Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Shaw, Gary S.

Abstract

Parkin is an RBR E3 ubiquitin ligase that has been implicated in both sporadic and familial Parkinson’s disease. Upon mitochondrial damage, parkin is activated step-wise to recruit and ligate ubiquitin to a substrate on the outer mitochondrial membrane. Disruption of this activation and ligation cascade is hypothesized to result in neuronal death related to Parkinson’s disease.

While structures of parkin for a number of these activation states exist, it is important to note they are not of full-length human parkin. These structures are often truncated and come from various non-human species to eliminate important, yet hard to quantify structural elements. Protein structure is important for deciphering function, but also only presents a partial story. To fully understand protein function, one must examine the changes that occur over time. Protein dynamics is the study of how proteins change over time.

To understand the intra- and inter-domain changes parkin undergoes during each step of activation, protein dynamics studies were undertaken in the slow to intermediate-fast time scales. The oligomeric state of parkin was also explored to further understand parkin ligation mechanisms. These approaches were used to create a more complete understanding of the functional- ity of parkin as an E3 ligase. Specifically, new models for each state of activation are proposed for the in vitro parkin pathway.

It was concluded that parkin acts monomerically throughout the various state changes. Further, parkin exhibits a number of conformational changes, not previously seen in the structures that help to elucidate its functionality. New structural models are proposed as a result of this work for a more complete model of parkin activation.

Summary for Lay Audience

Our bodies maintain a delicate balance of microbiological activities every day to maintain our health. There are thousands of proteins that act like molecular machines that carry out functions from DNA repair to clearing away old and malfunctioning parts of our cells. Many diseases happen when something goes wrong with these proteins. Parkinson's disease which is characterized by slow movements and tremors is one such disease. Parkinson's disease is a neurodegenerative disease that is caused by the death of brain cells that we depend on to coordinate and regulate our movements.

A protein called parkin helps to regulate and maintain neuronal health by tagging damaged mitochondria for degradation in neurons. This process allows for the clearing away of a damaged part of the neuron, to maintain its health and functionality. To accomplish this, parkin must be first activated to recruit the molecules it uses for tagging damaged mitochondria. When parkin malfunctions, this clearance pathway also malfunctions and results in the death of a neuron instead.

While parkin has been studied previously, there are still questions surrounding how it actually works. There are several models of parkin at the various stages of activation. This work uses a field called protein dynamics to look at how parking goes from one stage of activation to the next. Parkin dynamics is the study of how a protein, like parkin, changes shape and goes through changes, like activation, over time. Like a flipbook is just many pictures quickly put together, so too is the information derived from protein dynamics experiments.

This work was able to resolve that parkin acts alone and has a number of changes that it goes through at each step. These changes help researchers to understand how parkin works. Once scientists know how something works, they can understand better why it malfunctions. Once science understands how Parkinson's disease is caused, researchers can work on better treatments and even prevent the disease earlier on.

Available for download on Tuesday, January 17, 2023

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