Src-like adaptor protein regulates TCR expression on thymocytes by linking the ubiquitin ligase c-Cbl to the TCR complex.

PubMed

Myers, Margaret D; Sosinowski, Tomasz; Dragone, Leonard L; White, Carmen; Band, Hamid; Gu, Hua; Weiss, Arthur

2006-01-01

The adaptor molecule SLAP and E3 ubiquitin ligase c-Cbl each regulate expression of T cell receptor (TCR)-CD3 on thymocytes. Here we provide genetic and biochemical evidence that both molecules function in the same pathway. TCR-CD3 expression was similar in the absence of SLAP and/or c-Cbl. SLAP and c-Cbl were found to interact, and their expression together downregulated CD3epsilon. This required multiple domains in SLAP and the ring finger of c-Cbl. Furthermore, expression of SLAP and c-Cbl together induced TCRzeta ubiquitination and degradation, preventing the accumulation of fully assembled recycling TCR complexes. These studies indicate that SLAP links the E3 ligase activity of c-Cbl to the TCR, allowing for stage-specific regulation of TCR expression.

Protein degradation machinery is present broadly during early development in the sea urchin.

PubMed

Zazueta-Novoa, Vanesa; Wessel, Gary M

2014-07-01

Ubiquitin-dependent proteosome-mediated proteolysis is an important pathway of degradation that controls the timed destruction of cellular proteins in all tissues. All intracellular proteins and many extracellular proteins are continually being hydrolyzed to their constituent amino acids as a result of their recognition by E3 ligases for specific targeting of ubiquitination. Gustavus is a member of an ECS-type E3 ligase which interacts with Vasa, a DEAD-box RNA helicase, to regulate its localization during sea urchin embryonic development, and Gustavus mRNA accumulation is highly localized and dynamic during development. We tested if the core complex for Gustavus function was present in the embryo and if other SOCS box proteins also had restricted expression profiles that would inform future research. Expression patterns of the key members of the proteasomal function, such as the E3 core complex which interacts with Gustavus, and other E3-SOCS box proteins, are widely spread and dynamic in early development of the embryo suggesting broad core complex availability in the proteasome degradation pathway and temporal/spatial enrichments of various E3 ligase dependent targeting mechanisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Protein degradation machinery is present broadly during early development in the sea urchin

PubMed Central

Zazueta-Novoa, Vanesa; Wessel, Gary M.

2014-01-01

Ubiquitin-dependent proteosome-mediated proteolysis is an important pathway of degradation that controls the timed destruction of cellular proteins in all tissues. All intracellular proteins and many extracellular proteins are continually being hydrolyzed to their constituent amino acids as a result of their recognition by E3 ligases for specific targeting of ubiquitination. Gustavus is a member of an ECS-type E3 ligase which interacts with Vasa, a DEAD-box RNA helicase, to regulate its localization during sea urchin embryonic development, and Gustavus mRNA accumulation is highly localized and dynamic during development. We tested if the core complex for Gustavus function was present in the embryo and if other SOCS box proteins also had restricted expression profiles that would inform future research. Expression patterns of the key members of the proteasomal function, such as the E3 core complex which interacts with Gustavus, and other E3-SOCS box proteins, are widely spread and dynamic in early development of the embryo suggesting broad core complex availability in the proteasome degradation pathway and temporal/spatial enrichments of various E3 ligase dependent targeting mechanisms. PMID:24963879

Structure of a Glomulin-RBX1-CUL1 Complex: Inhibition of a RING E3 Ligase through Masking of Its E2-Binding Surface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duda, David M.; Olszewski, Jennifer L.; Tron, Adriana E.

2012-11-01

The approximately 300 human cullin-RING ligases (CRLs) are multisubunit E3s in which a RING protein, either RBX1 or RBX2, recruits an E2 to catalyze ubiquitination. RBX1-containing CRLs also can bind Glomulin (GLMN), which binds RBX1's RING domain, regulates the RBX1-CUL1-containing SCF{sup FBW7} complex, and is disrupted in the disease Glomuvenous Malformation. Here we report the crystal structure of a complex between GLMN, RBX1, and a fragment of CUL1. Structural and biochemical analyses reveal that GLMN adopts a HEAT-like repeat fold that tightly binds the E2-interacting surface of RBX1, inhibiting CRL-mediated chain formation by the E2 CDC34. The structure explains themore » basis for GLMN's selectivity toward RBX1 over RBX2, and how disease-associated mutations disrupt GLMN-RBX1 interactions. Our study reveals a mechanism for RING E3 ligase regulation, whereby an inhibitor blocks E2 access, and raises the possibility that other E3s are likewise controlled by cellular proteins that mask E2-binding surfaces to mediate inhibition.« less

A Cullin1-Based SCF E3 Ubiquitin Ligase Targets the InR/PI3K/TOR Pathway to Regulate Neuronal Pruning

PubMed Central

Wong, Jack Jing Lin; Wang, Cheng; Zhang, Heng; Kirilly, Daniel; Wu, Chunlai; Liou, Yih-Cherng; Wang, Hongyan; Yu, Fengwei

2013-01-01

Pruning that selectively eliminates unnecessary axons/dendrites is crucial for sculpting the nervous system during development. During Drosophila metamorphosis, dendrite arborization neurons, ddaCs, selectively prune their larval dendrites in response to the steroid hormone ecdysone, whereas mushroom body γ neurons specifically eliminate their axon branches within dorsal and medial lobes. However, it is unknown which E3 ligase directs these two modes of pruning. Here, we identified a conserved SCF E3 ubiquitin ligase that plays a critical role in pruning of both ddaC dendrites and mushroom body γ axons. The SCF E3 ligase consists of four core components Cullin1/Roc1a/SkpA/Slimb and promotes ddaC dendrite pruning downstream of EcR-B1 and Sox14, but independently of Mical. Moreover, we demonstrate that the Cullin1-based E3 ligase facilitates ddaC dendrite pruning primarily through inactivation of the InR/PI3K/TOR pathway. We show that the F-box protein Slimb forms a complex with Akt, an activator of the InR/PI3K/TOR pathway, and promotes Akt ubiquitination. Activation of the InR/PI3K/TOR pathway is sufficient to inhibit ddaC dendrite pruning. Thus, our findings provide a novel link between the E3 ligase and the InR/PI3K/TOR pathway during dendrite pruning. PMID:24068890

Structure and catalytic activation of the TRIM23 RING E3 ubiquitin ligase: DAWIDZIAK et al.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dawidziak, Daria M.; Sanchez, Jacint G.; Wagner, Jonathan M.

Tripartite motif (TRIM) proteins comprise a large family of RING-type ubiquitin E3 ligases that regulate important biological processes. An emerging general model is that TRIMs form elongated antiparallel coiled-coil dimers that prevent interaction of the two attendant RING domains. The RING domains themselves bind E2 conjugating enzymes as dimers, implying that an active TRIM ligase requires higher-order oligomerization of the basal coiled-coil dimers. Here, we report crystal structures of the TRIM23 RING domain in isolation and in complex with an E2–ubiquitin conjugate. Our results indicate that TRIM23 enzymatic activity requires RING dimerization, consistent with the general model of TRIM activation.

RNAi-Based Suppressor Screens Reveal Genetic Interactions Between the CRL2LRR-1 E3-Ligase and the DNA Replication Machinery in Caenorhabditis elegans.

PubMed

Ossareh-Nazari, Batool; Katsiarimpa, Anthi; Merlet, Jorge; Pintard, Lionel

2016-10-13

Cullin-RING E3-Ligases (CRLs), the largest family of E3 ubiquitin-Ligases, regulate diverse cellular processes by promoting ubiquitination of target proteins. The evolutionarily conserved Leucine Rich Repeat protein 1 (LRR-1) is a substrate-recognition subunit of a CRL2 LRR-1 E3-ligase. Here we provide genetic evidence supporting a role of this E3-enzyme in the maintenance of DNA replication integrity in Caenorhabditis elegans Through RNAi-based suppressor screens of lrr-1(0) and cul-2(or209ts) mutants, we identified two genes encoding components of the GINS complex, which is part of the Cdc45-MCM-GINS (CMG) replicative helicase, as well as CDC-7 and MUS-101, which drives the assembly of the CMG helicase during DNA replication. In addition, we identified the core components of the ATR/ATL-1 DNA replication checkpoint pathway (MUS-101, ATL-1, CLSP-1, CHK-1). These results suggest that the CRL2 LRR-1 E3-ligase acts to modify or degrade factor(s) that would otherwise misregulate the replisome, eventually leading to the activation of the DNA replication checkpoint. Copyright © 2016 Ossareh-Nazari et al.

Arabidopsis BPM Proteins Function as Substrate Adaptors to a CULLIN3-Based E3 Ligase to Affect Fatty Acid Metabolism in Plants[W

PubMed Central

Chen, Liyuan; Lee, Joo Hyun; Weber, Henriette; Tohge, Takayuki; Witt, Sandra; Roje, Sanja; Fernie, Alisdair R.; Hellmann, Hanjo

2013-01-01

Regulation of transcriptional processes is a critical mechanism that enables efficient coordination of the synthesis of required proteins in response to environmental and cellular changes. Transcription factors require accurate activity regulation because they play a critical role as key mediators assuring specific expression of target genes. In this work, we show that CULLIN3-based E3 ligases have the potential to interact with a broad range of ETHYLENE RESPONSE FACTOR (ERF)/APETALA2 (AP2) transcription factors, mediated by MATH-BTB/POZ (for Meprin and TRAF [tumor necrosis factor receptor associated factor] homolog)-Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) proteins. The assembly with an E3 ligase causes degradation of their substrates via the 26S proteasome, as demonstrated for the WRINKLED1 ERF/AP2 protein. Furthermore, loss of MATH-BTB/POZ proteins widely affects plant development and causes altered fatty acid contents in mutant seeds. Overall, this work demonstrates a link between fatty acid metabolism and E3 ligase activities in plants and establishes CUL3-based E3 ligases as key regulators in transcriptional processes that involve ERF/AP2 family members. PMID:23792371

The RanBP2/RanGAP1*SUMO1/Ubc9 SUMO E3 ligase is a disassembly machine for Crm1-dependent nuclear export complexes

PubMed Central

Ritterhoff, Tobias; Das, Hrishikesh; Hofhaus, Götz; Schröder, Rasmus R.; Flotho, Annette; Melchior, Frauke

2016-01-01

Continuous cycles of nucleocytoplasmic transport require disassembly of transport receptor/Ran-GTP complexes in the cytoplasm. A basic disassembly mechanism in all eukaryotes depends on soluble RanGAP and RanBP1. In vertebrates, a significant fraction of RanGAP1 stably interacts with the nucleoporin RanBP2 at a binding site that is flanked by FG-repeats and Ran-binding domains, and overlaps with RanBP2's SUMO E3 ligase region. Here, we show that the RanBP2/RanGAP1*SUMO1/Ubc9 complex functions as an autonomous disassembly machine with a preference for the export receptor Crm1. We describe three in vitro reconstituted disassembly intermediates, which show binding of a Crm1 export complex via two FG-repeat patches, cargo-release by RanBP2's Ran-binding domains and retention of free Crm1 at RanBP2 after Ran-GTP hydrolysis. Intriguingly, all intermediates are compatible with SUMO E3 ligase activity, suggesting that the RanBP2/RanGAP1*SUMO1/Ubc9 complex may link Crm1- and SUMO-dependent functions. PMID:27160050

The Replisome-Coupled E3 Ubiquitin Ligase Rtt101Mms22 Counteracts Mrc1 Function to Tolerate Genotoxic Stress

PubMed Central

Melnik, Andre; Wilson-Zbinden, Caroline; Schellhaas, René; Kastner, Lisa; Piwko, Wojciech; Dees, Martina; Picotti, Paola; Maric, Marija; Labib, Karim; Luke, Brian; Peter, Matthias

2016-01-01

Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1’s replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks. PMID:26849847

Inhibition of Vpx-Mediated SAMHD1 and Vpr-Mediated Host Helicase Transcription Factor Degradation by Selective Disruption of Viral CRL4 (DCAF1) E3 Ubiquitin Ligase Assembly.

PubMed

Wang, Hong; Guo, Haoran; Su, Jiaming; Rui, Yajuan; Zheng, Wenwen; Gao, Wenying; Zhang, Wenyan; Li, Zhaolong; Liu, Guanchen; Markham, Richard B; Wei, Wei; Yu, Xiao-Fang

2017-05-01

The lentiviral accessory proteins Vpx and Vpr are known to utilize CRL4 (DCAF1) E3 ligase to induce the degradation of the host restriction factor SAMHD1 or host helicase transcription factor (HLTF), respectively. Selective disruption of viral CRL4 (DCAF1) E3 ligase could be a promising antiviral strategy. Recently, we have determined that posttranslational modification (neddylation) of Cullin-4 is required for the activation of Vpx-CRL4 (DCAF1) E3 ligase. However, the mechanism of Vpx/Vpr-CRL4 (DCAF1) E3 ligase assembly is still poorly understood. Here, we report that zinc coordination is an important regulator of Vpx-CRL4 E3 ligase assembly. Residues in a conserved zinc-binding motif of Vpx were essential for the recruitment of the CRL4 (DCAF1) E3 complex and Vpx-induced SAMHD1 degradation. Importantly, altering the intracellular zinc concentration by treatment with the zinc chelator N , N , N '-tetrakis-(2'-pyridylmethyl)ethylenediamine (TPEN) potently blocked Vpx-mediated SAMHD1 degradation and inhibited wild-type SIVmac (simian immunodeficiency virus of macaques) infection of myeloid cells, even in the presence of Vpx. TPEN selectively inhibited Vpx and DCAF1 binding but not the Vpx-SAMHD1 interaction or Vpx virion packaging. Moreover, we have shown that zinc coordination is also important for the assembly of the HIV-1 Vpr-CRL4 E3 ligase. In particular, Vpr zinc-binding motif mutation or TPEN treatment efficiently inhibited Vpr-CRL4 (DCAF1) E3 ligase assembly and Vpr-mediated HLTF degradation or Vpr-induced G 2 cell cycle arrest. Collectively, our study sheds light on a conserved strategy by the viral proteins Vpx and Vpr to recruit host CRL4 (DCAF1) E3 ligase, which represents a target for novel anti-human immunodeficiency virus (HIV) drug development. IMPORTANCE The Vpr and its paralog Vpx are accessory proteins encoded by different human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) lentiviruses. To facilitate viral replication, Vpx has evolved to induce SAMHD1 degradation and Vpr to mediate HLTF degradation. Both Vpx and Vpr perform their functions by recruiting CRL4 (DCAF1) E3 ligase. In this study, we demonstrate that the assembly of the Vpx- or Vpr-CRL4 E3 ligase requires a highly conserved zinc-binding motif. This motif is specifically required for the DCAF1 interaction but not for the interaction of Vpx or Vpr with its substrate. Selective disruption of Vpx- or Vpr-CRL4 E3 ligase function was achieved by zinc sequestration using N , N , N '-tetrakis-(2'-pyridylmethyl)ethylenediamine (TPEN). At the same time, zinc sequestration had no effect on zinc-dependent cellular protein functions. Therefore, information obtained from this study may be important for novel anti-HIV drug development. Copyright © 2017 American Society for Microbiology.

TMEM129 is a Derlin-1 associated ERAD E3 ligase essential for virus-induced degradation of MHC-I.

PubMed

van den Boomen, Dick J H; Timms, Richard T; Grice, Guinevere L; Stagg, Helen R; Skødt, Karsten; Dougan, Gordon; Nathan, James A; Lehner, Paul J

2014-08-05

The US11 gene product of human cytomegalovirus promotes viral immune evasion by hijacking the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. US11 initiates dislocation of newly translocated MHC I from the ER to the cytosol for proteasome-mediated degradation. Despite the critical role for ubiquitin in this degradation pathway, the responsible E3 ligase is unknown. In a forward genetic screen for host ERAD components hijacked by US11 in near-haploid KBM7 cells, we identified TMEM129, an uncharacterized polytopic membrane protein. TMEM129 is essential and rate-limiting for US11-mediated MHC-I degradation and acts as a novel ER resident E3 ubiquitin ligase. TMEM129 contains an unusual cysteine-only RING with intrinsic E3 ligase activity and is recruited to US11 via Derlin-1. Together with its E2 conjugase Ube2J2, TMEM129 is responsible for the ubiquitination, dislocation, and subsequent degradation of US11-associated MHC-I. US11 engages two degradation pathways: a Derlin-1/TMEM129-dependent pathway required for MHC-I degradation and a SEL1L/HRD1-dependent pathway required for "free" US11 degradation. Our data show that TMEM129 is a novel ERAD E3 ligase and the central component of a novel mammalian ERAD complex.

The E3 Ligase APC/C-Cdh1 Is Required for Associative Fear Memory and Long-Term Potentiation in the Amygdala of Adult Mice

ERIC Educational Resources Information Center

Pick, Joseph E.; Malumbres, Marcos; Klann, Eric

2013-01-01

The anaphase promoting complex/cyclosome (APC/C) is an E3 ligase regulated by Cdh1. Beyond its role in controlling cell cycle progression, APC/C-Cdh1 has been detected in neurons and plays a role in long-lasting synaptic plasticity and long-term memory. Herein, we further examined the role of Cdh1 in synaptic plasticity and memory by generating…

Targeting Cullin–RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation

PubMed Central

Bulatov, Emil; Ciulli, Alessio

2015-01-01

In the last decade, the ubiquitin–proteasome system has emerged as a valid target for the development of novel therapeutics. E3 ubiquitin ligases are particularly attractive targets because they confer substrate specificity on the ubiquitin system. CRLs [Cullin–RING (really interesting new gene) E3 ubiquitin ligases] draw particular attention, being the largest family of E3s. The CRLs assemble into functional multisubunit complexes using a repertoire of substrate receptors, adaptors, Cullin scaffolds and RING-box proteins. Drug discovery targeting CRLs is growing in importance due to mounting evidence pointing to significant roles of these enzymes in diverse biological processes and human diseases, including cancer, where CRLs and their substrates often function as tumour suppressors or oncogenes. In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity. A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included. This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs. PMID:25886174

MicroRNA-300 Regulates the Ubiquitination of PTEN through the CRL4BDCAF13 E3 Ligase in Osteosarcoma Cells.

PubMed

Chen, Zhi; Zhang, Wei; Jiang, Kaibiao; Chen, Bin; Wang, Kun; Lao, Lifeng; Hou, Canglong; Wang, Fei; Zhang, Caiguo; Shen, Hongxing

2018-03-02

Cullins, critical members of the cullin-RING ubiquitin ligases (CRLs), are often aberrantly expressed in different cancers. However, the underlying mechanisms regarding aberrant expression of these cullins and the specific substrates of CRLs in different cancers are mostly unknown. Here, we demonstrate that overexpressed CUL4B in human osteosarcoma cells forms an E3 complex with DNA damage binding protein 1 (DDB1) and DDB1- and CUL4-associated factor 13 (DCAF13). In vitro and in vivo analyses indicated that the CRL4B DCAF13 E3 ligase specifically recognized the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) for degradation, and disruption of this E3 ligase resulted in PTEN accumulation. Further analyses indicated that miR-300 directly targeted the 3' UTR of CUL4B, and DNA hypermethylation of a CpG island in the miR-300 promoter region contributed to the downregulation of miR-300. Interestingly, ectopic expression of miR-300 or treatment with 5-AZA-2'-deoxycytidine, a DNA methylation inhibitor, decreased the stability of CRL4B DCAF13 E3 ligase and reduced PTEN ubiquitination. By applying in vitro screening to identify small molecules that specifically inhibit CUL4B-DDB1 interaction, we found that TSC01131 could greatly inhibit osteosarcoma cell growth and could disrupt the stability of the CRL4B DCAF13 E3 ligase. Collectively, our findings shed new light on the molecular mechanism of CUL4B function and might also provide a new avenue for osteosarcoma therapy. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Development of an efficient E. coli expression and purification system for a catalytically active, human Cullin3-RINGBox1 protein complex and elucidation of its quaternary structure with Keap1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Small, Evan; Eggler, Aimee; Mesecar, Andrew D., E-mail: amesecar@purdue.edu

2010-10-01

Research highlights: {yields} A novel expression strategy was used to purify Cul3-Rbx1 from E. coli. {yields} The Cul3-Rbx1 complex is fully active and catalyzes ubiquitination of Nrf2 in vitro. {yields} Cul3, Rbx1, and Keap1 form a complex with unique stoichiometry of 1:1:2. -- Abstract: The Cullin3-based E3 ubiquitin ligase complex is thought to play an important role in the cellular response to oxidative stress and xenobiotic assault. While limited biochemical studies of the ligase's role in these complex signaling pathways are beginning to emerge, structural studies are lagging far behind due to the inability to acquire sufficient quantities of full-length,more » highly pure and active Cullin3. Here we describe the design and construction of an optimized expression and purification system for the full-length, human Cullin3-RINGBox 1 (Rbx1) protein complex from Escherichia coli. The dual-expression system is comprised of codon-optimized Cullin3 and Rbx1 genes co-expressed from a single pET-Duet-1 plasmid. Rapid purification of the Cullin3-Rbx1 complex is achieved in two steps via an affinity column followed by size-exclusion chromatography. Approximately 15 mg of highly pure and active Cullin3-Rbx1 protein from 1 L of E. coli culture can be achieved. Analysis of the quaternary structure of the Cullin3-Rbx1 and Cullin3-Rbx1-Keap1 complexes by size-exclusion chromatography and analytical ultracentrifugation indicates a 1:1 stoichiometry for the Cullin3-Rbx1 complex (MW = 111 kDa), and a 1:1:2 stoichiometry for the Cullin3-Rbx1-Keap1 complex (MW = 280 kDa). This latter complex has a novel quaternary structural organization for cullin E3 ligases, and it is fully active based on an in vitro Cullin3-Rbx1-Keap1-Nrf2 ubiquitination activity assay that was developed and optimized in this study.« less

Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin.

PubMed

Lazarou, Michael; Jin, Seok Min; Kane, Lesley A; Youle, Richard J

2012-02-14

Mutations in the mitochondrial kinase PINK1 and the cytosolic E3 ligase Parkin can cause Parkinson's disease. Damaged mitochondria accumulate PINK1 on the outer membrane where, dependent on kinase activity, it recruits and activates Parkin to induce mitophagy, potentially maintaining organelle fidelity. How PINK1 recruits Parkin is unknown. We show that endogenous PINK1 forms a 700 kDa complex with the translocase of the outer membrane (TOM) selectively on depolarized mitochondria whereas PINK1 ectopically targeted to the outer membrane retains association with TOM on polarized mitochondria. Inducibly targeting PINK1 to peroxisomes or lysosomes, which lack a TOM complex, recruits Parkin and activates ubiquitin ligase activity on the respective organelles. Once there, Parkin induces organelle selective autophagy of peroxisomes but not lysosomes. We propose that the association of PINK1 with the TOM complex allows rapid reimport of PINK1 to rescue repolarized mitochondria from mitophagy, and discount mitochondrial-specific factors for Parkin translocation and activation. Copyright © 2012 Elsevier Inc. All rights reserved.

Regulation of the Hippo signaling pathway by ubiquitin modification.

PubMed

Kim, Youngeun; Jho, Eek-Hoon

2018-03-01

The Hippo signaling pathway plays an essential role in adult tissue homeostasis and organ size control. Abnormal regulation of Hippo signaling can be a cause for multiple types of human cancers. Since the awareness of the importance of the Hippo signaling in a wide range of biological fields has been continually grown, it is also understood that a thorough and well-rounded comprehension of the precise dynamics could provide fundamental insights for therapeutic applications. Several components in the Hippo signaling pathway are known to be targeted for proteasomal degradation via ubiquitination by E3 ligases. β-TrCP is a well-known E3 ligase of YAP/TAZ, which leads to the reduction of YAP/TAZ levels. The Hippo signaling pathway can also be inhibited by the E3 ligases (such as ITCH) which target LATS1/2 for degradation. Regulation via ubiquitination involves not only complex network of E3 ligases but also deubiquitinating enzymes (DUBs), which remove ubiquitin from its targets. Interestingly, non-degradative ubiquitin modifications are also known to play important roles in the regulation of Hippo signaling. Although there has been much advanced progress in the investigation of ubiquitin modifications acting as regulators of the Hippo signaling pathway, research done to date still remains inadequate due to the sheer complexity and diversity of the subject. Herein, we review and discuss recent developments that implicate ubiquitin-mediated regulatory mechanisms at multiple steps of the Hippo signaling pathway. [BMB Reports 2018; 51(3): 143-150].

The caenorhabditis elegans CDT-2 ubiquitin ligase is required for attenuation of EGFR signalling in vulva precursor cells

PubMed Central

2010-01-01

Background Attenuation of the EGFR (Epidermal Growth Factor Receptor) signalling cascade is crucial to control cell fate during development. A candidate-based RNAi approach in C. elegans identified CDT-2 as an attenuator of LET-23 (EGFR) signalling. Human CDT2 is a component of the conserved CDT2/CUL4/DDB1 ubiquitin ligase complex that plays a critical role in DNA replication and G2/M checkpoint. Within this complex, CDT2 is responsible for substrate recognition. This ubiquitin ligase complex has been shown in various organisms, including C. elegans, to target the replication-licensing factor CDT1, and the CDK inhibitor p21. However, no previous link to EGFR signalling has been identified. Results We have characterised CDT-2's role during vulva development and found that it is a novel attenuator of LET-23 signalling. CDT-2 acts redundantly with negative modulators of LET-23 signalling and CDT-2 or CUL-4 downregulation causes persistent expression of the egl-17::cfp transgene, a marker of LET-23 signalling during vulva development. In addition, we show that CDT-2 physically interacts with SEM-5 (GRB2), a known negative modulator of LET-23 signalling that directly binds LET-23, and provide genetic evidence consistent with CDT-2 functioning at or downstream of LET-23. Interestingly, both SEM-5 and CDT-2 were identified independently in a screen for genes involved in receptor-mediated endocytosis in oocytes, suggesting that attenuation of LET-23 by CDT-2 might be through regulation of endocytosis. Conclusions In this study, we have shown that CDT-2 and CUL-4, members of the CUL-4/DDB-1/CDT-2 E3 ubiquitin ligase complex attenuate LET-23 signalling in vulval precursor cells. In future, it will be interesting to investigate the potential link to endocytosis and to determine whether other signalling pathways dependent on endocytosis, e.g. LIN-12 (Notch) could be regulated by this ubiquitin ligase complex. This work has uncovered a novel function for the CUL-4/DDB-1/CDT-2 E3 ligase that may be relevant for its mammalian oncogenic activity. PMID:20977703

Sorting of a multi-subunit ubiquitin ligase complex in the endolysosome system

PubMed Central

Yang, Xi; Arines, Felichi Mae; Zhang, Weichao

2018-01-01

The yeast Dsc E3 ligase complex has long been recognized as a Golgi-specific protein ubquitination system. It shares a striking sequence similarity to the Hrd1 complex that plays critical roles in the ER-associated degradation pathway. Using biochemical purification and mass spectrometry, we identified two novel Dsc subunits, which we named as Gld1 and Vld1. Surprisingly, Gld1 and Vld1 do not coexist in the same complex. Instead, they compete with each other to form two functionally independent Dsc subcomplexes. The Vld1 subcomplex takes the AP3 pathway to reach the vacuole membrane, whereas the Gld1 subcomplex travels through the VPS pathway and is cycled between Golgi and endosomes by the retromer. Thus, instead of being Golgi-specific, the Dsc complex can regulate protein levels at three distinct organelles, namely Golgi, endosome, and vacuole. Our study provides a novel model of achieving multi-tasking for transmembrane ubiquitin ligases with interchangeable trafficking adaptors. PMID:29355480

Linear ubiquitin chains: enzymes, mechanisms and biology

PubMed Central

2017-01-01

Ubiquitination is a versatile post-translational modification that regulates a multitude of cellular processes. Its versatility is based on the ability of ubiquitin to form multiple types of polyubiquitin chains, which are recognized by specific ubiquitin receptors to induce the required cellular response. Linear ubiquitin chains are linked through Met 1 and have been established as important players of inflammatory signalling and apoptotic cell death. These chains are generated by a ubiquitin E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) that is thus far the only E3 ligase capable of forming linear ubiquitin chains. The complex consists of three subunits, HOIP, HOIL-1L and SHARPIN, each of which have specific roles in the observed biological functions of LUBAC. Furthermore, LUBAC has been found to be associated with OTULIN and CYLD, deubiquitinases that disassemble linear chains and counterbalance the E3 ligase activity of LUBAC. Gene mutations in HOIP, HOIL-1L and OTULIN are found in human patients who suffer from autoimmune diseases, and HOIL-1L mutations are also found in myopathy patients. In this paper, we discuss the mechanisms of linear ubiquitin chain generation and disassembly by their respective enzymes and review our current understanding of their biological functions and association with human diseases. PMID:28446710

Linear ubiquitin chains: enzymes, mechanisms and biology.

PubMed

Rittinger, Katrin; Ikeda, Fumiyo

2017-04-01

Ubiquitination is a versatile post-translational modification that regulates a multitude of cellular processes. Its versatility is based on the ability of ubiquitin to form multiple types of polyubiquitin chains, which are recognized by specific ubiquitin receptors to induce the required cellular response. Linear ubiquitin chains are linked through Met 1 and have been established as important players of inflammatory signalling and apoptotic cell death. These chains are generated by a ubiquitin E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) that is thus far the only E3 ligase capable of forming linear ubiquitin chains. The complex consists of three subunits, HOIP, HOIL-1L and SHARPIN, each of which have specific roles in the observed biological functions of LUBAC. Furthermore, LUBAC has been found to be associated with OTULIN and CYLD, deubiquitinases that disassemble linear chains and counterbalance the E3 ligase activity of LUBAC. Gene mutations in HOIP, HOIL-1L and OTULIN are found in human patients who suffer from autoimmune diseases, and HOIL-1L mutations are also found in myopathy patients. In this paper, we discuss the mechanisms of linear ubiquitin chain generation and disassembly by their respective enzymes and review our current understanding of their biological functions and association with human diseases. © 2017 The Authors.

Mapping the interactome of HPV E6 and E7 oncoproteins with the ubiquitin-proteasome system.

PubMed

Poirson, Juline; Biquand, Elise; Straub, Marie-Laure; Cassonnet, Patricia; Nominé, Yves; Jones, Louis; van der Werf, Sylvie; Travé, Gilles; Zanier, Katia; Jacob, Yves; Demeret, Caroline; Masson, Murielle

2017-10-01

Protein ubiquitination and its reverse reaction, deubiquitination, regulate protein stability, protein binding activity, and their subcellular localization. These reactions are catalyzed by the enzymes E1, E2, and E3 ubiquitin (Ub) ligases and deubiquitinases (DUBs). The Ub-proteasome system (UPS) is targeted by viruses for the sake of their replication and to escape host immune response. To identify novel partners of human papillomavirus 16 (HPV16) E6 and E7 proteins, we assembled and screened a library of 590 cDNAs related to the UPS by using the Gaussia princeps luciferase protein complementation assay. HPV16 E6 was found to bind to the homology to E6AP C terminus-type Ub ligase (E6AP), three really interesting new gene (RING)-type Ub ligases (MGRN1, LNX3, LNX4), and the DUB Ub-specific protease 15 (USP15). Except for E6AP, the binding of UPS factors did not require the LxxLL-binding pocket of HPV16 E6. LNX3 bound preferentially to all high-risk mucosal HPV E6 tested, whereas LNX4 bound specifically to HPV16 E6. HPV16 E7 was found to bind to several broad-complex tramtrack and bric-a-brac domain-containing proteins (such as TNFAIP1/KCTD13) that are potential substrate adaptors of Cullin 3-RING Ub ligases, to RING-type Ub ligases implicated in innate immunity (RNF135, TRIM32, TRAF2, TRAF5), to the substrate adaptor DCAF15 of Cullin 4-RING Ub ligase and to some DUBs (USP29, USP33). The binding to UPS factors did not require the LxCxE motif but rather the C-terminal region of HPV16 E7 protein. The identified UPS factors interacted with most of E7 proteins across different HPV types. This study establishes a strategy for the rapid identification of interactions between host or pathogen proteins and the human ubiquitination system. © 2017 Federation of European Biochemical Societies.

Regulation of AR Degradation and Function by Ubiquitylation

DTIC Science & Technology

2015-10-01

ubiquitylation and degradation remain to be established. Newly synthesized AR associates with an HSP90 chaperone complex, and an HSP90 associated E3 ubiquitin ...clearly additional cytoplasmic and/or nuclear ubiquitin ligases that regulate the normal turnover and degradation of the liganded AR. Indeed, multiple... ubiquitin ligases have been reported to interact with AR and regulate its transcriptional activities and/or degradation. Moreover, previous studies

PHD domain-mediated E3 ligase activity directs intramolecular sumoylation of an adjacent bromodomain required for gene silencing.

PubMed

Ivanov, Alexey V; Peng, Hongzhuang; Yurchenko, Vyacheslav; Yap, Kyoko L; Negorev, Dmitri G; Schultz, David C; Psulkowski, Elyse; Fredericks, William J; White, David E; Maul, Gerd G; Sadofsky, Moshe J; Zhou, Ming-Ming; Rauscher, Frank J

2007-12-14

Tandem PHD and bromodomains are often found in chromatin-associated proteins and have been shown to cooperate in gene silencing. Each domain can bind specifically modified histones: the mechanisms of cooperation between these domains are unknown. We show that the PHD domain of the KAP1 corepressor functions as an intramolecular E3 ligase for sumoylation of the adjacent bromodomain. The RING finger-like structure of the PHD domain is required for both Ubc9 binding and sumoylation and directs modification to specific lysine residues in the bromodomain. Sumoylation is required for KAP1-mediated gene silencing and functions by directly recruiting the SETDB1 histone methyltransferase and the CHD3/Mi2 component of the NuRD complex via SUMO-interacting motifs. Sumoylated KAP1 stimulates the histone methyltransferase activity of SETDB1. These data provide a mechanistic explanation for the cooperation of PHD and bromodomains in gene regulation and describe a function of the PHD domain as an intramolecular E3 SUMO ligase.

Analysis of the DNA joining repertoire of Chlorella virus DNA ligase and a new crystal structure of the ligase-adenylate intermediate.

PubMed

Odell, Mark; Malinina, Lucy; Sriskanda, Verl; Teplova, Marianna; Shuman, Stewart

2003-09-01

Chlorella virus DNA ligase is the smallest eukaryotic ATP-dependent DNA ligase known; it suffices for yeast cell growth in lieu of the essential yeast DNA ligase Cdc9. The Chlorella virus ligase-adenylate intermediate has an intrinsic nick sensing function and its DNA footprint extends 8-9 nt on the 3'-hydroxyl (3'-OH) side of the nick and 11-12 nt on the 5'-phosphate (5'-PO4) side. Here we establish the minimal length requirements for ligatable 3'-OH and 5'-PO4 strands at the nick (6 nt) and describe a new crystal structure of the ligase-adenylate in a state construed to reflect the configuration of the active site prior to nick recognition. Comparison with a previous structure of the ligase-adenylate bound to sulfate (a mimetic of the nick 5'-PO4) suggests how the positions and contacts of the active site components and the bound adenylate are remodeled by DNA binding. We find that the minimal Chlorella virus ligase is capable of catalyzing non-homologous end-joining reactions in vivo in yeast, a process normally executed by the structurally more complex cellular Lig4 enzyme. Our results suggest a model of ligase evolution in which: (i) a small 'pluripotent' ligase is the progenitor of the much larger ligases found presently in eukaryotic cells and (ii) gene duplications, variations within the core ligase structure and the fusion of new domains to the core structure (affording new protein-protein interactions) led to the compartmentalization of eukaryotic ligase function, i.e. by enhancing some components of the functional repertoire of the ancestral ligase while disabling others.

Activity‐Based Probes for HECT E3 Ubiquitin Ligases

PubMed Central

Byrne, Robert; Mund, Thomas

2017-01-01

Abstract Activity‐based probes (ABPs) have been used to dissect the biochemical/structural properties and cellular functions of deubiquitinases. However, their utility in studying cysteine‐based E3 ubiquitin ligases has been limited. In this study, we evaluate the use of ubiquitin‐ABPs (Ub‐VME and Ub‐PA) and a novel set of E2–Ub‐ABPs on a panel of HECT E3 ubiquitin ligases. Our in vitro data show that ubiquitin‐ABPs can label HECT domains. We also provide the first evidence that, in addition to the RBR E3 ubiquitin ligase Parkin, E2–Ub‐ABPs can also label the catalytic HECT domains of NEDD4, UBE3C, and HECTD1. Importantly, the endogenous proteasomal E3 ligase UBE3C was also successfully labelled by Ub‐PA and His‐UBE2D2–Ub‐ABP in lysate of cells grown under basal conditions. Our findings provide novel insights into the use of ABPs for the study of HECT E3 ubiquitin ligases. PMID:28425671

E3 ubiquitin ligases: key regulators of hormone signaling in plants.

PubMed

Kelley, Dior

2018-03-07

Ubiquitin-mediated control of protein stability is central to most aspects of plant hormone signaling. Attachment of ubiquitin to target proteins occurs via an enzymatic cascade with the final step being catalyzed by a family of enzymes known as E3 ubiquitin ligases, which have been classified based on their protein domains and structures. While E3 ubiquitin ligases are conserved among eukaryotes, in plants they are well-known to fulfill unique roles as central regulators of phytohormone signaling, including hormone perception and regulation of hormone biosynthesis. This review will highlight up-to-date findings that have refined well-known E3 ligase-substrate interactions and defined novel E3 ligase substrates that mediate numerous hormone signaling pathways. Additionally, examples of how particular E3 ligases may mediate hormone crosstalk will be discussed as an emerging theme. Looking forward, promising experimental approaches and methods that will provide deeper mechanistic insight into the roles of E3 ubiquitin ligases in plants will be considered. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Role of SKP1-CUL1-F-Box-Protein (SCF) E3 Ubiquitin Ligases in Skin Cancer

PubMed Central

Xie, Chuan-Ming; Wei, Wenyi; Sun, Yi

2013-01-01

Many biological processes such as cell proliferation, differentiation, and cell death depend precisely on the timely synthesis and degradation of key regulatory proteins. While protein synthesis can be regulated at multiple levels, protein degradation is mainly controlled by the ubiquitin—proteasome system (UPS), which consists of two distinct steps: (1) ubiquitylation of targeted protein by E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase, and (2) subsequent degradation by the 26S proteasome. Among all E3 ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) E3 ligases are the largest family and are responsible for the turnover of many key regulatory proteins. Aberrant regulation of SCF E3 ligases is associated with various human diseases, such as cancers, including skin cancer. In this review, we provide a comprehensive overview of all currently published data to define a promoting role of SCF E3 ligases in the development of skin cancer. The future directions in this area of research are also discussed with an ultimate goal to develop small molecule inhibitors of SCF E3 ligases as a novel approach for the treatment of human skin cancer. Furthermore, altered components or substrates of SCF E3 ligases may also be developed as the biomarkers for early diagnosis or predicting prognosis. PMID:23522382

Protein quality control at the inner nuclear membrane

PubMed Central

Khmelinskii, Anton; Blaszczak, Ewa; Pantazopoulou, Marina; Fischer, Bernd; Omnus, Deike J.; Le Dez, Gaëlle; Brossard, Audrey; Gunnarsson, Alexander; Barry, Joseph D.; Meurer, Matthias; Kirrmaier, Daniel; Boone, Charles; Huber, Wolfgang; Rabut, Gwenaël; Ljungdahl, Per O.; Knop, Michael

2015-01-01

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression1. The outer nuclear membrane is continuous with the endoplasmic reticulum (ER) and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by ER-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc72,3. However, little is known regarding protein quality control at the INM. Here we describe a protein degradation pathway at the INM mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi34. We report that the As complex functions together with the ubiquitin conjugating enzymes Ubc6andUbc7to degrade soluble and integral membrane proteins. Genetic evidence suggest that the Asi ubiquitin ligase defines a pathway distinct from but complementary to ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer (tFT)5, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquity ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalised integral membrane proteins, thus acting to maintain and safeguard the identity of the INM. PMID:25519137

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity.

PubMed

Koliopoulos, Marios G; Esposito, Diego; Christodoulou, Evangelos; Taylor, Ian A; Rittinger, Katrin

2016-06-01

TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N-terminal portion which comprises a canonical RING domain, one or two B-box domains and a coiled-coil region that mediates ligase dimerization. Self-association via the coiled-coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin-loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full-length protein. Our data reveal an unexpected diversity in the self-association mechanism of TRIMs that might be crucial for their biological function. © 2016 Francis Crick Institute. Published under the terms of the CC BY 4.0 license.

RING-Domain E3 Ligase-Mediated Host–Virus Interactions: Orchestrating Immune Responses by the Host and Antagonizing Immune Defense by Viruses

PubMed Central

Zhang, Yuexiu; Li, Lian-Feng; Munir, Muhammad; Qiu, Hua-Ji

2018-01-01

The RING-domain E3 ligases (RING E3s), a group of E3 ligases containing one or two RING finger domains, are involved in various cellular processes such as cell proliferation, immune regulation, apoptosis, among others. In the host, a substantial number of the RING E3s have been implicated to inhibit viral replication through regulating immune responses, including activation and inhibition of retinoic acid-inducible gene I-like receptors, toll-like receptors, and DNA receptor signaling pathways, modulation of cell-surface expression of major histocompatibility complex, and co-stimulatory molecules. During the course of evolution and adaptation, viruses encode RING E3s to antagonize host immune defense, such as the infected cell protein 0 of herpes simplex virus type 1, the non-structural protein 1 of rotavirus, and the K3 and K5 of Kaposi’s sarcoma-associated herpesvirus. In addition, recent studies suggest that viruses can hijack the host RING E3s to facilitate viral replication. Based on emerging and interesting discoveries, the RING E3s present novel links among the host and viruses. Herein, we focus on the latest research progresses in the RING E3s-mediated host–virus interactions and discuss the outlooks of the RING E3s for future research. PMID:29872431

Functional characterization of the Dsc E3 ligase complex in the citrus postharvest pathogen Penicillium digitatum.

PubMed

Ruan, Ruoxin; Chung, Kuang-Ren; Li, Hongye

2017-12-01

Sterol regulatory element binding proteins (SREBPs) are required for sterol homeostasis in eukaryotes. Activation of SREBPs is regulated by the Dsc E3 ligase complex in Schizosaccharomyces pombe and Aspergillus spp. Previous studies indicated that an SREBP-coding gene PdsreA is required for fungicide resistance and ergosterol biosynthesis in the citrus postharvest pathogen Penicillium digitatum. In this study, five genes, designated PddscA, PddscB, PddscC, PddscD, and PddscE encoding the Dsc E3 ligase complex were characterized to be required for fungicide resistance, ergosterol biosynthesis and CoCl 2 tolerance in P. digitatum. Each of the dsc genes was inactivated by target gene disruption and the resulted phenotypes were analyzed and compared. Genetic analysis reveals that, of five Dsc complex components, PddscB is the core subunit gene in P. digitatum. Although the resultant dsc mutants were able to infect citrus fruit and induce maceration lesions as the wild-type, the mutants rarely produced aerial mycelia on affected citrus fruit peels. P. digitatum Dsc proteins regulated not only the expression of genes involved in ergosterol biosynthesis but also that of PdsreA. Yeast two-hybrid assays revealed a direct interaction between the PdSreA protein and the Dsc proteins. Ectopic expression of the PdSreA N-terminus restored fungicide resistance in the dsc mutants. Our results provide important evidence to understand the mechanisms underlying SREBP activation and regulation of ergosterol biosynthesis in plant pathogenic fungi. Copyright © 2017 Elsevier GmbH. All rights reserved.

A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

DOE Office of Scientific and Technical Information (OSTI.GOV)

McMillan, Brian J.; Schnute, Björn; Ohlenhard, Nadja

Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. Here we present crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membranemore » proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.« less

A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

PubMed Central

McMillan, Brian J.; Schnute, Björn; Ohlenhard, Nadja; Zimmerman, Brandon; Miles, Laura; Beglova, Natalia; Klein, Thomas; Blacklow, Stephen C.

2015-01-01

Summary Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. We present here crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membrane proximal region and the other near the C-terminus. Together, these studies provide new insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential new target for therapeutic modulation of Notch signal transduction in disease. PMID:25747658

A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

DOE PAGES

McMillan, Brian J.; Schnute, Björn; Ohlenhard, Nadja; ...

2015-03-05

Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. Here we present crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membranemore » proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.« less

Implication of SUMO E3 ligases in nucleotide excision repair.

PubMed

Tsuge, Maasa; Kaneoka, Hidenori; Masuda, Yusuke; Ito, Hiroki; Miyake, Katsuhide; Iijima, Shinji

2015-08-01

Post-translational modifications alter protein function to mediate complex hierarchical regulatory processes that are crucial to eukaryotic cellular function. The small ubiquitin-like modifier (SUMO) is an important post-translational modification that affects transcriptional regulation, nuclear localization, and the maintenance of genome stability. Nucleotide excision repair (NER) is a very versatile DNA repair system that is essential for protection against ultraviolet (UV) irradiation. The deficiencies in NER function remarkably increase the risk of skin cancer. Recent studies have shown that several NER factors are SUMOylated, which influences repair efficiency. However, how SUMOylation modulates NER has not yet been elucidated. In the present study, we performed RNAi knockdown of SUMO E3 ligases and found that, in addition to PIASy, the polycomb protein Pc2 affected the repair of cyclobutane pyrimidine dimers. PIAS1 affected both the removal of 6-4 pyrimidine pyrimidone photoproducts and cyclobutane pyrimidine dimers, whereas other SUMO E3 ligases did not affect the removal of either UV lesion.

Functional characterization of DnSIZ1, a SIZ/PIAS-type SUMO E3 ligase from Dendrobium.

PubMed

Liu, Feng; Wang, Xiao; Su, Mengying; Yu, Mengyuan; Zhang, Shengchun; Lai, Jianbin; Yang, Chengwei; Wang, Yaqin

2015-09-17

SUMOylation is an important post-translational modification of eukaryotic proteins that involves the reversible conjugation of a small ubiquitin-related modifier (SUMO) polypeptide to its specific protein substrates, thereby regulating numerous complex cellular processes. The PIAS (protein inhibitor of activated signal transducers and activators of transcription [STAT]) and SIZ (scaffold attachment factor A/B/acinus/PIAS [SAP] and MIZ) proteins are SUMO E3 ligases that modulate SUMO conjugation. The characteristic features and SUMOylation mechanisms of SIZ1 protein in monocotyledon are poorly understood. Here, we examined the functions of a homolog of Arabidopsis SIZ1, a functional SIZ/PIAS-type SUMO E3 ligase from Dendrobium. In Dendrobium, the predicted DnSIZ1 protein has domains that are highly conserved among SIZ/PIAS-type proteins. DnSIZ1 is widely expressed in Dendrobium organs and has a up-regulated trend by treatment with cold, high temperature and wounding. The DnSIZ1 protein localizes to the nucleus and shows SUMO E3 ligase activity when expressed in an Escherichia coli reconstitution system. Moreover, ectopic expression of DnSIZ1 in the Arabidopsis siz1-2 mutant partially complements several phenotypes and results in enhanced levels of SUMO conjugates in plants exposed to heat shock conditions. We observed that DnSIZ1 acts as a negative regulator of flowering transition which may be via a vernalization-induced pathway. In addition, ABA-hypersensitivity of siz1-2 seed germination can be partially suppressed by DnSIZ1. Our results suggest that DnSIZ1 is a functional homolog of the Arabidopsis SIZ1 with SUMO E3 ligase activity and may play an important role in the regulation of Dendrobium stress responses, flowering and development.

Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface

DOE PAGES

Gorelik, Maryna; Orlicky, Stephen; Sartori, Maria A.; ...

2016-03-14

Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface.more » Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.« less

Functional characterization of EI24-induced autophagy in the degradation of RING-domain E3 ligases

PubMed Central

Devkota, Sushil; Jeong, Hyobin; Kim, Yunmi; Ali, Muhammad; Roh, Jae-il; Hwang, Daehee; Lee, Han-Woong

2016-01-01

ABSTRACT Historically, the ubiquitin-proteasome system (UPS) and autophagy pathways were believed to be independent; however, recent data indicate that these pathways engage in crosstalk. To date, the players mediating this crosstalk have been elusive. Here, we show experimentally that EI24 (EI24, autophagy associated transmembrane protein), a key component of basal macroautophagy/autophagy, degrades 14 physiologically important E3 ligases with a RING (really interesting new gene) domain, whereas 5 other ligases were not degraded. Based on the degradation results, we built a statistical model that predicts the RING E3 ligases targeted by EI24 using partial least squares discriminant analysis. Of 381 RING E3 ligases examined computationally, our model predicted 161 EI24 targets. Those targets are primarily involved in transcription, proteolysis, cellular bioenergetics, and apoptosis and regulated by TP53 and MTOR signaling. Collectively, our work demonstrates that EI24 is an essential player in UPS-autophagy crosstalk via degradation of RING E3 ligases. These results indicate a paradigm shift regarding the fate of E3 ligases. PMID:27541728

Inhibitors of ubiquitin E3 ligase as potential new antimalarial drug leads.

PubMed

Jain, Jagrati; Jain, Surendra K; Walker, Larry A; Tekwani, Babu L

2017-06-02

Protein ubiquitylation is an important post-translational regulation, which has been shown to be necessary for life cycle progression and survival of Plasmodium falciparum. Ubiquitin is a highly conserved 76 amino acid polypeptide, which attaches covalently to target proteins through combined action of three classes of enzymes namely, the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin-protein ligase (E3). Ubiquitin E1 and E2 are highly conserved within eukaryotes. However, the P. falciparum E3 ligase is substantially variable and divergent compared to the homologs from other eukaryotes, which make the E3 ligase a parasite-specific target. A set of selected E3 ubiquitin ligase inhibitors was tested in vitro against a chloroquine-sensitive P. falciparum D6 strain (PfD6) and a chloroquine-resistant P. falciparum W2 strain (PfW2). The inhibitors were also tested against Vero and transformed THP1 cells for cytotoxicity. The lead antimalarial E3 ubiquitin ligase inhibitors were further evaluated for the stage-specific antimalarial action and effects on cellular development of P. falciparum in vitro. Statistics analysis was done by two-way ANOVA followed by Tukey and Sidak multiple comparison test using GraphPad Prism 6. E3 ligase inhibitors namely, JNJ 26854165, HLI 373 and Nutlin 3 showed prominent antimalarial activity against PfD6 and PfW2. These inhibitors were considerably less cytotoxic to mammalian Vero cells. JNJ 26854165, HLI 373 and Nutlin 3 blocked the development of P. falciparum parasite at the trophozoite and schizont stages, resulting in accumulation of distorted trophozoites and immature schizonts. Interruption of trophozoites and schizont maturation by the antimalarial E3 ligase inhibitors suggest the role of ubiquitin/proteasome functions in the intraerythrocytic development of malaria parasite. The ubiquitin/proteasome functions may be critical for schizont maturation. Further investigations on the lead E3 ligase inhibitors shall provide better understanding regarding the importance of E3 ligase functions in the malaria parasite as a potential new antimalarial drug target and a new class of antimalarial drug leads.

Calcium Activates Nedd4 E3 Ubiquitin Ligases by Releasing the C2 Domain-mediated Auto-inhibition*

PubMed Central

Wang, Jian; Peng, Qisheng; Lin, Qiong; Childress, Chandra; Carey, David; Yang, Wannian

2010-01-01

Nedd4 E3 ligases are members of the HECT E3 ubiquitin ligase family and regulate ubiquitination-mediated protein degradation. In this report, we demonstrate that calcium releases the C2 domain-mediated auto-inhibition in both Nedd4-1 and Nedd4-2. Calcium disrupts binding of the C2 domain to the HECT domain. Consistent with this, calcium activates the E3 ubiquitin ligase activity of Nedd4. Elevation of intracellular calcium by ionomycin treatment, or activation of acetylcholine receptor or epidermal growth factor receptor by carbachol or epidermal growth factor stimulation induced activation of endogenous Nedd4 in vivo evaluated by assays of either Nedd4 E3 ligase activity or ubiquitination of Nedd4 substrate ENaC-β. The activation effect of calcium on Nedd4 E3 ligase activity was dramatically enhanced by a membrane-rich fraction, suggesting that calcium-mediated membrane translocation through the C2 domain might be an activation mechanism of Nedd4 in vivo. Our studies have revealed an activation mechanism of Nedd4 E3 ubiquitin ligases and established a connection of intracellular calcium signaling to regulation of protein ubiquitination. PMID:20172859

Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide

PubMed Central

Fischer, Eric S.; Böhm, Kerstin; Lydeard, John R.; Yang, Haidi; Stadler, Michael B.; Cavadini, Simone; Nagel, Jane; Serluca, Fabrizio; Acker, Vincent; Lingaraju, Gondichatnahalli M.; Tichkule, Ritesh B.; Schebesta, Michael; Forrester, William C.; Schirle, Markus; Hassiepen, Ulrich; Ottl, Johannes; Hild, Marc; Beckwith, Rohan E. J.; Harper, J. Wade; Jenkins, Jeremy L.; Thomä, Nicolas H.

2015-01-01

In the 1950s the drug thalidomide administered as a sedative to pregnant women led to the birth of thousands of children with multiple defects. Despite its teratogenicity, thalidomide and its derivatives lenalidomide and pomalidomide (together known as Immunomodulatory Drugs: IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-dysplasia. IMiDs target the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase and promote the ubiquitination of Ikaros/Aiolos transcription factors by CRL4CRBN. Here we present the crystal structure of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes CRBN as a CRL4CRBN substrate receptor, which enantioselectively binds IMiDs. Through an unbiased screen we identify the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN. Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN when recruiting Ikaros/Aiolos for degradation. This dual activity implies that small molecules can principally modulate a ligase to up- or down-regulate the ubiquitination of proteins. PMID:25043012

TRIM E3 ligases in HIV infection: can these intrinsic immunity factors be harnessed for novel vaccines or therapies?

PubMed

Ndung'u, Thumbi

2011-01-01

Tripartite motif-containing (TRIM) E3 ligases are a recently identified family of proteins with potent antiviral activity in mammalian cells. The prototype TRIM E3 ligase, TRIM5α was initially identified as a species-specific antiviral restriction factor but subsequent studies suggest some antiviral activity by several TRIM E3 ligases in human cells. However, the mechanisms of antiviral activity by these proteins and their transcriptional, translational and post-translational regulation are poorly understood. Furthermore, the contribution of TRIM E3 ligases to relative resistance or viral control in vivo is largely unknown. Emerging data from our laboratory and other groups suggests that these proteins may have antiviral activity in vivo and contribute to HIV pathogenesis. Considering the significant difficulties so far encountered in developing an effective HIV vaccine and with the use of antiretroviral therapies, it will be important to further investigate the potential of TRIM E3 ligases as novel prophylactics or therapies.

Overview of the membrane-associated RING-CH (MARCH) E3 ligase family.

PubMed

Bauer, Johannes; Bakke, Oddmund; Morth, J Preben

2017-09-25

E3 ligases are critical checkpoints for protein ubiquitination, a signal that often results in protein sorting and degradation but has also been linked to regulation of transcription and DNA repair. In line with their key role in cellular trafficking and cell-cycle control, malfunction of E3 ligases is often linked to human disease. Thus, they have emerged as prime drug targets. However, the molecular basis of action of membrane-bound E3 ligases is still unknown. Here, we review the current knowledge on the membrane-embedded MARCH E3 ligases (MARCH-1-6,7,8,11) with a focus on how the transmembrane regions can contribute via GxxxG-motifs to the selection and recognition of other membrane proteins as substrates for ubiquitination. Further understanding of the molecular parameters that govern target protein recognition of MARCH E3 ligases will contribute to development of strategies for therapeutic regulation of MARCH-induced ubiquitination. Copyright © 2016 Elsevier B.V. All rights reserved.

Latency-Associated Nuclear Antigen E3 Ubiquitin Ligase Activity Impacts Gammaherpesvirus-Driven Germinal Center B Cell Proliferation.

PubMed

Cerqueira, Sofia A; Tan, Min; Li, Shijun; Juillard, Franceline; McVey, Colin E; Kaye, Kenneth M; Simas, J Pedro

2016-09-01

Viruses have evolved mechanisms to hijack components of cellular E3 ubiquitin ligases, thus modulating the ubiquitination pathway. However, the biological relevance of such mechanisms for viral pathogenesis in vivo remains largely unknown. Here, we utilized murid herpesvirus 4 (MuHV-4) infection of mice as a model system to address the role of MuHV-4 latency-associated nuclear antigen (mLANA) E3 ligase activity in gammaherpesvirus latent infection. We show that specific mutations in the mLANA SOCS box (V199A, V199A/L202A, or P203A/P206A) disrupted mLANA's ability to recruit Elongin C and Cullin 5, thereby impairing the formation of the Elongin BC/Cullin 5/SOCS (EC5S(mLANA)) complex and mLANA's E3 ligase activity on host NF-κB and Myc. Although these mutations resulted in considerably reduced mLANA binding to viral terminal repeat DNA as assessed by electrophoretic mobility shift assay (EMSA), the mutations did not disrupt mLANA's ability to mediate episome persistence. In vivo, MuHV-4 recombinant viruses bearing these mLANA SOCS box mutations exhibited a deficit in latency amplification in germinal center (GC) B cells. These findings demonstrate that the E3 ligase activity of mLANA contributes to gammaherpesvirus-driven GC B cell proliferation. Hence, pharmacological inhibition of viral E3 ligase activity through targeting SOCS box motifs is a putative strategy to control gammaherpesvirus-driven lymphoproliferation and associated disease. The gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause lifelong persistent infection and play causative roles in several human malignancies. Colonization of B cells is crucial for virus persistence, and access to the B cell compartment is gained by virus-driven proliferation in germinal center (GC) B cells. Infection of B cells is predominantly latent, with the viral genome persisting as a multicopy episome and expressing only a small subset of viral genes. Here, we focused on latency-associated nuclear antigen (mLANA) encoded by murid herpesvirus-4 (MuHV-4), which exhibits homology in sequence, structure, and function to KSHV LANA (kLANA), thereby allowing the study of LANA-mediated pathogenesis in mice. Our experiments show that mLANA's E3 ubiquitin ligase activity is necessary for efficient expansion of latency in GC B cells, suggesting that the development of pharmacological inhibitors of LANA E3 ubiquitin ligase activity may allow strategies to interfere with gammaherpesvirus-driven lymphoproliferation and associated disease. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome

PubMed Central

McGinty, Robert K.; Henrici, Ryan C.; Tan, Song

2014-01-01

The Polycomb group of epigenetic enzymes represses expression of developmentally regulated genes in higher eukaryotes. This group includes the Polycomb repressive complex 1 (PRC1), which ubiquitylates nucleosomal histone H2A Lys119 using its E3 ubiquitin ligase subunits, Ring1B and Bmi1, together with an E2 ubiquitin-conjugating enzyme, UbcH5c. However, the molecular mechanism of nucleosome substrate recognition by PRC1 or other chromatin enzymes is unclear. Here we present the crystal structure of the Ring1B/Bmi1/UbcH5c E3-E2 complex (the PRC1 ubiquitylation module) bound to its nucleosome core particle substrate. The structure shows how a chromatin enzyme achieves substrate specificity by interacting with multiple nucleosome surfaces spatially distinct from the site of catalysis. Our structure further reveals an unexpected role for the ubiquitin E2 enzyme in substrate recognition, and provides insight into how the related histone H2A E3 ligase, BRCA1, interacts with and ubiquitylates the nucleosome. PMID:25355358

Detecting UV-lesions in the genome: The modular CRL4 ubiquitin ligase does it best!

PubMed

Scrima, Andrea; Fischer, Eric S; Lingaraju, Gondichatnahalli M; Böhm, Kerstin; Cavadini, Simone; Thomä, Nicolas H

2011-09-16

The DDB1-DDB2-CUL4-RBX1 complex serves as the primary detection device for UV-induced lesions in the genome. It simultaneously functions as a CUL4 type E3 ubiquitin ligase. We review the current understanding of this dual function ubiquitin ligase and damage detection complex. The DDB2 damage binding module is merely one of a large family of possible DDB1-CUL4 associated factors (DCAF), most of which are substrate receptors for other DDB1-CUL4 complexes. DDB2 and the Cockayne-syndrome A protein (CSA) function in nucleotide excision repair, whereas the remaining receptors operate in a wide range of other biological pathways. We will examine the modular architecture of DDB1-CUL4 in complex with DDB2, CSA and CDT2 focusing on shared architectural, targeting and regulatory principles. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Selective Proteasomal Degradation of the B′β Subunit of Protein Phosphatase 2A by the E3 Ubiquitin Ligase Adaptor Kelch-like 15*

PubMed Central

Oberg, Elizabeth A.; Nifoussi, Shanna K.; Gingras, Anne-Claude; Strack, Stefan

2012-01-01

Protein phosphatase 2A (PP2A), a ubiquitous and pleiotropic regulator of intracellular signaling, is composed of a core dimer (AC) bound to a variable (B) regulatory subunit. PP2A is an enzyme family of dozens of heterotrimers with different subcellular locations and cellular substrates dictated by the B subunit. B′β is a brain-specific PP2A regulatory subunit that mediates dephosphorylation of Ca2+/calmodulin-dependent protein kinase II and tyrosine hydroxylase. Unbiased proteomic screens for B′β interactors identified Cullin3 (Cul3), a scaffolding component of E3 ubiquitin ligase complexes, and the previously uncharacterized Kelch-like 15 (KLHL15). KLHL15 is one of ∼40 Kelch-like proteins, many of which have been identified as adaptors for the recruitment of substrates to Cul3-based E3 ubiquitin ligases. Here, we report that KLHL15-Cul3 specifically targets B′β to promote turnover of the PP2A subunit by ubiquitylation and proteasomal degradation. Comparison of KLHL15 and B′β tissue expression profiles suggests that the E3 ligase adaptor contributes to selective expression of the PP2A/B′β holoenzyme in the brain. We mapped KLHL15 residues critical for homodimerization as well as interaction with Cul3 and B′β. Explaining PP2A subunit selectivity, the divergent N terminus of B′β was found necessary and sufficient for KLHL15-mediated degradation, with Tyr-52 having an obligatory role. Although KLHL15 can interact with the PP2A/B′β heterotrimer, it only degrades B′β, thus promoting exchange with other regulatory subunits. E3 ligase adaptor-mediated control of PP2A holoenzyme composition thereby adds another layer of regulation to cellular dephosphorylation events. PMID:23135275

A screen for E3 ubiquitination ligases that genetically interact with the adaptor protein Cindr during Drosophila eye patterning

PubMed Central

Ketosugbo, Kwami F.; Bushnell, Henry L.

2017-01-01

Ubiquitination is a crucial post-translational modification that can target proteins for degradation. The E3 ubiquitin ligases are responsible for recognizing substrate proteins for ubiquitination, hence providing specificity to the process of protein degradation. Here, we describe a genetic modifier screen that identified E3 ligases that modified the rough-eye phenotype generated by expression of cindrRNAi transgenes during Drosophila eye development. In total, we identified 36 E3 ligases, as well as 4 Cullins, that modified the mild cindrRNA mis-patterning phenotype. This indicates possible roles for these E3s/Cullins in processes that require Cindr function, including cytoskeletal regulation, cell adhesion, cell signaling and cell survival. Three E3 ligases identified in our screen had previously been linked to regulating JNK signaling. PMID:29117266

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

PubMed Central

Mudhasani, Rajini; Tran, Julie P.; Retterer, Cary; Kota, Krishna P.; Whitehouse, Chris A.; Bavari, Sina

2016-01-01

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)FBXW11 E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCFFBXW11 E3 ligase. We further show that disrupting the assembly of the SCFFBXW11-NSs E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCFFBXW11-NSs E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCFFBXW11 complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection. PMID:26837067

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase.

PubMed

Mudhasani, Rajini; Tran, Julie P; Retterer, Cary; Kota, Krishna P; Whitehouse, Chris A; Bavari, Sina

2016-02-01

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)(FBXW11) E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCF(FBXW11) E3 ligase. We further show that disrupting the assembly of the SCF(FBXW11-NSs) E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCF(FBXW11-NSs) E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCF(FBXW11) complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.

AQP2 Abundance is Regulated by the E3-Ligase CHIP Via HSP70.

PubMed

Centrone, Mariangela; Ranieri, Marianna; Di Mise, Annarita; Berlingerio, Sante Princiero; Russo, Annamaria; Deen, Peter M T; Staub, Olivier; Valenti, Giovanna; Tamma, Grazia

2017-01-01

AQP2 expression is mainly controlled by vasopressin-dependent changes in protein abundance which is in turn regulated by AQP2 ubiquitylation and degradation, however the proteins involved in these processes are largely unknown. Here, we investigated the potential role of the CHIP E3 ligase in AQP2 regulation. MCD4 cells and kidney slices were used to study the involvement of the E3 ligase CHIP on AQP2 protein abundance by cell homogenization and immunoprecipitation followed by immunoblotting. We found that AQP2 complexes with CHIP in renal tissue. Expression of CHIP increased proteasomal degradation of AQP2 and HSP70 abundance, a molecular signature of HSP90 inhibition. Increased HSP70 level, secondary to CHIP expression, promoted ERK signaling resulting in increased AQP2 phosphorylation at S261. Phosphorylation of AQP2 at S256 and T269 were instead downregulated. Next, we investigated HSP70 interaction with AQP2, which is important for endocytosis. Compared with AQP2-wt, HSP70 binding decreased in AQP2-S256D and AQP2-S256D-S261D, while increased in AQP2-S256D-S261A. Surprisingly, expression of CHIP-delUbox, displaying a loss of E3 ligase activity, still induced AQP2 degradation, indicating that CHIP does not ubiquitylate and degrade AQP2 itself. Conversely, the AQP2 half-life was increased upon the expression of CHIP-delTPR a domain which binds Hsc70/HSP70 and HSP90. HSP70 has been reported to bind other E3 ligases such as MDM2. Notably, we found that co-expression of CHIP and MDM2 increased AQP2 degradation, whereas co-expression of CHIP with MDM2-delRING, an inactive form of MDM2, impaired AQP2 degradation. Our findings indicate CHIP as a master regulator of AQP2 degradation via HSP70 that has dual functions: (1) as chaperone for AQP2 and (2) as an anchoring protein for MDM2 E3 ligase, which is likely to be involved in AQP2 degradation. © 2017 The Author(s). Published by S. Karger AG, Basel.

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

DOE PAGES

Mudhasani, Rajini; Tran, Julie P.; Retterer, Cary; ...

2016-02-02

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)FBXW11 E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through anmore » alternate binding site to the SCFFBXW11 E3 ligase. We further show that disrupting the assembly of the SCFFBXW11-NSs E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCFFBXW11-NSs E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCFFBXW11 complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.« less

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mudhasani, Rajini; Tran, Julie P.; Retterer, Cary

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)FBXW11 E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through anmore » alternate binding site to the SCFFBXW11 E3 ligase. We further show that disrupting the assembly of the SCFFBXW11-NSs E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCFFBXW11-NSs E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCFFBXW11 complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.« less

PHD Domain-Mediated E3 Ligase Activity Directs Intramolecular Sumoylation of an Adjacent Bromodomain which is Required for Gene Silencing

PubMed Central

Ivanov, Alexey V.; Peng, Hongzhuang; Yurchenko, Vyacheslav; Yap, Kyoko L.; Negorev, Dmitri G.; Schultz, David C.; Psulkowski, Elyse; Fredericks, William J.; White, David E.; Maul, Gerd G.; Sadofsky, Moshe J.; Zhou, Ming-Ming; Rauscher, Frank J.

2015-01-01

SUMMARY Tandem PHD and bromodomains are often found in chromatin-associated proteins and have been shown to cooperate in gene silencing. Each domain can bind specifically modified histones: the mechanisms of cooperation between these domains are unknown. We show that the PHD domain of the KAP1 corepressor functions as an intramolecular E3 ligase for sumoylation of the adjacent bromodomain. The RING finger-like structure of the PHD domain is required for both Ubc9 binding and sumoylation and directs modification to specific lysine residues in the bromodomain. Sumoylation is required for KAP1-mediated gene silencing and functions by directly recruiting the SETDB1 histone methyltransferase and the CHD3/Mi2 component of the NuRD complex via SUMO interacting motifs. Sumoylated KAP1 stimulates the histone methyltransferase activity of SETDB1. These data provide a mechanistic explanation for the cooperation of PHD and bromodomains in gene regulation and describe a new function of the PHD domain as an intramolecular E3 SUMO ligase. PMID:18082607

Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery.

PubMed

Love, Kerry Routenberg; Pandya, Renuka K; Spooner, Eric; Ploegh, Hidde L

2009-04-17

Protein modification by ubiquitin (Ub) and ubiquitin-like modifiers (Ubl) requires the action of activating (E1), conjugating (E2), and ligating (E3) enzymes and is a key step in the specific destruction of proteins. Deubiquitinating enzymes (DUBs) deconjugate substrates modified with Ub/Ubl's and recycle Ub inside the cell. Genome mining based on sequence homology to proteins with known function has assigned many enzymes to this pathway without confirmation of either conjugating or DUB activity. Function-dependent methodologies are still the most useful for rapid identification or assessment of biological activity of expressed proteins from cells. Activity-based protein profiling uses chemical probes that are active-site-directed for the classification of protein activities in complex mixtures. Here we show that the design and use of an expanded set of Ub-based electrophilic probes allowed us to recover and identify members of each enzyme class in the ubiquitin-proteasome system, including E3 ligases and DUBs with previously unverified activity. We show that epitope-tagged Ub-electrophilic probes can be used as activity-based probes for E3 ligase identification by in vitro labeling and activity studies of purified enzymes identified from complex mixtures in cell lysate. Furthermore, the reactivity of our probe with the HECT domain of the E3 Ub ligase ARF-BP1 suggests that multiple cysteines may be in the vicinity of the E2-binding site and are capable of the transfer of Ub to self or to a substrate protein.

Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels.

PubMed

Hantouche, Christine; Williamson, Brittany; Valinsky, William C; Solomon, Joshua; Shrier, Alvin; Young, Jason C

2017-02-10

Cardiac long QT syndrome type 2 is caused by mutations in the human ether a go-go-related gene (hERG) potassium channel, many of which cause misfolding and degradation at the endoplasmic reticulum instead of normal trafficking to the cell surface. The Hsc70/Hsp70 chaperones assist the folding of the hERG cytosolic domains. Here, we demonstrate that the Hsp70 nucleotide exchange factor Bag1 promotes hERG degradation by the ubiquitin-proteasome system at the endoplasmic reticulum to regulate hERG levels and channel activity. Dissociation of hERG complexes containing Hsp70 and the E3 ubiquitin ligase CHIP requires the interaction of Bag1 with Hsp70, but this does not involve the Bag1 ubiquitin-like domain. The interaction with Bag1 then shifts hERG degradation to the membrane-anchored E3 ligase TRC8 and its E2-conjugating enzyme Ube2g2, as determined by siRNA screening. TRC8 interacts through the transmembrane region with hERG and decreases hERG functional expression. TRC8 also mediates degradation of the misfolded hERG-G601S disease mutant, but pharmacological stabilization of the mutant structure prevents degradation. Our results identify TRC8 as a previously unknown Hsp70-independent quality control E3 ligase for hERG. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Direct Role for Proliferating Cell Nuclear Antigen in Substrate Recognition by the E3 Ubiquitin Ligase CRL4Cdt2*

PubMed Central

Havens, Courtney G.; Shobnam, Nadia; Guarino, Estrella; Centore, Richard C.; Zou, Lee; Kearsey, Stephen E.; Walter, Johannes C.

2012-01-01

The E3 ubiquitin ligase Cullin-ring ligase 4-Cdt2 (CRL4Cdt2) is emerging as an important cell cycle regulator that targets numerous proteins for destruction in S phase and after DNA damage, including Cdt1, p21, and Set8. CRL4Cdt2 substrates contain a “PIP degron,” which consists of a canonical proliferating cell nuclear antigen (PCNA) interaction motif (PIP box) and an adjacent basic amino acid. Substrates use their PIP box to form a binary complex with PCNA on chromatin and the basic residue to recruit CRL4Cdt2 for substrate ubiquitylation. Using Xenopus egg extracts, we identify an acidic residue in PCNA that is essential to support destruction of all CRL4Cdt2 substrates. This PCNA residue, which adjoins the basic amino acid of the bound PIP degron, is dispensable for substrate binding to PCNA but essential for CRL4Cdt2 recruitment to chromatin. Our data show that the interaction of CRL4Cdt2 with substrates requires molecular determinants not only in the substrate degron but also on PCNA. The results illustrate a potentially general mechanism by which E3 ligases can couple ubiquitylation to the formation of protein-protein interactions. PMID:22303007

Parkin Regulates Mitosis and Genomic Stability through Cdc20/Cdh1.

PubMed

Lee, Seung Baek; Kim, Jung Jin; Nam, Hyun-Ja; Gao, Bowen; Yin, Ping; Qin, Bo; Yi, Sang-Yeop; Ham, Hyoungjun; Evans, Debra; Kim, Sun-Hyun; Zhang, Jun; Deng, Min; Liu, Tongzheng; Zhang, Haoxing; Billadeau, Daniel D; Wang, Liewei; Giaime, Emilie; Shen, Jie; Pang, Yuan-Ping; Jen, Jin; van Deursen, Jan M; Lou, Zhenkun

2015-10-01

Mutations in the E3 ubiquitin ligase Parkin have been linked to familial Parkinson's disease. Parkin has also been implicated in mitosis through mechanisms that are unclear. Here we show that Parkin interacts with anaphase promoting complex/cyclosome (APC/C) coactivators Cdc20 and Cdh1 to mediate the degradation of several key mitotic regulators independent of APC/C. We demonstrate that ordered progression through mitosis is orchestrated by two distinct E3 ligases through the shared use of Cdc20 and Cdh1. Furthermore, Parkin is phosphorylated and activated by polo-like kinase 1 (Plk1) during mitosis. Parkin deficiency results in overexpression of its substrates, mitotic defects, genomic instability, and tumorigenesis. These results suggest that the Parkin-Cdc20/Cdh1 complex is an important regulator of mitosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Multimeric complexes among ankyrin-repeat and SOCS-box protein 9 (ASB9), ElonginBC, and Cullin 5: insights into the structure and assembly of ECS-type Cullin-RING E3 ubiquitin ligases.

PubMed

Thomas, Jemima C; Matak-Vinkovic, Dijana; Van Molle, Inge; Ciulli, Alessio

2013-08-06

Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM-MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9-EloBC-Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM-MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.

