Molecular Mechanism of Mule in DNA Damage Response and Tumorigenesis

Summary

Principal Investigator: Qing Zhong
Affiliation: University of California
Country: USA
Abstract: DESCRIPTION (provided by applicant): Cancer remains a major killer that impacts a large number of new patients every year. One leading reason for cancer is an inappropriate response to DNA damage. Upon DNA damage, the cell will initiate apoptosis through the mitochondria apparatus when DNA damage is beyond repair. Once this cell death pathway is triggered, cytotoxic factors are released from mitochondria to activate caspases that induce apoptosis. An impaired apoptotic potential of damaged cells leads to continued cell division without restriction that frequently develops into cancer. Indeed, mis-regulation of apoptosis is associated with many cancers. Understanding the molecular mechanisms that regulate DNA damage signals and how they are transmitted to mitochondria to initiate apoptosis is therefore important for cancer research. Biochemical approaches are utilized to reconstitute DNA damage-induced apoptosis. Degradation of Mcl-1, a key anti-death protein, is required to trigger cytochrome c release from mitochondria upon DNA damage. Mcl-1 is degraded through the ubiquitin-proteasome pathway. In the ubiquitin-proteasome system, the ubiquitin ligases determine the specificity and timing of substrates destruction. A biochemical assay was established to search for such enzyme from human cell extracts, and a novel ubiquitin ligase Mule (Mcl-1 ubiquitin ligase e3) was cloned. Mule promotes ubiquitin modification of Mcl-1 through direct interaction through a BH3 domain, and Mule is indispensable for Mcl-1 mediated apoptotic pathway. Besides Mcl-1, Mule also ubiquitinates other substrates including p53 thus adding another intriguing link to the apoptosis pathway. In this proposal, the mechanism by which Mule activation and the differential regulation of Mule target different substrates in response to DNA damage signals will be investigated. The biological function of Mule in DNA damage response will be studied in Aim 1. Our newly generated Mule knockout mouse embryonic fibroblast cells will be utilized to study the function of Mule in DNA damage induced apoptosis and cell cycle checkpoints. We will also investigate the structure and function relationship of Mule, especially the requirement of the ubiquitin ligase activity of Mule for its function in DNA damage response in this aim. We will characterize the ubiquitination and degradation of Mcl-1 and p53 by Mule in Aim 2. Ubiquitin chain formation, E3 activity (Mule versus Mdm2), the contribution of Mcl-1 and p53 in Mule-dependent DNA damage response, and regulatory mechanisms of Mcl-1 and p53 ubiquitination will be investigated in this aim. In Aim 3, we will further characterize the interaction of Mule with its substrates;especially map the binding site of p53 to Mule. We will study how the interaction between Mule and substrates are regulated by DNA damage. More importantly, we will search for novel protein factors modulating Mule activity through tandem affinity purification. In Aim 4, we will put more focus on post- translation modification of Mule, especially phosphorylation of Mule in DNA damage response and discuss potential mechanisms involved in it. These experiments should reveal novel mechanisms mediating DNA damage signals and how they are transduced through Mule, thereby providing fundamental insights into regulatory networks controlling apoptosis and DNA damage response. A mechanistic understanding of this pathway will help us decipher how apoptosis is deregulated in cancer and potentially identify new targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Misregulation of apoptosis is considered to be one of the major mechanisms for tumorigenesis, especially upon exposure to exogenous DNA damaging agents and during normal physiological processes. This proposal will disclose a novel mechanism mediating DNA damage signals, thereby providing fundamental insights into regulatory networks controlling apoptosis. A mechanistic understanding of this pathway will help us decipher how apoptosis is deregulated in cancer and potentially identify new targets for therapeutic intervention.
Funding Period: -------------------- - --------------------
more information: NIH RePORT

Top Publications

  1. pmc Identification of Barkor as a mammalian autophagy-specific factor for Beclin 1 and class III phosphatidylinositol 3-kinase
    Qiming Sun
    Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
    Proc Natl Acad Sci U S A 105:19211-6. 2008
  2. doi Self-eating to remove cilia roadblock
    Zaiming Tang
    College of Life Sciences Zhejiang University Hangzhou, China Center for Autophagy Research Department of Internal Medicine University of Texas Southwestern Medical Center Dallas, TX USA
    Autophagy 10:379-81. 2014
  3. pmc Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites
    Zaiming Tang
    Center for Autophagy Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA
    Nature 502:254-7. 2013
  4. pmc A tethering coherent protein in autophagosome maturation
    Dandan Chen
    Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA
    Autophagy 8:985-6. 2012
  5. doi Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy
    Weiliang Fan
    State Key Lab of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, China
    Autophagy 6:614-21. 2010
  6. pmc Autophagosome targeting and membrane curvature sensing by Barkor/Atg14(L)
    Weiliang Fan
    Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
    Proc Natl Acad Sci U S A 108:7769-74. 2011
  7. pmc Membrane curvature response in autophagy
    Livia Wilz
    Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California at Berkeley, CA, USA
    Autophagy 7:1249-50. 2011
  8. pmc The RUN domain of rubicon is important for hVps34 binding, lipid kinase inhibition, and autophagy suppression
    Qiming Sun
    Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA
    J Biol Chem 286:185-91. 2011
  9. pmc Reconstitution of leucine-mediated autophagy via the mTORC1-Barkor pathway in vitro
    Xianghua Yan
    College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan, Hubei, China
    Autophagy 8:213-21. 2012
  10. pmc A mammalian autophagosome maturation mechanism mediated by TECPR1 and the Atg12-Atg5 conjugate
    Dandan Chen
    Division of Biochemistry, Biophysics, and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
    Mol Cell 45:629-41. 2012

