PINK1 drives Parkin self-association and HECT-like E3 activity upstream of mitochondrial binding

Genetic studies indicate that the mitochondrial kinase PINK1 and the RING-between-RING E3 ubiquitin ligase Parkin function in the same pathway. In concurrence, mechanistic studies show that PINK1 can recruit Parkin from the cytosol to the mitochondria, increase the ubiquitination activity of Parkin, and induce Parkin-mediated mitophagy. Here, we used a cell-free assay to recapitulate PINK1-dependent activation of Parkin ubiquitination of a validated mitochondrial substrate, mitofusin 1. We show that PINK1 activated the formation of a Parkin–ubiquitin thioester intermediate, a hallmark of HECT E3 ligases, both in vitro and in vivo. Parkin HECT-like ubiquitin ligase activity was essential for PINK1-mediated Parkin translocation to mitochondria and mitophagy. Using an inactive Parkin mutant, we found that PINK1 stimulated Parkin self-association and complex formation upstream of mitochondrial translocation. Self-association occurred independent of ubiquitination activity through the RING-between-RING domain, providing mechanistic insight into how PINK1 activates Parkin.

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The USP19 Deubiquitinase Regulates the Stability of c-IAP1 and c-IAP2

Journal of Biological Chemistry ◽

10.1074/jbc.m111.282020 ◽

2011 ◽

Vol 286 (41) ◽

pp. 35380-35387 ◽

Cited By ~ 53

Author(s):

Yide Mei ◽

Allison Alcivar Hahn ◽

Shimin Hu ◽

Xiaolu Yang

Keyword(s):

Deubiquitinating Enzyme ◽

Dependent Manner ◽

Deubiquitinating Enzymes ◽

E3 Ligases ◽

Cellular Processes ◽

Ubiquitin Ligase Activity ◽

Self Association ◽

The Stability

The inhibitors of apoptosis (IAPs) are critical regulators of apoptosis and other fundamental cellular processes. Many IAPs are RING domain-containing ubiquitin E3 ligases that control the stability of their interacting proteins. However, how IAP stability is regulated remains unclear. Here we report that USP19, a deubiquitinating enzyme, interacts with cellular IAP 1 (c-IAP1) and c-IAP2. Knockdown of USP19 decreases levels of both c-IAPs, whereas overexpression of USP19 results in a marked increase in c-IAP levels. USP19 effectively removes ubiquitin from c-IAPs in vitro, but it stabilizes c-IAPs in vivo mainly through deubiquitinase-independent mechanisms. The deubiquitinase activity is involved in the stabilization of USP19 itself, which is facilitated by USP19 self-association. Functionally, knockdown of USP19 enhances TNFα-induced caspase activation and apoptosis in a c-IAP1 and 2-dependent manner. These results suggest that the self-ubiquitin ligase activity of c-IAPs is inhibited by USP19 and implicate deubiquitinating enzymes in the regulation of IAP stability.

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E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

International Journal of Molecular Sciences ◽

10.3390/ijms22115712 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5712

Author(s):

Michał Tracz ◽

Ireneusz Górniak ◽

Andrzej Szczepaniak ◽

Wojciech Białek

Keyword(s):

Ubiquitin Ligase ◽

Conformational Changes ◽

Protein Interactions ◽

E3 Ubiquitin Ligase ◽

Lanthanide Ions ◽

E3 Ligases ◽

Fluorescence Measurements ◽

Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

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Diverse Effects of Mutations in Exon II of the von Hippel-Lindau (VHL) Tumor Suppressor Gene on the Interaction of pVHL with the Cytosolic Chaperonin and pVHL-Dependent Ubiquitin Ligase Activity

Molecular and Cellular Biology ◽

10.1128/mcb.22.6.1947-1960.2002 ◽

2002 ◽

Vol 22 (6) ◽

pp. 1947-1960 ◽

Cited By ~ 52

Author(s):

William J. Hansen ◽

Michael Ohh ◽

Javid Moslehi ◽

Keiichi Kondo ◽

William G. Kaelin ◽

...

Keyword(s):

