Discovery Oncology, Genentech, South San Francisco, California, 94080, USA.
Early Discovery Biochemistry, Genentech, South San Francisco, California, 94080, USA.
Nat Commun. 2018 Mar 21;9(1):1162. doi: 10.1038/s41467-018-03511-6.
Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.
活性探针（ABPs）广泛用于监测生物系统中酶家族的活性。从探针反应推断酶活性需要探针在其活性位点与酶反应；然而，探针标记位点很少被验证。在这里，我们提出了一种增强的化学蛋白质组学方法，用于评估去泛素化酶的活性和探针反应性，使用生物正交标记的 ABPs 和顺序在珠上消化方案来增强探针标记位点的鉴定。我们证实了去泛素化酶催化半胱氨酸残基的探针标记，并揭示了意想不到的去泛素化酶在非催化半胱氨酸残基上和非去泛素化酶蛋白上的标记。这样，我们确定 ZUFSP（ZUP1）是一种以前未注释的去泛素化酶，对切割 K63 连接链具有高选择性。ZUFSP与复制蛋白 A（RPA）复合物相互作用并调节其泛素化。我们的基于反应位点的化学蛋白质组学方法广泛适用于鉴定共价分子的反应位点，这可能会扩展我们对酶机制的理解。
