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A Analysis of protein-protein interactions of an mRNA degradation complex

by
Frances Gratacos
B.S. Industrial Biotechnology
University of Puerto Rico-Mayaguez - 2003




Thesis Advisor: Gary Brewer, Ph.D.
Graduate Program in Molecular Genetics,
Microbiology & Immunology

Microbiology Department Conference Room 747
RJWMS Research Tower
Piscataway

Monday, August 30, 2010
11:00 a.m.


Abstract

In mammals, many mRNAs encoding inflammatory cytokines are destabilized by AREs in their 3 UTRs. Rapid decay of these transcripts is associated with binding of proteins to the ARE. Previous studies have shown in vitro homodimerization of the AUBP AUF1, as well as interaction among different AUF1 isoforms. AUF1 oligomerization at the ARE site promotes the assembly of a large protein complex containing cap-dependent translation initiation factors and heat shock chaperones that recruit mRNA degradation enzymes. We call this multi-protein assembly AUBP and Signal Transduction-Regulated Complex (ASTRC). Monocyte adhesion at sites of tissue injury and inflammation results in ASTRC-mediated stabilization of cytokine mRNAs. We recently demonstrated that upon adhesion of monocytes to extracellular matrix, there are signaling-induced changes in ASTRC subunit composition and in the phosphorylation state of AUF1. We also identified chaperone Hsp27 as a novel AUBP within ASTRC. Hsp27 is a ubiquitous and multifunctional protein that mediates protein ubiquitination for proteosome-mediated degradation. Phosphorylation of Hsp27 promotes proteasome-dependent degradation of AUF1, resulting in stabilization of the Tumor necrosis factor- ARE-mRNA. Here, we investigated different binary protein-protein interactions between AUF1 itself and between other members of ASTRC, including: p37AUF1/p37AUF1 and p40AUF1/p40AUF1 homodimers, as well as p37AUF1/p40AUF1 heterodimers. We also examined effects of AUF1 phosphorylation on the p40AUF1/p40AUF1 homodimer and whether phosphorylation of Hsp27 alters its interaction with AUF1. For these studies we employed live-cell fluorescence resonance energy transfer (FRET) coupled with confocal microscopy. The characteristic of energy transfer as a proximity indicator between molecules helped us determine average distances between fluorescently tagged CFP/YFP fusion proteins of AUF1 and Hsp27. Our results confirmed homo-and heterodimerization of AUF1 isoforms that is not affected by phosphorylation. FRET also revealed effects of Hsp27 phosphorylation on its association with AUF1, which might impact accessibility of AUF1 to proteasomes, and hence, cytokine mRNA degradation.


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