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The Structural Proteomics of S-Nitrosylation: from Global Identification Ot Elucidating Protein Function Through Structural Bioinformatics

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The Structural Proteomics of S-Nitrosylation: from Global Identification Ot Elucidating Protein Function Through Structural Bioinformatics University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2011 The Structural Proteomics of S-Nitrosylation: From Global Identification ot Elucidating Protein Function Through Structural Bioinformatics Jennifer L. Greene University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, Bioinformatics Commons, and the Biophysics Commons Recommended Citation Greene, Jennifer L., "The Structural Proteomics of S-Nitrosylation: From Global Identification ot Elucidating Protein Function Through Structural Bioinformatics" (2011). Publicly Accessible Penn Dissertations. 513. https://repository.upenn.edu/edissertations/513 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/513 For more information, please contact [email protected]. The Structural Proteomics of S-Nitrosylation: From Global Identification ot Elucidating Protein Function Through Structural Bioinformatics Abstract ABSTRACT THE STRUCTURAL PROTEOMICS OF S-NITROSYLATION: FROM GLOBAL IDENTIFICATION TO ELUCIDATING PROTEIN FUNCTION THROUGH STRUCTURAL BIOINFORMATICS Jennifer L. Greene Harry Ischiropoulos, Ph.D. S-nitrosylation is the covalent addition of nitric oxide to reduced cysteine residues on proteins. It has been well documented that not all proteins are S-nitrosylated and more specifically, not all cysteine residues within an S-nitrosylated protein are modified. Therefore, it is very important to determine how this specificity is derived. Additionally, the mechanism by which nitric oxide can modify cysteines is still unclear. Even with the discovery of functional consequences of S-nitrosylation, there are still large deficits in our understanding and validation that it is a newly identified means of nitric xideo signaling within the body. These gaps in knowledge primarily exist due to a lack of tools necessary for identifying in vivo sites of S-nitrosylation. To this end, complementary mercury-based mass spectrometric approaches were developed for the identification of endogenous S-nitrosoproteomes. This resulted in the identification of 328 SNO-cysteines coordinated to 192 proteins in the mouse liver, 97% of which corresponded to novel targets of S-nitrosylation. Bioinformatic analysis of these targets then revealed that multiple mechanisms of S-nitrosylation may occur in vivo, one of which involving S-nitrosoglutathione (GSNO). To test this hypothesis, the SNO-proteome of mice incapable of metabolizing GSNO was resolved. Quantum mechanics/molecular mechanics calculations coupled with molecular dynamics simulations proposed a novel GSNO-mediated mechanism of transnitrosation. Basic residues in the surrounding cysteine microenvironment were shown to catalyze the formation of protein S-nitrosocysteine residues. Collectively, these data suggest that the specificity of cysteines targeted for S-nitrosylation is driven by the surrounding protein microenvironment. Additionally, with only 9 structures of S-nitrosylated proteins our present understanding of the structural consequences of S-nitrosylation is limited. Using an in vivo model, attempts were made to correlate changes in enzymatic activity as a function of S-nitrosylation. Normal mode analysis revealed local motions near the site of S-nitrosylation which may alter product release. In summary, this thesis utilized a global proteomic approach to craft a more targeted investigation into the specificity and molecular mechanism of S-nitrosylation. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biochemistry & Molecular Biophysics First Advisor Harry Ischiropoulos Keywords mass spectrometry, nitric oxide, proteomics, S-nitrosoglutathione, S-nitrosylation Subject Categories Biochemistry | Bioinformatics | Biophysics This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/513 THE STRUCTURAL PROTEOMICS OF S-NITROSYLATION: FROM GLOBAL IDENTIFICATION TO ELUCIDATING PROTEIN FUNCTION THROUGH STRUCTURAL BIOINFORMATICS Jennifer L. Greene A DISSERTATION in Biochemistry and Molecular Biophysics Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2011 ___________________________________ Supervisor of Dissertation: Harry Ischiropoulos, Ph.D., Research Professor of Pediatrics and Pharmacology ___________________________________ Graduate Group Chairperson: Kathryn M. Ferguson, Ph.D., Associate Professor, Physiology Dissertation Committee Doron C. Greenbaum, Ph.D., Assistant Professor, Department of Pharmacology Cecilia Tommos, Ph.D., Assistant Professor, Department of Biochemistry and Biophysics Roland