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Quantitative Profiling of Cysteine S‐nitrosylation Susceptibility in Cancer
Author(s) -
Zhou Yani,
Convertier Shalise,
Weerapana Eranthie
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.lb145
Subject(s) - cysteine , s nitrosylation , chemistry , serine , proteolysis , proteome , nitrosylation , biochemistry , phosphorylation , nitric oxide , organic chemistry , enzyme
We aimed to quantitatively measure the susceptibility of cysteine S‐nitrosylation (SNO), the transfer of a NO moiety to cysteine (Cys) on protein. SNO represents a form of redox modulation and plays an important role in diverse cellular processes. Recent developments of chemical methods to detect SNO have made it possible to identify SNO‐Cys sites. However, these methods do not monitor the extent of Cys to undergo nitrosylation in a complex proteome. We utilized activity‐based protein profiling to identify nitrosylated Cys and to measure the susceptibility of Cys SNO in cell lysates after treatment with NO donor. A gel based dose‐dependent experiment revealed that Cys residues on different proteins demonstrated diverse susceptibilities to SNO. Isotopic tandem orthogonal proteolysis‐activity‐based protein profiling (isoTOP‐ABPP) method was applied to further quantitatively monitor the SNO susceptibility. Several novel Cys SNO targets were identified and verified by overexpression in mammalian cells. One of these is the serine/threonine‐protein kinase 1 (AKT1) which plays a critical role in many different signaling pathways, and might contribute to tumor progression. Our study showed that one Cys on AKT1 is highly susceptible to SNO. We are exploring structural motifs that govern this selectivity, and further investigate the role of nitrosylation in modulating AKT1 activity in vivo .