Pablos Isabel, Machado Yoan, de Jesus Hugo C Ramos, Mohamud Yasir, Kappelhoff Reinhild, Lindskog Cecilia, Vlok Marli, Bell Peter A, Butler Georgina S, Grin Peter M, Cao Quynh T, Nguyen Jenny P, Solis Nestor, Abbina Srinivas, Rut Wioletta, Vederas John C, Szekely Laszlo, Szakos Attila, Drag Marcin, Kizhakkedathu Jayachandran N, Mossman Karen, Hirota Jeremy A, Jan Eric, Luo Honglin, Banerjee Arinjay, Overall Christopher M
Centre for Blood Research, Life Sciences Centre, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V6Z 1Y6, Canada; Center for Heart Lung Innovation, St. Paul's Hospital, Vancouver, BC V6Z 1Y6, Canada.
Cell Rep. 2021 Oct 26;37(4):109892. doi: 10.1016/j.celrep.2021.109892. Epub 2021 Oct 9.
The main viral protease (3CL) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CL by TAILS substrate-targeted N-terminomics. We identify more than 100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CL engaging substrates reveal how noncanonical cleavage sites, which diverge from SARS-CoV, guide substrate specificity. Cleaving the interactors of essential effector proteins, effectively stranding them from their binding partners, amplifies the consequences of proteolysis. We show that 3CL targets the Hippo pathway, including inactivation of MAP4K5, and key effectors of transcription, mRNA processing, and translation. We demonstrate that Spike glycoprotein directly binds galectin-8, with galectin-8 cleavage disengaging CALCOCO2/NDP52 to decouple antiviral-autophagy. Indeed, in post-mortem COVID-19 lung samples, NDP52 rarely colocalizes with galectin-8, unlike in healthy lungs. The 3CL substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design.
主要病毒蛋白酶（3CL）对于严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的复制不可或缺。我们通过TAILS底物靶向N端蛋白质组学描绘了3CL的人类蛋白质底物图谱。通过对感染SARS-CoV-2的细胞进行分析，我们在人肺和肾细胞中鉴定出100多种底物。3CL与底物相互作用的酶动力学和分子对接模拟揭示了与SARS-CoV不同的非经典切割位点如何指导底物特异性。切割必需效应蛋白的相互作用分子，有效地使其与结合伴侣分离，放大了蛋白水解的后果。我们发现3CL靶向Hippo信号通路，包括使丝裂原活化蛋白激酶4激酶5（MAP4K5）失活，以及转录、mRNA加工和翻译的关键效应分子。我们证明刺突糖蛋白直接结合半乳糖凝集素-8，半乳糖凝集素-8的切割使钙结合蛋白2/核点蛋白52（CALCOCO2/NDP52）解离，从而使抗病毒自噬解偶联。事实上，在新冠病毒肺炎（COVID-19）尸检肺组织样本中，与健康肺组织不同，NDP52很少与半乳糖凝集素-8共定位。3CL底物降解组建立了一个基础底物图谱，以加速对SARS-CoV-2病理学和药物设计的探索。