Multimeric Complexes among Ankyrin-Repeat and SOCS-box Protein 9 (ASB9), ElonginBC, and Cullin 5: Insights into the Structure and Assembly of ECS-type Cullin-RING E3 Ubiquitin Ligases

PubMed Central

2013-01-01

Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC–Cullin–SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM–MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9–EloBC–Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM–MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems. PMID:23837592

Skp1 in lung cancer: clinical significance and therapeutic efficacy of its small molecule inhibitors

PubMed Central

Li, Xin-Chun; Zhang, Jian; Wen, Zhe-Sheng; Huang, Zhi-Liang; Gao, Qin-Lei; Yang, Li-Na; Cheng, Yong-Xian; Tao, Sheng-Ce; Liu, Jinsong; Zhou, Guang-Biao

2015-01-01

Skp1 is an essential adaptor protein of the Skp1-Cul1-F-box protein complex and is able to stabilize the conformation of some ubiquitin E3 ligases. However, the role Skp1 plays during tumorigenesis remains unclear and Skp1-targeting agent is lacking. Here we showed that Skp1 was overexpressed in 36/64 (56.3%) of non-small cell lung cancers, and elevated Skp1 was associated with poor prognosis. By structure-based high-throughput virtual screening, we found some Skp1-targeting molecules including a natural compound 6-O-angeloylplenolin (6-OAP). 6-OAP bound Skp1 at sites critical to Skp1-Skp2 interaction, leading to dissociation and proteolysis of oncogenic E3 ligases NIPA, Skp2, and β-TRCP, and accumulation of their substrates Cyclin B1, P27 and E-Cadherin. 6-OAP induced prometaphase arrest and exerted potent anti-lung cancer activity in two murine models and showed low adverse effect. These results indicate that Skp1 is critical to lung cancer pathogenesis, and Skp1 inhibitor inactivates crucial oncogenic E3 ligases and exhibits significant therapeutic potentials. PMID:26474281

Recognition mechanism of p63 by the E3 ligase Itch

PubMed Central

Bellomaria, Alessia; Barbato, Gaetano; Melino, Gerry; Paci, Maurizio; Melino, Sonia

2012-01-01

The HECT-containing E3 ubiquitin ligase Itch mediates the degradation of several proteins, including p63 and p73, involved in cell specification and fate. Itch contains four WW domains, which are essential for recognition on the target substrate, which contains a short proline-rich sequence. Several signaling complexes containing these domains have been associated with human diseases such as muscular dystrophy, Alzheimer’s or Huntington’s diseases. To gain further insight into the structural determinants of the Itch-WW2 domain, we investigated its interaction with p63. We assigned, by 3D heteronuclear NMR experiments, the backbone and side chains of the uniformly ¹³C-¹⁵N-labeled Itch-WW2. In vitro interaction of Itch-WW2 domain with p63 was studied using its interactive p63 peptide, pep63. Pep63 is an 18-mer peptide corresponding to the region from 534–551 residue of p63, encompassing the PPxY motif that interacts with the Itch-WW domains, and we identified the residues involved in this molecular recognition. Moreover, here, a strategy of stabilization of the conformation of the PPxY peptide has been adopted, increasing the WW-ligand binding. We demonstrated that cyclization of pep63 leads to an increase of both the biological stability of the peptide and of the WW-ligand complex. Stable metal-binding complexes of the pep63 have been also obtained, and localized oxidative damage on Itch-WW2 domain has been induced, demonstrating the possibility of use of metal-pep63 complexes as models for the design of metal drugs to inhibit the Itch-WW-p63 recognition in vivo. Thus, our data suggest a novel strategy to study and inhibit the recognition mechanism of Itch E3-ligase. PMID:22935697

Insights into Cullin-RING E3 ubiquitin ligase recruitment: structure of the VHL-EloBC-Cul2 complex.

PubMed

Nguyen, Henry C; Yang, Haitao; Fribourgh, Jennifer L; Wolfe, Leslie S; Xiong, Yong

2015-03-03

The von Hippel-Lindau tumor suppressor protein (VHL) recruits a Cullin 2 (Cul2) E3 ubiquitin ligase to downregulate HIF-1α, an essential transcription factor for the hypoxia response. Mutations in VHL lead to VHL disease and renal cell carcinomas. Inhibition of this pathway to upregulate erythropoietin production is a promising new therapy to treat ischemia and chronic anemia. Here, we report the crystal structure of VHL bound to a Cul2 N-terminal domain, Elongin B, and Elongin C (EloC). Cul2 interacts with both the VHL BC box and cullin box and a novel EloC site. Comparison with other cullin E3 ligase structures shows that there is a conserved, yet flexible, cullin recognition module and that cullin selectivity is influenced by distinct electrostatic interactions. Our structure provides a structural basis for the study of the pathogenesis of VHL disease and rationale for the design of novel compounds that may modulate cullin-substrate receptor interactions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ubiquitin Ligases: Structure, Function, and Regulation.

PubMed

Zheng, Ning; Shabek, Nitzan

2017-06-20

Ubiquitin E3 ligases control every aspect of eukaryotic biology by promoting protein ubiquitination and degradation. At the end of a three-enzyme cascade, ubiquitin ligases mediate the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to specific substrate proteins. Early investigations of E3s of the RING (really interesting new gene) and HECT (homologous to the E6AP carboxyl terminus) types shed light on their enzymatic activities, general architectures, and substrate degron-binding modes. Recent studies have provided deeper mechanistic insights into their catalysis, activation, and regulation. In this review, we summarize the current progress in structure-function studies of ubiquitin ligases as well as exciting new discoveries of novel classes of E3s and diverse substrate recognition mechanisms. Our increased understanding of ubiquitin ligase function and regulation has provided the rationale for developing E3-targeting therapeutics for the treatment of human diseases.

Smad Ubiquitylation Regulatory Factor 1/2 (Smurf1/2) Promotes p53 Degradation by Stabilizing the E3 Ligase MDM2*

PubMed Central

Nie, Jing; Xie, Ping; Liu, Lin; Xing, Guichun; Chang, Zhijie; Yin, Yuxin; Tian, Chunyan; He, Fuchu; Zhang, Lingqiang

2010-01-01

The tumor suppressor p53 protein is tightly regulated by a ubiquitin-proteasomal degradation mechanism. Several E3 ubiquitin ligases, including MDM2 (mouse double minute 2), have been reported to play an essential role in the regulation of p53 stability. However, it remains unclear how the activity of these E3 ligases is regulated. Here, we show that the HECT-type E3 ligase Smurf1/2 (Smad ubiquitylation regulatory factor 1/2) promotes p53 degradation by enhancing the activity of the E3 ligase MDM2. We provide evidence that the role of Smurf1/2 on the p53 stability is not dependent on the E3 activity of Smurf1/2 but rather is dependent on the activity of MDM2. We find that Smurf1/2 stabilizes MDM2 by enhancing the heterodimerization of MDM2 with MDMX, during which Smurf1/2 interacts with MDM2 and MDMX. We finally provide evidence that Smurf1/2 regulates apoptosis through p53. To our knowledge, this is the first report to demonstrate that Smurf1/2 functions as a factor to stabilize MDM2 protein rather than as a direct E3 ligase in regulation of p53 degradation. PMID:20484049

Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases.

PubMed

Boomsma, Wouter; Nielsen, Sofie V; Lindorff-Larsen, Kresten; Hartmann-Petersen, Rasmus; Ellgaard, Lars

2016-01-01

The ubiquitin-proteasome system targets misfolded proteins for degradation. Since the accumulation of such proteins is potentially harmful for the cell, their prompt removal is important. E3 ubiquitin-protein ligases mediate substrate ubiquitination by bringing together the substrate with an E2 ubiquitin-conjugating enzyme, which transfers ubiquitin to the substrate. For misfolded proteins, substrate recognition is generally delegated to molecular chaperones that subsequently interact with specific E3 ligases. An important exception is San1, a yeast E3 ligase. San1 harbors extensive regions of intrinsic disorder, which provide both conformational flexibility and sites for direct recognition of misfolded targets of vastly different conformations. So far, no mammalian ortholog of San1 is known, nor is it clear whether other E3 ligases utilize disordered regions for substrate recognition. Here, we conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology of their ordered regions, and did not capture the unique disorder patterns that encode the functional mechanism of San1. However, by searching specifically for key features of the San1 sequence, such as long regions of intrinsic disorder embedded with short stretches predicted to be suitable for substrate interaction, we identified several E3 ligases with these characteristics. Our initial analysis revealed that another remarkable trait of San1 is shared with several candidate E3 ligases: long stretches of complete lysine suppression, which in San1 limits auto-ubiquitination. We encode these characteristic features into a San1 similarity-score, and present a set of proteins that are plausible candidates as San1 counterparts in humans. In conclusion, our work indicates that San1 is not a unique case, and that several other yeast and human E3 ligases have sequence properties that may allow them to recognize substrates by a similar mechanism as San1.

CTLs, a new class of RING-H2 ubiquitin ligases uncovered by YEELL, a motif close to the RING domain that is present across eukaryotes.

PubMed

Jiménez-López, Domingo; Aguilar-Henonin, Laura; González-Prieto, Juan Manuel; Aguilar-Hernández, Victor; Guzmán, Plinio

2018-01-01

RING ubiquitin E3 ligases enclose a RING domain for ubiquitin ligase activity and associated domains and/or conserved motifs outside the RING domain that collectively facilitate their classification and usually reveal some of key information related to mechanism of action. Here we describe a new family of E3 ligases that encodes a RING-H2 domain related in sequence to the ATL and BTL RING-H2 domains. This family, named CTL, encodes a motif designed as YEELL that expands 21 amino acids next to the RING-H2 domain that is present across most eukaryotic lineages. E3 ubiquitin ligase BIG BROTHER is a plant CTL that regulates organ size, and SUMO-targeted ubiquitin E3 ligase RNF111/ARKADIA is a vertebrate CTL. Basal animal and vertebrate, as well as fungi species, encode a single CTL gene that constraints the number of paralogs observed in vertebrates. Conversely, as previously described in ATL and BTL families in plants, CTL genes range from a single copy in green algae and 3 to 5 copies in basal species to 9 to 35 copies in angiosperms. Our analysis describes key structural features of a novel family of E3 ubiquitin ligases as an integral component of the set of core eukaryotic genes.

CTLs, a new class of RING-H2 ubiquitin ligases uncovered by YEELL, a motif close to the RING domain that is present across eukaryotes

PubMed Central

Jiménez-López, Domingo; Aguilar-Henonin, Laura; González-Prieto, Juan Manuel; Aguilar-Hernández, Victor

2018-01-01

RING ubiquitin E3 ligases enclose a RING domain for ubiquitin ligase activity and associated domains and/or conserved motifs outside the RING domain that collectively facilitate their classification and usually reveal some of key information related to mechanism of action. Here we describe a new family of E3 ligases that encodes a RING-H2 domain related in sequence to the ATL and BTL RING-H2 domains. This family, named CTL, encodes a motif designed as YEELL that expands 21 amino acids next to the RING-H2 domain that is present across most eukaryotic lineages. E3 ubiquitin ligase BIG BROTHER is a plant CTL that regulates organ size, and SUMO-targeted ubiquitin E3 ligase RNF111/ARKADIA is a vertebrate CTL. Basal animal and vertebrate, as well as fungi species, encode a single CTL gene that constraints the number of paralogs observed in vertebrates. Conversely, as previously described in ATL and BTL families in plants, CTL genes range from a single copy in green algae and 3 to 5 copies in basal species to 9 to 35 copies in angiosperms. Our analysis describes key structural features of a novel family of E3 ubiquitin ligases as an integral component of the set of core eukaryotic genes. PMID:29324855

The Chaperone-assisted E3 Ligase C Terminus of Hsc70-interacting Protein (CHIP) Targets PTEN for Proteasomal Degradation*

PubMed Central

Ahmed, Syed Feroj; Deb, Satamita; Paul, Indranil; Chatterjee, Anirban; Mandal, Tapashi; Chatterjee, Uttara; Ghosh, Mrinal K.

2012-01-01

The tumor suppressor, PTEN is key to the regulation of diverse cellular processes, making it a prime candidate to be tightly regulated. The PTEN level is controlled in a major way by E3 ligase-mediated degradation through the Ubiquitin-Proteasome System (UPS). Nedd 4-1, XIAP, and WWP2 have been shown to maintain PTEN turnover. Here, we report that CHIP, the chaperone-associated E3 ligase, induces ubiquitination and regulates the proteasomal turnover of PTEN. It was apparent from our findings that PTEN transiently associates with the molecular chaperones and thereby gets diverted to the degradation pathway through its interaction with CHIP. The TPR domain of CHIP and parts of the N-terminal domain of PTEN are required for their interaction. Overexpression of CHIP leads to elevated ubiquitination and a shortened half-life of endogenous PTEN. On the other hand, depletion of endogenous CHIP stabilizes PTEN. CHIP is also shown to regulate PTEN-dependent transcription presumably through its down-regulation. PTEN shared an inverse correlation with CHIP in human prostate cancer patient samples, thereby triggering the prospects of a more complex mode of PTEN regulation in cancer. PMID:22427670

Protein–Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP)*

PubMed Central

Narayan, Vikram; Landré, Vivien; Ning, Jia; Hernychova, Lenka; Muller, Petr; Verma, Chandra; Walkinshaw, Malcolm D.; Blackburn, Elizabeth A.; Ball, Kathryn L.

2015-01-01

CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control. PMID:26330542

Identification of HECT E3 ubiquitin ligase family genes involved in stem cell regulation and regeneration in planarians.

PubMed

Henderson, Jordana M; Nisperos, Sean V; Weeks, Joi; Ghulam, Mahjoobah; Marín, Ignacio; Zayas, Ricardo M

2015-08-15

E3 ubiquitin ligases constitute a large family of enzymes that modify specific proteins by covalently attaching ubiquitin polypeptides. This post-translational modification can serve to regulate protein function or longevity. In spite of their importance in cell physiology, the biological roles of most ubiquitin ligases remain poorly understood. Here, we analyzed the function of the HECT domain family of E3 ubiquitin ligases in stem cell biology and tissue regeneration in planarians. Using bioinformatic searches, we identified 17 HECT E3 genes that are expressed in the Schmidtea mediterranea genome. Whole-mount in situ hybridization experiments showed that HECT genes were expressed in diverse tissues and most were expressed in the stem cell population (neoblasts) or in their progeny. To investigate the function of all HECT E3 ligases, we inhibited their expression using RNA interference (RNAi) and determined that orthologs of huwe1, wwp1, and trip12 had roles in tissue regeneration. We show that huwe1 RNAi knockdown led to a significant expansion of the neoblast population and death by lysis. Further, our experiments showed that wwp1 was necessary for both neoblast and intestinal tissue homeostasis as well as uncovered an unexpected role of trip12 in posterior tissue specification. Taken together, our data provide insights into the roles of HECT E3 ligases in tissue regeneration and demonstrate that planarians will be a useful model to evaluate the functions of E3 ubiquitin ligases in stem cell regulation. Copyright © 2015 Elsevier Inc. All rights reserved.

Genetically engineered mouse models for functional studies of SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligases.

PubMed

Zhou, Weihua; Wei, Wenyi; Sun, Yi

2013-05-01

The SCF (SKP1 (S-phase-kinase-associated protein 1), Cullin-1, F-box protein) E3 ubiquitin ligases, the founding member of Cullin-RING ligases (CRLs), are the largest family of E3 ubiquitin ligases in mammals. Each individual SCF E3 ligase consists of one adaptor protein SKP1, one scaffold protein cullin-1 (the first family member of the eight cullins), one F-box protein out of 69 family members, and one out of two RING (Really Interesting New Gene) family proteins RBX1/ROC1 or RBX2/ROC2/SAG/RNF7. Various combinations of these four components construct a large number of SCF E3s that promote the degradation of many key regulatory proteins in cell-context, temporally, and spatially dependent manners, thus controlling precisely numerous important cellular processes, including cell cycle progression, apoptosis, gene transcription, signal transduction, DNA replication, maintenance of genome integrity, and tumorigenesis. To understand how the SCF E3 ligases regulate these cellular processes and embryonic development under in vivo physiological conditions, a number of mouse models with transgenic (Tg) expression or targeted deletion of components of SCF have been established and characterized. In this review, we will provide a brief introduction to the ubiquitin-proteasome system (UPS) and the SCF E3 ubiquitin ligases, followed by a comprehensive overview on the existing Tg and knockout (KO) mouse models of the SCF E3s, and discuss the role of each component in mouse embryogenesis, cell proliferation, apoptosis, carcinogenesis, as well as other pathogenic processes associated with human diseases. We will end with a brief discussion on the future directions of this research area and the potential applications of the knowledge gained to more effective therapeutic interventions of human diseases.

A Mental Retardation-linked Nonsense Mutation in Cereblon Is Rescued by Proteasome Inhibition*

PubMed Central

Xu, Guoqiang; Jiang, Xiaogang; Jaffrey, Samie R.

2013-01-01

A nonsense mutation in cereblon (CRBN) causes autosomal recessive nonsyndromic mental retardation. Cereblon is a substrate receptor for the Cullin-RING E3 ligase complex and couples the ubiquitin ligase to specific ubiquitination targets. The CRBN nonsense mutation (R419X) results in a protein lacking 24 amino acids at its C terminus. Although this mutation has been linked to mild mental retardation, the mechanism by which the mutation affects CRBN function is unknown. Here, we used biochemical and mass spectrometric approaches to explore the function of this mutant. We show that the protein retains its ability to assemble into a Cullin-RING E3 ligase complex and catalyzes the ubiquitination of CRBN-target proteins. However, we find that this mutant exhibits markedly increased levels of autoubiquitination and is more readily degraded by the proteasome than the wild type protein. We also show that the level of the mutant protein can be restored by a treatment of cells with a clinically utilized proteasome inhibitor, suggesting that this agent may be useful for the treatment of mental retardation associated with the CRBN R419X mutation. These data demonstrate that enhanced autoubiquitination and degradation account for the defect in CRBN activity that leads to mental retardation. PMID:23983124

The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension

PubMed Central

Smit, Judith J; Monteferrario, Davide; Noordermeer, Sylvie M; van Dijk, Willem J; van der Reijden, Bert A; Sixma, Titia K

2012-01-01

Activation of the NF-κB pathway requires the formation of Met1-linked ‘linear' ubiquitin chains on NEMO, which is catalysed by the Linear Ubiquitin Chain Assembly Complex (LUBAC) E3 consisting of HOIP, HOIL-1L and Sharpin. Here, we show that both LUBAC catalytic activity and LUBAC specificity for linear ubiquitin chain formation are embedded within the RING-IBR-RING (RBR) ubiquitin ligase subunit HOIP. Linear ubiquitin chain formation by HOIP proceeds via a two-step mechanism involving both RING and HECT E3-type activities. RING1-IBR catalyses the transfer of ubiquitin from the E2 onto RING2, to transiently form a HECT-like covalent thioester intermediate. Next, the ubiquitin is transferred from HOIP onto the N-terminus of a target ubiquitin. This transfer is facilitated by a unique region in the C-terminus of HOIP that we termed ‘Linear ubiquitin chain Determining Domain' (LDD), which may coordinate the acceptor ubiquitin. Consistent with this mechanism, the RING2-LDD region was found to be important for NF-κB activation in cellular assays. These data show how HOIP combines a general RBR ubiquitin ligase mechanism with unique, LDD-dependent specificity for producing linear ubiquitin chains. PMID:22863777

RNF166 Determines Recruitment of Adaptor Proteins during Antibacterial Autophagy.

PubMed

Heath, Robert J; Goel, Gautam; Baxt, Leigh A; Rush, Jason S; Mohanan, Vishnu; Paulus, Geraldine L C; Jani, Vijay; Lassen, Kara G; Xavier, Ramnik J

2016-11-22

Xenophagy is a form of selective autophagy that involves the targeting and elimination of intracellular pathogens through several recognition, recruitment, and ubiquitination events. E3 ubiquitin ligases control substrate selectivity in the ubiquitination cascade; however, systematic approaches to map the role of E3 ligases in antibacterial autophagy have been lacking. We screened more than 600 putative human E3 ligases, identifying E3 ligases that are required for adaptor protein recruitment and LC3-bacteria colocalization, critical steps in antibacterial autophagy. An unbiased informatics approach pinpointed RNF166 as a key gene that interacts with the autophagy network and controls the recruitment of ubiquitin as well as the autophagy adaptors p62 and NDP52 to bacteria. Mechanistic studies demonstrated that RNF166 catalyzes K29- and K33-linked polyubiquitination of p62 at residues K91 and K189. Thus, our study expands the catalog of E3 ligases that mediate antibacterial autophagy and identifies a critical role for RNF166 in this process. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The Blue Light-Dependent Polyubiquitination and Degradation of Arabidopsis Cryptochrome2 Requires Multiple E3 Ubiquitin Ligases.

PubMed

Liu, Qing; Wang, Qin; Liu, Bin; Wang, Wei; Wang, Xu; Park, Joon; Yang, Zhenming; Du, Xinglin; Bian, Mingdi; Lin, Chentao

2016-10-01

Cryptochromes are blue light receptors regulated by light-dependent ubiquitination and degradation in both plant and animal lineages. The Arabidopsis genome encodes two cryptochromes, CRY1 and CRY2, of which CRY2 undergoes blue light-dependent ubiquitination and 26S proteasome-dependent degradation. The molecular mechanism regulating blue light-dependent proteolysis of CRY2 is still not fully understood. We found that the F-box proteins ZEITLUPE (ZTL) and Lov Kelch Protein2 (LKP2), which mediate blue light suppression of degradation of the CRY2 signaling partner CIB1, are not required for the blue light-dependent CRY2 degradation. We further showed that the previously reported function of the COP1-SPA1 protein complex in blue light-dependent CRY2 degradation is more likely to be attributable to its cullin 4 (CUL4)-based E3 ubiquitin ligase activity than its activity as the cryptochrome signaling partner. However, the blue light-dependent CRY2 degradation is only partially impaired in the cul4 mutant, the cop1-5 null mutant and the spa1234 quadruple mutant, suggesting a possible involvement of additional E3 ubiquitin ligases in the regulation of CRY2. Consistent with this hypothesis, we demonstrated that the blue light-dependent CRY2 degradation is significantly impaired in the temperature-sensitive cul1 mutant allele (axr6-3), especially under the non-permissive temperature. Based on these and other results presented, we propose that photoexcited CRY2 undergoes Lys48-linked polyubiquitination catalyzed by the CUL4- and CUL1-based E3 ubiquitin ligases. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Ring finger protein 145 (RNF145) is a ubiquitin ligase for sterol-induced degradation of HMG-CoA reductase.

PubMed

Jiang, Lu-Yi; Jiang, Wei; Tian, Na; Xiong, Yan-Ni; Liu, Jie; Wei, Jian; Wu, Kai-Yue; Luo, Jie; Shi, Xiong-Jie; Song, Bao-Liang

2018-03-16

Cholesterol biosynthesis is tightly regulated in the cell. For example, high sterol concentrations can stimulate degradation of the rate-limiting cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, HMGCR). HMGCR is broken down by the endoplasmic reticulum membrane-associated protein complexes consisting of insulin-induced genes (Insigs) and the E3 ubiquitin ligase gp78. Here we found that HMGCR degradation is partially blunted in Chinese hamster ovary (CHO) cells lacking gp78 ( gp78 -KO). To identify other ubiquitin ligase(s) that may function together with gp78 in triggering HMGCR degradation, we performed a small-scale short hairpin RNA-based screening targeting endoplasmic reticulum-localized E3s. We found that knockdown of both ring finger protein 145 ( Rnf145 ) and gp78 genes abrogates sterol-induced degradation of HMGCR in CHO cells. We also observed that RNF145 interacts with Insig-1 and -2 proteins and ubiquitinates HMGCR. Moreover, the tetrapeptide sequence YLYF in the sterol-sensing domain and the Cys-537 residue in the RING finger domain were essential for RNF145 binding to Insigs and RNF145 E3 activity, respectively. Of note, amino acid substitutions in the YLYF or of Cys-537 completely abolished RNF145-mediated HMGCR degradation. In summary, our study reveals that RNF145, along with gp78, promotes HMGCR degradation in response to elevated sterol levels and identifies residues essential for RNF145 function. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Flying saucer1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed mucilage.

PubMed

Voiniciuc, Catalin; Dean, Gillian H; Griffiths, Jonathan S; Kirchsteiger, Kerstin; Hwang, Yeen Ting; Gillett, Alan; Dow, Graham; Western, Tamara L; Estelle, Mark; Haughn, George W

2013-03-01

Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca(2+) ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca(2+) or completely rescued using alkaline Ca(2+) chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1-yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells.

FLYING SAUCER1 Is a Transmembrane RING E3 Ubiquitin Ligase That Regulates the Degree of Pectin Methylesterification in Arabidopsis Seed Mucilage[W

PubMed Central

Voiniciuc, Cătălin; Dean, Gillian H.; Griffiths, Jonathan S.; Kirchsteiger, Kerstin; Hwang, Yeen Ting; Gillett, Alan; Dow, Graham; Western, Tamara L.; Estelle, Mark; Haughn, George W.

2013-01-01

Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca2+ ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca2+ or completely rescued using alkaline Ca2+ chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1–yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells. PMID:23482858

Recognition mechanism of p63 by the E3 ligase Itch: novel strategy in the study and inhibition of this interaction.

PubMed

Bellomaria, Alessia; Barbato, Gaetano; Melino, Gerry; Paci, Maurizio; Melino, Sonia

2012-10-01

The HECT-containing E3 ubiquitin ligase Itch mediates the degradation of several proteins, including p63 and p73, involved in cell specification and fate. Itch contains four WW domains, which are essential for recognition on the target substrate, which contains a short proline-rich sequence. Several signaling complexes containing these domains have been associated with human diseases such as muscular dystrophy, Alzheimer's or Huntington's diseases. To gain further insight into the structural determinants of the Itch-WW2 domain, we investigated its interaction with p63. We assigned, by 3D heteronuclear NMR experiments, the backbone and side chains of the uniformly (13)C-(15)N-labeled Itch-WW2. In vitro interaction of Itch-WW2 domain with p63 was studied using its interactive p63 peptide, pep63. Pep63 is an 18-mer peptide corresponding to the region from 534-551 residue of p63, encompassing the PPxY motif that interacts with the Itch-WW domains, and we identified the residues involved in this molecular recognition. Moreover, here, a strategy of stabilization of the conformation of the PPxY peptide has been adopted, increasing the WW-ligand binding. We demonstrated that cyclization of pep63 leads to an increase of both the biological stability of the peptide and of the WW-ligand complex. Stable metal-binding complexes of the pep63 have been also obtained, and localized oxidative damage on Itch-WW2 domain has been induced, demonstrating the possibility of use of metal-pep63 complexes as models for the design of metal drugs to inhibit the Itch-WW-p63 recognition in vivo. Thus, our data suggest a novel strategy to study and inhibit the recognition mechanism of Itch E3-ligase.

Enzyme reversal to explore the function of yeast E3 ubiquitin-ligases.

PubMed

MacDonald, Chris; Winistorfer, Stanley; Pope, Robert M; Wright, Michael E; Piper, Robert C

2017-07-01

The covalent attachment of ubiquitin onto proteins can elicit a variety of downstream consequences. Attachment is mediated by a large array of E3 ubiquitin ligases, each thought be subject to regulatory control and to have a specific repertoire of substrates. Assessing the biological roles of ligases, and in particular, identifying their biologically relevant substrates has been a persistent yet challenging question. In this study, we describe tools that may help achieve both of these goals. We describe a strategy whereby the activity of a ubiquitin ligase has been enzymatically reversed, accomplished by fusing it to a catalytic domain of an exogenous deubiquitinating enzyme. We present a library of 72 "anti-ligases" that appear to work in a dominant-negative fashion to stabilize their cognate substrates against ubiquitin-dependent proteasomal and lysosomal degradation. We then used the ligase-deubiquitinating enzyme (DUb) library to screen for E3 ligases involved in post-Golgi/endosomal trafficking. We identify ligases previously implicated in these pathways (Rsp5 and Tul1), in addition to ligases previously localized to endosomes (Pib1 and Vps8). We also document an optimized workflow for isolating and analyzing the "ubiquitome" of yeast, which can be used with mass spectrometry to identify substrates perturbed by expression of particular ligase-DUb fusions. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Ubiquitin-protein ligases in muscle wasting: multiple parallel pathways?

NASA Technical Reports Server (NTRS)

Lecker, Stewart H.; Goldberg, A. L. (Principal Investigator)

2003-01-01

PURPOSE OF REVIEW: Studies in a wide variety of animal models of muscle wasting have led to the concept that increased protein breakdown via the ubiquitin-proteasome pathway is responsible for the loss of muscle mass seen as muscle atrophy. The complexity of the ubiquitination apparatus has hampered our understanding of how this pathway is activated in atrophying muscles and which ubiquitin-conjugating enzymes in muscle are responsible. RECENT FINDINGS: Recent experiments have shown that two newly identified ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MURF-1, are critical in the development of muscle atrophy. Other in-vitro studies also implicated E2(14k) and E3alpha, of the N-end rule pathway, as playing an important role in the process. SUMMARY: It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as inhibitors of, these E3s.

RavN is a member of a previously unrecognized group of Legionella pneumophila E3 ubiquitin ligases

PubMed Central

Lin, Yi-Han; Evans, Timothy R.; Doms, Alexandra G.; Beauchene, Nicole A.; Hierro, Aitor

2018-01-01

The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway. The E3 ligase activity of RavN was located within its N-terminal region and was dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has undergone significant evolutionary alteration. Substitution of residues within the predicted E2 binding interface rendered RavN inactive, indicating that, despite significant structural changes, the mode of E2 recognition has remained conserved. Using hidden Markov model-based secondary structure analyses, we identified and experimentally validated four additional L. pneumophila effectors that were not previously recognized to possess E3 ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway. PMID:29415051

Chromatin-Bound Cullin-Ring Ligases: Regulatory Roles in DNA Replication and Potential Targeting for Cancer Therapy

PubMed Central

Jang, Sang-Min; Redon, Christophe E.; Aladjem, Mirit I.

2018-01-01

Cullin-RING (Really Interesting New Gene) E3 ubiquitin ligases (CRLs), the largest family of E3 ubiquitin ligases, are functional multi-subunit complexes including substrate receptors, adaptors, cullin scaffolds, and RING-box proteins. CRLs are responsible for ubiquitination of ~20% of cellular proteins and are involved in diverse biological processes including cell cycle progression, genome stability, and oncogenesis. Not surprisingly, cullins are deregulated in many diseases and instances of cancer. Recent studies have highlighted the importance of CRL-mediated ubiquitination in the regulation of DNA replication/repair, including specific roles in chromatin assembly and disassembly of the replication machinery. The development of novel therapeutics targeting the CRLs that regulate the replication machinery and chromatin in cancer is now an attractive therapeutic strategy. In this review, we summarize the structure and assembly of CRLs and outline their cellular functions and their diverse roles in cancer, emphasizing the regulatory functions of nuclear CRLs in modulating the DNA replication machinery. Finally, we discuss the current strategies for targeting CRLs against cancer in the clinic. PMID:29594129

The E3 ubiquitin-ligase SEVEN IN ABSENTIA like 7 mono-ubiquitinates glyceraldehyde-3-phosphate dehydrogenase 1 isoform in vitro and is required for its nuclear localization in Arabidopsis thaliana.

PubMed

Peralta, Diego A; Araya, Alejandro; Busi, Maria V; Gomez-Casati, Diego F

2016-01-01

The E3 ubiquitin-protein ligases are associated to various processes such as cell cycle control and diverse developmental pathways. Arabidopsis thaliana SEVEN IN ABSENTIA like 7, which has ubiquitin ligase activity, is located in the nucleus and cytosol and is expressed at several stages in almost all plant tissues suggesting an important role in plant functions. However, the mechanism underlying the regulation of this protein is unknown. Since we found that the SEVEN IN ABSENTIA like 7 gene expression is altered in plants with impaired mitochondria, and in plants deficient in the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase 1, we decided to study the possible interactions between both proteins as potential partners in plant signaling functions. We found that SEVEN IN ABSENTIA like 7 is able to interact in vitro with glyceraldehyde-3-phosphate dehydrogenase and that the Lys231 residue of the last is essential for this function. Following the interaction, a concomitant increase in the glyceraldehyde-3-phosphate dehydrogenase catalytic activity was observed. However, when SEVEN IN ABSENTIA like 7 was supplemented with E1 and E2 proteins to form a complete E1-E2-E3 modifier complex, we observed the mono-ubiquitination of glyceraldehyde-3-phosphate dehydrogenase 1 at the Lys76 residue and a dramatic decrease of its catalytic activity. Moreover, we found that localization of glyceraldehyde-3-phosphate dehydrogenase 1 in the nucleus is dependent on the expression SEVEN IN ABSENTIA like 7. These observations suggest that the association of both proteins might result in different biological consequences in plants either through affecting the glycolytic flux or via cytoplasm-nucleus relocation. Copyright © 2015 Elsevier Ltd. All rights reserved.

DWARF 53 acts as a repressor of strigolactone signalling in rice

NASA Astrophysics Data System (ADS)

Jiang, Liang; Liu, Xue; Xiong, Guosheng; Liu, Huihui; Chen, Fulu; Wang, Lei; Meng, Xiangbing; Liu, Guifu; Yu, Hong; Yuan, Yundong; Yi, Wei; Zhao, Lihua; Ma, Honglei; He, Yuanzheng; Wu, Zhongshan; Melcher, Karsten; Qian, Qian; Xu, H. Eric; Wang, Yonghong; Li, Jiayang

2013-12-01

Strigolactones (SLs) are a group of newly identified plant hormones that control plant shoot branching. SL signalling requires the hormone-dependent interaction of DWARF 14 (D14), a probable candidate SL receptor, with DWARF 3 (D3), an F-box component of the Skp-Cullin-F-box (SCF) E3 ubiquitin ligase complex. Here we report the characterization of a dominant SL-insensitive rice (Oryza sativa) mutant dwarf 53 (d53) and the cloning of D53, which encodes a substrate of the SCFD3 ubiquitination complex and functions as a repressor of SL signalling. Treatments with GR24, a synthetic SL analogue, cause D53 degradation via the proteasome in a manner that requires D14 and the SCFD3 ubiquitin ligase, whereas the dominant form of D53 is resistant to SL-mediated degradation. Moreover, D53 can interact with transcriptional co-repressors known as TOPLESS-RELATED PROTEINS. Our results suggest a model of SL signalling that involves SL-dependent degradation of the D53 repressor mediated by the D14-D3 complex.

Crystal Structure of the Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase Complex.

PubMed

Cardote, Teresa A F; Gadd, Morgan S; Ciulli, Alessio

2017-06-06

Cullin RING E3 ubiquitin ligases (CRLs) function in the ubiquitin proteasome system to catalyze the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins. CRLs are large dynamic complexes and attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. The atomic details of whole CRL assembly and interactions that dictate subunit specificity remain elusive. Here we present the crystal structure of a pentameric CRL2 VHL complex, composed of Cul2, Rbx1, Elongin B, Elongin C, and pVHL. The structure traps a closed state of full-length Cul2 and a new pose of Rbx1 in a trajectory from closed to open conformation. We characterize hotspots and binding thermodynamics at the interface between Cul2 and pVHL-EloBC and identify mutations that contribute toward a selectivity switch for Cul2 versus Cul5 recognition. Our findings provide structural and biophysical insights into the whole Cul2 complex that could aid future drug targeting. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

CD2-associated Protein (CD2AP) Enhances Casitas B Lineage Lymphoma-3/c (Cbl-3/c)-mediated Ret Isoform-specific Ubiquitination and Degradation via Its Amino-terminal Src Homology 3 Domains*

PubMed Central

Calco, Gina N.; Stephens, Olivia R.; Donahue, Laura M.; Tsui, Cynthia C.; Pierchala, Brian A.

2014-01-01

Ret is the receptor tyrosine kinase for the glial cell line-derived neurotrophic factor (GDNF) family of neuronal growth factors. Upon activation by GDNF, Ret is rapidly polyubiquitinated and degraded. This degradation process is isoform-selective, with the longer Ret51 isoform exhibiting different degradation kinetics than the shorter isoform, Ret9. In sympathetic neurons, Ret degradation is induced, at least in part, by a complex consisting of the adaptor protein CD2AP and the E3-ligase Cbl-3/c. Knockdown of Cbl-3/c using siRNA reduced the GDNF-induced ubiquitination and degradation of Ret51 in neurons and podocytes, suggesting that Cbl-3/c was a predominant E3 ligase for Ret. Coexpression of CD2AP with Cbl-3/c augmented the ubiquitination of Ret51 as compared with the expression of Cbl-3/c alone. Ret51 ubiquitination by the CD2AP·Cbl-3/c complex required a functional ring finger and TKB domain in Cbl-3/c. The SH3 domains of CD2AP were sufficient to drive the Cbl-3/c-dependent ubiquitination of Ret51, whereas the carboxyl-terminal coiled-coil domain of CD2AP was dispensable. Interestingly, activated Ret induced the degradation of CD2AP, but not Cbl-3/c, suggesting a potential inhibitory feedback mechanism. There were only two major ubiquitination sites in Ret51, Lys1060 and Lys1107, and the combined mutation of these lysines almost completely eliminated both the ubiquitination and degradation of Ret51. Ret9 was not ubiquitinated by the CD2AP·Cbl-3/c complex, suggesting that Ret9 was down-regulated by a fundamentally different mechanism. Taken together, these results suggest that only the SH3 domains of CD2AP were necessary to enhance the E3 ligase activity of Cbl-3/c toward Ret51. PMID:24425877

Functional characterization of Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligases in tumorigenesis

PubMed Central

Zhang, Jinfang; Wan, Lixin; Dai, Xiangpeng; Sun, Yi; Wei, Wenyi

2014-01-01

The Anaphase Promoting Complex/Cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that primarily governs cell cycle progression. APC/C is composed of at least 14 core subunits and recruits its substrates for ubiquitination via one of the two adaptor proteins, Cdc20 or Cdh1, in M or M/early G1 phase, respectively. Furthermore, recent studies have shed light on crucial functions for APC/C in maintaining genomic integrity, neuronal differentiation, cellular metabolism and tumorigenesis. To gain better insight into the in vivo physiological functions of APC/C in regulating various cellular processes, particularly development and tumorigenesis, a number of mouse models of APC/C core subunits, coactivators or inhibitors have been established and characterized. However, due to their essential role in cell cycle regulation, most of the germline knockout mice targeting the APC/C pathway are embryonic lethal, indicating the need for generating conditional knockout mouse models to assess the role in tumorigenesis for each APC/C signaling component in specific tissues. In this review, we will first provide a brief introduction of the ubiquitin-proteasome system (UPS) and the biochemical activities and cellular functions of the APC/C E3 ligase. We will then focus primarily on characterizing genetic mouse models used to understand the physiological roles of each APC/C signaling component in embryogenesis, cell proliferation, development and carcinogenesis. Finally, we discuss future research directions to further elucidate the physiological contributions of APC/C components during tumorigenesis and validate their potentials as a novel class of anti-cancer targets. PMID:24569229

Enzyme-substrate relationships in the ubiquitin system: approaches for identifying substrates of ubiquitin ligases.

PubMed

O'Connor, Hazel F; Huibregtse, Jon M

2017-09-01

Protein ubiquitylation is an important post-translational modification, regulating aspects of virtually every biochemical pathway in eukaryotic cells. Hundreds of enzymes participate in the conjugation and deconjugation of ubiquitin, as well as the recognition, signaling functions, and degradation of ubiquitylated proteins. Regulation of ubiquitylation is most commonly at the level of recognition of substrates by E3 ubiquitin ligases. Characterization of the network of E3-substrate relationships is a major goal and challenge in the field, as this expected to yield fundamental biological insights and opportunities for drug development. There has been remarkable success in identifying substrates for some E3 ligases, in many instances using the standard protein-protein interaction techniques (e.g., two-hybrid screens and co-immunoprecipitations paired with mass spectrometry). However, some E3s have remained refractory to characterization, while others have simply not yet been studied due to the sheer number and diversity of E3s. This review will discuss the range of tools and techniques that can be used for substrate profiling of E3 ligases.

Structural basis for catalytic activation by the human ZNF451 SUMO E3 ligase

DOE PAGES

Cappadocia, Laurent; Pichler, Andrea; Lima, Christopher D.

2015-11-02

E3 protein ligases enhance transfer of ubiquitin-like (Ubl) proteins from E2 conjugating enzymes to substrates by stabilizing the thioester-charged E2~Ubl in a closed configuration optimally aligned for nucleophilic attack. In this paper, we report biochemical and structural data that define the N-terminal domain of the Homo sapiens ZNF451 as the catalytic module for SUMO E3 ligase activity. The ZNF451 catalytic module contains tandem SUMO-interaction motifs (SIMs) bridged by a Pro-Leu-Arg-Pro (PLRP) motif. The first SIM and PLRP motif engage thioester-charged E2~SUMO while the next SIM binds a second molecule of SUMO bound to the back side of E2. We showmore » that ZNF451 is SUMO2 specific and that SUMO modification of ZNF451 may contribute to activity by providing a second molecule of SUMO that interacts with E2. Finally, our results are consistent with ZNF451 functioning as a bona fide SUMO E3 ligase.« less

Structural basis for catalytic activation by the human ZNF451 SUMO E3 ligase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cappadocia, Laurent; Pichler, Andrea; Lima, Christopher D.

E3 protein ligases enhance transfer of ubiquitin-like (Ubl) proteins from E2 conjugating enzymes to substrates by stabilizing the thioester-charged E2~Ubl in a closed configuration optimally aligned for nucleophilic attack. In this paper, we report biochemical and structural data that define the N-terminal domain of the Homo sapiens ZNF451 as the catalytic module for SUMO E3 ligase activity. The ZNF451 catalytic module contains tandem SUMO-interaction motifs (SIMs) bridged by a Pro-Leu-Arg-Pro (PLRP) motif. The first SIM and PLRP motif engage thioester-charged E2~SUMO while the next SIM binds a second molecule of SUMO bound to the back side of E2. We showmore » that ZNF451 is SUMO2 specific and that SUMO modification of ZNF451 may contribute to activity by providing a second molecule of SUMO that interacts with E2. Finally, our results are consistent with ZNF451 functioning as a bona fide SUMO E3 ligase.« less

The E3 ligase Ubr3 regulates Usher syndrome and MYH9 disorder proteins in the auditory organs of Drosophila and mammals.

PubMed

Li, Tongchao; Giagtzoglou, Nikolaos; Eberl, Daniel F; Jaiswal, Sonal Nagarkar; Cai, Tiantian; Godt, Dorothea; Groves, Andrew K; Bellen, Hugo J

2016-06-22

Myosins play essential roles in the development and function of auditory organs and multiple myosin genes are associated with hereditary forms of deafness. Using a forward genetic screen in Drosophila, we identified an E3 ligase, Ubr3, as an essential gene for auditory organ development. Ubr3 negatively regulates the mono-ubiquitination of non-muscle Myosin II, a protein associated with hearing loss in humans. The mono-ubiquitination of Myosin II promotes its physical interaction with Myosin VIIa, a protein responsible for Usher syndrome type IB. We show that ubr3 mutants phenocopy pathogenic variants of Myosin II and that Ubr3 interacts genetically and physically with three Usher syndrome proteins. The interactions between Myosin VIIa and Myosin IIa are conserved in the mammalian cochlea and in human retinal pigment epithelium cells. Our work reveals a novel mechanism that regulates protein complexes affected in two forms of syndromic deafness and suggests a molecular function for Myosin IIa in auditory organs.

Functional identification of MdSIZ1 as a SUMO E3 ligase in apple.

PubMed

Zhang, Rui-Fen; Guo, Ying; Li, Yuan-Yuan; Zhou, Li-Jie; Hao, Yu-Jin; You, Chun-Xiang

2016-07-01

SUMOylation, the conjugation of target proteins with SUMO (small ubiquitin-related modifier), is a type of post-translational modification in eukaryotes and involves the sequential action of activation (E1), conjugation (E2) and ligation (E3) enzymes. In Arabidopsis, the AtSIZ1 protein is a SUMO E3 ligase that promotes the conjugation of SUMO proteins to target substrates. Here, we isolated and identified a SUMO E3 ligase, MdSIZ1, in apple, which was similar to AtSIZ1. SUMOylation analysis showed that MdSIZ1 had SUMO E3 ligase activity in vitro and in vivo. SUMO conjugation was increased by high temperatures, low temperatures, and abscisic acid (ABA). The ectopic expression of MdSIZ1 in Arabidopsis siz1-2 mutant plants partially complemented the morphological mutant phenotype and enhanced the levels of SUMO conjugation. Taken together, these results suggest that MdSIZ1-mediated SUMO conjugation of target proteins is an important process that regulates the adaptation of apple plants to various environmental stresses. Copyright © 2016 Elsevier GmbH. All rights reserved.

The E3 ligase Ubr3 regulates Usher syndrome and MYH9 disorder proteins in the auditory organs of Drosophila and mammals

PubMed Central

Li, Tongchao; Giagtzoglou, Nikolaos; Eberl, Daniel F; Jaiswal, Sonal Nagarkar; Cai, Tiantian; Godt, Dorothea; Groves, Andrew K; Bellen, Hugo J

2016-01-01

Myosins play essential roles in the development and function of auditory organs and multiple myosin genes are associated with hereditary forms of deafness. Using a forward genetic screen in Drosophila, we identified an E3 ligase, Ubr3, as an essential gene for auditory organ development. Ubr3 negatively regulates the mono-ubiquitination of non-muscle Myosin II, a protein associated with hearing loss in humans. The mono-ubiquitination of Myosin II promotes its physical interaction with Myosin VIIa, a protein responsible for Usher syndrome type IB. We show that ubr3 mutants phenocopy pathogenic variants of Myosin II and that Ubr3 interacts genetically and physically with three Usher syndrome proteins. The interactions between Myosin VIIa and Myosin IIa are conserved in the mammalian cochlea and in human retinal pigment epithelium cells. Our work reveals a novel mechanism that regulates protein complexes affected in two forms of syndromic deafness and suggests a molecular function for Myosin IIa in auditory organs. DOI: http://dx.doi.org/10.7554/eLife.15258.001 PMID:27331610

Skp1: Implications in cancer and SCF-oriented anti-cancer drug discovery.

PubMed

Hussain, Muzammal; Lu, Yongzhi; Liu, Yong-Qiang; Su, Kai; Zhang, Jiancun; Liu, Jinsong; Zhou, Guang-Biao

2016-09-01

In the last decade, the ubiquitin proteasome system (UPS), in general, and E3 ubiquitin ligases, in particular, have emerged as valid drug targets for the development of novel anti-cancer therapeutics. Cullin RING Ligases (CRLs), which can be classified into eight groups (CRL1-8) and comprise approximately 200 members, represent the largest family of E3 ubiquitin ligases which facilitate the ubiquitination-derived proteasomal degradation of a myriad of functionally and structurally diverse substrates. S phase kinase-associated protein 1 (Skp1)-Cullin1-F-Box protein (SCF) complexes are the best characterized among CRLs, which play crucial roles in numerous cellular processes and physiological dysfunctions, such as in cancer biology. Currently, there is growing interest in developing SCF-targeting anti-cancer therapies for clinical application. Indeed, the research in this field has seen some progress in the form of cullin neddylation- and Skp2-inhibitors. However, it still remains an underdeveloped area and needs to design new strategies for developing improved form of therapy. In this review, we venture a novel strategy that rational pharmacological targeting of Skp1, a central regulator of SCF complexes, may provide a novel avenue for SCF-oriented anti-cancer therapy, expected: (i) to simultaneously address the critical roles that multiple SCF oncogenic complexes play in cancer biology, (ii) to selectively target cancer cells with minimal normal cell toxicity, and (iii) to offer multiple chemical series, via therapeutic interventions at the Skp1 binding interfaces in SCF complex, thereby maximizing chances of success for drug discovery. In addition, we also discuss the challenges that might be posed regarding rational pharmacological interventions against Skp1. Copyright © 2016 Elsevier Ltd. All rights reserved.

State of the APC/C: Organization, function, and structure

PubMed Central

McLean, Janel R.; Chaix, Denis; Ohi, Melanie D.; Gould, Kathleen L.

2016-01-01

The ubiquitin-proteasome protein degradation system is involved in many essential cellular processes including cell cycle regulation, cell differentiation, and the unfolded protein response.The anaphase-promoting complex/cyclosome (APC/C), an evolutionary conserved E3 ubiquitin ligase, was discovered 15 years ago because of its pivotal role in cyclin degradation and mitotic progression. Since then, we have learned that the APC/C is a very large, complex E3 ligase composed of 13 subunits, yielding a molecular machine of approximately 1 MDa. The intricate regulation of the APC/C is mediated by the Cdc20 family of activators, pseudosubstrate inhibitors, protein kinases and phosphatases and the spindle assembly checkpoint. The large size, complexity, and dynamic nature of the APC/C represent significant obstacles toward high-resolution structural techniques; however, over the last decade, there have been a number of lower resolution APC/C structures determined using single particle electron microscopy. These structures, when combined with data generated from numerous genetic and biochemical studies, have begun to shed light on how APC/C activity is regulated. Here, we discuss the most recent developments in the APC/C field concerning structure, substrate recognition, and catalysis. PMID:21261459

The E3 ubiquitin ligase mind bomb-2 (MIB2) protein controls B-cell CLL/lymphoma 10 (BCL10)-dependent NF-κB activation.

PubMed

Stempin, Cinthia C; Chi, Liying; Giraldo-Vela, Juan P; High, Anthony A; Häcker, Hans; Redecke, Vanessa

2011-10-28

B-cell CLL/lymphoma 10 (BCL10) is crucial for the activation of NF-κB in numerous immune receptor signaling pathways, including the T-cell receptor (TCR) and B-cell receptor signaling pathways. However, the molecular mechanisms that lead to signal transduction from BCL10 to downstream NF-κB effector kinases, such as TAK1 and components of the IKK complex, are not entirely understood. Here we used a proteomic approach and identified the E3 ligase MIB2 as a novel component of the activated BCL10 complex. In vitro translation and pulldown assays suggest direct interaction between BCL10 and MIB2. Overexpression experiments show that MIB2 controls BCL10-mediated activation of NF-κB by promoting autoubiquitination and ubiquitination of IKKγ/NEMO, as well as recruitment and activation of TAK1. Knockdown of MIB2 inhibited BCL10-dependent NF-κB activation. Together, our results identify MIB2 as a novel component of the activated BCL10 signaling complex and a missing link in the BCL10-dependent NF-κB signaling pathway.

The importance of regulatory ubiquitination in cancer and metastasis

PubMed Central

Gallo, L. H.; Ko, J.; Donoghue, D. J.

2017-01-01

ABSTRACT Ubiquitination serves as a degradation mechanism of proteins, but is involved in additional cellular processes such as activation of NFκB inflammatory response and DNA damage repair. We highlight the E2 ubiquitin conjugating enzymes, E3 ubiquitin ligases and Deubiquitinases that support the metastasis of a plethora of cancers. E3 ubiquitin ligases also modulate pluripotent cancer stem cells attributed to chemotherapy resistance. We further describe mutations in E3 ubiquitin ligases that support tumor proliferation and adaptation to hypoxia. Thus, this review describes how tumors exploit members of the vast ubiquitin signaling pathways to support aberrant oncogenic signaling for survival and metastasis. PMID:28166483

Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4–signalosome complex to regulate DNA repair and cell death

PubMed Central

Rao, Feng; Xu, Jing; Khan, A. Basit; Gadalla, Moataz M.; Cha, Jiyoung Y.; Xu, Risheng; Tyagi, Richa; Dang, Yongjun; Chakraborty, Anutosh; Snyder, Solomon H.

2014-01-01

Inositol polyphosphates containing an energetic pyrophosphate bond are formed primarily by a family of three inositol hexakisphosphate (IP6) kinases (IP6K1–3). The Cullin-RING ubiquitin ligases (CRLs) regulate diverse biological processes through substrate ubiquitylation. CRL4, comprising the scaffold Cullin 4A/B, the E2-interacting Roc1/2, and the adaptor protein damage-specific DNA-binding protein 1, is activated by DNA damage. Basal CRL4 activity is inhibited by binding to the COP9 signalosome (CSN). UV radiation and other stressors dissociate the complex, leading to E3 ligase activation, but signaling events that trigger signalosome dissociation from CRL4 have been unclear. In the present study, we show that, under basal conditions, IP6K1 forms a ternary complex with CSN and CRL4 in which IP6K1 and CRL4 are inactive. UV dissociates IP6K1 to generate IP7, which then dissociates CSN–CRL4 to activate CRL4. Thus, IP6K1 is a novel CRL4 subunit that transduces UV signals to mediate disassembly of the CRL4–CSN complex, thereby regulating nucleotide excision repair and cell death. PMID:25349427

BRCC36, A Novel Subunit of a BRCA1/2 E3 Ubiquitin Ligase Complex: Candidate Breast Cancer Susceptibility Gene

DTIC Science & Technology

2005-09-01

Hancock, A.K. Godwin, And Anthony T. Yeung. Enzymatic and Chemical Cleavage Methods to Identify Genetic Variation. In Molecular Diagnostics (Ed. G...R.G.H. Cotton, L. Hancock, A.K. Godwin, And Anthony T. Yeung. Enzymatic and Chemical Cleavage Methods to Identify Genetic Variation. In Molecular ... Diagnostics (Ed. G. Patrinos and W Ansorge) in press 2005. 9 Godwin, A.K., Ph.D. E-CONCLUSIONS: E.1. "BRCC36, a Novel Subunit of a BRCA1/2 E3 Ubiquitin

Rictor forms a complex with Cullin-1 to promote SGK1 ubiquitination and destruction

PubMed Central

Gao, Daming; Wan, Lixin; Inuzuka, Hiroyuki; Berg, Anders H.; Tseng, Alan; Zhai, Bo; Shaik, Shavali; Bennett, Eric; Tron, Adriana E.; Gasser, Jessica A.; Lau, Alan; Gygi, Steven; Harper, J. Wade; DeCaprio, James A.; Toker, Alex; Wei, Wenyi

2010-01-01

Summary The Rictor/mTOR complex (also known as mTORC2) plays a critical role in cellular homeostasis by phosphorylating AGC kinases such as Akt and SGK at their hydrophobic motifs to activate downstream signaling. However, the regulation of mTORC2 and whether it has additional function(s), remains largely unknown. Here we report that Rictor associates with Cullin-1 to form a functional E3 ubiquitin ligase. Rictor, but not Raptor or mTOR alone promotes SGK1 ubiquitination. Loss of Rictor/Cullin-1-mediated ubiquitination leads to increased SGK1 protein levels as detected in Rictor null cells. Moreover, as part of a feedback mechanism, phosphorylation of Rictor at T1135 by multiple AGC kinases disrupts the interaction between Rictor and Cullin-1 to impair SGK1 ubiquitination. These findings indicate that the Rictor/Cullin-1 E3 ligase activity is regulated by a specific signal relay cascade and that misregulation of this mechanism may contribute to the frequent overexpression of SGK1 in various human cancers. PMID:20832730

IKKε-mediated tumorigenesis requires K63-linked polyubiquitination by a cIAP1/cIAP2/TRAF2 E3 ubiquitin ligase complex

PubMed Central

Zhou, Alicia Y.; Shen, Rhine R.; Kim, Eejung; Lock, Ying J.; Xu, Ming; Chen, Zhijian J.; Hahn, William C.

2014-01-01

SUMMARY IκB kinase ε (IKKε, IKBKE) is a key regulator of innate immunity and a breast cancer oncogene, amplified in ~30% of breast cancers, that promotes malignant transformation through NF-κB activation. Here we show that IKKε is modified and regulated by K63-linked polyubiquitination at Lysine 30 and Lysine 401. TNFα and IL-1β stimulation induces IKKε K63-linked polyubiquitination over baseline levels in both macrophages and breast cancer cell lines, and this modification is essential for IKKε kinase activity, IKKε-mediated NF-κB activation and IKKε-induced malignant transformation. Disruption of K63-linked ubiquitination of IKKε does not affect its overall structure but impairs the recruitment of canonical NF-κB proteins. A cIAP1/cIAP2/TRAF2 E3 ligase complex binds to and ubiquitinates IKKε. Together, these observations demonstrate that K63-linked polyubiquitination regulates IKKε activity in both inflammatory and oncogenic contexts and suggests an alterative approach to target this breast cancer oncogene. PMID:23453969

Structure-Guided Design of Peptides as Tools to Probe the Protein-Protein Interaction between Cullin-2 and Elongin BC Substrate Adaptor in Cullin RING E3 Ubiquitin Ligases.

PubMed

Cardote, Teresa A F; Ciulli, Alessio

2017-09-21

Cullin RING E3 ubiquitin ligases (CRLs) are large dynamic multi-subunit complexes that control the fate of many proteins in cells. CRLs are attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. Herein we describe a structure-guided biophysical approach to probe the protein-protein interaction (PPI) between the Cullin-2 scaffold protein and the adaptor subunits Elongin BC within the context of the von Hippel-Lindau complex (CRL2 VHL ) using peptides. Two peptides were shown to bind at the targeted binding site on Elongin C, named the "EloC site", with micromolar dissociation constants, providing a starting point for future optimization. Our results suggest ligandability of the EloC binding site to short linear peptides, unveiling the opportunity and challenges to develop small molecules that have the potential to target selectively the Cul2-adaptor PPI within CRLs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Linking F-box protein 7 and parkin to neuronal degeneration in Parkinson's disease (PD).

PubMed

Zhou, Zhi Dong; Sathiyamoorthy, Sushmitha; Angeles, Dario C; Tan, Eng King

2016-04-18

Mutations of F-box protein 7 (FBXO7) and Parkin, two proteins in ubiquitin-proteasome system (UPS), are both implicated in pathogenesis of dopamine (DA) neuron degeneration in Parkinson's disease (PD). Parkin is a HECT/RING hybrid ligase that physically receives ubiquitin on its catalytic centre and passes ubiquitin onto its substrates, whereas FBXO7 is an adaptor protein in Skp-Cullin-F-box (SCF) SCF(FBXO7) ubiquitin E3 ligase complex to recognize substrates and mediate substrates ubiquitination by SCF(FBXO7) E3 ligase. Here, we discuss the overlapping pathophysiologic mechanisms and clinical features linking Parkin and FBXO7 with autosomal recessive PD. Both proteins play an important role in neuroprotective mitophagy to clear away impaired mitochondria. Parkin can be recruited to impaired mitochondria whereas cellular stress can promote FBXO7 mitochondrial translocation. PD-linked FBXO7 can recruit Parkin into damaged mitochondria and facilitate its aggregation. WT FBXO7, but not PD-linked FBXO7 mutants can rescue DA neuron degeneration in Parkin null Drosophila. A better understanding of the common pathophysiologic mechanisms of these two proteins could unravel specific pathways for targeted therapy in PD.

Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation.

PubMed

Kinsella, Sinéad; Fichtner, Michael; Watters, Orla; König, Hans-Georg; Prehn, Jochen H M

2018-05-02

Chronic pro-inflammatory signaling propagates damage to neural tissue and affects the rate of disease progression. Increased activation of Toll-like receptors (TLRs), master regulators of the innate immune response, is implicated in the etiology of several neuropathologies including amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease. Previously, we identified that the Bcl-2 family protein BH3-interacting domain death agonist (Bid) potentiates the TLR4-NF-κB pro-inflammatory response in glia, and specifically characterized an interaction between Bid and TNF receptor associated factor 6 (TRAF6) in microglia in response to TLR4 activation. We assessed the activation of mitogen-activated protein kinase (MAPK) and interferon regulatory factor 3 (IRF3) inflammatory pathways in response to TLR3 and TLR4 agonists in wild-type (wt) and bid-deficient microglia and macrophages, using Western blot and qPCR, focusing on the response of the E3 ubiquitin ligases Pellino 1 (Peli1) and TRAF3 in the absence of microglial and astrocytic Bid. Additionally, by Western blot, we investigated the Bid-dependent turnover of Peli1 and TRAF3 in wt and bid -/- microglia using the proteasome inhibitor Bortezomib. Interactions between the de-ubiquitinating Smad6-A20 and the E3 ubiquitin ligases, TRAF3 and TRAF6, were determined by FLAG pull-down in TRAF6-FLAG or Smad6-FLAG overexpressing wt and bid-deficient mixed glia. We elucidated a positive role of Bid in both TIR-domain-containing adapter-inducing interferon-β (TRIF)- and myeloid differentiation primary response 88 (MyD88)-dependent pathways downstream of TLR4, concurrently implicating TLR3-induced inflammation. We identified that Peli1 mRNA levels were significantly reduced in PolyI:C- and lipopolysaccharide (LPS)-stimulated bid-deficient microglia, suggesting disturbed IRF3 activation. Differential regulation of TRAF3 and Peli1, both essential E3 ubiquitin ligases facilitating TRIF-dependent signaling, was observed between wt and bid -/- microglia and astrocytes. bid deficiency resulted in increased A20-E3 ubiquitin ligase protein interactions in glia, specifically A20-TRAF6 and A20-TRAF3, implicating enhanced de-ubiquitination as the mechanism of action by which E3 ligase activity is perturbed. Furthermore, Smad6-facilitated recruitment of the de-ubiquitinase A20 to E3-ligases occurred in a bid-dependent manner. This study demonstrates that Bid promotes E3 ubiquitin ligase-mediated signaling downstream of TLR3 and TLR4 and provides further evidence for the potential of Bid inhibition as a therapeutic for the attenuation of the robust pro-inflammatory response culminating in TLR activation.

Smurf E3 ubiquitin ligases at the cross roads of oncogenesis and tumor suppression.

PubMed

David, Diana; Nair, S Asha; Pillai, M Radhakrishna

2013-01-01

Smad ubiquitin regulatory factors (Smurfs) belong to the HECT- family of E3 ubiquitin ligases and comprise mainly of two members, Smurf1 and Smurf2. Initially, Smurfs have been implicated in determining the competence of cells to respond to TGF-β/BMP signaling pathway. Nevertheless, the intrinsic catalytic activity has extended the repertoire of Smurf substrates beyond the TGF-β/BMP super family expanding its realm further to epigenetic modifications of histones governing the chromatin landscape. Through regulation of a large number of proteins in multiple cellular compartments, Smurfs regulate diverse cellular processes, including cell-cycle progression, cell proliferation, differentiation, DNA damage response, maintenance of genomic stability, and metastasis. As the genomic ablation of Smurfs leads to global changes in histone modifications and predisposition to a wide spectrum of tumors, Smurfs are also considered to have a novel tumor suppressor function. This review focuses on regulation network and biological functions of Smurfs in connection with its role in cancer progression. By providing a portrait of their protein targets, we intend to link the substrate specificity of Smurfs with their contribution to tumorigenesis. Since the regulation and biological functions of Smurfs are quite complex, understanding the oncogenic potential of these E3 ubiquitin ligases may facilitate the development of mechanism-based drugs in cancer treatment. Copyright © 2012 Elsevier B.V. All rights reserved.

KCTD2, an adaptor of Cullin3 E3 ubiquitin ligase, suppresses gliomagenesis by destabilizing c-Myc

PubMed Central

Kim, Eun-Jung; Kim, Sung-Hak; Jin, Xiong; Jin, Xun; Kim, Hyunggee

2017-01-01

Cullin3 E3 ubiquitin ligase ubiquitinates a wide range of substrates through substrate-specific adaptors Bric-a-brac, Tramtrack, and Broad complex (BTB) domain proteins. These E3 ubiquitin ligase complexes are involved in diverse cellular functions. Our recent study demonstrated that decreased Cullin3 expression induces glioma initiation and correlates with poor prognosis of patients with malignant glioma. However, the substrate recognition mechanism associated with tumorigenesis is not completely understood. Through yeast two-hybrid screening, we identified potassium channel tetramerization domain-containing 2 (KCTD2) as a BTB domain protein that binds to Cullin3. The interaction of Cullin3 and KCTD2 was verified using immunoprecipitation and immunofluorescence. Of interest, KCTD2 expression was markedly decreased in patient-derived glioma stem cells (GSCs) compared with non-stem glioma cells. Depletion of KCTD2 using a KCTD2-specific short-hairpin RNA in U87MG glioma cells and primary Ink4a/Arf-deficient murine astrocytes markedly increased self-renewal activity in addition with an increased expression of stem cell markers, and mouse in vivo intracranial tumor growth. As an underlying mechanism for these KCTD2-mediated phenotypic changes, we demonstrated that KCTD2 interacts with c-Myc, which is a key stem cell factor, and causes c-Myc protein degradation by ubiquitination. As a result, KCTD2 depletion acquires GSC features and affects aerobic glycolysis via expression changes in glycolysis-associated genes through c-Myc protein regulation. Of clinical significance was our finding that patients having a profile of KCTD2 mRNA-low and c-Myc gene signature-high, but not KCTD2 mRNA-low and c-Myc mRNA-high, are strongly associated with poor prognosis. This study describes a novel regulatory mode of c-Myc protein in malignant gliomas and provides a potential framework for glioma therapy by targeting c-Myc function. PMID:28060381

Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO.

PubMed

Reidick, Christina; El Magraoui, Fouzi; Meyer, Helmut E; Stenmark, Harald; Platta, Harald W

2014-12-23

The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes-autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept.

Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO

PubMed Central

Reidick, Christina; El Magraoui, Fouzi; Meyer, Helmut E.; Stenmark, Harald; Platta, Harald W.

2014-01-01

The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept. PMID:25545884

The Anaphase-Promoting Complex (APC) ubiquitin ligase affects chemosensory behavior in C. elegans.

PubMed

Wang, Julia; Jennings, Alexandra K; Kowalski, Jennifer R

2016-01-01

The regulation of fundamental aspects of neurobiological function has been linked to the ubiquitin signaling system (USS), which regulates the degradation and activity of proteins and is catalyzed by E1, E2, and E3 enzymes. The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that controls diverse developmental and signaling processes in post-mitotic neurons; however, potential roles for the APC in sensory function have yet to be explored. In this study, we examined the effect of the APC ubiquitin ligase on chemosensation in Caenorhabditis elegans by testing chemotaxis to the volatile odorants, diacetyl, pyrazine, and isoamyl alcohol, to which wild-type worms are attracted. Animals with loss of function mutations in either of two alleles (g48 and ye143) of the gene encoding the APC subunit EMB-27 APC6 showed increased chemotaxis towards diacetyl and pyrazine, odorants sensed by AWA neurons, but exhibited normal chemotaxis to isoamyl alcohol, which is sensed by AWC neurons. The statistically significant increase in chemotaxis in the emb-27 APC6 mutants suggests that the APC inhibits AWA-mediated chemosensation in C. elegans. Increased chemotaxis to pyrazine was also seen with mutants lacking another essential APC subunit, MAT-2 APC1; however, mat-2 APC1 mutants exhibited wild type responses to diacetyl. The difference in responsiveness of these two APC subunit mutants may be due to differential strength of these hypomorphic alleles or may indicate the presence of functional sub-complexes of the APC at work in this process. These findings are the first evidence for APC-mediated regulation of chemosensation and lay the groundwork for further studies aimed at identifying the expression levels, function, and targets of the APC in specific sensory neurons. Because of the similarity between human and C. elegans nervous systems, the role of the APC in sensory neurons may also advance our understanding of human sensory function and disease.

Structure of the Human FANCL RING-Ube2T Complex Reveals Determinants of Cognate E3-E2 Selection

PubMed Central

Hodson, Charlotte; Purkiss, Andrew; Miles, Jennifer Anne; Walden, Helen

2014-01-01

Summary The combination of an E2 ubiquitin-conjugating enzyme with an E3 ubiquitin-ligase is essential for ubiquitin modification of a substrate. Moreover, the pairing dictates both the substrate choice and the modification type. The molecular details of generic E3-E2 interactions are well established. Nevertheless, the determinants of selective, specific E3-E2 recognition are not understood. There are ∼40 E2s and ∼600 E3s giving rise to a possible ∼24,000 E3-E2 pairs. Using the Fanconi Anemia pathway exclusive E3-E2 pair, FANCL-Ube2T, we report the atomic structure of the FANCL RING-Ube2T complex, revealing a specific and extensive network of additional electrostatic and hydrophobic interactions. Furthermore, we show that these specific interactions are required for selection of Ube2T over other E2s by FANCL. PMID:24389026

The role of hybrid ubiquitin chains in the MyD88 and other innate immune signalling pathways.

PubMed

Cohen, Philip; Strickson, Sam

2017-07-01

The adaptor protein MyD88 is required for signal transmission by toll-like receptors and receptors of the interleukin-1 family of cytokines. MyD88 signalling triggers the formation of Lys63-linked and Met1-linked ubiquitin (K63-Ub, M1-Ub) chains within minutes. The K63-Ub chains, which are formed by the E3 ubiquitin ligases TRAF6, Pellino1 and Pellino2, activate TAK1, the master kinase that switches on mitogen-activated protein (MAP) kinase cascades and initiates activation of the canonical IκB kinase (IKK) complex. The M1-Ub chains, which are formed by the linear ubiquitin chain assembly complex (LUBAC), bind to the NEMO (NF-κB essential modulator) component of the IKK complex and are required for TAK1 to activate IKKs, but not MAP kinases. An essential E3 ligase-independent role of TRAF6 is to recruit LUBAC into the MyD88 signalling complex, where it recognises preformed K63-Ub chains attached to protein components of these complexes, such as IRAK1 (IL-1 receptor-associated kinase), producing ubiquitin chains containing both types of linkage, termed K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids, which is a feature of several innate immune signalling pathways, permits the co-recruitment of proteins that interact with either K63-Ub or M1-Ub chains. Two likely roles for K63/M1-Ub hybrids are to facilitate the TAK1-dependent activation of the IKK complex and to prevent the hyperactivation of these kinases by recruiting A20 and A20-binding inhibitor of NF-κB1 (ABIN1). These proteins restrict activation of the TAK1 and IKK complexes, probably by competing with them for binding to K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids may also regulate the rate at which the ubiquitin linkages in these chains are hydrolysed. The IKK-catalysed phosphorylation of some of its substrates permits their recognition by the E3 ligase SCF βTRCP , leading to their Lys48-linked ubiquitylation and proteasomal degradation. Innate immune signalling is therefore controlled by the formation and destruction of three different types of ubiquitin linkage.

The role of hybrid ubiquitin chains in the MyD88 and other innate immune signalling pathways

PubMed Central

Cohen, Philip; Strickson, Sam

2017-01-01

The adaptor protein MyD88 is required for signal transmission by toll-like receptors and receptors of the interleukin-1 family of cytokines. MyD88 signalling triggers the formation of Lys63-linked and Met1-linked ubiquitin (K63-Ub, M1-Ub) chains within minutes. The K63-Ub chains, which are formed by the E3 ubiquitin ligases TRAF6, Pellino1 and Pellino2, activate TAK1, the master kinase that switches on mitogen-activated protein (MAP) kinase cascades and initiates activation of the canonical IκB kinase (IKK) complex. The M1-Ub chains, which are formed by the linear ubiquitin chain assembly complex (LUBAC), bind to the NEMO (NF-κB essential modulator) component of the IKK complex and are required for TAK1 to activate IKKs, but not MAP kinases. An essential E3 ligase-independent role of TRAF6 is to recruit LUBAC into the MyD88 signalling complex, where it recognises preformed K63-Ub chains attached to protein components of these complexes, such as IRAK1 (IL-1 receptor-associated kinase), producing ubiquitin chains containing both types of linkage, termed K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids, which is a feature of several innate immune signalling pathways, permits the co-recruitment of proteins that interact with either K63-Ub or M1-Ub chains. Two likely roles for K63/M1-Ub hybrids are to facilitate the TAK1-dependent activation of the IKK complex and to prevent the hyperactivation of these kinases by recruiting A20 and A20-binding inhibitor of NF-κB1 (ABIN1). These proteins restrict activation of the TAK1 and IKK complexes, probably by competing with them for binding to K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids may also regulate the rate at which the ubiquitin linkages in these chains are hydrolysed. The IKK-catalysed phosphorylation of some of its substrates permits their recognition by the E3 ligase SCFβTRCP, leading to their Lys48-linked ubiquitylation and proteasomal degradation. Innate immune signalling is therefore controlled by the formation and destruction of three different types of ubiquitin linkage. PMID:28475177

Large-Scale, Lineage-Specific Expansion of a Bric-a-Brac/Tramtrack/Broad Complex Ubiquitin-Ligase Gene Family in Rice[W

PubMed Central

Gingerich, Derek J.; Hanada, Kousuke; Shiu, Shin-Han; Vierstra, Richard D.

2007-01-01

Selective ubiquitination of proteins is directed by diverse families of ubiquitin-protein ligases (or E3s) in plants. One important type uses Cullin-3 as a scaffold to assemble multisubunit E3 complexes containing one of a multitude of bric-a-brac/tramtrack/broad complex (BTB) proteins that function as substrate recognition factors. We previously described the 80-member BTB gene superfamily in Arabidopsis thaliana. Here, we describe the complete BTB superfamily in rice (Oryza sativa spp japonica cv Nipponbare) that contains 149 BTB domain–encoding genes and 43 putative pseudogenes. Amino acid sequence comparisons of the rice and Arabidopsis superfamilies revealed a near equal repertoire of putative substrate recognition module types. However, phylogenetic comparisons detected numerous gene duplication and/or loss events since the rice and Arabidopsis BTB lineages split, suggesting possible functional specialization within individual BTB families. In particular, a major expansion and diversification of a subset of BTB proteins containing Meprin and TRAF homology (MATH) substrate recognition sites was evident in rice and other monocots that likely occurred following the monocot/dicot split. The MATH domain of a subset appears to have evolved significantly faster than those in a smaller core subset that predates flowering plants, suggesting that the substrate recognition module in many monocot MATH-BTB E3s are diversifying to ubiquitinate a set of substrates that are themselves rapidly changing. Intriguing possibilities include pathogen proteins attempting to avoid inactivation by the monocot host. PMID:17720868

Identification of Arabidopsis MYB56 as a novel substrate for CRL3(BPM) E3 ligases.

PubMed

Chen, Liyuan; Bernhardt, Anne; Lee, JooHyun; Hellmann, Hanjo

2015-02-01

Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work, MYB56 is identified as a novel target of a CULLIN3 (CUL3)-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ (BPM) proteins, which function as substrate adaptors to the E3 ligase. Genetic studies have indicated that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T (FT), a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling flowering time in plants. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

Mechanism of TRIM25 Catalytic Activation in the Antiviral RIG-I Pathway

PubMed Central

Sanchez, Jacint G.; Chiang, Jessica J.; Sparrer, Konstantin M.J.; Alam, Steven L.; Chi, Michael; Roganowicz, Marcin D.; Sankaran, Banumathi; Gack, Michaela U.; Pornillos, Owen

2016-01-01

SUMMARY Antiviral response pathways induce interferon by higher-order assembly of signaling complexes called signalosomes. Assembly of the RIG-I signalosome is regulated by K63-linked polyubiquitin chains, which are synthesized by the E3 ubiquitin ligase, TRIM25. We have previously shown that the TRIM25 coiled-coil domain is a stable, antiparallel dimer that positions two catalytic RING domains on opposite ends of an elongated rod. We now show that the RING domain is a separate self-association motif that engages ubiquitin-conjugated E2 enzymes as a dimer. RING dimerization is required for catalysis, TRIM25-mediated RIG-I ubiquitination, interferon induction, and antiviral activity. We also provide evidence that RING dimerization and E3 ligase activity are promoted by binding of the TRIM25 SPRY domain to the RIG-I effector domain. These results indicate that TRIM25 actively participates in higher-order assembly of the RIG-I signalosome and helps to fine-tune the efficiency of the RIG-I-mediated antiviral response. PMID:27425606

The carboxyl terminus of FANCE recruits FANCD2 to the Fanconi Anemia (FA) E3 ligase complex to promote the FA DNA repair pathway.

PubMed

Polito, David; Cukras, Scott; Wang, Xiaozhe; Spence, Paige; Moreau, Lisa; D'Andrea, Alan D; Kee, Younghoon

2014-03-07

Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair.

The Carboxyl Terminus of FANCE Recruits FANCD2 to the Fanconi Anemia (FA) E3 Ligase Complex to Promote the FA DNA Repair Pathway*

PubMed Central

Polito, David; Cukras, Scott; Wang, Xiaozhe; Spence, Paige; Moreau, Lisa; D'Andrea, Alan D.; Kee, Younghoon

2014-01-01

Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair. PMID:24451376

The functional interplay between the HIF pathway and the ubiquitin system - more than a one-way road.

