Life-or-death decisions are critical for all organisms. The balance of the related biological processes determines the cell fate. Most if not all prominent human diseases are affected by the dysregulation of cell death. The mammalian cells mainly die through three types of cell death, namely apoptosis (type I), autophagy (type II) and necrosis (type III), in a programmed and regulated manner. Interestingly, emerging evidence suggest that autophagy also serves as a survival mechanism to inhibit apoptosis and necrosis. My lab studies how these programmed cell death pathways are regulated. We emphasize on the biochemical characterization of the key steps in these pathways in mammalian cells. Utilizing biochemical and genetic approaches, we seek to identify and characterize the key regulators. My long-term goal is to biochemically reconstitute these biological processes. The knowledge will help us to identify drug-able targets and screen small-molecular compounds to interfere these activities, which should provide novel therapeutic tools to human diseases including cancers and other aging related diseases.
Project 1: Mechanism of autophagy
Mechanism of autophagosome biogenesis: During autophagy, a double membrane autophagosome engulfs cargo such as damaged organelles, cytosol, and microorganisms and then fuses with lysosome where its internal components are degraded by acid hydrolases. The formation of autophagosome is driven by the combined effort of lipids and proteins. Phosphatidylinositol 3-phosphate (PtdIns(3)P) has been shown to be extremely important for autophagosome biogenesis. PtdIns(3)P production is characterized by the class III phosphatidylinositol 3-kinase (PI3KC3) that is evolutionary conserved from yeast to humans. We are the first group to purify and identify the human PI3KC3 complex, which contains seven components hVps34, p150/Vps30, Beclin 1/Atg6, UVRAG, Rubicon, Barkor/Atg14(L), and p40. Three of them, Barkor, Rubicon and p40, have not been previously linked to autophagy. Seven subunits form two mutually exclusive subcomplexes that are targeted to either autophagosome or endosome. We report that the Beclin 1-hVps34-p150 complex are targeted to autophagosome by Barkor/Atg14(L). Barkor positively regulates the PI3KC3 activity on the autophagosome membrane, through which it determines the specificity of PI3KC3 in the autophagic pathway. Moreover, we found that Barkor interacts with the autophagic membrane through its C-terminal 80 amino acids (named the BATS domain), and senses membrane curvature via an ALPS motif within the BATS domain. Hence, we propose that Barkor plays a dual role in autophagosome biogenesis by activating PI3KC3 on membrane and facilitating membrane curvature development, which are crucial for autophagosome biogenesis. The function of p40 is under investigation now. In the future study, we will focus on dissecting the signaling pathway regulating the PI3KC3 complex in autophgay, and we also aim to reconstitute the PI3KC3 lipid kinase activity and membrane deformation activity in vitro.
Selective degradation by autophagy: Autophagy degradation could be both non-selective and selective. We aim to identify selective autophagic substrates and cargo adaptors. For this purpose, we purified the cellular complex of LC3. In addition to p62 and FYCO-1, two well-known adaptors, we have also identified Keap1. Keap1 is crucial for oxidative stress response. Keap1 is a cargo adaptor but not likely a substrate. Keap1 interacts with p62 and LC3 and colocalizes with ubiquitin aggregates in a stress-inducible manner. Genetic ablation of Keap1 leads to the accumulation of ubiquitin aggregates, increased cytotoxicity of misfolded protein aggregates, and defective activation of autophagy. More interestingly, in the LC3 complex, in addition to p62, Keap1 and FYCO-1, we have also identified another set of interacting proteins, the biological significance of these interaction proteins are being studied in the lab.>
Endosome maturation by the endosomal PI3KC3 complex: The endosome-localized PI3KC3 subcomplex is important for endosome maturation. We found that two newly identified PI3KC3 subunits, Rubicon and UVRAG, are both localized to early endosomes. Rubicon functions as a negative regulator since it antagonizes UVRAG by sequestering it in the complex form. Interestingly, this sequestration is released by Rab7. GTP-bound Rab7 preferentially interacts with Rubicon and frees UVRAG.
