טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBen-Lulu Shani
SubjectProteomic and Functional Analysis of Protein Cysteine
Nitrosylation in Macrophages
DepartmentDepartment of Medicine
Supervisor Professor Moran Benhar
Full Thesis textFull thesis text - English Version


Abstract

Protein S-nitrosylation (SNO), the covalent attachment of nitric oxide (NO) to cysteine thiols, is emerging as a key mechanism by which NO regulates cell signaling. S-nitrosylation is a precisely targeted and rapidly reversible post-translational modification that allows cells to flexibly and specifically respond to changes in their environment.

Thioredoxins (Trxs) are ubiquitous proteins that play vital roles in many processes in living cells.  Trxs have a conserved Cys-Gly-Pro-Cys redox-active site that is essential for their function as oxidoreductases. Recent reports have shown that Trx denitrosylates multiple S-nitrosylated proteins under physiologically relevant conditions. Progress towards the identification of the complete set of Trx SNO-substrates is important in order to better define the range of NO-related cellular processes that are regulated by Trx.

The primary goals of this research were: (1) Identification of proteins and pathways in macrophages that are regulated by reversible S-nitrosylation; (2) Characterization of the scope and role of Trx-mediated denitrosylation in macrophages.

A known strategy to identify oxidized target proteins of Trx family members is mechanism-based kinetic trapping, using a mutant Trx protein in which the second cysteine in the active-site is eliminated (TrxC35S). In this study, we developed a Trx-based trapping as a powerful approach to identify macrophage proteins regulated by S-nitrosylation. We found that Trx(C35S) was able to trap many proteins in THP-1 cells challenged with S-nitrosocysteine, with trapping increased when cells were pretreated with inhibitors of Trx-reductase (TrxR). Trx(C35S) was also capable of capturing multiple nitrosylated proteins from macrophages activated by endotoxin and interferon-γ.

By coupling Trx-dependent substrate trapping with mass spectrometry-based proteomics we identified more than 400 putative nitroso-proteins in S-nitrosocysteine-treated THP-1 monocytic cells and about 200 nitrosylation substrates in stimulated macrophages. Validating the trapping approach, we identified several known SNO targets of Trx, such as caspase-3 and GAPDH. We also identified over 20 known SNO targets for which the denitrosylation mechanism was unknown. Importantly, the large majority of the identified proteins represent novel nitrosylation targets and they include many proteins that play key roles in macrophage homeostasis and inflammatory signaling.

Biochemical and functional experiments validated the proteomic results and further suggested a role for Trx in the denitrosylation and activation of inducible NO synthase and the protein kinase MEK1.

Our findings contribute to a better understanding of the macrophage S-nitrosoproteome and the impact of Trx-mediated denitrosylation on NO signaling and macrophage function.