Chemical biology approaches to probe protein networks for alleviation of trafficking defects in Parkinson’s disease

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2021

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Abstract

Parkinson’s disease (PD), a common neurodegenerative disorder, can result from defective proteostasis mechanisms that induce neuronal toxicity. A common pathological hallmark of PD is the disruption of protein transport and trafficking between organelles within the neuron. To date, there has been some success in identifying small drug-like molecules that can restore defective trafficking pathways. These compounds are often identified through phenotypic screening in cellular models of PD toxicity, but, for many, the specific target of the compound and its mechanism of action are not fully understood. One such compound, identified in a screening effort in the laboratory of Susan Lindquist, was recently shown to alleviate multiple phenotypic markers of PD toxicity. In particular, the compound was shown to alleviate markers of toxicity induced by aggregation of α-synuclein, a protein that is genetically linked to PD. The activity of this compound, which contains a characteristic N-arylbenzdiimidazole (NAB) scaffold, was shown to depend upon E3 ubiquitin ligase Nedd4. Nedd4 has been previously implicated in PD toxicity as it regulates α-synuclein aggregation and proteostasis through ubiquitin signaling, but the mechanism of NAB compounds as modulators of Nedd4 was not elucidated. To this end, a series of biochemical and biophysical analyses were employed to understand the binding and mechanism of NAB2, the most potent NAB derivative, as a ligand of Nedd4. These experiments revealed that NAB2 binds to Nedd4 with high apparent affinity in the nanomolar range but does not induce changes in Nedd4 enzymatic activity in vitro. As Nedd4 activity is dependent upon protein-protein interaction and is tightly regulated at a cellular level, a proteomics-based evaluation of ubiquitination was pursued to study the effect of NAB2 treatment on the global ubiquitylome. This analysis revealed that induction of α-synuclein toxicity dramatically remodels the ubiquitylome, and NAB2 treatment induces small but phenotypically relevant changes in protein ubiquitination. Through this effort, a previously unrecognized Nedd4 substrate, trafficking scaffold protein TFG, was identified to be ubiquitinated in a NAB-dependent manner.

To further expand our understanding of the NAB2 mechanism in alleviation of α-synuclein toxicity, an unbiased chemoproteomic approach was employed to identify NAB2 targets across the proteome. This effort revealed small GTPase Rab1a, a regulator of endoplasmic reticulum (ER)-to-Golgi body transport, as an additional putative target of the NAB scaffold. This result is particularly promising as ER-to-Golgi transport is disrupted in PD toxicity and restored by NAB2 treatment. Further analysis of NAB2/Rab1a binding indicate that NAB2 binding occurs in a nucleotide-dependent manner, and NAB2 treatment phenocopies Rab1a overexpression by improving the viability of α-synuclein toxic cells. While the functional link between Nedd4 and Rab1a is not yet clear, the efforts toward understanding the NAB mechanism of action have revealed a protein network involved in NAB2-dependent rescue of trafficking defects and PD toxicity. Cumulatively, these results expand our understanding, at a molecular level, of PD toxicity and small molecule rescue thereof, enabling future efforts to target these proteins for development or optimization of neuroprotective compounds.

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Hatstat, Anna Katherine (2021). Chemical biology approaches to probe protein networks for alleviation of trafficking defects in Parkinson’s disease. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/23008.

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