The Atg12-Atg5 conjugate and TECPR1 in autophagosome maturation: We identified TECPR1 as an Atg12-Atg5 interacting protein, and showed that the Atg12-Atg5-TECPR1 complex plays a key role in autophagosome maturation. TECPR1 localizes to lysosomes and recruits Atg12-Atg5 to autolysosomes. This protein interaction also facilitates the protein-lipid interaction between TECPR1 and PtdIns(3)P. Genetic analyses reveal that depletion of TECPR1 leads to accumulation of autophagic vacuoles and substrates including the lipidated LC3 form and p62. The autophagy flux is also defective in TECPR1 depleted cells. The Atg12-Atg5-TECPR1 complex might mediate the fusion between autophagosomes and lysosomes.
Project 2: Function of Mule in DNA damage and HDACi induced apoptosis
Apoptosis-inducing DNA damaging agents has been widely used as anti-cancer reagents. New emerging next-generation cancer drugs like histone deacetylase inhibitors (HDACi) also elicit apoptosis. The apoptotic mechanisms underlying these drugs are not well understood. My lab is working on one such critical mediator Mule in these drugs induced apoptosis. Mule is named as Mcl-1 ubiquitin ligase E3. Mule also targets multiple substrates in the DNA damage response. In this study, we found that Mule is crucial for DNA damage and HDACi induced apoptosis, which is due to the elevated HDAC2 activity in Mule null cells. Mule specifically targets HDAC2 for ubiquitination and degradation. The accumulation of HDAC2 in Mule null cells leads to severe defects in p53 acetylation, phosphorylation, accumulation, transcriptional activation and apoptosis upon stress. These phenotypes could be partially reversed by administration of HDACi, and fully rescued by lowering elevated HDAC2 protein levels in Mule null cells. This study identifies the Mule-HDAC2 pathway as key mediators in the apoptotic response to HDACi and DNA damage, and this knowledge might be important for the HDACi application in cancer treatment.
The molecular mechanism underlying the crosstalk among apoptosis, autophagy and necrosis are also being extensively studied in the lab.
Chen D, Fan W, Lu Y, Ding X, Chen S, Zhong Q. A mammalian autophagosome maturation mechanism mediated by TECPR1 and the Atg12-Atg5 conjugate. Mol Cell, 2012 In press.Zhang J, Kan S, Huang B, Hao Z, Mak TW, Zhong Q. Mule determines the apoptotic response to HDAC inhibitors by targeted ubiquitination and destruction of HDAC2. Genes Dev. 2011 Dec 15;25(24):2610-8.
Fan W, Nassiri A, Zhong Q. Autophagosome targeting and membrane curvature sensing by Barkor/Atg14(L).Proc Natl Acad Sci U S A. 2011 May 10; 108(19):7769-7774.
Sun Q, Westphal W, Wong K, Tan I, Zhong Q. Rubicon controls endosome maturation as a Rab7 effector. Proc Natl Acad Sci U S A. 2010 Nov 9; 107(45):19338-43.
Sun Q, Fan W, Chen K, Ding X, Chen S, Zhong Q. Identification of Barkor as a Mammalian Autophagy-Specific Factor for Beclin 1 and Class III Phosphatidyl-inositol 3-Kinase. Proc Natl Acad Sci U S A. 2008 Dec 9;105 (49):19211-6.
Zhong Q, Gao W, Du F, Wang X. Mule/ARF-BP1, a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis. Cell. 2005 121(7): 1085-1095.
Zhong Q, Chen C-F, Li S, Chen Y, Wang C-C, Xiao J, Chen P-L, Sharp ZD, and Lee WH: Association of BRCA1 with the hRad50-hMre11-p95 Complex and the DNA Damage Response. Science, 1999 Jul 30;285: 747-750.
Last Updated 2012-02-13