Tumor Suppressor ◽

Ubiquitin Ligase ◽

Hypoxia Inducible Factor ◽

Detectable Effect ◽

Missense Mutations ◽

Subsequent Formation ◽

Von Hippel Lindau ◽

Ubiquitin Ligase Activity

ABSTRACT We examined the biogenesis of the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL) in vitro and in vivo. pVHL formed a complex with the cytosolic chaperonin containing TCP-1 (CCT or TRiC) en route to assembly with elongin B/C and the subsequent formation of the VCB-Cul2 ubiquitin ligase. Blocking the interaction of pVHL with elongin B/C resulted in accumulation of pVHL within the CCT complex. pVHL present in purified VHL-CCT complexes, when added to rabbit reticulocyte lysate, proceeded to form VCB and VCB-Cul2. Thus, CCT likely functions, at least in part, by retaining VHL chains pending the availability of elongin B/C for final folding and/or assembly. Tumor-associated mutations within exon II of the VHL syndrome had diverse effects upon the stability and/or function of pVHL-containing complexes. First, a pVHL mutant lacking the entire region encoded by exon II did not bind to CCT and yet could still assemble into complexes with elongin B/C and elongin B/C-Cul2. Second, a number of tumor-derived missense mutations in exon II did not decrease CCT binding, and most had no detectable effect upon VCB-Cul2 assembly. Many exon II mutants, however, were found to be defective in the binding to and subsequent ubiquitination of hypoxia-inducible factor 1α (HIF-1α), a substrate of the VCB-Cul2 ubiquitin ligase. We conclude that the selection pressure to mutate VHL exon II during tumorigenesis does not relate to loss of CCT binding but may reflect quantitative or qualitative defects in HIF binding and/or in pVHL-dependent ubiquitin ligase activity.

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The APC11 RING-H2 Finger Mediates E2-Dependent Ubiquitination

Molecular Biology of the Cell ◽

10.1091/mbc.11.7.2315 ◽

2000 ◽

Vol 11 (7) ◽

pp. 2315-2325 ◽

Cited By ~ 114

Author(s):

Joel D. Leverson ◽

Claudio A.P. Joazeiro ◽

Andrew M. Page ◽

Han-kuei Huang ◽

Philip Hieter ◽

...

Keyword(s):

Ubiquitin Ligase ◽

26S Proteasome ◽

Anaphase Promoting Complex ◽

Mitotic Progression ◽

Protein Substrates ◽

Ubiquitin Ligase Activity ◽

Ubiquitin Conjugating Enzyme ◽

Ubiquitin Conjugating Enzymes

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that theSaccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.

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The CUL1 C-Terminal Sequence and ROC1 Are Required for Efficient Nuclear Accumulation, NEDD8 Modification, and Ubiquitin Ligase Activity of CUL1

Molecular and Cellular Biology ◽

10.1128/mcb.20.21.8185-8197.2000 ◽

2000 ◽

Vol 20 (21) ◽

pp. 8185-8197 ◽

Cited By ~ 86

Author(s):

Manabu Furukawa ◽

Yanping Zhang ◽

Joseph McCarville ◽

Tomohiko Ohta ◽

Yue Xiong

Keyword(s):

Nuclear Localization ◽

Ubiquitin Ligase ◽

Nuclear Import ◽

Ring Finger ◽

Nuclear Accumulation ◽

Protein Families ◽

Terminal Sequence ◽

Ubiquitin Ligase Activity

ABSTRACT Members of the cullin and RING finger ROC protein families form heterodimeric complexes to constitute a potentially large number of distinct E3 ubiquitin ligases. We report here that the highly conserved C-terminal sequence in CUL1 is dually required, both for nuclear localization and for modification by NEDD8. Disruption of ROC1 binding impaired nuclear accumulation of CUL1 and decreased NEDD8 modification in vivo but had no effect on NEDD8 modification of CUL1 in vitro, suggesting that ROC1 promotes CUL1 nuclear accumulation to facilitate its NEDD8 modification. Disruption of NEDD8 binding had no effect on ROC1 binding, nor did it affect nuclear localization of CUL1, suggesting that nuclear localization and NEDD8 modification of CUL1 are two separable steps, with nuclear import preceding and required for NEDD8 modification. Disrupting NEDD8 modification diminishes the IκBα ubiquitin ligase activity of CUL1. These results identify a pathway for regulation of CUL1 activity—ROC1 and the CUL1 C-terminal sequence collaboratively mediate nuclear accumulation and NEDD8 modification, facilitating assembly of active CUL1 ubiquitin ligase. This pathway may be commonly utilized for the assembly of other cullin ligases.

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SUMO-targeted ubiquitin ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin

Molecular Biology of the Cell ◽

10.1091/mbc.e15-12-0827 ◽

2016 ◽

Vol 27 (9) ◽

pp. 1500-1510 ◽

Cited By ~ 42

Author(s):

Kentaro Ohkuni ◽

Yoshimitsu Takahashi ◽

Alyona Fulp ◽

Josh Lawrimore ◽

Wei-Chun Au ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Genome Stability ◽

Critical Role ◽

Histone H3 ◽

E3 Ligases ◽

Physiological Conditions ◽

Histone H3 Variant ◽

Maintain Genome Stability

Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast and flies and tumorigenesis in human cells; thus defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast shows that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by the small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1, since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization than either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.

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Np95 Is a Histone-Binding Protein Endowed with Ubiquitin Ligase Activity

Molecular and Cellular Biology ◽

10.1128/mcb.24.6.2526-2535.2004 ◽

2004 ◽

Vol 24 (6) ◽

pp. 2526-2535 ◽

Cited By ~ 126

Author(s):

Elisabetta Citterio ◽

Roberto Papait ◽

Francesco Nicassio ◽

Manuela Vecchi ◽

Paola Gomiero ◽

...

