Peroxisomal fatty acid metabolism in genetic and age-related disorders

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Project Summary/Abstract Peroxisomes are ubiquitous eukaryotic organelles that form a focal point for multiple metabolic pathways. Lipid metabolism, and in particular, fatty acid transport related to it, depend heavily on peroxisomal membrane proteins that have specifically evolved for such purposes. The ATP-dependent cassette (ABC) transporters of ‘D’ subfamily’ reside in the peroxisomal membrane and are responsible for fatty acid import into the peroxisomes, defects in which process are related to various metabolic disorders. Mutations in ABCD1 cause X-linked adrenoleukodystrophy (X-ALD) which manifests as mild to severe central nervous system (CNS) demyelination. Dysfunction of ABCD3 and/or peroxisome biogenesis factors (PEXs) may cause Zellweger syndrome (ZS), a heterogeneous group of peroxisome assembly disorders. In addition to inherited diseases, reduced peroxisomal function is associated with aging and pathogenesis of age-related acquired diseases like diabetes, neurodegenerative disorders. Key gaps in understanding the function of ABCDs in metabolism and disease are due to lack of structural details, particularly of their conformational plasticity during substrate transport, and how this is compromised during aging and disease. My goal is to understand the involvement of peroxisomal fatty acid metabolism in aging and age-related neurodegenerative diseases, specifically Alzheimer’s disease and ultimately to lead a research group as an independent researcher in this area. I propose to elucidate the structural basis for function of ABCDs at atomic level using CryoEM in the absence and presence of substrate, and ATP (Aim 1a and 1b, mentored), coupling between ATPase and thioesterase activity, and fatty acid binding and transport (Aim 2a and 2b, mentored). I will determine atomic structures of ABCDs in complex with PEXs (Pex3 and Pex19) using CryoEM (Aim 3a, mentored) and assess alterations in peroxisomal morphology using Cryo-Electron Tomography; function and membrane composition of ABCD(1-3) knockdown cell lines, aged cells and Alzheimer’s disease (AD) model cell lines (Aim 3b, 3c, 3d mentored/ independent phase). These aims foster my long-term aim of studying peroxisome function and its relation to cellular physiology and disease, while obtaining training and experience in forefront tools of structural biophysics. For executing these aims, I have support of a very strong team of mentors, consultants and advisors. My mentor Prof. Robert Stroud is a pioneer in membrane protein structure and mechanism, my co- mentor Prof. Peter Walter is a pioneer in the unfolded protein response (UPR) and specializes in cell and organelle biology. My co-mentor Dr. Adam Frost is at the forefront of CryoEM/CryoET. Consultant Prof. Eric Verdin is a leading scientist in aging. Consultant Prof. Martin Kampmann is discovering new molecular players in AD. Scientific advisors Prof. Yifan Cheng pioneered CryoEM and Prof. Charles Craik has developed antibody and Fab libraries as tools. Together, this proposal will utilize their expertise in the respective fields, while leveraging my passion to study organellar biology through a structural lens.