PubMed

Günter, Julia; Ruiz-Serrano, Amalia; Pickel, Christina; Wenger, Roland H; Scholz, Carsten C

2017-07-15

The hypoxia inducible factor (HIF) pathway and the ubiquitin system represent major cellular processes that are involved in the regulation of a plethora of cellular signaling pathways and tissue functions. The ubiquitin system controls the ubiquitination of proteins, which is the covalent linkage of one or several ubiquitin molecules to specific targets. This ubiquitination is catalyzed by approximately 1000 different E3 ubiquitin ligases and can lead to different effects, depending on the type of internal ubiquitin chain linkage. The best-studied function is the targeting of proteins for proteasomal degradation. The activity of E3 ligases is antagonized by proteins called deubiquitinases (or deubiquitinating enzymes), which negatively regulate ubiquitin chains. This is performed in most cases by the catalytic removal of these chains from the targeted protein. The HIF pathway is regulated in an oxygen-dependent manner by oxygen-sensing hydroxylases. Covalent modification of HIFα subunits leads to the recruitment of an E3 ligase complex via the von Hippel-Lindau (VHL) protein and the subsequent polyubiquitination and proteasomal degradation of HIFα subunits, demonstrating the regulation of the HIF pathway by the ubiquitin system. This unidirectional effect of an E3 ligase on the HIF pathway is the best-studied example for the interplay between these two important cellular processes. However, additional regulatory mechanisms of the HIF pathway through the ubiquitin system are emerging and, more recently, also the reciprocal regulation of the ubiquitin system through components of the HIF pathway. Understanding these mechanisms and their relevance for the activity of each other is of major importance for the comprehensive elucidation of the oxygen-dependent regulation of cellular processes. This review describes the current knowledge of the functional bidirectional interplay between the HIF pathway and the ubiquitin system on the protein level. Copyright © 2017 Elsevier Inc. All rights reserved.

The Ubiquitin Ligase Component Siah1a Is Required for Completion of Meiosis I in Male Mice

PubMed Central

Dickins, Ross A.; Frew, Ian J.; House, Colin M.; O'Bryan, Moira K.; Holloway, Andrew J.; Haviv, Izhak; Traficante, Nadia; de Kretser, David M.; Bowtell, David D. L.

2002-01-01

The mammalian Siah genes encode highly conserved proteins containing a RING domain. As components of E3 ubiquitin ligase complexes, Siah proteins facilitate the ubiquitination and degradation of diverse protein partners including β-catenin, N-CoR, and DCC. We used gene targeting in mice to analyze the function of Siah1a during mammalian development and reveal novel roles in growth, viability, and fertility. Mutant animals have normal weights at term but are postnatally growth retarded, despite normal levels of pituitary growth hormone. Embryonic fibroblasts isolated from mutant animals grow normally. Most animals die before weaning, and few survive beyond 3 months. Serum gonadotropin levels are normal in Siah1a mutant mice; however, females are subfertile and males are sterile due to a block in spermatogenesis. Although spermatocytes in mutant mice display normal meiotic prophase and meiosis I spindle formation, they accumulate at metaphase to telophase of meiosis I and subsequently undergo apoptosis. The requirement of Siah1a for normal progression beyond metaphase I suggests that Siah1a may be part of a novel E3 complex acting late in the first meiotic division. PMID:11884614

BRCC36, A Novel Subunit of a BRCA1/2 E3 Ubiquitin Ligase Complex: Candidate Breast Cancer Susceptibility Gene

DTIC Science & Technology

2004-09-01

Variation. In Molecular Diagnostics (Ed. G. Patrinos and W Ansorge) in press 2004. 7 Godwin, A.K., Ph.D. E-CONCLUSIONS: E.1. "BRCC36, a Novel Subunit...R.G.H. Cotton, L. Hancock, A.K. Godwin, And Anthony T. Yeung. Enzymatic and Chemical Cleavage Methods to Identify Genetic Variation. In Molecular Diagnostics (Ed. G. Patrinos and W Ansorge) in press 2004 9

The E3 Ligase CHIP: Insights into Its Structure and Regulation

PubMed Central

Paul, Indranil; Ghosh, Mrinal K.

2014-01-01

The carboxy-terminus of Hsc70 interacting protein (CHIP) is a cochaperone E3 ligase containing three tandem repeats of tetratricopeptide (TPR) motifs and a C-terminal U-box domain separated by a charged coiled-coil region. CHIP is known to function as a central quality control E3 ligase and regulates several proteins involved in a myriad of physiological and pathological processes. Recent studies have highlighted varied regulatory mechanisms operating on the activity of CHIP which is crucial for cellular homeostasis. In this review article, we give a concise account of our current knowledge on the biochemistry and regulation of CHIP. PMID:24868554

Targeting Signaling to YAP for the Therapy of NF2

DTIC Science & Technology

2015-10-01

clear mechanism of Merlin’s tumor suppressor function. Our studies have shown that inactivation of Merlin/NF2 de-regulates the E3 ubiquitin ligase...Keywords NF2, E3 ubiquitin ligase, high throughput small molecule screening, targeted therapy. 6 Accomplishment Major goals and objectives

Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rockel, Beate; Schmaler, Tilo; Huang, Xiaohua

2014-07-25

Highlights: • Deneddylation rates of human erythrocyte and mouse fibroblast CSN are very similar. • 3D models of native human and mouse CSN reveal common architectures. • The cryo-structure of native mammalian CSN shows a horseshoe subunit arrangement. - Abstract: The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with themore » 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14 years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts are very similar, Flag-CSN pulldowns are a proper alternative to CSN preparation from erythrocytes.« less

Inhibitors of ubiquitin E3 ligase as potential new antimalarial drug leads

USDA-ARS?s Scientific Manuscript database

The ubiquitin/proteasome pathway is the principal system for degradation of proteins in eukaryotes. Ubiquitin is a highly conserved polypeptide that covalently attaches to target proteins through the combined action ofubiquitin-activating enzyme (E1), conjugating enzyme (E2) and a protein ligase (E...

A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function

PubMed Central

Liu, Yaobin; Huang, Xiangao; He, Xian; Zhou, Yanqing; Jiang, Xiaogang; Chen-Kiang, Selina; Jaffrey, Samie R.; Xu, Guoqiang

2015-01-01

The immunomodulatory drug (IMiD) thalidomide and its structural analogs lenalidomide and pomalidomide are highly effective in treating clinical indications. Thalidomide binds to cereblon (CRBN), a substrate receptor of the cullin-4 really interesting new gene (RING) E3 ligase complex. Here, we examine the effect of thalidomide and its analogs on CRBN ubiquitination and its functions in human cell lines. We find that the ubiquitin modification of CRBN includes K48-linked polyubiquitin chains and that thalidomide blocks the formation of CRBN-ubiquitin conjugates. Furthermore, we show that ubiquitinated CRBN is targeted for proteasomal degradation. Treatment of human myeloma cell lines such as MM1.S, OPM2, and U266 with thalidomide (100 μM) and its structural analog lenalidomide (10 μM) results in stabilization of CRBN and elevation of CRBN protein levels. This in turn leads to the reduced level of CRBN target proteins and enhances the sensitivity of human multiple myeloma cells to IMiDs. Our results reveal a novel mechanism by which thalidomide and its analogs modulate the CRBN function in cells. Through inhibition of CRBN ubiquitination, thalidomide and its analogs allow CRBN to accumulate, leading to the increased cullin-4 RING E3 ligase-mediated degradation of target proteins.—Liu, Y., Huang, X., He, X., Zhou, Y., Jiang, X., Chen-Kiang, S., Jaffrey, S. R., Xu, G. A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function. PMID:26231201

Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity*

PubMed Central

Ranaweera, Ruchira S.; Yang, Xiaolu

2013-01-01

The RING domain E3 ubiquitin ligase Mdm2 is the master regulator of the tumor suppressor p53. It targets p53 for proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Like most E3 ligases, Mdm2 can also ubiquitinate itself. How Mdm2 auto-ubiquitination may influence its substrate ubiquitin ligase activity is undefined. Here we show that auto-ubiquitination of Mdm2 is an activating event. Mdm2 that has been conjugated to polyubiquitin chains, but not to single ubiquitins, exhibits substantially enhanced activity to polyubiquitinate p53. Mechanistically, auto-ubiquitination of Mdm2 facilitates the recruitment of the E2 ubiquitin-conjugating enzyme. This occurs through noncovalent interactions between the ubiquitin chains on Mdm2 and the ubiquitin binding domain on E2s. Mutations that diminish the noncovalent interactions render auto-ubiquitination unable to stimulate Mdm2 substrate E3 activity. These results suggest a model in which polyubiquitin chains on an E3 increase the local concentration of E2 enzymes and permit the processivity of substrate ubiquitination. They also support the notion that autocatalysis may be a prevalent mode for turning on the activity of latent enzymes. PMID:23671280

Understanding Cullin-RING E3 Biology through Proteomics-based Substrate Identification*

PubMed Central

Harper, J. Wade; Tan, Meng-Kwang Marcus

2012-01-01

Protein turnover through the ubiquitin-proteasome pathway controls numerous developmental decisions and biochemical processes in eukaryotes. Central to protein ubiquitylation are ubiquitin ligases, which provide specificity in targeted ubiquitylation. With more than 600 ubiquitin ligases encoded by the human genome, many of which remain to be studied, considerable effort is being placed on the development of methods for identifying substrates of specific ubiquitin ligases. In this review, we describe proteomic technologies for the identification of ubiquitin ligase targets, with a particular focus on members of the cullin-RING E3 class of ubiquitin ligases, which use F-box proteins as substrate specific adaptor proteins. Various proteomic methods are described and are compared with genetic approaches that are available. The continued development of such methods is likely to have a substantial impact on the ubiquitin-proteasome field. PMID:22962057

Understanding cullin-RING E3 biology through proteomics-based substrate identification.

PubMed

Harper, J Wade; Tan, Meng-Kwang Marcus

2012-12-01

Protein turnover through the ubiquitin-proteasome pathway controls numerous developmental decisions and biochemical processes in eukaryotes. Central to protein ubiquitylation are ubiquitin ligases, which provide specificity in targeted ubiquitylation. With more than 600 ubiquitin ligases encoded by the human genome, many of which remain to be studied, considerable effort is being placed on the development of methods for identifying substrates of specific ubiquitin ligases. In this review, we describe proteomic technologies for the identification of ubiquitin ligase targets, with a particular focus on members of the cullin-RING E3 class of ubiquitin ligases, which use F-box proteins as substrate specific adaptor proteins. Various proteomic methods are described and are compared with genetic approaches that are available. The continued development of such methods is likely to have a substantial impact on the ubiquitin-proteasome field.

Degradation of phosphorylated p53 by viral protein-ECS E3 ligase complex.

PubMed

Sato, Yoshitaka; Kamura, Takumi; Shirata, Noriko; Murata, Takayuki; Kudoh, Ayumi; Iwahori, Satoko; Nakayama, Sanae; Isomura, Hiroki; Nishiyama, Yukihiro; Tsurumi, Tatsuya

2009-07-01

p53-signaling is modulated by viruses to establish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV) lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an ubiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component of the ECS (Elongin B/C-Cul2/5-SOCS-box protein) ubiquitin ligase complex targeting p53 for degradation. Intringuingly, C-terminal phosphorylation of p53 resulting from activated DNA damage response by viral lytic replication enhances its binding to BZLF1 protein. Purified BZLF1 protein-associated ECS could be shown to catalyze ubiquitination of phospho-mimetic p53 more efficiently than the wild-type in vitro. The compensation of p53 at middle and late stages of the lytic infection inhibits viral DNA replication and production during lytic infection, suggesting that the degradation of p53 is required for efficient viral propagation. Taken together, these findings demonstrate a role for the BZLF1 protein-associated ECS ligase complex in regulation of p53 phosphorylated by activated DNA damage signaling during viral lytic infection.

Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance.

PubMed

Payne, Felicity; Colnaghi, Rita; Rocha, Nuno; Seth, Asha; Harris, Julie; Carpenter, Gillian; Bottomley, William E; Wheeler, Eleanor; Wong, Stephen; Saudek, Vladimir; Savage, David; O'Rahilly, Stephen; Carel, Jean-Claude; Barroso, Inês; O'Driscoll, Mark; Semple, Robert

2014-09-01

Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5-6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase-deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress.

Hey bHLH Proteins Interact with a FBXO45 Containing SCF Ubiquitin Ligase Complex and Induce Its Translocation into the Nucleus.

PubMed

Salat, Daniela; Winkler, Anja; Urlaub, Henning; Gessler, Manfred

2015-01-01

The Hey protein family, comprising Hey1, Hey2 and HeyL in mammals, conveys Notch signals in many cell types. The helix-loop-helix (HLH) domain as well as the Orange domain, mediate homo- and heterodimerization of these transcription factors. Although distinct interaction partners have been identified so far, their physiological relevance for Hey functions is still largely unclear. Using a tandem affinity purification approach and mass spectrometry analysis we identified members of an ubiquitin E3-ligase complex consisting of FBXO45, PAM and SKP1 as novel Hey1 associated proteins. There is a direct interaction between Hey1 and FBXO45, whereas FBXO45 is needed to mediate indirect Hey1 binding to SKP1. Expression of Hey1 induces translocation of FBXO45 and PAM into the nucleus. Hey1 is a short-lived protein that is degraded by the proteasome, but there is no evidence for FBXO45-dependent ubiquitination of Hey1. On the contrary, Hey1 mediated nuclear translocation of FBXO45 and its associated ubiquitin ligase complex may extend its spectrum to additional nuclear targets triggering their ubiquitination. This suggests a novel mechanism of action for Hey bHLH factors.

Small molecule therapeutics targeting F-box proteins in cancer.

PubMed

Liu, Yuan; Mallampalli, Rama K

2016-02-01

The ubiquitin proteasome system (UPS) plays vital roles in maintaining protein equilibrium mainly through proteolytic degradation of targeted substrates. The archetypical SCF ubiquitin E3 ligase complex contains a substrate recognition subunit F-box protein that recruits substrates to the catalytic ligase core for its polyubiquitylation and subsequent proteasomal degradation. Several well-characterized F-box proteins have been demonstrated that are tightly linked to neoplasia. There is mounting information characterizing F-box protein-substrate interactions with the rationale to develop unique therapeutics for cancer treatment. Here we review that how F-box proteins function in cancer and summarize potential small molecule inhibitors for cancer therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Testing the Effects of SIAH Ubiquitin E3 Ligases on Lysine Acetyl Transferases.

PubMed

Hagenbucher, Jan; Stekman, Hilda; Rodriguez-Gil, Alfonso; Kracht, Michael; Schmitz, M Lienhard

2017-01-01

The family of seven-in-absentia (SIAH) ubiquitin E3 ligases functions in the control of numerous key signaling pathways. These enzymes belong to the RING (really interesting new gene) group of E3 ligases and mediate the attachment of ubiquitin chains to substrates, which then leads to their proteasomal degradation. Here, we describe a protocol that allows measuring SIAH-mediated ubiquitination and degradation of its client proteins as exemplified by acetyl transferases using simple overexpression experiments. The impact of SIAH expression on the relative amounts of target proteins and their mRNAs can be quantified by Western blotting and quantitative PCR (qPCR) as described here.

Allosteric auto-inhibition and activation of the Nedd4 family E3 ligase Itch.

PubMed

Zhu, Kang; Shan, Zelin; Chen, Xing; Cai, Yuqun; Cui, Lei; Yao, Weiyi; Wang, Zhen; Shi, Pan; Tian, Changlin; Lou, Jizhong; Xie, Yunli; Wen, Wenyu

2017-09-01

The Nedd4 family E3 ligases are key regulators of cell growth and proliferation and are often misregulated in human cancers and other diseases. The ligase activities of Nedd4 E3s are tightly controlled via auto-inhibition. However, the molecular mechanism underlying Nedd4 E3 auto-inhibition and activation is poorly understood. Here, we show that the WW domains proceeding the catalytic HECT domain play an inhibitory role by binding directly to HECT in the Nedd4 E3 family member Itch. Our structural and biochemical analyses of Itch reveal that the WW2 domain and a following linker allosterically lock HECT in an inactive state inhibiting E2-E3 transthiolation. Binding of the Ndfip1 adaptor or JNK1-mediated phosphorylation relieves the auto-inhibition of Itch in a WW2-dependent manner. Aberrant activation of Itch leads to migration defects of cortical neurons during development. Our study provides a new mechanism governing the regulation of Itch. © 2017 The Authors.

BRCC36, A Novel Subunit of a BRCA1 E3 Ubiquitin Ligase Complex, Candidates for BRCA3

DTIC Science & Technology

2006-06-01

ovarian tumours. Nat Genet 1995;9:439–43. 16. Hughes-Davies L, Huntsman D, Ruas M, et al. EMSY links the BRCA2 pathway to sporadic breast and ovarian...133–138. Campos B, Diez O, Domenech M, Baena M, Balmana J, Sanz J, Ramirez A, Alonso C, Baiget M. 2003. RNA analysis of eight BRCA1 and BRCA2

Identification of Arabidopsis MYB56 as a novel substrate for CRL3BPM E3 ligases.

PubMed

Chen, Liyuan; Bernhardt, Anne; Lee, JooHyun; Hellmann, Hanjo

2014-10-24

Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work MYB56 is identified as a novel target of a CULLIN3 (CUL3)-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ (BPM) proteins, which function as substrate adaptors to the E3 ligase. Genetic studies pointed out that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T (FT), a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling the flowering time point in plants. © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.

Characterization of a novel RING-type ubiquitin E3 ligase GhRING2 differentially expressed in cotton fiber

USDA-ARS?s Scientific Manuscript database

The ubiquitin-proteasome proteolysis pathway is responsible for the degradation of abnormal and short-lived proteins to regulate many important biochemical activities in eukaryotes. By employing affymetrix microarray analysis, we have identified a novel ubiquitin ligase E3 gene GhRING2 that is diffe...

Identification and Characterization of Components of a Putative Petunia S-Locus F-Box–Containing E3 Ligase Complex Involved in S-RNase–Based Self-Incompatibility[W

PubMed Central

Hua, Zhihua; Kao, Teh-hui

2006-01-01

Petunia inflata S-locus F-box (Pi SLF) is thought to function as a typical F-box protein in ubiquitin-mediated protein degradation and, along with Skp1, Cullin-1, and Rbx1, could compose an SCF complex mediating the degradation of nonself S-RNase but not self S-RNase. We isolated three P. inflata Skp1s (Pi SK1, -2, and -3), two Cullin-1s (Pi CUL1-C and -G), and an Rbx1 (Pi RBX1) cDNAs and found that Pi CUL1-G did not interact with Pi RBX1 and that none of the three Pi SKs interacted with Pi SLF2. We also isolated a RING-HC protein, S-RNase Binding Protein1 (Pi SBP1), almost identical to Petunia hybrida SBP1, which interacts with Pi SLFs, S-RNases, Pi CUL1-G, and an E2 ubiquitin-conjugating enzyme, suggesting that Pi CUL1-G, SBP1, and SLF may be components of a novel E3 ligase complex, with Pi SBP1 playing the roles of Skp1 and Rbx1. S-RNases interact more with nonself Pi SLFs than with self Pi SLFs, and Pi SLFs also interact more with nonself S-RNases than with self S-RNases. Bacterially expressed S1-, S2-, and S3-RNases are degraded by the 26S proteasomal pathway in a cell-free system, albeit not in an S-allele–specific manner. Native glycosylated S3-RNase is not degraded to any significant extent; however, deglycosylated S3-RNase is degraded as efficiently as the bacterially expressed S-RNases. Finally, S-RNases are ubiquitinated in pollen tube extracts, but whether this is mediated by the Pi SLF–containing E3 complex is unknown. PMID:17028207

The plant homeodomain fingers of fission yeast Msc1 exhibit E3 ubiquitin ligase activity.

PubMed

Dul, Barbara E; Walworth, Nancy C

2007-06-22

The DNA damage checkpoint pathway governs how cells regulate cell cycle progression in response to DNA damage. A screen for suppressors of a fission yeast chk1 mutant defective in the checkpoint pathway identified a novel Schizosaccharomyces pombe protein, Msc1. Msc1 contains 3 plant homeodomain (PHD) finger motifs, characteristically defined by a C4HC3 consensus similar to RING finger domains. PHD finger domains in viral proteins and in the cellular protein kinase MEKK1 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1) have been implicated as ubiquitin E3 protein ligases that affect protein stability. The close structural relationship of PHD fingers to RING fingers suggests that other PHD domain-containing proteins might share this activity. We show that each of the three PHD fingers of Msc1 can act as ubiquitin E3 ligases, reporting for the first time that PHD fingers from a nuclear protein exhibit E3 ubiquitin ligase activity. The function of the PHD fingers of Msc1 is needed to rescue the DNA damage sensitivity of a chk1Delta strain. Msc1 co-precipitates Rhp6, the S. pombe homologue of the human ubiquitin-conjugating enzyme Ubc2. Strikingly, deletion of msc1 confers complete suppression of the slow growth phenotype, UV and hydroxyurea sensitivities of an rhp6 deletion strain and restores deficient histone H3 methylation observed in the rhp6Delta mutant. We speculate that the target of the E3 ubiquitin ligase activity of Msc1 is likely to be a chromatin-associated protein.

The APC/C Ubiquitin Ligase: From Cell Biology to Tumorigenesis

PubMed Central

Penas, Clara; Ramachandran, Vimal; Ayad, Nagi George

2011-01-01

The ubiquitin proteasome system (UPS) is required for normal cell proliferation, vertebrate development, and cancer cell transformation. The UPS consists of multiple proteins that work in concert to target a protein for degradation via the 26S proteasome. Chains of an 8.5-kDa protein called ubiquitin are attached to substrates, thus allowing recognition by the 26S proteasome. Enzymes called ubiquitin ligases or E3s mediate specific attachment to substrates. Although there are over 600 different ubiquitin ligases, the Skp1–Cullin–F-box (SCF) complexes and the anaphase promoting complex/cyclosome (APC/C) are the most studied. SCF involvement in cancer has been known for some time while APC/C’s cancer role has recently emerged. In this review we will discuss the importance of APC/C to normal cell proliferation and development, underscoring its possible contribution to transformation. We will also examine the hypothesis that modulating a specific interaction of the APC/C may be therapeutically attractive in specific cancer subtypes. Finally, given that the APC/C pathway is relatively new as a cancer target, therapeutic interventions affecting APC/C activity may be beneficial in cancers that are resistant to classical chemotherapy. PMID:22655255

iRhom2 is essential for innate immunity to RNA virus by antagonizing ER- and mitochondria-associated degradation of VISA.

PubMed

Luo, Wei-Wei; Li, Shu; Li, Chen; Zheng, Zhou-Qin; Cao, Pan; Tong, Zhen; Lian, Huan; Wang, Su-Yun; Shu, Hong-Bing; Wang, Yan-Yi

2017-11-01

VISA (also known as MAVS, IPS-1 and Cardif) is an essential adaptor protein in innate immune response to RNA virus. The protein level of VISA is delicately regulated before and after viral infection to ensure the optimal activation and timely termination of innate antiviral response. It has been reported that several E3 ubiquitin ligases can mediate the degradation of VISA, but how the stability of VISA is maintained before and after viral infection remains enigmatic. In this study, we found that the ER-associated inactive rhomboid protein 2 (iRhom2) plays an essential role in mounting an efficient innate immune response to RNA virus by maintaining the stability of VISA through distinct mechanisms. In un-infected and early infected cells, iRhom2 mediates auto-ubiquitination and degradation of the E3 ubiquitin ligase RNF5 and impairs the assembly of VISA-RNF5-GP78 complexes, thereby antagonizes ER-associated degradation (ERAD) of VISA. In the late phase of viral infection, iRhom2 mediates proteasome-dependent degradation of the E3 ubiquitin ligase MARCH5 and impairs mitochondria-associated degradation (MAD) of VISA. Maintenance of VISA stability by iRhom2 ensures efficient innate antiviral response at the early phase of viral infection and ready for next round of response. Our findings suggest that iRhom2 acts as a checkpoint for the ERAD/MAD of VISA, which ensures proper innate immune response to RNA virus.

E3 ubiquitin ligase SP1 regulates peroxisome biogenesis in Arabidopsis

DOE PAGES

Pan, Ronghui; Satkovich, John; Hu, Jianping

2016-10-31

Peroxisomes are ubiquitous eukaryotic organelles that play pivotal roles in a suite of metabolic processes and often act coordinately with other organelles, such as chloroplasts and mitochondria. Peroxisomes import proteins to the peroxisome matrix by peroxins (PEX proteins), but how the function of the PEX proteins is regulated is poorly understood. In this study, we identified the Arabidopsis RING (really interesting new gene) type E3 ubiquitin ligase SP1 [suppressor of plastid protein import locus 1 (ppi1) 1] as a peroxisome membrane protein with a regulatory role in peroxisome protein import. SP1 interacts physically with the two components of the peroxisomemore » protein docking complex PEX13–PEX14 and the (RING)-finger peroxin PEX2. Loss of SP1 function suppresses defects of the pex14-2 and pex13-1 mutants, and SP1 is involved in the degradation of PEX13 and possibly PEX14 and all three RING peroxins. An in vivo ubiquitination assay showed that SP1 has the ability to promote PEX13 ubiquitination. Our study has revealed that, in addition to its previously reported function in chloroplast biogenesis, SP1 plays a role in peroxisome biogenesis. The same E3 ubiquitin ligase promotes the destabilization of components of two distinct protein-import machineries, indicating that degradation of organelle biogenesis factors by the ubiquitin–proteasome system may constitute an important regulatory mechanism in coordinating the biogenesis of metabolically linked organelles in eukaryotes.« less

iRhom2 is essential for innate immunity to RNA virus by antagonizing ER- and mitochondria-associated degradation of VISA

PubMed Central

Luo, Wei-Wei; Li, Shu; Cao, Pan; Tong, Zhen; Lian, Huan; Wang, Su-Yun; Shu, Hong-Bing

2017-01-01

VISA (also known as MAVS, IPS-1 and Cardif) is an essential adaptor protein in innate immune response to RNA virus. The protein level of VISA is delicately regulated before and after viral infection to ensure the optimal activation and timely termination of innate antiviral response. It has been reported that several E3 ubiquitin ligases can mediate the degradation of VISA, but how the stability of VISA is maintained before and after viral infection remains enigmatic. In this study, we found that the ER-associated inactive rhomboid protein 2 (iRhom2) plays an essential role in mounting an efficient innate immune response to RNA virus by maintaining the stability of VISA through distinct mechanisms. In un-infected and early infected cells, iRhom2 mediates auto-ubiquitination and degradation of the E3 ubiquitin ligase RNF5 and impairs the assembly of VISA-RNF5-GP78 complexes, thereby antagonizes ER-associated degradation (ERAD) of VISA. In the late phase of viral infection, iRhom2 mediates proteasome-dependent degradation of the E3 ubiquitin ligase MARCH5 and impairs mitochondria-associated degradation (MAD) of VISA. Maintenance of VISA stability by iRhom2 ensures efficient innate antiviral response at the early phase of viral infection and ready for next round of response. Our findings suggest that iRhom2 acts as a checkpoint for the ERAD/MAD of VISA, which ensures proper innate immune response to RNA virus. PMID:29155878

Linear ubiquitin assembly complex negatively regulates RIG-I and TRIM25 mediated type-I interferon induction

PubMed Central

Inn, Kyung-Soo; Gack, Michaela U.; Tokunaga, Fuminori; Shi, Mude; Wong, Lai-Yee; Iwai, Kazuhiro; Jung, Jae U.

2011-01-01

Summary Upon detection of viral RNA, retinoic acid inducible gene I (RIG-I) undergoes TRIM25-mediated Lys-63 linked ubiquitination, leading to type-I interferon (IFN) production. In this study, we demonstrate that the linear ubiquitin assembly complex (LUBAC), comprised of two RING-IBR-RING (RBR)-containing E3 ligases HOIL-1L and HOIP, independently targets TRIM25 and RIG-I to effectively suppress virus-induced IFN production. RBR E3 ligase domains of HOIL-1L and HOIP bind and induce proteosomal degradation of TRIM25, whereas the NZF domain of HOIL-1L competes with TRIM25 for RIG-I binding. Consequently, both actions by the HOIL-1L/HOIP LUBAC potently inhibit RIG-I ubiquitination and anti-viral activity, but in a mechanistically separate manner. Conversely, the genetic deletion or depletion of HOIL-1L and HOIP robustly enhances virus-induced type-I IFN production. Taken together, the HOIL-1L/HOIP LUBAC specifically suppresses RIG-I ubiquitination and activation by inducing TRIM25 degradation and inhibiting TRIM25 interaction with RIG-I, resulting in the comprehensive suppression of the IFN-mediated anti-viral signaling pathway. PMID:21292167

Linear ubiquitin assembly complex negatively regulates RIG-I- and TRIM25-mediated type I interferon induction.

PubMed

Inn, Kyung-Soo; Gack, Michaela U; Tokunaga, Fuminori; Shi, Mude; Wong, Lai-Yee; Iwai, Kazuhiro; Jung, Jae U

2011-02-04

Upon detection of viral RNA, retinoic acid-inducible gene I (RIG-I) undergoes TRIM25-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that the linear ubiquitin assembly complex (LUBAC), comprised of two RING-IBR-RING (RBR)-containing E3 ligases, HOIL-1L and HOIP, independently targets TRIM25 and RIG-I to effectively suppress virus-induced IFN production. RBR E3 ligase domains of HOIL-1L and HOIP bind and induce proteasomal degradation of TRIM25, whereas the NZF domain of HOIL-1L competes with TRIM25 for RIG-I binding. Consequently, both actions by the HOIL-1L/HOIP LUBAC potently inhibit RIG-I ubiquitination and antiviral activity, but in a mechanistically separate manner. Conversely, the genetic deletion or depletion of HOIL-1L and HOIP robustly enhances virus-induced type I IFN production. Taken together, the HOIL-1L/HOIP LUBAC specifically suppresses RIG-I ubiquitination and activation by inducing TRIM25 degradation and inhibiting TRIM25 interaction with RIG-I, resulting in the comprehensive suppression of the IFN-mediated antiviral signaling pathway. Copyright © 2011 Elsevier Inc. All rights reserved.

Shigella IpaH7.8 E3 ubiquitin ligase targets glomulin and activates inflammasomes to demolish macrophages

PubMed Central

Suzuki, Shiho; Mimuro, Hitomi; Kim, Minsoo; Ogawa, Michinaga; Ashida, Hiroshi; Toyotome, Takahito; Franchi, Luigi; Suzuki, Masato; Sanada, Takahito; Suzuki, Toshihiko; Tsutsui, Hiroko; Núñez, Gabriel; Sasakawa, Chihiro

2014-01-01

When nucleotide-binding oligomerization domain–like receptors (NLRs) sense cytosolic-invading bacteria, they induce the formation of inflammasomes and initiate an innate immune response. In quiescent cells, inflammasome activity is tightly regulated to prevent excess inflammation and cell death. Many bacterial pathogens provoke inflammasome activity and induce inflammatory responses, including cell death, by delivering type III secreted effectors, the rod component flagellin, and toxins. Recent studies indicated that Shigella deploy multiple mechanisms to stimulate NLR inflammasomes through type III secretion during infection. Here, we show that Shigella induces rapid macrophage cell death by delivering the invasion plasmid antigen H7.8 (IpaH7.8) enzyme 3 (E3) ubiquitin ligase effector via the type III secretion system, thereby activating the NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing 4 (NLRC4) inflammasomes and caspase-1 and leading to macrophage cell death in an IpaH7.8 E3 ligase-dependent manner. Mice infected with Shigella possessing IpaH7.8, but not with Shigella possessing an IpaH7.8 E3 ligase-null mutant, exhibited enhanced bacterial multiplication. We defined glomulin/flagellar-associated protein 68 (GLMN) as an IpaH7.8 target involved in IpaH7.8 E3 ligase-dependent inflammasome activation. This protein originally was identified through its association with glomuvenous malformations and more recently was described as a member of a Cullin ring ligase inhibitor. Modifying GLMN levels through overexpression or knockdown led to reduced or augmented inflammasome activation, respectively. Macrophages stimulated with lipopolysaccharide/ATP induced GLMN puncta that localized with the active form of caspase-1. Macrophages from GLMN+/− mice were more responsive to inflammasome activation than those from GLMN+/+ mice. Together, these results highlight a unique bacterial adaptation that hijacks inflammasome activation via interactions between IpaH7.8 and GLMN. PMID:25246571

The adenovirus E4-ORF3 protein functions as a SUMO E3 ligase for TIF-1γ sumoylation and poly-SUMO chain elongation.

PubMed

Sohn, Sook-Young; Hearing, Patrick

2016-06-14

The adenovirus (Ad) early region 4 (E4)-ORF3 protein regulates diverse cellular processes to optimize the host environment for the establishment of Ad replication. E4-ORF3 self-assembles into multimers to form a nuclear scaffold in infected cells and creates distinct binding interfaces for different cellular target proteins. Previous studies have shown that the Ad5 E4-ORF3 protein induces sumoylation of multiple cellular proteins and subsequent proteasomal degradation of some of them, but the detailed mechanism of E4-ORF3 function remained unknown. Here, we investigate the role of E4-ORF3 in the sumoylation process by using transcription intermediary factor (TIF)-1γ as a substrate. Remarkably, we discovered that purified E4-ORF3 protein stimulates TIF-1γ sumoylation in vitro, demonstrating that E4-ORF3 acts as a small ubiquitin-like modifier (SUMO) E3 ligase. Furthermore, E4-ORF3 significantly increases poly-SUMO3 chain formation in vitro in the absence of substrate, showing that E4-ORF3 has SUMO E4 elongase activity. An E4-ORF3 mutant, which is defective in protein multimerization, exhibited severely decreased activity, demonstrating that E4-ORF3 self-assembly is required for these activities. Using a SUMO3 mutant, K11R, we found that E4-ORF3 facilitates the initial acceptor SUMO3 conjugation to TIF-1γ as well as poly-SUMO chain elongation. The E4-ORF3 protein displays no SUMO-targeted ubiquitin ligase activity in our assay system. These studies reveal the mechanism by which E4-ORF3 targets specific cellular proteins for sumoylation and proteasomal degradation and provide significant insight into how a small viral protein can play a role as a SUMO E3 ligase and E4-like SUMO elongase to impact a variety of cellular responses.

MAVS ubiquitination by the E3 ligase TRIM25 and degradation by the proteasome is involved in type I interferon production after activation of the antiviral RIG-I-like receptors

PubMed Central

2012-01-01

Background During a viral infection, the intracellular RIG-I-like receptors (RLRs) sense viral RNA and signal through the mitochondrial antiviral signaling adaptor MAVS (also known as IPS-1, Cardif and VISA) whose activation triggers a rapid production of type I interferons (IFN) and of pro-inflammatory cytokines through the transcription factors IRF3/IRF7 and NF-κB, respectively. While MAVS is essential for this signaling and known to operate through the scaffold protein NEMO and the protein kinase TBK1 that phosphorylates IRF3, its mechanism of action and regulation remain unclear. Results We report here that RLR activation triggers MAVS ubiquitination on lysine 7 and 10 by the E3 ubiquitin ligase TRIM25 and marks it for proteasomal degradation concomitantly with downstream signaling. Inhibition of this MAVS degradation with a proteasome inhibitor does not affect NF-κB signaling but it hampers IRF3 activation, and NEMO and TBK1, two essential mediators in type I IFN production, are retained at the mitochondria. Conclusions These results suggest that MAVS functions as a recruitment platform that assembles a signaling complex involving NEMO and TBK1, and that the proteasome-mediated MAVS degradation is required to release the signaling complex into the cytosol, allowing IRF3 phosphorylation by TBK1. PMID:22626058

MAVS ubiquitination by the E3 ligase TRIM25 and degradation by the proteasome is involved in type I interferon production after activation of the antiviral RIG-I-like receptors.

PubMed

Castanier, Céline; Zemirli, Naima; Portier, Alain; Garcin, Dominique; Bidère, Nicolas; Vazquez, Aimé; Arnoult, Damien

2012-05-24

During a viral infection, the intracellular RIG-I-like receptors (RLRs) sense viral RNA and signal through the mitochondrial antiviral signaling adaptor MAVS (also known as IPS-1, Cardif and VISA) whose activation triggers a rapid production of type I interferons (IFN) and of pro-inflammatory cytokines through the transcription factors IRF3/IRF7 and NF-κB, respectively. While MAVS is essential for this signaling and known to operate through the scaffold protein NEMO and the protein kinase TBK1 that phosphorylates IRF3, its mechanism of action and regulation remain unclear. We report here that RLR activation triggers MAVS ubiquitination on lysine 7 and 10 by the E3 ubiquitin ligase TRIM25 and marks it for proteasomal degradation concomitantly with downstream signaling. Inhibition of this MAVS degradation with a proteasome inhibitor does not affect NF-κB signaling but it hampers IRF3 activation, and NEMO and TBK1, two essential mediators in type I IFN production, are retained at the mitochondria. These results suggest that MAVS functions as a recruitment platform that assembles a signaling complex involving NEMO and TBK1, and that the proteasome-mediated MAVS degradation is required to release the signaling complex into the cytosol, allowing IRF3 phosphorylation by TBK1.

The FBXO7 homologue nutcracker and binding partner PI31 in Drosophila melanogaster models of Parkinson's disease.

PubMed

Merzetti, Eric M; Dolomount, Lindsay A; Staveley, Brian E

2017-01-01

Parkinsonian-pyramidal syndrome (PPS) is an early onset form of Parkinson's disease (PD) that shows degeneration of the extrapyramidal region of the brain to result in a severe form of PD. The toxic protein build-up has been implicated in the onset of PPS. Protein removal is mediated by an intracellular proteasome complex: an E3 ubiquitin ligase, the targeting component, is essential for function. FBXO7 encodes the F-box component of the SCF E3 ubiquitin ligase linked to familial forms of PPS. The Drosophila melanogaster homologue nutcracker (ntc) and a binding partner, PI31, have been shown to be active in proteasome function. We show that altered expression of either ntc or PI31 in dopaminergic neurons leads to a decrease in longevity and locomotor ability, phenotypes both associated with models of PD. Furthermore, expression of ntc-RNAi in an established α-synuclein-dependent model of PD rescues the phenotypes of diminished longevity and locomotor control.

F-box protein interactions with the hallmark pathways in cancer.

PubMed

Randle, Suzanne J; Laman, Heike

2016-02-01

F-box proteins (FBP) are the substrate specifying subunit of Skp1-Cul1-FBP (SCF)-type E3 ubiquitin ligases and are responsible for directing the ubiquitination of numerous proteins essential for cellular function. Due to their ability to regulate the expression and activity of oncogenes and tumour suppressor genes, FBPs themselves play important roles in cancer development and progression. In this review, we provide a comprehensive overview of FBPs and their targets in relation to their interaction with the hallmarks of cancer cell biology, including the regulation of proliferation, epigenetics, migration and invasion, metabolism, angiogenesis, cell death and DNA damage responses. Each cancer hallmark is revealed to have multiple FBPs which converge on common signalling hubs or response pathways. We also highlight the complex regulatory interplay between SCF-type ligases and other ubiquitin ligases. We suggest six highly interconnected FBPs affecting multiple cancer hallmarks, which may prove sensible candidates for therapeutic intervention. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Ubiquitin ligase activity of TFIIH and the transcriptional response to DNA damage.

PubMed

Takagi, Yuichiro; Masuda, Claudio A; Chang, Wei-Hau; Komori, Hirofumi; Wang, Dong; Hunter, Tony; Joazeiro, Claudio A P; Kornberg, Roger D

2005-04-15

Core transcription factor (TF) IIH purified from yeast possesses an E3 ubiquitin (Ub) ligase activity, which resides, at least in part, in a RING finger (RNF) domain of the Ssl1 subunit. Yeast strains mutated in the Ssl1 RNF domain are sensitive to ultraviolet (UV) light and to methyl methanesulfonate (MMS). This increased sensitivity to DNA-damaging agents does not reflect a deficiency in nucleotide excision repair. Rather, it correlates with reduced transcriptional induction of genes involved in DNA repair, suggesting that the E3 Ub ligase activity of TFIIH mediates the transcriptional response to DNA damage.

Mechanism of TRIM25 Catalytic Activation in the Antiviral RIG-I Pathway.

PubMed

Sanchez, Jacint G; Chiang, Jessica J; Sparrer, Konstantin M J; Alam, Steven L; Chi, Michael; Roganowicz, Marcin D; Sankaran, Banumathi; Gack, Michaela U; Pornillos, Owen

2016-08-02

Antiviral response pathways induce interferon by higher-order assembly of signaling complexes called signalosomes. Assembly of the RIG-I signalosome is regulated by K63-linked polyubiquitin chains, which are synthesized by the E3 ubiquitin ligase, TRIM25. We have previously shown that the TRIM25 coiled-coil domain is a stable, antiparallel dimer that positions two catalytic RING domains on opposite ends of an elongated rod. We now show that the RING domain is a separate self-association motif that engages ubiquitin-conjugated E2 enzymes as a dimer. RING dimerization is required for catalysis, TRIM25-mediated RIG-I ubiquitination, interferon induction, and antiviral activity. We also provide evidence that RING dimerization and E3 ligase activity are promoted by binding of the TRIM25 SPRY domain to the RIG-I effector domain. These results indicate that TRIM25 actively participates in higher-order assembly of the RIG-I signalosome and helps to fine-tune the efficiency of the RIG-I-mediated antiviral response. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Mechanism of TRIM25 Catalytic Activation in the Antiviral RIG-I Pathway

DOE PAGES

Sanchez, Jacint G.; Chiang, Jessica J.; Sparrer, Konstantin M. J.; ...

2016-07-14

Antiviral response pathways induce interferon by higher-order assembly of signaling complexes called signalosomes. Assembly of the RIG-I signalosome is regulated by K63-linked polyubiquitin chains, which are synthesized by the E3 ubiquitin ligase, TRIM25. We have previously shown that the TRIM25 coiled-coil domain is a stable, antiparallel dimer that positions two catalytic RING domains on opposite ends of an elongated rod. We now show that the RING domain is a separate self-association motif that engages ubiquitin-conjugated E2 enzymes as a dimer. RING dimerization is required for catalysis, TRIM25-mediated RIG-I ubiquitination, interferon induction, and antiviral activity. We also provide evidence that RINGmore » dimerization and E3 ligase activity are promoted by binding of the TRIM25 SPRY domain to the RIG-I effector domain. These results indicate that TRIM25 actively participates in higher-order assembly of the RIG-I signalosome and helps to fine-tune the efficiency of the RIG-I-mediated antiviral response.« less

Structure of an APC3–APC16 Complex: Insights into Assembly of the Anaphase-Promoting Complex/Cyclosome

DOE PAGES

Yamaguchi, Masaya; Yu, Shanshan; Qiao, Renping; ...

2014-12-06

The anaphase-promoting complex/cyclosome (APC/C) is a massive E3 ligase that controls mitosis by catalyzing ubiquitination of key cell cycle regulatory proteins. The APC/C assembly contains two subcomplexes: the “Platform” centers around a cullin-RING-like E3 ligase catalytic core; the “Arc Lamp” is a hub that mediates transient association with regulators and ubiquitination substrates. The Arc Lamp contains the small subunits APC16, CDC26, and APC13, and tetratricopeptide repeat (TPR) proteins (APC7, APC3, APC6, and APC8) that homodimerize and stack with quasi-2-fold symmetry. Within the APC/C complex, APC3 serves as center for regulation. APC3's TPR motifs recruit substrate-binding coactivators, CDC20 and CDH1, viamore » their C-terminal conserved Ile-Arg (IR) tail sequences. Human APC3 also binds APC16 and APC7 and contains a > 200-residue loop that is heavily phosphorylated during mitosis, although the basis for APC3 interactions and whether loop phosphorylation is required for ubiquitination are unclear. Here, we map the basis for human APC3 assembly with APC16 and APC7, report crystal structures of APC3Δloop alone and in complex with the C-terminal domain of APC16, and test roles of APC3's loop and IR tail binding surfaces in APC/C-catalyzed ubiquitination. The structures show how one APC16 binds asymmetrically to the symmetric APC3 dimer and, together with biochemistry and prior data, explain how APC16 recruits APC7 to APC3, show how APC3's C-terminal domain is rearranged in the full APC/C assembly, and visualize residues in the IR tail binding cleft important for coactivator-dependent ubiquitination. Overall, the results provide insights into assembly, regulation, and interactions of TPR proteins and the APC/C.« less

Slip slidin’ away of mitosis with CRL2Zyg11

PubMed Central

2016-01-01

The spindle assembly checkpoint arrests mitotic cells by preventing degradation of cyclin B1 by the anaphase-promoting complex/cyclosome, but some cells evade this checkpoint and slip out of mitosis. Balachandran et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201601083) show that the E3 ligase CRL2ZYG11 degrades cyclin B1, allowing mitotic slippage. PMID:27810907

Characterization and Promoter Analysis of a Cotton Ring-Type Ubiquitin Ligase (E3) Gene

USDA-ARS?s Scientific Manuscript database

A cotton fiber cDNA, GhRING1, and its corresponding gene have been cloned and characterized. The GhRING1 gene encodes a RING-type ubiquitin ligase (E3) containing 337 amino acids (aa). The GhRING1 protein contains a RING finger motif with conserved cysteine and histine residues at the C-terminus a...

DNA-binding regulates site-specific ubiquitination of IRF-1.

PubMed

Landré, Vivien; Pion, Emmanuelle; Narayan, Vikram; Xirodimas, Dimitris P; Ball, Kathryn L

2013-02-01

Understanding the determinants for site-specific ubiquitination by E3 ligase components of the ubiquitin machinery is proving to be a challenge. In the present study we investigate the role of an E3 ligase docking site (Mf2 domain) in an intrinsically disordered domain of IRF-1 [IFN (interferon) regulatory factor-1], a short-lived IFNγ-regulated transcription factor, in ubiquitination of the protein. Ubiquitin modification of full-length IRF-1 by E3 ligases such as CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and MDM2 (murine double minute 2), which dock to the Mf2 domain, was specific for lysine residues found predominantly in loop structures that extend from the DNA-binding domain, whereas no modification was detected in the more conformationally flexible C-terminal half of the protein. The E3 docking site was not available when IRF-1 was in its DNA-bound conformation and cognate DNA-binding sequences strongly suppressed ubiquitination, highlighting a strict relationship between ligase binding and site-specific modification at residues in the DNA-binding domain. Hyperubiquitination of a non-DNA-binding mutant supports a mechanism where an active DNA-bound pool of IRF-1 is protected from polyubiquitination and degradation.

Ubiquitinylation of α-Synuclein by Carboxyl Terminus Hsp70-Interacting Protein (CHIP) Is Regulated by Bcl-2-Associated Athanogene 5 (BAG5)

PubMed Central

Chau, Hien; Lozano, Andres M.; Hyman, Bradley T.; McLean, Pamela J.

2011-01-01

Parkinson's disease (PD) is a common neurodegenerative condition in which abnormalities in protein homeostasis, or proteostasis, may lead to accumulation of the protein α-synuclein (α-syn). Mutations within or multiplications of the gene encoding α-syn are known to cause genetic forms of PD and polymorphisms in the gene are recently established risk factors for idiopathic PD. α-syn is a major component of Lewy bodies, the intracellular proteinaceous inclusions which are pathological hallmarks of most forms of PD. Recent evidence demonstrates that α-syn can self associate into soluble oligomeric species and implicates these α-syn oligomers in cell death. We have previously shown that carboxyl terminus of Hsp70-interacting protein (CHIP), a co-chaperone molecule with E3 ubiquitin ligase activity, may reduce the levels of toxic α-syn oligomers. Here we demonstrate that α-syn is ubiquitinylated by CHIP both in vitro and in cells. We find that the products from ubiquitinylation by CHIP include both monoubiquitinylated and polyubiquitinylated forms of α-syn. We also demonstrate that CHIP and α-syn exist within a protein complex with the co-chaperone bcl-2-associated athanogene 5 (BAG5) in brain. The interaction of CHIP with BAG5 is mediated by Hsp70 which binds to the tetratricopeptide repeat domain of CHIP and the BAG domains of BAG5. The Hsp70-mediated association of BAG5 with CHIP results in inhibition of CHIP E3 ubiquitin ligase activity and subsequently reduces α-syn ubiquitinylation. Furthermore, we use a luciferase-based protein-fragment complementation assay of α-syn oligomerization to investigate regulation of α-syn oligomers by CHIP in living cells. We demonstrate that BAG5 mitigates the ability of CHIP to reduce α-syn oligomerization and that non-ubiquitinylated α-syn has an increased propensity for oligomerization. Thus, our results identify CHIP as an E3 ubiquitin ligase of α-syn and suggest a novel function for BAG5 as a modulator of CHIP E3 ubiquitin ligase activity with implications for CHIP-mediated regulation of α-syn oligomerization. PMID:21358815

Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms.

PubMed

Dove, Katja K; Stieglitz, Benjamin; Duncan, Emily D; Rittinger, Katrin; Klevit, Rachel E

2016-08-01

RING-in-between-RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub-conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT-type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING-type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub-binding site on HHARI RING2 important for its recruitment to RING1-bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs. © 2016 The Authors.

Liver Cytochrome P450 3A Ubiquitination in Vivo by gp78/Autocrine Motility Factor Receptor and C Terminus of Hsp70-interacting Protein (CHIP) E3 Ubiquitin Ligases

PubMed Central

Kim, Sung-Mi; Acharya, Poulomi; Engel, Juan C.; Correia, Maria Almira

2010-01-01

CYP3A4 is a dominant human liver cytochrome P450 enzyme engaged in the metabolism and disposition of >50% of clinically relevant drugs and held responsible for many adverse drug-drug interactions. CYP3A4 and its mammalian liver CYP3A orthologs are endoplasmic reticulum (ER)-anchored monotopic proteins that undergo ubiquitin (Ub)-dependent proteasomal degradation (UPD) in an ER-associated degradation (ERAD) process. These integral ER proteins are ubiquitinated in vivo, and in vitro studies have identified the ER-integral gp78 and the cytosolic co-chaperone, CHIP (C terminus of Hsp70-interacting protein), as the relevant E3 Ub-ligases, along with their cognate E2 Ub-conjugating enzymes UBC7 and UbcH5a, respectively. Using lentiviral shRNA templates targeted against each of these Ub-ligases, we now document that both E3s are indeed physiologically involved in CYP3A ERAD/UPD in cultured rat hepatocytes. Accordingly, specific RNAi resulted in ≈80% knockdown of each hepatic Ub-ligase, with a corresponding ≈2.5-fold CYP3A stabilization. Surprisingly, however, such stabilization resulted in increased levels of functionally active CYP3A, thereby challenging the previous notion that E3 recognition and subsequent ERAD of CYP3A proteins required ab initio their structural and/or functional inactivation. Furthermore, coexpression in HepG2 cells of both CYP3A4 and gp78, but not its functionally inactive RING-finger mutant, resulted in enhanced CYP3A4 loss greater than that in corresponding cells expressing only CYP3A4. Stabilization of a functionally active CYP3A after RNAi knockdown of either of the E3s, coupled with the increased CYP3A4 loss on gp78 or CHIP coexpression, suggests that ERAD-associated E3 Ub-ligases can influence clinically relevant drug metabolism by effectively regulating the physiological CYP3A content and consequently its function. PMID:20819951

Mindbomb 1, an E3 ubiquitin ligase, forms a complex with RYK to activate Wnt/β-catenin signaling

PubMed Central

Berndt, Jason D.; Aoyagi, Atsushi; Yang, Peitzu; Anastas, Jamie N.; Tang, Lan

2011-01-01

Receptor-like tyrosine kinase (RYK) functions as a transmembrane receptor for the Wnt family of secreted protein ligands. Although RYK undergoes endocytosis in response to Wnt, the mechanisms that regulate its internalization and concomitant activation of Wnt signaling are unknown. We discovered that RYK both physically and functionally interacts with the E3 ubiquitin ligase Mindbomb 1 (MIB1). Overexpression of MIB1 promotes the ubiquitination of RYK and reduces its steady-state levels at the plasma membrane. Moreover, we show that MIB1 is sufficient to activate Wnt/β-catenin (CTNNB1) signaling and that this activity depends on endogenous RYK. Conversely, in loss-of-function studies, both RYK and MIB1 are required for Wnt-3A–mediated activation of CTNNB1. Finally, we identify the Caenorhabditis elegans orthologue of MIB1 and demonstrate a genetic interaction between ceMIB and lin-18/RYK in vulva development. These findings provide insights into the mechanisms of Wnt/RYK signaling and point to novel targets for the modulation of Wnt signaling. PMID:21875946

Dlg3 Trafficking and Apical Tight Junction Formation Is Regulated by Nedd4 and Nedd4-2 E3 Ubiquitin Ligases

PubMed Central

Van Campenhout, Claude A.; Eitelhuber, Andrea; Gloeckner, Christian J.; Giallonardo, Patrizia; Gegg, Moritz; Oller, Heide; Grant, Seth G.N.; Krappmann, Daniel; Ueffing, Marius; Lickert, Heiko

2011-01-01

Summary The Drosophila Discs large (Dlg) scaffolding protein acts as a tumor suppressor regulating basolateral epithelial polarity and proliferation. In mammals, four Dlg homologs have been identified; however, their functions in cell polarity remain poorly understood. Here, we demonstrate that the X-linked mental retardation gene product Dlg3 contributes to apical-basal polarity and epithelial junction formation in mouse organizer tissues, as well as to planar cell polarity in the inner ear. We purified complexes associated with Dlg3 in polarized epithelial cells, including proteins regulating directed trafficking and tight junction formation. Remarkably, of the four Dlg family members, Dlg3 exerts a distinct function by recruiting the ubiquitin ligases Nedd4 and Nedd4-2 through its PPxY motifs. We found that these interactions are required for Dlg3 monoubiquitination, apical membrane recruitment, and tight junction consolidation. Our findings reveal an unexpected evolutionary diversification of the vertebrate Dlg family in basolateral epithelium formation. PMID:21920314

The E3 ubiquitin ligase CHIP selectively regulates mutant epidermal growth factor receptor by ubiquitination and degradation.

PubMed

Chung, Chaeuk; Yoo, Geon; Kim, Tackhoon; Lee, Dahye; Lee, Choong-Sik; Cha, Hye Rim; Park, Yeon Hee; Moon, Jae Young; Jung, Sung Soo; Kim, Ju Ock; Lee, Jae Cheol; Kim, Sun Young; Park, Hee Sun; Park, Myoungrin; Park, Dong Il; Lim, Dae-Sik; Jang, Kang Won; Lee, Jeong Eun

2016-10-14

Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) is a decisive factor for the therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The stability of mutant EGFR is maintained by various regulators, including heat shock protein 90 (Hsp90). The C terminus of Hsc70-interacting protein (CHIP) is a Hsp70/Hsp90 co-chaperone and exhibits E3 ubiquitin ligase activity. The high-affinity Hsp90-CHIP complex recognizes and selectively regulates their client proteins. CHIP also works with its own E3 ligase activity independently of Hsp70/Hsp90. Here, we investigated the role of CHIP in regulating EGFR in lung adenocarcinoma and also evaluated the specificity of CHIP's effects on mutant EGFR. In HEK 293T cells transfected with either WT EGFR or EGFR mutants, the overexpression of CHIP selectively decreased the expression of certain EGFR mutants (G719S, L747_E749del A750P and L858R) but not WT EGFR. In a pull-down assay, CHIP selectively interacted with EGFR mutants and simultaneously induced their ubiquitination and proteasomal degradation. The expressions of mutant EGFR in PC9 and H1975 were diminished by CHIP, while the expression of WT EGFR in A549 was nearly not affected. In addition, CHIP overexpression inhibited cell proliferation and xenograft's tumor growth of EGFR mutant cell lines, but not WT EGFR cell lines. EGFR mutant specific ubiquitination by CHIP may provide a crucial regulating mechanism for EGFR in lung adenocarcinoma. Our results suggest that CHIP can be novel therapeutic target for overcoming the EGFR TKI resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

Tripartite motif ligases catalyze polyubiquitin chain formation through a cooperative allosteric mechanism.

PubMed

Streich, Frederick C; Ronchi, Virginia P; Connick, J Patrick; Haas, Arthur L

2013-03-22

Ligation of polyubiquitin chains to proteins is a fundamental post-translational modification, often resulting in targeted degradation of conjugated proteins. Attachment of polyubiquitin chains requires the activities of an E1 activating enzyme, an E2 carrier protein, and an E3 ligase. The mechanism by which polyubiquitin chains are formed remains largely speculative, especially for RING-based ligases. The tripartite motif (TRIM) superfamily of ligases functions in many cellular processes including innate immunity, cellular localization, development and differentiation, signaling, and cancer progression. The present results show that TRIM ligases catalyze polyubiquitin chain formation in the absence of substrate, the rates of which can be used as a functional readout of enzyme function. Initial rate studies under biochemically defined conditions show that TRIM32 and TRIM25 are specific for the Ubc5 family of E2-conjugating proteins and, along with TRIM5α, exhibit cooperative kinetics with respect to Ubc5 concentration, with submicromolar [S]0.5 and Hill coefficients of 3-5, suggesting they possess multiple binding sites for their cognate E2-ubiquitin thioester. Mutation studies reveal a second, non-canonical binding site encompassing the C-terminal Ubc5α-helix. Polyubiquitin chain formation requires TRIM subunit oligomerization through the conserved coiled-coil domain, but can be partially replaced by fusing the catalytic domain to GST to promote dimerization. Other results suggest that TRIM32 assembles polyubiquitin chains as a Ubc5-linked thioester intermediate. These results represent the first detailed mechanistic study of TRIM ligase activity and provide a functional context for oligomerization observed in the superfamily.

Deinococcus radiodurans RNA ligase exemplifies a novel ligase clade with a distinctive N-terminal module that is important for 5'-PO4 nick sealing and ligase adenylylation but dispensable for phosphodiester formation at an adenylylated nick.

PubMed

Raymond, Amy; Shuman, Stewart

2007-01-01

Deinococcus radiodurans RNA ligase (DraRnl) is a template-directed ligase that seals nicked duplexes in which the 3'-OH strand is RNA. DraRnl is a 342 amino acid polypeptide composed of a C-terminal adenylyltransferase domain fused to a distinctive 126 amino acid N-terminal module (a putative OB-fold). An alanine scan of the C domain identified 9 amino acids essential for nick ligation, which are located within nucleotidyltransferase motifs I, Ia, III, IIIa, IV and V. Seven mutants were dysfunctional by virtue of defects in ligase adenylylation: T163A, H167A, G168A, K186A, E230A, F281A and E305A. Four of these were also defective in phosphodiester formation at a preadenylylated nick: G168A, E230A, F281A and E305A. Two nick sealing-defective mutants were active in ligase adenylylation and sealing a preadenylylated nick, thereby implicating Ser185 and Lys326 in transfer of AMP from the enzyme to the nick 5'-PO(4). Whereas deletion of the N-terminal domain suppressed overall nick ligation and ligase adenylylation, it did not compromise sealing at a preadenylylated nick. Mutational analysis of 15 residues of the N domain identified Lys26, Gln31 and Arg79 as key constituents. Structure-activity relationships at the essential residues were determined via conservative substitutions. We propose that DraRnl typifies a new clade of polynucleotide ligases. DraRnl homologs are detected in several eukaryal proteomes.

Deinococcus radiodurans RNA ligase exemplifies a novel ligase clade with a distinctive N-terminal module that is important for 5′-PO4 nick sealing and ligase adenylylation but dispensable for phosphodiester formation at an adenylylated nick

PubMed Central

Raymond, Amy; Shuman, Stewart

2007-01-01

Deinococcus radiodurans RNA ligase (DraRnl) is a template-directed ligase that seals nicked duplexes in which the 3′-OH strand is RNA. DraRnl is a 342 amino acid polypeptide composed of a C-terminal adenylyltransferase domain fused to a distinctive 126 amino acid N-terminal module (a putative OB-fold). An alanine scan of the C domain identified 9 amino acids essential for nick ligation, which are located within nucleotidyltransferase motifs I, Ia, III, IIIa, IV and V. Seven mutants were dysfunctional by virtue of defects in ligase adenylylation: T163A, H167A, G168A, K186A, E230A, F281A and E305A. Four of these were also defective in phosphodiester formation at a preadenylylated nick: G168A, E230A, F281A and E305A. Two nick sealing-defective mutants were active in ligase adenylylation and sealing a preadenylylated nick, thereby implicating Ser185 and Lys326 in transfer of AMP from the enzyme to the nick 5′-PO4. Whereas deletion of the N-terminal domain suppressed overall nick ligation and ligase adenylylation, it did not compromise sealing at a preadenylylated nick. Mutational analysis of 15 residues of the N domain identified Lys26, Gln31 and Arg79 as key constituents. Structure–activity relationships at the essential residues were determined via conservative substitutions. We propose that DraRnl typifies a new clade of polynucleotide ligases. DraRnl homologs are detected in several eukaryal proteomes. PMID:17204483

RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment

DOE PAGES

Hodge, Curtis D.; Ismail, Ismail H.; Edwards, Ross A.; ...

2016-02-22

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. In this paper, we defined the activated RNF8-Ubc13~ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13more » active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. Finally, these findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.« less

Decoding the Ubiquitin-Mediated Pathway of Arthropod Disease Vectors

PubMed Central

Choy, Anthony; Severo, Maiara S.; Sun, Ruobai; Girke, Thomas; Gillespie, Joseph J.; Pedra, Joao H. F.

2013-01-01

Protein regulation by ubiquitin has been extensively described in model organisms. However, characterization of the ubiquitin machinery in disease vectors remains mostly unknown. This fundamental gap in knowledge presents a concern because new therapeutics are needed to control vector-borne diseases, and targeting the ubiquitin machinery as a means for disease intervention has been already adopted in the clinic. In this study, we employed a bioinformatics approach to uncover the ubiquitin-mediated pathway in the genomes of Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus, Ixodes scapularis, Pediculus humanus and Rhodnius prolixus. We observed that (1) disease vectors encode a lower percentage of ubiquitin-related genes when compared to Drosophila melanogaster, Mus musculus and Homo sapiens but not Saccharomyces cerevisiae; (2) overall, there are more proteins categorized as E3 ubiquitin ligases when compared to E2-conjugating or E1-activating enzymes; (3) the ubiquitin machinery within the three mosquito genomes is highly similar; (4) ubiquitin genes are more than doubled in the Chagas disease vector (R. prolixus) when compared to other arthropod vectors; (5) the deer tick I. scapularis and the body louse (P. humanus) genomes carry low numbers of E1-activating enzymes and HECT-type E3 ubiquitin ligases; (6) R. prolixus have low numbers of RING-type E3 ubiquitin ligases; and (7) C. quinquefasciatus present elevated numbers of predicted F-box E3 ubiquitin ligases, JAB and UCH deubiquitinases. Taken together, these findings provide novel opportunities to study the interaction between a pathogen and an arthropod vector. PMID:24205097

Characterisation of the Cullin-3 mutation that causes a severe form of familial hypertension and hyperkalaemia

PubMed Central

Schumacher, Frances-Rose; Siew, Keith; Zhang, Jinwei; Johnson, Clare; Wood, Nicola; Cleary, Sarah E; Al Maskari, Raya S; Ferryman, James T; Hardege, Iris; Figg, Nichola L; Enchev, Radoslav; Knebel, Axel; O’Shaughnessy, Kevin M; Kurz, Thimo

2015-01-01

Deletion of exon 9 from Cullin-3 (CUL3, residues 403–459: CUL3Δ403–459) causes pseudohypoaldosteronism type IIE (PHA2E), a severe form of familial hyperkalaemia and hypertension (FHHt). CUL3 binds the RING protein RBX1 and various substrate adaptors to form Cullin-RING-ubiquitin-ligase complexes. Bound to KLHL3, CUL3-RBX1 ubiquitylates WNK kinases, promoting their ubiquitin-mediated proteasomal degradation. Since WNK kinases activate Na/Cl co-transporters to promote salt retention, CUL3 regulates blood pressure. Mutations in both KLHL3 and WNK kinases cause PHA2 by disrupting Cullin-RING-ligase formation. We report here that the PHA2E mutant, CUL3Δ403–459, is severely compromised in its ability to ubiquitylate WNKs, possibly due to altered structural flexibility. Instead, CUL3Δ403–459 auto-ubiquitylates and loses interaction with two important Cullin regulators: the COP9-signalosome and CAND1. A novel knock-in mouse model of CUL3WT/Δ403–459 closely recapitulates the human PHA2E phenotype. These mice also show changes in the arterial pulse waveform, suggesting a vascular contribution to their hypertension not reported in previous FHHt models. These findings may explain the severity of the FHHt phenotype caused by CUL3 mutations compared to those reported in KLHL3 or WNK kinases. PMID:26286618

Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins.

PubMed

Saritas-Yildirim, Banu; Pliner, Hannah A; Ochoa, Angelica; Silva, Elena M

2015-01-01

Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.

Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications.

PubMed

Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

2014-01-01

Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling.

Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications

PubMed Central

Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

2014-01-01

Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling. PMID:25628960

E3 ubiquitin ligase gene CMPG1-V from Haynaldia villosa L. contributes to powdery mildew resistance in common wheat (Triticum aestivum L.).

PubMed

Zhu, Yanfei; Li, Yingbo; Fei, Fei; Wang, Zongkuan; Wang, Wei; Cao, Aizhong; Liu, Yuan; Han, Shuang; Xing, Liping; Wang, Haiyan; Chen, Wei; Tang, Sanyuan; Huang, Xiahe; Shen, Qianhua; Xie, Qi; Wang, Xiue

2015-10-01

Powdery mildew is one of the most devastating wheat fungal diseases. A diploid wheat relative, Haynaldia villosa L., is highly resistant to powdery mildew, and its genetic resource of resistances, such as the Pm21 locus, is now widely used in wheat breeding. Here we report the cloning of a resistance gene from H. villosa, designated CMPG1-V, that encodes a U-box E3 ubiquitin ligase. Expression of the CMPG1-V gene was induced in the leaf and stem of H. villosa upon inoculation with Blumeria graminis f. sp. tritici (Bgt) fungus, and the presence of Pm21 is essential for its rapid induction of expression. CMPG1-V has conserved key residues for E3 ligase, and possesses E3 ligase activity in vitro and in vivo. CMPG1-V is localized in the nucleus, endoplasmic reticulum, plasma membrane and partially in trans-Golgi network/early endosome vesicles. Transgenic wheat over-expressing CMPG1-V showed improved broad-spectrum powdery mildew resistance at seedling and adult stages, associated with an increase in expression of salicylic acid-responsive genes, H2 O2 accumulation, and cell-wall protein cross-linking at the Bgt infection sites, and the expression of CMPG1-V in H. villosa was increased when treated with salicylic acid, abscisic acid and H2 O2 . These results indicate the involvement of E3 ligase in defense responses to Bgt fungus in wheat, particularly in broad-spectrum disease resistance, and suggest association of reactive oxidative species and the phytohormone pathway with CMPG1-V-mediated powdery mildew resistance. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Ralstonia solanacearum novel E3 ubiquitin ligase (NEL) effectors RipAW and RipAR suppress pattern-triggered immunity in plants.

PubMed

Nakano, Masahito; Oda, Kenji; Mukaihara, Takafumi

2017-07-01

Ralstonia solanacearum is the causal agent of bacterial wilt in solanaceous crops. This pathogen injects more than 70 effector proteins into host plant cells via the Hrp type III secretion system to cause a successful infection. However, the function of these effectors in plant cells, especially in the suppression of plant immunity, remains largely unknown. In this study, we characterized two Ralstonia solanacearum effectors, RipAW and RipAR, which share homology with the IpaH family of effectors from animal and plant pathogenic bacteria, that have a novel E3 ubiquitin ligase (NEL) domain. Recombinant RipAW and RipAR show E3 ubiquitin ligase activity in vitro. RipAW and RipAR localized to the cytoplasm of plant cells and significantly suppressed pattern-triggered immunity (PTI) responses such as the production of reactive oxygen species and the expression of defence-related genes when expressed in leaves of Nicotiana benthamiana. Mutation in the conserved cysteine residue in the NEL domain of RipAW completely abolished the E3 ubiquitin ligase activity in vitro and the ability to suppress PTI responses in plant leaves. These results indicate that RipAW suppresses plant PTI responses through the E3 ubiquitin ligase activity. Unlike other members of the IpaH family of effectors, RipAW and RipAR had no leucine-rich repeat motifs in their amino acid sequences. A conserved C-terminal region of RipAW is indispensable for PTI suppression. Transgenic Arabidopsis plants expressing RipAW and RipAR showed increased disease susceptibility, suggesting that RipAW and RipAR contribute to bacterial virulence in plants.

Targeting endogenous proteins for degradation through the affinity-directed protein missile system.

PubMed

Fulcher, Luke J; Hutchinson, Luke D; Macartney, Thomas J; Turnbull, Craig; Sapkota, Gopal P

2017-05-01

Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel-Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins. © 2017 The Authors.

Targeting endogenous proteins for degradation through the affinity-directed protein missile system

PubMed Central

Fulcher, Luke J.; Hutchinson, Luke D.; Macartney, Thomas J.; Turnbull, Craig

2017-01-01

Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel–Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins. PMID:28490657

Identification of a primary target of thalidomide teratogenicity.

PubMed

Ito, Takumi; Ando, Hideki; Suzuki, Takayuki; Ogura, Toshihiko; Hotta, Kentaro; Imamura, Yoshimasa; Yamaguchi, Yuki; Handa, Hiroshi

2010-03-12

Half a century ago, thalidomide was widely prescribed to pregnant women as a sedative but was found to be teratogenic, causing multiple birth defects. Today, thalidomide is still used in the treatment of leprosy and multiple myeloma, although how it causes limb malformation and other developmental defects is unknown. Here, we identified cereblon (CRBN) as a thalidomide-binding protein. CRBN forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1) and Cul4A that is important for limb outgrowth and expression of the fibroblast growth factor Fgf8 in zebrafish and chicks. Thalidomide initiates its teratogenic effects by binding to CRBN and inhibiting the associated ubiquitin ligase activity. This study reveals a basis for thalidomide teratogenicity and may contribute to the development of new thalidomide derivatives without teratogenic activity.

Ubiquitin enzymes in the regulation of immune responses.

PubMed

Ebner, Petra; Versteeg, Gijs A; Ikeda, Fumiyo

2017-08-01

Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses.

Targeting Non-proteolytic Protein Ubiquitination for the Treatment of Diffuse Large B Cell Lymphoma.

PubMed

Yang, Yibin; Kelly, Priscilla; Shaffer, Arthur L; Schmitz, Roland; Yoo, Hee Min; Liu, Xinyue; Huang, Da Wei; Webster, Daniel; Young, Ryan M; Nakagawa, Masao; Ceribelli, Michele; Wright, George W; Yang, Yandan; Zhao, Hong; Yu, Xin; Xu, Weihong; Chan, Wing C; Jaffe, Elaine S; Gascoyne, Randy D; Campo, Elias; Rosenwald, Andreas; Ott, German; Delabie, Jan; Rimsza, Lisa; Staudt, Louis M

2016-04-11

Chronic active B cell receptor (BCR) signaling, a hallmark of the activated B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), engages the CARD11-MALT1-BCL10 (CBM) adapter complex to activate IκB kinase (IKK) and the classical NF-κB pathway. Here we show that the CBM complex includes the E3 ubiquitin ligases cIAP1 and cIAP2, which are essential mediators of BCR-dependent NF-κB activity in ABC DLBCL. cIAP1/2 attach K63-linked polyubiquitin chains on themselves and on BCL10, resulting in the recruitment of IKK and the linear ubiquitin chain ligase LUBAC, which is essential for IKK activation. SMAC mimetics target cIAP1/2 for destruction, and consequently suppress NF-κB and selectively kill BCR-dependent ABC DLBCL lines, supporting their clinical evaluation in patients with ABC DLBCL. Copyright © 2016 Elsevier Inc. All rights reserved.

Ras-sensitive IMP modulation of the Raf/MEK/ERK cascade through KSR1.

PubMed

Matheny, Sharon A; White, Michael A

2006-01-01

The E3 ubiquitin ligase IMP (impedes mitogenic signal propagation) was isolated as a novel Ras effector that negatively regulates ERK1/2 activation. Current evidence suggests that IMP limits the functional assembly of Raf/MEK complexes by inactivation of the KSR1 adaptor/scaffold protein. Interaction with Ras-GTP stimulates IMP autoubiquitination to relieve limitations on KSR function. The elevated sensitivity of IMP-depleted cells to ERK1/2 pathway activation suggests IMP acts as a signal threshold regulator by imposing reversible restrictions on the assembly of functional Raf/MEK/ERK kinase modules. These observations challenge commonly held concepts of signal transmission by Ras to the MAPK pathway and provide evidence for the role of amplitude modulation in tuning cellular responses to ERK1/2 pathway engagement. Here we describe details of the methods, including RNA interference, ubiquitin ligase assays, and protein complex analysis, that can be used to display the Ras-sensitive contribution of IMP to KSR-dependent modulation of the Raf/MEK/ERK pathway.

The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy.

PubMed

Sun, Aiqin; Wei, Jing; Childress, Chandra; Shaw, John H; Peng, Ke; Shao, Genbao; Yang, Wannian; Lin, Qiong

2017-03-04

The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy.

Overexpression of GhSARP1 encoding a E3 ligase from cotton reduce the tolerance to salt in transgenic Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Yongchang; Zhang, Xinyu; Zhu, Shouhong

Ubiquitination plays a very important role in the response to abiotic stresses of plant. To identify key regulators of salt stress, a gene GhSARP1(Salt-Associated Ring finger Protein)encoding C3H2C3-type E3 ligase, was cloned from cotton. Transcription level of GhSARP1 was high in leaf, flower and fiber of 24,27 and 27DPA (Days Post-Anthesis), but low in root and stem. Except PEG6000 treatment, the expression of GhSARP1 was down-regulated by NaCl, cold and ABA after being treated for 1 h. GhSARP1-GFP fusion protein located on the plasma membrane, which was dependent on trans-membrane motif. In vitro ubiquitination assay showed that GhSARP1 had E3 ligase activity.more » Heterogeneous overexpression of GhSARP1reduced salt tolerance of transgenic Arabidopsis in germination and post-germination stage. Our results suggested that the GhSARP1 might negatively regulate the response to salt stress mediated by the ubiquitination in cotton. - Highlights: • GhSARP1 expression was regulated by various abiotic stresses. • GhSARP1 have E3 ligase activity in vitro and locate on the plasma membrane. • Overexpression of GhSARP1 in Arabidopsis reduced the salt tolerance.« less

Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP*

PubMed Central

Narayan, Vikram; Pion, Emmanuelle; Landré, Vivien; Müller, Petr; Ball, Kathryn L.

2011-01-01

Characteristically for a regulatory protein, the IRF-1 tumor suppressor turns over rapidly with a half-life of between 20–40 min. This allows IRF-1 to reach new steady state protein levels swiftly in response to changing environmental conditions. Whereas CHIP (C terminus of Hsc70-interacting protein), appears to chaperone IRF-1 in unstressed cells, formation of a stable IRF-1·CHIP complex is seen under specific stress conditions. Complex formation, in heat- or heavy metal-treated cells, is accompanied by a decrease in IRF-1 steady state levels and an increase in IRF-1 ubiquitination. CHIP binds directly to an intrinsically disordered domain in the central region of IRF-1 (residues 106–140), and this site is sufficient to form a stable complex with CHIP in cells and to compete in trans with full-length IRF-1, leading to a reduction in its ubiquitination. The study reveals a complex relationship between CHIP and IRF-1 and highlights the role that direct binding or “docking” of CHIP to its substrate(s) can play in its mechanism of action as an E3 ligase. PMID:20947504

DDB1 Stimulates Viral Transcription of Hepatitis B Virus via HBx-Independent Mechanisms.

