4 research outputs found
DEFINING THE FUNCTIONS OF HUMAN SIRTUIN 2 IN CELLULAR HOUSEKEEPING AND DURING VIRAL INFECTION
The cellular proteome is regulated at multiple levels, including transcription, translation, post-translational modifications, and degradation. This regulatory complexity allows for finely-tuned maintenance of intracellular homeostasis. NAD+- dependent deacetylases sirtuins (SIRTs) are key regulators of such processes. They are positioned to receive information about intracellular energy states and to relay this information to downstream pathways by modulating their substrates. Sirtuins have been linked to various human diseases, including cancer, neurodegeneration, and viral infection. More recently, we found that sirtuins act in antiviral response against several DNA and RNA viruses. However, how sirtuins exert these functions remains largely unknown. In my thesis work, I integrated virology, proteomics, bioinformatics, and biochemistry to investigate one of the least characterized sirtuins, the cytoplasmic SIRT2, and its cooperative functions with nuclear SIRT1. First, I describe how proteomics approaches using label-free and metabolic-labeling mass spectrometry can be applied to studying protein functions through investigation of protein-protein interactions. Next, I used these methods to investigate SIRT2, its dynamic protein interactions, and the impact of SIRT2 on the proteome of human fibroblasts. From these studies, I discovered that SIRT2 is positioned to function in intracellular trafficking through its interactions and localization at the intersection of the ER-Golgi trafficking pathways, as well as through regulation of proteins in membrane trafficking routes. Next, to assess its roles during infection, we tested the impact of sirtuin activation or inhibition on viral protein levels at different stages of infection with human cytomegalovirus (HCMV). A reduction in early and late HCMV protein levels was observed upon treatment with small molecules that activate sirtuins. This sirtuin antiviral function is evolutionarily conserved, as we show that the sirtuin homologue in E.Coli, CobB, acts against bacteriophage infections. Finally, we provide mechanistic insights into the individual and cooperative functions of SIRT1 and SIRT2 during HCMV infection. We show that simultaneous inhibition of SIRT1 and SIRT2 decreases HCMV replication in part through a p53-dependent mechanism. I also characterize SIRT2 interactions and localization during HCMV infection time course, which point to a possible contribution to regulation of viral gene expression and virion assembly
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Transcriptional Elongation of HSV Immediate Early Genes by the Super Elongation Complex Drives Lytic Infection and Reactivation from Latency
The cellular transcriptional coactivator HCF-1 is required for initiation of herpes simplex virus (HSV) lytic infection and for reactivation from latency in sensory neurons. HCF-1 stabilizes the viral Immediate Early (IE) genes enhancer complex and mediates chromatin transitions to promote IE transcription initiation. In infected cells, HCF-1 was also found to be associated with a network of transcription elongation components including the Super Elongation Complex (SEC). IE genes exhibit characteristics of genes controlled by transcriptional elongation and the SEC-P-TEFb complex is specifically required to drive the levels of productive IE mRNAs. Significantly, compounds that enhance the levels of SEC-P-TEFb also potently stimulated HSV reactivation from latency both in a sensory ganglia model system and in vivo. Thus, transcriptional elongation of HSV IE genes is a key limiting parameter governing both the initiation of HSV infection and reactivation of latent genomes
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Proteomics-Based Methods for Discovery, Quantification, and Validation of Protein–Protein Interactions
Proteins play a fundamental role in establishing the diversity of cellular processes in health or disease systems. This diversity is accomplished by a vast array of protein functions. In fact, a protein rarely has a single function. The majority of proteins are involved in numerous cellular processes, and these multiple functions are made possible by interactions with other molecules. The complexity of interactions is substantially increased by the spatial and temporal diversity of proteins. For example, proteins can be part of distinct complexes within different subcellular compartments or at different stages of the cell cycle. Post-translational modifications can regulate and further expand the ability of proteins to establish localization- or temporal-dependent interactions. This complexity and functional divergence of interactions is further increased by the simultaneous presence of stable, transient, direct, and indirect protein interactions. Thus, an understanding of protein functions cannot be fully accomplished without knowledge of its interactions. Characterizing these interactions is therefore critical to understanding the biology of health and disease systems