PROTEIN S-NITROSYLATION: A SWITCH FOR REDOX-­SIGNALING IN CARDIOVASCULARSYSTEM

In cellular physiology, nitric oxide plays an important role in the regulation of redox signaling through the cGMP-dependent pathway. It also signals through an oxidative post-translational modification of cysteine thiols, S-nitrosylation (SNO, also known as S-nitrosation), which in turn regulates protein function. This modification is implicated in the cardiovascular system in both normal and disease states. However, the SNO modification is labile and thus, it’s been challenging to detect, especially coupled with mass spectrometry-based analysis. This thesis combines two parts; a method to detect the SNO of cysteine (chapter 2 and 3) and its applications to redox-proteomics with subsequent functional analysis to investigate the role of this modification in the heart (chapter 4). First, a method to utilize a newly released tag, iodoTMT (TMT=tandem mass tag), was optimized to identify SNO-proteins. Next, sub-populations of SNO-cysteine which have different chemical environments were revealed. Based on this observation, a proteomics workflow was developed to maximize the coverage of SNO-proteome using a dual-labeling strategy and tested in GSNO reductase knock out mouse hearts, where SNO should be maximal. This technique was applied to profile SNO sites in a disease model of interest, Duchenne muscular dystrophy (DMD). DMD is a muscle disorder that induces severe skeletal and cardiac dysfunction and ultimately cell death. In this thesis, a DMD mouse model where transient receptor potential canonical channel type-6 (TRPC6) gene was deficient was used, since TRPC6 was reported to be linked with the DMD phenotype in cardiomyocytes, underlying excessive Ca2+ entry. And changes in Ca2+ concentration were also reported to be linked to nitric oxide production in previous studies. Here, the impact of TRPC6 on DMD on muscle functionality in vivo was determined as SNO-proteins were identified and compared to DMD and control hearts. Taken together the functional relevance of TRPC6/SNO in DMD hearts, a set of potential targets for DMD was proposed. The findings presented in this thesis provide technical detail for the detection of SNO, a modification on cysteine thiols and new insights into the regulation of the cardiovascular system via the critical cysteine redox-modification

Dual Labeling Biotin Switch Assay to Reduce Bias Derived From Different Cysteine Subpopulations

RATIONALE: S-nitrosylation (SNO), an oxidative post-translational modification of cysteine residues, responds to changes in the cardiac redox-environment. Classic biotin switch assay and its derivatives are the most common methods used for detecting SNO. In this approach, the labile SNO group is selectively replaced with a single stable tag. To date, a variety of thiol-reactive tags have been introduced. However, these methods have not produced a consistent dataset which suggests an incomplete capture by a single tag and potentially the presence of different cysteine subpopulations. OBJECTIVE: To investigate potential labeling bias in the existing methods with a single tag to detect SNO, explore if there are distinct cysteine subpopulations, and then, develop a strategy to maximize the coverage of SNO proteome. METHODS AND RESULTS: We obtained SNO-modified cysteine datasets for wild-type and S-nitrosoglutathione reductase (GSNOR) knock-out mouse hearts (GSNOR is a negative regulator of GSNO production) and NO-induced human embryonic kidney cell using two labeling reagents; the cysteine-reactive pyridyldithiol and iodoacetyl based tandem mass tags. Comparison revealed that <30% of the SNO-modified residues were detected by both tags, while the remaining SNO sites were only labeled by one reagent. Characterization of the two distinct subpopulations of SNO residues indicated that pyridyldithiol reagent preferentially labels cysteine residues that are more basic and hydrophobic. Based on this observation, we proposed a parallel dual labeling strategy followed by an optimized proteomics workflow. This enabled the profiling of 493 SNO-sites in GSNOR knock-out hearts. CONCLUSIONS: Using a protocol comprising two tags for dual labeling maximizes overall detection of SNO by reducing the previously unrecognized labeling bias derived from different cysteine subpopulations