9c5e
From Proteopedia
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Covalent Complex Between Parkin Catalytic (Rcat) Domain and Ubiquitin
Structural highlights
FunctionPRKN_DROME E3 ubiquitin-protein ligase which accepts ubiquitin from E2 ubiquitin-conjugating enzymes in the form of a thioester and then directly transfers the ubiquitin to targeted substrates, such as Paris, Marf, Opa1, Miro, pnut, Septin1, Tom20 and porin (PubMed:16002472, PubMed:17456438, PubMed:20194754, PubMed:23770917, PubMed:24192653, PubMed:24901221, PubMed:25474007, PubMed:27906179, PubMed:31714929, PubMed:32047033, PubMed:32138754). Mediates monoubiquitination as well as 'Lys-6', 'Lys-11', 'Lys-48'-linked and 'Lys-63'-linked polyubiquitination of substrates, depending on the context (PubMed:18957282, PubMed:23650379, PubMed:24901221, PubMed:25474007, PubMed:27906179, PubMed:31714929, PubMed:32047033). Protects against mitochondrial dysfunction during cellular stress, by acting downstream of Pink1, to coordinate mitochondrial quality control mechanisms that remove and replace dysfunctional mitochondrial components (PubMed:12642658, PubMed:15073152, PubMed:16672980, PubMed:16672981, PubMed:17123504, PubMed:18230723, PubMed:18443288, PubMed:18799731, PubMed:18957282, PubMed:20194754, PubMed:20496123, PubMed:23509287, PubMed:24192653, PubMed:24901221, PubMed:25474007, PubMed:27906179, PubMed:29497364, PubMed:32047033). Depending on the severity of mitochondrial damage and/or dysfunction, activity ranges from preventing apoptosis and stimulating mitochondrial biogenesis to regulating mitochondrial dynamics and eliminating severely damaged mitochondria via mitophagy (PubMed:12642658, PubMed:15073152, PubMed:16002472, PubMed:16672980, PubMed:16672981, PubMed:17123504, PubMed:18230723, PubMed:18443288, PubMed:18799731, PubMed:18957282, PubMed:20194754, PubMed:20496123, PubMed:23509287, PubMed:24192653, PubMed:24901221, PubMed:25474007, PubMed:27906179, PubMed:29497364, PubMed:32047033). Appears to be particularly important in maintaining the physiology and function of cells with high energy demands that are undergoing stress or altered metabolic environment, including spermatids, muscle cells and neurons such as the dopaminergic (DA) neurons (PubMed:12642658, PubMed:15073152, PubMed:16002472, PubMed:16672980, PubMed:17123504, PubMed:18799731, PubMed:20483372, PubMed:22396657, PubMed:24901221, PubMed:28435104, PubMed:29497364, PubMed:31714929). Activation and recruitment onto the outer membrane of damaged/dysfunctional mitochondria (OMM) requires Pink1-mediated phosphorylation of both park and ubiquitin (PubMed:18230723, PubMed:18799731, PubMed:18957282, PubMed:20194754, PubMed:22396657, PubMed:24901221, PubMed:25474007, PubMed:27906179). In depolarized mitochondria, mediates the decision between mitophagy or preventing apoptosis by inducing either the poly- or monoubiquitination of porin/VDAC; polyubiquitination of porin promotes mitophagy, while monoubiquitination of porin decreases mitochondrial calcium influx which ultimately inhibits apoptosis (PubMed:32047033). When cellular stress results in irreversible mitochondrial damage, promotes the autophagic degradation of dysfunctional depolarized mitochondria (mitophagy) by promoting the ubiquitination of mitochondrial proteins (PubMed:16672980, PubMed:16672981, PubMed:18957282, PubMed:20194754, PubMed:23509287, PubMed:24192653, PubMed:25474007, PubMed:29497364). Preferentially assembles 'Lys-6'-, 'Lys-11'- and 'Lys-63'-linked polyubiquitin chains following mitochondrial damage, leading to mitophagy (PubMed:23650379, PubMed:32047033). In developing tissues, inhibits JNK-mediated apoptosis by negatively regulating bsk transcription (PubMed:16002472, PubMed:20496123). The Pink1-park pathway also promotes fission and/or inhibits fusion of damaged mitochondria by mediating the ubiquitination and subsequent degradation of proteins involved in mitochondrial fusion/fission