Autophagy is a conserved cellular response which serves to recycle macromolecules during nutrient limitation. In mammals, it involves the sequestration of cytoplasmic material into double-membraned vesicles termed autophagosomes, which are transported to the lysosome, degraded and recycled. In recent years, aberrant autophagy has been implicated in the etiology of several pathological human states including neurodegeneration, infection and cancer. A number of genes have been identified which are responsible for proper autophagosomal biogenesis and function. One of these genes, beclin-1, has been shown to function as a tumor suppressor in mice and resides at a genomic position frequently mutated in human cancers. The Beclin-1 protein is thought to activate autophagy by modulating the activity of the lipid-kinase, Vps34. However, it also interacts with the cells suicide machinery by binding the anti-apoptotic protein Bcl-2. Through this interaction, Bcl-2 is thought to exert an inhibitory effect on autophagy. The precise molecular mechanisms by which Beclin-1 interacts with and regulates Bcl-2 and Vps34 are presently unclear. So are the mechanisms by which the three proteins co-regulate each other, as well as autophagy and apoptosis.
We initiated structural and cell biological studies of Beclin-1 in order to gain insights into its molecular function. Using X-ray crystallography and biochemical assays we identified a Bcl-2 Homology 3 (BH3) domain in Beclin-1. By analyzing the interaction surface between the Beclin-1 BH3 domain and a close homolog of Bcl-2, Bcl-XL, we were able to show that the Beclin-1 binds a hydrophobic groove of Bcl-XL in a similar manner as other pro-apoptotic “BH3-only” proteins. Mutations in the BH3 domain of Beclin-1 lower its affinity for Bcl-XL, and disrupt association with Bcl-XL. This analysis implied that Beclin-1 is a novel BH3-only protein. Since all previously identified BH3-only proteins are thought to initiate apoptotic cell death, we proposed that Beclin-1 might have a cryptic pro-apoptotic function and that this function might be related to its ability to coordinate autophagy and apoptosis, as well as its tumor suppressor function.
Our cell biological studies revealed a previously unidentified phenotype upon RNA interference (RNAi) mediated suppression of Beclin-1. Suppression of Beclin-1 led to massive accumulation of large, phase-lucent, intracellular vacuoles, which were identical to those observed upon suppression of human Vps34. These vacuoles had characteristics of late-endosomes, and were likely due to depletion of phoshatidyinositol-3-phosphate (PtdIns(3)P) at endosomal membranes due to destabilization of the Vps34 holoenzyme. Surprisingly, autophagy appeared to remain intact in both Beclin-1 and Vps34 suppressed cells. We observed numerous autophagic structures and normal autophagic proteolysis in both cases, suggesting that PtdIns(3)P may be dispensable for autophagy in some mammalian cell types. We did, however, observe defects in the recruitment of two important autophagy-associated proteins, suggesting that Beclin-1/Vps34/PtdIns(3)P may have a role in selective, rather than non-selective, autophagocytosis. We speculate that this role for Beclin-1 in selective autophagy might be related to its ability to suppress tumors.
|School Location:||United States -- New Jersey|
|Source:||DAI-B 71/11, Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology|
|Keywords:||Autophagy, Intracellular vacuoles, PI3-kinase, Protein Beclin-1, Vps34|
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