PubMed

Kim, Woohyun; Lee, Sooyoung; Son, Yeongnam; Ko, Chunkyu; Ryu, Wang-Shick

2016-11-01

HBx, a small regulatory protein of hepatitis B virus (HBV), augments viral DNA replication by stimulating viral transcription. Among numerous reported HBx-binding proteins, DDB1 has drawn attention, because DDB1 acts as a substrate receptor of the Cul4-DDB1 ubiquitin E3 ligase. Previous work reported that the DDB1-HBx interaction is indispensable for HBx-stimulated viral DNA replication, suggesting that the Cul4-DDB1 ubiquitin E3 ligase might target cellular restriction factors for ubiquitination and proteasomal degradation. To gain further insight into the DDB1-HBx interaction, we generated HBx mutants deficient for DDB1 binding (i.e., R96A, L98A, and G99A) and examined whether they support HBx-stimulated viral DNA replication. In contrast to data from previous reports, our results showed that the HBx mutants deficient for DDB1 binding supported viral DNA replication to nearly wild-type levels, revealing that the DDB1-HBx interaction is largely dispensable for HBx-stimulated viral DNA replication. Instead, we found that DDB1 directly stimulates viral transcription regardless of HBx expression. Through an HBV infection study, importantly, we demonstrated that DDB1 stimulates viral transcription from covalently closed circular DNA, a physiological template for viral transcription. Overall, we concluded that DDB1 stimulates viral transcription via a mechanism that does not involve an interaction with HBx. DDB1 constitutes a cullin-based ubiquitin E3 ligase, where DDB1 serves as an adaptor linking the cullin scaffold to the substrate receptor. Previous findings that the DDB1-binding ability of HBx is essential for HBx-stimulated viral DNA replication led to the hypothesis that HBx could downregulate host restriction factors that limit HBV replication through the cullin ubiquitin E3 ligase that requires the DDB1-HBx interaction. Consistent with this hypothesis, recent work identified Smc5/6 as a host restriction factor that is regulated by the viral cullin ubiquitin E3 ligase. In contrast, here we found that the DDB1-HBx interaction is largely dispensable for HBx-stimulated viral DNA replication. Instead, our results clearly showed that DDB1, regardless of HBx expression, enhances viral transcription. Overall, besides its role in the viral cullin ubiquitin E3 ligase, DDB1 itself stimulates viral transcription via HBx-independent mechanisms. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Bul Proteins, a Nonredundant, Antagonistic Family of Ubiquitin Ligase Regulatory Proteins

PubMed Central

Novoselova, Tatiana V.; Zahira, Kiran; Rose, Ruth-Sarah

2012-01-01

Like other Nedd4 ligases, Saccharomyces cerevisiae E3 Rsp5p utilizes adaptor proteins to interact with some substrates. Previous studies have indentified Bul1p and Bul2p as adaptor proteins that facilitate the ligase-substrate interaction. Here, we show the identification of a third member of the Bul family, Bul3p, the product of two adjacent open reading frames separated by a stop codon that undergoes readthrough translation. Combinatorial analysis of BUL gene deletions reveals that they regulate some, but not all, of the cellular pathways known to involve Rsp5p. Surprisingly, we find that Bul proteins can act antagonistically to regulate the same ubiquitin-dependent process, and the nature of this antagonistic activity varies between different substrates. We further show, using in vitro ubiquitination assays, that the Bul proteins have different specificities for WW domains and that the two forms of Bul3p interact differently with Rsp5p, potentially leading to alternate functional outcomes. These data introduce a new level of complexity into the regulatory interactions that take place between Rsp5p and its adaptors and substrates and suggest a more critical role for the Bul family of proteins in controlling adaptor-mediated ubiquitination. PMID:22307975

Dissecting the function of Cullin-RING ubiquitin ligase complex genes in planarian regeneration.

PubMed

Strand, Nicholas S; Allen, John M; Ghulam, Mahjoobah; Taylor, Matthew R; Munday, Roma K; Carrillo, Melissa; Movsesyan, Artem; Zayas, Ricardo M

2018-01-15

The ubiquitin system plays a role in nearly every aspect of eukaryotic cell biology. The enzymes responsible for transferring ubiquitin onto specific substrates are the E3 ubiquitin ligases, a large and diverse family of proteins, for which biological roles and target substrates remain largely undefined. Studies using model organisms indicate that ubiquitin signaling mediates key steps in developmental processes and tissue regeneration. Here, we used the freshwater planarian, Schmidtea mediterranea, to investigate the role of Cullin-RING ubiquitin ligase (CRL) complexes in stem cell regulation during regeneration. We identified six S. mediterranea cullin genes, and used RNAi to uncover roles for homologs of Cullin-1, -3 and -4 in planarian regeneration. The cullin-1 RNAi phenotype included defects in blastema formation, organ regeneration, lesions, and lysis. To further investigate the function of cullin-1-mediated cellular processes in planarians, we examined genes encoding the adaptor protein Skp1 and F-box substrate-recognition proteins that are predicted to partner with Cullin-1. RNAi against skp1 resulted in phenotypes similar to cullin-1 RNAi, and an RNAi screen of the F-box genes identified 19 genes that recapitulated aspects of cullin-1 RNAi, including ones that in mammals are involved in stem cell regulation and cancer biology. Our data provides evidence that CRLs play discrete roles in regenerative processes and provide a platform to investigate how CRLs regulate stem cells in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

The E3 ubiquitin ligase ZNRF2 is a substrate of mTORC1 and regulates its activation by amino acids

PubMed Central

Hoxhaj, Gerta; Caddye, Edward; Najafov, Ayaz; Houde, Vanessa P; Johnson, Catherine; Dissanayake, Kumara; Toth, Rachel; Campbell, David G; Prescott, Alan R; MacKintosh, Carol

2016-01-01

The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1. DOI: http://dx.doi.org/10.7554/eLife.12278.001 PMID:27244671

The E3 ligase Mule protects the heart against oxidative stress and mitochondrial dysfunction through Myc-dependent inactivation of Pgc-1α and Pink1.

PubMed

Dadson, Keith; Hauck, Ludger; Hao, Zhenyue; Grothe, Daniela; Rao, Vivek; Mak, Tak W; Billia, Filio

2017-02-02

Cardiac homeostasis requires proper control of protein turnover. Protein degradation is principally controlled by the Ubiquitin-Proteasome System. Mule is an E3 ubiquitin ligase that regulates cellular growth, DNA repair and apoptosis to maintain normal tissue architecture. However, Mule's function in the heart has yet to be described. In a screen, we found reduced Mule expression in left ventricular samples from end-stage heart failure patients. Consequently, we generated conditional cardiac-specific Mule knockout (Mule  fl/fl(y) ;mcm) mice. Mule ablation in adult Mule  fl/fl(y) ;mcm mice prevented myocardial c-Myc polyubiquitination, leading to c-Myc accumulation and subsequent reduced expression of Pgc-1α, Pink1, and mitochondrial complex proteins. Furthermore, these mice developed spontaneous cardiac hypertrophy, left ventricular dysfunction, and early mortality. Co-deletion of Mule and c-Myc rescued this phenotype. Our data supports an indispensable role for Mule in cardiac homeostasis through the regulation of mitochondrial function via maintenance of Pgc-1α and Pink1 expression and persistent negative regulation of c-Myc.

The story so far: post-translational regulation of peroxisome proliferator-activated receptors by ubiquitination and SUMOylation

PubMed Central

Wadosky, Kristine M.

2012-01-01

Many studies have implicated the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptor transcription factors in regulating cardiac substrate metabolism and ATP generation. Recently, evidence from a variety of cell culture and organ systems has implicated ubiquitin and small ubiquitin-like modifier (SUMO) conjugation as post-translational modifications that regulate the activity of PPAR transcription factors and their coreceptors/coactivators. Here we introduce the ubiquitin and SUMO conjugation systems and extensively review how they have been shown to regulate all three PPAR isoforms (PPARα, PPARβ/δ, and PPARγ) in addition to the retinoid X receptor and PPARγ coactivator-1α subunits of the larger PPAR transcription factor complex. We then present how the specific ubiquitin (E3) ligases have been implicated and review emerging evidence that post-translational modifications of PPARs with ubiquitin and/or SUMO may play a role in cardiac disease. Because PPAR activity is perturbed in a variety of forms of heart disease and specific proteins regulate this process (E3 ligases), this may be a fruitful area of investigation with respect to finding new therapeutic targets. PMID:22037188

The Kinase Activity of Calcineurin B-like Interacting Protein Kinase 26 (CIPK26) Influences Its Own Stability and that of the ABA-regulated Ubiquitin Ligase, Keep on Going (KEG)

PubMed Central

Lyzenga, Wendy J.; Sullivan, Victoria; Liu, Hongxia; Stone, Sophia L.

2017-01-01

The Really Interesting New Gene (RING)-type E3 ligase, Keep on Going (KEG) plays a critical role in Arabidopsis growth after germination and the connections between KEG and hormone signaling pathways are expanding. With regards to abscisic acid (ABA) signaling, KEG targets ABA-responsive transcription factors abscisic acid insensitive 5, ABF1 and ABF3 for ubiquitination and subsequent degradation through the 26S proteasome. Regulation of E3 ligases through self-ubiquitination is common to RING-type E3 ligases and ABA promotes KEG self-ubiquitination and degradation. ABA-mediated degradation of KEG is phosphorylation-dependent; however, upstream signaling proteins that may regulate KEG stability have not been characterized. In this report, we show that CBL-Interacting Protein Kinase (CIPK) 26 can phosphorylate KEG in vitro. Using both in vitro and in planta degradation assays we provide evidence which suggests that the kinase activity of CIPK26 promotes the degradation of KEG. Furthermore, we found that the kinase activity of CIPK26 also influences its own stability; a constitutively active version is more stable than a wild type or a kinase dead version. Our results suggest a reciprocal regulation model wherein an activated and stable CIPK26 phosphorylates KEG to promote degradation of the E3. PMID:28443108

Molecular characterization of atrogin-1/F-box protein-32 (FBXO32) and F-box protein 25 (FBXO25) in rainbow trout (Oncorhynchus mykiss); expression across tissues in response to feed deprivation

USDA-ARS?s Scientific Manuscript database

The characteristic increase in protein catabolism during muscle atrophy is largely the result of an increase in E3 ubiquitin ligase expression, specifically that of atrogin-1, or FBXO32, which functions to polyubiquitinate proteins. In rainbow trout, the cDNA sequences of two E3 ubiquitin ligase F-...

SCFSlmb E3 ligase-mediated degradation of Expanded is inhibited by the Hippo pathway in Drosophila

PubMed Central

Zhang, Hongtao; Li, Changqing; Chen, Hanqing; Wei, Chuanxian; Dai, Fei; Wu, Honggang; Dui, Wen; Deng, Wu-Min; Jiao, Renjie

2015-01-01

Deregulation of the evolutionarily conserved Hippo pathway has been implicated in abnormal development of animals and in several types of cancer. One mechanism of Hippo pathway regulation is achieved by controlling the stability of its regulatory components. However, the executive E3 ligases that are involved in this process, and how the process is regulated, remain poorly defined. In this study, we identify, through a genetic candidate screen, the SCFSlmb E3 ligase as a novel negative regulator of the Hippo pathway in Drosophila imaginal tissues via mediation of the degradation of Expanded (Ex). Mechanistic study shows that Slmb-mediated degradation of Ex is inhibited by the Hippo signaling. Considering the fact that Hippo signaling suppresses the transcription of ex, we propose that the Hippo pathway employs a double security mechanism to ensure fine-tuned homeostasis during development. PMID:25522691

E1B-55K mediated regulation of RNF4 STUbL promotes HAdV gene expression.

PubMed

Müncheberg, Sarah; Hay, Ron T; Ip, Wing H; Meyer, Tina; Weiß, Christina; Brenke, Jara; Masser, Sawinee; Hadian, Kamyar; Dobner, Thomas; Schreiner, Sabrina

2018-04-25

HAdV E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in non-permissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 Ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established.RNF4, a cellular SUMO-targeted Ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM, and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNAi resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies. IMPORTANCE Daxx is a PML-NB-associated transcription factor, which was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 Ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4 and E1B-55K targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor, which is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention. Copyright © 2018 American Society for Microbiology.

Purification of SUMO conjugating enzymes and kinetic analysis of substrate conjugation

PubMed Central

Yunus, Ali A.; Lima, Christopher D.

2009-01-01

SUMO conjugation to protein substrates requires the concerted action of a dedicated E2 ubiquitin conjugation enzyme (Ubc9) and associated E3 ligases. Although Ubc9 can directly recognize and modify substrate lysine residues that occur within a consensus site for SUMO modification, E3 ligases can redirect specificity and enhance conjugation rates during SUMO conjugation in vitro and in vivo. In this chapter, we will describe methods utilized to purify SUMO conjugating enzymes and model substrates which can be used for analysis of SUMO conjugation in vitro. We will also describe methods to extract kinetic parameters during E3-dependent or E3-independent substrate conjugation. PMID:19107417

Ubiquitin enzymes in the regulation of immune responses

PubMed Central

Ebner, Petra; Versteeg, Gijs A.; Ikeda, Fumiyo

2017-01-01

Abstract Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses. PMID:28524749

Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes.

PubMed

Ramadan, Abdelaziz; Nemoto, Keiichirou; Seki, Motoaki; Shinozaki, Kazuo; Takeda, Hiroyuki; Takahashi, Hirotaka; Sawasaki, Tatsuya

2015-11-10

Protein ubiquitination is a ubiquitous mechanism in eukaryotes. In Arabidopsis, ubiquitin modification is mainly mediated by two ubiquitin activating enzymes (E1s), 37 ubiquitin conjugating enzymes (E2s), and more than 1300 predicted ubiquitin ligase enzymes (E3s), of which ~470 are RING-type E3s. A large proportion of the RING E3's gene products have yet to be characterised in vitro, likely because of the laborious work involved in large-scale cDNA cloning and protein expression, purification, and characterisation. In addition, several E2s, which might be necessary for the activity of certain E3 ligases, cannot be expressed by Escherichia coli or cultured insect cells and, therefore, remain uncharacterised. Using the RIKEN Arabidopsis full-length cDNA library (RAFL) with the 'split-primer' PCR method and a wheat germ cell-free system, we established protein libraries of Arabidopsis E2 and RING E3 enzymes. We expressed 35 Arabidopsis E2s including six enzymes that have not been previously expressed, and 204 RING proteins, most of which had not been functionally characterised. Thioester assays using dithiothreitol (DTT) showed DTT-sensitive ubiquitin thioester formation for all E2s expressed. In expression assays of RING proteins, 31 proteins showed high molecular smears, which are probably the result of their functional activity. The activities of another 27 RING proteins were evaluated with AtUBC10 and/or a group of different E2s. All the 27 RING E3s tested showed ubiquitin ligase activity, including 17 RING E3s. Their activities are reported for the first time. The wheat germ cell-free system used in our study, which is a eukaryotic expression system and more closely resembles the endogenous expression of plant proteins, is very suitable for expressing Arabidopsis E2s and RING E3s in their functional form. In addition, the protein libraries described here can be used for further understanding E2-E3 specificities and as platforms for protein-protein interaction screening.

The APC/C E3 Ligase Complex Activator FZR1 Restricts BRAF Oncogenic Function.

PubMed

Wan, Lixin; Chen, Ming; Cao, Juxiang; Dai, Xiangpeng; Yin, Qing; Zhang, Jinfang; Song, Su-Jung; Lu, Ying; Liu, Jing; Inuzuka, Hiroyuki; Katon, Jesse M; Berry, Kelsey; Fung, Jacqueline; Ng, Christopher; Liu, Pengda; Song, Min Sup; Xue, Lian; Bronson, Roderick T; Kirschner, Marc W; Cui, Rutao; Pandolfi, Pier Paolo; Wei, Wenyi

2017-04-01

BRAF drives tumorigenesis by coordinating the activation of the RAS/RAF/MEK/ERK oncogenic signaling cascade. However, upstream pathways governing BRAF kinase activity and protein stability remain undefined. Here, we report that in primary cells with active APC FZR1 , APC FZR1 earmarks BRAF for ubiquitination-mediated proteolysis, whereas in cancer cells with APC-free FZR1, FZR1 suppresses BRAF through disrupting BRAF dimerization. Moreover, we identified FZR1 as a direct target of ERK and CYCLIN D1/CDK4 kinases. Phosphorylation of FZR1 inhibits APC FZR1 , leading to elevation of a cohort of oncogenic APC FZR1 substrates to facilitate melanomagenesis. Importantly, CDK4 and/or BRAF/MEK inhibitors restore APC FZR1 E3 ligase activity, which might be critical for their clinical effects. Furthermore, FZR1 depletion cooperates with AKT hyperactivation to transform primary melanocytes, whereas genetic ablation of Fzr1 synergizes with Pten loss, leading to aberrant coactivation of BRAF/ERK and AKT signaling in mice. Our findings therefore reveal a reciprocal suppression mechanism between FZR1 and BRAF in controlling tumorigenesis. Significance: FZR1 inhibits BRAF oncogenic functions via both APC-dependent proteolysis and APC-independent disruption of BRAF dimers, whereas hyperactivated ERK and CDK4 reciprocally suppress APC FZR1 E3 ligase activity. Aberrancies in this newly defined signaling network might account for BRAF hyperactivation in human cancers, suggesting that targeting CYCLIN D1/CDK4, alone or in combination with BRAF/MEK inhibition, can be an effective anti-melanoma therapy. Cancer Discov; 7(4); 424-41. ©2017 AACR. See related commentary by Zhang and Bollag, p. 356 This article is highlighted in the In This Issue feature, p. 339 . ©2017 American Association for Cancer Research.

C-terminus of HSC70-Interacting Protein (CHIP) Inhibits Adipocyte Differentiation via Ubiquitin- and Proteasome-Mediated Degradation of PPARγ

PubMed Central

Kim, Jung-Hoon; Shin, Soyeon; Seo, Jinho; Lee, Eun-Woo; Jeong, Manhyung; Lee, Min-sik; Han, Hyun-Ji; Song, Jaewhan

2017-01-01

PPARγ (Peroxisome proliferator-activated receptor γ) is a nuclear receptor involved in lipid homeostasis and related metabolic diseases. Acting as a transcription factor, PPARγ is a master regulator for adipocyte differentiation. Here, we reveal that CHIP (C-terminus of HSC70-interacting protein) suppresses adipocyte differentiation by functioning as an E3 ligase of PPARγ. CHIP directly binds to and induces ubiquitylation of the PPARγ protein, leading to proteasome-dependent degradation. Stable overexpression or knockdown of CHIP inhibited or promoted adipogenesis, respectively, in 3T3-L1 cells. On the other hand, a CHIP mutant defective in E3 ligase could neither regulate PPARγ protein levels nor suppress adipogenesis, indicating the importance of CHIP-mediated ubiquitylation of PPARγ in adipocyte differentiation. Lastly, a CHIP null embryo fibroblast exhibited augmented adipocyte differentiation with increases in PPARγ and its target protein levels. In conclusion, CHIP acts as an E3 ligase of PPARγ, suppressing PPARγ-mediated adipogenesis. PMID:28059128

RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheren, Jamie E.; Kassenbrock, C. Kenneth, E-mail: ken.kassenbrock@ucdenver.edu; Department of Biology, Colorado State University, Fort Collins, CO 80523-1878

2013-11-01

Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequencemore » (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.« less

Probes of Ubiquitin E3 ligases distinguish different stages of Parkin activation

PubMed Central

Pao, Kuan-Chuan; Stanley, Mathew; Han, Cong; Lai, Yu-Chiang; Murphy, Paul; Balk, Kristin; Wood, Nicola T.; Corti, Olga; Corvol, Jean-Christophe; Muqit, Miratul M.K.; Virdee, Satpal

2016-01-01

E3 ligases represent an important class of enzymes, yet there are currently no chemical probes to profile their activity. We develop a new class of activity-based probe by reengineering of a ubiquitin-charged E2 conjugating enzyme and demonstrate their utility by profiling the transthiolation activity of the RING-in-between-RING (RBR) E3 ligase Parkin in vitro and in cellular extracts. Our study provides valuable insight into the roles, and cellular hierarchy, of distinct phosphorylation events in Parkin activation. We also profile Parkin patient disease-associated mutations and strikingly demonstrate that they largely mediate their effect by altering transthiolation activity. Furthermore, our probes enable direct and quantitative measurement of endogenous Parkin activity revealing that endogenous Parkin is activated in neuronal cell lines (≥75 %) in response to mitochondrial depolarization. This new technology also holds promise as a novel biomarker of PINK1-Parkin signalling as demonstrated by compatibility with Parkinson’s disease patient-derived samples. PMID:26928937

Histone deacetylase and Cullin3-REN(KCTD11) ubiquitin ligase interplay regulates Hedgehog signalling through Gli acetylation.

PubMed

Canettieri, Gianluca; Di Marcotullio, Lucia; Greco, Azzura; Coni, Sonia; Antonucci, Laura; Infante, Paola; Pietrosanti, Laura; De Smaele, Enrico; Ferretti, Elisabetta; Miele, Evelina; Pelloni, Marianna; De Simone, Giuseppina; Pedone, Emilia Maria; Gallinari, Paola; Giorgi, Alessandra; Steinkühler, Christian; Vitagliano, Luigi; Pedone, Carlo; Schinin, M Eugenià; Screpanti, Isabella; Gulino, Alberto

2010-02-01

Hedgehog signalling is crucial for development and is deregulated in several tumours, including medulloblastoma. Regulation of the transcriptional activity of Gli (glioma-associated oncogene) proteins, effectors of the Hedgehog pathway, is poorly understood. We show here that Gli1 and Gli2 are acetylated proteins and that their HDAC-mediated deacetylation promotes transcriptional activation and sustains a positive autoregulatory loop through Hedgehog-induced upregulation of HDAC1. This mechanism is turned off by HDAC1 degradation through an E3 ubiquitin ligase complex formed by Cullin3 and REN, a Gli antagonist lost in human medulloblastoma. Whereas high HDAC1 and low REN expression in neural progenitors and medulloblastomas correlates with active Hedgehog signalling, loss of HDAC activity suppresses Hedgehog-dependent growth of neural progenitors and tumour cells. Consistent with this, abrogation of Gli1 acetylation enhances cellular proliferation and transformation. These data identify an integrated HDAC- and ubiquitin-mediated circuitry, where acetylation of Gli proteins functions as an unexpected key transcriptional checkpoint of Hedgehog signalling.

RING-type E3 ligases: Master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination

PubMed Central

Metzger, Meredith B.; Pruneda, Jonathan N.; Klevit, Rachel E.; Weissman, Allan M.

2013-01-01

RING finger domain and RING finger-like ubiquitin ligases (E3s), such as U-box proteins, constitute the vast majority of known E3s. RING-type E3s function together with ubiquitin-conjugating enzymes (E2s) to mediate ubiquitination and are implicated in numerous cellular processes. In part because of their importance in human physiology and disease, these proteins and their cellular functions represent an intense area of study. Here we review recent advances in RING-type E3 recognition of substrates, their cellular regulation, and their varied architecture. Additionally, recent structural insights into RING-type E3 function, with a focus on important interactions with E2s and ubiquitin, are reviewed. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. PMID:23747565

The D113N mutation in the RING E3 ubiquitin protein ligase gene is not associated with ex vivo susceptibility to common anti-malarial drugs in African Plasmodium falciparum isolates.

PubMed

Gendrot, Mathieu; Foguim, Francis Tsombeng; Robert, Marie Gladys; Amalvict, Rémy; Mosnier, Joel; Benoit, Nicolas; Madamet, Marylin; Pradines, Bruno

2018-03-12

Plasmodium falciparum resistance to artemisinin-based combination therapy has emerged and spread in Southeast Asia. In areas where artemisinin resistance is emerging, the efficacy of combination is now based on partner drugs. In this context, the identification of novel markers of resistance is essential to monitor the emergence and spread of resistance to these partner drugs. The ubiquitylation pathway could be a possible target for anti-malarial compounds and might be involved in resistance. Polymorphisms in the E3 ubiquitin-protein ligase (PF3D7_0627300) gene could be associated with decreased in vitro susceptibility to anti-malarial drugs. Plasmodium falciparum isolates were collected from patients hospitalized in France with imported malaria from a malaria-endemic country from January 2015 to December 2016 and, more particularly, from African French-speaking countries. In total, 215 isolates were successfully sequenced for the E3 ubiquitin-protein ligase gene and assessed for ex vivo susceptibility to anti-malarial drugs. The D113N mutation in the RING E3 ubiquitin-protein ligase gene was present in 147 out of the 215 samples (68.4%). The IC 50 values for the ten anti-malarial drugs were not significantly different between the wild-type and mutant parasites (p values between 0.225 and 0.933). There was no significant difference in terms of the percentage of parasites with decreased susceptibility between the D113 wild-type and the 133N mutated P. falciparum strains (p values between 0.541 and 1). The present data confirmed the absence of the association between polymorphisms in the RING E3 ubiquitin-protein ligase gene and the ex vivo susceptibility to common anti-malarial drugs in African P. falciparum isolates.

The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

PubMed Central

Halter, David; Collart, Martine A.; Panasenko, Olesya O.

2014-01-01

Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome. PMID:24465968

Overexpression of the Rice SUMO E3 Ligase Gene OsSIZ1 in Cotton Enhances Drought and Heat Tolerance, and Substantially Improves Fiber Yields in the Field under Reduced Irrigation and Rainfed Conditions

PubMed Central

Mishra, Neelam; Sun, Li; Zhu, Xunlu; Smith, Jennifer; Prakash Srivastava, Anurag; Yang, Xiaojie; Pehlivan, Necla; Esmaeili, Nardana; Luo, Hong; Shen, Guoxin; Jones, Don; Auld, Dick; Burke, John

2017-01-01

The Arabidopsis SUMO E3 ligase gene AtSIZ1 plays important roles in plant response to abiotic stresses as loss of function in AtSIZ1 leads to increased sensitivity to drought, heat and salt stresses. Overexpression of the AtSIZ1 rice homolog, OsSIZ1, leads to increased heat and drought tolerance in bentgrass, suggesting that the function of the E3 ligase SIZ1 is highly conserved in plants and it plays a critical role in abiotic stress responses. To test the possibility that the SUMO E3 ligase could be used to engineer drought- and heat-tolerant crops, the rice gene OsSIZ1 was overexpressed in cotton. We report here that overexpression of OsSIZ1 in cotton results in higher net photosynthesis and better growth than wild-type cotton under drought and thermal stresses in growth chamber and greenhouse conditions. Additionally, this tolerance to abiotic stresses was correlated with higher fiber yield in both controlled-environment and field trials carried out under reduced irrigation and rainfed conditions. These results suggest that OsSIZ1 is a viable candidate gene to improve crop yields under water-limited and rainfed agricultural production systems. PMID:28340002

A Large Complement of the Predicted Arabidopsis ARM Repeat Proteins Are Members of the U-Box E3 Ubiquitin Ligase Family1[w

PubMed Central

Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L.; Salt, Jennifer N.; Goring, Daphne R.

2004-01-01

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis. PMID:14657406

A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family.

PubMed

Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L; Salt, Jennifer N; Goring, Daphne R

2004-01-01

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis.

Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin

PubMed Central

Im, Eunju; Yoo, Lang; Hyun, Minju; Shin, Woo Hyun

2016-01-01

Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the pars compacta of the substantia nigra and accumulation of ubiquitinated proteins in aggregates called Lewy bodies. Several mutated genes have been found in familial PD patients, including SNCA (α-synuclein), PARK2 (parkin), PINK1, PARK7 (DJ-1), LRRK2 and ATP13A2. Many pathogenic mutations of PARK2, which encodes the ubiquitin E3 ligase parkin, result in loss of function, leading to accumulation of parkin substrates and consequently contributing to dopaminergic cell death. ISG15 is a member of the ubiquitin-like modifier family and is induced by stimulation with type I interferons. Similar to ubiquitin and ubiquitination, covalent conjugation of ISG15 to target proteins (ISGylation) regulates their biochemical properties. In this study, we identified parkin as a novel target of ISGylation specifically mediated by the ISG15-E3 ligase HERC5. In addition, we identified two ISGylation sites, Lys-349 and Lys-369, in the in-between-ring domain of parkin. ISGylation of these sites promotes parkin's ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation and increases its cytoprotective effect. In conclusion, covalent ISG15 conjugation is a novel mode of modulating parkin activity, and alteration in this pathway may be associated with PD pathogenesis. PMID:27534820

Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin.

PubMed

Im, Eunju; Yoo, Lang; Hyun, Minju; Shin, Woo Hyun; Chung, Kwang Chul

2016-08-01

Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the pars compacta of the substantia nigra and accumulation of ubiquitinated proteins in aggregates called Lewy bodies. Several mutated genes have been found in familial PD patients, including SNCA (α-synuclein), PARK2 (parkin), PINK1, PARK7 (DJ-1), LRRK2 and ATP13A2 Many pathogenic mutations of PARK2, which encodes the ubiquitin E3 ligase parkin, result in loss of function, leading to accumulation of parkin substrates and consequently contributing to dopaminergic cell death. ISG15 is a member of the ubiquitin-like modifier family and is induced by stimulation with type I interferons. Similar to ubiquitin and ubiquitination, covalent conjugation of ISG15 to target proteins (ISGylation) regulates their biochemical properties. In this study, we identified parkin as a novel target of ISGylation specifically mediated by the ISG15-E3 ligase HERC5. In addition, we identified two ISGylation sites, Lys-349 and Lys-369, in the in-between-ring domain of parkin. ISGylation of these sites promotes parkin's ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation and increases its cytoprotective effect. In conclusion, covalent ISG15 conjugation is a novel mode of modulating parkin activity, and alteration in this pathway may be associated with PD pathogenesis. © 2016 The Authors.

Parkin New Cargos: a New ROS Independent Role for Parkin in Regulating Cell Division.

PubMed

Stieg, David C; Cooper, Katrina F

2016-01-01

Cell cycle progression requires the destruction of key cell cycle regulators by the multi-subunit E3 ligase called the anaphase promoting complex (APC/C). As the cell progresses through the cell cycle, the APC/C is sequentially activated by two highly conserved co-activators called Cdc20 and Cdh1. Importantly, APC/C Cdc20 is required to degrade substrates in G2/M whereas APC Cdh1 drives the cells into G1. Recently, Parkin, a monomeric E3 ligase that is required for ubiquitin-mediated mitophagy following mitochondrial stress, was shown to both bind and be activated by Cdc20 or Cdh1 during the cell cycle. This mitotic role for Parkin does not require an activating phosphorylation by its usual kinase partner PINK. Rather, mitotic Parkin activity requires phosphorylation on a different serine by the polo-like kinase Plk1. Interestingly, although Parkin Cdc20 and Parkin Cdh1 activity is independent of the APC/C, it mediates degradation of an overlapping subset of substrates. However, unlike the APC/C, Parkin is not necessary for cell cycle progression. Despite this, loss of Parkin activity accelerates genome instability and tumor growth in xenograft models. These findings provide a mechanism behind the previously described, but poorly understood, tumor suppressor role for Parkin. Taken together, studies suggest that the APC/C and Parkin have similar and unique roles to play in cell division, possibly being dependent upon the different subcellular address of these two ligases.

PolySUMOylation by Siz2 and Mms21 triggers relocation of DNA breaks to nuclear pores through the Slx5/Slx8 STUbL

PubMed Central

Horigome, Chihiro; Bustard, Denise E.; Marcomini, Isabella; Delgoshaie, Neda; Tsai-Pflugfelder, Monika; Cobb, Jennifer A.; Gasser, Susan M.

2016-01-01

High-resolution imaging shows that persistent DNA damage in budding yeast localizes in distinct perinuclear foci for repair. The signals that trigger DNA double-strand break (DSB) relocation or determine their destination are unknown. We show here that DSB relocation to the nuclear envelope depends on SUMOylation mediated by the E3 ligases Siz2 and Mms21. In G1, a polySUMOylation signal deposited coordinately by Mms21 and Siz2 recruits the SUMO targeted ubiquitin ligase Slx5/Slx8 to persistent breaks. Both Slx5 and Slx8 are necessary for damage relocation to nuclear pores. When targeted to an undamaged locus, however, Slx5 alone can mediate relocation in G1-phase cells, bypassing the requirement for polySUMOylation. In contrast, in S-phase cells, monoSUMOylation mediated by the Rtt107-stabilized SMC5/6–Mms21 E3 complex drives DSBs to the SUN domain protein Mps3 in a manner independent of Slx5. Slx5/Slx8 and binding to pores favor repair by ectopic break-induced replication and imprecise end-joining. PMID:27056668

The Red Light Receptor Phytochrome B Directly Enhances Substrate-E3 Ligase Interactions to Attenuate Ethylene Responses.

PubMed

Shi, Hui; Shen, Xing; Liu, Renlu; Xue, Chang; Wei, Ning; Deng, Xing Wang; Zhong, Shangwei

2016-12-05

Plants germinating under subterranean darkness assume skotomorphogenesis, a developmental program strengthened by ethylene in response to mechanical pressure of soil. Upon reaching the surface, light triggers a dramatic developmental transition termed de-etiolation that requires immediate termination of ethylene responses. Here, we report that light activation of photoreceptor phyB results in rapid degradation of EIN3, the master transcription factor in the ethylene signaling pathway. As a result, light rapidly and efficiently represses ethylene actions. Specifically, phyB directly interacts with EIN3 in a light-dependent manner and also physically associates with F box protein EBFs. The light-activated association of phyB, EIN3, and EBF1/EBF2 proteins stimulates robust EIN3 degradation by SCF EBF1/EBF2 E3 ligases. We reveal that phyB manipulates substrate-E3 ligase interactions in a light-dependent manner, thus directly controlling the stability of EIN3. Our findings illustrate a mechanistic model of how plants transduce light information to immediately turn off ethylene signaling for de-etiolation initiation. Copyright © 2016 Elsevier Inc. All rights reserved.

The zinc-binding region (ZBR) fragment of Emi2 can inhibit APC/C by targeting its association with the coactivator Cdc20 and UBE2C-mediated ubiquitylation

PubMed Central

Shoji, Shisako; Muto, Yutaka; Ikeda, Mariko; He, Fahu; Tsuda, Kengo; Ohsawa, Noboru; Akasaka, Ryogo; Terada, Takaho; Wakiyama, Motoaki; Shirouzu, Mikako; Yokoyama, Shigeyuki

2014-01-01

Anaphase-promoting complex or cyclosome (APC/C) is a multisubunit ubiquitin ligase E3 that targets cell-cycle regulators. Cdc20 is required for full activation of APC/C in M phase, and mediates substrate recognition. In vertebrates, Emi2/Erp1/FBXO43 inhibits APC/C-Cdc20, and functions as a cytostatic factor that causes long-term M phase arrest of mature oocytes. In this study, we found that a fragment corresponding to the zinc-binding region (ZBR) domain of Emi2 inhibits cell-cycle progression, and impairs the association of Cdc20 with the APC/C core complex in HEK293T cells. Furthermore, we revealed that the ZBR fragment of Emi2 inhibits in vitro ubiquitin chain elongation catalyzed by the APC/C cullin-RING ligase module, the ANAPC2–ANAPC11 subcomplex, in combination with the ubiquitin chain-initiating E2, E2C/UBE2C/UbcH10. Structural analyses revealed that the Emi2 ZBR domain uses different faces for the two mechanisms. Thus, the double-faced ZBR domain of Emi2 antagonizes the APC/C function by inhibiting both the binding with the coactivator Cdc20 and ubiquitylation mediated by the cullin-RING ligase module and E2C. In addition, the tail region between the ZBR domain and the C-terminal RL residues [the post-ZBR (PZ) region] interacts with the cullin subunit, ANAPC2. In the case of the ZBR fragment of the somatic paralogue of Emi2, Emi1/FBXO5, these inhibitory activities against cell division and ubiquitylation were not observed. Finally, we identified two sets of key residues in the Emi2 ZBR domain that selectively exert each of the dual Emi2-specific modes of APC/C inhibition, by their mutation in the Emi2 ZBR domain and their transplantation into the Emi1 ZBR domain. PMID:25161877

Destabilization of strigolactone receptor DWARF14 by binding of ligand and E3-ligase signaling effector DWARF3

PubMed Central

Zhao, Li-Hua; Zhou, X Edward; Yi, Wei; Wu, Zhongshan; Liu, Yue; Kang, Yanyong; Hou, Li; de Waal, Parker W; Li, Suling; Jiang, Yi; Scaffidi, Adrian; Flematti, Gavin R; Smith, Steven M; Lam, Vinh Q; Griffin, Patrick R; Wang, Yonghong; Li, Jiayang; Melcher, Karsten; Xu, H Eric

2015-01-01

Strigolactones (SLs) are endogenous hormones and exuded signaling molecules in plant responses to low levels of mineral nutrients. Key mediators of the SL signaling pathway in rice include the α/β-fold hydrolase DWARF 14 (D14) and the F-box component DWARF 3 (D3) of the ubiquitin ligase SCFD3 that mediate ligand-dependent degradation of downstream signaling repressors. One perplexing feature is that D14 not only functions as the SL receptor but is also an active enzyme that slowly hydrolyzes diverse natural and synthetic SLs including GR24, preventing the crystallization of a binary complex of D14 with an intact SL as well as the ternary D14/SL/D3 complex. Here we overcome these barriers to derive a structural model of D14 bound to intact GR24 and identify the interface that is required for GR24-mediated D14-D3 interaction. The mode of GR24-mediated signaling, including ligand recognition, hydrolysis by D14, and ligand-mediated D14-D3 interaction, is conserved in structurally diverse SLs. More importantly, D14 is destabilized upon the binding of ligands and D3, thus revealing an unusual mechanism of SL recognition and signaling, in which the hormone, the receptor, and the downstream effectors are systematically destabilized during the signal transduction process. PMID:26470846

Blocking an N-terminal acetylation–dependent protein interaction inhibits an E3 ligase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scott, Daniel C.; Hammill, Jared T.; Min, Jaeki

N-terminal acetylation is an abundant modification influencing protein functions. Because ~80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation–dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors aremore » highly selective with respect to other protein acetyl-amide–binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2–E3 ligases.« less

Targeted Degradation of Proteins Localized in Subcellular Compartments by Hybrid Small Molecules.

PubMed

Okuhira, Keiichiro; Shoda, Takuji; Omura, Risa; Ohoka, Nobumichi; Hattori, Takayuki; Shibata, Norihito; Demizu, Yosuke; Sugihara, Ryo; Ichino, Asato; Kawahara, Haruka; Itoh, Yukihiro; Ishikawa, Minoru; Hashimoto, Yuichi; Kurihara, Masaaki; Itoh, Susumu; Saito, Hiroyuki; Naito, Mikihiko

2017-03-01

Development of novel small molecules that selectively degrade pathogenic proteins would provide an important advance in targeted therapy. Recently, we have devised a series of hybrid small molecules named SNIPER (specific and nongenetic IAP-dependent protein ERaser) that induces the degradation of target proteins via the ubiquitin-proteasome system. To understand the localization of proteins that can be targeted by this protein knockdown technology, we examined whether SNIPER molecules are able to induce degradation of cellular retinoic acid binding protein II (CRABP-II) proteins localized in subcellular compartments of cells. CRABP-II is genetically fused with subcellular localization signals, and they are expressed in the cells. SNIPER(CRABP) with different IAP-ligands, SNIPER(CRABP)-4 with bestatin and SNIPER(CRABP)-11 with MV1 compound, induce the proteasomal degradation of wild-type (WT), cytosolic, nuclear, and membrane-localized CRABP-II proteins, whereas only SNIPER(CRABP)-11 displayed degradation activity toward the mitochondrial CRABP-II protein. The small interfering RNA-mediated silencing of cIAP1 expression attenuated the knockdown activity of SNIPER(CRABP) against WT and cytosolic CRABP-II proteins, indicating that cIAP1 is the E3 ligase responsible for degradation of these proteins. Against membrane-localized CRABP-II protein, cIAP1 is also a primary E3 ligase in the cells, but another E3 ligase distinct from cIAP2 and X-linked inhibitor of apoptosis protein (XIAP) could also be involved in the SNIPER(CRABP)-11-induced degradation. However, for the degradation of nuclear and mitochondrial CRABP-II proteins, E3 ligases other than cIAP1, cIAP2, and XIAP play a role in the SNIPER-mediated protein knockdown. These results indicate that SNIPER can target cytosolic, nuclear, membrane-localized, and mitochondrial proteins for degradation, but the responsible E3 ligase is different, depending on the localization of the target protein. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

A complex ligase ribozyme evolved in vitro from a group I ribozyme domain

NASA Technical Reports Server (NTRS)

Jaeger, L.; Wright, M. C.; Joyce, G. F.; Bada, J. L. (Principal Investigator)

1999-01-01

Like most proteins, complex RNA molecules often are modular objects made up of distinct structural and functional domains. The component domains of a protein can associate in alternative combinations to form molecules with different functions. These observations raise the possibility that complex RNAs also can be assembled from preexisting structural and functional domains. To test this hypothesis, an in vitro evolution procedure was used to isolate a previously undescribed class of complex ligase ribozymes, starting from a pool of 10(16) different RNA molecules that contained a constant region derived from a large structural domain that occurs within self-splicing group I ribozymes. Attached to this constant region were three hypervariable regions, totaling 85 nucleotides, that gave rise to the catalytic motif within the evolved catalysts. The ligase ribozymes catalyze formation of a 3',5'-phosphodiester linkage between adjacent template-bound oligonucleotides, one bearing a 3' hydroxyl and the other a 5' triphosphate. Ligation occurs in the context of a Watson-Crick duplex, with a catalytic rate of 0.26 min(-1) under optimal conditions. The constant region is essential for catalytic activity and appears to retain the tertiary structure of the group I ribozyme. This work demonstrates that complex RNA molecules, like their protein counterparts, can share common structural domains while exhibiting distinct catalytic functions.

Arabidopsis ANGUSTIFOLIA3 (AN3) is associated with the promoter of CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) to regulate light-mediated stomatal development.

PubMed

Meng, Lai-Sheng; Li, Cong; Xu, Meng-Ke; Sun, Xu-Dong; Wan, Wen; Cao, Xiao-Ying; Zhang, Jin-Lin; Chen, Kun-Ming

2018-04-12

Light signals are perceived by multiple photoreceptors that converge to suppress the RING E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) for the regulation of stomatal development. Thus, COP1 is a point of integration between light signaling and stomatal patterning. However, how light signaling is collected into COP1 for the production and spacing of stomata is still unknown. Here, we report that the loss-of-function mutant of ANGUSTIFOLIA3 (AN3) delays asymmetric cell division, which leads to decreased stomatal index. Furthermore, overexpression of AN3 accelerates asymmetric cell division, which results in clusters of stomata. In addition, the stomatal development through AN3 regulation is mediated by light signaling. Finally, we find that an3 is a light-signaling mutant, and that AN3 protein is light regulated. Self-activation by AN3 contributes to the control of AN3 expression. Thus, AN3 is a point of collection between light signaling and stomatal patterning. Target-gene analysis indicates that AN3 is associated with COP1 promoter for the regulation of light-controlling stomatal development. Together, these components for regulating stomatal development form an AN3-COP1-E3 ubiquitin ligase complex, allowing the integration of light signaling into the production and spacing of stomata. © 2018 John Wiley & Sons Ltd.

The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy

PubMed Central

Sun, Aiqin; Wei, Jing; Childress, Chandra; Shaw, John H.; Peng, Ke; Shao, Genbao; Yang, Wannian; Lin, Qiong

2017-01-01

ABSTRACT The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy. PMID:28085563

Identification and Characterization of MCM3 as a Kelch-like ECH-associated Protein 1 (KEAP1) Substrate*

PubMed Central

Mulvaney, Kathleen M.; Matson, Jacob P.; Siesser, Priscila F.; Tamir, Tigist Y.; Goldfarb, Dennis; Jacobs, Timothy M.; Cloer, Erica W.; Harrison, Joseph S.; Vaziri, Cyrus; Cook, Jeanette G.; Major, Michael B.

2016-01-01

KEAP1 is a substrate adaptor protein for a CUL3-based E3 ubiquitin ligase. Ubiquitylation and degradation of the antioxidant transcription factor NRF2 is considered the primary function of KEAP1; however, few other KEAP1 substrates have been identified. Because KEAP1 is altered in a number of human pathologies and has been proposed as a potential therapeutic target therein, we sought to better understand KEAP1 through systematic identification of its substrates. Toward this goal, we combined parallel affinity capture proteomics and candidate-based approaches. Substrate-trapping proteomics yielded NRF2 and the related transcription factor NRF1 as KEAP1 substrates. Our targeted investigation of KEAP1-interacting proteins revealed MCM3, an essential subunit of the replicative DNA helicase, as a new substrate. We show that MCM3 is ubiquitylated by the KEAP1-CUL3-RBX1 complex in cells and in vitro. Using ubiquitin remnant profiling, we identify the sites of KEAP1-dependent ubiquitylation in MCM3, and these sites are on predicted exposed surfaces of the MCM2–7 complex. Unexpectedly, we determined that KEAP1 does not regulate total MCM3 protein stability or subcellular localization. Our analysis of a KEAP1 targeting motif in MCM3 suggests that MCM3 is a point of direct contact between KEAP1 and the MCM hexamer. Moreover, KEAP1 associates with chromatin in a cell cycle-dependent fashion with kinetics similar to the MCM2–7 complex. KEAP1 is thus poised to affect MCM2–7 dynamics or function rather than MCM3 abundance. Together, these data establish new functions for KEAP1 within the nucleus and identify MCM3 as a novel substrate of the KEAP1-CUL3-RBX1 E3 ligase. PMID:27621311

Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases.

PubMed

Wiborg, Jakob; O'Shea, Charlotte; Skriver, Karen

2008-08-01

The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp(266) in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis-trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3-E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.

RING E3 ligases: key regulatory elements are involved in abiotic stress responses in plants

PubMed Central

Cho, Seok Keun; Ryu, Moon Young; Kim, Jong Hum; Hong, Jeong Soo; Oh, Tae Rin; Kim, Woo Taek; Yang, Seong Wook

2017-01-01

Plants are constantly exposed to a variety of abiotic stresses, such as drought, heat, cold, flood, and salinity. To survive under such unfavorable conditions, plants have evolutionarily developed their own resistant-mechanisms. For several decades, many studies have clarified specific stress response pathways of plants through various molecular and genetic studies. In particular, it was recently discovered that ubiquitin proteasome system (UPS), a regulatory mechanism for protein turn over, is greatly involved in the stress responsive pathways. In the UPS, many E3 ligases play key roles in recognizing and tethering poly-ubiquitins on target proteins for subsequent degradation by the 26S proteasome. Here we discuss the roles of RING ligases that have been defined in related to abiotic stress responses in plants. PMID:28712388

Merkel cell polyomavirus small T antigen induces genome instability by E3 ubiquitin ligase targeting.

PubMed

Kwun, H J; Wendzicki, J A; Shuda, Y; Moore, P S; Chang, Y

2017-12-07

The formation of a bipolar mitotic spindle is an essential process for the equal segregation of duplicated DNA into two daughter cells during mitosis. As a result of deregulated cellular signaling pathways, cancer cells often suffer a loss of genome integrity that might etiologically contribute to carcinogenesis. Merkel cell polyomavirus (MCV) small T (sT) oncoprotein induces centrosome overduplication, aneuploidy, chromosome breakage and the formation of micronuclei by targeting cellular ligases through a sT domain that also inhibits MCV large T oncoprotein turnover. These results provide important insight as to how centrosome number and chromosomal stability can be affected by the E3 ligase targeting capacity of viral oncoproteins such as MCV sT, which may contribute to Merkel cell carcinogenesis.

NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR

PubMed Central

Kainulainen, Markus; Lau, Simone; Samuel, Charles E.; Hornung, Veit

2016-01-01

ABSTRACT Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. IMPORTANCE Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs requires E3 ubiquitin ligase complexes of the SCF (Skp1, Cul1, F-box protein) type to destroy PKR. SCF-type complexes can engage variant ubiquitination substrate recognition subunits, and we found the F-box proteins FBXW11 and β-TRCP1 to be relevant for the action of NSs against PKR. Thus, we identified the host cell factors that are critically needed by Rift Valley fever virus to uphold its replication against the potent antiviral kinase PKR. PMID:27122577

Endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 controls B-cell immunity through degradation of the death receptor CD95/Fas

PubMed Central

Kong, Sinyi; Yang, Yi; Xu, Yuanming; Wang, Yajun; Zhang, Yusi; Melo-Cardenas, Johanna; Xu, Xiangping; Gao, Beixue; Thorp, Edward B.; Zhang, Donna D.; Zhang, Bin; Song, Jianxun; Zhang, Kezhong; Zhang, Jianning; Zhang, Jinping; Li, Huabin; Fang, Deyu

2016-01-01

Humoral immunity involves multiple checkpoints during B-cell development, maturation, and activation. The cell death receptor CD95/Fas-mediated apoptosis plays a critical role in eliminating the unwanted activation of B cells by self-reactive antigens and in maintaining B-cell homeostasis through activation-induced B-cell death (AICD). The molecular mechanisms controlling AICD remain largely undefined. Herein, we show that the E3 ubiquitin ligase Hrd1 protected B cells from activation-induced cell death by degrading the death receptor Fas. Hrd1-null B cells exhibited high Fas expression during activation and rapidly underwent Fas-mediated apoptosis, which could be largely inhibited by FasL neutralization. Fas mutation in Hrd1 KO mice abrogated the increase in B-cell AICD. We identified Hrd1 as the first E3 ubiquitin ligase of the death receptor Fas and Hrd1-mediated Fas destruction as a molecular mechanism in regulating B-cell immunity. PMID:27573825

Endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 controls B-cell immunity through degradation of the death receptor CD95/Fas.

PubMed

Kong, Sinyi; Yang, Yi; Xu, Yuanming; Wang, Yajun; Zhang, Yusi; Melo-Cardenas, Johanna; Xu, Xiangping; Gao, Beixue; Thorp, Edward B; Zhang, Donna D; Zhang, Bin; Song, Jianxun; Zhang, Kezhong; Zhang, Jianning; Zhang, Jinping; Li, Huabin; Fang, Deyu

2016-09-13

Humoral immunity involves multiple checkpoints during B-cell development, maturation, and activation. The cell death receptor CD95/Fas-mediated apoptosis plays a critical role in eliminating the unwanted activation of B cells by self-reactive antigens and in maintaining B-cell homeostasis through activation-induced B-cell death (AICD). The molecular mechanisms controlling AICD remain largely undefined. Herein, we show that the E3 ubiquitin ligase Hrd1 protected B cells from activation-induced cell death by degrading the death receptor Fas. Hrd1-null B cells exhibited high Fas expression during activation and rapidly underwent Fas-mediated apoptosis, which could be largely inhibited by FasL neutralization. Fas mutation in Hrd1 KO mice abrogated the increase in B-cell AICD. We identified Hrd1 as the first E3 ubiquitin ligase of the death receptor Fas and Hrd1-mediated Fas destruction as a molecular mechanism in regulating B-cell immunity.

Multiple interactions drive adaptor-mediated recruitment of the ubiquitin ligase rsp5 to membrane proteins in vivo and in vitro.

PubMed

Sullivan, James A; Lewis, Michael J; Nikko, Elina; Pelham, Hugh R B

2007-07-01

Recognition of membrane proteins by the Nedd4/Rsp5 ubiquitin ligase family is a critical step in their targeting to the multivesicular body pathway. Some substrates contain "PY" motifs (PPxY), which bind to WW domains in the ligase. Others lack PY motifs and instead rely on adaptors that recruit the ligase to them. To investigate the mechanism of adaptor-mediated ubiquitination, we have characterized the interactions between the adaptor Bsd2, the ubiquitin ligase Rsp5, and the membrane proteins Cps1, Tre1, and Smf1 from Saccharomyces cerevisiae. We have reconstituted adaptor-mediated modification of Cps1 and Tre1 in vitro, and we show that two PY motifs in Bsd2 and two WW domains (WW2 and WW3) in Rsp5 are crucial for this. The binding of a weak noncanonical DMAPSY motif in Bsd2 to WW3 is an absolute requirement for Bsd2 adaptor function. We show that sorting of the manganese transporter Smf1, which requires both Bsd2 and Tre1, depends upon two PY motifs in Bsd2 and one motif in Tre1 but only two WW domains in Rsp5. We suggest that sequential assembly of first a Bsd2/Rsp5 complex, then a Tre1/Bsd2/Rsp5 complex followed by a rearrangement of PY-WW interactions is required for the ubiquitination of Smf1.

Multiple Interactions Drive Adaptor-Mediated Recruitment of the Ubiquitin Ligase Rsp5 to Membrane Proteins In Vivo and In Vitro

PubMed Central

Sullivan, James A.; Lewis, Michael J.; Nikko, Elina

2007-01-01

Recognition of membrane proteins by the Nedd4/Rsp5 ubiquitin ligase family is a critical step in their targeting to the multivesicular body pathway. Some substrates contain “PY” motifs (PPxY), which bind to WW domains in the ligase. Others lack PY motifs and instead rely on adaptors that recruit the ligase to them. To investigate the mechanism of adaptor-mediated ubiquitination, we have characterized the interactions between the adaptor Bsd2, the ubiquitin ligase Rsp5, and the membrane proteins Cps1, Tre1, and Smf1 from Saccharomyces cerevisiae. We have reconstituted adaptor-mediated modification of Cps1 and Tre1 in vitro, and we show that two PY motifs in Bsd2 and two WW domains (WW2 and WW3) in Rsp5 are crucial for this. The binding of a weak noncanonical DMAPSY motif in Bsd2 to WW3 is an absolute requirement for Bsd2 adaptor function. We show that sorting of the manganese transporter Smf1, which requires both Bsd2 and Tre1, depends upon two PY motifs in Bsd2 and one motif in Tre1 but only two WW domains in Rsp5. We suggest that sequential assembly of first a Bsd2/Rsp5 complex, then a Tre1/Bsd2/Rsp5 complex followed by a rearrangement of PY–WW interactions is required for the ubiquitination of Smf1. PMID:17429078

Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration.

PubMed

Del Prete, Dolores; Rice, Richard C; Rajadhyaksha, Anjali M; D'Adamio, Luciano

2016-08-12

The amyloid precursor protein (APP), whose mutations cause Alzheimer disease, plays an important in vivo role and facilitates transmitter release. Because the APP cytosolic region (ACR) is essential for these functions, we have characterized its brain interactome. We found that the ACR interacts with proteins that regulate the ubiquitin-proteasome system, predominantly with the E3 ubiquitin-protein ligases Stub1, which binds the NH2 terminus of the ACR, and CRL4(CRBN), which is formed by Cul4a/b, Ddb1, and Crbn, and interacts with the COOH terminus of the ACR via Crbn. APP shares essential functions with APP-like protein-2 (APLP2) but not APP-like protein-1 (APLP1). Noteworthy, APLP2, but not APLP1, interacts with Stub1 and CRL4(CRBN), pointing to a functional pathway shared only by APP and APLP2. In vitro ubiquitination/ubiquitome analysis indicates that these E3 ligases are enzymatically active and ubiquitinate the ACR residues Lys(649/650/651/676/688) Deletion of Crbn reduces ubiquitination of Lys(676) suggesting that Lys(676) is physiologically ubiquitinated by CRL4(CRBN) The ACR facilitated in vitro ubiquitination of presynaptic proteins that regulate exocytosis, suggesting a mechanism by which APP tunes transmitter release. Other dementia-related proteins, namely Tau and apoE, interact with and are ubiquitinated via the ACR in vitro This, and the evidence that CRBN and CUL4B are linked to intellectual disability, prompts us to hypothesize a pathogenic mechanism, in which APP acts as a modulator of E3 ubiquitin-protein ligase(s), shared by distinct neuronal disorders. The well described accumulation of ubiquitinated protein inclusions in neurodegenerative diseases and the link between the ubiquitin-proteasome system and neurodegeneration make this concept plausible. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The glomuvenous malformation protein Glomulin binds Rbx1 and regulates cullin RING ligase-mediated turnover of Fbw7.

PubMed

Tron, Adriana E; Arai, Takehiro; Duda, David M; Kuwabara, Hiroshi; Olszewski, Jennifer L; Fujiwara, Yuko; Bahamon, Brittany N; Signoretti, Sabina; Schulman, Brenda A; DeCaprio, James A

2012-04-13

Fbw7, a substrate receptor for Cul1-RING-ligase (CRL1), facilitates the ubiquitination and degradation of several proteins, including Cyclin E and c-Myc. In spite of much effort, the mechanisms underlying Fbw7 regulation are mostly unknown. Here, we show that Glomulin (Glmn), a protein found mutated in the vascular disorder glomuvenous malformation (GVM), binds directly to the RING domain of Rbx1 and inhibits its E3 ubiquitin ligase activity. Loss of Glmn in a variety of cells, tissues, and GVM lesions results in decreased levels of Fbw7 and increased levels of Cyclin E and c-Myc. The increased turnover of Fbw7 is dependent on CRL and proteasome activity, indicating that Glmn modulates the E3 activity of CRL1(Fbw7). These data reveal an unexpected functional connection between Glmn and Rbx1 and demonstrate that defective regulation of Fbw7 levels contributes to GVM. Copyright © 2012 Elsevier Inc. All rights reserved.

The Glomuvenous Malformation Protein Glomulin Binds Rbx1 and Regulates Cullin RING Ligase-Mediated Turnover of Fbw7

PubMed Central

Tron, Adriana E.; Arai, Takehiro; Duda, David M.; Kuwabara, Hiroshi; Olszewski, Jennifer L.; Fujiwara, Yuko; Bahamon, Brittany N.; Signoretti, Sabina; Schulman, Brenda A.; DeCaprio, James A.

2012-01-01

SUMMARY Fbw7, a substrate receptor for Cul1-RING-ligase (CRL1), facilitates the ubiquitination and degradation of several proteins including Cyclin E and c-Myc. In spite of much effort, the mechanisms underlying Fbw7 regulation are mostly unknown. Here we show that Glomulin (Glmn), a protein found mutated in the vascular disorder Glomuvenous Malformation (GVM), binds directly to the RING domain of Rbx1 and inhibits its E3 ubiquitin ligase activity. Loss of Glmn in a variety of cells, tissues and GVM lesions results in decreased levels of Fbw7 and increased levels of Cyclin E and c-Myc. The increased turnover of Fbw7 is dependent on CRL and proteasome activity indicating that Glmn modulates the E3 activity of CRL1Fbw7. These data reveal an unexpected functional connection between Glmn and Rbx1 and demonstrate that defective regulation of Fbw7 levels contributes to GVM. PMID:22405651

Structure of the Siz/PIAS SUMO E3 ligase Siz1 and determinants required for SUMO modification of PCNA

PubMed Central

Yunus, Ali A.; Lima, Christopher D.

2009-01-01

Summary Siz1 is a founding member of the Siz/PIAS RING family of SUMO E3 ligases. The x-ray structure of an active Siz1 ligase revealed an elongated tripartite architecture comprised of an N-terminal PINIT domain, a central zinc-containing RING-like SP-RING domain, and a C-terminal domain we term the SP-CTD. Structure-based mutational analysis and biochemical studies show that the SP-RING and SP-CTD are required for activation of the E2~SUMO thioester while the PINIT domain is essential for redirecting SUMO conjugation to the proliferating cell nuclear antigen (PCNA) at lysine 164, a non-consensus lysine residue that is not modified by the SUMO E2 in the absence of Siz1. Mutational analysis of Siz1 and PCNA revealed surfaces on both proteins that are required for efficient SUMO modification of PCNA in vitro and in vivo. PMID:19748360

Developing structure-activity relationships from an HTS hit for inhibition of the Cks1-Skp2 protein-protein interaction.

PubMed

Singh, Rajinder; Sran, Arvinder; Carroll, David C; Huang, Jianing; Tsvetkov, Lyuben; Zhou, Xiulan; Sheung, Julie; McLaughlin, John; Issakani, Sarkiz D; Payan, Donald G; Shaw, Simon J

2015-11-15

Structure-activity relationships have been developed around 5-bromo-8-toluylsulfonamidoquinoline 1 a hit compound in an assay for the interaction of the E3 ligase Skp2 with Cks1, part of the SCF ligase complex. Disruption of this protein-protein interaction results in higher levels of CDK inhibitor p27, which can act as a tumor suppressor. The results of the SAR developed highlight the relationship between the sulfonamide and quinoline nitrogen, while also suggesting that an aryl substituent at the 5-position of the quinoline ring contributes to the potency in the interaction assay. Compounds showing potency in the interaction assay result in greater levels of p27 and have been shown to inhibit cell growth of two p27 sensitive tumor cell lines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Both sides of the same coin: Rac1 splicing regulating by EGF signaling.

PubMed

Fu, Xiang-Dong

2017-04-01

EGF, a well-studied mitogen for cancer cells, is revealed to induce an E3 ubiquitin ligase adaptor SPSB1, which recruits the Elongin B/C-Collin complex to trigger ubiquitylation of the negative splicing regulator hnRNP A1. This event is synergized with EGF-activated SR proteins to alter alternative splicing of a key small GTPase Rac1 to enhance cell migration, highlighting converging EGF signals on both negative and positive splicing regulators to jointly promote a key cancer pathway.

The Role of Ubiquitin E3 Ligase SCF-SKP2 in Prostate Cancer Development

DTIC Science & Technology

2007-02-01

2004; 303:1371-4. 26. Nag A, Bondar T, Shiv S, Raychaudhuri P. The xeroderma pigmentosum group E gene product DDB2 is a specific target of cullin 4A...ubiquitin ligases. Nat Rev Mol Cell Biol 2005; 6:9-20. 2. Nag A, Bondar T, Shiv S, Raychaudhuri P. The xeroderma pigmentosum group E gene product DDB2 is... xeroderma pigmentosum group E patient and the subsequent inability to bind DDB1 (ref. 16). This motif is present in most of the WDR proteins we found (see

E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6

PubMed Central

Hibbert, Richard G.; Huang, Anding; Boelens, Rolf; Sixma, Titia K.

2011-01-01

In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyse either monoubiquitination or ubiquitin chain formation. The E2/E3 complex Rad6/Rad18 exclusively monoubiquitinates the proliferating cell nuclear antigen (PCNA) to signal for “error prone” DNA damage tolerance, whereas a different set of conjugation enzymes is required for ubiquitin chain formation on PCNA. Here we show that human E2 enzyme Rad6b is intrinsically capable of catalyzing ubiquitin chain formation. This activity is prevented during PCNA ubiquitination by the interaction of Rad6 with E3 enzyme Rad18. Using NMR and X-ray crystallography we show that the R6BD of Rad18 inhibits this activity by competing with ubiquitin for a noncovalent “backside” binding site on Rad6. Our findings provide mechanistic insights into how E3 enzymes can regulate the ubiquitin conjugation process. PMID:21422291

COP1, a negative regulator of photomorphogenesis, positively regulates plant disease resistance via double-stranded RNA binding proteins

PubMed Central

Lim, Gah-Hyun; Zhu, Shifeng; Clavel, Marion; Yu, Keshun; Navarre, Duroy; Kachroo, Aardra; Deragon, Jean-Marc

2018-01-01

The E3 ubiquitin ligase COP1 (Constitutive Photomorphogenesis 1) is a well known component of the light-mediated plant development that acts as a repressor of photomorphogenesis. Here we show that COP1 positively regulates defense against turnip crinkle virus (TCV) and avrRPM1 bacteria by contributing to stability of resistance (R) protein HRT and RPM1, respectively. HRT and RPM1 levels and thereby pathogen resistance is significantly reduced in the cop1 mutant background. Notably, the levels of at least two double-stranded RNA binding (DRB) proteins DRB1 and DRB4 are reduced in the cop1 mutant background suggesting that COP1 affects HRT stability via its effect on the DRB proteins. Indeed, a mutation in either drb1 or drb4 resulted in degradation of HRT. In contrast to COP1, a multi-subunit E3 ligase encoded by anaphase-promoting complex (APC) 10 negatively regulates DRB4 and TCV resistance but had no effect on DRB1 levels. We propose that COP1-mediated positive regulation of HRT is dependent on a balance between COP1 and negative regulators that target DRB1 and DRB4. PMID:29513740

SCFJFK is a bona fide E3 ligase for ING4 and a potent promoter of the angiogenesis and metastasis of breast cancer

PubMed Central

Yan, Ruorong; He, Lin; Li, Zhongwu; Han, Xiao; Liang, Jing; Si, Wenzhe; Chen, Zhe; Li, Lei; Xie, Guojia; Li, Wanjin; Wang, Peiyan; Lei, Liandi; Zhang, Hongquan; Pei, Fei; Cao, Dengfeng

2015-01-01

Loss of function/dysregulation of inhibitor of growth 4 (ING4) and hyperactivation of NF-κB are frequent events in many types of human malignancies. However, the molecular mechanisms underlying these remarkable aberrations are not understood. Here, we report that ING4 is physically associated with JFK. We demonstrated that JFK targets ING4 for ubiquitination and degradation through assembly of an Skp1–Cul1–F-box (SCF) complex. We showed that JFK-mediated ING4 destabilization leads to the hyperactivation of the canonical NF-κB pathway and promotes angiogenesis and metastasis of breast cancer. Significantly, the expression of JFK is markedly up-regulated in breast cancer, and the level of JFK is negatively correlated with that of ING4 and positively correlated with an aggressive clinical behavior of breast carcinomas. Our study identified SCFJFK as a bona fide E3 ligase for ING4 and unraveled the JFK–ING4–NF-κB axis as an important player in the development and progression of breast cancer, supporting the pursuit of JFK as a potential target for breast cancer intervention. PMID:25792601

CAR Suppresses Hepatic Gluconeogenesis by Facilitating the Ubiquitination and Degradation of PGC1α

PubMed Central

Gao, Jie; Yan, Jiong; Xu, Meishu; Ren, Songrong

2015-01-01

The constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α) are master regulators of drug metabolism and gluconeogenesis, respectively. In supporting the cross talk between drug metabolism and energy metabolism, activation of CAR has been shown to suppress hepatic gluconeogenesis and ameliorate hyperglycemia in vivo, but the underlying molecular mechanism remains elusive. In this study, we demonstrated that CAR suppressed hepatic gluconeogenic gene expression through posttranslational regulation of the subcellular localization and degradation of PGC1α. Activated CAR translocated into the nucleus and served as an adaptor protein to recruit PGC1α to the Cullin1 E3 ligase complex for ubiquitination. The interaction between CAR and PGC1α also led to their sequestration within the promyelocytic leukemia protein-nuclear bodies, where PGC1α and CAR subsequently underwent proteasomal degradation. Taken together, our findings revealed an unexpected function of CAR in recruiting an E3 ligase and targeting the gluconeogenic activity of PGC1α. Both drug metabolism and gluconeogenesis are energy-demanding processes. The negative regulation of PGC1α by CAR may represent a cellular adaptive mechanism to accommodate energy-restricted conditions. PMID:26407237

CAR Suppresses Hepatic Gluconeogenesis by Facilitating the Ubiquitination and Degradation of PGC1α.

PubMed

Gao, Jie; Yan, Jiong; Xu, Meishu; Ren, Songrong; Xie, Wen

2015-11-01

The constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α) are master regulators of drug metabolism and gluconeogenesis, respectively. In supporting the cross talk between drug metabolism and energy metabolism, activation of CAR has been shown to suppress hepatic gluconeogenesis and ameliorate hyperglycemia in vivo, but the underlying molecular mechanism remains elusive. In this study, we demonstrated that CAR suppressed hepatic gluconeogenic gene expression through posttranslational regulation of the subcellular localization and degradation of PGC1α. Activated CAR translocated into the nucleus and served as an adaptor protein to recruit PGC1α to the Cullin1 E3 ligase complex for ubiquitination. The interaction between CAR and PGC1α also led to their sequestration within the promyelocytic leukemia protein-nuclear bodies, where PGC1α and CAR subsequently underwent proteasomal degradation. Taken together, our findings revealed an unexpected function of CAR in recruiting an E3 ligase and targeting the gluconeogenic activity of PGC1α. Both drug metabolism and gluconeogenesis are energy-demanding processes. The negative regulation of PGC1α by CAR may represent a cellular adaptive mechanism to accommodate energy-restricted conditions.

Degradation of Hof1 by SCFGrr1 is important for actomyosin contraction during cytokinesis in yeast

PubMed Central

Blondel, Marc; Bach, Stéphane; Bamps, Sophie; Dobbelaere, Jeroen; Wiget, Philippe; Longaretti, Céline; Barral, Yves; Meijer, Laurent; Peter, Matthias

2005-01-01

SCF-type (SCF: Skp1–Cullin–F-box protein complex) E3 ligases regulate ubiquitin-dependent degradation of many cell cycle regulators, mainly at the G1/S transition. Here, we show that SCFGrr1 functions during cytokinesis by degrading the PCH protein Hof1. While Hof1 is required early in mitosis to assemble a functional actomyosin ring, it is specifically degraded late in mitosis and remains unstable during the entire G1 phase of the cell cycle. Degradation of Hof1 depends on its PEST motif and a functional 26S proteasome. Interestingly, degradation of Hof1 is independent of APCCdh1, but instead requires the SCFGrr1 E3 ligase. Grr1 is recruited to the mother–bud neck region after activation of the mitotic-exit network, and interacts with Hof1 in a PEST motif-dependent manner. Our results also show that downregulation of Hof1 at the end of mitosis is necessary to allow efficient contraction of the actomyosin ring and cell separation during cytokinesis. SCFGrr1-mediated degradation of Hof1 may thus represent a novel mechanism to couple exit from mitosis with initiation of cytokinesis. PMID:15775961

FANCL ubiquitinates β-catenin and enhances its nuclear function.

PubMed

Dao, Kim-Hien T; Rotelli, Michael D; Petersen, Curtis L; Kaech, Stefanie; Nelson, Whitney D; Yates, Jane E; Hanlon Newell, Amy E; Olson, Susan B; Druker, Brian J; Bagby, Grover C

2012-07-12

Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of β-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates β-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, β-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate β-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34(+) stem and progenitor cells results in fewer β-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/β-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss.

Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70.

PubMed

Boboila, Cristian; Jankovic, Mila; Yan, Catherine T; Wang, Jing H; Wesemann, Duane R; Zhang, Tingting; Fazeli, Alex; Feldman, Lauren; Nussenzweig, Andre; Nussenzweig, Michel; Alt, Frederick W

2010-02-16

Class switch recombination (CSR) in B lymphocytes is initiated by introduction of multiple DNA double-strand breaks (DSBs) into switch (S) regions that flank immunoglobulin heavy chain (IgH) constant region exons. CSR is completed by joining a DSB in the donor S mu to a DSB in a downstream acceptor S region (e.g., S gamma1) by end-joining. In normal cells, many CSR junctions are mediated by classical nonhomologous end-joining (C-NHEJ), which employs the Ku70/80 complex for DSB recognition and XRCC4/DNA ligase 4 for ligation. Alternative end-joining (A-EJ) mediates CSR, at reduced levels, in the absence of C-NHEJ, even in combined absence of Ku70 and ligase 4, demonstrating an A-EJ pathway totally distinct from C-NHEJ. Multiple DSBs are introduced into S mu during CSR, with some being rejoined or joined to each other to generate internal switch deletions (ISDs). In addition, S-region DSBs can be joined to other chromosomes to generate translocations, the level of which is increased by absence of a single C-NHEJ component (e.g., XRCC4). We asked whether ISD and S-region translocations occur in the complete absence of C-NHEJ (e.g., in Ku70/ligase 4 double-deficient B cells). We found, unexpectedly, that B-cell activation for CSR generates substantial ISD in both S mu and S gamma1 and that ISD in both is greatly increased by the absence of C-NHEJ. IgH chromosomal translocations to the c-myc oncogene also are augmented in the combined absence of Ku70 and ligase 4. We discuss the implications of these findings for A-EJ in normal and abnormal DSB repair.

HACE1-dependent protein degradation provides cardiac protection in response to haemodynamic stress

NASA Astrophysics Data System (ADS)

Zhang, Liyong; Chen, Xin; Sharma, Parveen; Moon, Mark; Sheftel, Alex D.; Dawood, Fayez; Nghiem, Mai P.; Wu, Jun; Li, Ren-Ke; Gramolini, Anthony O.; Sorensen, Poul H.; Penninger, Josef M.; Brumell, John H.; Liu, Peter P.

2014-03-01

The HECT E3 ubiquitin ligase HACE1 is a tumour suppressor known to regulate Rac1 activity under stress conditions. HACE1 is increased in the serum of patients with heart failure. Here we show that HACE1 protects the heart under pressure stress by controlling protein degradation. Hace1 deficiency in mice results in accelerated heart failure and increased mortality under haemodynamic stress. Hearts from Hace1-/- mice display abnormal cardiac hypertrophy, left ventricular dysfunction, accumulation of LC3, p62 and ubiquitinated proteins enriched for cytoskeletal species, indicating impaired autophagy. Our data suggest that HACE1 mediates p62-dependent selective autophagic turnover of ubiquitinated proteins by its ankyrin repeat domain through protein-protein interaction, which is independent of its E3 ligase activity. This would classify HACE1 as a dual-function E3 ligase. Our finding that HACE1 has a protective function in the heart in response to haemodynamic stress suggests that HACE1 may be a potential diagnostic and therapeutic target for heart disease.

The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in rice.

PubMed

Park, Chan-Ho; Chen, Songbiao; Shirsekar, Gautam; Zhou, Bo; Khang, Chang Hyun; Songkumarn, Pattavipha; Afzal, Ahmed J; Ning, Yuese; Wang, Ruyi; Bellizzi, Maria; Valent, Barbara; Wang, Guo-Liang

2012-11-01

Although the functions of a few effector proteins produced by bacterial and oomycete plant pathogens have been elucidated in recent years, information for the vast majority of pathogen effectors is still lacking, particularly for those of plant-pathogenic fungi. Here, we show that the avirulence effector AvrPiz-t from the rice blast fungus Magnaporthe oryzae preferentially accumulates in the specialized structure called the biotrophic interfacial complex and is then translocated into rice (Oryza sativa) cells. Ectopic expression of AvrPiz-t in transgenic rice suppresses the flg22- and chitin-induced generation of reactive oxygen species (ROS) and enhances susceptibility to M. oryzae, indicating that AvrPiz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Interaction assays show that AvrPiz-t suppresses the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 and that, in return, APIP6 ubiquitinates AvrPiz-t in vitro. Interestingly, agroinfection assays reveal that AvrPiz-t and AvrPiz-t Interacting Protein 6 (APIP6) are both degraded when coexpressed in Nicotiana benthamiana. Silencing of APIP6 in transgenic rice leads to a significant reduction of flg22-induced ROS generation, suppression of defense-related gene expression, and enhanced susceptibility of rice plants to M. oryzae. Taken together, our results reveal a mechanism in which a fungal effector targets the host ubiquitin proteasome system for the suppression of PAMP-triggered immunity in plants.

Enhanced ubiquitination of cytoskeletal proteins in pressure overloaded myocardium is accompanied by changes in specific E3 ligases.

