Article

Identification of S-nitrosylated targets of thioredoxin using a quantitative proteomic approach.

Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
Biochemistry (impact factor: 3.42). 08/2010; 49(32):6963-9. DOI:10.1021/bi100619k
Source: PubMed

ABSTRACT 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.

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Keywords

46 new substrates
 
cell culture
 
cellular functions
 
cognate substrates
 
common mechanism utilized
 
cysteine denitrosylases
 
denitrosylase substrates
 
diverse cellular processes
 
Jurkat cells
 
large-scale identification
 
major protein denitrosylase
 
mammalian cells
 
multiple S-nitrosylated proteins
 
principal findings
 
quantitative proteomics
 
redox homeostasis
 
results point
 
Reversible protein cysteine nitrosylation
 
stable isotope labeling
 
thioredoxin 1
 

Moran Benhar