The autophagy-lysosome degradation mechanism contributes to the maintenance of cellular homeostasis by removing harmful cytoplasmic materials, such as damaged organelles and unfolded protein aggregates. The accumulation of these materials is among the primary causes of human pathologies, such as neurodegeneration, heart disease, cancer, and infection. Autophagy is initiated by the formation of specialized double-membrane vesicles termed autophagosomes, which sequester and transport such materials to lysosomes for degradation. How autophagy proteins function to generate autophagosomes and how cytotoxic materials are selectively targeted by autophagosomes are unknown. The proposed research seeks to identify key molecular mechanisms underlying autophagosome formation using a combination of biochemical and structural tools. The projects are focused on mechanisms of the autophagy-specific ubiquitin-like proteins (Ubls), Atg8 and Atg12. These Ubls play pivotal roles in autophagosome formation by controlling the unique membrane dynamics.
In Aim 1, we will identify and characterize key interactions in the E1-E2-E3 cascade that catalyze the Atg8 lipid conjugation. The E1-E2 and E2-E3 communication will be studied at a biophysical level. Molecular structures containing components of the unique E2-E3 will be determined by X-ray crystallography.
In Aim 2, we will determine the membrane and substrate recognition mechanism for Atg8 lipidation. NMR and fluorescence spectroscopy will used to identify structural mechanisms of the recognition.
In Aim 3, we will define the structural and biochemical requirements of Atg8-mediated membrane dynamics using a newly developed membrane tethering assay, NMR, and biochemical experiments. The results from these aims will provide mechanistic understanding of the functions of autophagic Ubls and will be a large step toward comprehensive description of autophagosome formation. The impact of the work will be not only on the autophagy field but also on a broad range of biology.

Public Health Relevance

Autophagy is the cellular degradation-recycling system that plays a crucial role in cellular homeostasis. This project seeks to understand the molecular mechanisms of autophagic ubiquitin-like proteins that mediate formation of unique vesicles called autophagosomes. The results from the research will be valuable information for development of new therapeutic strategies involving intervention into the autophagy pathway.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM092740-04
Application #
8586315
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Gindhart, Joseph G
Project Start
2010-12-01
Project End
2015-11-30
Budget Start
2013-12-01
Budget End
2014-11-30
Support Year
4
Fiscal Year
2014
Total Cost
$341,100
Indirect Cost
$161,100
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Metlagel, Zoltan; Otomo, Chinatsu; Ohashi, Kazuto et al. (2014) Structural insights into E2-E3 interaction for LC3 lipidation. Autophagy 10:522-3
Otomo, Chinatsu; Metlagel, Zoltan; Takaesu, Giichi et al. (2013) Structure of the human ATG12~ATG5 conjugate required for LC3 lipidation in autophagy. Nat Struct Mol Biol 20:59-66