Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
Biochemistry. 2010 Aug 17;49(32):6963-9. doi: 10.1021/bi100619k.
Reversible protein cysteine nitrosylation (S-nitrosylation) is a common mechanism utilized in signal transduction and other diverse cellular processes. Protein denitrosylation is largely mediated by cysteine denitrosylases, but the functional scope and significance of these enzymes are incompletely defined, in part due to limited information on their cognate substrates. Here, using Jurkat cells, we employed stable isotope labeling by amino acids in cell culture (SILAC), coupled to the biotin switch technique and mass spectrometry, to identify 46 new substrates of one denitrosylase, thioredoxin 1. These substrates are involved in a wide range of cellular functions including cytoskeletal organization, cellular metabolism, signal transduction, and redox homeostasis. We also identified multiple S-nitrosylated proteins that are not substrates of thioredoxin 1. A verification of our principal findings was made in a second cell type (RAW264.7 cells). Our results point to thioredoxin 1 as a major protein denitrosylase in mammalian cells and demonstrate the utility of quantitative proteomics for large-scale identification of denitrosylase substrates.
蛋白质半胱氨酸硝基化（S-硝基化）的可逆反应是信号转导和其他多种细胞过程中常用的一种机制。蛋白质去硝基化主要由半胱氨酸去硝基酶介导，但这些酶的功能范围和意义尚未完全确定，部分原因是对其同源底物的信息有限。在这里，我们使用 Jurkat 细胞，采用细胞培养中的稳定同位素标记氨基酸（SILAC），结合生物素开关技术和质谱法，鉴定了一种去硝基酶，即硫氧还蛋白 1 的 46 个新底物。这些底物涉及广泛的细胞功能，包括细胞骨架组织、细胞代谢、信号转导和氧化还原稳态。我们还鉴定了多个不是硫氧还蛋白 1 底物的 S-硝基化蛋白。在第二种细胞类型（RAW264.7 细胞）中对我们的主要发现进行了验证。我们的结果表明，硫氧还蛋白 1 是哺乳动物细胞中主要的蛋白质去硝基酶，并证明了定量蛋白质组学在大规模鉴定去硝基酶底物方面的实用性。