Detail Information

Publications14

  1. pmc Identification of Barkor as a mammalian autophagy-specific factor for Beclin 1 and class III phosphatidylinositol 3-kinase
    Qiming Sun
    Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
    Proc Natl Acad Sci U S A 105:19211-6. 2008
    ....
  2. doi Self-eating to remove cilia roadblock
    Zaiming Tang
    College of Life Sciences Zhejiang University Hangzhou, China Center for Autophagy Research Department of Internal Medicine University of Texas Southwestern Medical Center Dallas, TX USA
    Autophagy 10:379-81. 2014
    ..These findings therefore suggest an unexpected link among autophagy, ciliogenesis, ciliopathy, and cancers. ..
  3. pmc Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites
    Zaiming Tang
    Center for Autophagy Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA
    Nature 502:254-7. 2013
    ..This work reveals that removal of OFD1 by autophagy at centriolar satellites represents a general mechanism to promote ciliogenesis in mammalian cells. These findings define a newly recognized role of autophagy in organelle biogenesis. ..
  4. pmc A tethering coherent protein in autophagosome maturation
    Dandan Chen
    Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA
    Autophagy 8:985-6. 2012
    ..We propose that the interaction between TECPR1 and ATG12-ATG5 initiates the fusion between the autophagosome and lysosome, and TECPR1 is a TEthering Coherent PRotein in autophagosome maturation...
  5. doi Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy
    Weiliang Fan
    State Key Lab of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, China
    Autophagy 6:614-21. 2010
    ..Therefore, this study assigns a novel positive role of Keap1 in upregulating p62-mediated autophagic clearance of ubiquitin aggregates...
  6. pmc Autophagosome targeting and membrane curvature sensing by Barkor/Atg14(L)
    Weiliang Fan
    Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
    Proc Natl Acad Sci U S A 108:7769-74. 2011
    ....
  7. pmc Membrane curvature response in autophagy
    Livia Wilz
    Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California at Berkeley, CA, USA
    Autophagy 7:1249-50. 2011
    ..Consequently, this study uncovered an exciting new role for the PtdIns3KC3 complex as a potential inducer of autophagosome formation...
  8. pmc The RUN domain of rubicon is important for hVps34 binding, lipid kinase inhibition, and autophagy suppression
    Qiming Sun
    Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA
    J Biol Chem 286:185-91. 2011
    ..Furthermore, a Rubicon RUN domain deletion mutant fails to complement the autophagy deficiency in Rubicon-depleted cells. Hence, these results reveal a critical role of the Rubicon RUN domain in PI3KC3 and autophagy regulation...
  9. pmc Reconstitution of leucine-mediated autophagy via the mTORC1-Barkor pathway in vitro
    Xianghua Yan
    College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan, Hubei, China
    Autophagy 8:213-21. 2012
    ....
  10. pmc A mammalian autophagosome maturation mechanism mediated by TECPR1 and the Atg12-Atg5 conjugate
    Dandan Chen
    Division of Biochemistry, Biophysics, and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
    Mol Cell 45:629-41. 2012
    ....
  11. pmc Rubicon controls endosome maturation as a Rab7 effector
    Qiming Sun
    Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
    Proc Natl Acad Sci U S A 107:19338-43. 2010
    ..Hence, Rubicon serves as a previously unknown Rab7 effector to ensure the proper progression of the endocytic pathway...
  12. pmc Regulation of Beclin 1 in autophagy
    Qiming Sun
    University of California, Berkeley, Department of Molecular and Cell Biology, Berkeley, CA 94720, USA
    Autophagy 5:713-6. 2009
    ..Barkor shares considerable sequence homology with Atg14 in yeast, representing an evolutionary conserved autophagy specific regulatory step in early autophagosome formation...
  13. pmc Mule determines the apoptotic response to HDAC inhibitors by targeted ubiquitination and destruction of HDAC2
    Jing Zhang
    Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA
    Genes Dev 25:2610-8. 2011
    ..Taken together, our results reveal a critical regulatory mechanism of HDAC2 by Mule and suggest this pathway determines the cellular response to HDACis and DNA damage...