PubMed

Balasubramanian, Sundaravadivel; Mani, Santhoshkumar; Shiraishi, Hirokazu; Johnston, Rebecca K; Yamane, Kentaro; Willey, Christopher D; Cooper, George; Tuxworth, William J; Kuppuswamy, Dhandapani

2006-10-01

Ubiquitin conjugation of proteins is critical for cell homeostasis and contributes to both cell survival and death. Here we studied ubiquitination of proteins in pressure overloaded (PO) myocardium in the context of cardiomyocyte survival. Analysis using a feline right ventricular pressure overload (RVPO) model revealed a robust and transient increase in ubiquitination of proteins present in the Triton X-100-insoluble fraction in 24 to 48 h PO myocardium, and confocal micrographs indicate this increase in ubiquitination occurs subsarcolemmaly near the intercalated disc area of cardiomyocytes. The ubiquitination was accompanied by changes in E3 ligases including Cbl, E6AP, Mdm2 and cIAP in the same period of PO, although atrophy-related E3 ligases, MuRF1 and MuRF3 were unaltered. Furthermore, Cbl displayed a substantial increase in both levels of expression and tyrosine phosphorylation in 48 h PO myocardium. Confocal studies revealed enrichment of Cbl at the intercalated discs of 48 h PO cardiomyocytes, as evidenced by its colocalization with N-cadherin. Although apoptosis was observed in 48 h PO myocardium by TUNEL staining, cardiomyocytes showing ubiquitin staining were not positive for TUNEL staining. Furthermore, 48 h PO resulted in the phosphorylation of inhibitor of nuclear factor kappa B (IkappaB), suggesting its ubiquitin-mediated degradation and the nuclear localization of NFkappaB for the expression of specific cell survival factors such as cIAPs. Together these data indicate that increased levels of E3 ligases that regulate cell homeostasis and promote cell survival could ubiquitinate multiple cytoskeletal protein targets and that these events that occur during the early phase of PO may contribute to both cardiomyocyte survival and hypertrophy.

RING E3 ligases: key regulatory elements are involved in abiotic stress responses in plants.

PubMed

Cho, Seok Keun; Ryu, Moon Young; Kim, Jong Hum; Hong, Jeong Soo; Oh, Tae Rin; Kim, Woo Taek; Yang, Seong Wook

2017-08-01

Plants are constantly exposed to a variety of abiotic stresses, such as drought, heat, cold, flood, and salinity. To survive under such unfavorable conditions, plants have evolutionarily developed their own resistant-mechanisms. For several decades, many studies have clarified specific stress response pathways of plants through various molecular and genetic studies. In particular, it was recently discovered that ubiquitin proteasome system (UPS), a regulatory mechanism for protein turn over, is greatly involved in the stress responsive pathways. In the UPS, many E3 ligases play key roles in recognizing and tethering poly-ubiquitins on target proteins for subsequent degradation by the 26S proteasome. Here we discuss the roles of RING ligases that have been defined in related to abiotic stress responses in plants. [BMB Reports 2017; 50(8): 393-400].

Deficiency of UBE2T, the E2 Ubiquitin Ligase Necessary for FANCD2 and FANCI Ubiquitination, Causes FA-T Subtype of Fanconi Anemia.

PubMed

Rickman, Kimberly A; Lach, Francis P; Abhyankar, Avinash; Donovan, Frank X; Sanborn, Erica M; Kennedy, Jennifer A; Sougnez, Carrie; Gabriel, Stacey B; Elemento, Olivier; Chandrasekharappa, Settara C; Schindler, Detlev; Auerbach, Arleen D; Smogorzewska, Agata

2015-07-07

Fanconi anemia (FA) is a rare bone marrow failure and cancer predisposition syndrome resulting from pathogenic mutations in genes encoding proteins participating in the repair of DNA interstrand crosslinks (ICLs). Mutations in 17 genes (FANCA-FANCS) have been identified in FA patients, defining 17 complementation groups. Here, we describe an individual presenting with typical FA features who is deficient for the ubiquitin-conjugating enzyme (E2), UBE2T. UBE2T is known to interact with FANCL, the E3 ubiquitin-ligase component of the multiprotein FA core complex, and is necessary for the monoubiquitination of FANCD2 and FANCI. Proband fibroblasts do not display FANCD2 and FANCI monoubiquitination, do not form FANCD2 foci following treatment with mitomycin C, and are hypersensitive to crosslinking agents. These cellular defects are complemented by expression of wild-type UBE2T, demonstrating that deficiency of the protein UBE2T can lead to Fanconi anemia. UBE2T gene gains an alias of FANCT. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Spatiotemporal Regulation of the Anaphase-Promoting Complex in Mitosis

PubMed Central

Sivakumar, Sushama; Gorbsky, Gary J

2015-01-01

The appropriate timing of events that lead to chromosome segregation during mitosis and cytokinesis is essential to prevent aneuploidy, and defects in these processes can contribute to tumorigenesis. Key mitotic regulators are controlled through ubiquitylation and proteasome-mediated degradation. The Anaphase-Promoting Complex or Cyclosome (APC/C) is an E3 ubiquitin ligase that has a crucial function in the regulation of the mitotic cell cycle, particularly at the onset of anaphase and during mitotic exit. Co-activator proteins, inhibitor proteins, protein kinases and phosphatases interact with the APC/C to temporally and spatially control its activity and thus ensure accurate timing of mitotic events. PMID:25604195

TRIM41-Mediated Ubiquitination of Nucleoprotein Limits Influenza A Virus Infection.

PubMed

Patil, Girish; Zhao, Mengmeng; Song, Kun; Hao, Wenzhuo; Bouchereau, Daniel; Wang, Lingyan; Li, Shitao

2018-06-13

Influenza A virus (IAV) is a highly transmissible respiratory pathogen and a major cause of morbidity and mortality around the world. Nucleoprotein (NP) is an abundant IAV protein essential for multiple steps of viral life cycle. Our recent proteomic study of the IAV-host interaction network found that the tripartite motif containing 41 (TRIM41), a ubiquitin E3 ligase, interacted with NP. However, the role of TRIM41 in IAV infection is unknown. Here, we report that TRIM41 interacts with NP through its SPRY domain. Furthermore, TRIM41 is constitutively expressed in lung epithelial cells and overexpression of TRIM41 inhibits IAV infection. Conversely, RNA interference (RNAi) and knockout of TRIM41 increase host susceptibility to IAV infection. As a ubiquitin E3 ligase, TRIM41 ubiquitinates NP in vitro and in cells. The TRIM41 mutant lacking E3 ligase activity fails to inhibit IAV infection, suggesting that the E3 ligase activity is indispensable for TRIM41 antiviral function. Mechanistic analysis further revealed that the polyubiquitination leads to NP protein degradation and viral inhibition. Taken together, TRIM41 is a constitutively expressed intrinsic IAV restriction factor that targets NP for ubiquitination and protein degradation. IMPORTANCE Influenza control strategies rely on annual immunization and require frequent updates of the vaccine, which are not always a foolproof process. Furthermore, the current antivirals are also losing effectiveness as new viral strains are often refractory to conventional treatments. Thus, there is an urgent need to find new antiviral mechanisms and develop therapeutic drugs based on these mechanisms. Targeting the virus-host interface is an emerging new strategy because host factors controlling viral replication activity will be ideal candidates and cellular proteins are less likely to mutate under drug-mediated selective pressure. Here, we show that the ubiquitin E3 ligase TRIM41 is an intrinsic host restriction factor to IAV. TRIM41 directly binds the viral nucleoprotein and targets it for ubiquitination and proteasomal degradation, thereby limiting viral infection. Exploitation of this natural defense pathway may open new avenues to develop influenza antivirals. Copyright © 2018 American Society for Microbiology.

Electronic structure and optical properties of noncentrosymmetric LiGaSe2: Experimental measurements and DFT band structure calculations

NASA Astrophysics Data System (ADS)

Lavrentyev, A. A.; Gabrelian, B. V.; Vu, V. T.; Ananchenko, L. N.; Isaenko, L. I.; Yelisseyev, A. P.; Khyzhun, O. Y.

2017-04-01

We report on measurements of X-ray photoelectron (XP) spectra for pristine and Ar+ ion-irradiated surfaces of LiGaSe2 single crystal grown by Bridgman-Stockbarger method. Electronic structure of the LiGaSe2 compound is studied from a theoretical and experimental viewpoint. In particular, total and partial densities of states of LiGaSe2 are investigated by density functional theory (DFT) calculations employing the augmented plane wave + local orbitals (APW + lo) method and they are verified by data of X-ray spectroscopy measurements. The DFT calculations indicate that the main contributors to the valence band of LiGaSe2 are the Se 4p states, which contribute mainly at the top and in the upper portion of the valence band, with also essential contributions of these states in the lower portion of the band. Other substantial contributions to the valence band of LiGaSe2 emerge from the Ga 4s and Ga 4p states contributing mainly at the lower ant upper portions of the valence band, respectively. With respect to the conduction band, the calculations indicate that its bottom is composed mainly from contributions of the unoccupied Ga s and Se p states. The present calculations are confirmed experimentally when comparing the XP valence-band spectrum of the LiGaS2 single crystal on a common energy scale with the X-ray emission bands representing the energy distribution of the Ga 4p and Se 4p states. Measurements of the fundamental absorption edges at room temperature reveal that bandgap value, Eg, of LiGaSe2 is equal to 3.47 eV and the Eg value increases up to 3.66 eV when decreasing temperature to 80 K. The main optical characteristics of the LiGaSe2 compound are clarified by the DFT calculations.

BRCC36, a Novel Subunit of a BRCA1 E3 Ubiquitin Ligase Complex: Candidates for BRCA3

DTIC Science & Technology

2008-06-01

Biocomputing 1:724. Hughes-Davies L, Huntsman D, Ruas M, Fuks F, Bye J, Chin SF, Milner J, Brown LA, Hsu F, Gilks B and others. 2003. EMSY links the...16. Hughes-Davies L, Huntsman D, Ruas M, et al. EMSY links the BRCA2 pathway to sporadic breast and ovarian cancer. Cell 2003;115:523–35. 17...BRCA1 germ- line mutations involving splice donor sites. Genet Test 8:133–138. Campos B, Diez O, Domenech M, Baena M, Balmana J, Sanz J, Ramirez A

Identification of factors required for m6 A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI.

PubMed

Růžička, Kamil; Zhang, Mi; Campilho, Ana; Bodi, Zsuzsanna; Kashif, Muhammad; Saleh, Mária; Eeckhout, Dominique; El-Showk, Sedeer; Li, Hongying; Zhong, Silin; De Jaeger, Geert; Mongan, Nigel P; Hejátko, Jan; Helariutta, Ykä; Fray, Rupert G

2017-07-01

N6-adenosine methylation (m 6 A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m 6 A writer proteins in Arabidopsis thaliana. The components required for m 6 A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m 6 A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m 6 A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m 6 A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Apple RING E3 ligase MdMIEL1 inhibits anthocyanin accumulation by ubiquitinating and degrading MdMYB1 protein.

PubMed

An, Jian-Ping; Liu, Xin; Li, Hao-Hao; You, Chun-Xiang; Wang, Xiao-Fei; Hao, Yu-Jin

2017-11-01

MdMYB1 is an important regulator for anthocyanin accumulation in apple (Malus × domestica). Here, an apple RING E3 ligase, MdMIEL1, was screened out as a partner of MdMYB1 with a yeast two-hybrid approach. Pull-down, bimolecular fluorescence complementation and coimmunoprecipitation assays further verified the interaction between MdMIEL1 and MdMYB1 proteins. Subsequently, in vitro and in vivo experiments indicated that MdMIEL1 functioned as a ubiquitin E3 ligase to ubiquitinate MdMYB1 protein, followed by degradation through a 26S proteasome pathway. Furthermore, transgenic studies in apple calli and Arabidopsis demonstrated that MdMIEL1 negatively regulated anthocyanin accumulation by modulating the degradation of MdMYB1 protein. Taken together, our findings provide a new insight into the molecular mechanism by which MdMIEL1 negatively regulates anthocyanin biosynthesis by ubiquitinating and degrading MdMYB1 protein. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The β-catenin E3 ubiquitin ligase SIAH-1 is regulated by CSN5/JAB1 in CRC cells.

PubMed

Jumpertz, Sandra; Hennes, Thomas; Asare, Yaw; Vervoorts, Jörg; Bernhagen, Jürgen; Schütz, Anke K

2014-09-01

COP9 signalosome subunit 5 (CSN5) plays a decisive role in cellular processes such as cell cycle regulation and apoptosis via promoting protein degradation, gene transcription, and nuclear export. CSN5 regulates cullin-RING-E3 ligase (CRL) activity through its deNEDDylase function. It is overexpressed in several tumor entities, but its role in colorectal cancer (CRC) is poorly understood. Wnt/β-catenin signaling is aberrant in most CRC cells, resulting in increased levels of oncogenic β-catenin and thus tumor progression. Under physiological conditions, β-catenin levels are tightly regulated by continuous proteasomal degradation. We recently showed that knockdown of CSN5 in model and CRC cells results in decreased (phospho)-β-catenin levels. Reduced β-catenin levels were associated with an attenuated proliferation rate of different CRC cell types after CSN5 knockdown. The canonical Wnt pathway involves degradation of β-catenin by a β-TrCP1-containing E3 ligase, but is mostly non-functional in CRC cells. We thus hypothesized that alternative β-catenin degradation mediated by SIAH-1 (seven in absentia homolog-1), is responsible for the effect of CSN5 on β-catenin signaling in CRC cells. We found that SIAH-1 plays an essential role in β-catenin degradation in HCT116 CRC cells and that CSN5 affects β-catenin target gene expression in these cells. Of note, CSN5 affected SIAH-1 mRNA and SIAH-1 protein levels. Moreover, β-catenin and SIAH-1 form protein complexes with CSN5 in HCT116 cells. Lastly, we demonstrate that CSN5 promotes SIAH-1 degradation in HCT116 and SW480 cells and that this is associated with its deNEDDylase activity. In conclusion, we have identified a CSN5/β-catenin/SIAH-1 interaction network that might control β-catenin degradation in CRC cells. Copyright © 2014 Elsevier Inc. All rights reserved.

The Rice E3-Ubiquitin Ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 Modulates the Expression of ROOT MEANDER CURLING, a Gene Involved in Root Mechanosensing, through the Interaction with Two ETHYLENE-RESPONSE FACTOR Transcription Factors1

PubMed Central

Lourenço, Tiago F.; Serra, Tânia S.; Cordeiro, André M.; Swanson, Sarah J.; Gilroy, Simon; Saibo, Nelson J.M.; Oliveira, M. Margarida

2015-01-01

Plant roots can sense and respond to a wide diversity of mechanical stimuli, including touch and gravity. However, little is known about the signal transduction pathways involved in mechanical stimuli responses in rice (Oryza sativa). This work shows that rice root responses to mechanical stimuli involve the E3-ubiquitin ligase rice HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 (OsHOS1), which mediates protein degradation through the proteasome complex. The morphological analysis of the roots in transgenic RNA interference::OsHOS1 and wild-type plants, exposed to a mechanical barrier, revealed that the OsHOS1 silencing plants keep a straight root in contrast to wild-type plants that exhibit root curling. Moreover, it was observed that the absence of root curling in response to touch can be reverted by jasmonic acid. The straight root phenotype of the RNA interference::OsHOS1 plants was correlated with a higher expression rice ROOT MEANDER CURLING (OsRMC), which encodes a receptor-like kinase characterized as a negative regulator of rice root curling mediated by jasmonic acid. Using the yeast two-hybrid system and bimolecular fluorescence complementation assays, we showed that OsHOS1 interacts with two ETHYLENE-RESPONSE FACTOR transcription factors, rice ETHYLENE-RESPONSIVE ELEMENT BINDING PROTEIN1 (OsEREBP1) and rice OsEREBP2, known to regulate OsRMC gene expression. In addition, we showed that OsHOS1 affects the stability of both transcription factors in a proteasome-dependent way, suggesting that this E3-ubiquitin ligase targets OsEREBP1 and OsEREBP2 for degradation. Our results highlight the function of the proteasome in rice response to mechanical stimuli and in the integration of these signals, through hormonal regulation, into plant growth and developmental programs. PMID:26381316

Endoplasmic Reticulum Protein Quality Control Is Determined by Cooperative Interactions between Hsp/c70 Protein and the CHIP E3 Ligase*

PubMed Central

Matsumura, Yoshihiro; Sakai, Juro; Skach, William R.

2013-01-01

The C terminus of Hsp70 interacting protein (CHIP) E3 ligase functions as a key regulator of protein quality control by binding the C-terminal (M/I)EEVD peptide motif of Hsp/c70(90) with its N-terminal tetratricopeptide repeat (TPR) domain and facilitating polyubiquitination of misfolded client proteins via its C-terminal catalytic U-box. Using CFTR as a model client, we recently showed that the duration of the Hsc70-client binding cycle is a primary determinant of stability. However, molecular features that control CHIP recruitment to Hsp/c70, and hence the fate of the Hsp/c70 client, remain unknown. To understand how CHIP recognizes Hsp/c70, we utilized a dominant negative mutant in which loss of a conserved proline in the U-box domain (P269A) eliminates E3 ligase activity. In a cell-free reconstituted ER-associated degradation system, P269A CHIP inhibited Hsc70-dependent CFTR ubiquitination and degradation in a dose-dependent manner. Optimal inhibition required both the TPR and the U-box, indicating cooperativity between the two domains. Neither the wild type nor the P269A mutant changed the extent of Hsc70 association with CFTR nor the dissociation rate of the Hsc70-CFTR complex. However, the U-box mutation stimulated CHIP binding to Hsc70 while promoting CHIP oligomerization. CHIP binding to Hsc70 binding was also stimulated by the presence of an Hsc70 client with a preference for the ADP-bound state. Thus, the Hsp/c70 (M/I)EEVD motif is not a simple anchor for the TPR domain. Rather CHIP recruitment involves reciprocal allosteric interactions between its TPR and U-box domains and the substrate-binding and C-terminal domains of Hsp/c70. PMID:23990462

RNF185, a Novel Mitochondrial Ubiquitin E3 Ligase, Regulates Autophagy through Interaction with BNIP1

PubMed Central

Tang, Fei; Wang, Bin; Li, Na; Wu, Yanfang; Jia, Junying; Suo, Talin; Chen, Quan; Liu, Yong-Jun; Tang, Jie

2011-01-01

Autophagy is an evolutionarily conserved catabolic process that allows recycling of cytoplasmic organelles, such as mitochondria, to offer a bioenergetically efficient pathway for cell survival. Considerable progress has been made in characterizing mitochondrial autophagy. However, the dedicated ubiquitin E3 ligases targeting mitochondria for autophagy have not been revealed. Here we show that human RNF185 is a mitochondrial ubiquitin E3 ligase that regulates selective mitochondrial autophagy in cultured cells. The two C-terminal transmembrane domains of human RNF185 mediate its localization to mitochondrial outer membrane. RNF185 stimulates LC3II accumulation and the formation of autophagolysosomes in human cell lines. We further identified the Bcl-2 family protein BNIP1 as one of the substrates for RNF185. Human BNIP1 colocalizes with RNF185 at mitochondria and is polyubiquitinated by RNF185 through K63-based ubiquitin linkage in vivo. The polyubiquitinated BNIP1 is capable of recruiting autophagy receptor p62, which simultaneously binds both ubiquitin and LC3 to link ubiquitination and autophagy. Our study might reveal a novel RNF185-mediated mechanism for modulating mitochondrial homeostasis through autophagy. PMID:21931693

Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance

PubMed Central

Payne, Felicity; Colnaghi, Rita; Rocha, Nuno; Seth, Asha; Harris, Julie; Carpenter, Gillian; Bottomley, William E.; Wheeler, Eleanor; Wong, Stephen; Saudek, Vladimir; Savage, David; O’Rahilly, Stephen; Carel, Jean-Claude; Barroso, Inês; O’Driscoll, Mark; Semple, Robert

2014-01-01

Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5–6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase–deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress. PMID:25105364

Trypanosoma brucei RNA Editing Complex

PubMed Central

O'Hearn, Sean F.; Huang, Catherine E.; Hemann, Mike; Zhelonkina, Alevtina; Sollner-Webb, Barbara

2003-01-01

Maturation of Trypanosoma brucei mitochondrial mRNA involves massive posttranscriptional insertion and deletion of uridine residues. This RNA editing utilizes an enzymatic complex with seven major proteins, band I through band VII. We here use RNA interference (RNAi) to examine the band II and band V proteins. Band II is found essential for viability; it is needed to maintain the normal structure of the editing complex and to retain the band V ligase protein. Previously, band III was found essential for certain activities, including maintenance of the editing complex and retention of the band IV ligase protein. Thus, band II and band V form a protein pair with features analogous to the band III/band IV ligase pair. Since band V is specific for U insertion and since band IV is needed for U deletion, their parallel organization suggests that the editing complex has a pseudosymmetry. However, unlike the essential band IV ligase, RNAi to band V has only a morphological but no growth rate effect, suggesting that it is stimulatory but nonessential. Indeed, in vitro analysis of band V RNAi cell extract demonstrates that band IV can seal U insertion when band V is lacking. Thus, band IV ligase is the first activity of the basic editing complex shown able to serve in both forms of editing. Our studies also indicate that the U insertional portion may be less central in the editing complex than the corresponding U deletional portion. PMID:14560033

Cytoplasmic destruction of p53 by the endoplasmic reticulum-resident ubiquitin ligase ‘Synoviolin'

PubMed Central

Yamasaki, Satoshi; Yagishita, Naoko; Sasaki, Takeshi; Nakazawa, Minako; Kato, Yukihiro; Yamadera, Tadayuki; Bae, Eunkyung; Toriyama, Sayumi; Ikeda, Rie; Zhang, Lei; Fujitani, Kazuko; Yoo, Eunkyung; Tsuchimochi, Kaneyuki; Ohta, Tomohiko; Araya, Natsumi; Fujita, Hidetoshi; Aratani, Satoko; Eguchi, Katsumi; Komiya, Setsuro; Maruyama, Ikuro; Higashi, Nobuyo; Sato, Mitsuru; Senoo, Haruki; Ochi, Takahiro; Yokoyama, Shigeyuki; Amano, Tetsuya; Kim, Jaeseob; Gay, Steffen; Fukamizu, Akiyoshi; Nishioka, Kusuki; Tanaka, Keiji; Nakajima, Toshihiro

2007-01-01

Synoviolin, also called HRD1, is an E3 ubiquitin ligase and is implicated in endoplasmic reticulum -associated degradation. In mammals, Synoviolin plays crucial roles in various physiological and pathological processes, including embryogenesis and the pathogenesis of arthropathy. However, little is known about the molecular mechanisms of Synoviolin in these actions. To clarify these issues, we analyzed the profile of protein expression in synoviolin-null cells. Here, we report that Synoviolin targets tumor suppressor gene p53 for ubiquitination. Synoviolin sequestrated and metabolized p53 in the cytoplasm and negatively regulated its cellular level and biological functions, including transcription, cell cycle regulation and apoptosis. Furthermore, these p53 regulatory functions of Synoviolin were irrelevant to other E3 ubiquitin ligases for p53, such as MDM2, Pirh2 and Cop1, which form autoregulatory feedback loops. Our results provide novel insights into p53 signaling mediated by Synoviolin. PMID:17170702

Cytoplasmic destruction of p53 by the endoplasmic reticulum-resident ubiquitin ligase 'Synoviolin'.

PubMed

Yamasaki, Satoshi; Yagishita, Naoko; Sasaki, Takeshi; Nakazawa, Minako; Kato, Yukihiro; Yamadera, Tadayuki; Bae, Eunkyung; Toriyama, Sayumi; Ikeda, Rie; Zhang, Lei; Fujitani, Kazuko; Yoo, Eunkyung; Tsuchimochi, Kaneyuki; Ohta, Tomohiko; Araya, Natsumi; Fujita, Hidetoshi; Aratani, Satoko; Eguchi, Katsumi; Komiya, Setsuro; Maruyama, Ikuro; Higashi, Nobuyo; Sato, Mitsuru; Senoo, Haruki; Ochi, Takahiro; Yokoyama, Shigeyuki; Amano, Tetsuya; Kim, Jaeseob; Gay, Steffen; Fukamizu, Akiyoshi; Nishioka, Kusuki; Tanaka, Keiji; Nakajima, Toshihiro

2007-01-10

Synoviolin, also called HRD1, is an E3 ubiquitin ligase and is implicated in endoplasmic reticulum -associated degradation. In mammals, Synoviolin plays crucial roles in various physiological and pathological processes, including embryogenesis and the pathogenesis of arthropathy. However, little is known about the molecular mechanisms of Synoviolin in these actions. To clarify these issues, we analyzed the profile of protein expression in synoviolin-null cells. Here, we report that Synoviolin targets tumor suppressor gene p53 for ubiquitination. Synoviolin sequestrated and metabolized p53 in the cytoplasm and negatively regulated its cellular level and biological functions, including transcription, cell cycle regulation and apoptosis. Furthermore, these p53 regulatory functions of Synoviolin were irrelevant to other E3 ubiquitin ligases for p53, such as MDM2, Pirh2 and Cop1, which form autoregulatory feedback loops. Our results provide novel insights into p53 signaling mediated by Synoviolin.

Burn-induced increase in atrogin-1 and MuRF-1 in skeletal muscle is glucocorticoid independent but downregulated by IGF-I.

PubMed

Lang, Charles H; Huber, Danuta; Frost, Robert A

2007-01-01

The present study determined whether thermal injury increases the expression of the ubiquitin (Ub) E3 ligases referred to as muscle ring finger (MuRF)-1 and muscle atrophy F-box (MAFbx; aka atrogin-1), which are muscle specific and responsible for the increased protein breakdown observed in other catabolic conditions. After 48 h of burn injury (40% total body surface area full-thickness scald burn) gastrocnemius weight was reduced, and this change was associated with an increased mRNA abundance for atrogin-1 and MuRF-1 (3.1- to 8-fold, respectively). Similarly, burn increased polyUb mRNA content in the gastrocnemius twofold. In contrast, there was no burn-induced atrophy of the soleus and no significant change in atrogin-1, MuRF-1, or polyUb mRNA. Burns also did not alter E3 ligase expression in heart. Four hours after administration of the anabolic agent insulin-like growth factor (IGF)-I to burned rats, the mRNA content of atrogin-1 and polyUb in gastrocnemius had returned to control values and the elevation in MuRF-1 was reduced 50%. In contrast, leucine did not alter E3 ligase expression. In a separate study, in vivo administration of the proteasome inhibitor Velcade prevented burn-induced loss of muscle mass determined at 48 h. Finally, administration of the glucocorticoid receptor antagonist RU-486 did not prevent burn-induced atrophy of the gastrocnemius or the associated elevation in atrogin-1, MuRF-1, or polyUb. In summary, the acute muscle wasting accompanying thermal injury is associated with a glucocorticoid-independent increase in the expression of several Ub E3 ligases that can be downregulated by IGF-I.

Degradation Signals for Ubiquitin-Proteasome Dependent Cytosolic Protein Quality Control (CytoQC) in Yeast

PubMed Central

Maurer, Matthew J.; Spear, Eric D.; Yu, Allen T.; Lee, Evan J.; Shahzad, Saba; Michaelis, Susan

2016-01-01

Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic “degron library” in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3. About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones. PMID:27172186

Fab-based inhibitors reveal ubiquitin independent functions for HIV Vif neutralization of APOBEC3 restriction factors

PubMed Central

Smith, Amber M.; Hultquist, Judd F.; Caretta Cartozo, Nathalie; Campbell, Melody G.; Burton, Lily; La Greca, Florencia; McGregor, Michael J.; Ta, Hai M.; Bartholomeeusen, Koen; Peterlin, B. Matija; Krogan, Nevan J.; Sevillano, Natalia

2018-01-01

The lentiviral protein Viral Infectivity Factor (Vif) counteracts the antiviral effects of host APOBEC3 (A3) proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs) to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminates how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity. PMID:29304101

Crystallization and preliminary crystallographic analysis of d-alanine-d-alanine ligase from Streptococcus mutans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Yong-Zhi; Sheng, Yu; Li, Lan-Fen

2007-09-01

A potential target for antibiotic drug design, d-alanine-d-alanine ligase from S. mutans, was expressed in E. coli, purified and crystallized. Diffraction data were collected to 2.4 Å resolution. d-Alanine-d-alanine ligase is encoded by the gene ddl (SMU-599) in Streptococcus mutans. This ligase plays a very important role in cell-wall biosynthesis and may be a potential target for drug design. To study the structure and function of this ligase, the gene ddl was amplified from S. mutans genomic DNA and cloned into the expression vector pET28a. The protein was expressed in soluble form in Escherichia coli strain BL21 (DE3). Homogeneous proteinmore » was obtained using a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Purified protein was crystallized and the cube-shaped crystal diffracted to 2.4 Å. The crystal belongs to space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 79.50, c = 108.97 Å. There is one molecule per asymmetric unit.« less

Distinct Functional Domains of Ubc9 Dictate Cell Survival and Resistance to Genotoxic Stress

PubMed Central

van Waardenburg, Robert C. A. M.; Duda, David M.; Lancaster, Cynthia S.; Schulman, Brenda A.; Bjornsti, Mary-Ann

2006-01-01

Covalent modification with SUMO alters protein function, intracellular localization, or protein-protein interactions. Target recognition is determined, in part, by the SUMO E2 enzyme, Ubc9, while Siz/Pias E3 ligases may facilitate select interactions by acting as substrate adaptors. A yeast conditional Ubc9P123L mutant was viable at 36°C yet exhibited enhanced sensitivity to DNA damage. To define functional domains in Ubc9 that dictate cellular responses to genotoxic stress versus those necessary for cell viability, a 1.75-Å structure of yeast Ubc9 that demonstrated considerable conservation of backbone architecture with human Ubc9 was solved. Nevertheless, differences in side chain geometry/charge guided the design of human/yeast chimeras, where swapping domains implicated in (i) binding residues within substrates that flank canonical SUMOylation sites, (ii) interactions with the RanBP2 E3 ligase, and (iii) binding of the heterodimeric E1 and SUMO had distinct effects on cell growth and resistance to DNA-damaging agents. Our findings establish a functional interaction between N-terminal and substrate-binding domains of Ubc9 and distinguish the activities of E3 ligases Siz1 and Siz2 in regulating cellular responses to genotoxic stress. PMID:16782883

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Zheng; Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, 1838 Guangzhou Dadao Bei, Guangzhou 510515; Zhou, Yuning

Highlights: Black-Right-Pointing-Pointer Rictor associates with FBXW7 to form an E3 complex. Black-Right-Pointing-Pointer Knockdown of rictor decreases ubiquitination of c-Myc and cylin E. Black-Right-Pointing-Pointer Knockdown of rictor increases protein levels of c-Myc and cylin E. Black-Right-Pointing-Pointer Overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Black-Right-Pointing-Pointer Rictor regulation of c-Myc and cyclin E requires FBXW7. -- Abstract: Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation.more » Here, we show that rictor associates with FBXW7 to form an E3 complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor-FBXW7 complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of c-Myc and cyclin E while overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7 blunts the effects of rictor, suggesting that rictor regulation of c-Myc and cyclin E requires FBXW7. Our findings identify rictor as an important component of FBXW7 E3 ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation. Importantly, our results suggest that elevated growth factor signaling may contribute to decrease rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.« less

GNIP1 E3 ubiquitin ligase is a novel player in regulating glycogen metabolism in skeletal muscle.

PubMed

Montori-Grau, Marta; Pedreira-Casahuga, Robert; Boyer-Díaz, Zoé; Lassot, Iréna; García-Martínez, Celia; Orozco, Anna; Cebrià, Judith; Osorio-Conles, Oscar; Chacón, Matilde R; Vendrell, Joan; Vázquez-Carrera, Manuel; Desagher, Solange; Jiménez-Chillarón, Josep Carles; Gómez-Foix, Anna Ma

2018-06-01

Glycogenin-interacting protein 1 (GNIP1) is a tripartite motif (TRIM) protein with E3 ubiquitin ligase activity that interacts with glycogenin. These data suggest that GNIP1 could play a major role in the control of glycogen metabolism. However, direct evidence based on functional analysis remains to be obtained. The aim of this study was 1) to define the expression pattern of glycogenin-interacting protein/Tripartite motif containing protein 7 (GNIP/TRIM7) isoforms in humans, 2) to test their ubiquitin E3 ligase activity, and 3) to analyze the functional effects of GNIP1 on muscle glucose/glycogen metabolism both in human cultured cells and in vivo in mice. We show that GNIP1 was the most abundant GNIP/TRIM7 isoform in human skeletal muscle, whereas in cardiac muscle only TRIM7 was expressed. GNIP1 and TRIM7 had autoubiquitination activity in vitro and were localized in the Golgi apparatus and cytosol respectively in LHCN-M2 myoblasts. GNIP1 overexpression increased glucose uptake in LHCN-M2 myotubes. Overexpression of GNIP1 in mouse muscle in vivo increased glycogen content, glycogen synthase (GS) activity and phospho-GSK-3α/β (Ser21/9) and phospho-Akt (Ser473) content, whereas decreased GS phosphorylation in Ser640. These modifications led to decreased blood glucose levels, lactate levels and body weight, without changing whole-body insulin or glucose tolerance in mouse. GNIP1 is an ubiquitin ligase with a markedly glycogenic effect in skeletal muscle. Copyright © 2018 Elsevier Inc. All rights reserved.

Substrate phosphorylation and feedback regulation in JFK-promoted p53 destabilization.

PubMed

Sun, Luyang; Shi, Lei; Wang, Feng; Huangyang, Peiwei; Si, Wenzhe; Yang, Jie; Yao, Zhi; Shang, Yongfeng

2011-02-11

The p53 tumor suppressor plays a central role in integrating cellular responses to various stresses. Tight regulation of p53 is thus essential for the maintenance of genome integrity and normal cell proliferation. Previously, we reported that JFK, the only Kelch domain-containing F-box protein in human, promotes ubiquitination and degradation of p53 and that unlike the other E3 ligases for p53, all of which possess an intrinsic ubiquitin ligase activity, JFK destabilizes p53 through the assembly of a Skp1-Cul1-F-box complex. Here, we report that the substrate recognition by JFK requires phosphorylation of p53 in its central core region by CSN (COP9 signalosome)-associated kinase. Significantly, inhibition of CSN-associated kinase activity or knockdown of CSN5 impairs JFK-promoted p53 degradation, enhances p53-dependent transcription, and promotes cell growth suppression, G(1) arrest, and apoptosis. Moreover, we showed that JFK is transcriptionally regulated by p53 and forms an auto-regulatory negative feedback loop with p53. These data may shed new light on the functional connection between CSN, Skp1-Cul1-F-box ubiquitin ligase, and p53 and provide a molecular mechanism for the regulation of JFK-promoted p53 degradation.

Substrate Phosphorylation and Feedback Regulation in JFK-promoted p53 Destabilization*

PubMed Central

Sun, Luyang; Shi, Lei; Wang, Feng; Huangyang, Peiwei; Si, Wenzhe; Yang, Jie; Yao, Zhi; Shang, Yongfeng

2011-01-01

The p53 tumor suppressor plays a central role in integrating cellular responses to various stresses. Tight regulation of p53 is thus essential for the maintenance of genome integrity and normal cell proliferation. Previously, we reported that JFK, the only Kelch domain-containing F-box protein in human, promotes ubiquitination and degradation of p53 and that unlike the other E3 ligases for p53, all of which possess an intrinsic ubiquitin ligase activity, JFK destabilizes p53 through the assembly of a Skp1-Cul1-F-box complex. Here, we report that the substrate recognition by JFK requires phosphorylation of p53 in its central core region by CSN (COP9 signalosome)-associated kinase. Significantly, inhibition of CSN-associated kinase activity or knockdown of CSN5 impairs JFK-promoted p53 degradation, enhances p53-dependent transcription, and promotes cell growth suppression, G1 arrest, and apoptosis. Moreover, we showed that JFK is transcriptionally regulated by p53 and forms an auto-regulatory negative feedback loop with p53. These data may shed new light on the functional connection between CSN, Skp1-Cul1-F-box ubiquitin ligase, and p53 and provide a molecular mechanism for the regulation of JFK-promoted p53 degradation. PMID:21127074

The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 protein forms an SCF ubiquitin ligase and regulates hypocotyl elongation.

PubMed

Franciosini, Anna; Lombardi, Benedetta; Iafrate, Silvia; Pecce, Valeria; Mele, Giovanni; Lupacchini, Leonardo; Rinaldi, Gianmarco; Kondou, Youichi; Gusmaroli, Giuliana; Aki, Shiori; Tsuge, Tomohiko; Deng, Xing-Wang; Matsui, Minami; Vittorioso, Paola; Costantino, Paolo; Serino, Giovanna

2013-09-01

The regulation of protein turnover by the ubiquitin proteasome system (UPS) is a major posttranslational mechanism in eukaryotes. One of the key components of the UPS, the COP9 signalosome (CSN), regulates 'cullin-ring' E3 ubiquitin ligases. In plants, CSN participates in diverse cellular and developmental processes, ranging from light signaling to cell cycle control. In this work, we isolated a new plant-specific CSN-interacting F-box protein, which we denominated CFK1 (COP9 INTERACTING F-BOX KELCH 1). We show that, in Arabidopsis thaliana, CFK1 is a component of a functional ubiquitin ligase complex. We also show that CFK1 stability is regulated by CSN and by proteasome-dependent proteolysis, and that light induces accumulation of the CFK1 transcript in the hypocotyl. Analysis of CFK1 knockdown, mutant, and overexpressing seedlings indicates that CFK1 promotes hypocotyl elongation by increasing cell size. Reduction of CSN levels enhances the short hypocotyl phenotype of CFK1-depleted seedlings, while complete loss of CSN activity suppresses the long-hypocotyl phenotype of CFK1-overexpressing seedlings. We propose that CFK1 (and its regulation by CSN) is a novel component of the cellular mechanisms controlling hypocotyl elongation.

Ubiquitin in Motion: Structural Studies of the Ubiquitin-Conjugating Enzyme~Ubiquitin Conjugate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pruneda, Jonathan N.; Stoll, Kate E.; Bolton, Laura J.

2011-03-15

Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for the transfer of Ub to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2~Ub conjugate. Therefore, full characterization of the structure and dynamics of E2~Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2~Ub conjugates of two human enzymes, UbcH5c~Ub and Ubc13~Ub,more » in solution as determined by nuclear magnetic resonance and small-angle X-ray scattering. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: the UbcH5c~Ub conjugate populates an array of extended conformations, and the population of Ubc13~Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces helix 2 of Ubc13. Finally, we propose that the varied conformations adopted by Ub represent available binding modes of the E2~Ub species and thus provide insight into the diverse E2~Ub protein interactome, particularly with regard to interaction with Ub ligases.« less

Two Distinct Types of E3 Ligases Work in Unison to Regulate Substrate Ubiquitylation.

PubMed

Scott, Daniel C; Rhee, David Y; Duda, David M; Kelsall, Ian R; Olszewski, Jennifer L; Paulo, Joao A; de Jong, Annemieke; Ovaa, Huib; Alpi, Arno F; Harper, J Wade; Schulman, Brenda A

2016-08-25

Hundreds of human cullin-RING E3 ligases (CRLs) modify thousands of proteins with ubiquitin (UB) to achieve vast regulation. Current dogma posits that CRLs first catalyze UB transfer from an E2 to their client substrates and subsequent polyubiquitylation from various linkage-specific E2s. We report an alternative E3-E3 tagging cascade: many cellular NEDD8-modified CRLs associate with a mechanistically distinct thioester-forming RBR-type E3, ARIH1, and rely on ARIH1 to directly add the first UB and, in some cases, multiple additional individual monoubiquitin modifications onto CRL client substrates. Our data define ARIH1 as a component of the human CRL system, demonstrate that ARIH1 can efficiently and specifically mediate monoubiquitylation of several CRL substrates, and establish principles for how two distinctive E3s can reciprocally control each other for simultaneous and joint regulation of substrate ubiquitylation. These studies have broad implications for CRL-dependent proteostasis and mechanisms of E3-mediated UB ligation. Copyright © 2016 Elsevier Inc. All rights reserved.

The Human Ligase IIIα-XRCC1 Protein Complex Performs DNA Nick Repair after Transient Unwrapping of Nucleosomal DNA*

PubMed Central

Rashid, Ishtiaque; Tomkinson, Alan E.; Pederson, David S.

2017-01-01

Reactive oxygen species generate potentially cytotoxic and mutagenic lesions in DNA, both between and within the nucleosomes that package DNA in chromatin. The vast majority of these lesions are subject to base excision repair (BER). Enzymes that catalyze the first three steps in BER can act at many sites in nucleosomes without the aid of chromatin-remodeling agents and without irreversibly disrupting the host nucleosome. Here we show that the same is true for a protein complex comprising DNA ligase IIIα and the scaffolding protein X-ray repair cross-complementing protein 1 (XRCC1), which completes the fourth and final step in (short-patch) BER. Using in vitro assembled nucleosomes containing discretely positioned DNA nicks, our evidence indicates that the ligase IIIα-XRCC1 complex binds to DNA nicks in nucleosomes only when they are exposed by periodic, spontaneous partial unwrapping of DNA from the histone octamer; that the scaffolding protein XRCC1 enhances the ligation; that the ligation occurs within a complex that ligase IIIα-XRCC1 forms with the host nucleosome; and that the ligase IIIα-XRCC1-nucleosome complex decays when ligation is complete, allowing the host nucleosome to return to its native configuration. Taken together, our results illustrate ways in which dynamic properties intrinsic to nucleosomes may contribute to the discovery and efficient repair of base damage in chromatin. PMID:28184006

NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR.

PubMed

Kainulainen, Markus; Lau, Simone; Samuel, Charles E; Hornung, Veit; Weber, Friedemann

2016-07-01

Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs requires E3 ubiquitin ligase complexes of the SCF (Skp1, Cul1, F-box protein) type to destroy PKR. SCF-type complexes can engage variant ubiquitination substrate recognition subunits, and we found the F-box proteins FBXW11 and β-TRCP1 to be relevant for the action of NSs against PKR. Thus, we identified the host cell factors that are critically needed by Rift Valley fever virus to uphold its replication against the potent antiviral kinase PKR. Copyright © 2016 Kainulainen et al.

Both Ubiquitin Ligases FBXW8 and PARK2 Are Sequestrated into Insolubility by ATXN2 PolyQ Expansions, but Only FBXW8 Expression Is Dysregulated

PubMed Central

Halbach, Melanie Vanessa; Stehning, Tanja; Damrath, Ewa; Jendrach, Marina; Şen, Nesli Ece; Başak, A. Nazlı; Auburger, Georg

2015-01-01

The involvement of the ubiquitin-proteasome system (UPS) in the course of various age-associated neurodegenerative diseases is well established. The single RING finger type E3 ubiquitin-protein ligase PARK2 is mutated in a Parkinson’s disease (PD) variant and was found to interact with ATXN2, a protein where polyglutamine expansions cause Spinocerebellar ataxia type 2 (SCA2) or increase the risk for Levodopa-responsive PD and for the motor neuron disease Amyotrophic lateral sclerosis (ALS). We previously reported evidence for a transcriptional induction of the multi-subunit RING finger Skp1/Cul/F-box (SCF) type E3 ubiquitin-protein ligase complex component FBXW8 in global microarray profiling of ATXN2-expansion mouse cerebellum and demonstrated its role for ATXN2 degradation in vitro. Now, we documented co-localization in vitro and co-immunoprecipitations both in vitro and in vivo, which indicate associations of FBXW8 with ATXN2 and PARK2. Both FBXW8 and PARK2 proteins are driven into insolubility by expanded ATXN2. Whereas the FBXW8 transcript upregulation by ATXN2- expansion was confirmed also in qPCR of skin fibroblasts and blood samples of SCA2 patients, a FBXW8 expression dysregulation was not observed in ATXN2-deficient mice, nor was a PARK2 transcript dysregulation observed in any samples. Jointly, all available data suggest that the degradation of wildtype and mutant ATXN2 is dependent on FBXW8, and that ATXN2 accumulation selectively modulates FBXW8 levels, while PARK2 might act indirectly through FBXW8. The effects of ATXN2-expansions on FBXW8 expression in peripheral tissues like blood may become useful for clinical diagnostics. PMID:25790475

Stringent Control of NFE2L3 (Nuclear Factor, Erythroid 2-Like 3; NRF3) Protein Degradation by FBW7 (F-box/WD Repeat-containing Protein 7) and Glycogen Synthase Kinase 3 (GSK3)*

PubMed Central

Kannan, Meenakshi B.; Dodard-Friedman, Isadore; Blank, Volker

2015-01-01

The NFE2L3 transcription factor has been implicated in various cellular processes, including carcinogenesis, stress response, differentiation, and inflammation. Previously it has been shown that NFE2L3 has a rapid turnover and is stabilized by proteasomal inhibitors. The mechanisms regulating the degradation of this protein have not been investigated. Here we report ubiquitination of NFE2L3 and demonstrate that F-box/WD repeat-containing protein 7 (FBW7 or FBWX7), a component of Skp1, Cullin 1, F-box containing complex (SCF)-type E3 ligase, is the E3 ligase mediating the degradation of NFE2L3. We showed that FBW7 interacts with NFE2L3 and that dimerization of FBW7 is required for the degradation of the transcription factor. We also demonstrate that the kinase glycogen synthase kinase 3 (GSK3) mediates the FBW7-dependent ubiquitination of NFE2L3. We show phosphorylation of NFE2L3 by GSK3 and its significance in the regulation of NFE2L3 by the tumor suppressor FBW7. FBW7 abrogated NFE2L3-mediated repression of the NAD(P)H:quinone oxidoreductase 1 (NQO1) gene antioxidant response element (ARE). Our findings reveal FBW7 and GSK3 as novel regulators of the NFE2L3 transcription factor and a potential mechanism by which FBW7 might regulate detoxification and the cellular response to stress. PMID:26306035

UV-B induction of the E3 ligase ARIADNE12 depends on CONSTITUTIVELY PHOTOMORPHOGENIC 1

PubMed Central

Xie, Lisi; Lang-Mladek, Christina; Richter, Julia; Nigam, Neha; Hauser, Marie-Theres

2015-01-01

The UV-B inducible ARIADNE12 (ARI12) gene of Arabidopsis thaliana is a member of the RING-between-RING (RBR) family of E3 ubiquitin ligases for which a novel ubiquitination mechanism was identified in mammalian homologs. This RING-HECT hybrid mechanism needs a conserved cysteine which is replaced by serine in ARI12 and might affect the E3 ubiquitin ligase activity. We have shown that under photomorphogenic UV-B, ARI12 is a downstream target of the classical ultraviolet B (UV-B) UV RESISTANCE LOCUS 8 (UVR8) pathway. However, under high fluence rate of UV-B ARI12 was induced independently of UVR8 and the UV-A/blue light and red/far-red photoreceptors. A key component of several light signaling pathways is CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). Upon UV-B COP1 is trapped in the nucleus through interaction with UVR8 permitting the activation of genes that regulate the biosynthesis of UV-B protective metabolites and growth adaptations. To clarify the role of COP1 in the regulation of ARI12 mRNA expression and ARI12 protein stability, localization and interaction with COP1 was assessed with and without UV-B. We found that COP1 controls ARI12 in white light, low and high fluence rate of UV-B. Furthermore we show that ARI12 is indeed an E3 ubiquitin ligase which is mono-ubiquitinated, a prerequisite for the RING-HECT hybrid mechanism. Finally, genetic analyses with transgenes expressing a genomic pmARI12:ARI12-GFP construct confirm the epistatic interaction between COP1 and ARI12 in growth responses to high fluence rate UV-B. PMID:25817546

Interaction between RING1 (R1) and the Ubiquitin-like (UBL) Domains Is Critical for the Regulation of Parkin Activity*

PubMed Central

Ham, Su Jin; Lee, Soo Young; Song, Saera; Chung, Ju-Ryung; Choi, Sekyu; Chung, Jongkyeong

2016-01-01

Parkin is an E3 ligase that contains a ubiquitin-like (UBL) domain in the N terminus and an R1-in-between-ring-RING2 motif in the C terminus. We showed that the UBL domain specifically interacts with the R1 domain and negatively regulates Parkin E3 ligase activity, Parkin-dependent mitophagy, and Parkin translocation to the mitochondria. The binding between the UBL domain and the R1 domain was suppressed by carbonyl cyanide m-chlorophenyl hydrazone treatment or by expression of PTEN-induced putative kinase 1 (PINK1), an upstream kinase that phosphorylates Parkin at the Ser-65 residue of the UBL domain. Moreover, we demonstrated that phosphorylation of the UBL domain at Ser-65 prevents its binding to the R1 domain and promotes Parkin activities. We further showed that mitochondrial translocation of Parkin, which depends on phosphorylation at Ser-65, and interaction between the R1 domain and a mitochondrial outer membrane protein, VDAC1, are suppressed by binding of the UBL domain to the R1 domain. Interestingly, Parkin with missense mutations associated with Parkinson disease (PD) in the UBL domain, such as K27N, R33Q, and A46P, did not translocate to the mitochondria and induce E3 ligase activity by m-chlorophenyl hydrazone treatment, which correlated with the interaction between the R1 domain and the UBL domain with those PD mutations. These findings provide a molecular mechanism of how Parkin recruitment to the mitochondria and Parkin activation as an E3 ubiquitin ligase are regulated by PINK1 and explain the previously unknown mechanism of how Parkin mutations in the UBL domain cause PD pathogenesis. PMID:26631732

Interaction between RING1 (R1) and the Ubiquitin-like (UBL) Domains Is Critical for the Regulation of Parkin Activity.

PubMed

Ham, Su Jin; Lee, Soo Young; Song, Saera; Chung, Ju-Ryung; Choi, Sekyu; Chung, Jongkyeong

2016-01-22

Parkin is an E3 ligase that contains a ubiquitin-like (UBL) domain in the N terminus and an R1-in-between-ring-RING2 motif in the C terminus. We showed that the UBL domain specifically interacts with the R1 domain and negatively regulates Parkin E3 ligase activity, Parkin-dependent mitophagy, and Parkin translocation to the mitochondria. The binding between the UBL domain and the R1 domain was suppressed by carbonyl cyanide m-chlorophenyl hydrazone treatment or by expression of PTEN-induced putative kinase 1 (PINK1), an upstream kinase that phosphorylates Parkin at the Ser-65 residue of the UBL domain. Moreover, we demonstrated that phosphorylation of the UBL domain at Ser-65 prevents its binding to the R1 domain and promotes Parkin activities. We further showed that mitochondrial translocation of Parkin, which depends on phosphorylation at Ser-65, and interaction between the R1 domain and a mitochondrial outer membrane protein, VDAC1, are suppressed by binding of the UBL domain to the R1 domain. Interestingly, Parkin with missense mutations associated with Parkinson disease (PD) in the UBL domain, such as K27N, R33Q, and A46P, did not translocate to the mitochondria and induce E3 ligase activity by m-chlorophenyl hydrazone treatment, which correlated with the interaction between the R1 domain and the UBL domain with those PD mutations. These findings provide a molecular mechanism of how Parkin recruitment to the mitochondria and Parkin activation as an E3 ubiquitin ligase are regulated by PINK1 and explain the previously unknown mechanism of how Parkin mutations in the UBL domain cause PD pathogenesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

SCF(JFK) is a bona fide E3 ligase for ING4 and a potent promoter of the angiogenesis and metastasis of breast cancer.

PubMed

Yan, Ruorong; He, Lin; Li, Zhongwu; Han, Xiao; Liang, Jing; Si, Wenzhe; Chen, Zhe; Li, Lei; Xie, Guojia; Li, Wanjin; Wang, Peiyan; Lei, Liandi; Zhang, Hongquan; Pei, Fei; Cao, Dengfeng; Sun, Luyang; Shang, Yongfeng

2015-03-15

Loss of function/dysregulation of inhibitor of growth 4 (ING4) and hyperactivation of NF-κB are frequent events in many types of human malignancies. However, the molecular mechanisms underlying these remarkable aberrations are not understood. Here, we report that ING4 is physically associated with JFK. We demonstrated that JFK targets ING4 for ubiquitination and degradation through assembly of an Skp1-Cul1-F-box (SCF) complex. We showed that JFK-mediated ING4 destabilization leads to the hyperactivation of the canonical NF-κB pathway and promotes angiogenesis and metastasis of breast cancer. Significantly, the expression of JFK is markedly up-regulated in breast cancer, and the level of JFK is negatively correlated with that of ING4 and positively correlated with an aggressive clinical behavior of breast carcinomas. Our study identified SCF(JFK) as a bona fide E3 ligase for ING4 and unraveled the JFK-ING4-NF-κB axis as an important player in the development and progression of breast cancer, supporting the pursuit of JFK as a potential target for breast cancer intervention. © 2015 Yan et al.; Published by Cold Spring Harbor Laboratory Press.

FANCL ubiquitinates β-catenin and enhances its nuclear function

PubMed Central

Rotelli, Michael D.; Petersen, Curtis L.; Kaech, Stefanie; Nelson, Whitney D.; Yates, Jane E.; Hanlon Newell, Amy E.; Olson, Susan B.; Druker, Brian J.; Bagby, Grover C.

2012-01-01

Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of β-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates β-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, β-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate β-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34+ stem and progenitor cells results in fewer β-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/β-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss. PMID:22653977

Tuning BRCA1 and BARD1 activity to investigate RING ubiquitin ligase mechanisms.

PubMed

Stewart, Mikaela D; Duncan, Emily D; Coronado, Ernesto; DaRosa, Paul A; Pruneda, Jonathan N; Brzovic, Peter S; Klevit, Rachel E

2017-03-01

The tumor-suppressor protein BRCA1 works with BARD1 to catalyze the transfer of ubiquitin onto protein substrates. The N-terminal regions of BRCA1 and BARD1 that contain their RING domains are responsible for dimerization and ubiquitin ligase activity. This activity is a common feature among hundreds of human RING domain-containing proteins. RING domains bind and activate E2 ubiquitin-conjugating enzymes to promote ubiquitin transfer to substrates. We show that the identity of residues at specific positions in the RING domain can tune activity levels up or down. We report substitutions that create a structurally intact BRCA1/BARD1 heterodimer that is inactive in vitro with all E2 enzymes. Other substitutions in BRCA1 or BARD1 RING domains result in hyperactivity, revealing that both proteins have evolved attenuated activity. Loss of attenuation results in decreased product specificity, providing a rationale for why nature has tuned BRCA1 activity. The ability to tune BRCA1 provides powerful tools for understanding its biological functions and provides a basis to assess mechanisms for rescuing the activity of cancer-associated variations. Beyond the applicability to BRCA1, we show the identity of residues at tuning positions that can be used to predict and modulate the activity of an unrelated RING E3 ligase. These findings provide valuable insights into understanding the mechanism and function of RING E3 ligases like BRCA1. © 2017 The Protein Society.

Lack of discrimination between DNA ligases I and III by two classes of inhibitors, anthracyclines and distamycins.

PubMed

Montecucco, A; Lestingi, M; Rossignol, J M; Elder, R H; Ciarrocchi, G

1993-04-06

We have measured the effects of eight distamycin and two anthracycline derivatives on polynucleotide joining and self-adenylating activities of human DNA ligase I and rat DNA ligases I and III. All test drugs show good inhibitory activity against the three enzymes in the poly[d(A-T)] joining assay. Several distamycins also inhibit the DNA-independent self-adenylation reaction catalysed by the human enzyme and, to a lesser extent, by rat DNA ligases. These results confirm that anthracyclines and distamycins express their inhibitory action against DNA joining activities mainly via specific interactions with the substrate, and suggest that the three test DNA ligases utilize similar, if not identical, mechanisms of recognition and interaction with DNA-drug complexes. Our findings also indicate that distamycins have a greater affinity for human DNA ligase I than for rat enzymes, suggesting that, in this respect, rat DNA ligase I is more similar to rat DNA ligase III than to human DNA ligase I.

MurD enzymes: some recent developments.

PubMed

Šink, Roman; Barreteau, Hélène; Patin, Delphine; Mengin-Lecreulx, Dominique; Gobec, Stanislav; Blanot, Didier

2013-12-01

The synthesis of the peptide stem of bacterial peptidoglycan involves four enzymes, the Mur ligases (MurC, D, E and F). Among them, MurD is responsible for the ATP-dependent addition of d-glutamic acid to UDP-MurNAc-l-Ala, a reaction which involves acyl-phosphate and tetrahedral intermediates. Like most enzymes of peptidoglycan biosynthesis, MurD constitutes an attractive target for the design and synthesis of new antibacterial agents. Escherichia coli MurD has been the first Mur ligase for which the tridimensional (3D) structure was solved. Thereafter, several co-crystal structures with different ligands or inhibitors were released. In the present review, we will deal with work performed on substrate specificity, reaction mechanism and 3D structure of E. coli MurD. Then, a part of the review will be devoted to recent work on MurD orthologs from species other than E. coli and to cellular organization of Mur ligases and in vivo regulation of the MurD activity. Finally, we will review the different classes of MurD inhibitors that have been designed and assayed to date with the hope of obtaining new antibacterial compounds.

A MARCH6 and IDOL E3 Ubiquitin Ligase Circuit Uncouples Cholesterol Synthesis from Lipoprotein Uptake in Hepatocytes

PubMed Central

Loregger, Anke; Cook, Emma Claire Laura; Nelson, Jessica Kristin; Moeton, Martina; Sharpe, Laura Jane; Engberg, Susanna; Karimova, Madina; Lambert, Gilles; Brown, Andrew John

2015-01-01

Cholesterol synthesis and lipoprotein uptake are tightly coordinated to ensure that the cellular level of cholesterol is adequately maintained. Hepatic dysregulation of these processes is associated with pathological conditions, most notably cardiovascular disease. Using a genetic approach, we have recently identified the E3 ubiquitin ligase MARCH6 as a regulator of cholesterol biosynthesis, owing to its ability to promote degradation of the rate-limiting enzymes 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) and squalene epoxidase (SQLE). Here, we present evidence for MARCH6 playing a multifaceted role in the control of cholesterol homeostasis in hepatocytes. We identify MARCH6 as an endogenous inhibitor of the sterol regulatory element binding protein (SREBP) transcriptional program. Accordingly, loss of MARCH6 increases expression of SREBP-regulated genes involved in cholesterol biosynthesis and lipoprotein uptake. Unexpectedly, this is associated with a decrease in cellular lipoprotein uptake, induced by enhanced lysosomal degradation of the low-density lipoprotein receptor (LDLR). Finally, we provide evidence that induction of the E3 ubiquitin ligase IDOL represents the molecular mechanism underlying this MARCH6-induced phenotype. Our study thus highlights a MARCH6-dependent mechanism to direct cellular cholesterol accretion that relies on uncoupling of cholesterol synthesis from lipoprotein uptake. PMID:26527619

Visualizing the complex functions and mechanisms of the anaphase promoting complex/cyclosome (APC/C)

PubMed Central

Alfieri, Claudio; Zhang, Suyang

2017-01-01

The anaphase promoting complex or cyclosome (APC/C) is a large multi-subunit E3 ubiquitin ligase that orchestrates cell cycle progression by mediating the degradation of important cell cycle regulators. During the two decades since its discovery, much has been learnt concerning its role in recognizing and ubiquitinating specific proteins in a cell-cycle-dependent manner, the mechanisms governing substrate specificity, the catalytic process of assembling polyubiquitin chains on its target proteins, and its regulation by phosphorylation and the spindle assembly checkpoint. The past few years have witnessed significant progress in understanding the quantitative mechanisms underlying these varied APC/C functions. This review integrates the overall functions and properties of the APC/C with mechanistic insights gained from recent cryo-electron microscopy (cryo-EM) studies of reconstituted human APC/C complexes. PMID:29167309

Mutations in CUL4B, which encodes a ubiquitin E3 ligase subunit, cause an X-linked mental retardation syndrome associated with aggressive outbursts, seizures, relative macrocephaly, central obesity, hypogonadism, pes cavus, and tremor.

PubMed

Tarpey, Patrick S; Raymond, F Lucy; O'Meara, Sarah; Edkins, Sarah; Teague, Jon; Butler, Adam; Dicks, Ed; Stevens, Claire; Tofts, Calli; Avis, Tim; Barthorpe, Syd; Buck, Gemma; Cole, Jennifer; Gray, Kristian; Halliday, Kelly; Harrison, Rachel; Hills, Katy; Jenkinson, Andrew; Jones, David; Menzies, Andrew; Mironenko, Tatiana; Perry, Janet; Raine, Keiran; Richardson, David; Shepherd, Rebecca; Small, Alexandra; Varian, Jennifer; West, Sofie; Widaa, Sara; Mallya, Uma; Moon, Jenny; Luo, Ying; Holder, Susan; Smithson, Sarah F; Hurst, Jane A; Clayton-Smith, Jill; Kerr, Bronwyn; Boyle, Jackie; Shaw, Marie; Vandeleur, Lucianne; Rodriguez, Jayson; Slaugh, Rachel; Easton, Douglas F; Wooster, Richard; Bobrow, Martin; Srivastava, Anand K; Stevenson, Roger E; Schwartz, Charles E; Turner, Gillian; Gecz, Jozef; Futreal, P Andrew; Stratton, Michael R; Partington, Michael

2007-02-01

We have identified three truncating, two splice-site, and three missense variants at conserved amino acids in the CUL4B gene on Xq24 in 8 of 250 families with X-linked mental retardation (XLMR). During affected subjects' adolescence, a syndrome emerged with delayed puberty, hypogonadism, relative macrocephaly, moderate short stature, central obesity, unprovoked aggressive outbursts, fine intention tremor, pes cavus, and abnormalities of the toes. This syndrome was first described by Cazebas et al., in a family that was included in our study and that carried a CUL4B missense variant. CUL4B is a ubiquitin E3 ligase subunit implicated in the regulation of several biological processes, and CUL4B is the first XLMR gene that encodes an E3 ubiquitin ligase. The relatively high frequency of CUL4B mutations in this series indicates that it is one of the most commonly mutated genes underlying XLMR and suggests that its introduction into clinical diagnostics should be a high priority.

Aurora Kinase A Promotes AR Degradation via the E3 Ligase CHIP.

PubMed

Sarkar, Sukumar; Brautigan, David L; Larner, James M

2017-08-01

Reducing the levels of the androgen receptor (AR) is one of the most viable approaches to combat castration-resistant prostate cancer. Previously, we observed that proteasomal-dependent degradation of AR in response to 2-methoxyestradiol (2-ME) depends primarily on the E3 ligase C-terminus of HSP70-interacting protein (STUB1/CHIP). Here, 2-ME stimulation activates CHIP by phosphorylation via Aurora kinase A (AURKA). Aurora A kinase inhibitors and RNAi knockdown of Aurora A transcript selectively blocked CHIP phosphorylation and AR degradation. Aurora A kinase is activated by 2-ME in the S-phase as well as during mitosis, and phosphorylates CHIP at S273. Prostate cancer cells expressing an S273A mutant of CHIP have attenuated AR degradation upon 2-ME treatment compared with cells expressing wild-type CHIP, supporting the idea that CHIP phosphorylation by Aurora A activates its E3 ligase activity for the AR. These results reveal a novel 2-ME→Aurora A→CHIP→AR pathway that promotes AR degradation via the proteasome that may offer novel therapeutic opportunities for prostate cancer. Mol Cancer Res; 15(8); 1063-72. ©2017 AACR . ©2017 American Association for Cancer Research.

Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation*♦

PubMed Central

Yamano, Koji; Queliconi, Bruno B.; Koyano, Fumika; Saeki, Yasushi; Hirokawa, Takatsugu; Tanaka, Keiji; Matsuda, Noriyuki

2015-01-01

Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser65 by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity. PMID:26260794

Building a pseudo-atomic model of the anaphase-promoting complex.

PubMed

Kulkarni, Kiran; Zhang, Ziguo; Chang, Leifu; Yang, Jing; da Fonseca, Paula C A; Barford, David

2013-11-01

The anaphase-promoting complex (APC/C) is a large E3 ubiquitin ligase that regulates progression through specific stages of the cell cycle by coordinating the ubiquitin-dependent degradation of cell-cycle regulatory proteins. Depending on the species, the active form of the APC/C consists of 14-15 different proteins that assemble into a 20-subunit complex with a mass of approximately 1.3 MDa. A hybrid approach of single-particle electron microscopy and protein crystallography of individual APC/C subunits has been applied to generate pseudo-atomic models of various functional states of the complex. Three approaches for assigning regions of the EM-derived APC/C density map to specific APC/C subunits are described. This information was used to dock atomic models of APC/C subunits, determined either by protein crystallography or homology modelling, to specific regions of the APC/C EM map, allowing the generation of a pseudo-atomic model corresponding to 80% of the entire complex.

LIN-23, an E3 Ubiquitin Ligase Component, Is Required for the Repression of CDC-25.2 Activity during Intestinal Development in Caenorhabditis elegans.

PubMed

Son, Miseol; Kawasaki, Ichiro; Oh, Bong-Kyeong; Shim, Yhong-Hee

2016-11-30

Caenorhabditis elegans ( C. elegans ) utilizes two different cell-cycle modes, binucleations during the L1 larval stage and endoreduplications at four larval moltings, for its postembryonic intestinal development. Previous genetic studies indicated that CDC-25.2 is specifically required for binucleations at the L1 larval stage and is repressed before endoreduplications. Furthermore, LIN-23, the C. elegans β-TrCP ortholog, appears to function as a repressor of CDC-25.2 to prevent excess intestinal divisions. We previously reported that intestinal hyperplasia in lin-23(e1883) mutants was effectively suppressed by the RNAi depletion of cdc-25.2 . Nevertheless, LIN-23 targeting CDC-25.2 for ubiquitination as a component of E3 ubiquitin ligase has not yet been tested. In this study, LIN-23 is shown to be the major E3 ubiquitin ligase component, recognizing CDC-25.2 to repress their activities for proper transition of cell-cycle modes during the C. elegans postembryonic intestinal development. In addition, for the first time that LIN-23 physically interacts with both CDC-25.1 and CDC-25.2 and facilitates ubiquitination for timely regulation of their activities during the intestinal development.

It's all about talking: two-way communication between proteasomal and lysosomal degradation pathways via ubiquitin.

PubMed

Liebl, Martina P; Hoppe, Thorsten

2016-08-01

Selective degradation of proteins requires a fine-tuned coordination of the two major proteolytic pathways, the ubiquitin-proteasome system (UPS) and autophagy. Substrate selection and proteolytic activity are defined by a plethora of regulatory cofactors influencing each other. Both proteolytic pathways are initiated by ubiquitylation to mark substrate proteins for degradation, although the size and/or topology of the modification are different. In this context E3 ubiquitin ligases, ensuring the covalent attachment of activated ubiquitin to the substrate, are of special importance. The regulation of E3 ligase activity, competition between different E3 ligases for binding E2 conjugation enzymes and substrates, as well as their interplay with deubiquitylating enzymes (DUBs) represent key events in the cross talk between the UPS and autophagy. The coordination between both degradation routes is further influenced by heat shock factors and ubiquitin-binding proteins (UBPs) such as p97, p62, or optineurin. Mutations in enzymes and ubiquitin-binding proteins or a general decline of both proteolytic systems during aging result in accumulation of damaged and aggregated proteins. Thus further mechanistic understanding of how UPS and autophagy communicate might allow therapeutic intervention especially against age-related diseases. Copyright © 2016 the American Physiological Society.

Structurally complex and highly active RNA ligases derived from random RNA sequences

NASA Technical Reports Server (NTRS)

Ekland, E. H.; Szostak, J. W.; Bartel, D. P.

1995-01-01

Seven families of RNA ligases, previously isolated from random RNA sequences, fall into three classes on the basis of secondary structure and regiospecificity of ligation. Two of the three classes of ribozymes have been engineered to act as true enzymes, catalyzing the multiple-turnover transformation of substrates into products. The most complex of these ribozymes has a minimal catalytic domain of 93 nucleotides. An optimized version of this ribozyme has a kcat exceeding one per second, a value far greater than that of most natural RNA catalysts and approaching that of comparable protein enzymes. The fact that such a large and complex ligase emerged from a very limited sampling of sequence space implies the existence of a large number of distinct RNA structures of equivalent complexity and activity.

USP19-Mediated Deubiquitination Facilitates the Stabilization of HRD1 Ubiquitin Ligase.

PubMed

Harada, Kumi; Kato, Masako; Nakamura, Nobuhiro

2016-11-02

In the endoplasmic reticulum (ER), misfolded and unfolded proteins are eliminated by a process called ER-associated protein degradation (ERAD) in order to maintain cell homeostasis. In the ERAD pathway, several ER-localized E3 ubiquitin ligases target ERAD substrate proteins for ubiquitination and subsequent proteasomal degradation. However, little is known about how the functions of the ERAD ubiquitin ligases are regulated. Recently, USP19, an ER-anchored deubiquitinating enzyme (DUB), has been suggested to be involved in the regulation of ERAD. In this study, HRD1, an ERAD ubiquitin ligase, is shown to be a novel substrate for USP19. We demonstrate that USP19 rescues HRD1 from proteasomal degradation by deubiquitination of K48-linked ubiquitin chains. In addition, the altered expression of USP19 affects the steady-state levels of HRD1. These results suggest that USP19 regulates the stability of HRD1 and provide insight into the regulatory mechanism of the ERAD ubiquitin ligases.

Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination.

PubMed

Ahmed, M Rafiuddin; Zhan, Xuanzhi; Song, Xiufeng; Kook, Seunghyi; Gurevich, Vsevolod V; Gurevich, Eugenia V

2011-05-10

Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmunoprecipitation of endogenous proteins from brain tissue and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both nonvisual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology.

A lysine-to-arginine mutation on NEDD8 markedly reduces the activity of cullin RING E3 ligase through the impairment of neddylation cascades

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sui, Yiyan; Liu, Yaobin; Xu, Guoqiang, E-mail: gux2002@suda.edu.cn

2015-06-12

Neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) is a ubiquitin-like modifier, which forms covalent conjugates on lysines of its substrates. This post-translational modification, neddylation, plays important roles in tumor cell proliferation and viability. Ubiquitin can form diverse polyubiquitin chains, on its seven lysines, which play important functions in various biological processes. However, the roles of lysines in NEDD8 have not been explored. Here, we generated nine NEDD8 point mutants, each with one lysine replaced by an arginine, to study the putative function of lysines in NEDD8. Our experiments discover that Lys27 in NEDD8 is a critical residue for protein neddylation. Replacement ofmore » this residue with arginine almost completely eliminates the conjugation of NEDD8 to its substrates. Furthermore, we find that the K27R mutant impairs NEDD8 conjugation to the E2 enzyme, which normally forms thioester bonds for further transferring NEDD8 to its ligases and substrates. Therefore, this mutation completely inhibits global protein neddylation, including neddylation of cullin family proteins, resulting in decreased activity of cullin-RING E3 ligases. This work sheds new light on the roles of NEDD8 lysines on neddylation cascades and provides a dominant negative mutant for the study of neddylation and its biological functions. - Highlights: • Lys27 in NEDD8 is critical for protein neddylation. • NEDD8 K27R mutant impairs the NEDD8 conjugation. • NEDD8 K27R mutant significantly reduces the activity of cullin-RING E3 ligases.« less

Myeloid leukemia factor 1 stabilizes tumor suppressor C/EBPα to prevent Trib1-driven acute myeloid leukemia.

PubMed

Nakamae, Ikuko; Kato, Jun-Ya; Yokoyama, Takashi; Ito, Hidenori; Yoneda-Kato, Noriko

2017-09-12

C/EBPα is a key transcription factor regulating myeloid differentiation and leukemogenesis. The Trib1-COP1 complex is an E3 ubiquitin ligase that targets C/EBPα for degradation, and its overexpression specifically induces acute myeloid leukemia (AML). Here we show that myeloid leukemia factor 1 (MLF1) stabilizes C/EBPα protein levels by inhibiting the ligase activity of the Trib1-COP1 complex. MLF1 directly interacts with COP1 in the nucleus and interferes with the formation of the Trib1-COP1 complex, thereby blocking its ability to polyubiquitinate C/EBPα for degradation. MLF1 overexpression suppressed the Trib1-induced growth advantage in a murine bone marrow (BM) culture and Trib1-induced AML development in BM-transplanted mouse models. MLF1 was expressed in hematopoietic stem cells and myeloid progenitors (common myeloid progenitors and granulocyte-macrophage progenitors) in normal hematopoiesis, which is consistent with the distribution of C/EBPα. An MLF1 deficiency conferred a more immature phenotype on Trib1-induced AML development. A higher expression ratio of Trib1 to MLF1 was a key determinant for AML development in mouse models, which was also confirmed in human patient samples with acute leukemia. These results indicate that MLF1 is a positive regulator that is critical for C/EBPα stability in the early phases of hematopoiesis and leukemogenesis.

Myeloid leukemia factor 1 stabilizes tumor suppressor C/EBPα to prevent Trib1-driven acute myeloid leukemia

PubMed Central

Nakamae, Ikuko; Kato, Jun-ya; Yokoyama, Takashi; Ito, Hidenori

2017-01-01

C/EBPα is a key transcription factor regulating myeloid differentiation and leukemogenesis. The Trib1-COP1 complex is an E3 ubiquitin ligase that targets C/EBPα for degradation, and its overexpression specifically induces acute myeloid leukemia (AML). Here we show that myeloid leukemia factor 1 (MLF1) stabilizes C/EBPα protein levels by inhibiting the ligase activity of the Trib1-COP1 complex. MLF1 directly interacts with COP1 in the nucleus and interferes with the formation of the Trib1-COP1 complex, thereby blocking its ability to polyubiquitinate C/EBPα for degradation. MLF1 overexpression suppressed the Trib1-induced growth advantage in a murine bone marrow (BM) culture and Trib1-induced AML development in BM-transplanted mouse models. MLF1 was expressed in hematopoietic stem cells and myeloid progenitors (common myeloid progenitors and granulocyte-macrophage progenitors) in normal hematopoiesis, which is consistent with the distribution of C/EBPα. An MLF1 deficiency conferred a more immature phenotype on Trib1-induced AML development. A higher expression ratio of Trib1 to MLF1 was a key determinant for AML development in mouse models, which was also confirmed in human patient samples with acute leukemia. These results indicate that MLF1 is a positive regulator that is critical for C/EBPα stability in the early phases of hematopoiesis and leukemogenesis. PMID:29296815

Ligand-mediated protein degradation reveals functional conservation among sequence variants of the CUL4-type E3 ligase substrate receptor cereblon.

PubMed

Akuffo, Afua A; Alontaga, Aileen Y; Metcalf, Rainer; Beatty, Matthew S; Becker, Andreas; McDaniel, Jessica M; Hesterberg, Rebecca S; Goodheart, William E; Gunawan, Steven; Ayaz, Muhammad; Yang, Yan; Karim, Md Rezaul; Orobello, Morgan E; Daniel, Kenyon; Guida, Wayne; Yoder, Jeffrey A; Rajadhyaksha, Anjali M; Schönbrunn, Ernst; Lawrence, Harshani R; Lawrence, Nicholas J; Epling-Burnette, Pearlie K

2018-04-20

Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN's activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN's nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN's E3 ubiquitin-conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBN's substrate-recruiting function. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Ubiquitin-dependent Regulation of Phospho-AKT Dynamics by the Ubiquitin E3 Ligase, NEDD4-1, in the Insulin-like Growth Factor-1 Response*

PubMed Central

Fan, Chuan-Dong; Lum, Michelle A.; Xu, Chao; Black, Jennifer D.; Wang, Xinjiang

2013-01-01

AKT is a critical effector kinase downstream of the PI3K pathway that regulates a plethora of cellular processes including cell growth, death, differentiation, and migration. Mechanisms underlying activated phospho-AKT (pAKT) translocation to its action sites remain unclear. Here we show that NEDD4-1 is a novel E3 ligase that specifically regulates ubiquitin-dependent trafficking of pAKT in insulin-like growth factor (IGF)-1 signaling. NEDD4-1 physically interacts with AKT and promotes HECT domain-dependent ubiquitination of exogenous and endogenous AKT. NEDD4-1 catalyzes K63-type polyubiquitin chain formation on AKT in vitro. Plasma membrane binding is the key step for AKT ubiquitination by NEDD4-1 in vivo. Ubiquitinated pAKT translocates to perinuclear regions, where it is released into the cytoplasm, imported into the nucleus, or coupled with proteasomal degradation. IGF-1 signaling specifically stimulates NEDD4-1-mediated ubiquitination of pAKT, without altering total AKT ubiquitination. A cancer-derived plasma membrane-philic mutant AKT(E17K) is more effectively ubiquitinated by NEDD4-1 and more efficiently trafficked into the nucleus compared with wild type AKT. This study reveals a novel mechanism by which a specific E3 ligase is required for ubiquitin-dependent control of pAKT dynamics in a ligand-specific manner. PMID:23195959

Activity of a C-terminal plant homeodomain (PHD) of Msc1 is essential for function.

