1成功催化了ATZ的泛素化修饰。本研究提供了一种体外获得Z型突变体ATZ纯化蛋白的方法，并确认了STUB1介导ATZ的泛素化修饰功能，推进了对α-1抗胰蛋白酶Z型突变体蛋白在细胞内降解过程的调控机制的理解。

Abstract:

The α-1 antitrypsin Z-mutant protein (ATZ) is the primary cause of α-1 antitrypsin deficiency (AATD). Studying the ubiquitination modification and degradation of ATZ protein is importance for developing treatments for AATD. STUB1 is an important E3 ubiquitin ligase that regulates ubiquitination modification of various proteins. However, whether STUB1 in involved in the ubiquitination modification of ATZ has not been fully elucidated. In this study, the ATZ and STUB1 coding genes were first cloned into the pET28a plasmid, constructing 2 protein expression plasmids. The recombinant plasmids were then transferred into the Escherichia coli for expression. With the optimization of induction temperature and IPTG dosage, the recombinant proteins were successfully expressed. The target proteins were then efficiently purified from cell lysates using metal-chelating affinity chromatography, and the accuracy of the amino acid sequence was verified through protein mass spectrometry analysis. Using the purified ATZ and STUB1, we established an in vitro ubiquitination reaction system. Experimental results showed that, in the presence of ATP, E1 ubiquitin-activating enzyme, and E2 ubiquitin-conjugating enzyme, STUB1 catalyzed the ubiquitination modification of ATZ. This study provides a method for obtaining the ATZ protein in vitro, elucidates the mechanism of STUB1 mediating ATZ ubiquitination, thereby advancing our understanding of the intracellular degradation mechanism of the α-1 antitrypsin Z-mutant.