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Discovery and Characterization of Novel Bioactive Peptides and a Natural Erralpha Ligand Discovery and Characterization of Novel Bioactive Peptides and a Natural ERRalpha Ligand The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Schwaid, Adam. 2013. Discovery and Characterization of Novel Bioactive Peptides and a Natural ERRalpha Ligand. Doctoral dissertation, Harvard University. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:11181064 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Discovery and Characterization of Novel Bioactive Peptides and a Natural ERR α Ligand A dissertation presented by Adam Schwaid to The Department of Chemistry and Chemical Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Chemistry Harvard University Cambridge, Massachusetts August, 2013 © 2013 Adam Schwaid All rights reserved. Dissertation Advisor: Professor Alan Saghatelian Adam Schwaid Discovery and Characterization of Novel Bioactive Peptides and a Natural ERR α Ligand Abstract Metabolites and peptides have a central role in biology that is often overlooked. Despite the importance of metabolites in key protein-metabolite interactions (PMIs), the extent and identity of these interactions is not known. Likewise, the extent to which short open reading frames (sORFs) in the genome are translated into peptides has also been an elusive question. This dissertation describes the development and application of methods to elucidate unknown molecules and interactions critical to understanding biology, and the subsequent characterization of the biological roles of these discoveries in cells and mice. A liquid chromatography-mass spectrometry (LC-MS)-based metabolomics approach was used to discover that cholesterol is a ligand for Estrogen Related Receptor alpha (ERR α), an orphan nuclear receptor critical in numerous biological processes including metabolism, bone growth, and certain cancers. Despite intense study over the 25 years since this nuclear receptor was discovered there is no known endogenous ligand for this nuclear receptor. The discovery of cholesterol as a natural ERR α ligand allows for the understanding of how ERR α driven pathways are regulated and enables the modulation of receptor activity levels through control of cholesterol levels. In addition, I also helped pioneer the development of a peptidomics strategy to discover novel sORF-encoded polypeptides (SEPs). Using our approach, we identified iii 86 novel SEPs. To further the discovery and characterization of these molecules, I collaborated on the development of a chemoproteomics approach to discover cysteine- containing SEPs (ccSEPs), leading to the identification of a further 17 SEPs. In total, 103 novel SEP, representing 103 novel human genes with unknown functions are now known. To characterize SEPs, I developed a new workflow that relies on transcriptomics to characterize the functions of novel SEPs, and found that SEPs regulate gene expression. Based on changes in gene expression, SEPs can be assigned to several putative pathways. In one case, this analysis revealed that overexpression of the SEP results in a gene expression profile associated with addition of TNF α, which was confirmed by further biochemical characterization indicating this SEP promotes inflammation. More importantly, by establishing this approach, I have demonstrated a general strategy for elucidating the functions of SEPs. iv Acknowledgements I owe a great deal of thanks to Alan Saghatelian for his guidance and mentoring over the course of my Ph.D. If it were not for his willingness to take me under his wing I would never have had this opportunity to learn and grow as a scientist. His tutelage has allowed me to develop the skills to pose interesting scientific questions and answer them experimentally. Alan has fostered a uniquely collegial, collaborative, and friendly atmosphere in his laboratory that encourages the free flow of ideas and promotes teamwork amongst his students. I would also like to thank my colleagues in the Saghatelian lab, from whom I learn every day. I would like to thank Sarah Slavoff for teaching me everything I know about experimental molecular biology, and frequent scientific conversations about varied topics—both relevant and irrelevant to our research. I would like to thank Andrew Mitchell who first mentored me when I joined the Saghatelian lab, and taught me how to ask the right questions when tackling mountains of information; Yui Vinayavekhin for her friendly welcome into the Saghatelian lab and her fastidious experimental advice; Anna Mari Lone for her tutelage in peptidomics, and good natured sense of humor; Amanda McFedries for her expertise in protein expression and all aspects of biochemistry—in addition to her keen insights into prospective business ventures; Tejia Zhang for her expertise in metabolomics and advice regarding experiments; Jiao Ma for her dedication, and quick mastery of new topics; Edwin Homan for his insights on mass spectrometry and science in general; Bogdan Budnik and John Neveau for advice on mass spectrometry and proteomics; Whitney Nolte for v frequent and unerring advice regarding graduate school and beyond, and Mathias Leidl for keeping lab lively and convivial. Additionally, I would like to thank my colleagues in the Verdine Lab and Gregory Verdine for their counsel during the course of my Master’s work. The support of my family and friends was crucial to the completion of this dissertation. Their encouragement kept me going during the most challenging periods of my studies. In particular I would like to thank Steve Hershman for his prescient advice in the face of limited information. Most importantly, Julie French’s unwavering faith and understanding in me formed the cornerstone of my strength without which my studies would have crumbled. vi Table of Contents Chapter 1: Methods for the Elucidation of Protein-Small Molecule Interactions ............. 1 1.1 Introduction ............................................................................................................ 2 1.2 Small molecule-to-protein ...................................................................................... 3 1.2.1 Small-molecule affinity methods ...................................................................... 3 1.2.1 Proteomic target identification ......................................................................... 7 1.2.1 Chemoproteomic target identification ............................................................ 12 1.3 Protein-to-small molecule .................................................................................... 16 1.3.1 Biophysical identification of small molecule binders ...................................... 16 1.3.2 Affinity-based identification of small molecule binders .................................. 18 1.4 Conclusions ......................................................................................................... 23 1.5 References .......................................................................................................... 24 Chapter 2: Cholesterol is a Natural ERR α Ligand ........................................................ 28 2.1 Introduction .......................................................................................................... 29 2.2 Discovery of an endogenous ERR α Binder ......................................................... 30 2.3 Structural Analysis of ERR α Cholesterol Binding ................................................ 36 2.4 Cholesterol regulates ERR α Transcription ........................................................... 38 2.5 Cholesterol regulates ERR α activity in Osteoclastogenesis ................................ 39 2.6 Cholesterol agonism reveals a novel role for ERR α in atherosclerotic foam cell formation .................................................................................................................... 44 2.7 Cholesterol functions as an ERR α agonist in vivo ............................................... 45 2.8 Conclusion ........................................................................................................... 48 2.9 Methods ............................................................................................................... 49 2.10 References ........................................................................................................ 56 Chapter 3: Discovery and Characterization of sORF Encoded Peptides....................... 59 3.1 Introduction .......................................................................................................... 60 3.2 Discovering SEPs Encoded by Annotated Transcripts ........................................ 62 3.3 SEPs are Derived from Unannotated Transcripts ................................................ 66 3.4 SEP Translation is Initiated at Non-AUG Codons ................................................ 68 3.5 Supporting SEP length assignments ................................................................... 69 3.6 Cellular Concentrations of SEPs.......................................................................... 71 3.7 Heterologous Expression of SEPs ....................................................................... 72 3.8 SEPs Exhibit Subcellular Localization
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