PubMed

Qiu, Xinxing; Dul, Barbara E; Walworth, Nancy C

2010-11-19

Msc1, a member of the Jarid1 family of putative histone demethylases, is required for chromosome stability in fission yeast. Msc1 associates with the Swr1 complex that facilitates deposition of histone H2A.Z into chromatin. To assess the function of Msc1 in the Swr1 complex, domains of Msc1 necessary for interaction with Swr1 were identified. The C-terminal plant homeodomain (PHD) 2 and PHD3 of Msc1 are sufficient to confer association with Swr1 and allow Msc1 to function in the context of kinetochore mutants. On the other hand, a mutant with a single amino acid substitution in PHD2 within the full-length Msc1 protein retains the ability to bind to Swr1 but eliminates the function of Msc1 in combination with kinetochore mutants. Thus, Swr1 association is critical but not sufficient for Msc1 function. An activity of Msc1 that depends on the cysteine residue within PHD2 of Msc1 is likewise critical for function. On the basis of our observation that the PHDs of Msc1 act as E3 ubiquitin ligases and that mutations of cysteine residues within those domains abolish ligase activity, we speculate that the ability of Msc1 to facilitate ubiquitin transfer is critical for the function it mediates through its association with Swr1.

Activity of a C-terminal Plant Homeodomain (PHD) of Msc1 Is Essential for Function*

PubMed Central

Qiu, Xinxing; Dul, Barbara E.; Walworth, Nancy C.

2010-01-01

Msc1, a member of the Jarid1 family of putative histone demethylases, is required for chromosome stability in fission yeast. Msc1 associates with the Swr1 complex that facilitates deposition of histone H2A.Z into chromatin. To assess the function of Msc1 in the Swr1 complex, domains of Msc1 necessary for interaction with Swr1 were identified. The C-terminal plant homeodomain (PHD) 2 and PHD3 of Msc1 are sufficient to confer association with Swr1 and allow Msc1 to function in the context of kinetochore mutants. On the other hand, a mutant with a single amino acid substitution in PHD2 within the full-length Msc1 protein retains the ability to bind to Swr1 but eliminates the function of Msc1 in combination with kinetochore mutants. Thus, Swr1 association is critical but not sufficient for Msc1 function. An activity of Msc1 that depends on the cysteine residue within PHD2 of Msc1 is likewise critical for function. On the basis of our observation that the PHDs of Msc1 act as E3 ubiquitin ligases and that mutations of cysteine residues within those domains abolish ligase activity, we speculate that the ability of Msc1 to facilitate ubiquitin transfer is critical for the function it mediates through its association with Swr1. PMID:20858896

The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells.

PubMed

Paolino, Magdalena; Choidas, Axel; Wallner, Stephanie; Pranjic, Blanka; Uribesalgo, Iris; Loeser, Stefanie; Jamieson, Amanda M; Langdon, Wallace Y; Ikeda, Fumiyo; Fededa, Juan Pablo; Cronin, Shane J; Nitsch, Roberto; Schultz-Fademrecht, Carsten; Eickhoff, Jan; Menninger, Sascha; Unger, Anke; Torka, Robert; Gruber, Thomas; Hinterleitner, Reinhard; Baier, Gottfried; Wolf, Dominik; Ullrich, Axel; Klebl, Bert M; Penninger, Josef M

2014-03-27

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a 'pill' that awakens the innate immune system to kill cancer metastases.

RYBP stimulates PRC1 to shape chromatin-based communication between Polycomb repressive complexes

PubMed Central

Rose, Nathan R; King, Hamish W; Blackledge, Neil P; Fursova, Nadezda A; Ember, Katherine JI; Fischer, Roman; Kessler, Benedikt M; Klose, Robert J

2016-01-01

Polycomb group (PcG) proteins function as chromatin-based transcriptional repressors that are essential for normal gene regulation during development. However, how these systems function to achieve transcriptional regulation remains very poorly understood. Here, we discover that the histone H2AK119 E3 ubiquitin ligase activity of Polycomb repressive complex 1 (PRC1) is defined by the composition of its catalytic subunits and is highly regulated by RYBP/YAF2-dependent stimulation. In mouse embryonic stem cells, RYBP plays a central role in shaping H2AK119 mono-ubiquitylation at PcG targets and underpins an activity-based communication between PRC1 and Polycomb repressive complex 2 (PRC2) which is required for normal histone H3 lysine 27 trimethylation (H3K27me3). Without normal histone modification-dependent communication between PRC1 and PRC2, repressive Polycomb chromatin domains can erode, rendering target genes susceptible to inappropriate gene expression signals. This suggests that activity-based communication and histone modification-dependent thresholds create a localized form of epigenetic memory required for normal PcG chromatin domain function in gene regulation. DOI: http://dx.doi.org/10.7554/eLife.18591.001 PMID:27705745

Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

PubMed Central

Kazlauskaite, Agne; Kelly, Van; Johnson, Clare; Baillie, Carla; Hastie, C. James; Peggie, Mark; Macartney, Thomas; Woodroof, Helen I.; Alessi, Dario R.; Pedrioli, Patrick G. A.; Muqit, Miratul M. K.

2014-01-01

Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser65) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser65 phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Miro1. We observe that Parkin efficiently ubiquitylates Miro1 at highly conserved lysine residues, 153, 230, 235, 330 and 572, upon phosphorylation by PINK1. We have further established an E2-ubiquitin discharge assay to assess Parkin activity and observe robust discharge of ubiquitin-loaded UbcH7 E2 ligase upon phosphorylation of Parkin at Ser65 by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser65 phosphorylation may activate Parkin E3 ligase activity. For the first time, to the best of our knowledge, we report the effect of Parkin disease-associated mutations in substrate-based assays using full-length untagged recombinant Parkin. Our mutation analysis indicates an essential role for the catalytic cysteine Cys431 and reveals fundamental new knowledge on how mutations may confer pathogenicity via disruption of Miro1 ubiquitylation, free ubiquitin chain formation or by impacting Parkin's ability to discharge ubiquitin from a loaded E2. This study provides further evidence that phosphorylation of Parkin at Ser65 is critical for its activation. It also provides evidence that Miro1 is a direct Parkin substrate. The assays and reagents developed in this study will be important to uncover new insights into Parkin biology as well as aid in the development of screens to identify small molecule Parkin activators for the treatment of Parkinson's disease. PMID:24647965

DLG1 is an anchor for the E3 ligase MARCH2 at sites of cell-cell contact

PubMed Central

Cao, Zhifang; Huett, Alan; Kuballa, Petric; Giallourakis, Cosmas; Xavier, Ramnik J.

2008-01-01

PDZ domain containing molecular scaffolds play a central role in organizing synaptic junctions. Observations in Drosophila and mammalian cells have implicated that ubiquitination and endosomal trafficking, of molecular scaffolds are critical to the development and maintenance of cell-cell junctions and cell polarity. To elucidate if there is a connection between these pathways, we applied an integrative genomic strategy, which combined comparative genomics and proteomics with cell biological assays. Given the importance of ubiquitin in regulating endocytic processes, we first identified the subset of E3 ligases with conserved PDZ binding motifs. Among this subset, the MARCH family ubiquitin ligases account for the largest family and MARCH2 has been previously implicated in endosomal trafficking. Next, we tested in an unbiased fashion, if MARCH2 binds PDZ proteins in vivo using a modified tandem affinity purification strategy followed by mass spectrometry. Of note, DLG1 was co-purified from MARCH2, with subsequent confirmation that MARCH2 interacts with full-length DLG1 in a PDZ domain dependent manner. Furthermore, we demonstrated that MARCH2 co-localized with DLG1 at sites of cell-cell contact. In addition, loss of the MARCH2 PDZ binding motif led to loss of MARCH2 localization at cell-cell contact sites and MARCH2 appeared to localize away from cell-cell junctions. In in vivo ubiquitination assays we show that MARCH2 promotes DLG1 ubiquitination Overall, these results suggest that PDZ ligands with E3 ligase activity may link PDZ domain containing tumor suppressors to endocytic pathways and cell polarity determination. PMID:17980554

The E3 ubiquitin ligase, HECTD1, is involved in ABCA1-mediated cholesterol export from macrophages.

PubMed

Aleidi, Shereen M; Yang, Alryel; Sharpe, Laura J; Rao, Geetha; Cochran, Blake J; Rye, Kerry-Anne; Kockx, Maaike; Brown, Andrew J; Gelissen, Ingrid C

2018-04-01

The ABC lipid transporters, ABCA1 and ABCG1, are essential for maintaining lipid homeostasis in cells such as macrophages by exporting excess cholesterol to extracellular acceptors. These transporters are highly regulated at the post-translational level, including protein ubiquitination. Our aim was to investigate the role of the E3 ubiquitin ligase HECTD1, recently identified as associated with ABCG1, on ABCG1 and ABCA1 protein levels and cholesterol export function. Here, we show that HECTD1 protein is widely expressed in a range of human and murine primary cells and cell lines, including macrophages, neuronal cells and insulin secreting β-cells. siRNA knockdown of HECTD1 unexpectedly decreased overexpressed ABCG1 protein levels and cell growth, but increased native ABCA1 protein in CHO-K1 cells. Knockdown of HECTD1 in unloaded THP-1 macrophages did not affect ABCG1 but significantly increased ABCA1 protein levels, in wild-type as well as THP-1 cells that do not express ABCG1. Cholesterol export from macrophages to apoA-I over time was increased after knockdown of HECTD1, however these effects were not sustained in cholesterol-loaded cells. In conclusion, we have identified a new candidate, the E3 ubiquitin ligase HECTD1, that may be involved in the regulation of ABCA1-mediated cholesterol export from unloaded macrophages to apoA-I. The exact mechanism by which this ligase affects this pathway remains to be elucidated. Copyright © 2018 Elsevier B.V. All rights reserved.

The E3 ubiquitin ligases β-TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation induced by the Akt inhibitor API-1, resulting in apoptosis.

PubMed

Ren, Hui; Koo, Junghui; Guan, Baoxiang; Yue, Ping; Deng, Xingming; Chen, Mingwei; Khuri, Fadlo R; Sun, Shi-Yong

2013-11-22

The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis. API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction. API-1 induces GSK3-dependent, β-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis.

The E3 ubiquitin ligases β-TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation induced by the Akt inhibitor API-1, resulting in apoptosis

PubMed Central

2013-01-01

Background The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis. Results API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction. Conclusions API-1 induces GSK3-dependent, β-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis. PMID:24261825

Ubiquitin chain specificities of E6AP E3 ligase and its HECT domain.

PubMed

Kobayashi, Fuminori; Nishiuchi, Takumi; Takaki, Kento; Konno, Hiroki

2018-02-05

Ubiquitination of target proteins is accomplished by isopeptide bond formation between the carboxy group of the C-terminal glycine (Gly) residue of ubiquitin (Ub) and the ɛ-amino group of lysine (Lys) on the target proteins. The formation of an isopeptide bond between Ubs that gives rise to a poly-Ub chain on the target proteins and the types of poly-Ub chains formed depend on which of the seven Lys residues or N-terminal methionine (Met) residue on Ub is used for chain elongation. To understand the linkage specificity mechanism of Ub chains on E3, the previous study established an assay to monitor the formation of a free diubiquitin chain (Ub 2 chain synthesis assay) by HECT type E3 ligase. In this study, we investigated Ub 2 chain specificity using E6AP HECT domain. We here demonstrate the importance of the N-terminal domain of full length E6AP for Ub 2 chain specificity. Copyright © 2017 Elsevier Inc. All rights reserved.

Optimal experimental design in an epidermal growth factor receptor signalling and down-regulation model.

PubMed

Casey, F P; Baird, D; Feng, Q; Gutenkunst, R N; Waterfall, J J; Myers, C R; Brown, K S; Cerione, R A; Sethna, J P

2007-05-01

We apply the methods of optimal experimental design to a differential equation model for epidermal growth factor receptor signalling, trafficking and down-regulation. The model incorporates the role of a recently discovered protein complex made up of the E3 ubiquitin ligase, Cbl, the guanine exchange factor (GEF), Cool-1 (beta -Pix) and the Rho family G protein Cdc42. The complex has been suggested to be important in disrupting receptor down-regulation. We demonstrate that the model interactions can accurately reproduce the experimental observations, that they can be used to make predictions with accompanying uncertainties, and that we can apply ideas of optimal experimental design to suggest new experiments that reduce the uncertainty on unmeasurable components of the system.

Deregulation of F-box proteins and its consequence on cancer development, progression and metastasis

PubMed Central

Heo, Jinho; Eki, Rebeka; Abbas, Tarek

2015-01-01

F-box proteins are substrate receptors of the SCF (SKP1-Cullin 1-F-box protein) E3 ubiquitin ligase that play important roles in a number of physiological processes and activities. Through their ability to assemble distinct E3 ubiquitin ligases and target key regulators of cellular activities for ubiquitylation and degradation, this versatile group of proteins is able to regulate the abundance of cellular proteins whose deregulated expression or activity contributes to disease. In this review, we describe the important roles of select F-box proteins in regulating cellular activities, the perturbation of which contributes to the initiation and progression of a number of human malignancies. PMID:26432751

Proteolysis targeting peptide (PROTAP) strategy for protein ubiquitination and degradation.

PubMed

Zheng, Jing; Tan, Chunyan; Xue, Pengcheng; Cao, Jiakun; Liu, Feng; Tan, Ying; Jiang, Yuyang

2016-02-19

Ubiquitination proteasome pathway (UPP) is the most important and selective way to degrade proteins in vivo. Here, a novel proteolysis targeting peptide (PROTAP) strategy, composed of a target protein binding peptide, a linker and a ubiquitin E3 ligase recognition peptide, was designed to recruit both target protein and E3 ligase and then induce polyubiquitination and degradation of the target protein through UPP. In our study, the PROTAP strategy was proved to be a general method with high specificity using Bcl-xL protein as model target in vitro and in cells, which indicates that the strategy has great potential for in vivo application. Copyright © 2016 Elsevier Inc. All rights reserved.

Autoubiquitination of feline E3 ubiquitin ligase BCA2.

PubMed

Wang, Weiran; Qu, Meng; Wang, Jiawen; Zhang, Xin; Zhang, Haihong; Wu, Jiaxin; Yu, Bin; Wu, Hui; Kong, Wei; Yu, Xianghui

2018-01-05

BCA2/RNF115/Rabring7 is a RING type E3 ubiquitin ligase that is overexpressed in human breast tumors and is important for regulating breast cancer cell migration. In the present investigation, feline BCA2 (fBCA2) was identified and characterized. Compared with its human counterpart, the fBCA2 cDNA was confirmed to be 918 base pairs in length showing 92.6% consensus and identity positions, encoding a protein of 305 amino acids with 96.7% consensus and 93.1% identity positions. The fBCA2 protein contains a RING domain at the C-terminus, which was found to be essential for its autoubiquitination. Copyright © 2017. Published by Elsevier B.V.

Diggin’ on U(biquitin): A Novel Method for the Identification of Physiological E3 Ubiquitin Ligase Substrates

PubMed Central

Rubel, Carrie E.; Schisler, Jonathan C.; Hamlett, Eric D.; DeKroon, Robert M.; Gautel, Mathias; Alzate, Oscar; Patterson, Cam

2013-01-01

The ubiquitin-proteasome system (UPS) plays a central role in maintaining protein homeostasis, emphasized by a myriad of diseases that are associated with altered UPS function such as cancer, muscle-wasting, and neurodegeneration. Protein ubiquitination plays a central role in both the promotion of proteasomal degradation as well as cellular signaling through regulation of the stability of transcription factors and other signaling molecules. Substrate specificity is a critical regulatory step of ubiquitination and is mediated by ubiquitin ligases. Recent studies implicate ubiquitin ligases in multiple models of cardiac diseases such as cardiac hypertrophy, atrophy, and ischemia/reperfusion injury, both in a cardioprotective and maladaptive role. Therefore, identifying physiological substrates of cardiac ubiquitin ligases provides both mechanistic insights into heart disease as well as possible therapeutic targets. Current methods identifying substrates for ubiquitin ligases rely heavily upon non-physiologic in vitro methods, impeding the unbiased discovery of physiological substrates in relevant model systems. Here we describe a novel method for identifying ubiquitin ligase substrates utilizing Tandem Ubiquitin Binding Entities (TUBE) technology, two-dimensional differential in gel electrophoresis (2-D DIGE), and mass spectrometry, validated by the identification of both known and novel physiological substrates of the ubiquitin ligase MuRF1 in primary cardiomyocytes. This method can be applied to any ubiquitin ligase, both in normal and disease model systems, in order to identify relevant physiological substrates under various biological conditions, opening the door to a clearer mechanistic understanding of ubiquitin ligase function and broadening their potential as therapeutic targets. PMID:23695782

Phylogenetic analysis of the SINA/SIAH ubiquitin E3 ligase family in Metazoa.

PubMed

Pepper, Ian J; Van Sciver, Robert E; Tang, Amy H

2017-08-07

The RAS signaling pathway is a pivotal developmental pathway that controls many fundamental biological processes including cell proliferation, differentiation, movement and apoptosis. Drosophila Seven-IN-Absentia (SINA) is a ubiquitin E3 ligase that is the most downstream signaling "gatekeeper" whose biological activity is essential for proper RAS signal transduction. Vertebrate SINA homologs (SIAHs) share a high degree of amino acid identity with that of Drosophila SINA. SINA/SIAH is the most conserved signaling component in the canonical EGFR/RAS/RAF/MAPK signal transduction pathway. Vertebrate SIAH1, 2, and 3 are the three orthologs to invertebrate SINA protein. SINA and SIAH1 orthologs are found in all major taxa of metazoans. These proteins have four conserved functional domains, known as RING (Really Interesting New Gene), SZF (SIAH-type zinc finger), SBS (substrate binding site) and DIMER (Dimerization). In addition to the siah1 gene, most vertebrates encode two additional siah genes (siah2 and siah3) in their genomes. Vertebrate SIAH2 has a highly divergent and extended N-terminal sequence, while its RING, SZF, SBS and DIMER domains maintain high amino acid identity/similarity to that of SIAH1. But unlike vertebrate SIAH1 and SIAH2, SIAH3 lacks a functional RING domain, suggesting that SIAH3 may be an inactive E3 ligase. The SIAH3 subtree exhibits a high degree of amino acid divergence when compared to the SIAH1 and SIAH2 subtrees. We find that SIAH1 and SIAH2 are expressed in all human epithelial cell lines examined thus far, while SIAH3 is only expressed in a limited subset of cancer cell lines. Through phylogenetic analyses of metazoan SINA and SIAH E3 ligases, we identified many invariant and divergent amino acid residues, as well as the evolutionarily conserved functional motifs in this medically relevant gene family. Our phylomedicinal study of this unique metazoan SINA/SIAH protein family has provided invaluable evolution-based support towards future effort to design logical, potent, and durable anti-SIAH-based anticancer strategies against oncogenic K-RAS-driven metastatic human cancers. Thus, this method of evolutionary study should be of interest in cancer biology.

Atomic structure of the APC/C and its mechanism of protein ubiquitination

PubMed Central

Yang, Jing; McLaughlin, Stephen H.; Barford, David

2015-01-01

The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex, and interphase inhibitor Emi1 ensures the correct order and timing of distinct cell cycle transitions. Here, we used cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module, and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of experiments to further investigate APC/C functions in vivo. PMID:26083744

Ube2w and ataxin-3 coordinately regulate the ubiquitin ligase CHIP

PubMed Central

Scaglione, K. Matthew; Zavodszky, Eszter; Todi, Sokol V.; Patury, Srikanth; Xu, Ping; Rodríguez-Lebrón, Edgardo; Fischer, Svetlana; Konen, John; Djarmati, Ana; Peng, Junmin; Gestwicki, Jason E.; Paulson, Henry L.

2011-01-01

Summary The mechanisms by which ubiquitin ligases are regulated remain poorly understood. Here we describe a series of molecular events that coordinately regulate CHIP, a neuroprotective E3 implicated in protein quality control. Through their opposing activities, the initiator E2, Ube2w, and the specialized deubiquitinating enzyme (DUB), ataxin-3, participate in initiating, regulating and terminating the CHIP ubiquitination cycle. Monoubiquitination of CHIP by Ube2w stabilizes the interaction between CHIP and ataxin-3, which through its DUB activity limits the length of chains attached to CHIP substrates. Upon completion of substrate ubiquitination ataxin-3 deubiquitinates CHIP, effectively terminating the reaction. Our results suggest that functional pairing of E3s with ataxin-3 or similar DUBs represents an important point of regulation in ubiquitin-dependent protein quality control. In addition, the results shed light on disease pathogenesis in SCA3, a neurodegenerative disorder caused by polyglutamine expansion in ataxin-3. PMID:21855799

A C2HC zinc finger is essential for the RING-E2 interaction of the ubiquitin ligase RNF125

PubMed Central

Bijlmakers, Marie-José; Teixeira, João M. C.; Boer, Roeland; Mayzel, Maxim; Puig-Sàrries, Pilar; Karlsson, Göran; Coll, Miquel; Pons, Miquel; Crosas, Bernat

2016-01-01

The activity of RING ubiquitin ligases (E3s) depends on an interaction between the RING domain and ubiquitin conjugating enzymes (E2), but posttranslational events or additional structural elements, yet largely undefined, are frequently required to enhance or regulate activity. Here, we show for the ubiquitin ligase RNF125 that, in addition to the RING domain, a C2HC Zn finger (ZnF) is crucial for activity, and a short linker sequence (Li2120-128) enhances activity. The contribution of these regions was first shown with truncated proteins, and the essential role of the ZnF was confirmed with mutations at the Zn chelating Cys residues. Using NMR, we established that the C2HC ZnF/Li2120-128 region is crucial for binding of the RING domain to the E2 UbcH5a. The partial X-ray structure of RNF125 revealed the presence of extensive intramolecular interactions between the RING and C2HC ZnF. A mutation at one of the contact residues in the C2HC ZnF, a highly conserved M112, resulted in the loss of ubiquitin ligase activity. Thus, we identified the structural basis for an essential role of the C2HC ZnF and conclude that this domain stabilizes the RING domain, and is therefore required for binding of RNF125 to an E2. PMID:27411375

RNF41 interacts with the VPS52 subunit of the GARP and EARP complexes.

PubMed

Masschaele, Delphine; De Ceuninck, Leentje; Wauman, Joris; Defever, Dieter; Stenner, Frank; Lievens, Sam; Peelman, Frank; Tavernier, Jan

2017-01-01

RNF41 (Ring Finger Protein 41) is an E3 ubiquitin ligase involved in the intracellular sorting and function of a diverse set of substrates. Next to BRUCE and Parkin, RNF41 can directly ubiquitinate ErbB3, IL-3, EPO and RARα receptors or downstream signaling molecules such as Myd88, TBK1 and USP8. In this way it can regulate receptor signaling and routing. To further elucidate the molecular mechanism behind the role of RNF41 in intracellular transport we performed an Array MAPPIT (Mammalian Protein-Protein Interaction Trap) screen using an extensive set of proteins derived from the human ORFeome collection. This paper describes the identification of VPS52, a subunit of the GARP (Golgi-Associated Retrograde Protein) and the EARP (Endosome-Associated Recycling Protein) complexes, as a novel interaction partner of RNF41. Through interaction via their coiled coil domains, RNF41 ubiquitinates and relocates VPS52 away from VPS53, a common subunit of the GARP and EARP complexes, towards RNF41 bodies.

p63 threonine phosphorylation signals the interaction with the WW domain of the E3 ligase Itch

PubMed Central

Melino, Sonia; Bellomaria, Alessia; Nepravishta, Ridvan; Paci, Maurizio; Melino, Gerry

2014-01-01

Both in epithelial development as well as in epithelial cancers, the p53 family member p63 plays a crucial role acting as a master transcriptional regulator. P63 steady state protein levels are regulated by the E3 ubiquitin ligase Itch, via a physical interaction between the PPxY consensus sequence (PY motif) of p63 and one of the 4 WW domains of Itch; this substrate recognition process leads to protein-ubiquitylation and p63 proteasomal degradation. The interaction of the WW domains, a highly compact protein-protein binding module, with the short proline-rich sequences is therefore a crucial regulatory event that may offer innovative potential therapeutic opportunity. Previous molecular studies on the Itch-p63 recognition have been performed in vitro using the Itch-WW2 domain and the peptide interacting fragment of p63 (pep63), which includes the PY motif. Itch-WW2-pep63 interaction is also stabilized in vitro by the conformational constriction of the S-S cyclization in the p63 peptide. The PY motif of p63, as also for other proteins, is characterized by the nearby presence of a (T/S)P motif, which is a potential recognition site of the WW domain of the IV group present in the prolyl-isomerase Pin1. In this study, we demonstrate, by in silico and spectroscopical studies using both the linear pep63 and its cyclic form, that the threonine phosphorylation of the (T/S)PPPxY motif may represent a crucial regulatory event of the Itch-mediated p63 ubiquitylation, increasing the Itch-WW domains-p63 recognition event and stabilizing in vivo the Itch-WW-p63 complex. Moreover, our studies confirm that the subsequently trans/cis proline isomerization of (T/S)P motif by the Pin1 prolyl-isomerase, could modulate the E3-ligase interaction, and that the (T/S)pPtransPPxY motif represent the best conformer for the ItchWW-(T/S)PPPxY motif recognition. PMID:25485500

p63 threonine phosphorylation signals the interaction with the WW domain of the E3 ligase Itch.

PubMed

Melino, Sonia; Bellomaria, Alessia; Nepravishta, Ridvan; Paci, Maurizio; Melino, Gerry

2014-01-01

Both in epithelial development as well as in epithelial cancers, the p53 family member p63 plays a crucial role acting as a master transcriptional regulator. P63 steady state protein levels are regulated by the E3 ubiquitin ligase Itch, via a physical interaction between the PPxY consensus sequence (PY motif) of p63 and one of the 4 WW domains of Itch; this substrate recognition process leads to protein-ubiquitylation and p63 proteasomal degradation. The interaction of the WW domains, a highly compact protein-protein binding module, with the short proline-rich sequences is therefore a crucial regulatory event that may offer innovative potential therapeutic opportunity. Previous molecular studies on the Itch-p63 recognition have been performed in vitro using the Itch-WW2 domain and the peptide interacting fragment of p63 (pep63), which includes the PY motif. Itch-WW2-pep63 interaction is also stabilized in vitro by the conformational constriction of the S-S cyclization in the p63 peptide. The PY motif of p63, as also for other proteins, is characterized by the nearby presence of a (T/S)P motif, which is a potential recognition site of the WW domain of the IV group present in the prolyl-isomerase Pin1. In this study, we demonstrate, by in silico and spectroscopical studies using both the linear pep63 and its cyclic form, that the threonine phosphorylation of the (T/S)PPPxY motif may represent a crucial regulatory event of the Itch-mediated p63 ubiquitylation, increasing the Itch-WW domains-p63 recognition event and stabilizing in vivo the Itch-WW-p63 complex. Moreover, our studies confirm that the subsequently trans/cis proline isomerization of (T/S)P motif by the Pin1 prolyl-isomerase, could modulate the E3-ligase interaction, and that the (T/S)pPtransPPxY motif represent the best conformer for the ItchWW-(T/S)PPPxY motif recognition.

A Plasmodium yoelii HECT-like E3 ubiquitin ligase regulates parasite growth and virulence.

PubMed

Nair, Sethu C; Xu, Ruixue; Pattaradilokrat, Sittiporn; Wu, Jian; Qi, Yanwei; Zilversmit, Martine; Ganesan, Sundar; Nagarajan, Vijayaraj; Eastman, Richard T; Orandle, Marlene S; Tan, John C; Myers, Timothy G; Liu, Shengfa; Long, Carole A; Li, Jian; Su, Xin-Zhuan

2017-08-09

Infection of mice with strains of Plasmodium yoelii parasites can result in different pathology, but molecular mechanisms to explain this variation are unclear. Here we show that a P. yoelii gene encoding a HECT-like E3 ubiquitin ligase (Pyheul) influences parasitemia and host mortality. We genetically cross two lethal parasites with distinct disease phenotypes, and identify 43 genetically diverse progeny by typing with microsatellites and 9230 single-nucleotide polymorphisms. A genome-wide quantitative trait loci scan links parasite growth and host mortality to two major loci on chromosomes 1 and 7 with LOD (logarithm of the odds) scores = 6.1 and 8.1, respectively. Allelic exchange of partial sequences of Pyheul in the chromosome 7 locus and modification of the gene expression alter parasite growth and host mortality. This study identifies a gene that may have a function in parasite growth, virulence, and host-parasite interaction, and therefore could be a target for drug or vaccine development.Many strains of Plasmodium differ in virulence, but factors that control these distinctions are not known. Here the authors comparatively map virulence loci using the offspring from a P. yoelii YM and N67 genetic cross, and identify a putative HECT E3 ubiquitin ligase that may explain the variance.

PUB22 and PUB23 U-BOX E3 ligases directly ubiquitinate RPN6, a 26S proteasome lid subunit, for subsequent degradation in Arabidopsis thaliana.

PubMed

Cho, Seok Keun; Bae, Hansol; Ryu, Moon Young; Wook Yang, Seong; Kim, Woo TaeK

2015-09-04

Drought stress strongly affects plant growth and development, directly connected with crop yields, accordingly. However, related to the function of U-BOX E3 ligases, the underlying molecular mechanisms of desiccation stress response in plants are still largely unknown. Here we report that PUB22 and PUB23, two U-box E3 ligase homologs, tether ubiquitins to 19S proteasome regulatory particle (RP) subunit RPN6, leading to its degradation. RPN6 was identified as an interacting substrate of PUB22 by yeast two-hybrid screening, and in vitro pull-down assay confirmed that RPN6 interacts not only with PUB22, but also with PUB23. Both PUB22 and PUB23 were able to conjugate ubiquitins on RPN6 in vitro. Furthermore, RPN6 showed a shorter protein half-life in PUB22 overexpressing plants than in wild-type, besides RPN6 was significantly stabilized in pub22pub23 double knockout plants. Taken together, these results solidify a notion that PUB22 and PUB23 can alter the activity of 26S proteasome in response to drought stress. Copyright © 2015 Elsevier Inc. All rights reserved.

Cloning and characterization of carboxyl terminus of heat shock cognate 70-interacting protein gene from the silkworm, Bombyx mori.

PubMed

Ohsawa, Takeshi; Fujimoto, Shota; Tsunakawa, Akane; Shibano, Yuka; Kawasaki, Hideki; Iwanaga, Masashi

2016-11-01

Carboxyl terminus of heat shock cognate 70-interacting protein (CHIP) is an evolutionarily conserved E3 ubiquitin ligase across different eukaryotic species and is known to play a key role in protein quality control. CHIP has two distinct functional domains, an N-terminal tetratricopeptide repeat (TPR) and a C-terminal U-box domain, which are required for the ubiquitination of numerous labile client proteins that are chaperoned by heat shock proteins (HSPs) and heat shock cognate proteins (HSCs). During our screen for CHIP-like proteins in the Bombyx mori databases, we found a novel silkworm gene, Bombyx mori CHIP. Phylogenetic analysis showed that BmCHIP belongs to Lepidopteran lineages. Quantitative reverse transcription-PCR analysis indicated that BmCHIP was relatively highly expressed in the gonad and fat body. A pull-down experiment and auto-ubiquitination assay showed that BmCHIP interacted with BmHSC70 and had E3 ligase activity. Additionally, immunohistochemical analysis revealed that BmCHIP was partially co-localized with ubiquitin in BmN4 cells. These data support that BmCHIP plays an important role in the ubiquitin proteasome system as an E3 ubiquitin ligase in B. mori. Copyright © 2016 Elsevier Inc. All rights reserved.

SCAR Mediates Light-Induced Root Elongation in Arabidopsis through Photoreceptors and Proteasomes[W][OA

PubMed Central

Dyachok, Julia; Zhu, Ling; Liao, Fuqi; He, Ji; Huq, Enamul; Blancaflor, Elison B.

2011-01-01

The ARP2/3 complex, a highly conserved nucleator of F-actin, and its activator, the SCAR complex, are essential for growth in plants and animals. In this article, we present a pathway through which roots of Arabidopsis thaliana directly perceive light to promote their elongation. The ARP2/3-SCAR complex and the maintenance of longitudinally aligned F-actin arrays are crucial components of this pathway. The involvement of the ARP2/3-SCAR complex in light-regulated root growth is supported by our finding that mutants of the SCAR complex subunit BRK1/HSPC300, or other individual subunits of the ARP2/3-SCAR complex, showed a dramatic inhibition of root elongation in the light, which mirrored reduced growth of wild-type roots in the dark. SCAR1 degradation in dark-grown wild-type roots by constitutive photomorphogenic 1 (COP1) E3 ligase and 26S proteasome accompanied the loss of longitudinal F-actin and reduced root growth. Light perceived by the root photoreceptors, cryptochrome and phytochrome, suppressed COP1-mediated SCAR1 degradation. Taken together, our data provide a biochemical explanation for light-induced promotion of root elongation by the ARP2/3-SCAR complex. PMID:21972261

An Arabidopsis E3 Ligase, SHOOT GRAVITROPISM9, Modulates the Interaction between Statoliths and F-Actin in Gravity Sensing[W][OA

PubMed Central

Nakamura, Moritaka; Toyota, Masatsugu; Tasaka, Masao; Morita, Miyo Terao

2011-01-01

Higher plants use the sedimentation of amyloplasts in statocytes as statolith to sense the direction of gravity during gravitropism. In Arabidopsis thaliana inflorescence stem statocyte, amyloplasts are in complex movement; some show jumping-like saltatory movement and some tend to sediment toward the gravity direction. Here, we report that a RING-type E3 ligase SHOOT GRAVITROPISM9 (SGR9) localized to amyloplasts modulates amyloplast dynamics. In the sgr9 mutant, which exhibits reduced gravitropism, amyloplasts did not sediment but exhibited increased saltatory movement. Amyloplasts sometimes formed a cluster that is abnormally entangled with actin filaments (AFs) in sgr9. By contrast, in the fiz1 mutant, an ACT8 semidominant mutant that induces fragmentation of AFs, amyloplasts, lost saltatory movement and sedimented with nearly statically. Both treatment with Latrunculin B, an inhibitor of AF polymerization, and the fiz1 mutation rescued the gravitropic defect of sgr9. In addition, fiz1 decreased saltatory movement and induced amyloplast sedimentation even in sgr9. Our results suggest that amyloplasts are in equilibrium between sedimentation and saltatory movement in wild-type endodermal cells. Furthermore, this equilibrium is the result of the interaction between amyloplasts and AFs modulated by the SGR9. SGR9 may promote detachment of amyloplasts from AFs, allowing the amyloplasts to sediment in the AFs-dependent equilibrium of amyloplast dynamics. PMID:21602290

Derlin-1 promotes ubiquitylation and degradation of the epithelial Na+ channel, ENaC.

PubMed

You, Hui; Ge, Yamei; Zhang, Jian; Cao, Yizhi; Xing, Jing; Su, Dongming; Huang, Yujie; Li, Min; Qu, Shen; Sun, Fei; Liang, Xiubin

2017-03-15

Ubiquitylation of the epithelial Na + channel (ENaC) plays a critical role in cellular functions, including transmembrane transport of Na + , Na + and water balance, and blood pressure stabilization. Published studies have suggested that ENaC subunits are targets of ER-related degradation (ERAD) in yeast systems. However, the molecular mechanism underlying proteasome-mediated degradation of ENaC subunits remains to be established. Derlin-1, an E3 ligase mediator, links recognized target proteins to ubiquitin-mediated proteasomal degradation in the cytosol. In the present study, we found that derlin-1 suppressed the expression of ENaC at the protein level and that the subunit α-ENaC (also known as SCNN1A) physically interacted with derlin-1 at the membrane-anchored domains or the loop regions, and that derlin-1 initiated α-ENaC retrotranslocation. In addition, HUWE1, an endoplasmic reticulum (ER)-resident E3 ubiquitin ligase, was recruited and promoted K11-linked polyubiquitylation of α-ENaC and, hence, formation of an α-ENaC ubiquitin-mediated degradation complex. These findings suggest that derlin-1 promotes ENaC ubiquitylation and enhances ENaC ubiquitin- mediated proteasome degradation. The derlin-1 pathway therefore may represent a significant early checkpoint in the recognition and degradation of ENaC in mammalian cells. © 2017. Published by The Company of Biologists Ltd.

ITCH directly K63-ubiquitinates the NOD2 binding protein, RIP2, to influence inflammatory signaling pathways

PubMed Central

Tao, MingFang; Scacheri, Peter C.; Marinis, Jill M.; Harhaj, Edward W.; Matesic, Lydia E.; Abbott, Derek W.

2009-01-01

Background: The inability to coordinate the signaling pathways that lead to proper cytokine responses characterizes the pathogenesis of inflammatory diseases such as Crohn's Disease. The Crohn's Disease susceptibility protein, NOD2, helps coordinate cytokine responses upon intracellular exposure to bacteria, and this signal coordination by NOD2 is accomplished, in part, through K63-linked polyubiquitin chains that create binding surfaces for the scaffolding of signaling complexes. Results: In this work, we show that the NOD2 signaling partner, RIP2, is directly K63 polyubiquitinated by ITCH, an E3 ubiquitin ligase which when lost genetically, causes widespread inflammatory disease at mucosal surfaces. We show that ITCH is responsible for RIP2 polyubiquitination in response to infection with listeria monocytogenes. We further show that NOD2 can bind polyubiquitinated RIP2, and while ITCH E3 ligase activity is required for optimal NOD2:RIP2-induced p38 and JNK activation, ITCH inhibits NOD2:RIP2-induced NFκB activation. This effect can be seen independently at the whole genome level by microarray analysis of MDP-treated Itch−/− primary macrophages. Conclusions: These findings suggest that ITCH helps regulate NOD2-dependent signal transduction pathways and as such, may be involved in the pathogenesis of NOD2-mediated inflammatory disease. PMID:19592251

Molecular mechanism of ER stress-induced pre-emptive quality control involving association of the translocon, Derlin-1, and HRD1.

PubMed

Kadowaki, Hisae; Satrimafitrah, Pasjan; Takami, Yasunari; Nishitoh, Hideki

2018-05-09

The maintenance of endoplasmic reticulum (ER) homeostasis is essential for cell function. ER stress-induced pre-emptive quality control (ERpQC) helps alleviate the burden to a stressed ER by limiting further protein loading. We have previously reported the mechanisms of ERpQC, which includes a rerouting step and a degradation step. Under ER stress conditions, Derlin family proteins (Derlins), which are components of ER-associated degradation, reroute specific ER-targeting proteins to the cytosol. Newly synthesized rerouted polypeptides are degraded via the cytosolic chaperone Bag6 and the AAA-ATPase p97 in the ubiquitin-proteasome system. However, the mechanisms by which ER-targeting proteins are rerouted from the ER translocation pathway to the cytosolic degradation pathway and how the E3 ligase ubiquitinates ERpQC substrates remain unclear. Here, we show that ERpQC substrates are captured by the carboxyl-terminus region of Derlin-1 and ubiquitinated by the HRD1 E3 ubiquitin ligase prior to degradation. Moreover, HRD1 forms a large ERpQC-related complex composed of Sec61α and Derlin-1 during ER stress. These findings indicate that the association of the degradation factor HRD1 with the translocon and the rerouting factor Derlin-1 may be necessary for the smooth and effective clearance of ERpQC substrates.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan, Ronghui; Satkovich, John; Hu, Jianping

Peroxisomes are ubiquitous eukaryotic organelles that play pivotal roles in a suite of metabolic processes and often act coordinately with other organelles, such as chloroplasts and mitochondria. Peroxisomes import proteins to the peroxisome matrix by peroxins (PEX proteins), but how the function of the PEX proteins is regulated is poorly understood. In this study, we identified the Arabidopsis RING (really interesting new gene) type E3 ubiquitin ligase SP1 [suppressor of plastid protein import locus 1 (ppi1) 1] as a peroxisome membrane protein with a regulatory role in peroxisome protein import. SP1 interacts physically with the two components of the peroxisomemore » protein docking complex PEX13–PEX14 and the (RING)-finger peroxin PEX2. Loss of SP1 function suppresses defects of the pex14-2 and pex13-1 mutants, and SP1 is involved in the degradation of PEX13 and possibly PEX14 and all three RING peroxins. An in vivo ubiquitination assay showed that SP1 has the ability to promote PEX13 ubiquitination. Our study has revealed that, in addition to its previously reported function in chloroplast biogenesis, SP1 plays a role in peroxisome biogenesis. The same E3 ubiquitin ligase promotes the destabilization of components of two distinct protein-import machineries, indicating that degradation of organelle biogenesis factors by the ubiquitin–proteasome system may constitute an important regulatory mechanism in coordinating the biogenesis of metabolically linked organelles in eukaryotes.« less

Differential response of skeletal muscles to mTORC1 signaling during atrophy and hypertrophy

PubMed Central

2013-01-01

Background Skeletal muscle mass is determined by the balance between protein synthesis and degradation. Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of protein translation and has been implicated in the control of muscle mass. Inactivation of mTORC1 by skeletal muscle-specific deletion of its obligatory component raptor results in smaller muscles and a lethal dystrophy. Moreover, raptor-deficient muscles are less oxidative through changes in the expression PGC-1α, a critical determinant of mitochondrial biogenesis. These results suggest that activation of mTORC1 might be beneficial to skeletal muscle by providing resistance to muscle atrophy and increasing oxidative function. Here, we tested this hypothesis by deletion of the mTORC1 inhibitor tuberous sclerosis complex (TSC) in muscle fibers. Method Skeletal muscles of mice with an acute or a permanent deletion of raptor or TSC1 were examined using histological, biochemical and molecular biological methods. Response of the muscles to changes in mechanical load and nerve input was investigated by ablation of synergistic muscles or by denervation . Results Genetic deletion or knockdown of raptor, causing inactivation of mTORC1, was sufficient to prevent muscle growth and enhance muscle atrophy. Conversely, short-term activation of mTORC1 by knockdown of TSC induced muscle fiber hypertrophy and atrophy-resistance upon denervation, in both fast tibialis anterior (TA) and slow soleus muscles. Surprisingly, however, sustained activation of mTORC1 by genetic deletion of Tsc1 caused muscle atrophy in all but soleus muscles. In contrast, oxidative capacity was increased in all muscles examined. Consistently, TSC1-deficient soleus muscle was atrophy-resistant whereas TA underwent normal atrophy upon denervation. Moreover, upon overloading, plantaris muscle did not display enhanced hypertrophy compared to controls. Biochemical analysis indicated that the atrophy response of muscles was based on the suppressed phosphorylation of PKB/Akt via feedback inhibition by mTORC1 and subsequent increased expression of the E3 ubiquitin ligases MuRF1 and atrogin-1/MAFbx. In contrast, expression of both E3 ligases was not increased in soleus muscle suggesting the presence of compensatory mechanisms in this muscle. Conclusions Our study shows that the mTORC1- and the PKB/Akt-FoxO pathways are tightly interconnected and differentially regulated depending on the muscle type. These results indicate that long-term activation of the mTORC1 signaling axis is not a therapeutic option to promote muscle growth because of its strong feedback induction of the E3 ubiquitin ligases involved in protein degradation. PMID:23497627

Conversion from CUL4-based COP1–SPA E3 apparatus to UVR8–COP1–SPA complexes underlies a distinct biochemical function of COP1 under UV-B

PubMed Central

Huang, Xi; Ouyang, Xinhao; Yang, Panyu; Lau, On Sun; Chen, Liangbi; Wei, Ning; Deng, Xing Wang

2013-01-01

The evolutionarily conserved CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) is a RING and WD40 protein that functions as a substrate receptor of CULLIN4–DAMAGED DNA BINDING PROTEIN 1 (CUL4–DDB1)–based E3 ubiquitin ligases in both plants and animals. In Arabidopsis, COP1 is a central repressor of photomorphogenesis in the form of COP1–SUPPRESSOR OF PHYA (SPA) complex(es). CUL4–DDB1–COP1–SPA suppresses the photomorphogenic program by targeting the transcription factor ELONGATED HYPOCOTYL 5 for degradation. Intriguingly, under photomorphogenic UV-B light, COP1 reverses its repressive role and promotes photomorphogenesis. However, the mechanism by which COP1 is functionally switched is still obscure. Here, we demonstrate that UV-B triggers the physical and functional disassociation of the COP1–SPA core complex(es) from CUL4–DDB1 and the formation of a unique complex(es) containing the UV-B receptor UV RESISTANCE LOCUS 8 (UVR8). The establishment of this UV-B–dependent COP1 complex(es) is associated with its positive modulation of ELONGATED HYPOCOTYL 5 stability and activity, which sheds light on the mechanism of COP1’s promotive action in UV-B–induced photomorphogenesis. PMID:24067658

Biochemical and Structural Characterisation of DNA Ligases from Bacteria and Archaea.

PubMed

Pergolizzi, Giulia; Wagner, Gerd K; Bowater, Richard Peter

2016-08-31

DNA ligases are enzymes that seal breaks in the backbones of DNA, leading to them being essential for the survival of all organisms. DNA ligases have been studied from many different types of cells and organisms and shown to have diverse sizes and sequences, with well conserved specific sequences that are required for enzymatic activity. A significant number of DNA ligases have been isolated or prepared in recombinant forms and, here, we review their biochemical and structural characterisation. All DNA ligases contain an essential lysine that transfers an adenylate group from a co-factor to the 5'-phosphate of the DNA end that will ultimately be joined to the 3'-hydroxyl of the neighbouring DNA strand. The essential DNA ligases in bacteria use nicotinamide adenine dinucleotide ( β -NAD + ) as their co-factor whereas those that are essential in other cells use adenosine-5'-triphosphate (ATP) as their co-factor. This observation suggests that the essential bacterial enzyme could be targeted by novel antibiotics and the complex molecular structure of β -NAD + affords multiple opportunities for chemical modification. Several recent studies have synthesised novel derivatives and their biological activity against a range of DNA ligases has been evaluated as inhibitors for drug discovery and/or non-natural substrates for biochemical applications. Here, we review the recent advances that herald new opportunities to alter the biochemical activities of these important enzymes. The recent development of modified derivatives of nucleotides highlights that the continued combination of structural, biochemical and biophysical techniques will be useful in targeting these essential cellular enzymes. ©2016 The Author(s).

Characterization of Elements Regulating the Nuclear-to-Cytoplasmic Translocation of ICP0 in Late Herpes Simplex Virus 1 Infection.

PubMed

Samrat, Subodh Kumar; Ha, Binh L; Zheng, Yi; Gu, Haidong

2018-01-15

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It targets several host factors for proteasomal degradation and subsequently activates viral expression. ICP0 has a nuclear localization sequence and functions in the nucleus early during infection. However, later in infection, ICP0 is found solely in the cytoplasm. The molecular mechanism and biological function of the ICP0 nuclear-to-cytoplasmic translocation are not well understood. In this study, we sought to characterize elements important for this translocation. We found that (i) in human embryonic lung fibroblast (HEL) cells, ICP0 C-terminal residues 741 to 775 were necessary but not sufficient for the nuclear-to-cytoplasmic translocation; (ii) the loss of ICP0 E3 ubiquitin ligase activity, which led to defective viral replication in nonpermissive cells, also caused mutant ICP0 to be retained in the nucleus of HEL cells; (iii) in permissive U2OS cells, however, ICP0 lacking E3 ligase activity was translocated to the cytoplasm at a pace faster than that of wild-type ICP0, suggesting that nuclear retention of ICP0 occurs in an ICP0 E3 ligase-dependent manner; and (iv) the ICP0 C terminus and late viral proteins cooperate in order to overcome nuclear retention and stimulate ICP0 cytoplasmic translocation. Taken together, less ICP0 nuclear retention may contribute to the permissiveness of U2OS cells to HSV-1 in the absence of functional ICP0. IMPORTANCE A distinct characteristic for eukaryotes is the compartmentalization of cell metabolic pathways, which allows greater efficiency and specificity of cellular functions. ICP0 of HSV-1 is a multifunctional viral protein that travels through different compartments as infection progresses. Its main regulatory functions are carried out in the nucleus, but it is translocated to the cytoplasm late during HSV-1 infection. To understand the biological significance of cytoplasmic ICP0 in HSV-1 infection, we investigated the potential players involved in this nuclear-to-cytoplasmic translocation. We found that there is a nuclear retention force in an ICP0 E3 ubiquitin ligase-dependent manner. In addition, we identified the C terminus of ICP0 as a cis element cooperating with late viral proteins to overcome the nuclear retention and stimulate the nuclear-to-cytoplasmic translocation of ICP0. Copyright © 2018 American Society for Microbiology.

The ECS(SPSB) E3 ubiquitin ligase is the master regulator of the lifetime of inducible nitric-oxide synthase.

PubMed

Matsumoto, Kazuma; Nishiya, Tadashi; Maekawa, Satoshi; Horinouchi, Takahiro; Ogasawara, Kouetsu; Uehara, Takashi; Miwa, Soichi

2011-05-27

The ubiquitin-proteasome pathway is an important regulatory system for the lifetime of inducible nitric-oxide synthase (iNOS), a high-output isoform compared to neuronal NOS (nNOS) and endothelial NOS (eNOS), to prevent overproduction of NO that could trigger detrimental effects such as cytotoxicity. Two E3 ubiquitin ligases, Elongin B/C-Cullin-5-SPRY domain- and SOCS box-containing protein [ECS(SPSB)] and the C-terminus of Hsp70-interacting protein (CHIP), recently have been reported to target iNOS for proteasomal degradation. However, the significance of each E3 ubiquitin ligase for the proteasomal degradation of iNOS remains to be determined. Here, we show that ECS(SPSB) specifically interacted with iNOS, but not nNOS and eNOS, and induced the subcellular redistribution of iNOS from dense regions to diffused expression as well as the ubiquitination and proteasomal degradation of iNOS, whereas CHIP neither interacted with iNOS nor had any effects on the subcellular localization, ubiquitination, and proteasomal degradation of iNOS. These results differ from previous reports. Furthermore, the lifetime of the iNOS(N27A) mutant, a form of iNOS that does not bind to ECS(SPSB), was substantially extended in macrophages. These results demonstrate that ECS(SPSB), but not CHIP, is the master regulator of the iNOS lifetime. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification of a Cullin5-ElonginB-ElonginC E3 complex in degradation of feline immunodeficiency virus Vif-mediated feline APOBEC3 proteins.

PubMed

Wang, Jiawen; Zhang, Wenyan; Lv, Mingyu; Zuo, Tao; Kong, Wei; Yu, Xianghui

2011-12-01

Various feline APOBEC3 (fA3) proteins exhibit broad antiviral activities against a wide range of viruses, such as feline immunodeficiency virus (FIV), feline foamy virus (FFV), and feline leukemia virus (FeLV), as well as those of other species. This activity can be counteracted by the FIV Vif protein, but the mechanism by which FIV Vif suppresses fA3s is unknown. In the present study, we demonstrated that FIV Vif could act via a proteasome-dependent pathway to overcome fA3s. FIV Vif interacted with feline cellular proteins Cullin5 (Cul5), ElonginB, and ElonginC to form an E3 complex to induce degradation of fA3s. Both the dominant-negative Cul5 mutant and a C-terminal hydrophilic replacement ElonginC mutant potently disrupted the FIV Vif activity against fA3s. Furthermore, we identified a BC-box motif in FIV Vif that was essential for the recruitment of E3 ubiquitin ligase and also required for FIV Vif-mediated degradation of fA3s. Moreover, despite the lack of either a Cul5-box or a HCCH zinc-binding motif, FIV Vif specifically selected Cul5. Therefore, FIV Vif may interact with Cul5 via a novel mechanism. These finding imply that SOCS proteins may possess distinct mechanisms to bind Cul5 during formation of the Elongin-Cullin-SOCS box complex.

Epigallocatechin-3-gallate(EGCG) suppresses melanoma cell growth and metastasis by targeting TRAF6 activity.

PubMed

Zhang, Jianglin; Lei, Zhou; Huang, Zunnan; Zhang, Xu; Zhou, Youyou; Luo, Zhongling; Zeng, Weiqi; Su, Juan; Peng, Cong; Chen, Xiang

2016-11-29

TRAF6 (TNF Receptor-Associated Factor 6) is an E3 ubiquitin ligase that contains a Ring domain, induces K63-linked polyubiquitination, and plays a critical role in signaling transduction. Our previous results demonstrated that TRAF6 is overexpressed in melanoma and that TRAF6 knockdown dramatically attenuates tumor cell growth and metastasis. In this study, we found that EGCG can directly bind to TRAF6, and a computational model of the interaction between EGCG and TRAF6 revealed that EGCG probably interacts with TRAF6 at the residues of Gln54, Gly55, Asp57 ILe72, Cys73 and Lys96. Among these amino acids, mutation of Gln54, Asp57, ILe72 in TRAF6 could destroy EGCG bound to TRAF6, furthermore, our results demonstrated that EGCG significantly attenuates interaction between TRAF6 and UBC13(E2) and suppresses TRAF6 E3 ubiquitin ligase activity in vivo and in vitro. Additionally, the phosphorylation of IκBα, p-TAK1 expression are decreased and the nuclear translocation of p65 and p50 is blocked by treatment with EGCG, leading to inactivation of the NF-κB pathway. Moreover, EGCG significantly inhibits cell growth as well as the migration and invasion of melanoma cells. Taken together, these findings show that EGCG is a novel E3 ubiquitin ligase inhibitor that could be used to target TRAF6 for chemotherapy or the prevention of melanoma.

Conserved structural and functional aspects of the tripartite motif gene family point towards therapeutic applications in multiple diseases.

PubMed

Gushchina, Liubov V; Kwiatkowski, Thomas A; Bhattacharya, Sayak; Weisleder, Noah L

2018-05-01

The tripartite motif (TRIM) gene family is a highly conserved group of E3 ubiquitin ligase proteins that can establish substrate specificity for the ubiquitin-proteasome complex and also have proteasome-independent functions. While several family members were studied previously, it is relatively recent that over 80 genes, based on sequence homology, were grouped to establish the TRIM gene family. Functional studies of various TRIM genes linked these proteins to modulation of inflammatory responses showing that they can contribute to a wide variety of disease states including cardiovascular, neurological and musculoskeletal diseases, as well as various forms of cancer. Given the fundamental role of the ubiquitin-proteasome complex in protein turnover and the importance of this regulation in most aspects of cellular physiology, it is not surprising that TRIM proteins display a wide spectrum of functions in a variety of cellular processes. This broad range of function and the highly conserved primary amino acid sequence of family members, particularly in the canonical TRIM E3 ubiquitin ligase domain, complicates the development of therapeutics that specifically target these proteins. A more comprehensive understanding of the structure and function of TRIM proteins will help guide therapeutic development for a number of different diseases. This review summarizes the structural organization of TRIM proteins, their domain architecture, common and unique post-translational modifications within the family, and potential binding partners and targets. Further discussion is provided on efforts to target TRIM proteins as therapeutic agents and how our increasing understanding of the nature of TRIM proteins can guide discovery of other therapeutics in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

Revisiting the NaCl cotransporter regulation by with-no-lysine kinases

PubMed Central

Bazúa-Valenti, Silvana

2015-01-01

The renal thiazide-sensitive Na+-Cl− cotransporter (NCC) is the salt transporter in the distal convoluted tubule. Its activity is fundamental for defining blood pressure levels. Decreased NCC activity is associated with salt-remediable arterial hypotension with hypokalemia (Gitelman disease), while increased activity results in salt-sensitive arterial hypertension with hyperkalemia (pseudohypoaldosteronism type II; PHAII). The discovery of four different genes causing PHAII revealed a complex multiprotein system that regulates the activity of NCC. Two genes encode for with-no-lysine (K) kinases WNK1 and WNK4, while two encode for kelch-like 3 (KLHL3) and cullin 3 (CUL3) proteins that form a RING type E3 ubiquitin ligase complex. Extensive research has shown that WNK1 and WNK4 are the targets for the KLHL3-CUL3 complex and that WNKs modulate the activity of NCC by means of intermediary Ste20-type kinases known as SPAK or OSR1. The understanding of the effect of WNKs on NCC is a complex issue, but recent evidence discussed in this review suggests that we could be reaching the end of the dark ages regarding this matter. PMID:25788573

Molecular trade-offs in RNA ligases affected the modular emergence of complex ribozymes at the origin of life

PubMed Central

Weinberg, Marc S.; Michod, Richard E.

2017-01-01

In the RNA world hypothesis complex, self-replicating ribozymes were essential. For the emergence of an RNA world, less is known about the early processes that accounted for the formation of complex, long catalysts from small passively formed molecules. The functional role of small sequences has not been fully explored and, here, a possible role for smaller ligases is demonstrated. An established RNA polymerase model, the R18, was truncated from the 3′ end to generate smaller molecules. All the molecules were investigated for self-ligation functions with a set of oligonucleotide substrates without predesigned base pairing. The smallest molecule that exhibited self-ligation activity was a 40-nucleotide RNA. It also demonstrated the greatest functional flexibility as it was more general in the kinds of substrates it ligated to itself although its catalytic efficiency was the lowest. The largest ribozyme (R18) ligated substrates more selectively and with greatest efficiency. With increase in size and predicted structural stability, self-ligation efficiency improved, while functional flexibility decreased. These findings reveal that molecular size could have increased from the activity of small ligases joining oligonucleotides to their own end. In addition, there is a size-associated molecular-level trade-off that could have impacted the evolution of RNA-based life. PMID:28989747

Molecular trade-offs in RNA ligases affected the modular emergence of complex ribozymes at the origin of life

NASA Astrophysics Data System (ADS)

Dhar, Nisha; Weinberg, Marc S.; Michod, Richard E.; Durand, Pierre M.

2017-09-01

In the RNA world hypothesis complex, self-replicating ribozymes were essential. For the emergence of an RNA world, less is known about the early processes that accounted for the formation of complex, long catalysts from small passively formed molecules. The functional role of small sequences has not been fully explored and, here, a possible role for smaller ligases is demonstrated. An established RNA polymerase model, the R18, was truncated from the 3' end to generate smaller molecules. All the molecules were investigated for self-ligation functions with a set of oligonucleotide substrates without predesigned base pairing. The smallest molecule that exhibited self-ligation activity was a 40-nucleotide RNA. It also demonstrated the greatest functional flexibility as it was more general in the kinds of substrates it ligated to itself although its catalytic efficiency was the lowest. The largest ribozyme (R18) ligated substrates more selectively and with greatest efficiency. With increase in size and predicted structural stability, self-ligation efficiency improved, while functional flexibility decreased. These findings reveal that molecular size could have increased from the activity of small ligases joining oligonucleotides to their own end. In addition, there is a size-associated molecular-level trade-off that could have impacted the evolution of RNA-based life.

Dynamic interactions of proteins in complex networks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Appella, E.; Anderson, C.

2009-10-01

Recent advances in techniques such as NMR and EPR spectroscopy have enabled the elucidation of how proteins undergo structural changes to act in concert in complex networks. The three minireviews in this series highlight current findings and the capabilities of new methodologies for unraveling the dynamic changes controlling diverse cellular functions. They represent a sampling of the cutting-edge research presented at the 17th Meeting of Methods in Protein Structure Analysis, MPSA2008, in Sapporo, Japan, 26-29 August, 2008 (http://www.iapsap.bnl.gov). The first minireview, by Christensen and Klevit, reports on a structure-based yeast two-hybrid method for identifying E2 ubiquitin-conjugating enzymes that interact withmore » the E3 BRCA1/BARD1 heterodimer ligase to generate either mono- or polyubiquitinated products. This method demonstrated for the first time that the BRCA1/BARD1 E3 can interact with 10 different E2 enzymes. Interestingly, the interaction with multiple E2 enzymes displayed unique ubiquitin-transfer properties, a feature expected to be common among other RING and U-box E3s. Further characterization of new E3 ligases and the E2 enzymes that interact with them will greatly enhance our understanding of ubiquitin transfer and facilitate studies of roles of ubiquitin and ubiquitin-like proteins in protein processing and trafficking. Stein et al., in the second minireview, describe recent progress in defining the binding specificity of different peptide-binding domains. The authors clearly point out that transient peptide interactions mediated by both post-translational modifications and disordered regions ensure a high level of specificity. They postulate that a regulatory code may dictate the number of combinations of domains and post-translational modifications needed to achieve the required level of interaction specificity. Moreover, recognition alone is not enough to obtain a stable complex, especially in a complex cellular environment. Increasing evidence indicates that disordered domains can acquire structural features that modulate the binding and strength of the signaling cascade. Whereas the first two minireviews describe ways in which protein interactions are modulated, the third, by Tompa, focuses on the importance of protein disorder in a subset of amyloid proteins. It is apparent that within this group, part of the polypeptide chain remains disordered during amyloid formation. Moreover, the disordered segments have different amino acid composition and physicochemical characteristics, which suggests that they may play a role in amyloid stability. The disordered region may serve as a linker to connect the ordered core and a globular domain, maintaining the stability and structure of the globular domain and minimizing protein refolding upon amyloid formation. As techniques in protein chemistry advance, we are learning more and more about the mechanisms that regulate and are regulated by protein interactions. The three minireviews in this series offer a glimpse of the complex dynamics fundamental to protein-protein interactions. In the future, we expect that the knowledge gained will help to augment our ability to control complex pathologies and treat diverse diseases states.« less

Molecular characterization of a CpTRIM35-like protein and its splice variants from whitespotted bamboo shark (Chiloscyllium plagiosum)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Xinshang, E-mail: sanmaosound@163.com; Zhao, Heng, E-mail: hengzhao2000@gmail.com; Chen, Yeyu, E-mail: cyyleaf@126.com

2014-10-24

Highlights: • A TRIM gene and three splice variants were firstly cloned from elasmobranch fish. • The genes were constitutively expressed with high levels in spleen and kidney. • The gene products were distributed in cytoplasm alone or cytoplasm and nucleus. • As E3 ubiquitin ligases, the proteins differed in immune responses to challenges. - Abstract: The tripartite motif (TRIM) proteins play important roles in a broad range of biological processes, including apoptosis, cell proliferation and innate immunity response. In this study, a TRIM gene and its three splice variants were cloned from an elasmobranch fish—whitespotted bamboo shark (Chiloscyllium plagiosummore » Bennett). Phylogenetic analysis indicated that the gene was closely related to TRIM35 homologs, thus termed CpTRIM35-like. Deduced CpTRIM35 has a RBCC-PRY/SPRY structure typical of TRIM proteins, and its splice variants (CpTRIM35-1–3) have different truncations at the C-terminus. The gene products were constitutively expressed in adult sharks with the highest levels in spleen and kidney. The different subcellular locations, upregulation upon LPS and poly I:C stimulation, and significant E3 ubiquitin ligase activities suggested their different roles in immune responses as an E3 ubiquitin ligase. This is the first TRIM protein ever characterized in elasmobranch fish.« less

CBL family E3 ubiquitin ligases control JAK2 ubiquitination and stability in hematopoietic stem cells and myeloid malignancies

PubMed Central

Lv, Kaosheng; Jiang, Jing; Donaghy, Ryan; Riling, Christopher R.; Cheng, Ying; Chandra, Vemika; Rozenova, Krasimira; An, Wei; Mohapatra, Bhopal C.; Goetz, Benjamin T.; Pillai, Vinodh; Han, Xu; Todd, Emily A.; Jeschke, Grace R.; Langdon, Wallace Y.; Kumar, Suresh; Hexner, Elizabeth O.

2017-01-01

Janus kinase 2 (JAK2) is a central kinase in hematopoietic stem/progenitor cells (HSPCs), and its uncontrolled activation is a prominent oncogenic driver of hematopoietic neoplasms. However, molecular mechanisms underlying the regulation of JAK2 have remained elusive. Here we report that the Casitas B-cell lymphoma (CBL) family E3 ubiquitin ligases down-regulate JAK2 stability and signaling via the adaptor protein LNK/SH2B3. We demonstrated that depletion of CBL/CBL-B or LNK abrogated JAK2 ubiquitination, extended JAK2 half-life, and enhanced JAK2 signaling and cell growth in human cell lines as well as primary murine HSPCs. Built on these findings, we showed that JAK inhibitor (JAKi) significantly reduced aberrant HSPCs and mitigated leukemia development in a mouse model of aggressive myeloid leukemia driven by loss of Cbl and Cbl-b. Importantly, primary human CBL mutated (CBLmut) leukemias exhibited increased JAK2 protein levels and signaling and were hypersensitive to JAKi. Loss-of-function mutations in CBL E3 ubiquitin ligases are found in a wide range of myeloid malignancies, which are diseases without effective treatment options. Hence, our studies reveal a novel signaling axis that regulates JAK2 in normal and malignant HSPCs and suggest new therapeutic strategies for treating CBLmut myeloid malignancies. PMID:28611190

Nuclear TRIM25 Specifically Targets Influenza Virus Ribonucleoproteins to Block the Onset of RNA Chain Elongation.

PubMed

Meyerson, Nicholas R; Zhou, Ligang; Guo, Yusong R; Zhao, Chen; Tao, Yizhi J; Krug, Robert M; Sawyer, Sara L

2017-11-08

TRIM25 is an E3 ubiquitin ligase that activates RIG-I to promote the antiviral interferon response. The NS1 protein from all strains of influenza A virus binds TRIM25, although not all virus strains block the interferon response, suggesting alternative mechanisms for TRIM25 action. Here we present a nuclear role for TRIM25 in specifically restricting influenza A virus replication. TRIM25 inhibits viral RNA synthesis through a direct mechanism that is independent of its ubiquitin ligase activity and the interferon pathway. This activity can be inhibited by the viral NS1 protein. TRIM25 inhibition of viral RNA synthesis results from its binding to viral ribonucleoproteins (vRNPs), the structures containing individual viral RNA segments, the viral polymerase, and multiple viral nucleoproteins. TRIM25 binding does not inhibit initiation of capped-RNA-primed viral mRNA synthesis by the viral polymerase. Rather, the onset of RNA chain elongation is inhibited because TRIM25 prohibits the movement of RNA into the polymerase complex. Copyright © 2017 Elsevier Inc. All rights reserved.

Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination

PubMed Central

Ahmed, M. Rafiuddin; Zhan, Xuanzhi; Song, Xiufeng; Kook, Seunghyi; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

2011-01-01

Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmuno-precipitation of endogenous proteins from brain tissue, and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both non-visual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology. PMID:21466165

The E3 ubiquitin ligase Trim7 mediates c-Jun/AP-1 activation by Ras signalling

PubMed Central

Chakraborty, Atanu; Diefenbacher, Markus E.; Mylona, Anastasia; Kassel, Olivier; Behrens, Axel

2015-01-01

The c-Jun/AP-1 transcription factor controls key cellular behaviours, including proliferation and apoptosis, in response to JNK and Ras/MAPK signalling. While the JNK pathway has been well characterised, the mechanism of activation by Ras was elusive. Here we identify the uncharacterised ubiquitin ligase Trim7 as a critical component of AP-1 activation via Ras. We found that MSK1 directly phosphorylates Trim7 in response to direct activation by the Ras–Raf–MEK–ERK pathway, and this modification stimulates Trim7 E3 ubiquitin ligase activity. Trim7 mediates Lys63-linked ubiquitination of the AP-1 coactivator RACO-1, leading to RACO-1 protein stabilisation. Consequently, Trim7 depletion reduces RACO-1 levels and AP-1-dependent gene expression. Moreover, transgenic overexpression of Trim7 increases lung tumour burden in a Ras-driven cancer model, and knockdown of Trim7 in established xenografts reduces tumour growth. Thus, phosphorylation-ubiquitination crosstalk between MSK1, Trim7 and RACO-1 completes the long sought-after mechanism linking growth factor signalling and AP-1 activation. PMID:25851810

Multiple degradation pathways regulate versatile CIP/KIP CDK inhibitors.

PubMed

Starostina, Natalia G; Kipreos, Edward T

2012-01-01

The mammalian CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors (CKIs) comprises three proteins--p21(Cip1/WAF1), p27(Kip1), and p57(Kip2)--that bind and inhibit cyclin-CDK complexes, which are key regulators of the cell cycle. CIP/KIP CKIs have additional independent functions in regulating transcription, apoptosis and actin cytoskeletal dynamics. These divergent functions are performed in distinct cellular compartments and contribute to the seemingly contradictory observation that the CKIs can both suppress and promote cancer. Multiple ubiquitin ligases (E3s) direct the proteasome-mediated degradation of p21, p27 and p57. This review analyzes recent data highlighting our current understanding of how distinct E3 pathways regulate subpopulations of the CKIs to control their diverse functions. Copyright © 2011 Elsevier Ltd. All rights reserved.

Proteomic analysis of the gamma human papillomavirus type 197 E6 and E7 associated cellular proteins

PubMed Central

Grace, Miranda; Munger, Karl

2016-01-01

Gamma HPV197 was the most frequently identified HPV when human skin cancer specimens were analyzed by deep sequencing. To gain insight into the biological activities of HPV197, we investigated the cellular interactomes of HPV197 E6 and E7. HPV197 E6 protein interacts with a broad spectrum of cellular LXXLL domain proteins, including UBE3A and MAML1. HPV197 E6 also binds and inhibits the TP53 tumor suppressor and interacts with the CCR4-NOT ubiquitin ligase and deadenylation complex. Despite lacking a canonical retinoblastoma (RB1) tumor suppressor binding site, HPV197 E7 binds RB1 and activates E2F transcription. Hence, HPV197 E6 and E7 proteins interact with a similar set of cellular proteins as E6 and E7 proteins encoded by HPVs that have been linked to human carcinogenesis and/or have transforming activities in vitro. PMID:27771561

Signaling threshold regulation by the Ras effector IMP.

PubMed

Matheny, Sharon A; White, Michael A

2009-04-24

The Ras effector and E3 ligase family member IMP (impedes mitogenic signal propagation) acts as a steady-state resistor within the Raf-MEK-ERK kinase module. IMP concentrations are directly regulated by Ras, through induction of autoubiquitination, to permit productive Raf-MEK complex assembly. Inhibition of Raf-MEK pathway activation by IMP occurs through the inactivation of KSR, a scaffold/adapter protein that couples activated Raf to its substrate MEK1. The capacity of IMP to inhibit signal propagation through Raf to MEK is, in part, a consequence of disrupting KSR1 homo-oligomerization and c-Raf-B-Raf hetero-oligomerization. These observations suggest that IMP functions as a threshold modulator, controlling sensitivity of the cascade to stimulus by directly limiting the assembly of functional KSR1-dependent Raf-MEK complexes.

Last stop on the road to repair: structure of E. coli DNA ligase bound to nicked DNA-adenylate.

PubMed

Nandakumar, Jayakrishnan; Nair, Pravin A; Shuman, Stewart

2007-04-27

NAD(+)-dependent DNA ligases (LigA) are ubiquitous in bacteria and essential for growth. Their distinctive substrate specificity and domain organization vis-a-vis human ATP-dependent ligases make them outstanding targets for anti-infective drug discovery. We report here the 2.3 A crystal structure of Escherichia coli LigA bound to an adenylylated nick, which captures LigA in a state poised for strand closure and reveals the basis for nick recognition. LigA envelopes the DNA within a protein clamp. Large protein domain movements and remodeling of the active site orchestrate progression through the three chemical steps of the ligation reaction. The structure inspires a strategy for inhibitor design.

Protein Neddylation: Beyond Cullin-RING Ligases

PubMed Central

Enchev, Radoslav I.; Schulman, Brenda A.; Peter, Matthias

2016-01-01

NEDD8 is a ubiquitin-like protein that activates the largest ubiquitin E3 ligase family, the cullin RING ligases. Many non-cullin neddylation targets have been proposed in recent years. However, overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery, which makes validating potential NEDD8 targets challenging. Here we re-evaluate these studies in light of the current understanding of the neddylation pathway, and suggest criteria for the identification of genuine neddylation substrates under homeostatic conditions. We describe the biological processes that might be regulated by non-cullin neddylation, and the utility of neddylation inhibitors for research and as potential therapies. Understanding the biological significance of non-cullin neddylation is an exciting research prospect primed to reveal fundamental insights. PMID:25531226

Dual-Color Click Beetle Luciferase Heteroprotein Fragment Complementation Assays

PubMed Central

Villalobos, Victor; Naik, Snehal; Bruinsma, Monique; Dothager, Robin S.; Pan, Mei-Hsiu; Samrakandi, Mustapha; Moss, Britney; Elhammali, Adnan; Piwnica-Worms, David

2010-01-01

Summary Understanding the functional complexity of protein interactions requires mapping biomolecular complexes within the cellular environment over biologically-relevant time scales. Herein we describe a novel set of reversible, multicolored heteroprotein complementation fragments based on various firefly and click beetle luciferases that utilize the same substrate, D-luciferin. Luciferase heteroprotein fragment complementation systems enabled dual-color quantification of two discreet pairs of interacting proteins simultaneously or two distinct proteins interacting with a third shared protein in live cells. Using real-time analysis of click beetle green and click beetle red luciferase heteroprotein fragment complementation applied to β-TrCP, an E3-ligase common to the regulation of both β-catenin and IκBα, GSK3β was identified as a novel candidate kinase regulating IκBα processing. These dual-color protein interaction switches may enable directed dynamic analysis of a variety of protein interactions in living cells. PMID:20851351

The Arabidopsis RING-Type E3 Ligase TEAR1 Controls Leaf Development by Targeting the TIE1 Transcriptional Repressor for Degradation[OPEN

PubMed Central

Zhang, Jinzhe; Wei, Baoye; Yuan, Rongrong; Yu, Hao

2017-01-01

The developmental plasticity of leaf size and shape is important for leaf function and plant survival. However, the mechanisms by which plants form diverse leaves in response to environmental conditions are not well understood. Here, we identified TIE1-ASSOCIATED RING-TYPE E3 LIGASE1 (TEAR1) and found that it regulates leaf development by promoting the degradation of TCP INTERACTOR-CONTAINING EAR MOTIF PROTEIN1 (TIE1), an important repressor of CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors, which are key for leaf development. TEAR1 contains a typical C3H2C3-type RING domain and has E3 ligase activity. We show that TEAR1 interacts with the TCP repressor TIE1, which is ubiquitinated in vivo and degraded by the 26S proteasome system. We demonstrate that TEAR1 is colocalized with TIE1 in nuclei and negatively regulates TIE1 protein levels. Overexpression of TEAR1 rescued leaf defects caused by TIE1 overexpression, whereas disruption of TEAR1 resulted in leaf phenotypes resembling those caused by TIE1 overexpression or TCP dysfunction. Deficiency in TEAR partially rescued the leaf defects of TCP4 overexpression line and enhanced the wavy leaf phenotypes of jaw-5D. We propose that TEAR1 positively regulates CIN-like TCP activity to promote leaf development by mediating the degradation of the TCP repressor TIE1. PMID:28100709

Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects.

PubMed

Callegari, Sylvie; Oeljeklaus, Silke; Warscheid, Bettina; Dennerlein, Sven; Thumm, Michael; Rehling, Peter; Dudek, Jan

2017-01-02

The E3 ubiquitin ligase PARK2 and the mitochondrial protein kinase PINK1 are required for the initiation of mitochondrial damage-induced mitophagy. Together, PARK2 and PINK1 generate a phospho-ubiquitin signal on outer mitochondrial membrane proteins that triggers recruitment of the autophagy machinery. This paper describes the detection of a defined 500-kDa phospho-ubiquitin-rich PARK2 complex that accumulates on mitochondria upon treatment with the membrane uncoupler CCCP. Formation of this complex is dependent on the presence of PINK1 and is absent in mutant forms of PARK2, whereby mitophagy is also arrested. These results signify a functional signaling complex that is essential for the progression of mitophagy. The visualization of the PARK2 signaling complex represents a novel marker for this critical step in mitophagy and can be used to monitor mitophagy progression in PARK2 mutants and to uncover additional upstream factors required for PARK2-mediated mitophagy signaling.

