S -Nitrosylation of Parkin Regulates Ubiquitination and Compromises Parkin's Protective Function
Kenny K. K. ChungBobby ThomasXiaojie LiOlga PletnikováJuan C. TroncosoLaura MarshValina L. DawsonTed M. Dawson
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Abstract:
Parkin is an E3 ubiquitin ligase involved in the ubiquitination of proteins that are important in the survival of dopamine neurons in Parkinson's disease (PD). We show that parkin is S-nitrosylated in vitro, as well as in vivo in a mouse model of PD and in brains of patients with PD and diffuse Lewy body disease. Moreover, S-nitrosylation inhibits parkin's ubiquitin E3 ligase activity and its protective function. The inhibition of parkin's ubiquitin E3 ligase activity by S-nitrosylation could contribute to the degenerative process in these disorders by impairing the ubiquitination of parkin substrates.Keywords:
S-Nitrosylation
Ubiquitin-Protein Ligases
(2011). The ubiquitin ligase parkin modulates the execution of autophagy. Autophagy: Vol. 7, No. 8, pp. 919-921.
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E3 ubiquitin ligases of which there are >600 putative in humans, constitute a family of highly heterogeneous proteins and protein complexes that are the ultimate enzymes responsible for the recruitment of an ubiquitin loaded E2 ubiquitin-conjugating enzyme, recognise the appropriate protein substrate and directly or indirectly transfer the ubiquitin load onto the substrate. The aftermath of an E3 ligase activity is usually the formation of an isopeptide bond between the free carboxylate group of ubiquitin’s C-terminal Gly76 and an ε-amino group of the substrate’s Lys, even though non-canonical ubiquitylation on non-amine groups of target proteins have been observed. E3 ligases are grouped into four distinct families: HECT, RING-finger/U-box, RBR and PHD-finger. E3 ubiquitin ligases play critical roles in subcellular signalling cascades in eukaryotes. Dysfunctional E3 ubiquitin ligases therefore tend to inflict dramatic effects on human health and may result in the development of various diseases including Parkinson’s, Amyotrophic Lateral Sclerosis, Alzheimer’s, cancer, etc. Being regulators of numerous cellular processes, some E3 ubiquitin ligases have become potential targets for therapy. This chapter will present a comprehensive review of up-to-date findings in E3 ligases, their role in the pathology of disease and therapeutic potential for future drug development.
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Ubiquitin is a covalent protein tag that alters the stability or behavior of a growing list of proteins. Covalent attachment of ubiquitin to target proteins occurs through a cascade of enzymes: Ubiquitin is charged by a ubiquitin-activating enzyme (E1), and transferred to a ubiquitin-conjugating enzyme (E2). Then, transfer of ubiquitin from E2 to a target protein is brokered by a ubiquitin ligase (E3). A critical aspect of E3 function is the selection of a particular E2 to accomplish ubiquitination of a substrate. We examined the requirements for correct E2-E3 specificity in the RING-H2 ubiquitin ligase Hrd1p, an ER-localized protein known to use primarily Ubc7p for its function. Versions of Hrd1p containing the RING motif from homologous E3s were unable to carry out Hrd1p function, revealing a requirement for the specific Hrd1p RING motif in vivo. An in vitro assay revealed that these RING motifs were sufficient to function as ubiquitin ligases, but that they did not display the E2 specificity predicted from in vivo results. We further refined the in vitro assay of Hrd1p function by demanding not only ubiquitin ligase activity, but also specific activity that recapitulated both the E2 specificity and RING selectivity observed in vivo. Doing so revealed that correct E2 engagement by Hrd1p required the presence of portions of the Hrd1p soluble cytoplasmic domain outside the RING motif, the placement of the Hrd1p ubiquitin ligase in the ER membrane, and presentation of Ubc7p in the cytosolic context. We confirmed that these conditions supported the ubiquitination of Hrd1p itself, and the transfer of ubiquitin to the prototype substrate Hmg2p-GFP, validating Hrd1p self-ubiquitination as a viable assay of ligase function. During these studies we observed enhanced Ubc7p-dependent ubiquitination in the presence of soluble Cue1p, which interacts with Ubc7p in vivo. Soluble Cue1p promoted the transfer of ubiquitin from Ubc7p to other ubiquitin molecules in solution. We also observed that this stimulation of Ubc7p by Cue1p and the anchoring of Ubc7p to the ER membrane by Cue1p were both necessary for Ubc7p function in vivo. Ubc7p activation by Cue1p was observed at the ER and with Ubc7p relocated to a cytosolic E3. In total, these studies have substantially improved and expanded our understanding of how Hrd1p functions to degrade proteins in the ER
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Post-translational modification of proteins by ubiquitin is a central regulatory
process in all eukaryotic cells. Substrate selection and type of modification are
events catalyzed by the E3 ligase, a component of the ubiquitin pathway.
Several ubiquitin E3 ligases are implicated in cancer and other disease states,
underlying the need for mechanistic insight of these enzymes.
Parkinson’s disease is a neurodegenerative disorder characterised by the loss
of dopaminergic neurons from the substantia nigra, the presence of Lewy
Bodies, and pathogenic aggregates rich in ubiquitin. Autosomal Recessive
Juvenile Parkinsonism (AR-JP), which is one of the most common familial forms
of the disease, is directly linked to mutations in the Parkin gene (PARK2).
Parkin is a RING E3 and catalyses a range of ubiquitination events (mono, multi
mono, K48- and K63- linked poly) in concert with several E2s on a variety of
substrates, including itself. Furthermore, Parkin is capable of binding the 26S
proteasome and mediates selective degradation of target substrates.
The data presented will demonstrate that the Ubiquitin-like domain (UblD) of
Parkin functions to inhibit its auto-ubiquitination via a novel mechanism.
Pathogenic Parkin mutations disrupt this inhibition and result in a constitutively
active molecule. The inhibition is mediated by an intra-molecular interaction
between UblD and the C-terminus of Parkin, and Lysine 48 on UblD participates
in this interaction. The study also uncovered unique UblD/Ubiquitin Binding
Regions (UBRs) on the C-terminus of Parkin that play a novel role in its RING
E3 ligase activity. The observations provide critical mechanistic insights into the
myriad functions of Parkin and the underlying basis of Parkinson’s disease.
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Abstract A large family of E3 ligases that contain both substrate recruitment and RING domains confer specificity within the ubiquitylation cascade. Regulation of RING E3s depends on modulating their ability to stabilise the RING bound E2~ubiquitin conjugate in the activated (or closed) conformation. Here we report the structure of the Ark2C RING bound to both a regulatory ubiquitin molecule and an activated E2~ubiquitin conjugate. The structure shows that the RING domain and non-covalently bound ubiquitin molecule together make contacts that stabilise the activated conformation of the conjugate, revealing why ubiquitin is a key regulator of Ark2C activity. We also identify a charged loop N-terminal to the RING domain that enhances activity by interacting with both the regulatory ubiquitin and ubiquitin conjugated to the E2. In addition, the structure suggests how Lys48-linked ubiquitin chains might be assembled by Ark2C and UbcH5b. Together this study identifies features common to RING E3s, as well elements that are unique to Ark2C and related E3s, which enhance assembly of ubiquitin chains.
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