Sharma Arun Kumar, Mal Soumya, Sahu Sanjaya Kumar, Bagchi Shreya, Majumder Debayan, Chakravorty Debangana, Saha Sudipto, Kundu Manikuntala, Basu Joyoti
Department of Chemical Sciences, Bose Institute, Kolkata, India.
Department of Biological Sciences, Bose Institute, Kolkata, India.
FEBS J. 2025 Jan;292(1):94-114. doi: 10.1111/febs.17261. Epub 2024 Sep 17.
Peptidyl prolyl isomerases (PPIases) are well-conserved protein-folding enzymes that moonlight as regulators of bacterial virulence. Peptidyl prolyl isomerase A, PPiA (Rv0009) is a secretory protein of Mycobacterium tuberculosis that possesses sequence and structural similarity to eukaryotic cyclophilins. In this study, we validated the interaction of PPiA with stimulator of interferon genes (STING) using both, Escherichia coli-based and mammalian in vitro expression systems. In vitro pull-down assays confirmed that the cytosolic domain of STING interacts with PPiA, and moreover, we found that PPiA could induce dimerization of STING in macrophages. In silico docking analyses suggested that the PXXP (PDP) motif of PPiA is crucial for interaction with STING, and concordantly, mutations in the PDP domain (PPiA MUT-II) abrogated this interaction, as well as the ability of PPiA to facilitate STING dimerization. In agreement with these observations, fluorescence microscopy demonstrated that STING and wild-type PPiA, but not PPiA MUT-II, could colocalize when expressed in HEK293 cells. Highlighting the importance of the PDP domain further, PPiA, but not PPiA MUT-II could activate Tank binding kinase 1 (TBK1)-interferon regulatory factor 3 (IRF3) signaling to promote the release of interferon-beta (IFNβ). PPiA, but not PPiA MUT-II expressed in Mycobacterium smegmatis induced IFNβ release and facilitated bacterial survival in macrophages in a STING-dependent manner. The PPiA-induced release of IFNβ was c-GAS independent. We conclude that PPiA is a previously undescribed mycobacterial regulator of STING-dependent type I interferon production from macrophages.
肽基脯氨酰异构酶（PPIases）是一类保守性良好的蛋白质折叠酶，同时还兼职细菌毒力调节剂。肽基脯氨酰异构酶A，即PPiA（Rv0009）是结核分枝杆菌的一种分泌蛋白，与真核亲环蛋白具有序列和结构相似性。在本研究中，我们使用基于大肠杆菌的体外表达系统和哺乳动物体外表达系统，验证了PPiA与干扰素基因刺激因子（STING）之间的相互作用。体外下拉试验证实，STING的胞质结构域与PPiA相互作用，此外，我们发现PPiA可诱导巨噬细胞中STING二聚化。计算机对接分析表明，PPiA的PXXP（PDP）基序对于与STING的相互作用至关重要，与此一致的是，PDP结构域（PPiA MUT-II）中的突变消除了这种相互作用，以及PPiA促进STING二聚化的能力。与这些观察结果一致，荧光显微镜显示，当在HEK293细胞中表达时，STING和野生型PPiA可以共定位，但PPiA MUT-II则不能。进一步强调PDP结构域的重要性，PPiA而非PPiA MUT-II可以激活Tank结合激酶1（TBK1）-干扰素调节因子3（IRF3）信号通路，以促进干扰素-β（IFNβ）的释放。耻垢分枝杆菌中表达的PPiA而非PPiA MUT-II以STING依赖的方式诱导IFNβ释放并促进细菌在巨噬细胞中的存活。PPiA诱导的IFNβ释放不依赖于c-GAS。我们得出结论，PPiA是一种先前未被描述的分枝杆菌调节剂，可调节巨噬细胞中依赖STING的I型干扰素产生。