L. Dunbrack, Jr, Ph.D., Adjunct Associate Professor, Department of Biochemistry and Biophysics Ian A. Blair, Ph.D., Professor, Department of Pharmacology Gideon Dreyfuss, Ph.D., Professor, Department of Biochemistry and Biophysics Andrew Gow, Ph.D., Associate Professor, Department of Pharmacology and Toxicology THE STRUCTURAL PROTEOMICS OF S-NITROSYLATION: FROM GLOBAL IDENTIFICATION TO ELUCIDATING PROTEIN FUNCTION THROUGH STRUCTURAL BIOINFORMATICS COPYRIGHT 2011 Jennifer Lynn Greene iii “I was leaving the South to fling myself into the unknown. I was taking a part of the South to transplant in alien soil, to see if it could grow differently, if it could drink of new and cool rains, bend in strange winds, respond to the warmth of other suns, and, perhaps, to bloom.” -Richard Wright To my grandparents, Flenard and Jessie, who planted the seed and my mother, Melvelyn, who relentlessly nurtured it. iv ACKNOWLEDGMENTS I would first like to thank the entire Ischiropoulos Laboratory. I joined this lab at a time when I was unsure about my own future in science. The collaborative spirit and good nature of everyone in the lab, past and present, helped bring my scientific dreams back to life. I would like to thank my advisor, Dr. Harry Ischiropoulos, for his constant support and encouragement. Under his tutelage, I have become a far better scientist than I could have imagined. I would like to thank Richard Lightfoot, Drs. Margarita Tenopoulou and Paschalis-Thomas Doulias. Karthik Raju, Kristen Malkus, Marissa Martinez, and past members: Drs. Elpida Tsika, Todd Greco, and Christie Bruno. I would also like to thank the members of the Vanderkooi laboratory for being my lab home for my first 3 years here at Penn. I would like to thank the members of my committee for their advice and support. I’d also like to thank my friends and family, especially my mother Mel, my sister Nikoia, and my brother Bernard. And lastly, I’d like to thank my fiancé, Ebenezer. Without your love, I don’t think I would have made it. Sola fide v ABSTRACT THE STRUCTURAL PROTEOMICS OF S-NITROSYLATION: FROM GLOBAL IDENTIFICATION TO ELUCIDATING PROTEIN FUNCTION THROUGH STRUCTURAL BIOINFORMATICS Jennifer L. Greene Harry Ischiropoulos, Ph.D. S-nitrosylation is the covalent addition of nitric oxide to reduced cysteine residues on proteins. It has been well documented that not all proteins are S-nitrosylated and more specifically, not all cysteine residues within an S-nitrosylated protein are modified. Therefore, it is very important to determine how this specificity is derived. Additionally, the mechanism by which nitric oxide can modify cysteines is still unclear. Even with the discovery of functional consequences of S-nitrosylation, there are still large deficits in our understanding and validation that it is a newly identified means of nitric oxide signaling within the body. These gaps in knowledge primarily exist due to a lack of tools necessary for identifying in vivo sites of S-nitrosylation. To this end, complementary mercury-based mass spectrometric approaches were developed for the identification of endogenous S- nitrosoproteomes. This resulted in the identification of 328 SNO-cysteines coordinated to 192 proteins in the mouse liver, 97% of which corresponded to novel targets of S- nitrosylation. Bioinformatic analysis of these targets then revealed that multiple mechanisms of S-nitrosylation may occur in vivo, one of which involving S- vi nitrosoglutathione (GSNO). To test this hypothesis, the SNO-proteome of mice incapable of metabolizing GSNO was resolved. Quantum mechanics/molecular mechanics calculations coupled with molecular dynamics simulations proposed a novel GSNO- mediated mechanism of transnitrosation. Basic residues in the surrounding cysteine microenvironment were shown to catalyze the formation of protein S-nitrosocysteine residues. Collectively, these data suggest that the specificity of cysteines targeted for S- nitrosylation is driven by the surrounding protein microenvironment. Additionally, with only 9 structures of S-nitrosylated proteins our present understanding of the structural consequences of S-nitrosylation is limited. Using an in vivo model, attempts were made to correlate changes in enzymatic activity as a function of S-nitrosylation. Normal mode analysis revealed local motions near the site of S- nitrosylation which may alter product release. In summary, this thesis utilized a global proteomic approach to craft a more targeted investigation into the specificity and molecular mechanism of S-nitrosylation. vii TABLE OF CONTENTS TITLE PAGE……………………………………………………………………………...i DEDICATION…………………………………………………………………………..iii ACKNOWLEDGMENTS…………………………………………………………….....iv ABSTRACT……………………………………………………………………………...v TABLE OF CONTENTS………………………………………………………………..vii
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