Testing the HYPE: FIC-mediated Adenylylation/Ampylation in Eukaryotic Signaling
Abstract
Post-translational modifications (PTMs) are integral regulators of protein function in the cell. Decades of research on PTMs such as phosphorylation, glycosylation, methylation, acetylation and ubiquitination, to name a few, have provided great insight into cellular signal transduction. In comparison, adenylylation, more recently called AMPylation- which entails the covalent addition of an AMP (adenosine monophosphate) to target protein- remains less understood. Recently, a family of enzymes, defined by the presence of a Fic domain, was found to function as a new class of adenylyltransferases. These enzymes are characterized by the presence of a conserved HxFx(D/E)GN(G/K)RxxR sequence motif, which forms the Fic active core. While bacterial Fic proteins are known to regulate pathogenesis by inhibiting host-signaling cascades, studies on eukaryotic Fic proteins conducted by us and others, suggest a role in cellular stress responses. This thesis entails a cellular and enzymatic characterization of the sole human Fic protein, HYPE/FicD and the identification of three novel substrates of HYPE-mediated adenylylation: an Hsp70 molecular chaperone, BiP; α-Synuclein, the protein whose aggregation leads to Parkinson’s disease; and Vimentin, a building block of intermediate filaments. We show that HYPE is upregulated upon endoplasmic reticulum (ER) stress and is critical for maintaining ER homeostasis by regulating a key pathway called the unfolded protein response (UPR). Specifically, we elucidate that HYPE localizes to the ER lumen where it adenylylates BiP, a key sentinel for UPR regulation and protein folding. In vitro, adenylylation enhances BiP’s ATPase activity. Further, we determine the kinetic parameters governing HYPE-mediated adenylylation of BiP, and offer insights for the HYPE-BiP interaction. Finally, our in vitro biochemical assays, tissue culture based cellular assays and in vivo studies reveal that HYPE targets a diverse range of protein substrates, suggesting that adenylylation represents a highly versatile mode of regulating cellular signaling in humans. The goal of this scientific endeavor is to enrich the understanding of this novel PTM in eukaryotes, which has already given us an appreciation of the multifarious nature of HYPE’s physiological substrates and keen insights for future studies directed at targeting adenylylation for disease therapy.
Degree
Ph.D.
Advisors
Mattoo, Purdue University.
Subject Area
Molecular biology|Cellular biology|Biochemistry
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