Semester

Fall

Date of Graduation

2012

Document Type

Thesis

Degree Type

MS

College

School of Medicine

Department

Biochemistry

Committee Chair

Maxim Sokolov

Committee Co-Chair

Peter Mathers

Committee Member

Visvanathan Ramamurthy

Committee Member

Peter Stoilov

Committee Member

Eric Tucker

Abstract

Chaperonins are ubiquitous molecular chaperones that are found in all animal kingdoms. They all share a common structure and function to assist the folding of other proteins. All chaperonins consist of two stacked rings, which come together to form a central cavity where folding can take place. The eukaryotic cytosolic chaperonin containing TCP1 (CCT) is a member of the group II chaperonins, which are defined as having octameric or nonameric rings composed of more than one type of subunit.;CCT is an essential part of eukaryotic cell function, and it has been estimated that it folds up to 10% of newly synthesized cytosolic proteins (3). Some of the proteins that have been shown to depend on CCT for their biogenesis include cytoskeletal proteins (alpha-, and beta-actin, alpha-, beta-, and gamma-tubulin), histone deacetylases (HDAC3, Set3p, and Hos2p), cell cycle regulators (Cdc20p, and Cdc55p), and proteins possessing the seven-bladed beta-propeller WD40 domain, including the beta-subunit of the heterotrimeric guanine nucleotide-binding protein (G-protein) complex (4, 5). Heterotrimeric G proteins are a highly conserved group of molecules involved in a great number of signaling processes. They are composed of three subunits (alpha-, beta-, and gamma), which are bound together in the inactive form. In this heterotrimeric state, guanosine di-phosphate (GDP) is bound to the G alpha-subunit. Upon activation, the alpha-subunit exchanges GDP for guanosine tri-phosphate (GTP). This exchange triggers Gbetagamma to dissociate from Galpha and both Galpha-GTP and Gbetagamma are then free to activate downstream effectors. This form of signal transduction is so common that G-proteins regulate or modulate nearly every cellular and physiological process. Because it is so critical for normal cellular function, dysregulation of G-protein signaling is associated with many human diseases (6) and more than half of the current pharmaceutical therapies target a component of the G-protein pathway (7). While much is known about the signal transduction, far less is known about the biogenesis and assembly of G-proteins.;The overall objective of my research has been to elucidate the mechanisms of the chaperonin containing Tailless Complex Polypeptide 1 (CCT) with respect to heterotrimeric guanine nucleotide-binding protein (G protein) signaling. The CCT co-factor, phosducin-like protein (PhLP) is required for Gbeta biosynthesis, and therefore its mechanism is critical for our understanding of this process. In the first chapter of my thesis, I take a structure-based approach to investigate the interaction between PhLP and Gbeta. Currently there are no available structures of this complex, nor is there a single published structure of PhLP. Therefore, I used a homology modeling approach to generate the first known 3D structure of PhLP. Then in order to gain insight into this interaction, I docked PhLP to the Gbetagamma and compared it to the crystal structure of phosducin (Pdc), a closely related protein that also forms a complex with Gbetagamma. The second chapter is centered on a truncated splice isoform of PhLP, referred to as PhLPSHORT (PhLPs), which has been shown to be a potent inhibitor of CCT. A recent paper published by my lab showed that PhLPs expression led to a decrease in alpha-, beta-, and gamma-subunits of the G-protein transducin at the protein level. Later experiments showed that this decrease occurred at the mRNA level as well. While the decrease in Gbeta and Ggamma wasn't surprising, the decrease in Galpha was unexpected since it is not a known substrate of CCT. In order to find a connection between CCT and Galpha, I looked for any perturbations of gene expression that occurred in transgenic mice constitutively expressing PhLPs using microarray results. Furthermore, because these mice express PhLPs at an early age I repeated these experiments in inducible transgenic mouse lines that we developed to express PhLPs in adult mice. Taken together, my research provides additional data to refine the mechanism of Gbeta biosynthesis and suggests that there is an additional level of regulatory control over G-proteins that connects the expression of all three subunits. (Abstract shortened by UMI.).

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