Browsing School, Graduate by Subject "ULK1"
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The Characterization of the PARK10 Gene USP24 in Autophagy and MitophagyParkinson’s disease (PD) is the second most common neurodegenerative disease in the world and there is currently no cure, just treatments to mask symptoms. PD results in loss or damage to dopaminergic (DA) neurons within the substantia nigra pars compacta. Autophagy, a lysosome-dependent degradation pathway, is essential for neuronal survival. Dysregulation of this pathway has been linked to several neurodegenerative diseases. Defects in autophagy and mitochondrial specific autophagy (mitophagy) have been implicated in PD. The goal of this project was to characterize the function of the deubiquitinating enzyme (DUB) ubiquitin-specific peptidase 24 (USP24) in autophagy as well as in mitophagy. USP24 is located on chromosome 1p of the PARK10 locus that has been associated with late-onset PD. As a DUB USP24 is responsible for removing ubiquitin chains from target substrates, thus altering their stability and function. The precise role of USP24 in PD is not yet known. My data demonstrate that USP24 is a negative regulator of autophagy that is downstream of MTROC1 and acts via the class III PtdIns3K and ULK1 complexes. Specifically, USP24 negatively regulates ubiquitination levels and stability of the ULK1 protein, thus decreasing autophagy flux. I have also shown that USP24 is able to regulate autophagy in induced pluripotent stem cells (iPSC)-derived DA neurons with no adverse effects. In fact, knockdown of USP24 led to increased neurite length in aged iPSC-DA neurons, suggesting a potential neuroprotective effect. Furthermore, in addition to ULK1, USP24 may regulate levels of the PD-associated Parkin protein. Recruitment of Parkin to the outer mitochondrial membrane is necessary for initiation of mitophagy. My data demonstrate that similarly to ULK1, stability of Parkin is increased following USP24 knockdown, suggesting that USP24 may negatively regulate Parkin-dependent mitophagy. Together, my data provide a better understanding of USP24 function in regulation of autophagy, mitophagy and PD. In addition, the data suggest that USP24 could be a possible new therapeutic target for PD.
Viral and Cellular Determinants of Picornavirus-mediated Autophagy InductionMacro-autophagy is a basal cellular process that involves the degradation and turnover of cytosolic components, including elimination of damaged organelles and cytosolic cargo. In response to cell stressors, such as but not limited to, starvation and infection from xenobiotics, autophagy is upregulated. The process is controlled by the upstream autophagy signaling ULK complex, composed of the kinases ULK1 or ULK2, and the scaffold proteins ATG13, RB1CC1, and ATG101. This complex serves as a nexus for signaling pathways from nutrient sensitive kinase complexes such as MTORC1 or AMPK. Poliovirus (PV) has been shown to induce autophagy in infected cells, but the mechanism of initiation has not been completely elucidated. Furthermore, the host cellular factors that are involved in this virus-induced autophagy are unknown. We recently have shown that PV does not require the ULK1/2 complex for replication or autophagic signal induction during infection, demonstrating a novel ULK1/2-independent autophagic signaling pathway. We show that knockdown of RB1CC1, a vital scaffold protein for the ULK1/2 complexes, has no effect on PV replication and does not impede the ability of the virus to induce autophagic signals. Furthermore, PV mediates the elimination of this complex during infection in a mechanism that is not dependent on the proteasome. PV causes the cleavage of an autophagic cargo receptor SQSTM1, which was previously described in CV-B3, and therefore impairs our ability to measure bona fide autophagy during infection. We have also found that several members of the Enterovirus genus: Enterovirus D68 (EV-D68), Coxsackievirus B3 (CV-B3), and Rhinovirus A1 (RV-A1) also do not require the ULK complex for replication or with their respective effects on autophagy. We have evidence that suggests that the BECN1 complex, downstream of the ULK complex, is dispensable for PV. Exogenous expression of viral proteins 2BC and 3A from PV and CV-B3 increase the presence of LC3+ puncta but show no acidification of autophagosomes, suggesting the presence of a secondary acidification signal. We discuss the implications of these findings in regards to the ability of picornaviruses to reformat the induction process for their own benefit.