Arkley Research Labs, Arkley BioTek, LLC, 4444 Decatur Blvd., Indianapolis, IN 46241, USA.
Eli Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA.
Int J Mol Sci. 2022 Apr 21;23(9):4604. doi: 10.3390/ijms23094604.
Oxidation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by reactive oxygen species such as HO activate pleiotropic signaling pathways is associated with pathophysiological cell fate decisions. Oxidized GAPDH binds chaperone proteins with translocation of the complex to the nucleus and mitochondria initiating autophagy and cellular apoptosis. In this study, we establish the mechanism by which HO-oxidized GAPDH subunits undergo a subunit conformational rearrangement. HO oxidizes both the catalytic cysteine and a vicinal cysteine (four residues downstream) to their respective sulfenic acids. A 'two-cysteine switch' is activated, whereby the sulfenic acids irreversibly condense to an intrachain thiosulfinic ester resulting in a major metastable subunit conformational rearrangement. All four subunits of the homotetramer are uniformly and independently oxidized by HO, and the oxidized homotetramer is stabilized at low temperatures. Over time, subunits unfold forming disulfide-linked aggregates with the catalytic cysteine oxidized to a sulfinic acid, resulting from thiosulfinic ester hydrolysis via the highly reactive thiosulfonic ester intermediate. Molecular Dynamic Simulations provide additional mechanistic insights linking GAPDH subunit oxidation with generating a putative signaling conformer. The low-temperature stability of the HO-oxidized subunit conformer provides an operable framework to study mechanisms associated with gain-of-function activities of oxidized GAPDH to identify novel targets for the treatment of neurodegenerative diseases.
活性氧物质（如 HO）氧化甘油醛-3-磷酸脱氢酶（GAPDH）会激活多种信号通路，与病理生理细胞命运决定有关。氧化的 GAPDH 与伴侣蛋白结合，将复合物易位到细胞核和线粒体，启动自噬和细胞凋亡。在这项研究中，我们建立了 HO 氧化的 GAPDH 亚基发生亚基构象重排的机制。HO 氧化催化半胱氨酸和相邻的半胱氨酸（下游四个残基）形成各自的亚磺酰基。激活了“双半胱氨酸开关”，其中亚磺酰基不可逆地缩合形成链内硫代磺酸酯，导致主要的亚稳定亚基构象重排。四聚体的四个亚基都被 HO 均匀且独立地氧化，并且氧化的四聚体在低温下稳定。随着时间的推移，亚基展开形成二硫键连接的聚集体，其中催化半胱氨酸被氧化成亚磺酰基，这是由于通过高反应性的硫代磺酸酯中间物水解形成硫代磺酸酯。分子动力学模拟提供了更多的机制见解，将 GAPDH 亚基氧化与生成假定的信号构象联系起来。HO 氧化亚基构象的低温稳定性为研究与氧化 GAPDH 的功能获得活性相关的机制提供了可行的框架，以确定治疗神经退行性疾病的新靶标。
