Biochemistry, Biophysics, and Structural Biology Commons^™

The Study Of The Structure And Dynamics Of Parkin Activation, Elaine Aisha Freeman

Electronic Thesis and Dissertation Repository

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. …

New Perspectives On Phosphorylation State In The Parkin Ubiquitination Cascade, Karen Dunkerley

Electronic Thesis and Dissertation Repository

The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of parkin itself, both mediated by the OMM kinase, PINK1. However, targeted mitochondrial proteins have little structural or sequence similarity, with the commonality between substrates being proximity to the OMM. The objective of this thesis was to identify the molecular consequences of parkin phosphorylation, interaction with pUb and how this promotes ubiquitination activity of known Ub-acceptor proteins and parkin itself.

The three-dimensional structure of …

Conformational Arrangements Of Ubch7-Ubiquitin With Ospg And Parkin, Tara E. C. Condos

Electronic Thesis and Dissertation Repository

The E2-ubiquitin conjugate is a key regulator of ubiquitination and is therefore an important component of cellular homeostasis. Disruptions to proper E2-ubiquitin functioning have implications in diseases such as shigellosis and Parkinson’s disease discussed here. E2-ubiquitin conjugates like UbcH7-ubiquitin are extremely dynamic and can adopt multiple conformations in solution or bound to target proteins. However, the conformational arrangements that UbcH7-ubiquitin adopts while free in solution, bound to the shigellosis-associated kinase OspG or to the Parkinson’s disease-related E3 ligase parkin are unknown. Also unknown, is a mechanistic explanation for how UbcH7-ubiquitin interactions with OspG and parkin are associated with disease. Here, …

Modulating Parkin E3 Ubiquitin Ligase Activity Using Phospho-Ubiquitin Variants, Susanna George

Electronic Thesis and Dissertation Repository

Parkin is a Parkinson’s disease-linked E3 ubiquitin (Ub) ligase that promotes mitophagy by ubiquitination of mitochondrial outer membrane proteins. Phosphorylation of Ub at Ser65 by the PTEN-induced putative kinase 1 activates parkin. The role of other Ub phosphorylation sites and the associated kinases remain unknown. We optimized genetic code expansion to produce pure site-specfically phosphorylated Ub (pUb) variants (pUb^S7, pUb^S12, pUb^S20, pUb^S65) and investigated their activity in a key neurodegenerative pathway. Purification of pUb^S7 revealed a +3 frameshifted protein (Ub ∆7) that was successfully purified away from the pUb. Parkin was …

Parkin Misfolding, Dysfunction, And Degradation In Parkinson's Disease, Alexander S. Mccarton

Electronic Thesis and Dissertation Repository

Mutations in the gene encoding parkin (PARK2) result in familial early onset forms of Parkinson’s disease (PD) based on the loss of parkin’s E3 ubiquitin ligase function. Protein misfolding is a common molecular feature of most neurodegenerative diseases, including PD. To test whether parkin misfolding also plays a role in the more common spontaneous PD, we established and functionally characterized a parkin yeast model. We found that oxidative and protein folding stress, parkin point mutations and truncations, and parkin’s interaction with the PD-associated kinase PINK1 profoundly alter parkin’s subcellular localization and toxicity. Notably, these conditions also induce parkin fragmentation, degradation, …

Structure Of C-Terminal Domain Of Parkin, Ibr-Ring2, Yeong Ju Noh

Electronic Thesis and Dissertation Repository

Parkin is an E3 ubiquitin ligase which degrades misfolded proteins and prevents the formation of abnormal protein aggregates often formed in Parkinson’s disease. The main goal of this thesis was to perform structural analysis on the IBR(In-Between-RING)-RING2 (Really Interesting New Gene) domain of parkin. After determining the three-dimensional solution structure of the protein by NMR spectroscopy, the RING2 domain was identified to be similar to the IBR domain, showing that it is not a canonical RING domain. The catalytic cysteine on RING2 was also shown to be solvent exposed, supporting the recently proposed RING/HECT hybrid mechanism of parkin as an …

Characterization Of The Interaction Between The Parkin Ubiquitin-Like Domain And Ataxin-3 Ubiquitin Interacting Domains, Jane J. Bai

Electronic Thesis and Dissertation Repository

The ubiquitin signaling pathway (USP) consists of hundreds of enzymes which are tightly regulated for proper maintenance of intracellular protein level homeostasis. The main goals of this thesis were to characterize the interaction of two proteins involved in the USP, the E3 ubiquitin ligase called parkin, and the Deubiquitinating (DUB) enzyme, ataxin-3. The effect of disease-state substitutions in the parkin ubiquitin-like domain (UbLD) on the interaction with ataxin-3 was investigated through NMR ¹H-¹⁵N HSQC titration experiments and affinity binding assays. The three UIM regions in ataxin-3bind the hydrophobic patch of parkin UbLD (K_D = 680 μM) …