Upon initial characterization of the PARKIN gene open-reading frame, a proteindomain homology search revealed that the most likely function of the PARKIN protein involves E3 ligase activity due to the presence of two conserved RING fingers near the C-terminus and homology to the ubiquitin molecule itself near the N-terminus. RING-finger domains are found in several other known E3 ligase proteins such as the MDM2 protein which targets p53 for degradation and the BRCA1 protein which functions in a number of ubiquitin-dependent pathways (11). E3 ubiquitin ligases are thought to comprise an exceptionally large family of proteins in mammals, perhaps one thousand or more encoded in the human genome, and are classified into three distinct types: N-end rule E3s; E3s containing the HECT (Homology to E6AP C-Terminus) domain; and E3's containing RING fingers (by far the most common E3-ligase protein). In an initial functional assessment of enzymatic activity, PARKIN interacted with the E2 ubiquitin ligase UbcH7 and UbcH8 and promoted ubiquitination in, in vitro assays while mutations associated with PD abrogated the E3-ligase-like activity (54). In understanding the function of PARKIN in the pathogenesis of PD, the role of PARKIN as an E3 ligase became the focus of research.
The RING finger motifs identified in the PARKIN protein are defined as a linear series of conserved cysteine and histidine residues, Cys-X2-Cys-X9-39-Cys-X1-3-Cys/ His-X2-Cys-X4-48-Cys-X2-Cys, where X can be any amino acid (55). Although there is high conservation in the cysteine and histidine residues, there is disparate conservation between the remaining sequence structures among proteins containing RING fingers. RING fingers bind two zinc atoms in a cross-brace arrangement, and are known to be essential protein protein interaction domains (55). PARKIN contains two RING finger domains (otherwise known as a TRIAD motif) where the second RING finger binds only a single zinc atom with resemblance to a zinc-ribbon motif and may be principally responsible for E3 ligase activity (56). Many RING-finger-containing proteins are known to possess autoubiquitination properties in addition to binding to an often daunting number of cellular targets as evident by BRCA1-related research. The suggestion is that the RING-finger classification of E3 ligases could potentially imply a role in diverse cellular processes for the host protein thereby preventing an obvious understanding of PARKIN function.
While the presence of two RING-finger domains within PARKIN suggests functionality in ubiquitin-dependent pathways, the presence of an ubiquitin-like (UbL) domain near the N-terminus strongly suggests the ability of PARKIN to interact directly with the proteasome. As opposed to RING-finger domains, UbL domains are rarely encoded in the human proteasome, present in just 62 proteins, although only ~25% of these proteins contain the residues necessary to directly bind the proteasome (57). The PARKIN UbL encodes the rare proteasome-interacting motif and might be thought of in a conventional way to facilitate the delivery of either autoubiquitinated protein or ubiquitinated substrate protein to the proteasome for degradation.
Since the UbL domain is also present in non-E3 ligase proteins, such as ataxin-1 or zinc-finger-containing transcription factors (57), the interaction of PARKIN's UbL domain and the proteasome may have altogether novel biochemical significance apart from models where PARKIN simply chaperones proteins to the proteasome. Indeed, the vast majority of E3 ligases accomplish efficient degradation of target substrates without a UbL domain. While the classification of PARKIN as an E3 ligase based on protein homology helped to focus downstream experimentation, a broad number of diverse cellular roles may still be attributed to the encoded protein domains thereby requiring the identification of physiological substrates and biochemical pathways to understand the role of PARKIN in neurodegeneration.
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