Russell Theresa M, Tang Xiaoling, Goldstein Jason M, Bagarozzi Dennis, Johnson Barbara J B
Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Fort Collins, CO, USA.
Division of Scientific Resources, Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Atlanta, GA, USA.
Mol Microbiol. 2016 Feb;99(3):586-96. doi: 10.1111/mmi.13251. Epub 2015 Nov 19.
HtrA serine proteases are highly conserved and essential ATP-independent proteases with chaperone activity. Bacteria express a variable number of HtrA homologues that contribute to the virulence and pathogenicity of bacterial pathogens. Lyme disease spirochetes possess a single HtrA protease homologue, Borrelia burgdorferi HtrA (BbHtrA). Previous studies established that, like the human orthologue HtrA1, BbHtrA is proteolytically active against numerous extracellular proteins in vitro. In this study, we utilized size exclusion chromatography and blue native polyacrylamide gel electrophoresis (BN-PAGE) to demonstrate BbHtrA oligomeric structures that were substrate independent and salt sensitive. Examination of the influence of transition metals on the activity of BbHtrA revealed that this protease is inhibited by Zn(2+) > Cu(2+) > Mn(2+). Extending this analysis to two other HtrA proteases, E. coli DegP and HtrA1, revealed that all three HtrA proteases were reversibly inhibited by ZnCl2 at all micro molar concentrations examined. Commercial inhibitors for HtrA proteases are not available and physiologic HtrA inhibitors are unknown. Our observation of conserved zinc inhibition of HtrA proteases will facilitate structural and functional studies of additional members of this important class of proteases.
HtrA丝氨酸蛋白酶是高度保守的、必不可少的不依赖ATP的蛋白酶，具有伴侣活性。细菌表达数量可变的HtrA同源物，这些同源物有助于细菌病原体的毒力和致病性。莱姆病螺旋体拥有单一的HtrA蛋白酶同源物，即伯氏疏螺旋体HtrA（BbHtrA）。先前的研究表明，与人类同源物HtrA1一样，BbHtrA在体外对多种细胞外蛋白质具有蛋白水解活性。在本研究中，我们利用尺寸排阻色谱法和蓝色天然聚丙烯酰胺凝胶电泳（BN-PAGE）来证明BbHtrA的寡聚结构，该结构不依赖底物且对盐敏感。对过渡金属对BbHtrA活性影响的研究表明，该蛋白酶受Zn(2+) > Cu(2+) > Mn(2+)的抑制。将该分析扩展到另外两种HtrA蛋白酶，即大肠杆菌DegP和HtrA1，发现在所有检测的微摩尔浓度下，所有三种HtrA蛋白酶都被ZnCl2可逆抑制。目前尚无HtrA蛋白酶的商业抑制剂，生理性HtrA抑制剂也未知。我们观察到HtrA蛋白酶存在保守的锌抑制作用，这将有助于对这一重要蛋白酶类别的其他成员进行结构和功能研究。
