. Numerous studies, including critical work from our lab, has revealed the fundamental mechanisms by which proteins encoded by two Parkinson?s Disease (PD) genes ? the PINK1 protein kinase and PARKIN ubiquitin (Ub) ligase ? promote the ubiquitylation and autophagic capture of damaged mitochondria to promote their clearance by mitophagy. Recently, we have merged a quantitative proteomics platform with stem cell- derived, induced neurons (iNeurons) harboring pathway mutations to elucidate PARKIN and PINK1 ubiquitylation targets under endogenous conditions, and have determined the role of the mitochondrial deubiquitylase USP30 and the p97 segregase in PARKIN and mitophagic flux regulation. Yet, our understanding of the extent to which other proteins mutated in PD collaborate with the PARKIN-PINK1 system to contribute to disease etiology remains limited, as is our understanding of how the PINK1 activation threshold on the mitochondrial translocon is mechanistically controlled. Here, we propose a series of experiments that address both of these knowledge gaps. First, among the most compelling genes to emerge from our recent mitophagic flux CRISPR screen is FBXO7, a gene mutated in PD (PARK15) and a member of the F-Box family of proteins that forms an SCF Ub ligase. FBXO7?s critical functions and targets, as well as how its mutation predisposes to PD, are unknown. Through interaction proteomics, we find that FBXO7 associates with multiple regulatory components of the proteasome, and propose that FBXO7 may play a central role by integrating mitophagy and proteasomal control mechanisms to support organelle homeostasis.
In Aim 1, we will use our iNeuron system to examine FBXO7?s role in mitophagic flux using an array of quantitative assays that examine sequential steps in the pathway, and we will genetically and functionally dissect ubiquitylation targets and regulatory mechanisms as an initial step toward understanding how patient mutations in FBXO7 may contribute to PD. Second, our preliminary data, and work in the field, indicate that both PINK1 and USP30 are physically associated with the mitochondrial translocon, placing the translocon at the nexus of PARKIN regulation. Our data show that USP30 has a role in controlling both the threshold for PARKIN activation by removing Ub from the translocon and also may have a previously unappreciated role in import quality control at the translocon itself.
In Aim 2, we will systematically examine translocon components and ubiquitylation for their roles in setting the threshold for PARKIN activation via Ub phosphorylation. In parallel, we will elucidate how USP30 functions in this newly recognized Import Quality Control (IQC) pathway for removal of Ub chains from translocon import substrates. Finally, our work has led to the first visualization of PINK1 in association with the translocon using single-particle electron microscopy, and we seek to further develop a biochemical and structural understanding of how this complex is assembled and regulated. Together, these focused mechanistic studies on how these key molecules intersect with the PARKIN system will provide a deeper understanding of mitochondrial quality control.
Parkinson?s Disease leads to loss of dopaminergic neurons and many of the genes linked to the disease play roles in organelle quality control. Here we explore the mechanisms of action of two disease genes ? FBXO7 and PINK1 ? in mitochondrial quality control using systematic cell biological and proteomic approaches.
| Guarani, Virginia; McNeill, Elizabeth M; Paulo, Joao A et al. (2015) QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology. Elife 4: |
| Guarani, Virginia; Paulo, Joao; Zhai, Bo et al. (2014) TIMMDC1/C3orf1 functions as a membrane-embedded mitochondrial complex I assembly factor through association with the MCIA complex. Mol Cell Biol 34:847-61 |
| Lydeard, John R; Schulman, Brenda A; Harper, J Wade (2013) Building and remodelling Cullin-RING E3 ubiquitin ligases. EMBO Rep 14:1050-61 |
