Dataset Information

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation.

ABSTRACT: The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early-onset Parkinson's disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin's N-terminal ubiquitin-like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phospho-ubiquitin-loaded C-terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re-modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity.

SUBMITTER: Condos TE

PROVIDER: S-EPMC6276879 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation.

Condos Tara Ec TE Dunkerley Karen M KM Freeman E Aisha EA Barber Kathryn R KR Aguirre Jacob D JD Chaugule Viduth K VK Xiao Yiming Y Konermann Lars L Walden Helen H Shaw Gary S GS

The EMBO journal 20181116 23

The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early-onset Parkinson's disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin's N-terminal ubiquitin-like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We ...[more]

PMID: 30446597

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Project description:Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.

Project description:BACKGROUND:Systemic lupus erythematosus (SLE) is an autoimmune inflammatory rheumatic disease. SLE susceptibility is affected by multiple genetic elements, environmental factors, and their interactions. We aimed in this study to statistically and functionally characterize a gene-gene interaction (epistasis) recently documented to affect SLE risk. METHODS:Two single-nucleotide polymorphisms, rs2230926 in TNFAIP3 (A20) gene and rs131654 in UBE2L3 (UBCH7) gene, were genotyped in all 3525 Korean participants, and their SLE risk association and epistasis were statistically analyzed by calculating odds ratio (OR), 95% confidence interval (CI), and P values in genotype comparisons between 1318 SLE patients and 2207 healthy controls. Furthermore, their effects on gene functions were assessed by comparatively examining separate and combined effects of TNFAIP3 and UBE2L3 knockdowns on NF-?B transcription factor activity in human cells. RESULTS:SLE susceptibility is associated with TNFAIP3 rs2230926 (OR?=?1.9, 95% CI 1.6-2.4, P =?8.6?×?10-11) and UBE2L3 rs131654 (OR?=?1.2, 95% CI 1.1-1.4, P =?1.1?×?10-4) in a Korean population of this study. Their risk-associated alleles synergistically elevate SLE susceptibility in both multivariate logistic regression analysis (ORinteraction =?1.6, P =?0.0028) and genotype-stratified analysis (ORinteraction =?2.4), confirming the synergistic TNFAIP3-UBE2L3 interaction in SLE risk. Additionally, the SLE-susceptible alleles confer decreased TNFAIP3 expression (P =?1.1?×?10-6, n =?610) and increased UBE2L3 expression (P =?9.5?×?10-11, n =?475), respectively, in B cell analysis of the International HapMap Project individuals with adjustment for ethnicity. Furthermore, when compared with TNFAIP3 non-knockdown and UBE2L3 knockdown in human HeLa cells, TNFAIP3 knockdown and UBE2L3 non-knockdown synergistically increase three cytokines, CCL2, CXCL8 (IL8), and IL6, all regulated by NF-?B in the human TNFR signaling pathway. CONCLUSIONS:A synergistic interaction between TNFAIP3 and UBE2L3 genes is observed in SLE risk, as being evident in comparison of genotype distributions between SLE patients and controls. Additionally, the synergistic gene-gene interaction is functionally validated, as TNFAIP3 reduction and UBE2L3 augment exert synergism in activation of NF-?B and subsequent induction of inflammatory cytokines. Accordingly, SLE inflammation and risk could be synergistically alleviated by TNFAIP3 upregulation and UBE2L3 downregulation.

Dataset Information

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation.

Publications

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation.

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