such as Marf, Opa1 and fzo (PubMed:17123504, PubMed:18230723, PubMed:18443288, PubMed:18799731, PubMed:20194754, PubMed:23650379, PubMed:24192653, PubMed:24901221, PubMed:29497364). This prevents the refusion of unhealthy mitochondria with the healthy mitochondrial network and/or initiates mitochondrial fragmentation facilitating their later engulfment by autophagosomes (PubMed:17123504, PubMed:18230723, PubMed:18443288, PubMed:18799731, PubMed:20194754, PubMed:23650379, PubMed:24192653, PubMed:24901221, PubMed:29497364). Regulates motility of damaged mitochondria by phosphorylating Miro which likely promotes its park-dependent degradation by the proteasome; in motor neurons, this inhibits mitochondrial intracellular anterograde transport along the axons which probably increases the chance of the mitochondria being eliminated in the soma (PubMed:22396657). The Pink1-park pathway is also involved in mitochondrial regeneration processes such as promoting mitochondrial biogenesis, activating localized mitochondrial repair, promoting selective turnover of mitochondrial proteins and initiating the mitochondrial import of endogenous proteins (PubMed:16672980, PubMed:20496123, PubMed:20869429, PubMed:23509287, PubMed:23650379, PubMed:24192653, PubMed:25565208, PubMed:29497364). Involved in mitochondrial biogenesis via the ubiquitination of transcriptional repressor Paris which leads to its subsequent proteasomal degradation and allows activation of the transcription factor srl (PubMed:23509287, PubMed:29497364, PubMed:32138754). Promotes localized mitochondrial repair by activating the translation of specific nuclear-encoded mitochondrial RNAs (nc-mtRNAs) on the mitochondrial surface, including several key electron transport chain component nc-mtRNAs (PubMed:23509287, PubMed:25565208).[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] Publication Abstract from PubMedParkin is an E3 ubiquitin ligase implicated in early-onset forms of Parkinson's disease. It catalyzes a transthiolation reaction by accepting ubiquitin (Ub) from an E2 conjugating enzyme, forming a short-lived thioester intermediate, and transfers Ub to mitochondrial membrane substrates to signal mitophagy. A major impediment to the development of Parkinsonism therapeutics is the lack of structural and mechanistic detail for the essential, short-lived transthiolation intermediate. It is not known how Ub is recognized by the catalytic Rcat domain in parkin that enables Ub transfer from an E2~Ub conjugate to the catalytic site and the structure of the transthiolation complex is undetermined. Here, we capture the catalytic intermediate for the Rcat domain of parkin in complex with ubiquitin (Rcat-Ub) and determine its structure using NMR-based chemical shift perturbation experiments. We show that a previously unidentified alpha-helical region near the Rcat domain is unmasked as a recognition motif for Ub and guides the C-terminus of Ub toward the parkin catalytic site. Further, we apply a combination of guided AlphaFold modeling, chemical cross-linking, and single turnover assays to establish and validate a model of full-length parkin in complex with UbcH7, its donor Ub, and phosphoubiquitin, trapped in the process of transthiolation. Identification of this catalytic intermediate and orientation of Ub with respect to the Rcat domain provides important structural insights into Ub transfer by this E3 ligase and explains how the previously enigmatic Parkinson's pathogenic mutation T415N alters parkin activity. Capturing the catalytic intermediates of parkin ubiquitination.,Connelly EM, Rintala-Dempsey AC, Gundogdu M, Freeman EA, Koszela J, Aguirre JD, Zhu G, Kamarainen O, Tadayon R, Walden H, Shaw GS Proc Natl Acad Sci U S A. 2024 Aug 6;121(32):e2403114121. doi: , 10.1073/pnas.2403114121. Epub 2024 Jul 30. PMID:39078678[30] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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