The wavy growth 3 E3 ligase family controls the gravitropic response in Arabidopsis roots.

PubMed

Sakai, Tatsuya; Mochizuki, Susumu; Haga, Ken; Uehara, Yukiko; Suzuki, Akane; Harada, Akiko; Wada, Takuji; Ishiguro, Sumie; Okada, Kiyotaka

2012-04-01

Regulation of the root growth pattern is an important control mechanism during plant growth and propagation. To better understand alterations in root growth direction in response to environmental stimuli, we have characterized an Arabidopsis thaliana mutant, wavy growth 3 (wav3), whose roots show a short-pitch pattern of wavy growth on inclined agar medium. The wav3 mutant shows a greater curvature of root bending in response to gravity, but a smaller curvature in response to light, suggesting that it is a root gravitropism-enhancing mutation. This wav3 phenotype also suggests that enhancement of the gravitropic response in roots strengthens root tip impedance after contact with the agar surface and/or causes an increase in subsequent root bending in response to obstacle-touching stimulus in these mutants. WAV3 encodes a protein with a RING finger domain, and is mainly expressed in root tips. RING-containing proteins often function as an E3 ubiquitin ligase, and the WAV3 protein shows such activity in vitro. There are three genes homologous to WAV3 in the Arabidopsis genome [EMBRYO SAC DEVELOPMENT ARREST 40 (EDA40), WAVH1 and WAVH2 ], and wav3 wavh1 wavh2 triple mutants show marked root gravitropism abnormalities. This genetic study indicates that WAV3 functions positively rather than negatively in root gravitropism, and that enhancement of the gravitropic response in wav3 roots is dependent upon the function of WAVH2 in the absence of WAV3. Hence, our results demonstrate that the WAV3 family of proteins are E3 ligases that are required for root gravitropism in Arabidopsis. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

E3 ubiquitin ligase RFWD2 controls lung branching through protein-level regulation of ETV transcription factors.

PubMed

Zhang, Yan; Yokoyama, Shigetoshi; Herriges, John C; Zhang, Zhen; Young, Randee E; Verheyden, Jamie M; Sun, Xin

2016-07-05

The mammalian lung is an elaborate branching organ, and it forms following a highly stereotypical morphogenesis program. It is well established that precise control at the transcript level is a key genetic underpinning of lung branching. In comparison, little is known about how regulation at the protein level may play a role. Ring finger and WD domain 2 (RFWD2, also termed COP1) is an E3 ubiquitin ligase that modifies specific target proteins, priming their degradation via the ubiquitin proteasome system. RFWD2 is known to function in the adult in pathogenic processes such as tumorigenesis. Here, we show that prenatal inactivation of Rfwd2 gene in the lung epithelium led to a striking halt in branching morphogenesis shortly after secondary branch formation. This defect is accompanied by distalization of the lung epithelium while growth and cellular differentiation still occurred. In the mutant lung, two E26 transformation-specific (ETS) transcription factors essential for normal lung branching, ETS translocation variant 4 (ETV4) and ETV5, were up-regulated at the protein level, but not at the transcript level. Introduction of Etv loss-of-function alleles into the Rfwd2 mutant background attenuated the branching phenotype, suggesting that RFWD2 functions, at least in part, through degrading ETV proteins. Because a number of E3 ligases are known to target factors important for lung development, our findings provide a preview of protein-level regulatory network essential for lung branching morphogenesis.

Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay.

PubMed

Gul, Sheraz; Brown, Richard; May, Earl; Mazzulla, Marie; Smyth, Martin G; Berry, Colin; Morby, Andrew; Powell, David J

2004-11-01

DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the K(m) values for NAD+ (2.75+/-0.1 microM) and the acridinium-ester-labelled DNA substrate (2.5+/-0.2 nM). A study of the pH-dependencies of kcat, K(m) and kcat/K(m) has revealed values of kinetically influential ionizations within the enzyme-substrate complexes (kcat) and free enzyme (kcat/K(m)). In each case, the curves were shown to be composed of one kinetically influential ionization, for k(cat), pK(a)=6.6+/-0.1 and kcat/K(m), pK(a)=7.1+/-0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30+/-0.86 microM for doxorubicin and 1.40+/-0.07 microM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 microl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.

Trafficking of the transcription factor Nrf2 to promyelocytic leukemia-nuclear bodies: implications for degradation of NRF2 in the nucleus.

PubMed

Malloy, Melanie Theodore; McIntosh, Deneshia J; Walters, Treniqka S; Flores, Andrea; Goodwin, J Shawn; Arinze, Ifeanyi J

2013-05-17

Ubiquitylation of Nrf2 by the Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase complex targets Nrf2 for proteasomal degradation in the cytoplasm and is an extensively studied mechanism for regulating the cellular level of Nrf2. Although mechanistic details are lacking, reports abound that Nrf2 can also be degraded in the nucleus. Here, we demonstrate that Nrf2 is a target for sumoylation by both SUMO-1 and SUMO-2. HepG2 cells treated with As2O3, which enhances attachment of SUMO-2/3 to target proteins, increased SUMO-2/3-modification (polysumoylation) of Nrf2. We show that Nrf2 traffics, in part, to promyelocytic leukemia-nuclear bodies (PML-NBs). Cell fractions harboring key components of PML-NBs did not contain biologically active Keap1 but contained modified Nrf2 as well as RING finger protein 4 (RNF4), a poly-SUMO-specific E3 ubiquitin ligase. Overexpression of wild-type RNF4, but not the catalytically inactive mutant, decreased the steady-state levels of Nrf2, measured in the PML-NB-enriched cell fraction. The proteasome inhibitor MG-132 interfered with this decrease, resulting in elevated levels of polysumoylated Nrf2 that was also ubiquitylated. Wild-type RNF4 accelerated the half-life (t½) of Nrf2, measured in PML-NB-enriched cell fractions. These results suggest that RNF4 mediates polyubiquitylation of polysumoylated Nrf2, leading to its subsequent degradation in PML-NBs. Overall, this work identifies Nrf2 as a target for sumoylation and provides a novel mechanism for its degradation in the nucleus, independent of Keap1.

The E3 ubiquitin ligase Mule acts through the ATM-p53 axis to maintain B lymphocyte homeostasis.

PubMed

Hao, Zhenyue; Duncan, Gordon S; Su, Yu-Wen; Li, Wanda Y; Silvester, Jennifer; Hong, Claire; You, Han; Brenner, Dirk; Gorrini, Chiara; Haight, Jillian; Wakeham, Andrew; You-Ten, Annick; McCracken, Susan; Elia, Andrew; Li, Qinxi; Detmar, Jacqui; Jurisicova, Andrea; Hobeika, Elias; Reth, Michael; Sheng, Yi; Lang, Philipp A; Ohashi, Pamela S; Zhong, Qing; Wang, Xiaodong; Mak, Tak W

2012-01-16

Cellular homeostasis is controlled by pathways that balance cell death with survival. Mcl-1 ubiquitin ligase E3 (Mule) is an E3 ubiquitin ligase that targets the proapoptotic molecule p53 for polyubiquitination and degradation. To elucidate the role of Mule in B lymphocyte homeostasis, B cell-specific Mule knockout (BMKO) mice were generated using the Cre-LoxP recombination system. Analysis of BMKO mice showed that Mule was essential for B cell development, proliferation, homeostasis, and humoral immune responses. p53 transactivation was increased by two- to fourfold in Mule-deficient B cells at steady state. Genetic ablation of p53 in BMKO mice restored B cell development, proliferation, and homeostasis. p53 protein was increased in resting Mule-deficient mouse embryonic fibroblasts (MEFs) and embryonic stem (ES) cells. Loss of Mule in both MEFs and B cells at steady state resulted in increased levels of phospho-ataxia telangiectasia mutated (ATM) and the ATM substrate p53. Under genotoxic stress, BMKO B cells were resistant to apoptosis, and control MEFs exhibited evidence of a physical interaction between Mule and phospho-ATM. Phospho-ATM, phospho-p53, and Brca1 levels were reduced in Mule-deficient B cells and MEFs subjected to genotoxic stress. Thus, Mule regulates the ATM-p53 axis to maintain B cell homeostasis under both steady-state and stress conditions.

The E3 ubiquitin ligase NEDD4 induces endocytosis and lysosomal sorting of connexin 43 to promote loss of gap junctions.

PubMed

Totland, Max Z; Bergsland, Christian H; Fykerud, Tone A; Knudsen, Lars M; Rasmussen, Nikoline L; Eide, Peter W; Yohannes, Zeremariam; Sørensen, Vigdis; Brech, Andreas; Lothe, Ragnhild A; Leithe, Edward

2017-09-01

Intercellular communication via gap junctions has an important role in controlling cell growth and in maintaining tissue homeostasis. Connexin 43 (Cx43; also known as GJA1) is the most abundantly expressed gap junction channel protein in humans and acts as a tumor suppressor in multiple tissue types. Cx43 is often dysregulated at the post-translational level during cancer development, resulting in loss of gap junctions. However, the molecular basis underlying the aberrant regulation of Cx43 in cancer cells has remained elusive. Here, we demonstrate that the oncogenic E3 ubiquitin ligase NEDD4 regulates the Cx43 protein level in HeLa cells, both under basal conditions and in response to protein kinase C activation. Furthermore, overexpression of NEDD4, but not a catalytically inactive form of NEDD4, was found to result in nearly complete loss of gap junctions and increased lysosomal degradation of Cx43 in both HeLa and C33A cervical carcinoma cells. Collectively, the data provide new insights into the molecular basis underlying the regulation of gap junction size and represent the first evidence that an oncogenic E3 ubiquitin ligase promotes loss of gap junctions and Cx43 degradation in human carcinoma cells. © 2017. Published by The Company of Biologists Ltd.

The E3 Ligase CHIP Mediates p21 Degradation to Maintain Radioresistance

PubMed Central

Biswas, Kuntal; Sarkar, Sukumar; Du, Kangping; Brautigan, David L.; Abbas, Tarek; Larner, James M.

2017-01-01

Lung cancer resists radiation therapy, making it one of the deadliest forms of cancer. Here we show that human lung cancer cell lines can be rendered sensitive to ionizing radiation (IR) by RNAi knockdown of C-terminus of Hsc70-interacting protein (CHIP/STUB1), a U-box-type E3 ubiquitin ligase that targets a number of stress-induced proteins. Mechanistically ubiquitin-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor p21 protein is reduced by CHIP knockdown, leading to enhanced senescence of cells in response to exposure to IR. Cellular senescence and sensitivity to IR is prevented by CRISPR/Cas9-mediated deletion of the p21 gene (CDKN1A) in CHIP knockdown cells. Conversely, over-expression of CHIP potentiates p21 degradation and promotes greater radioresistance of lung cancer cells. In vitro and cell-based assays demonstrate that p21 is a novel and direct ubiquitylation substrate of CHIP that also requires the CHIP-associated chaperone heat shock protein 70 (HSP70). These data reveal that the inhibition of the E3 ubiquitin ligase CHIP promotes radiosensitivity; thus, suggesting a novel strategy for the treatment of lung cancer. Implications The CHIP-HSP70-p21 ubiquitylation/degradation axis identified here could be exploited to enhance the efficacy of radiotherapy in patients with non-small cell lung cancer. PMID:28232384

The E3 Ligase CHIP Mediates p21 Degradation to Maintain Radioresistance.

PubMed

Biswas, Kuntal; Sarkar, Sukumar; Du, Kangping; Brautigan, David L; Abbas, Tarek; Larner, James M

2017-06-01

Lung cancer resists radiotherapy, making it one of the deadliest forms of cancer. Here, we show that human lung cancer cell lines can be rendered sensitive to ionizing radiation (IR) by RNAi knockdown of C-terminus of Hsc70-interacting protein (CHIP/STUB1), a U-box-type E3 ubiquitin ligase that targets a number of stress-induced proteins. Mechanistically, ubiquitin-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor, p21 protein, is reduced by CHIP knockdown, leading to enhanced senescence of cells in response to exposure to IR. Cellular senescence and sensitivity to IR is prevented by CRISPR/Cas9-mediated deletion of the p21 gene ( CDKN1A) in CHIP knockdown cells. Conversely, overexpression of CHIP potentiates p21 degradation and promotes greater radioresistance of lung cancer cells. In vitro and cell-based assays demonstrate that p21 is a novel and direct ubiquitylation substrate of CHIP that also requires the CHIP-associated chaperone HSP70. These data reveal that the inhibition of the E3 ubiquitin ligase CHIP promotes radiosensitivity, thus suggesting a novel strategy for the treatment of lung cancer. Implications: The CHIP-HSP70-p21 ubiquitylation/degradation axis identified here could be exploited to enhance the efficacy of radiotherapy in patients with non-small cell lung cancer. Mol Cancer Res; 15(6); 651-9. ©2017 AACR . ©2017 American Association for Cancer Research.

Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface.

PubMed

Williamson, Adele; Rothweiler, Ulli; Leiros, Hanna Kirsti Schrøder

2014-11-01

DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.

The Unique Morgue Ubiquitination Protein Is Conserved in a Diverse but Restricted Set of Invertebrates

PubMed Central

Zhou, Ying; Carpenter, Zachary W.; Brennan, Gregory

2009-01-01

Drosophila Morgue is a unique ubiquitination protein that facilitates programmed cell death and associates with DIAP1, a critical cell death inhibitor with E3 ubiquitin ligase activity. Morgue possesses a unique combination of functional domains typically associated with distinct types of ubiquitination enzymes. This includes an F box characteristic of the substrate-binding subunit in Skp, Cullin, and F box (SCF)-type ubiquitin E3 ligase complexes and a variant ubiquitin E2 conjugase domain where the active site cysteine is replaced by a glycine. Morgue also contains a single C4-type zinc finger motif. This architecture suggests potentially novel ubiquitination activities for Morgue. In this study, we address the evolutionary origins of this distinctive protein utilizing a combination of bioinformatics and molecular biology approaches. We find that Morgue exhibits widespread but restricted phylogenetic distribution among metazoans. Morgue proteins were identified in a wide range of Protostome phyla, including Arthropoda, Annelida, Mollusca, Nematoda, and Platyhelminthes. However, with one potential exception, Morgue was not detected in Deuterostomes, including Chordates, Hemichordates, or Echinoderms. Morgue was also not found in Ctenophora, Cnidaria, Placozoa, or Porifera. Characterization of Morgue sequences within specific animal lineages suggests that gene deletion or acquisition has occurred during divergence of nematodes and that at least one arachnid expresses an atypical form of Morgue consisting only of the variant E2 conjugase domain. Analysis of the organization of several morgue genes suggests that exon-shuffling events have contributed to the evolution of the Morgue protein. These results suggest that Morgue mediates conserved and distinctive ubiquitination functions in specific cell death pathways. PMID:19602541

The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells.

PubMed

Shao, Genbao; Wang, Ranran; Sun, Aiqin; Wei, Jing; Peng, Ke; Dai, Qian; Yang, Wannian; Lin, Qiong

2018-02-19

EGFR-dependent cell migration plays an important role in lung cancer progression. Our previous study observed that the HECT E3 ubiquitin ligase NEDD4 is significantly correlated with tumor metastasis and required for migration and invasion signaling of EGFR in gastric cancer cells. However, how NEDD4 promotes the EGFR-dependent lung cancer cell migration is unknown. This study is to elucidate the mechanism by which NEDD4 mediates the EGFR lung cancer migration signaling. Lentiviral vector-loaded NEDD4 shRNA was used to deplete endogenous NEDD4 in lung cancer cell lines. Effects of the NEDD4 knockdown on the EGFR-dependent or independent lung cancer cell migration were determined using the wound-healing and transwell assays. Association of NEDD4 with activated EGFR was assayed by co-immunoprecipitation. Co-expression of NEDD4 with EGFR or PTEN was determined by immunohistochemical (IHC) staining in 63 lung adenocarcinoma tissue samples. Effects of NEDD4 ectopic expression or knockdown on PTEN ubiquitination and down-regulation, AKT activation and lysosomal secretion were examined using the GST-Uba pulldown assay, immunoblotting, immunofluorescent staining and a human cathepsin B ELISA assay respectively. The specific cathepsin B inhibitor CA-074Me was used for assessing the role of cathepsin B in lung cancer cell migration. Knockdown of NEDD4 significantly reduced EGF-stimulated cell migration in non-small cell lung carcinoma (NSCLC) cells. Co-immunoprecipitation assay found that NEDD4 is associated with EGFR complex upon EGF stimulation, and IHC staining indicates that NEDD4 is co-expressed with EGFR in lung adenocarcinoma tumor tissues, suggesting that NEDD4 might mediate lung cancer cell migration by interaction with the EGFR signaling complex. Interestingly, NEDD4 promotes the EGF-induced cathepsin B secretion, possibly through lysosomal exocytosis, as overexpression of the ligase-dead mutant of NEDD4 impedes lysosomal secretion, and knockdown of NEDD4 significantly reduced extracellular amount of cathepsin B induced by EGF. Consistent with the role of NEDD4, cathepsin B is pivotal for both basal and the EGF-stimulated lung cancer cell migration. Our studies propose a novel mechanism underlying the EGFR-promoted lung cancer cell migration that is mediated by NEDD4 through regulation of cathepsin B secretion. NEDD4 mediates the EGFR lung cancer cell migration signaling through promoting lysosomal secretion of cathepsin B.

The Tomato U-Box Type E3 Ligase PUB13 Acts With Group III Ubiquitin E2 Enzymes to Modulate FLS2-Mediated Immune Signaling

PubMed Central

Zhou, Bangjun; Zeng, Lirong

2018-01-01

In Arabidopsis and rice, the ubiquitin ligase PUB13-mediated protein degradation plays a significant role in plant pattern-triggered immunity (PTI) and flowering time control. The Arabidopsis PUB13 has been shown to attenuate the pattern recognition receptor FLS2-mediated immune signaling by ubiquitinating FLS2 and consequently promoting its degradation by the 26S proteasome. Nevertheless, the cognate ubiquitin-conjugating enzymes (E2) with which PUB13 acts to modulate FLS2-mediated PTI are unknown. To address this question, we investigate here the tomato (Solanum lycopersicum) homolog of PUB13, SlPUB13 by utilizing the recently characterized complete set of tomato E2s. Of the 13 groups of tomato E2s, only members in group III are found to interact and act with SlPUB13. Knocking-down of the group III E2 genes enhances callose deposition and induction of the RbohB gene in the immunity-associated, early oxidative burst after flg22 treatment. The group III E2s are also found to work with SlPUB13 to ubiquitinate FLS2 in vitro and are required for PUB13-mediated degradation of FLS2 in vivo upon flg22 treatment, suggesting an essential role for group III E2s in the modulation of FLS2-mediated immune signaling by PUB13. Additionally, another immunity-associated E3, NtCMPG1 is shown to also work specifically with members of group III E2 in the in vitro ubiquitination assay, which implies the group III E2 enzymes may cooperate with many E3 ligases to regulate different aspects of PTI. Taken together, these data corroborate the notion that group III E2 enzymes play an important role in PTI and build a foundation for further functional and mechanistic characterization of tomato PUB13.

Ubiquitylation of a Melanosomal Protein by HECT-E3 Ligases Serves as Sorting Signal for Lysosomal DegradationD⃞

PubMed Central

Lévy, Frédéric; Muehlethaler, Katja; Salvi, Suzanne; Peitrequin, Anne-Lise; Lindholm, Cecilia K.; Cerottini, Jean-Charles; Rimoldi, Donata

2005-01-01

The production of pigment by melanocytic cells of the skin involves a series of enzymatic reactions that take place in specialized organelles called melanosomes. Melan-A/MART-1 is a melanocytic transmembrane protein with no enzymatic activity that accumulates in vesicles at the trans side of the Golgi and in melanosomes. We show here that, in melanoma cells, Melan-A associates with two homologous to E6-AP C-terminus (HECT)-E3 ubiquitin ligases, NEDD4 and Itch, and is ubiquitylated. Both NEDD4 and Itch participate in the degradation of Melan-A. A mutant Melan-A lacking ubiquitin-acceptor residues displays increased half-life and, in pigmented cells, accumulates in melanosomes. These results suggest that ubiquitylation regulates the lysosomal sorting and degradation of Melan-A/MART-1 from melanosomes in melanocytic cells. PMID:15703212

K48-linked KLF4 ubiquitination by E3 ligase Mule controls T-cell proliferation and cell cycle progression.

PubMed

Hao, Zhenyue; Sheng, Yi; Duncan, Gordon S; Li, Wanda Y; Dominguez, Carmen; Sylvester, Jennifer; Su, Yu-Wen; Lin, Gloria H Y; Snow, Bryan E; Brenner, Dirk; You-Ten, Annick; Haight, Jillian; Inoue, Satoshi; Wakeham, Andrew; Elford, Alisha; Hamilton, Sara; Liang, Yi; Zúñiga-Pflücker, Juan C; He, Housheng Hansen; Ohashi, Pamela S; Mak, Tak W

2017-01-13

T-cell proliferation is regulated by ubiquitination but the underlying molecular mechanism remains obscure. Here we report that Lys-48-linked ubiquitination of the transcription factor KLF4 mediated by the E3 ligase Mule promotes T-cell entry into S phase. Mule is elevated in T cells upon TCR engagement, and Mule deficiency in T cells blocks proliferation because KLF4 accumulates and drives upregulation of its transcriptional targets E2F2 and the cyclin-dependent kinase inhibitors p21 and p27. T-cell-specific Mule knockout (TMKO) mice develop exacerbated experimental autoimmune encephalomyelitis (EAE), show impaired generation of antigen-specific CD8 + T cells with reduced cytokine production, and fail to clear LCMV infections. Thus, Mule-mediated ubiquitination of the novel substrate KLF4 regulates T-cell proliferation, autoimmunity and antiviral immune responses in vivo.

H2B ubiquitination: Conserved molecular mechanism, diverse physiologic functions of the E3 ligase during meiosis.

PubMed

Wang, Liying; Cao, Chunwei; Wang, Fang; Zhao, Jianguo; Li, Wei

2017-09-03

RNF20/Bre1 mediated H2B ubiquitination (H2Bub) has various physiologic functions. Recently, we found that H2Bub participates in meiotic recombination by promoting chromatin relaxation during meiosis. We then analyzed the phylogenetic relationships among the E3 ligase for H2Bub, its E2 Rad6 and their partner WW domain-containing adaptor with a coiled-coil (WAC) or Lge1, and found that the molecular mechanism underlying H2Bub is evolutionarily conserved from yeast to mammals. However, RNF20 has diverse physiologic functions in different organisms, which might be caused by the evolutionary divergency of their domain/motif architectures. In the current extra view, we not only elucidate the evolutionarily conserved molecular mechanism underlying H2Bub, but also discuss the diverse physiologic functions of RNF20 during meiosis.

New strategy for renal fibrosis: Targeting Smad3 proteins for ubiquitination and degradation.

PubMed

Wang, Xin; Feng, Shaozhen; Fan, Jinjin; Li, Xiaoyan; Wen, Qiong; Luo, Ning

2016-09-15

Smad3 is a critical signaling protein in renal fibrosis. Proteolysis targeting chimeric molecules (PROTACs) are small molecules designed to degrade target proteins via ubiquitination. They have three components: (1) a recognition motif for E3 ligase; (2) a linker; and (3) a ligand for the target protein. We aimed to design a new PROTAC to prevent renal fibrosis by targeting Smad3 proteins and using hydroxylated pentapeptide of hypoxia-inducible factor-1α as the recognition motif for von Hippel-Lindau (VHL) ubiquitin ligase (E3). Computer-aided drug design was used to find a specific ligand targeting Smad3. Surface plasmon resonance (SPR) was used to verify and optimize screening results. Synthesized PROTAC was validated by two-stage mass spectrometry. The PROTAC's specificity for VHL (E3 ligase) was proved with two human renal carcinoma cell lines, 786-0 (VHL(-)) and ACHN (VHL(+)), and its anti-fibrosis effect was tested in renal fibrosis cell models. Thirteen small molecular compounds (SMCs) were obtained from the Enamine library using GLIDE molecular docking program. SPR results showed that #8 SMC (EN300-72284) combined best with Smad3 (KD=4.547×10(-5)M). Mass spectrometry showed that synthesized PROTAC had the correct peptide molecular weights. Western blot showed Smad3 was degraded by PROTAC with whole-cell lysate of ACHN but not 786-0. Degradation, but not ubiquitination, of Smad3 was inhibited by proteasome inhibitor MG132. The upregulation of fibronectin and Collagen I induced by TGF-β1 in both renal fibroblast and mesangial cells were inhibited by PROTAC. The new PROTAC might prevent renal fibrosis by targeting Smad3 for ubiquitination and degradation. Copyright © 2016 Elsevier Inc. All rights reserved.

Degradation of human Lipin-1 by BTRC E3 ubiquitin ligase.

PubMed

Ishimoto, Kenji; Hayase, Ayaka; Kumagai, Fumiko; Kawai, Megumi; Okuno, Hiroko; Hino, Nobumasa; Okada, Yoshiaki; Kawamura, Takeshi; Tanaka, Toshiya; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Tachibana, Keisuke; Doi, Takefumi

2017-06-17

Lipin-1 has dual functions in the regulation of lipid and energy metabolism according to its subcellular localization, which is tightly controlled. However, it is unclear how Lipin-1 degradation is regulated. Here, we demonstrate that Lipin-1 is degraded through its DSGXXS motif. We show that Lipin-1 interacts with either of two E3 ubiquitin ligases, BTRC or FBXW11, and that this interaction is DSGXXS-dependent and mediates the attachment of polyubiquitin chains. Further, we demonstrate that degradation of Lipin-1 is regulated by BTRC in the cytoplasm and on membranes. These novel insights into the regulation of human Lipin-1 stability will be useful in planning further studies to elucidate its metabolic processes. Copyright © 2017 Elsevier Inc. All rights reserved.

Suppression of Arabidopsis RING E3 ubiquitin ligase AtATL78 increases tolerance to cold stress and decreases tolerance to drought stress.

PubMed

Kim, Soo Jin; Kim, Woo Taek

2013-08-19

AtATL78 is an Arabidopsis RING E3 ubiquitin ligase. RT-PCR and promoter-GUS assays revealed that AtATL78 was up-regulated by cold stress and down-regulated by drought. AtATL78 was localized at the plasma-membrane. Suppression of AtATL78 increased tolerance to cold stress but decreased tolerance to drought. Our data suggests that AtATL78 is a negative regulator of cold stress response and a positive regulator of drought stress response in Arabidopsis. These results further suggest that AtATL78 plays opposing roles in cold and drought stress responses. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Footprinting of Chlorella virus DNA ligase bound at a nick in duplex DNA.

PubMed

Odell, M; Shuman, S

1999-05-14

The 298-amino acid ATP-dependent DNA ligase of Chlorella virus PBCV-1 is the smallest eukaryotic DNA ligase known. The enzyme has intrinsic specificity for binding to nicked duplex DNA. To delineate the ligase-DNA interface, we have footprinted the enzyme binding site on DNA and the DNA binding site on ligase. The size of the exonuclease III footprint of ligase bound a single nick in duplex DNA is 19-21 nucleotides. The footprint is asymmetric, extending 8-9 nucleotides on the 3'-OH side of the nick and 11-12 nucleotides on the 5'-phosphate side. The 5'-phosphate moiety is essential for the binding of Chlorella virus ligase to nicked DNA. Here we show that the 3'-OH moiety is not required for nick recognition. The Chlorella virus ligase binds to a nicked ligand containing 2',3'-dideoxy and 5'-phosphate termini, but cannot catalyze adenylation of the 5'-end. Hence, the 3'-OH is important for step 2 chemistry even though it is not itself chemically transformed during DNA-adenylate formation. A 2'-OH cannot substitute for the essential 3'-OH in adenylation at a nick or even in strand closure at a preadenylated nick. The protein side of the ligase-DNA interface was probed by limited proteolysis of ligase with trypsin and chymotrypsin in the presence and absence of nicked DNA. Protease accessible sites are clustered within a short segment from amino acids 210-225 located distal to conserved motif V. The ligase is protected from proteolysis by nicked DNA. Protease cleavage of the native enzyme prior to DNA addition results in loss of DNA binding. These results suggest a bipartite domain structure in which the interdomain segment either comprises part of the DNA binding site or undergoes a conformational change upon DNA binding. The domain structure of Chlorella virus ligase inferred from the solution experiments is consistent with the structure of T7 DNA ligase determined by x-ray crystallography.

CNOT4-Mediated Ubiquitination of Influenza A Virus Nucleoprotein Promotes Viral RNA Replication

PubMed Central

Lin, Yu-Chen; Jeng, King-Song

2017-01-01

ABSTRACT Influenza A virus (IAV) RNA segments are individually packaged with viral nucleoprotein (NP) and RNA polymerases to form a viral ribonucleoprotein (vRNP) complex. We previously reported that NP is a monoubiquitinated protein which can be deubiquitinated by a cellular ubiquitin protease, USP11. In this study, we identified an E3 ubiquitin ligase, CNOT4 (Ccr4-Not transcription complex subunit 4), which can ubiquitinate NP. We found that the levels of viral RNA, protein, viral particles, and RNA polymerase activity in CNOT4 knockdown cells were lower than those in the control cells upon IAV infection. Conversely, overexpression of CNOT4 rescued viral RNP activity. In addition, CNOT4 interacted with the NP in the cell. An in vitro ubiquitination assay also showed that NP could be ubiquitinated by in vitro-translated CNOT4, but ubiquitination did not affect the protein stability of NP. Significantly, CNOT4 increased NP ubiquitination, whereas USP11 decreased it. Mass spectrometry analysis of ubiquitinated NP revealed multiple ubiquitination sites on the various lysine residues of NP. Three of these, K184, K227, and K273, are located on the RNA-binding groove of NP. Mutations of these sites to arginine reduced viral RNA replication. These results indicate that CNOT4 is a ubiquitin ligase of NP, and ubiquitination of NP plays a positive role in viral RNA replication. PMID:28536288

New Functions of APC/C Ubiquitin Ligase in the Nervous System and Its Role in Alzheimer's Disease.

PubMed

Fuchsberger, Tanja; Lloret, Ana; Viña, Jose

2017-05-14

The E3 ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C) regulates important processes in cells, such as the cell cycle, by targeting a set of substrates for degradation. In the last decade, APC/C has been related to several major functions in the nervous system, including axon guidance, synaptic plasticity, neurogenesis, and neuronal survival. Interestingly, some of the identified APC/C substrates have been related to neurodegenerative diseases. There is an accumulation of some degradation targets of APC/C in Alzheimer's disease (AD) brains, which suggests a dysregulation of the protein complex in the disorder. Moreover, recently evidence has been provided for an inactivation of APC/C in AD. It has been shown that oligomers of the AD-related peptide, Aβ, induce degradation of the APC/C activator subunit cdh1, in vitro in neurons in culture and in vivo in the mouse hippocampus. Furthermore, in the AD mouse model APP/PS1, lower cdh1 levels were observed in pyramidal neurons in CA1 when compared to age-matched wildtype mice. In this review, we provide a complete list of APC/C substrates that are involved in the nervous system and we discuss their functions. We also summarize recent studies that show neurobiological effects in cdh1 knockout mouse models. Finally, we discuss the role of APC/C in the pathophysiology of AD.

A Decade of Boon or Burden: What Has the CHIP Ever Done for Cellular Protein Quality Control Mechanism Implicated in Neurodegeneration and Aging?

PubMed Central

Joshi, Vibhuti; Amanullah, Ayeman; Upadhyay, Arun; Mishra, Ribhav; Kumar, Amit; Mishra, Amit

2016-01-01

Cells regularly synthesize new proteins to replace old and abnormal proteins for normal cellular functions. Two significant protein quality control pathways inside the cellular milieu are ubiquitin proteasome system (UPS) and autophagy. Autophagy is known for bulk clearance of cytoplasmic aggregated proteins, whereas the specificity of protein degradation by UPS comes from E3 ubiquitin ligases. Few E3 ubiquitin ligases, like C-terminus of Hsc70-interacting protein (CHIP) not only take part in protein quality control pathways, but also plays a key regulatory role in other cellular processes like signaling, development, DNA damage repair, immunity and aging. CHIP targets misfolded proteins for their degradation through proteasome, as well as autophagy; simultaneously, with the help of chaperones, it also regulates folding attempts for misfolded proteins. The broad range of CHIP substrates and their associations with multiple pathologies make it a key molecule to work upon and focus for future therapeutic interventions. E3 ubiquitin ligase CHIP interacts and degrades many protein inclusions formed in neurodegenerative diseases. The presence of CHIP at various nodes of cellular protein-protein interaction network presents this molecule as a potential candidate for further research. In this review, we have explored a wide range of functionality of CHIP inside cells by a detailed presentation of its co-chaperone, E3 and E4 enzyme like functions, with central focus on its protein quality control roles in neurodegenerative diseases. We have also raised many unexplored but expected fundamental questions regarding CHIP functions, which generate hopes for its future applications in research, as well as drug discovery. PMID:27757073

Specificity determinants for lysine incorporation in Staphylococcus aureus peptidoglycan as revealed by the structure of a MurE enzyme ternary complex.

PubMed

Ruane, Karen M; Lloyd, Adrian J; Fülöp, Vilmos; Dowson, Christopher G; Barreteau, Hélène; Boniface, Audrey; Dementin, Sébastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Gobec, Stanislav; Dessen, Andréa; Roper, David I

2013-11-15

Formation of the peptidoglycan stem pentapeptide requires the insertion of both L and D amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between L-lysine and D,L-diaminopimelic acid, the predominant amino acid that replaces L-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of L-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for L-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic L-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.

Structure/function implications in a dynamic complex of the intrinsically disordered Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase

PubMed Central

Mittag, Tanja; Marsh, Joseph; Grishaev, Alexander; Orlicky, Stephen; Lin, Hong; Sicheri, Frank; Tyers, Mike; Forman-Kay, Julie D.

2010-01-01

Summary Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations utilizing experimental NMR and small-angle x-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated. PMID:20399186

The E3 ligase ube3a is required for learning in Drosophila melanogaster.

PubMed

Chakraborty, Moumita; Paul, Blesson K; Nayak, Tanmoyita; Das, Aniruddha; Jana, Nihar R; Bhutani, Supriya

2015-06-19

Angelman syndrome and autism are neurodevelopmental disorders linked to mutations and duplications of an E3 ligase called ube3a respectively. Since cognitive deficits and learning disabilities are hallmark symptoms of both these disorders, we investigated a role for dube3a in the learning ability of flies using the aversive phototaxis suppression assay. We show that down and up-regulation of dube3a are both detrimental to learning in larvae and adults. Using conditional gene expression we found that dube3a is required for normal brain development and during adulthood. Furthermore, we suggest that dube3a could be interacting with other learning and memory genes such as derailed. Along with firmly establishing dube3a as a gene that is required for learning, our work also opens avenues for further understanding the role played by this gene in brain development and behavior. Copyright © 2015 Elsevier Inc. All rights reserved.

Discovery of a Keap1-dependent peptide PROTAC to knockdown Tau by ubiquitination-proteasome degradation pathway.

PubMed

Lu, Mengchen; Liu, Tian; Jiao, Qiong; Ji, Jianai; Tao, Mengmin; Liu, Yijun; You, Qidong; Jiang, Zhengyu

2018-02-25

Induced protein degradation by PROTACs has emerged as a promising strategy to target nonenzymatic proteins inside the cell. The aim of this study was to identify Keap1, a substrate adaptor protein for ubiquitin E3 ligase involved in oxidative stress regulation, as a novel candidate for PROTACs that can be applied in the degradation of the nonenzymatic protein Tau. A peptide PROTAC by recruiting Keap1-Cul3 ubiquitin E3 ligase was developed and applied in the degradation of intracellular Tau. Peptide 1 showed strong in vitro binding with Keap1 and Tau. With proper cell permeability, peptide 1 was found to colocalize with cellular Keap1 and resulted in the coimmunoprecipitation of Tau and Keap1. The results of flow cytometry and western blotting assays showed that peptide 1 can downregulate the intracellular Tau level in both time- and concentration-dependent manner. The application of Keap1 siRNA silencing and the proteasome inhibitor MG132 confirmed that peptide 1 could promote the Keap1-dependent poly-ubiquitination and proteasome-dependent degradation of Tau. The results suggested that using PROTACs to recruit Keap1 to induce the degradation of Tau may show promising character in the treatment of neurodegenerative disease. Besides, our research demonstrated that Keap1 should be a promising E3 ligase adaptor to be used in the design of novel PROTACs. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

RtcB is the RNA ligase component of an Escherichia coli RNA repair operon.

PubMed

Tanaka, Naoko; Shuman, Stewart

2011-03-11

RNA 2',3'-cyclic phosphate ends play important roles in RNA metabolism as substrates for RNA ligases during tRNA restriction-repair and tRNA splicing. Diverse bacteria from multiple phyla encode a two-component RNA repair cassette, comprising Pnkp (polynucleotide kinase-phosphatase-ligase) and Hen1 (RNA 3'-terminal ribose 2'-O-methyltransferase), that heals and then seals broken tRNAs with 2',3'-cyclic phosphate and 5'-OH ends. The Pnkp-Hen1 repair operon is absent in the majority of bacterial species, thereby raising the prospect that other RNA repair systems might be extant. A candidate component is RNA 3'-phosphate cyclase, a widely distributed enzyme that transforms RNA 3'-monophosphate termini into 2',3'-cyclic phosphates but cannot seal the ends it produces. Escherichia coli RNA cyclase (RtcA) is encoded in a σ(54)-regulated operon with RtcB, a protein of unknown function. Taking a cue from Pnkp-Hen1, we purified E. coli RtcB and tested it for RNA ligase activity. We report that RtcB per se seals broken tRNA-like stem-loop structures with 2',3'-cyclic phosphate and 5'-OH ends to form a splice junction with a 2'-OH, 3',5'-phosphodiester. We speculate that: (i) RtcB might afford bacteria a means to recover from stress-induced RNA damage; and (ii) RtcB homologs might catalyze tRNA repair or splicing reactions in archaea and eukarya.

The Banana Fruit SINA Ubiquitin Ligase MaSINA1 Regulates the Stability of MaICE1 to be Negatively Involved in Cold Stress Response.

PubMed

Fan, Zhong-Qi; Chen, Jian-Ye; Kuang, Jian-Fei; Lu, Wang-Jin; Shan, Wei

2017-01-01

The regulation of ICE1 protein stability is important to ensure effective cold stress response, and is extensively studied in Arabidopsis . Currently, how ICE1 stability in fruits under cold stress is controlled remains largely unknown. Here, we reported the possible involvement of a SEVEN IN ABSENTIA (SINA) ubiquitin ligase MaSINA1 from banana fruit in affecting MaICE1 stability. MaSINA1 was identified based on a yeast two-hybrid screening using MaICE1 as bait. Further yeast two-hybrid, pull-down, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (CoIP) assays confirmed that MaSINA1 interacted with MaICE1. The expression of MaSINA1 was repressed by cold stress. Subcellular localization analysis in tobacco leaves showed that MaSINA1 was localized predominantly in the nucleus. In vitro ubiquitination assay showed that MaSINA1 possessed E3 ubiquitin ligase activity. More importantly, in vitro and semi- in vivo experiments indicated that MaSINA1 can ubiquitinate MaICE1 for the 26S proteasome-dependent degradation, and therefore suppressed the transcriptional activation of MaICE1 to MaNAC1, an important regulator of cold stress response of banana fruit. Collectively, our data reveal a mechanism in banana fruit for control of the stability of ICE1 and for the negative regulation of cold stress response by a SINA E3 ligase via the ubiquitin proteasome system.

Pathogenic variants in E3 ubiquitin ligase RLIM/RNF12 lead to a syndromic X-linked intellectual disability and behavior disorder.

PubMed

Frints, Suzanna G M; Ozanturk, Aysegul; Rodríguez Criado, Germán; Grasshoff, Ute; de Hoon, Bas; Field, Michael; Manouvrier-Hanu, Sylvie; E Hickey, Scott; Kammoun, Molka; Gripp, Karen W; Bauer, Claudia; Schroeder, Christopher; Toutain, Annick; Mihalic Mosher, Theresa; Kelly, Benjamin J; White, Peter; Dufke, Andreas; Rentmeester, Eveline; Moon, Sungjin; Koboldt, Daniel C; van Roozendaal, Kees E P; Hu, Hao; Haas, Stefan A; Ropers, Hans-Hilger; Murray, Lucinda; Haan, Eric; Shaw, Marie; Carroll, Renee; Friend, Kathryn; Liebelt, Jan; Hobson, Lynne; De Rademaeker, Marjan; Geraedts, Joep; Fryns, Jean-Pierre; Vermeesch, Joris; Raynaud, Martine; Riess, Olaf; Gribnau, Joost; Katsanis, Nicholas; Devriendt, Koen; Bauer, Peter; Gecz, Jozef; Golzio, Christelle; Gontan, Cristina; Kalscheuer, Vera M

2018-05-04

RLIM, also known as RNF12, is an X-linked E3 ubiquitin ligase acting as a negative regulator of LIM-domain containing transcription factors and participates in X-chromosome inactivation (XCI) in mice. We report the genetic and clinical findings of 84 individuals from nine unrelated families, eight of whom who have pathogenic variants in RLIM (RING finger LIM domain-interacting protein). A total of 40 affected males have X-linked intellectual disability (XLID) and variable behavioral anomalies with or without congenital malformations. In contrast, 44 heterozygous female carriers have normal cognition and behavior, but eight showed mild physical features. All RLIM variants identified are missense changes co-segregating with the phenotype and predicted to affect protein function. Eight of the nine altered amino acids are conserved and lie either within a domain essential for binding interacting proteins or in the C-terminal RING finger catalytic domain. In vitro experiments revealed that these amino acid changes in the RLIM RING finger impaired RLIM ubiquitin ligase activity. In vivo experiments in rlim mutant zebrafish showed that wild type RLIM rescued the zebrafish rlim phenotype, whereas the patient-specific missense RLIM variants failed to rescue the phenotype and thus represent likely severe loss-of-function mutations. In summary, we identified a spectrum of RLIM missense variants causing syndromic XLID and affecting the ubiquitin ligase activity of RLIM, suggesting that enzymatic activity of RLIM is required for normal development, cognition and behavior.

Chemical approaches to targeted protein degradation through modulation of the ubiquitin-proteasome pathway.

PubMed

Collins, Ian; Wang, Hannah; Caldwell, John J; Chopra, Raj

2017-03-15

Manipulation of the ubiquitin-proteasome system to achieve targeted degradation of proteins within cells using chemical tools and drugs has the potential to transform pharmacological and therapeutic approaches in cancer and other diseases. An increased understanding of the molecular mechanism of thalidomide and its analogues following their clinical use has unlocked small-molecule modulation of the substrate specificity of the E3 ligase cereblon (CRBN), which in turn has resulted in the advancement of new immunomodulatory drugs (IMiDs) into the clinic. The degradation of multiple context-specific proteins by these pleiotropic small molecules provides a means to uncover new cell biology and to generate future drug molecules against currently undruggable targets. In parallel, the development of larger bifunctional molecules that bring together highly specific protein targets in complexes with CRBN, von Hippel-Lindau, or other E3 ligases to promote ubiquitin-dependent degradation has progressed to generate selective chemical compounds with potent effects in cells and in vivo models, providing valuable tools for biological target validation and with future potential for therapeutic use. In this review, we survey recent breakthroughs achieved in these two complementary methods and the discovery of new modes of direct and indirect engagement of target proteins with the proteasome. We discuss the experimental characterisation that validates the use of molecules that promote protein degradation as chemical tools, the preclinical and clinical examples disclosed to date, and the future prospects for this exciting area of chemical biology. © 2017 The Author(s).

Recognition of p63 by the E3 ligase ITCH: Effect of an ectodermal dysplasia mutant.

PubMed

Bellomaria, A; Barbato, Gaetano; Melino, G; Paci, M; Melino, Sonia

2010-09-15

The E3 ubiquitin ligase Itch mediates the degradation of the p63 protein. Itch contains four WW domains which are pivotal for the substrate recognition process. Indeed, this domain is implicated in several signalling complexes crucially involved in human diseases including Muscular Dystrophy, Alzheimer's Disease and Huntington Disease. WW domains are highly compact protein-protein binding modules that interact with short proline-rich sequences. The four WW domains present in Itch belong to the Group I type, which binds polypeptides with a PY motif characterized by a PP xY consensus sequence, where x can be any residue. Accordingly, the Itch-p63 interaction results from a direct binding of Itch-WW2 domain with the PY motif of p63. Here, we report a structural analysis of the Itch-p63 interaction by fluorescence, CD and NMR spectroscopy. Indeed, we studied the in vitro interaction between Itch-WW2 domain and p63(534-551), an 18-mer peptide encompassing a fragment of the p63 protein including the PY motif. In addition, we evaluated the conformation and the interaction with Itch-WW2 of a site specific mutant of p63, I549T, that has been reported in both Hay-Wells syndrome and Rapp-Hodgkin syndrome. Based on our results, we propose an extended PP xY motif for the Itch recognition motif (P-P-P-Y-x(4)-[ST]-[ILV]), which includes these C-terminal residues to the PP xY motif.

RNF41 interacts with the VPS52 subunit of the GARP and EARP complexes

PubMed Central

Masschaele, Delphine; De Ceuninck, Leentje; Wauman, Joris; Defever, Dieter; Stenner, Frank; Lievens, Sam; Peelman, Frank; Tavernier, Jan

2017-01-01

RNF41 (Ring Finger Protein 41) is an E3 ubiquitin ligase involved in the intracellular sorting and function of a diverse set of substrates. Next to BRUCE and Parkin, RNF41 can directly ubiquitinate ErbB3, IL-3, EPO and RARα receptors or downstream signaling molecules such as Myd88, TBK1 and USP8. In this way it can regulate receptor signaling and routing. To further elucidate the molecular mechanism behind the role of RNF41 in intracellular transport we performed an Array MAPPIT (Mammalian Protein-Protein Interaction Trap) screen using an extensive set of proteins derived from the human ORFeome collection. This paper describes the identification of VPS52, a subunit of the GARP (Golgi-Associated Retrograde Protein) and the EARP (Endosome-Associated Recycling Protein) complexes, as a novel interaction partner of RNF41. Through interaction via their coiled coil domains, RNF41 ubiquitinates and relocates VPS52 away from VPS53, a common subunit of the GARP and EARP complexes, towards RNF41 bodies. PMID:28542518

RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination.

PubMed

Choudhury, Nila Roy; Heikel, Gregory; Trubitsyna, Maryia; Kubik, Peter; Nowak, Jakub Stanislaw; Webb, Shaun; Granneman, Sander; Spanos, Christos; Rappsilber, Juri; Castello, Alfredo; Michlewski, Gracjan

2017-11-08

TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. However, there is scarce knowledge about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. Here, we reveal that the RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25's endogenous RNA targets and protein binding partners. We demonstrate that TRIM25 controls the levels of Zinc Finger Antiviral Protein (ZAP). Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase activity towards itself (autoubiquitination) and its physiologically relevant target ZAP. Our results suggest that many other proteins with the PRY/SPRY domain could have yet uncharacterized RNA-binding potential. Together, our data reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor in their enzymatic activity.

Further insight into BRUTUS domain composition and functionality

PubMed Central

Matthiadis, Anna; Long, Terri A.

2016-01-01

ABSTRACT BRUTUS (BTS) is a hemerythrin (HHE) domain containing E3 ligase that facilitates the degradation of POPEYE-like (PYEL) proteins in a proteasomal-dependent manner. Deletion of BTS HHE domains enhances BTS stability in the presence of iron and also complements loss of BTS function, suggesting that the HHE domains are critical for protein stability but not for enzymatic function. The RING E3 domain plays an essential role in BTS' capacity to both interact with PYEL proteins and to act as an E3 ligase. Here we show that removal of the RING domain does not complement loss of BTS function. We conclude that enzymatic activity of BTS via the RING domain is essential for response to iron deficiency in plants. Further, we analyze possible BTS domain structure evolution and predict that the combination of domains found in BTS is specific to photosynthetic organisms, potentially indicative of a role for BTS and its orthologs in mitigating the iron-related challenges presented by photosynthesis. PMID:27359166

Further insight into BRUTUS domain composition and functionality.

PubMed

Matthiadis, Anna; Long, Terri A

2016-08-02

BRUTUS (BTS) is a hemerythrin (HHE) domain containing E3 ligase that facilitates the degradation of POPEYE-like (PYEL) proteins in a proteasomal-dependent manner. Deletion of BTS HHE domains enhances BTS stability in the presence of iron and also complements loss of BTS function, suggesting that the HHE domains are critical for protein stability but not for enzymatic function. The RING E3 domain plays an essential role in BTS' capacity to both interact with PYEL proteins and to act as an E3 ligase. Here we show that removal of the RING domain does not complement loss of BTS function. We conclude that enzymatic activity of BTS via the RING domain is essential for response to iron deficiency in plants. Further, we analyze possible BTS domain structure evolution and predict that the combination of domains found in BTS is specific to photosynthetic organisms, potentially indicative of a role for BTS and its orthologs in mitigating the iron-related challenges presented by photosynthesis.

Carboxyl Terminus of HSC70-interacting Protein (CHIP) Down-regulates NF-κB-inducing Kinase (NIK) and Suppresses NIK-induced Liver Injury*

PubMed Central

Jiang, Bijie; Shen, Hong; Chen, Zheng; Yin, Lei; Zan, Linsen; Rui, Liangyou

2015-01-01

Ser/Thr kinase NIK (NF-κB-inducing kinase) mediates the activation of the noncanonical NF-κB2 pathway, and it plays an important role in regulating immune cell development and liver homeostasis. NIK levels are extremely low in quiescent cells due to ubiquitin/proteasome-mediated degradation, and cytokines stimulate NIK activation through increasing NIK stability; however, regulation of NIK stability is not fully understood. Here we identified CHIP (carboxyl terminus of HSC70-interacting protein) as a new negative regulator of NIK. CHIP contains three N-terminal tetratricopeptide repeats (TPRs), a middle dimerization domain, and a C-terminal U-box. The U-box domain contains ubiquitin E3 ligase activity that promotes ubiquitination of CHIP-bound partners. We observed that CHIP bound to NIK via its TPR domain. In both HEK293 and primary hepatocytes, overexpression of CHIP markedly decreased NIK levels at least in part through increasing ubiquitination and degradation of NIK. Accordingly, CHIP suppressed NIK-induced activation of the noncanonical NF-κB2 pathway. CHIP also bound to TRAF3, and CHIP and TRAF3 acted coordinately to efficiently promote NIK degradation. The TPR but not the U-box domain was required for CHIP to promote NIK degradation. In mice, hepatocyte-specific overexpression of NIK resulted in liver inflammation and injury, leading to death, and liver-specific expression of CHIP reversed the detrimental effects of hepatic NIK. Our data suggest that CHIP/TRAF3/NIK interactions recruit NIK to E3 ligase complexes for ubiquitination and degradation, thus maintaining NIK at low levels. Defects in CHIP regulation of NIK may result in aberrant NIK activation in the liver, contributing to live injury, inflammation, and disease. PMID:25792747

Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation

PubMed Central

Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan MF; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul MK

2015-01-01

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser65)—which lies within its ubiquitin-like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of ubiquitinPhospho-Ser65. PMID:26116755

Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation.

PubMed

Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan M F; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul M K

2015-08-01

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser(65))--which lies within its ubiquitin-like domain (Ubl)--and indirectly through phosphorylation of ubiquitin at Ser(65). How Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin(Phospho-Ser65) binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser(65) by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin(Phospho-Ser65), thereby promoting Parkin Ser(65) phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser(65) phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin(Phospho-Ser65) to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser(65). Finally, purified Parkin maximally phosphorylated at Ser(65) in vitro cannot be further activated by the addition of ubiquitin(Phospho-Ser65). Our results thus suggest that a major role of ubiquitin(Phospho-Ser65) is to promote PINK1-mediated phosphorylation of Parkin at Ser(65), leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser(65)-binding pocket on the surface of Parkin that is critical for the ubiquitin(Phospho-Ser65) interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin(Phospho-Ser65), which could aid in the development of Parkin activators that mimic the effect of ubiquitin(Phospho-Ser65). © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

CDC-25.2, a C. elegans ortholog of cdc25, is essential for the progression of intestinal divisions.

PubMed

Lee, Yong-Uk; Son, Miseol; Kim, Jiyoung; Shim, Yhong-Hee; Kawasaki, Ichiro

2016-01-01

Intestinal divisions in Caenorhabditis elegans take place in 3 stages: (1) cell divisions during embryogenesis, (2) binucleations at the L1 stage, and (3) endoreduplications at the end of each larval stage. Here, we report that CDC-25.2, a C. elegans ortholog of Cdc25, is required for these specialized division cycles between the 16E cell stage and the onset of endoreduplication. Results of our genetic analyses suggest that CDC-25.2 regulates intestinal cell divisions and binucleations by counteracting WEE-1.3 and by activating the CDK-1/CYB-1 complex. CDC-25.2 activity is then repressed by LIN-23 E3 ubiquitin ligase before the onset of intestinal endoreduplication, and this repression is maintained by LIN-35, the C. elegans ortholog of Retinoblastoma (Rb). These findings indicate that timely regulation of CDC-25.2 activity is essential for the progression of specialized division cycles and development of the C. elegans intestine.

CDC-25.2, a C. elegans ortholog of cdc25, is essential for the progression of intestinal divisions

PubMed Central

Lee, Yong-Uk; Son, Miseol; Kim, Jiyoung; Shim, Yhong-Hee; Kawasaki, Ichiro

2016-01-01

ABSTRACT Intestinal divisions in Caenorhabditis elegans take place in 3 stages: (1) cell divisions during embryogenesis, (2) binucleations at the L1 stage, and (3) endoreduplications at the end of each larval stage. Here, we report that CDC-25.2, a C. elegans ortholog of Cdc25, is required for these specialized division cycles between the 16E cell stage and the onset of endoreduplication. Results of our genetic analyses suggest that CDC-25.2 regulates intestinal cell divisions and binucleations by counteracting WEE-1.3 and by activating the CDK-1/CYB-1 complex. CDC-25.2 activity is then repressed by LIN-23 E3 ubiquitin ligase before the onset of intestinal endoreduplication, and this repression is maintained by LIN-35, the C. elegans ortholog of Retinoblastoma (Rb). These findings indicate that timely regulation of CDC-25.2 activity is essential for the progression of specialized division cycles and development of the C. elegans intestine. PMID:27104746

Arabidopsis C3HC4-RING finger E3 ubiquitin ligase AtAIRP4 positively regulates stress-responsive abscisic acid signaling.

PubMed

Yang, Liang; Liu, Qiaohong; Liu, Zhibin; Yang, Hao; Wang, Jianmei; Li, Xufeng; Yang, Yi

2016-01-01

Degradation of proteins via the ubiquitin system is an important step in many stress signaling pathways in plants. E3 ligases recognize ligand proteins and dictate the high specificity of protein degradation, and thus, play a pivotal role in ubiquitination. Here, we identified a gene, named Arabidopsis thaliana abscisic acid (ABA)-insensitive RING protein 4 (AtAIRP4), which is induced by ABA and other stress treatments. AtAIRP4 encodes a cellular protein with a C3HC4-RING finger domain in its C-terminal side, which has in vitro E3 ligase activity. Loss of AtAIRP4 leads to a decrease in sensitivity of root elongation and stomatal closure to ABA, whereas overexpression of this gene in the T-DNA insertion mutant atairp4 effectively recovered the ABA-associated phenotypes. AtAIRP4 overexpression plants were hypersensitive to salt and osmotic stresses during seed germination, and showed drought avoidance compared with the wild-type and atairp4 mutant plants. In addition, the expression levels of ABA- and drought-induced marker genes in AtAIRP4 overexpression plants were markedly higher than those in the wild-type and atairp4 mutant plants. Hence, these results indicate that AtAIRP4 may act as a positive regulator of ABA-mediated drought avoidance and a negative regulator of salt tolerance in Arabidopsis. © 2015 The Authors. Journal of Integrative Plant Biology published by Wiley Publishing Asia Pty Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.

Biotin and fluorescent labeling of RNA using T4 RNA ligase.

PubMed Central

Richardson, R W; Gumport, R I

1983-01-01

Biotin, fluorescein, and tetramethylrhodamine derivatives of P1-(6-aminohex-1-yl)-P2-(5'-adenosine) pyrophosphate were synthesized and used as substrates with T4 RNA ligase. In the absence of ATP, the non-adenylyl portion of these substrates is transferred to the 3'-hydroxyl of an RNA acceptor to form a phosphodiester bond and the AMP portion is released. E. coli and D. melanogaster 5S RNA, yeast tRNAPhe, (Ap)3C, and (Ap)3A serve as acceptors with yields of products varying from 50 to 100%. Biotin-labeled oligonucleotides are bound selectively and quantitatively to avidin-agarose and may be eluted with 6 M guanidine hydrochloride, pH 2.5. Fluorescein and tetramethylrhodamine-labeled oligonucleotides are highly fluorescent and show no quenching due to attachment to the acceptor. The diverse structures of the appended groups and of the chain lengths and compositions of the acceptor RNAs show that T4 RNA ligase will be a useful modification reagent for the addition of various functional groups to the 3'-terminus of RNA molecules. Images PMID:6194506

The role of E3 ubiquitin-ligases MuRF-1 and MAFbx in loss of skeletal muscle mass.

PubMed

Rom, Oren; Reznick, Abraham Z

2016-09-01

The ubiquitin-proteasome system (UPS) is the main regulatory mechanism of protein degradation in skeletal muscle. The ubiquitin-ligase enzymes (E3s) have a central role in determining the selectivity and specificity of the UPS. Since their identification in 2001, the muscle specific E3s, muscle RING finger-1 (MuRF-1) and muscle atrophy F-box (MAFbx), have been shown to be implicated in the regulation of skeletal muscle atrophy in various pathological and physiological conditions. This review aims to explore the involvement of MuRF-1 and MAFbx in catabolism of skeletal muscle during various pathologies, such as cancer cachexia, sarcopenia of aging, chronic kidney disease (CKD), diabetes, and chronic obstructive pulmonary disease (COPD). In addition, the effects of various lifestyle and modifiable factors (e.g. nutrition, exercise, cigarette smoking, and alcohol) on MuRF-1 and MAFbx regulation will be discussed. Finally, evidence of potential strategies to protect against skeletal muscle wasting through inhibition of MuRF-1 and MAFbx expression will be explored. Copyright © 2015 Elsevier Inc. All rights reserved.

RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex

DOE PAGES

Brown, Nicholas G.; VanderLinden, Ryan; Watson, Edmond R.; ...

2015-03-30

For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2~Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaboratesmore » with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING–E2~Ub catalytic modules such as APC11–UBCH10~Ub collide with distally tethered disordered substrates remains poorly understood. In this paper, we report structural mechanisms of UBCH10 recruitment to APC CDH1 and substrate ubiquitination. Unexpectedly, in addition to binding APC11’s RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APC CDH1–UBCH10~Ub–substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin–RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin–RING–E2 interactions establish APC’s specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. Finally, we propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3–E2~Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.« less

Hacking RNA: Hakai promotes tumorigenesis by switching on the RNA-binding function of PSF

PubMed Central

Figueroa, Angélica; Fujita, Yasuyuki; Gorospe, Myriam

2009-01-01

Hakai, an E3 ubiquitin ligase for the E-cadherin complex, plays a crucial role in lowering cell-cell contacts in epithelial cells, a hallmark feature of tumor progression. Recently, Hakai was also found to interact with PSF (PTB-associated splicing factor). While PSF can function as a DNA-binding protein with a tumor suppressive function, its association with Hakai promotes PSF’s RNA-binding ability and post-transcriptional influence on target mRNAs. Hakai overexpression enhanced the binding of PSF to mRNAs encoding cancer-related proteins, while knockdown of Hakai reduced the RNA-binding ability of PSF. Furthermore, the knockdown of PSF suppressed Hakai-induced cell proliferation. Thus, Hakai can affect the oncogenic phenotype both by altering E-cadherin-based intercellular adhesions and by increasing PSF’s ability to bind RNAs that promote cancer-related gene expression. PMID:19855157

Effects of partner proteins on BCA2 RING ligase activity

PubMed Central

2012-01-01

Background BCA2 is an E3 ligase linked with hormone responsive breast cancers. We have demonstrated previously that the RING E3 ligase BCA2 has autoubiquitination activity and is a very unstable protein. Previously, only Rab7, tetherin, ubiquitin and UBC9 were known to directly interact with BCA2. Methods Here, additional BCA2 binding proteins were found using yeast two-hybrid and bacterial-II-hybrid screening techniques with Human breast and HeLa cDNA libraries. Co-expression of these proteins was analyzed through IHC of TMAs. Investigation of the molecular interactions and effects were examined through a series of in vivo and in vitro assays. Results Ten unique BCA2 interacting proteins were identified, two of which were hHR23a and 14-3-3sigma. Both hHR23a and 14-3-3sigma are co-expressed with BCA2 in breast cancer cell lines and patient breast tumors (n = 105). hHR23a and BCA2 expression was significantly correlated (P = < 0.0001 and P = 0.0113) in both nucleus and cytoplasm. BCA2 expression showed a statistically significant correlation with tumor grade. High cytoplasmic hHR23a trended towards negative nodal status. Binding to BCA2 by hHR23a and 14-3-3sigma was confirmed in vitro using tagged partner proteins and BCA2. hHR23a and 14-3-3sigma effect the autoubiquitination and auto-degradation activity of BCA2. Ubiquitination of hHR23a-bound BCA2 was found to be dramatically lower than that of free BCA2, suggesting that hHR23a promotes the stabilization of BCA2 by inactivating its autoubiquitination activity, without degradation of hHR23a. On the other hand, phosphorylated BCA2 protein is stabilized by interaction with 14-3-3sigma both with and without proteasome inhibitor MG-132 suggesting that BCA2 is regulated by multiple degradation pathways. Conclusions The interaction between BCA2 and hHR23a in breast cancer cells stabilizes BCA2. High expression of BCA2 is correlated with grade in breast cancer, suggesting regulation of this E3 ligase is important to cancer progression. PMID:22315970

Clustering and negative feedback by endocytosis in planar cell polarity signaling is modulated by ubiquitinylation of prickle.

PubMed

Cho, Bomsoo; Pierre-Louis, Gandhy; Sagner, Andreas; Eaton, Suzanne; Axelrod, Jeffrey D

2015-05-01

The core components of the planar cell polarity (PCP) signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. We find that the E3 ubiquitin ligase complex Cullin1(Cul1)/SkpA/Supernumerary limbs(Slimb) regulates the stability of one of the peripheral membrane components, Prickle (Pk). Excess Pk disrupts PCP feedback and prevents asymmetry. We show that Pk participates in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang) and Flamingo (Fmi), and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. Our results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling.

The human RNA-binding protein and E3 ligase MEX-3C binds the MEX-3-recognition element (MRE) motif with high affinity.

PubMed

Yang, Lingna; Wang, Chongyuan; Li, Fudong; Zhang, Jiahai; Nayab, Anam; Wu, Jihui; Shi, Yunyu; Gong, Qingguo

2017-09-29

MEX-3 is a K-homology (KH) domain-containing RNA-binding protein first identified as a translational repressor in Caenorhabditis elegans , and its four orthologs (MEX-3A-D) in human and mouse were subsequently found to have E3 ubiquitin ligase activity mediated by a RING domain and critical for RNA degradation. Current evidence implicates human MEX-3C in many essential biological processes and suggests a strong connection with immune diseases and carcinogenesis. The highly conserved dual KH domains in MEX-3 proteins enable RNA binding and are essential for the recognition of the 3'-UTR and post-transcriptional regulation of MEX-3 target transcripts. However, the molecular mechanisms of translational repression and the consensus RNA sequence recognized by the MEX-3C KH domain are unknown. Here, using X-ray crystallography and isothermal titration calorimetry, we investigated the RNA-binding activity and selectivity of human MEX-3C dual KH domains. Our high-resolution crystal structures of individual KH domains complexed with a noncanonical U-rich and a GA-rich RNA sequence revealed that the KH1/2 domains of human MEX-3C bound MRE10, a 10-mer RNA (5'-CAGAGUUUAG-3') consisting of an eight-nucleotide MEX-3-recognition element (MRE) motif, with high affinity. Of note, we also identified a consensus RNA motif recognized by human MEX-3C. The potential RNA-binding sites in the 3'-UTR of the human leukocyte antigen serotype ( HLA-A2 ) mRNA were mapped with this RNA-binding motif and further confirmed by fluorescence polarization. The binding motif identified here will provide valuable information for future investigations of the functional pathways controlled by human MEX-3C and for predicting potential mRNAs regulated by this enzyme. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Spontaneous Seizures and Altered Gene Expression in GABA Signaling Pathways in a mind bomb Mutant Zebrafish

PubMed Central

Hortopan, Gabriela A.; Dinday, Matthew T.; Baraban, Scott C.

2010-01-01

Disruption of E3 ubiquitin ligase activity in immature zebrafish mind bomb mutants, leads to a failure in Notch signaling, excessive numbers of neurons, and depletion of neural progenitor cells. This neurogenic phenotype is associated with defects in neural patterning and brain development. Because developmental brain abnormalities are recognized as an important feature of childhood neurological disorders such as epilepsy and autism, we determined whether zebrafish mutants with grossly abnormal brain structure exhibit spontaneous electrical activity that resembles the long-duration, high-amplitude multi-spike discharges reported in immature zebrafish exposed to convulsant drugs. Electrophysiological recordings from agar immobilized mind bomb mutants at three days postfertilization (dpf) confirmed the occurrence of electrographic seizure activity; seizure-like behaviors were also noted during locomotion video tracking of freely behaving mutants. To identify genes differentially expressed in the mind bomb mutant and provide insight into molecular pathways that may mediate these epileptic phenotypes, a transcriptome analysis was performed using microarray. Interesting candidate genes were further analyzed using conventional reverse transcriptasepolymerase chain reaction (RT-PCR) and real-time quantitative PCR (qPCR), as well as whole-mount in situ hybridization. Approximately 150 genes, some implicated in development, transcription, cell metabolism and signal transduction, are differentially regulated including down-regulation of several genes necessary for GABA-mediated signaling. These findings identify a collection of gene transcripts that may be responsible for the abnormal electrical discharge and epileptic activities observed in a mind bomb zebrafish mutant. This work may have important implications for neurological and neurodevelopmental disorders associated with mutations in ubiquitin ligase activity. Notch is an essential component of an evolutionarily conserved signal transduction cascade mediating development. In neuroectoderm, where cells have the potential to become neurons, activated Notch inhibits proneural gene expression in a process referred to as lateral inhibition. In the absence of Notch-mediated lateral inhibition, too many early-born cells differentiate into neurons (Chitnis et al., 1995; de la Pompa et al., 1997). Recent studies identified several E3 ligases that modulate Notch signaling through ubiquitin-dependent protein degradation and endocytosis (Lai, 2002). Ubiquitination, which occurs when an E3 ligase enzyme binds to both substrate and an E2 thioesterified protein (Deshaies and Joazeiro, 2009), is a key mechanism regulating many cellular processes. Mutation or small deletions within the ubiquitin E3A ligase gene in humans has been linked to autism spectrum disorders (Glessner et al., 2009) and Angelman syndrome, a neurogenetic disorder characterized by developmental delay, severe intellectual disability, absent speech, exuberant behavior, motor impairment, and epilepsy (Clayton-Smith and Laan, 2003). PMID:20943912

Skp1 prolyl 4-hydroxylase of dictyostelium mediates glycosylation-independent and -dependent responses to O2 without affecting Skp1 stability.

PubMed

Zhang, Dongmei; van der Wel, Hanke; Johnson, Jennifer M; West, Christopher M

2012-01-13

Cytoplasmic prolyl 4-hydroxylases (PHDs) have a primary role in O(2) sensing in animals via modification of the transcriptional factor subunit HIFα, resulting in its polyubiquitination by the E3(VHL)ubiquitin (Ub) ligase and degradation in the 26 S proteasome. Previously thought to be restricted to animals, a homolog (P4H1) of HIFα-type PHDs is expressed in the social amoeba Dictyostelium where it also exhibits characteristics of an O(2) sensor for development. Dictyostelium lacks HIFα, and P4H1 modifies a different protein, Skp1, an adaptor of the SCF class of E3-Ub ligases related to the E3(VHL)Ub ligase that targets animal HIFα. Normally, the HO-Skp1 product of the P4H1 reaction is capped by a GlcNAc sugar that can be subsequently extended to a pentasaccharide by novel glycosyltransferases. To analyze the role of glycosylation, the Skp1 GlcNAc-transferase locus gnt1 was modified with a missense mutation to block catalysis or a stop codon to truncate the protein. Despite the accumulation of the hydroxylated form of Skp1, Skp1 was not destabilized based on metabolic labeling. However, hydroxylation alone allowed for partial correction of the high O(2) requirement of P4H1-null cells, therefore revealing both glycosylation-independent and glycosylation-dependent roles for hydroxylation. Genetic complementation of the latter function required an enzymatically active form of Gnt1. Because the effect of the gnt1 deficiency depended on P4H1, and Skp1 was the only protein labeled when the GlcNAc-transferase was restored to mutant extracts, Skp1 apparently mediates the cellular functions of both P4H1 and Gnt1. Although Skp1 stability itself is not affected by hydroxylation, its modification may affect the stability of targets of Skp1-dependent Ub ligases.

Diversity in the architecture of ATLs, a family of plant ubiquitin-ligases, leads to recognition and targeting of substrates in different cellular environments.

PubMed

Aguilar-Hernández, Victor; Aguilar-Henonin, Laura; Guzmán, Plinio

2011-01-01

Ubiquitin-ligases or E3s are components of the ubiquitin proteasome system (UPS) that coordinate the transfer of ubiquitin to the target protein. A major class of ubiquitin-ligases consists of RING-finger domain proteins that include the substrate recognition sequences in the same polypeptide; these are known as single-subunit RING finger E3s. We are studying a particular family of RING finger E3s, named ATL, that contain a transmembrane domain and the RING-H2 finger domain; none of the member of the family contains any other previously described domain. Although the study of a few members in A. thaliana and O. sativa has been reported, the role of this family in the life cycle of a plant is still vague. To provide tools to advance on the functional analysis of this family we have undertaken a phylogenetic analysis of ATLs in twenty-four plant genomes. ATLs were found in all the 24 plant species analyzed, in numbers ranging from 20-28 in two basal species to 162 in soybean. Analysis of ATLs arrayed in tandem indicates that sets of genes are expanding in a species-specific manner. To get insights into the domain architecture of ATLs we generated 75 pHMM LOGOs from 1815 ATLs, and unraveled potential protein-protein interaction regions by means of yeast two-hybrid assays. Several ATLs were found to interact with DSK2a/ubiquilin through a region at the amino-terminal end, suggesting that this is a widespread interaction that may assist in the mode of action of ATLs; the region was traced to a distinct sequence LOGO. Our analysis provides significant observations on the evolution and expansion of the ATL family in addition to information on the domain structure of this class of ubiquitin-ligases that may be involved in plant adaptation to environmental stress.

Identification of essential sequences for cellular localization in the muscle-specific ubiquitin E3 ligase MAFbx/Atrogin 1.

PubMed

Julie, Lagirand-Cantaloube; Sabrina, Batonnet-Pichon; Marie-Pierre, Leibovitch; Leibovitch, Serge A

2012-02-17

In skeletal muscle atrophy, upregulation and nuclear accumulation of the Ubiquitin E3 ligase MAFbx is essential for accelerated muscle protein loss, but the nuclear/cytoplasmic shuttling of MAFbx is undefined. Here we found that MAFbx contains two functional nuclear localization signals (NLS). Mutation or deletion of only one NLS induced cytoplasmic localization of MAFbx. We identified a non-classical NES located in the leucine charged domain (LCD) of MAFbx, which is leptomycin B insensitive. We demonstrated that mutation (L169Q) in LLXXL motif of LCD suppressed cytoplasmic retention of MAFbx. Nucleocytoplasmic shuttling of MAFbx represents a novel mechanism for targeting its substrates and its cytosolic partners in muscle atrophy. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Autoregulation of Parkin activity through its ubiquitin-like domain

PubMed Central

Chaugule, Viduth K; Burchell, Lynn; Barber, Kathryn R; Sidhu, Ateesh; Leslie, Simon J; Shaw, Gary S; Walden, Helen

2011-01-01

Parkin is an E3-ubiquitin ligase belonging to the RBR (RING–InBetweenRING–RING family), and is involved in the neurodegenerative disorder Parkinson's disease. Autosomal recessive juvenile Parkinsonism, which is one of the most common familial forms of the disease, is directly linked to mutations in the parkin gene. However, the molecular mechanisms of Parkin dysfunction in the disease state remain to be established. We now demonstrate that the ubiquitin-like domain of Parkin functions to inhibit its autoubiquitination. Moreover pathogenic Parkin mutations disrupt this autoinhibition, resulting in a constitutively active molecule. In addition, we show that the mechanism of autoregulation involves ubiquitin binding by a C-terminal region of Parkin. Our observations provide important molecular insights into the underlying basis of Parkinson's disease, and in the regulation of RBR E3-ligase activity. PMID:21694720

Iron-Binding E3 Ligase Mediates Iron Response in Plants by Targeting Basic Helix-Loop-Helix Transcription Factors1[OPEN

PubMed Central

Selote, Devarshi; Samira, Rozalynne; Matthiadis, Anna; Gillikin, Jeffrey W.; Long, Terri A.

2015-01-01

Iron uptake and metabolism are tightly regulated in both plants and animals. In Arabidopsis (Arabidopsis thaliana), BRUTUS (BTS), which contains three hemerythrin (HHE) domains and a Really Interesting New Gene (RING) domain, interacts with basic helix-loop-helix transcription factors that are capable of forming heterodimers with POPEYE (PYE), a positive regulator of the iron deficiency response. BTS has been shown to have E3 ligase capacity and to play a role in root growth, rhizosphere acidification, and iron reductase activity in response to iron deprivation. To further characterize the function of this protein, we examined the expression pattern of recombinant ProBTS::β-GLUCURONIDASE and found that it is expressed in developing embryos and other reproductive tissues, corresponding with its apparent role in reproductive growth and development. Our findings also indicate that the interactions between BTS and PYE-like (PYEL) basic helix-loop-helix transcription factors occur within the nucleus and are dependent on the presence of the RING domain. We provide evidence that BTS facilitates 26S proteasome-mediated degradation of PYEL proteins in the absence of iron. We also determined that, upon binding iron at the HHE domains, BTS is destabilized and that this destabilization relies on specific residues within the HHE domains. This study reveals an important and unique mechanism for plant iron homeostasis whereby an E3 ubiquitin ligase may posttranslationally control components of the transcriptional regulatory network involved in the iron deficiency response. PMID:25452667

An E3 Ligase Affects the NLR Receptor Stability and Immunity to Powdery Mildew1

PubMed Central

Chang, Cheng; Gu, Cheng; Tang, Sanyuan

2016-01-01

Following the detection of pathogen cognate effectors, plant Nod-like receptors (NLRs) trigger isolate-specific immunity that is generally associated with cell death. The regulation of NLR stability is important to ensure effective immunity. In barley (Hordeum vulgare), the allelic Mildew locus A (MLA) receptors mediate isolate-specific disease resistance against powdery mildew fungus (Blumeria graminis f. sp. hordei). Currently, how MLA stability is controlled remains unknown. Here, we identified an MLA-interacting RING-type E3 ligase, MIR1, that interacts with several MLAs. We showed that the carboxyl-terminal TPR domain of MIR1 mediates the interaction with the coiled-coil domain-containing region of functional MLAs, such as MLA1, MLA6, and MLA10, but not with that of the nonfunctional MLA18-1. MIR1 can ubiquitinate the amino-terminal region of MLAs in vitro and promotes the proteasomal degradation of MLAs in vitro and in planta. Both proteasome inhibitor treatment and virus-induced gene silencing-mediated MIR1 silencing significantly increased MLA abundance in barley transgenic lines. Furthermore, overexpression of MIR1 specifically compromised MLA-mediated disease resistance in barley, while coexpression of MIR1 and MLA10 attenuated MLA10-induced cell death signaling in Nicotiana benthamiana. Together, our data reveal a mechanism for the control of the stability of MLA immune receptors and for the attenuation of MLA-triggered defense signaling by a RING-type E3 ligase via the ubiquitin proteasome system. PMID:27780896

The single-subunit RING-type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling.

