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“Study of the function of autophagy in adipocyte differentiation and the regulation of autophagy by Beclin 1”

by
Rebecca Baerga
B.S., 2003
Pontifical Catholic University of Puerto Rico
Ponce, Puerto Rico

Thesis Advisor: Shengkan “Victor” Jin, Ph.D.

Graduate Program in Cellular
& Molecular Pharmacology

RWJMS, Room V-10

Friday, August 29, 2008
2:00 p.m.


Abstract

The process of autophagy is a complex catabolic program for lysosomal degradation of proteins and other subcellular constituents. It is important for normal growth control and is defective in a number of cancers. Beclin 1 gene is the first mammalian gene identified involved in autophagy. Mono-allelic deletions of Beclin 1 gene are identified in 40-75% of human sporadic breast, ovarian and prostate cancers. Beclin 1 knockout mice were recently generated. Homozygous Beclin 1 knockout mice are embryonic lethal, while the heterozygous Beclin 1 knockout mice develop various tumors. These mouse genetic results provide compelling evidence that autophagy plays an important role in preventing tumorigenesis. This work also defined a novel class of tumor suppressor genes which are involved in autophagy and its regulation. However, exactly why the Beclin 1 gene and autophagy are critical for tumor suppression remains unknown.

In order to understand the molecular mechanism of how Beclin 1 is involved in autophagy, we created a cell line expressing a Flag-HA-tag Beclin 1 protein. Western blot analysis revealed that after the induction of autophagy resulting from starvation, Beclin 1 underwent a molecular weight shift. These results suggest that the protein undergoes a post-translational modification when autophagy is activated. We then opted to purify the Beclin 1 protein and its interacting partners by immunoprecipitation. Two proteins that interact specifically with Beclin 1 were purified and further identified by mass spectrometry. They are two kinases homologous to yeast Vps34 and Vps15. Furthermore, the region of the post-translational modification site was defined with a monoclonal antibody that specifically recognizes the non-modified form of Beclin-1, but not the modified one. Site-directed mutagenesis revealed the region of the post-translational modification of Beclin 1. Expression of a subset of the mutants of Beclin 1 resulted in the constitutive activation of autophagy. These mutants provide a molecular tool to study the impact of autophagy activation on cancer therapy and cancer prevention.

While investigating the role of Beclin 1 in autophagy, we noticed that there are high levels of unmodified inactive Beclin 1 protein in fat tissue. This led us to question if autophagy plays a particular role in adipocyte differentiation. A mature mammalian adipocyte has a unique structure in which nearly the entire cell volume is occupied by one large lipid droplet while other cellular components, including the nucleus and cytoplasm, localize peripherally and occupy minimal space. Clearly the differentiation of a mature adipocyte requires highly coordinated and massive cellular remodeling processes, which are poorly defined up to date. Autophagy is a cellular membrane trafficking process that leads to the lysosomal degradation of cytoplasmic components. We studied the role of autophagy in adipocyte differentiation and demonstrated that autophagy deficiency impairs this process. We found that primary mouse embryonic fibroblasts (MEFs) derived from autophagy deficient atg5-/- mice exhibit a defect in differentiation into adipocytes. These atg5-/- MEFs initially appear normal as they accumulate small lipid droplets, but the majority of these cells stall at this early differentiation stage and fail to undergo further morphological transformation. Massive autophagy activation is observed when wild type primary MEFs undergo differentiation into adipocytes, as indicated by increased levels of MAP-LC3 cleavage and increased numbers of autophagic vacuoles. Consistent with these in vitro results, defects in adipocyte differentiation are observed in vivo. Neonatal atg5-/- mice have less subcutaneous perilipin A-positive adipocytes and exhibit abnormalities in brown fat cells. Our studies provide cellular and genetic evidence that autophagy is a critical cellular process for normal adipocyte differentiation.


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