Autophagy is a conserved mechanism for the clearance of inclusions, damaged organelles, and all other unneeded or harmful materials that cannot be digested by proteasomes. In humans, autophagy has major roles in cancer, Parkinson?s disease, intracellular infection, neurodegeneration, and aging. Autophagy involves the formation of a unique cup-shaped double membrane, the phagophore, which expands, engulfs cargo, and closes to form the autophagosome. How membranes are remodeled into the cup shape of the phagophore is a central and intractable question in autophagy. We will use structural biology, computer simulations, and multi- color 3D superresolution imaging of the cup to unravel the long-standing mystery of the origin and shaping of the cup. Autophagy initiation is driven by the activation and targeting of the ULK1/Atg1 protein kinase complex, and the class III phosphatidylinositol kinase complex I (PI3KC3-C1).
The specific aims of the project are to understand 1) nucleation of the phagophore, and 2) regulation of the pre-autophagosomal structure.
Aim 1 begins with the hypothesis that the unique S-shaped geometry of the scaffolding protein Atg17, discovered by this laboratory, drives remodeling of membrane into cups. This hypothesis predicts that in all eukaryotes, an S-shape or dimer of crescents should exist as part of the core autophagy initiation machinery. We will test the hypothesis that ULK1 subunit FIP200 has this role in mammalian autophagy using x-ray diffraction, electron microscopy, theoretical modeling, and cell imaging, including 3D STORM microscopy capable of visualizing these nanoscale cups in cells. We will work out whether and how ATG13, ATG101, and ULK1 cooperate in this process, and how they are organized structurally in space and time. We will examine the role of PI3KC3-C1 in cup formation through in vitro reconstitution, electron microscopy, and fluoresence imaging of synthetic and cellular systems.
Aim 2 will examine how the initiation machinery is targeted to the right location and switched on at the correct time in cells. We will characterize the molecular and structural mechanisms for targeting ULK1 to ER exit sites, where starvation-induced phagophore nucleation occurs. We will determine the core regulatory circuitry that controls PI3KC3-C1 activity. We will test the hypothesis that the VPS15 protein kinase regulates the assembly and stability of PI3KC3-C1, and subsequently regulates VPS34 kinase activity in an allosteric and non-catalytic manner. Using cryo-EM, mass spectrometry, biochemistry, and cell imaging, we will determine whether and how this circuitry is used in the regulation of PI3KC3-C1 activity by ULK1 phosphorylation and by the binding of the proteins NRBF2, Bcl-2, and AMBRA1. We will also explore the hypothesis that PI3KC3 can be regulated at the level of membrane binding through the BECN1 BARA domain, using as a model system the PI3KC3-C2-Rubicon complex.
Autophagy is an ancient and conserved mechanism for cell survival under starvation and stress, with profound connections to aging, cancer, neurodegenerative diseases, and other complex diseases. Autophagy involves the formation of a unique cup shaped membrane, and is initiated by the ULK1/Atg1 and class III PI 3-kinase complexes. Using novel protein engineering, structural, biochemical and cell biological approaches, we will show how the cup shaped membrane is formed and how the process is switched on and off.
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|Bahrami, Amir Houshang; Lin, Mary G; Ren, Xuefeng et al. (2017) Scaffolding the cup-shaped double membrane in autophagy. PLoS Comput Biol 13:e1005817|
|Stjepanovic, Goran; Baskaran, Sulochanadevi; Lin, Mary G et al. (2017) Unveiling the role of VPS34 kinase domain dynamics in regulation of the autophagic PI3K complex. Mol Cell Oncol 4:e1367873|
|Stjepanovic, Goran; Baskaran, Sulochanadevi; Lin, Mary G et al. (2017) Vps34 Kinase Domain Dynamics Regulate the Autophagic PI 3-Kinase Complex. Mol Cell 67:528-534.e3|
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|Lin, Mary G; Hurley, James H (2016) Structure and function of the ULK1 complex in autophagy. Curr Opin Cell Biol 39:61-8|
|Young, Lindsey N; Cho, Kelvin; Lawrence, Rosalie et al. (2016) Dynamics and architecture of the NRBF2-containing phosphatidylinositol 3-kinase complex I of autophagy. Proc Natl Acad Sci U S A 113:8224-9|
|Davies, Christopher W; Stjepanovic, Goran; Hurley, James H (2015) How the Atg1 complex assembles to initiate autophagy. Autophagy 11:185-6|
|Köfinger, Jürgen; Ragusa, Michael J; Lee, Il-Hyung et al. (2015) Solution structure of the Atg1 complex: implications for the architecture of the phagophore assembly site. Structure 23:809-818|
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