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Molecular Mechanisms ◽

Important Determinant ◽

Histone H3 ◽

Ring Finger ◽

Protein Domain ◽

Ubiquitin Ligase Activity

ABSTRACT Np95 is an important determinant in cell cycle progression. Its expression is tightly regulated and becomes detectable shortly before the entry of cells into S phase. Accordingly, Np95 is absolutely required for the G1/S transition. Its continued expression throughout the S/G2/M phases further suggests additional roles. Indeed, Np95 has been implicated in DNA damage response. Here, we show that Np95 is tightly bound to chromatin in vivo and that it binds to histones in vivo and in vitro. The binding to histones is direct and shows a remarkable preference for histone H3 and its N-terminal tail. A novel protein domain, the SRA-YDG domain, contained in Np95 is indispensable both for the interaction with histones and for chromatin binding in vivo. Np95 contains a RING finger. We show that this domain confers E3 ubiquitin ligase activity on Np95, which is specific for core histones, in vitro. Finally, Np95 shows specific E3 activity for histone H3 when the endogenous core octamer, coimmunoprecipitating with Np95, is used as a substrate. Histone ubiquitination is an important determinant in the regulation of chromatin structure and gene transcription. Thus, the demonstration that Np95 is a chromatin-associated ubiquitin ligase suggests possible molecular mechanisms for its action as a cell cycle regulator.

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The E3 Ubiquitin Ligase TBK1 Mediates the Degradation of Multiple Picornavirus VP3 Proteins by Phosphorylation and Ubiquitination

Journal of Virology ◽

10.1128/jvi.01438-19 ◽

2019 ◽

Vol 93 (23) ◽

Cited By ~ 4

Author(s):

Dan Li ◽

Wenping Yang ◽

Jingjing Ren ◽

Yi Ru ◽

Keshan Zhang ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Molecular Mechanisms ◽

E3 Ubiquitin Ligase ◽

Adaptor Protein ◽

Ubiquitin Ligase Activity ◽

Lysine Residues ◽

Innate Antiviral Immunity

ABSTRACT TANK-binding kinase 1 (TBK1) is essential for interferon beta (IFN-β) production and innate antiviral immunity. However, other, additional functions of TBK1 have remained elusive. Here, we showed that TBK1 is an E3 ubiquitin ligase that undergoes self-ubiquitylation in vitro in the presence of the E2 enzyme UbcH5c. Further evidence showed that TBK1 could also be self-ubiquitylated in vivo. Importantly, multiple picornavirus VP3 proteins were degraded by TBK1 through its kinase and E3 ubiquitin ligase activity. Mechanistically, TBK1 phosphorylated multiple picornavirus VP3 proteins at serine residues and ubiquitinated them via K63-linked ubiquitination at lysine residues. In addition, the C426 and C605 residues of TBK1 were not essential for TBK1 innate immunity activity; however, these residues were required for degradation of multiple picornavirus VP3 proteins and for its E3 ubiquitin ligase activity. Hence, our findings identified a novel role of TBK1 in regulating the virus life cycle and provided new insights into the molecular mechanisms of TBK1-mediated antiviral response. IMPORTANCE TBK1 is an important adaptor protein required for innate immune response to viruses, but its other functions were unknown. In this study, we found that TBK1 is an E3 ubiquitin ligase that undergoes self-ubiquitylation in vitro in the presence of the E2 enzyme UbcH5c. In addition, multiple picornavirus VP3 proteins were degraded by TBK1 through its kinase and E3 ubiquitin ligase activity. Our report provides evidence that TBK1 plays a role in viral protein degradation.

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The RBCC GeneRFP2(Leu5) Encodes a Novel Transmembrane E3 Ubiquitin Ligase Involved in ERAD

Molecular Biology of the Cell ◽

10.1091/mbc.e06-03-0248 ◽

2007 ◽

Vol 18 (5) ◽

pp. 1670-1682 ◽

Cited By ~ 71

Author(s):

Mikael Lerner ◽

Martin Corcoran ◽

Diana Cepeda ◽

Michael L. Nielsen ◽

Roman Zubarev ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Fluorescent Protein ◽

Transmembrane Domain ◽

Yellow Fluorescent Protein ◽

E3 Ubiquitin Ligase ◽

Coiled Coil ◽

Ring Finger ◽

Ubiquitin Ligase Activity

RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-δ, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.

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Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084120 ◽

1978 ◽

Vol 36 (2) ◽

pp. 650-651

Author(s):

Raul I. Garcia ◽

Evelyn A. Flynn ◽

George Szabo

Keyword(s):

Direct Injection ◽

Skin Pigmentation ◽

Freeze Fracture ◽

Pigment Granule ◽

Time Lapse ◽

Ultrastructural Level ◽

Lanthanum Tracer ◽

A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

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