PubMed

Bueso, Eduardo; Rodriguez, Lesia; Lorenzo-Orts, Laura; Gonzalez-Guzman, Miguel; Sayas, Enric; Muñoz-Bertomeu, Jesús; Ibañez, Carla; Serrano, Ramón; Rodriguez, Pedro L

2014-12-01

Membrane-delimited events play a crucial role for ABA signaling and PYR/PYL/RCAR ABA receptors, clade A PP2Cs and SnRK2/CPK kinases modulate the activity of different plasma membrane components involved in ABA action. Therefore, the turnover of PYR/PYL/RCARs in the proximity of plasma membrane might be a step that affects receptor function and downstream signaling. In this study we describe a single-subunit RING-type E3 ubiquitin ligase RSL1 that interacts with the PYL4 and PYR1 ABA receptors at the plasma membrane. Overexpression of RSL1 reduces ABA sensitivity and rsl1 RNAi lines that impair expression of several members of the RSL1/RFA gene family show enhanced sensitivity to ABA. RSL1 bears a C-terminal transmembrane domain that targets the E3 ligase to plasma membrane. Accordingly, bimolecular fluorescent complementation (BiFC) studies showed the RSL1-PYL4 and RSL1-PYR1 interaction is localized to plasma membrane. RSL1 promoted PYL4 and PYR1 degradation in vivo and mediated in vitro ubiquitylation of the receptors. Taken together, these results suggest ubiquitylation of ABA receptors at plasma membrane is a process that might affect their function via effect on their half-life, protein interactions or trafficking. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Covalent ISG15 conjugation to CHIP promotes its ubiquitin E3 ligase activity and inhibits lung cancer cell growth in response to type I interferon.

PubMed

Yoo, Lang; Yoon, A-Rum; Yun, Chae-Ok; Chung, Kwang Chul

2018-01-24

The carboxyl terminus of Hsp70-interacting protein (CHIP) acts as a ubiquitin E3 ligase and a link between the chaperones Hsp70/90 and the proteasome system, playing a vital role in maintaining protein homeostasis. CHIP regulates a number of proteins involved in a myriad of physiological and pathological processes, but the underlying mechanism of action via posttranslational modification has not been extensively explored. In this study, we investigated a novel modulatory mode of CHIP and its effect on CHIP enzymatic activity. ISG15, an ubiquitin-like modifier, is induced by type I interferon (IFN) stimulation and can be conjugated to target proteins (ISGylation). Here we demonstrated that CHIP may be a novel target of ISGylation in HEK293 cells stimulated with type I IFN. We also found that Lys143/144/145 and Lys287 residues in CHIP are important for and target residues of ISGylation. Moreover, ISGylation promotes the E3 ubiquitin ligase activity of CHIP, subsequently causing a decrease in levels of oncogenic c-Myc, one of its many ubiquitination targets, in A549 lung cancer cells and inhibiting A549 cell and tumor growth. In conclusion, the present study demonstrates that covalent ISG15 conjugation produces a novel CHIP regulatory mode that enhances the tumor-suppressive activity of CHIP, thereby contributing to the antitumor effect of type I IFN.

Viral Ubiquitin Ligase Stimulates Selective Host MicroRNA Expression by Targeting ZEB Transcriptional Repressors

PubMed Central

Kim, Ju Youn; Leader, Andrew; Stoller, Michelle L.; Coen, Donald M.; Wilson, Angus C.

2017-01-01

Infection with herpes simplex virus-1 (HSV-1) brings numerous changes in cellular gene expression. Levels of most host mRNAs are reduced, limiting synthesis of host proteins, especially those involved in antiviral defenses. The impact of HSV-1 on host microRNAs (miRNAs), an extensive network of short non-coding RNAs that regulate mRNA stability/translation, remains largely unexplored. Here we show that transcription of the miR-183 cluster (miR-183, miR-96, and miR-182) is selectively induced by HSV-1 during productive infection of primary fibroblasts and neurons. ICP0, a viral E3 ubiquitin ligase expressed as an immediate-early protein, is both necessary and sufficient for this induction. Nuclear exclusion of ICP0 or removal of the RING (really interesting new gene) finger domain that is required for E3 ligase activity prevents induction. ICP0 promotes the degradation of numerous host proteins and for the most part, the downstream consequences are unknown. Induction of the miR-183 cluster can be mimicked by depletion of host transcriptional repressors zinc finger E-box binding homeobox 1 (ZEB1)/δ-crystallin enhancer binding factor 1 (δEF1) and zinc finger E-box binding homeobox 2 (ZEB2)/Smad-interacting protein 1 (SIP1), which we establish as new substrates for ICP0-mediated degradation. Thus, HSV-1 selectively stimulates expression of the miR-183 cluster by ICP0-mediated degradation of ZEB transcriptional repressors. PMID:28783105

BPM-CUL3 E3 ligase modulates thermotolerance by facilitating negative regulatory domain-mediated degradation of DREB2A in Arabidopsis.

PubMed

Morimoto, Kyoko; Ohama, Naohiko; Kidokoro, Satoshi; Mizoi, Junya; Takahashi, Fuminori; Todaka, Daisuke; Mogami, Junro; Sato, Hikaru; Qin, Feng; Kim, June-Sik; Fukao, Yoichiro; Fujiwara, Masayuki; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2017-10-03

DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A (DREB2A) acts as a key transcription factor in both drought and heat stress tolerance in Arabidopsis and induces the expression of many drought- and heat stress-inducible genes. Although DREB2A expression itself is induced by stress, the posttranslational regulation of DREB2A, including protein stabilization, is required for its transcriptional activity. The deletion of a 30-aa central region of DREB2A known as the negative regulatory domain (NRD) transforms DREB2A into a stable and constitutively active form referred to as DREB2A CA. However, the molecular basis of this stabilization and activation has remained unknown for a decade. Here we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the Cullin3 (CUL3)-based E3 ligase, as DREB2A-interacting proteins. We observed that DREB2A and BPMs interact in the nuclei, and that the NRD of DREB2A is sufficient for its interaction with BPMs. BPM -knockdown plants exhibited increased DREB2A accumulation and induction of DREB2A target genes under heat and drought stress conditions. Genetic analysis indicated that the depletion of BPM expression conferred enhanced thermotolerance via DREB2A stabilization. Thus, the BPM-CUL3 E3 ligase is likely the long-sought factor responsible for NRD-dependent DREB2A degradation. Through the negative regulation of DREB2A stability, BPMs modulate the heat stress response and prevent an adverse effect of excess DREB2A on plant growth. Furthermore, we found the BPM recognition motif in various transcription factors, implying a general contribution of BPM-mediated proteolysis to divergent cellular responses via an accelerated turnover of transcription factors.

UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis.

PubMed

Xie, Youming; Varshavsky, Alexander

2002-12-01

The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.

Cytoplasmic FANCA-FANCC Complex Interacts and Stabilizes the Cytoplasm-dislocalized Leukemic Nucleophosmin Protein (NPMc)*

PubMed Central

Du, Wei; Li, Jie; Sipple, Jared; Chen, Jianjun; Pang, Qishen

2010-01-01

Eight of the Fanconi anemia (FA) proteins form a core complex that activates the FA pathway. Some core complex components also form subcomplexes for yet-to-be-elucidated functions. Here, we have analyzed the interaction between a cytoplasmic FA subcomplex and the leukemic nucleophosmin (NPMc). Exogenous NPMc was degraded rapidly in FA acute myeloid leukemia bone marrow cells. Knockdown of FANCA or FANCC in leukemic OCI/AML3 cells induced rapid degradation of endogenous NPMc. NPMc degradation was mediated by the ubiquitin-proteasome pathway involving the IBR-type RING-finger E3 ubiquitin ligase IBRDC2, and genetic correction of FA-A or FA-C lymphoblasts prevented NPMc ubiquitination. Moreover, cytoplasmic FANCA and FANCC formed a cytoplasmic complex and interacted with NPMc. Using a patient-derived FANCC mutant and a nuclearized FANCC, we demonstrated that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability. Finally, depletion of FANCA and FANCC in NPMc-positive leukemic cells significantly increased inflammation and chemoresistance through NF-κB activation. Our findings not only reveal the molecular mechanism involving cytoplasmic retention of NPMc but also suggest cytoplasmic function of FANCA and FANCC in NPMc-related leukemogenesis. PMID:20864535

Cardiac systolic dysfunction in doxorubicin-challenged rats is associated with upregulation of MuRF2 and MuRF3 E3 ligases

PubMed Central

da Silva, Marcia Gracindo; Mattos, Elisabete; Camacho-Pereira, Juliana; Domitrovic, Tatiana; Galina, Antonio; Costa, Mauro W; Kurtenbach, Eleonora

2012-01-01

Doxorubicin (DOXO) is an efficient and low-cost chemotherapeutic agent. The use of DOXO is limited by its side effects, including cardiotoxicity, that may progress to cardiac failure as a result of multifactorial events that have not yet been fully elucidated. In the present study, the effects of DOXO at two different doses were analyzed to identify early functional and molecular markers of cardiac distress. One group of rats received 7.5 mg/kg of DOXO (low-dose group) and was followed for 20 weeks. A subset of these animals was then subjected to an additional cycle of DOXO treatment, generating a cumulative dose of 20 mg/kg (high-dose group). Physiological and biochemical parameters were assessed in both treatment groups and in a control group that received saline. Systolic dysfunction was observed only in the high-dose group. Mitochondrial function analysis showed a clear reduction in oxidative cellular respiration for animals in both DOXO treatment groups, with evidence of complex I damage being observed. Transcriptional analysis by quantitative polymerase chain reaction revealed an increase in atrial natriuretic peptide transcript in the high-dose group, which is consistent with cardiac failure. Analysis of transcription levels of key components of the cardiac ubiquitin-proteasome system found that the ubiquitin E3 ligase muscle ring finger 1 (MuRF1) was upregulated in both the low- and high-dose DOXO groups. MuRF2 and MuRF3 were also upregulated in the high-dose group but not in the low-dose group. This molecular profile may be useful as an early physiological and energetic cardiac failure indicator for testing therapeutic interventions in animal models. PMID:23620696

Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels.

PubMed

Fu, Ssu-Ju; Jeng, Chung-Jiuan; Ma, Chia-Hao; Peng, Yi-Jheng; Lee, Chi-Ming; Fang, Ya-Ching; Lee, Yi-Ching; Tang, Sung-Chun; Hu, Meng-Chun; Tang, Chih-Yung

2017-03-01

Voltage-gated Ca V 2.1 channels comprise a pore-forming α 1A subunit with auxiliary α 2 δ and β subunits. Ca V 2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the Ca V 2.1 subunit are associated with the cerebellar disease episodic ataxia type 2 (EA2). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of Ca V 2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human Ca V 2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel Ca V 2.1-binding partner. In neurons, RNF138 and Ca V 2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of Ca V 2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the Ca V 2.1 protein level and enhances Ca V 2.1 protein stability. Disrupting endogenous RNF138 function also effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of Ca V 2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on Ca V 2.1 WT functional expression, which can be attributed to defective membrane trafficking of Ca V 2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of Ca V 2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human Ca V 2.1 subunits. SIGNIFICANCE STATEMENT Loss-of-function mutations in the human Ca V 2.1 subunit are linked to episodic ataxia type 2 (EA2), a dominantly inherited disease characterized by paroxysmal attacks of ataxia and nystagmus. EA2-causing mutants may exert dominant-negative effects on the Ca V 2.1 wild-type subunit via aberrant proteasomal degradation. The molecular nature of the Ca V 2.1 ubiquitin-proteasome degradation pathway is currently unknown. The present study reports the first identification of an E3 ubiquitin ligase for Ca V 2.1, RNF138. Ca V 2.1 protein stability is dynamically regulated by RNF138 and auxiliary α 2 δ and β subunits. We provide a proof of concept that protecting the human Ca V 2.1 subunit from excessive proteasomal degradation with specific interruption of endogenous RNF138 function may partially contribute to the future development of a novel therapeutic strategy for EA2 patients. Copyright © 2017 the authors 0270-6474/17/372485-19$15.00/0.

Ataxia and hypogonadism caused by the loss of ubiquitin ligase activity of the U box protein CHIP.

PubMed

Shi, Chang-He; Schisler, Jonathan C; Rubel, Carrie E; Tan, Song; Song, Bo; McDonough, Holly; Xu, Lei; Portbury, Andrea L; Mao, Cheng-Yuan; True, Cadence; Wang, Rui-Hao; Wang, Qing-Zhi; Sun, Shi-Lei; Seminara, Stephanie B; Patterson, Cam; Xu, Yu-Ming

2014-02-15

Gordon Holmes syndrome (GHS) is a rare Mendelian neurodegenerative disorder characterized by ataxia and hypogonadism. Recently, it was suggested that disordered ubiquitination underlies GHS though the discovery of exome mutations in the E3 ligase RNF216 and deubiquitinase OTUD4. We performed exome sequencing in a family with two of three siblings afflicted with ataxia and hypogonadism and identified a homozygous mutation in STUB1 (NM_005861) c.737C→T, p.Thr246Met, a gene that encodes the protein CHIP (C-terminus of HSC70-interacting protein). CHIP plays a central role in regulating protein quality control, in part through its ability to function as an E3 ligase. Loss of CHIP function has long been associated with protein misfolding and aggregation in several genetic mouse models of neurodegenerative disorders; however, a role for CHIP in human neurological disease has yet to be identified. Introduction of the Thr246Met mutation into CHIP results in a loss of ubiquitin ligase activity measured directly using recombinant proteins as well as in cell culture models. Loss of CHIP function in mice resulted in behavioral and reproductive impairments that mimic human ataxia and hypogonadism. We conclude that GHS can be caused by a loss-of-function mutation in CHIP. Our findings further highlight the role of disordered ubiquitination and protein quality control in the pathogenesis of neurodegenerative disease and demonstrate the utility of combining whole-exome sequencing with molecular analyses and animal models to define causal disease polymorphisms.

Ataxia and hypogonadism caused by the loss of ubiquitin ligase activity of the U box protein CHIP

PubMed Central

Shi, Chang-He; Schisler, Jonathan C.; Rubel, Carrie E.; Tan, Song; Song, Bo; McDonough, Holly; Xu, Lei; Portbury, Andrea L.; Mao, Cheng-Yuan; True, Cadence; Wang, Rui-Hao; Wang, Qing-Zhi; Sun, Shi-Lei; Seminara, Stephanie B.; Patterson, Cam; Xu, Yu-Ming

2014-01-01

Gordon Holmes syndrome (GHS) is a rare Mendelian neurodegenerative disorder characterized by ataxia and hypogonadism. Recently, it was suggested that disordered ubiquitination underlies GHS though the discovery of exome mutations in the E3 ligase RNF216 and deubiquitinase OTUD4. We performed exome sequencing in a family with two of three siblings afflicted with ataxia and hypogonadism and identified a homozygous mutation in STUB1 (NM_005861) c.737C→T, p.Thr246Met, a gene that encodes the protein CHIP (C-terminus of HSC70-interacting protein). CHIP plays a central role in regulating protein quality control, in part through its ability to function as an E3 ligase. Loss of CHIP function has long been associated with protein misfolding and aggregation in several genetic mouse models of neurodegenerative disorders; however, a role for CHIP in human neurological disease has yet to be identified. Introduction of the Thr246Met mutation into CHIP results in a loss of ubiquitin ligase activity measured directly using recombinant proteins as well as in cell culture models. Loss of CHIP function in mice resulted in behavioral and reproductive impairments that mimic human ataxia and hypogonadism. We conclude that GHS can be caused by a loss-of-function mutation in CHIP. Our findings further highlight the role of disordered ubiquitination and protein quality control in the pathogenesis of neurodegenerative disease and demonstrate the utility of combining whole-exome sequencing with molecular analyses and animal models to define causal disease polymorphisms. PMID:24113144

Binding of nucleotides by T4 DNA ligase and T4 RNA ligase: optical absorbance and fluorescence studies.

PubMed Central

Cherepanov, A V; de Vries, S

2001-01-01

The interaction of nucleotides with T4 DNA and RNA ligases has been characterized using ultraviolet visible (UV-VIS) absorbance and fluorescence spectroscopy. Both enzymes bind nucleotides with the K(d) between 0.1 and 20 microM. Nucleotide binding results in a decrease of absorbance at 260 nm due to pi-stacking with an aromatic residue, possibly phenylalanine, and causes red-shifting of the absorbance maximum due to hydrogen bonding with the exocyclic amino group. T4 DNA ligase is shown to have, besides the catalytic ATP binding site, another noncovalent nucleotide binding site. ATP bound there alters the pi-stacking of the nucleotide in the catalytic site, increasing its optical extinction. The K(d) for the noncovalent site is approximately 1000-fold higher than for the catalytic site. Nucleotides quench the protein fluorescence showing that a tryptophan residue is located in the active site of the ligase. The decrease of absorbance around 298 nm suggests that the hydrogen bonding interactions of this tryptophan residue are weakened in the ligase-nucleotide complex. The excitation/emission properties of T4 RNA ligase indicate that its ATP binding pocket is in contact with solvent, which is excluded upon binding of the nucleotide. Overall, the spectroscopic analysis reveals important similarities between T4 ligases and related nucleotidyltransferases, despite the low sequence similarity. PMID:11721015

RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for Interstrand Crosslink Repair and Human Health.

PubMed

Feeney, Laura; Muñoz, Ivan M; Lachaud, Christophe; Toth, Rachel; Appleton, Paul L; Schindler, Detlev; Rouse, John

2017-06-01

Defects in the repair of DNA interstrand crosslinks (ICLs) are associated with the genome instability syndrome Fanconi anemia (FA). Here we report that cells with mutations in RFWD3, an E3 ubiquitin ligase that interacts with and ubiquitylates replication protein A (RPA), show profound defects in ICL repair. An amino acid substitution in the WD40 repeats of RFWD3 (I639K) found in a new FA subtype abolishes interaction of RFWD3 with RPA, thereby preventing RFWD3 recruitment to sites of ICL-induced replication fork stalling. Moreover, single point mutations in the RPA32 subunit of RPA that abolish interaction with RFWD3 also inhibit ICL repair, demonstrating that RPA-mediated RFWD3 recruitment to stalled replication forks is important for ICL repair. We also report that unloading of RPA from sites of ICL induction is perturbed in RFWD3-deficient cells. These data reveal important roles for RFWD3 localization in protecting genome stability and preserving human health. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

A High-Throughput Cell-Based Screen Identified a 2-[(E)-2-Phenylvinyl]-8-Quinolinol Core Structure That Activates p53

PubMed Central

Bechill, John; Zhong, Rong; Zhang, Chen; Solomaha, Elena

2016-01-01

p53 function is frequently inhibited in cancer either through mutations or by increased degradation via MDM2 and/or E6AP E3-ubiquitin ligases. Most agents that restore p53 expression act by binding MDM2 or E6AP to prevent p53 degradation. However, fewer compounds directly bind to and activate p53. Here, we identified compounds that shared a core structure that bound p53, caused nuclear localization of p53 and caused cell death. To identify these compounds, we developed a novel cell-based screen to redirect p53 degradation to the Skip-Cullin-F-box (SCF) ubiquitin ligase complex in cells expressing high levels of p53. In a multiplexed assay, we coupled p53 targeted degradation with Rb1 targeted degradation in order to identify compounds that prevented p53 degradation while not inhibiting degradation through the SCF complex or other proteolytic machinery. High-throughput screening identified several leads that shared a common 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that stabilized p53. Surface plasmon resonance analysis indicated that these compounds bound p53 with a KD of 200 ± 52 nM. Furthermore, these compounds increased p53 nuclear localization and transcription of the p53 target genes PUMA, BAX, p21 and FAS in cancer cells. Although p53-null cells had a 2.5±0.5-fold greater viability compared to p53 wild type cells after treatment with core compounds, loss of p53 did not completely rescue cell viability suggesting that compounds may target both p53-dependent and p53-independent pathways to inhibit cell proliferation. Thus, we present a novel, cell-based high-throughput screen to identify a 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that bound to p53 and increased p53 activity in cancer cells. These compounds may serve as anti-neoplastic agents in part by targeting p53 as well as other potential pathways. PMID:27124407

Nitric Oxide-GAPDH Transcriptional Signaling Mediates Behavioral Actions of Cocaine.

PubMed

Harraz, Maged M; Snyder, Solomon H

2015-01-01

Psychotropic actions of cocaine are generally thought to involve its blockade of monoamine transporters leading to increased synaptic levels of monoamines, especially dopamine. Subsequent intracellular events have been less well characterized. We describe a signaling system wherein lower behavioral stimulant doses of cocaine, as well as higher neurotoxic doses, activate a cascade wherein nitric oxide nitrosylates glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to generate a complex with the ubiquitin-E3-ligase Siah1 which translocates to the nucleus. With lower cocaine doses, nuclear GAPDH augments CREB signaling, while at higher doses p53 signaling is enhanced. The drug CGP3466B very potently blocks GAPDH nitrosylation, hindering both signaling cascades and inhibits both behavioral activating and neurotoxic effects of cocaine. This system affords potentially novel approaches to the therapy of cocaine abuse.

Molecular basis of APC/C regulation by the spindle assembly checkpoint

PubMed Central

Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

2016-01-01

In the dividing eukaryotic cell the spindle assembly checkpoint (SAC) ensures each daughter cell inherits an identical set of chromosomes. The SAC coordinates the correct attachment of sister chromatid kinetochores to the mitotic spindle with activation of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome separation. In response to unattached kinetochores, the SAC generates the mitotic checkpoint complex (MCC), a multimeric assembly that inhibits the APC/C, delaying chromosome segregation. Here, using cryo-electron microscopy we determined the near-atomic resolution structure of an APC/C-MCC complex (APC/CMCC). We reveal how degron-like sequences of the MCC subunit BubR1 block degron recognition sites on Cdc20, the APC/C coactivator subunit (Cdc20APC/C) responsible for substrate interactions. BubR1 also obstructs binding of UbcH10 (APC/C’s initiating E2) to repress APC/C ubiquitination activity. Conformational variability of the complex allows for UbcH10 association, and we show from a structure of APC/CMCC in complex with UbcH10 how the Cdc20 subunit intrinsic to the MCC (Cdc20MCC) is ubiquitinated, a process that results in APC/C reactivation when the SAC is silenced. PMID:27509861

A novel mechanism for regulation of the type I IFN response by herpesvirus deconjugases.

PubMed

Gupta, Soham; Ylä-Anttila, Päivi; Masucci, Maria G

2018-04-11

Upon infection, viral nucleic acids are recognized by germline-encoded pattern-recognition receptors (PRRs), and cytosolic retinoic acid-inducible gene I (RIG-I)-like helicases (RLHs) that initiate signaling pathways resulting in the production of type I IFN and pro-inflammatory cytokines. Binding of RIG-I to viral nucleic acids triggers the formation of the RIG-I signalosome where RIG-I is ubiquitinated by the TRIM25 ligase and, with the help of 14-3-3 scaffolds, further translocated to mitochondrial anti-viral signalling proteins (MAVS). Subsequent ubiquitination-mediated events trigger transcriptional activation of the effectors of innate immunity. We have found a new mechanism by which herpesviruses interfere with this signalling pathway to favour the establishment of latency and promote virus replication. The cysteine protease encoded in the conserved N-terminal domain of the herpesvirus large tegument protein binds to 14-3-3 proteins and forms a tri-molecular complex with TRIM25, promoting the activation and autoubiquitination of the ligase. RIG-I is recruited to the complex but its ubiquitination is drastically reduced, which effectively inactivates downstream signalling and blocks the type I IFN response.

Inhibition of Siah2 ubiquitin ligase by vitamin K3 (menadione) attenuates hypoxia and MAPK signaling and blocks melanoma tumorigenesis

PubMed Central

Shah, Meera; Stebbins, John L.; Dewing, Antimone; Qi, Jianfei; Pellecchia, Maurizio; Ronai, Ze’ev A.

2010-01-01

Summary The E3 ubiquitin ligase Siah2 has been implicated in the regulation of the hypoxia response, as well as in the control of Ras, JNK/p38/NF-κB signaling pathways. Both Ras/mitogen-activated protein kinase (MAPK) and hypoxia pathways are important for melanoma development and progression, pointing to the possible use of Siah2 as target for treatment of this tumor type. In the present study, we have established a high-throughput electro-chemiluninescent-based assay in order to screen and identify inhibitors of Siah2 ubiquitin ligase activity. Of 1840 compounds screened, we identified and characterized menadione (MEN) as a specific inhibitor of Siah2 ligase activity. MEN attenuated Siah2 self-ubiquitination, and increased expression of its substrates PHD3 and Sprouty2, with concomitant decrease in levels of HIF-1α and pERK, the respective downstream effectors. MEN treatment no longer affected PHD3 or Sprouty2 in Siah-KO cells, pointing to its Siah-dependent effects. Further, MEN inhibition of Siah2 was not attenuated by free radical scavenger, suggesting it is ROS-independent. Significantly, growth of xenograft melanoma tumors was inhibited following the administration of MEN or its derivative. These findings reveal an efficient platform for the identification of Siah inhibitors while identifying and characterizing MEN as Siah inhibitor that attenuates hypoxia and MAPK signaling, and inhibits melanoma tumorigenesis. PMID:19712206

Homology modeling and docking analyses of M. leprae Mur ligases reveals the common binding residues for structure based drug designing to eradicate leprosy.

PubMed

Shanmugam, Anusuya; Natarajan, Jeyakumar

2012-06-01

Multi drug resistance capacity for Mycobacterium leprae (MDR-Mle) demands the profound need for developing new anti-leprosy drugs. Since most of the drugs target a single enzyme, mutation in the active site renders the antibiotic ineffective. However, structural and mechanistic information on essential bacterial enzymes in a pathway could lead to the development of antibiotics that targets multiple enzymes. Peptidoglycan is an important component of the cell wall of M. leprae. The biosynthesis of bacterial peptidoglycan represents important targets for the development of new antibacterial drugs. Biosynthesis of peptidoglycan is a multi-step process that involves four key Mur ligase enzymes: MurC (EC:6.3.2.8), MurD (EC:6.3.2.9), MurE (EC:6.3.2.13) and MurF (EC:6.3.2.10). Hence in our work, we modeled the three-dimensional structure of the above Mur ligases using homology modeling method and analyzed its common binding features. The residues playing an important role in the catalytic activity of each of the Mur enzymes were predicted by docking these Mur ligases with their substrates and ATP. The conserved sequence motifs significant for ATP binding were predicted as the probable residues for structure based drug designing. Overall, the study was successful in listing significant and common binding residues of Mur enzymes in peptidoglycan pathway for multi targeted therapy.

Inhibition of Siah2 ubiquitin ligase by vitamin K3 (menadione) attenuates hypoxia and MAPK signaling and blocks melanoma tumorigenesis.

PubMed

Shah, Meera; Stebbins, John L; Dewing, Antimone; Qi, Jianfei; Pellecchia, Maurizio; Ronai, Ze'ev A

2009-12-01

The E3 ubiquitin ligase Siah2 has been implicated in the regulation of the hypoxia response, as well as in the control of Ras, JNK/p38/NF-kappaB signaling pathways. Both Ras/mitogen-activated protein kinase (MAPK) and hypoxia pathways are important for melanoma development and progression, pointing to the possible use of Siah2 as target for treatment of this tumor type. In the present study, we have established a high-throughput electro-chemiluninescent-based assay in order to screen and identify inhibitors of Siah2 ubiquitin ligase activity. Of 1840 compounds screened, we identified and characterized menadione (MEN) as a specific inhibitor of Siah2 ligase activity. MEN attenuated Siah2 self-ubiquitination, and increased expression of its substrates PHD3 and Sprouty2, with concomitant decrease in levels of HIF-1alpha and pERK, the respective downstream effectors. MEN treatment no longer affected PHD3 or Sprouty2 in Siah-KO cells, pointing to its Siah-dependent effects. Further, MEN inhibition of Siah2 was not attenuated by free radical scavenger, suggesting it is ROS-independent. Significantly, growth of xenograft melanoma tumors was inhibited following the administration of MEN or its derivative. These findings reveal an efficient platform for the identification of Siah inhibitors while identifying and characterizing MEN as Siah inhibitor that attenuates hypoxia and MAPK signaling, and inhibits melanoma tumorigenesis.

The COP9 Signalosome regulates seed germination by facilitating protein degradation of RGL2 and ABI5

PubMed Central

Li, Bosheng; Bücker, Birte; Keil, Philipp; Zhang, Shaoman; Li, Jigang; Kang, Dingming; Liu, Jie; Dong, Jie; Deng, Xing Wang; Irish, Vivian

2018-01-01

The control of seed germination and seed dormancy are critical for the successful propagation of plant species, and are important agricultural traits. Seed germination is tightly controlled by the balance of gibberellin (GA) and abscisic acid (ABA), and is influenced by environmental factors. The COP9 Signalosome (CSN) is a conserved multi-subunit protein complex that is best known as a regulator of the Cullin-RING family of ubiquitin E3 ligases (CRLs). Multiple viable mutants of the CSN showed poor germination, except for csn5b-1. Detailed analyses showed that csn1-10 has a stronger seed dormancy, while csn5a-1 mutants exhibit retarded seed germination in addition to hyperdormancy. Both csn5a-1 and csn1-10 plants show defects in the timely removal of the germination inhibitors: RGL2, a repressor of GA signaling, and ABI5, an effector of ABA responses. We provide genetic evidence to demonstrate that the germination phenotype of csn1-10 is caused by over-accumulation of RGL2, a substrate of the SCF (CRL1) ubiquitin E3 ligase, while the csn5a-1 phenotype is caused by over-accumulation of RGL2 as well as ABI5. The genetic data are consistent with the hypothesis that CSN5A regulates ABI5 by a mechanism that may not involve CSN1. Transcriptome analyses suggest that CSN1 has a more prominent role than CSN5A during seed maturation, but CSN5A plays a more important role than CSN1 during seed germination, further supporting the functional distinction of these two CSN genes. Our study delineates the molecular targets of the CSN complex in seed germination, and reveals that CSN5 has additional functions in regulating ABI5, thus the ABA signaling pathway. PMID:29462139

Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rutz, Natalja; Heilbronn, Regine; Weger, Stefan, E-mail: stefan.weger@charite.de

2015-08-28

Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 inducedmore » polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.« less

Mahogunin-mediated α-tubulin ubiquitination via noncanonical K6 linkage regulates microtubule stability and mitotic spindle orientation

PubMed Central

Srivastava, D; Chakrabarti, O

2014-01-01

Mahogunin ring finger-1 (MGRN1) is a cytosolic ubiquitin ligase whose disruption or interaction with some isoforms of cytosolically exposed prion protein leads to spongiform neurodegeneration and also lack of which results in reduced embryonic viability due to mispatterning of the left–right (LR) axis during development. Here we demonstrate an interaction between the cytoskeletal protein α-tubulin and MGRN1. In cultured cell systems, loss of the ubiquitin E3 ligase activity of MGRN1 results in spindle misorientation and decreased α-tubulin polymerization, an effect also seen in primary cells. α-Tubulin was post-translationally modified by MGRN1 via noncanonical K6-linked polyubiquitination. This was significant because expression of catalytically inactive MGRN1 and/or ubiquitin mutant capable of only monoubiquitination resulted in similar mitotic spindle misorientation. The modulatory effect of MGRN1 was specific for α-tubulin and similar changes could not be detected in β- or γ-tubulin. However, catalytic inactivation of MGRN1 did not abrogate monoubiquitination of α-tubulin, thus unraveling a unique dual mode of ubiquitination by an unknown E3 ligase and MGRN1. MGRN1-mediated α-tubulin modification, and hence its stability, may highlight a key event in the LR patterning during embryogenesis. PMID:24556679

Efficient Parvovirus Replication Requires CRL4Cdt2-Targeted Depletion of p21 to Prevent Its Inhibitory Interaction with PCNA

PubMed Central

Pintel, David J.

2014-01-01

Infection by the autonomous parvovirus minute virus of mice (MVM) induces a vigorous DNA damage response in host cells which it utilizes for its efficient replication. Although p53 remains activated, p21 protein levels remain low throughout the course of infection. We show here that efficient MVM replication required the targeting for degradation of p21 during this time by the CRL4Cdt2 E3-ubiquitin ligase which became re-localized to MVM replication centers. PCNA provides a molecular platform for substrate recognition by the CRL4Cdt2 E3-ubiquitin ligase and p21 targeting during MVM infection required its interaction both with Cdt2 and PCNA. PCNA is also an important co-factor for MVM replication which can be antagonized by p21 in vitro. Expression of a stable p21 mutant that retained interaction with PCNA inhibited MVM replication, while a stable p21 mutant which lacked this interaction did not. Thus, while interaction with PCNA was important for targeting p21 to the CRL4Cdt2 ligase re-localized to MVM replication centers, efficient viral replication required subsequent depletion of p21 to abrogate its inhibition of PCNA. PMID:24699724

Efficient parvovirus replication requires CRL4Cdt2-targeted depletion of p21 to prevent its inhibitory interaction with PCNA.

PubMed

Adeyemi, Richard O; Fuller, Matthew S; Pintel, David J

2014-04-01

Infection by the autonomous parvovirus minute virus of mice (MVM) induces a vigorous DNA damage response in host cells which it utilizes for its efficient replication. Although p53 remains activated, p21 protein levels remain low throughout the course of infection. We show here that efficient MVM replication required the targeting for degradation of p21 during this time by the CRL4Cdt2 E3-ubiquitin ligase which became re-localized to MVM replication centers. PCNA provides a molecular platform for substrate recognition by the CRL4Cdt2 E3-ubiquitin ligase and p21 targeting during MVM infection required its interaction both with Cdt2 and PCNA. PCNA is also an important co-factor for MVM replication which can be antagonized by p21 in vitro. Expression of a stable p21 mutant that retained interaction with PCNA inhibited MVM replication, while a stable p21 mutant which lacked this interaction did not. Thus, while interaction with PCNA was important for targeting p21 to the CRL4Cdt2 ligase re-localized to MVM replication centers, efficient viral replication required subsequent depletion of p21 to abrogate its inhibition of PCNA.

Structural basis of lentiviral subversion of a cellular protein degradation pathway

NASA Astrophysics Data System (ADS)

Schwefel, David; Groom, Harriet C. T.; Boucherit, Virginie C.; Christodoulou, Evangelos; Walker, Philip A.; Stoye, Jonathan P.; Bishop, Kate N.; Taylor, Ian A.

2014-01-01

Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4+ T cells by reducing the cellular deoxynucleoside 5'-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell's ubiquitination machinery to destroy the cell's protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell's normal protein degradation pathway to inactivate the cellular viral defence system.

Parkin, A Top Level Manager in the Cell’s Sanitation Department

PubMed Central

Rankin, Carolyn A; Roy, Ambrish; Zhang, Yang; Richter, Mark

2011-01-01

Parkin belongs to a class of multiple RING domain proteins designated as RBR (RING, in between RING, RING) proteins. In this review we examine what is known regarding the structure/function relationship of the Parkin protein. Parkin contains three RING domains plus a ubiquitin-like domain and an in-between-RING (IBR) domain. RING domains are rich in cysteine amino acids that act as ligands to bind zinc ions. RING domains may interact with DNA or with other proteins and perform a wide range of functions. Some function as E3 ubiquitin ligases, participating in attachment of ubiquitin chains to signal proteasome degradation; however, ubiquitin may be attached for purposes other than proteasome degradation. It was determined that the C-terminal most RING, RING2, is essential for Parkin to function as an E3 ubiquitin ligase and a number of substrates have been identified. However, Parkin also participates in a number of other fiunctions, such as DNA repair, microtubule stabilization, and formation of aggresomes. Some functions, such as participation in a multi-protein complex implicated in NMDA activity at the post synaptic density, do not require ubiquitination of substrate molecules. Recent observations of RING proteins suggest their function may be regulated by zinc ion binding. We have modeled the three RING domains of Parkin and have identified a new set of RING2 ligands. This set allows for binding of two rather than just one zinc ion, opening the possibility that the number of zinc ions bound acts as a molecular switch to modulate Parkin function. PMID:21633666

Syk-mediated tyrosine phosphorylation of mule promotes TNF-induced JNK activation and cell death.

PubMed

Lee, C K; Yang, Y; Chen, C; Liu, J

2016-04-14

The transcription factor Miz1 negatively regulates TNF-induced JNK activation and cell death by suppressing TRAF2 K63-polyubiquitination; upon TNF stimulation, the suppression is relieved by Mule/ARF-BP1-mediated Miz1 ubiquitination and subsequent degradation. It is not known how Mule is activated by TNF. Here we report that TNF activates Mule by inducing the dissociation of Mule from its inhibitor ARF. ARF binds to and thereby inhibits the E3 ligase activity of Mule in the steady state. TNF induces tyrosine phosphorylation of Mule, which subsequently dissociates from ARF and becomes activated. Inhibition of Mule phosphorylation by silencing of the Spleen Tyrosine Kinase (Syk) prevents its dissociation from ARF, thereby inhibiting Mule E3 ligase activity and TNF-induced JNK activation and cell death. Our data provides a missing link in TNF signaling pathway that leads to JNK activation and cell death.

E3 ubiquitin ligase Cbl-b in innate and adaptive immunity

PubMed Central

Liu, Qingjun; Zhou, Hong; Langdon, Wallace Y; Zhang, Jian

2014-01-01

Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING finger E3 ubiquitin-protein ligase, has been demonstrated to play a crucial role in establishing the threshold for T-cell activation and controlling peripheral T-cell tolerance via multiple mechanisms. Accumulating evidence suggests that Cbl-b also regulates innate immune responses and plays an important role in host defense to pathogens. Understanding the signaling pathways regulated by Cbl-b in innate and adaptive immune cells is therefore essential for efficient manipulation of Cbl-b in emerging immunotherapies for human disorders such as autoimmune diseases, allergic inflammation, infections, and cancer. In this article, we review the latest developments in the molecular structural basis of Cbl-b function, the regulation of Cbl-b expression, the signaling mechanisms of Cbl-b in immune cells, as well as the biological function of Cbl-b in physiological and pathological immune responses in animal models and human diseases. PMID:24875217

Structure and Function of the Mind bomb E3 ligase in the context of Notch Signal Transduction

PubMed Central

Guo, Bingqian; McMillan, Brian J.; Blacklow, Stephen C.

2016-01-01

The Notch signaling pathway has a critical role in cell fate determination and tissue homeostasis in a variety of different lineages. In the context of normal Notch signaling, the Notch receptor of the “signal-receiving” cell is activated in trans by a Notch ligand from a neighboring “signal-sending” cell. Genetic studies in several model organisms have established that ubiquitination of the Notch ligand, and its regulated endocytosis, is essential for transmission of this activation signal. In mammals, this ubiquitination step is dependent on the protein Mind bomb 1 (Mib1), a large multi-domain RING-type E3 ligase, and its direct interaction with the intracellular tails of Notch ligand molecules. Here, we discuss our current understanding of Mind bomb structure and mechanism in the context of Notch signaling and beyond. PMID:27285058

Shigella IpaH0722 E3 Ubiquitin Ligase Effector Targets TRAF2 to Inhibit PKC–NF-κB Activity in Invaded Epithelial Cells

PubMed Central

Ashida, Hiroshi; Nakano, Hiroyasu; Sasakawa, Chihiro

2013-01-01

NF-κB plays a central role in modulating innate immune responses to bacterial infections. Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation. The invasion of and subsequent replication of Shigella within epithelial cells is recognized by various pathogen recognition receptors as pathogen-associated molecular patterns. These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway. Here, we show the inhibition of the NF-κB activation by the delivery of the IpaH E3 ubiquitin ligase family member IpaH0722 using Shigella's type III secretion system. IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation. PMID:23754945

Crystal structure of the UBR-box from UBR6/FBXO11 reveals domain swapping mediated by zinc binding.

PubMed

Muñoz-Escobar, Juliana; Kozlov, Guennadi; Gehring, Kalle

2017-10-01

The UBR-box is a 70-residue zinc finger domain present in the UBR family of E3 ubiquitin ligases that directly binds N-terminal degradation signals in substrate proteins. UBR6, also called FBXO11, is an UBR-box containing E3 ubiquitin ligase that does not bind N-terminal signals. Here, we present the crystal structure of the UBR-box domain from human UBR6. The dimeric crystal structure reveals a unique form of domain swapping mediated by zinc coordination, where three independent protein chains come together to regenerate the topology of the monomeric UBR-box fold. Analysis of the structure suggests that the absence of N-terminal residue binding arises from the lack of an amino acid binding pocket. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

The Ubiquitin-associated Domain of Cellular Inhibitor of Apoptosis Proteins Facilitates Ubiquitylation*

PubMed Central

Budhidarmo, Rhesa; Day, Catherine L.

2014-01-01

The cellular inhibitor of apoptosis (cIAP) proteins are essential RING E3 ubiquitin ligases that regulate apoptosis and inflammatory responses. cIAPs contain a ubiquitin-associated (UBA) domain that binds ubiquitin and is implicated in the regulation of cell survival and proteasomal degradation. Here we show that mutation of the MGF and LL motifs in the UBA domain of cIAP1 caused unfolding and increased cIAP1 multimonoubiquitylation. By developing a UBA mutant that disrupted ubiquitin binding but not the structure of the UBA domain, we found that the UBA domain enhances cIAP1 and cIAP2 ubiquitylation. We demonstrate that the UBA domain binds to the UbcH5b∼Ub conjugate, and this promotes RING domain-dependent monoubiquitylation. This study establishes ubiquitin-binding modules, such as the UBA domain, as important regulatory modules that can fine tune the activity of E3 ligases. PMID:25065467

The E3 ligase c-Cbl regulates dendritic cell activation

PubMed Central

Chiou, Shin-Heng; Shahi, Payam; Wagner, Ryan T; Hu, Hongbo; Lapteva, Natalia; Seethammagari, Mamatha; Sun, Shao-Cong; Levitt, Jonathan M; Spencer, David M

2011-01-01

The activation of innate and adaptive immunity is always balanced by inhibitory signalling mechanisms to maintain tissue integrity. We have identified the E3 ligase c-Cbl––known for its roles in regulating lymphocyte signalling––as a modulator of dendritic cell activation. In c-Cbl-deficient dendritic cells, Toll-like receptor-induced expression of proinflammatory factors, such as interleukin-12, is increased, correlating with a greater potency of dendritic-cell-based vaccines against established tumours. This proinflammatory phenotype is accompanied by an increase in nuclear factor (NF)-κB activity. In addition, c-Cbl deficiency reduces both p50 and p105 levels, which have been shown to modulate the stimulatory function of NF-κB. Our data indicate that c-Cbl has a crucial, RING-domain-dependent role in regulating dendritic cell maturation, probably by facilitating the regulatory function of p105 and/or p50. PMID:21799517

Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases

PubMed Central

Zhelkovsky, Alexander M.; McReynolds, Larry A.

2014-01-01

RNA and DNA ligases catalyze the formation of a phosphodiester bond between the 5′-phosphate and 3′-hydroxyl ends of nucleic acids. In this work, we describe the ability of the thermophilic RNA ligase MthRnl from Methanobacterium thermoautotrophicum to recognize and modify the 3′-terminal phosphate of RNA and single-stranded DNA (ssDNA). This ligase can use an RNA 3′p substrate to generate an RNA 2′,3′-cyclic phosphate or convert DNA3′p to ssDNA3′pp5′A. An RNA ligase from the Thermus scotoductus bacteriophage TS2126 and a predicted T4 Rnl1-like protein from Thermovibrio ammonificans, TVa, were also able to adenylate ssDNA 3′p. These modifications of RNA and DNA 3′-phosphates are similar to the activities of RtcA, an RNA 3′-phosphate cyclase. The initial step involves adenylation of the enzyme by ATP, which is then transferred to either RNA 3′p or DNA 3′p to generate the adenylated intermediate. For RNA 3′pp5′A, the third step involves attack of the adjacent 2′ hydroxyl to generate the RNA 2′,3′-cyclic phosphate. These steps are analogous to those in classical 5′ phosphate ligation. MthRnl and TS2126 RNA ligases were not able to modify a 3′p in nicked double-stranded DNA. However, T4 DNA ligase and RtcA can use 3′-phosphorylated nicks in double-stranded DNA to produce a 3′-adenylated product. These 3′-terminal phosphate-adenylated intermediates are substrates for deadenylation by yeast 5′Deadenylase. Our findings that classic ligases can duplicate the adenylation and phosphate cyclization activity of RtcA suggests that they have an essential role in metabolism of nucleic acids with 3′-terminal phosphates. PMID:25324547

Mechanism of Ubiquitination and Deubiquitination in the Fanconi Anemia Pathway.

PubMed

van Twest, Sylvie; Murphy, Vincent J; Hodson, Charlotte; Tan, Winnie; Swuec, Paolo; O'Rourke, Julienne J; Heierhorst, Jörg; Crismani, Wayne; Deans, Andrew J

2017-01-19

Monoubiquitination and deubiquitination of FANCD2:FANCI heterodimer is central to DNA repair in a pathway that is defective in the cancer predisposition syndrome Fanconi anemia (FA). The "FA core complex" contains the RING-E3 ligase FANCL and seven other essential proteins that are mutated in various FA subtypes. Here, we purified recombinant FA core complex to reveal the function of these other proteins. The complex contains two spatially separate FANCL molecules that are dimerized by FANCB and FAAP100. FANCC and FANCE act as substrate receptors and restrict monoubiquitination to the FANCD2:FANCI heterodimer in only a DNA-bound form. FANCA and FANCG are dispensable for maximal in vitro ubiquitination. Finally, we show that the reversal of this reaction by the USP1:UAF1 deubiquitinase only occurs when DNA is disengaged. Our work reveals the mechanistic basis for temporal and spatial control of FANCD2:FANCI monoubiquitination that is critical for chemotherapy responses and prevention of Fanconi anemia. Copyright © 2017 Elsevier Inc. All rights reserved.

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1

DOE PAGES

Lu, Dihong; Ni, Weimin; Stanley, Bruce A.; ...

2016-03-03

The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown. Here we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type,more » but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development. In conclusion, our results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.« less

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Dihong; Ni, Weimin; Stanley, Bruce A.

The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown. Here we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type,more » but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development. In conclusion, our results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.« less

Phosphorylated hydroxyethylamines as novel inhibitors of the bacterial cell wall biosynthesis enzymes MurC to MurF.

PubMed

Sova, Matej; Kovac, Andreja; Turk, Samo; Hrast, Martina; Blanot, Didier; Gobec, Stanislav

2009-12-01

Enzymes involved in the biosynthesis of bacterial peptidoglycan represent important targets for development of new antibacterial drugs. Among them, Mur ligases (MurC to MurF) catalyze the formation of the final cytoplasmic precursor UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid. We present the design, synthesis and biological evaluation of a series of phosphorylated hydroxyethylamines as new type of small-molecule inhibitors of Mur ligases. We show that the phosphate group attached to the hydroxyl moiety of the hydroxyethylamine core is essential for good inhibitory activity. The IC(50) values of these inhibitors were in the micromolar range, which makes them a promising starting point for the development of multiple inhibitors of Mur ligases as potential antibacterial agents. In addition, 1-(4-methoxyphenylsulfonamido)-3-morpholinopropan-2-yl dihydrogen phosphate 7a was discovered as one of the best inhibitors of MurE described so far.

Put a RING on it: regulation and inhibition of RNF8 and RNF168 RING finger E3 ligases at DNA damage sites

PubMed Central

Bartocci, Cristina; Denchi, Eros Lazzerini

2013-01-01

RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligases comprise a large family of enzymes that in combination with an E2 ubiquitin-conjugating enzyme, modify target proteins by attaching ubiquitin moieties. A number of RING E3s play an essential role in the cellular response to DNA damage highlighting a crucial contribution for ubiquitin-mediated signaling to the genome surveillance pathway. Among the RING E3s, RNF8 and RNF168 play a critical role in the response to double stranded breaks, one of the most deleterious types of DNA damage. These proteins act as positive regulators of the signaling cascade that initiates at DNA lesions. Inactivation of these enzymes is sufficient to severely impair the ability of cells to respond to DNA damage. Given their central role in the pathway, several layers of regulation act at this nodal signaling point. Here we will summarize current knowledge on the roles of RNF8 and RNF168 in maintaining genome integrity with particular emphasis on recent insights into the multiple layers of regulation that act on these enzymes to fine-tune the cellular response to DNA lesions. PMID:23847653

The E3 ubiquitin ligase NEDD4 enhances killing of membrane-perturbing intracellular bacteria by promoting autophagy

PubMed Central

Pei, Gang; Buijze, Hellen; Liu, Haipeng; Moura-Alves, Pedro; Goosmann, Christian; Brinkmann, Volker; Kawabe, Hiroshi; Dorhoi, Anca; Kaufmann, Stefan H. E.

2017-01-01

ABSTRACT The E3 ubiquitin ligase NEDD4 has been intensively studied in processes involved in viral infections, such as virus budding. However, little is known about its functions in bacterial infections. Our investigations into the role of NEDD4 in intracellular bacterial infections demonstrate that Mycobacterium tuberculosis and Listeria monocytogenes, but not Mycobacterium bovis BCG, replicate more efficiently in NEDD4 knockdown macrophages. In parallel, NEDD4 knockdown or knockout impaired basal macroautophagy/autophagy, as well as infection-induced autophagy. Conversely, NEDD4 expression promoted autophagy in an E3 catalytic activity-dependent manner, thereby restricting intracellular Listeria replication. Mechanistic studies uncovered that endogenous NEDD4 interacted with BECN1/Beclin 1 and this interaction increased during Listeria infection. Deficiency of NEDD4 resulted in elevated K48-linkage ubiquitination of endogenous BECN1. Further, NEDD4 mediated K6- and K27- linkage ubiquitination of BECN1, leading to elevated stability of BECN1 and increased autophagy. Thus, NEDD4 participates in killing of intracellular bacterial pathogens via autophagy by sustaining the stability of BECN1. PMID:29251248

An E3 Ubiquitin Ligase-BAG Protein Module Controls Plant Innate Immunity and Broad-Spectrum Disease Resistance.

PubMed

You, Quanyuan; Zhai, Keran; Yang, Donglei; Yang, Weibing; Wu, Jingni; Liu, Junzhong; Pan, Wenbo; Wang, Jianjun; Zhu, Xudong; Jian, Yikun; Liu, Jiyun; Zhang, Yingying; Deng, Yiwen; Li, Qun; Lou, Yonggen; Xie, Qi; He, Zuhua

2016-12-14

Programmed cell death (PCD) and immunity in plants are tightly controlled to promote antimicrobial defense while preventing autoimmunity. However, the mechanisms contributing to this immune homeostasis are poorly understood. Here, we isolated a rice mutant ebr1 (enhanced blight and blast resistance 1) that shows enhanced broad-spectrum bacterial and fungal disease resistance, but displays spontaneous PCD, autoimmunity, and stunted growth. EBR1 encodes an E3 ubiquitin ligase that interacts with OsBAG4, which belongs to the BAG (Bcl-2-associated athanogene) family that functions in cell death, growth arrest, and immune responses in mammals. EBR1 directly targets OsBAG4 for ubiquitination-mediated degradation. Elevated levels of OsBAG4 in rice are necessary and sufficient to trigger PCD and enhanced disease resistance to pathogenic infection, most likely by activating pathogen-associated molecular patterns-triggered immunity (PTI). Together, our study suggests that an E3-BAG module orchestrates innate immune homeostasis and coordinates the trade-off between defense and growth in plants. Copyright © 2016 Elsevier Inc. All rights reserved.

PINK1 is degraded through the N-end rule pathway

PubMed Central

Yamano, Koji; Youle, Richard J

2013-01-01

PINK1, a mitochondrial serine/threonine kinase, is the product of a gene mutated in an autosomal recessive form of Parkinson disease. PINK1 is constitutively degraded by an unknown mechanism and stabilized selectively on damaged mitochondria where it can recruit the E3 ligase PARK2/PARKIN to induce mitophagy. Here, we show that, under steady-state conditions, endogenous PINK1 is constitutively and rapidly degraded by E3 ubiquitin ligases UBR1, UBR2 and UBR4 through the N-end rule pathway. Following precursor import into mitochondria, PINK1 is cleaved in the transmembrane segment by a mitochondrial intramembrane protease PARL generating an N-terminal destabilizing amino acid and then retrotranslocates from mitochondria to the cytosol for N-end recognition and proteasomal degradation. Thus, sequential actions of mitochondrial import, PARL-processing, retrotranslocation and recognition by N-end rule E3 enzymes for the ubiquitin proteosomal degradation defines the rapid PINK1 turnover. PINK1 steady-state elimination by the N-end rule identifies a novel organelle to cytoplasm turnover pathway that yields a mechanism to flag damaged mitochondria for autophagic elimination. PMID:24121706

Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress.

PubMed

Han, Ziying; Sagum, Cari A; Takizawa, Fumio; Ruthel, Gordon; Berry, Corbett T; Kong, Jing; Sunyer, J Oriol; Freedman, Bruce D; Bedford, Mark T; Sidhu, Sachdev S; Sudol, Marius; Harty, Ronald N

2017-10-15

Ebola virus (EBOV) is a member of the Filoviridae family and the cause of hemorrhagic fever outbreaks. The EBOV VP40 (eVP40) matrix protein is the main driving force for virion assembly and budding. Indeed, expression of eVP40 alone in mammalian cells results in the formation and budding of virus-like particles (VLPs) which mimic the budding process and morphology of authentic, infectious EBOV. To complete the budding process, eVP40 utilizes its PPXY L-domain motif to recruit a specific subset of host proteins containing one or more modular WW domains that then function to facilitate efficient production and release of eVP40 VLPs. In this report, we identified additional host WW-domain interactors by screening for potential interactions between mammalian proteins possessing one or more WW domains and WT or PPXY mutant peptides of eVP40. We identified the HECT family E3 ubiquitin ligase WWP1 and all four of its WW domains as strong interactors with the PPXY motif of eVP40. The eVP40-WWP1 interaction was confirmed by both peptide pulldown and coimmunoprecipitation assays, which also demonstrated that modular WW domain 1 of WWP1 was most critical for binding to eVP40. Importantly, the eVP40-WWP1 interaction was found to be biologically relevant for VLP budding since (i) small interfering RNA (siRNA) knockdown of endogenous WWP1 resulted in inhibition of eVP40 VLP egress, (ii) coexpression of WWP1 and eVP40 resulted in ubiquitination of eVP40 and a subsequent increase in eVP40 VLP egress, and (iii) an enzymatically inactive mutant of WWP1 (C890A) did not ubiquitinate eVP40 or enhance eVP40 VLP egress. Last, our data show that ubiquitination of eVP40 by WWP1 enhances egress of VLPs and concomitantly decreases cellular levels of higher-molecular-weight oligomers of eVP40. In sum, these findings contribute to our fundamental understanding of the functional interplay between host E3 ligases, ubiquitination, and regulation of EBOV VP40-mediated egress. IMPORTANCE Ebola virus (EBOV) is a high-priority, emerging human pathogen that can cause severe outbreaks of hemorrhagic fever with high mortality rates. As there are currently no approved vaccines or treatments for EBOV, a better understanding of the biology and functions of EBOV-host interactions that promote or inhibit viral budding is warranted. Here, we describe a physical and functional interaction between EBOV VP40 (eVP40) and WWP1, a host E3 ubiquitin ligase that ubiquitinates VP40 and regulates VLP egress. This viral PPXY-host WW domain-mediated interaction represents a potential new target for host-oriented inhibitors of EBOV egress. Copyright © 2017 American Society for Microbiology.

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

PubMed Central

Unciuleac, Mihaela-Carmen; Shuman, Stewart

2015-01-01

The proteome of the amoebo-flagellate protozoan Naegleria gruberi is rich in candidate RNA repair enzymes, including 15 putative RNA ligases, one of which, NgrRnl, is a eukaryal homolog of Deinococcus radiodurans RNA ligase, DraRnl. Here we report that purified recombinant NgrRnl seals nicked 3′-OH/5′-PO4 duplexes in which the 3′-OH strand is RNA. It does so via the “classic” ligase pathway, entailing reaction with ATP to form a covalent NgrRnl–AMP intermediate, transfer of AMP to the nick 5′-PO4, and attack of the RNA 3′-OH on the adenylylated nick to form a 3′–5′ phosphodiester. Unlike members of the four known families of ATP-dependent RNA ligases, NgrRnl lacks a carboxy-terminal appendage to its nucleotidyltransferase domain. Instead, it contains a defining amino-terminal domain that we show is important for 3′-OH/5′-PO4 nick-sealing and ligase adenylylation, but dispensable for phosphodiester synthesis at a preadenylylated nick. We propose that NgrRnl, DraRnl, and their homologs from diverse bacteria, viruses, and unicellular eukarya comprise a new “Rnl5 family” of nick-sealing ligases with a signature domain organization. PMID:25740837

E3Net: a system for exploring E3-mediated regulatory networks of cellular functions.

PubMed

Han, Youngwoong; Lee, Hodong; Park, Jong C; Yi, Gwan-Su

2012-04-01

Ubiquitin-protein ligase (E3) is a key enzyme targeting specific substrates in diverse cellular processes for ubiquitination and degradation. The existing findings of substrate specificity of E3 are, however, scattered over a number of resources, making it difficult to study them together with an integrative view. Here we present E3Net, a web-based system that provides a comprehensive collection of available E3-substrate specificities and a systematic framework for the analysis of E3-mediated regulatory networks of diverse cellular functions. Currently, E3Net contains 2201 E3s and 4896 substrates in 427 organisms and 1671 E3-substrate specific relations between 493 E3s and 1277 substrates in 42 organisms, extracted mainly from MEDLINE abstracts and UniProt comments with an automatic text mining method and additional manual inspection and partly from high throughput experiment data and public ubiquitination databases. The significant functions and pathways of the extracted E3-specific substrate groups were identified from a functional enrichment analysis with 12 functional category resources for molecular functions, protein families, protein complexes, pathways, cellular processes, cellular localization, and diseases. E3Net includes interactive analysis and navigation tools that make it possible to build an integrative view of E3-substrate networks and their correlated functions with graphical illustrations and summarized descriptions. As a result, E3Net provides a comprehensive resource of E3s, substrates, and their functional implications summarized from the regulatory network structures of E3-specific substrate groups and their correlated functions. This resource will facilitate further in-depth investigation of ubiquitination-dependent regulatory mechanisms. E3Net is freely available online at http://pnet.kaist.ac.kr/e3net.

Arabidopsis RING E3 ubiquitin ligase AtATL80 is negatively involved in phosphate mobilization and cold stress response in sufficient phosphate growth conditions.

PubMed

Suh, Ji Yeon; Kim, Woo Taek

2015-08-07

Phosphate (Pi) remobilization in plants is critical to continuous growth and development. AtATL80 is a plasma membrane (PM)-localized RING E3 ubiquitin (Ub) ligase that belongs to the Arabidopsis Tóxicos en Levadura (ATL) family. AtATL80 was upregulated by long-term low Pi (0-0.02 mM KH2PO4) conditions in Arabidopsis seedlings. AtATL80-overexpressing transgenic Arabidopsis plants (35S:AtATL80-sGFP) displayed increased phosphorus (P) accumulation in the shoots and lower biomass, as well as reduced P-utilization efficiency (PUE) under high Pi (1 mM KH2PO4) conditions compared to wild-type plants. The loss-of-function atatl80 mutant line exhibited opposite phenotypic traits. The atatl80 mutant line bolted earlier than wild-type plants, whereas AtATL80-overexpressors bloomed significantly later and produced lower seed yields than wild-type plants under high Pi conditions. Thus, AtATL80 is negatively correlated not only with P content and PUE, but also with biomass and seed yield in Arabidopsis. In addition, AtATL80-overexpressors were significantly more sensitive to cold stress than wild-type plants, while the atatl80 mutant line exhibited an increased tolerance to cold stress. Taken together, our results suggest that AtATL80, a PM-localized ATL-type RING E3 Ub ligase, participates in the Pi mobilization and cold stress response as a negative factor in Arabidopsis. Copyright © 2015 Elsevier Inc. All rights reserved.

The Ubiquitin E3 Ligase PRU1 Regulates WRKY6 Degradation to Modulate Phosphate Homeostasis in Response to Low-Pi Stress in Arabidopsis.

PubMed

Ye, Qing; Wang, Hui; Su, Tong; Wu, Wei-Hua; Chen, Yi-Fang

2018-03-22

Since phosphorus is an essential nutrient for plants, plants have evolved a number of adaptive mechanisms to respond to changes in phosphate (Pi) supply. Previously, we reported that the transcription factor WRKY6 modulates Pi homeostasis by down-regulating PHOSPHATE 1 (PHO1) expression, and that WRKY6 is degraded during Pi starvation in Arabidopsis thaliana. However, the molecular mechanism underlying low-Pi-induced WRKY6 degradation was unknown. Here, we report that a ubiquitin E3 ligase, PHOSPHATE RESPONSE UBIQUITIN E3 LIGASE 1 (PRU1), modulates WRKY6 protein levels in response to low-Pi stress. A pru1 mutant was more sensitive than the wild type to Pi-deficient conditions, exhibiting a reduced Pi contents in the shoot, similar to the pho1-2 mutant and WRKY6-overexpressing line. PRU1 interacted with WRKY6 in vitro and in vivo. Under low-Pi stress, the ubiquitination and subsequent degradation of WRKY6, as well as the consequential enhancement of PHO1 expression, were impaired in pru1. PRU1 complementation lines displayed no obvious differences compared to wild-type plants. Further genetic analysis showed that disruption of WRKY6 abolished the low-Pi sensitivity of pru1, indicating that WRKY6 functioned downstream of PRU1. Taken together, this study uncovers a mechanism by which PRU1 modulates Pi homeostasis, through regulating the abundance of WRKY6 in response to low-Pi stress in Arabidopsis. © 2018 American Society of Plant Biologists. All rights reserved.

The Ubiquitin Receptor DA1 Interacts with the E3 Ubiquitin Ligase DA2 to Regulate Seed and Organ Size in Arabidopsis[C][W

PubMed Central

Xia, Tian; Li, Na; Dumenil, Jack; Li, Jie; Kamenski, Andrei; Bevan, Michael W.; Gao, Fan; Li, Yunhai

2013-01-01

Seed size in higher plants is determined by the coordinated growth of the embryo, endosperm, and maternal tissue. Several factors that act maternally to regulate seed size have been identified, such as AUXIN RESPONSE FACTOR2, APETALA2, KLUH, and DA1, but the genetic and molecular mechanisms of these factors in seed size control are almost totally unknown. We previously demonstrated that the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligase ENHANCER1 OF DA1 (EOD1)/BIG BROTHER to regulate the final size of seeds in Arabidopsis thaliana. Here, we describe another RING-type protein with E3 ubiquitin ligase activity, encoded by DA2, which regulates seed size by restricting cell proliferation in the maternal integuments of developing seeds. The da2-1 mutant forms large seeds, while overexpression of DA2 decreases seed size of wild-type plants. Overexpression of rice (Oryza sativa) GRAIN WIDTH AND WEIGHT2, a homolog of DA2, restricts seed growth in Arabidopsis. Genetic analyses show that DA2 functions synergistically with DA1 to regulate seed size, but does so independently of EOD1. Further results reveal that DA2 interacts physically with DA1 in vitro and in vivo. Therefore, our findings define the genetic and molecular mechanisms of three ubiquitin-related proteins DA1, DA2, and EOD1 in seed size control and indicate that they are promising targets for crop improvement. PMID:24045020

The RING Finger E3 Ligase SpRing is a Positive Regulator of Salt Stress Signaling in Salt-Tolerant Wild Tomato Species.

PubMed

Qi, Shilian; Lin, Qingfang; Zhu, Huishan; Gao, Fenghua; Zhang, Wenhao; Hua, Xuejun

2016-03-01

Protein ubiquitination in plants plays critical roles in many biological processes, including adaptation to abiotic stresses. Previously, RING finger E3 ligase has been characterized during salt stress response in several plant species, but little is known about its function in tomato. Here, we report that SpRing, a stress-inducible gene, is involved in salt stress signaling in wild tomato species Solanum pimpinellifolium 'PI365967'. In vitro ubiquitination assay revealed that SpRing is an E3 ubiquitin ligase and the RING finger conserved region is required for its activity. SpRing is expressed in all tissues of wild tomato and up-regulated by salt, drought and osmotic stresses, but repressed by low temperature. Green fluorescent protein (GFP) fusion analysis showed that SpRing is localized at the endoplasmic reticulum. Silencing of SpRing through a virus-induced gene silencing approach led to increased sensitivity to salt stress in wild tomato. Overexpression of SpRing in Arabidopsis thaliana resulted in enhanced salt tolerance during seed germination and early seedling development. The expression levels of certain key stress-related genes are altered both in SpRing-overexpressing Arabidopsis plants and virus-induced gene silenced tomato seedlings. Taken together, our results indicate that SpRing is involved in salt stress and functions as a positive regulator of salt tolerance. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: an antibiotic target.

PubMed

Pendini, Nicole R; Yap, Min Y; Traore, D A K; Polyak, Steven W; Cowieson, Nathan P; Abell, Andrew; Booker, Grant W; Wallace, John C; Wilce, Jacqueline A; Wilce, Matthew C J

2013-06-01

The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present small-angle X-ray scattering data of SaBPL in complex with its biotin-carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl-5'-AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery. © 2013 The Protein Society.

Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: An antibiotic target

PubMed Central

Pendini, Nicole R; Yap, Min Y; Polyak, Steven W; Cowieson, Nathan P; Abell, Andrew; Booker, Grant W; Wallace, John C; Wilce, Jacqueline A; Wilce, Matthew C J

2013-01-01

The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present small-angle X-ray scattering data of SaBPL in complex with its biotin-carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl-5′-AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery. PMID:23559560

An SH2 domain-based tyrosine kinase assay using biotin ligase modified with a terbium(III) complex.

PubMed

Sueda, Shinji; Shinboku, Yuki; Kusaba, Takeshi

2013-01-01

Src homology 2 (SH2) domains are modules of approximately 100 amino acids and are known to bind phosphotyrosine-containing sequences with high affinity and specificity. In the present work, we developed an SH2 domain-based assay for Src tyrosine kinase using a unique biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Here, an SH2 domain from lymphocyte-specific tyrosine kinase was genetically fused to a truncated BCCP, and the resulting fusion protein was labeled through biotinylation with BPL carrying multiple copies of a luminescent Tb(3+) complex. The labeled SH2 fusion proteins were employed to detect a phosphorylated peptide immobilized on the surface of the microtiter plate, where the phosphorylated peptide was produced by phosphorylation to the substrate peptide by Src tyrosine kinase. Our assay allows for a reliable determination of the activity of Src kinase lower than 10 pg/μL by a simple procedure.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hodge, Curtis D.; Ismail, Ismail H.; Edwards, Ross A.

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. In this paper, we defined the activated RNF8-Ubc13~ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13more » active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. Finally, these findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.« less

In vitro assembly of semi-artificial molecular machine and its use for detection of DNA damage.

PubMed

Minchew, Candace L; Didenko, Vladimir V

2012-01-11

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc). This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage. Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks), fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO(4) blunt-ended break (DNase I-type breaks), if T4 DNA ligase is present in the solution. When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO(4) blunt-ended breaks, the ligase reacts with 5'PO(4) DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO(4) blunt-ended DNA breaks. This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues. Copyright © 2012 Journal of Visualized Experiments

Methylated DNMT1 and E2F1 are targeted for proteolysis by L3MBTL3 and CRL4DCAF5 ubiquitin ligase.

PubMed

Leng, Feng; Yu, Jiekai; Zhang, Chunxiao; Alejo, Salvador; Hoang, Nam; Sun, Hong; Lu, Fei; Zhang, Hui

2018-04-24

Many non-histone proteins are lysine methylated and a novel function of this modification is to trigger the proteolysis of methylated proteins. Here, we report that the methylated lysine 142 of DNMT1, a major DNA methyltransferase that preserves epigenetic inheritance of DNA methylation patterns during DNA replication, is demethylated by LSD1. A novel methyl-binding protein, L3MBTL3, binds the K142-methylated DNMT1 and recruits a novel CRL4 DCAF5 ubiquitin ligase to degrade DNMT1. Both LSD1 and PHF20L1 act primarily in S phase to prevent DNMT1 degradation by L3MBTL3-CRL4 DCAF5 . Mouse L3MBTL3/MBT-1 deletion causes accumulation of DNMT1 protein, increased genomic DNA methylation, and late embryonic lethality. DNMT1 contains a consensus methylation motif shared by many non-histone proteins including E2F1, a key transcription factor for S phase. We show that the methylation-dependent E2F1 degradation is also controlled by L3MBTL3-CRL4 DCAF5 . Our studies elucidate for the first time a novel mechanism by which the stability of many methylated non-histone proteins are regulated.

Autophagy degrades hypoxia inducible factors

PubMed Central

DePavia, Adela; Jonasch, Eric; Liu, Xian-De

2016-01-01

ABSTRACT Hypoxia inducible factors are subjected to degradation by the ubiquitin-proteasome system (UPS), macroautophagy, and chaperone-mediated autophagy. The E3 ligases, ubiquitination, autophagy receptor proteins, and oxygen are determinants that direct hypoxia-inducible factors to different degradation pathways. PMID:27308629

Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

PubMed Central

Kazlauskaite, Agne; Kondapalli, Chandana; Gourlay, Robert; Campbell, David G.; Ritorto, Maria Stella; Hofmann, Kay; Alessi, Dario R.; Knebel, Axel; Trost, Matthias; Muqit, Miratul M. K.

2014-01-01

We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser65 within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser65 that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser65 in ubiquitin lies in a similar motif to Ser65 in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser65 of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser65-phosphorylated ubiquitin (ubiquitinPhospho−Ser65) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser65 (ΔUbl-Parkin) is robustly activated by ubiquitinPhospho−Ser65, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser65 (UblPhospho−Ser65) can also activate ΔUbl-Parkin similarly to ubiquitinPhospho−Ser65. Thirdly, we establish that ubiquitinPhospho−Ser65, but not non-phosphorylated ubiquitin or UblPhospho−Ser65, activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser65 and ubiquitin at Ser65, since only mutation of both proteins at Ser65 completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser65, but also by phosphorylating ubiquitin at Ser65. We propose that phosphorylation of Parkin at Ser65 serves to prime the E3 ligase enzyme for activation by ubiquitinPhospho−Ser65, suggesting that small molecules that mimic ubiquitinPhospho−Ser65 could hold promise as novel therapies for Parkinson's disease. PMID:24660806

Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1.

PubMed

Harrison, Joseph S; Cornett, Evan M; Goldfarb, Dennis; DaRosa, Paul A; Li, Zimeng M; Yan, Feng; Dickson, Bradley M; Guo, Angela H; Cantu, Daniel V; Kaustov, Lilia; Brown, Peter J; Arrowsmith, Cheryl H; Erie, Dorothy A; Major, Michael B; Klevit, Rachel E; Krajewski, Krzysztof; Kuhlman, Brian; Strahl, Brian D; Rothbart, Scott B

2016-09-06

The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance.

Aging Triggers Cytoplasmic Depletion and Nuclear Translocation of the E3 Ligase Mahogunin: A Function for Ubiquitin in Neuronal Survival.

PubMed

Benvegnù, Stefano; Mateo, María Inés; Palomer, Ernest; Jurado-Arjona, Jerónimo; Dotti, Carlos G

2017-05-04

A decline in proteasome function is causally connected to neuronal aging and aging-associated neuropathologies. By using hippocampal neurons in culture and in vivo, we show that aging triggers a reduction and a cytoplasm-to-nucleus redistribution of the E3 ubiquitin ligase mahogunin (MGRN1). Proteasome impairment induces MGRN1 monoubiquitination, the key post-translational modification for its nuclear entry. One potential mechanism for MGRN1 monoubiquitination is via progressive deubiquitination at the proteasome of polyubiquitinated MGRN1. Once in the nucleus, MGRN1 potentiates the transcriptional cellular response to proteotoxic stress. Inhibition of MGRN1 impairs ATF3-mediated neuronal responsiveness to proteosomal stress and increases neuronal stress, while increasing MGRN1 ameliorates signs of neuronal aging, including cognitive performance in old animals. Our results imply that, among others, the strength of neuronal survival in a proteasomal deterioration background, like during aging, depends on the fine-tuning of ubiquitination-deubiquitination. Copyright © 2017 Elsevier Inc. All rights reserved.

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

PubMed Central

Lohman, Gregory J. S.; Zhang, Yinhua; Zhelkovsky, Alexander M.; Cantor, Eric J.; Evans, Thomas C.

2014-01-01

Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA. PMID:24203707

Structure of nascent replicative form DNA of coliphage M13

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dasgupta, S.; Mitra, S.

Nascent replicative form type II (RFII) DNA of coliphage M13 synthesized in an Escherichia coli mutant deficient in the 5' ..-->.. 3' exonuclease associated with DNA polymerase I contains ribonucleotides that are retained in the covalently closed RFI DNA sealed in vitro by the joint action of T5 phage DNA polymerase and T4 phage DNA ligase. These RFI molecules are labile to alkali and RNase H, unlike the RFI produced either in vivo or from RFII with E. coli DNA polymerase I and E. coli DNA ligase. The ribonucleotides are located at one site and predominantly in one strand ofmore » the nascent RF DNA. Furthermore, these molecules contain multiple small gaps, randomly located, and one large gap in the intracistronic region.« less

A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation*

PubMed Central

Zattas, Dimitrios; Berk, Jason M.; Kreft, Stefan G.; Hochstrasser, Mark

2016-01-01

Specific proteins are modified by ubiquitin at the endoplasmic reticulum (ER) and are degraded by the proteasome, a process referred to as ER-associated protein degradation. In Saccharomyces cerevisiae, two principal ER-associated protein degradation ubiquitin ligases (E3s) reside in the ER membrane, Doa10 and Hrd1. The membrane-embedded Doa10 functions in the degradation of substrates in the ER membrane, nuclear envelope, cytoplasm, and nucleoplasm. How most E3 ligases, including Doa10, recognize their protein substrates remains poorly understood. Here we describe a previously unappreciated but highly conserved C-terminal element (CTE) in Doa10; this cytosolically disposed 16-residue motif follows the final transmembrane helix. A conserved CTE asparagine residue is required for ubiquitylation and degradation of a subset of Doa10 substrates. Such selectivity suggests that the Doa10 CTE is involved in substrate discrimination and not general ligase function. Functional conservation of the CTE was investigated in the human ortholog of Doa10, MARCH6 (TEB4), by analyzing MARCH6 autoregulation of its own degradation. Mutation of the conserved Asn residue (N890A) in the MARCH6 CTE stabilized the normally short lived enzyme to the same degree as a catalytically inactivating mutation (C9A). We also report the localization of endogenous MARCH6 to the ER using epitope tagging of the genomic MARCH6 locus by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing. These localization and CTE analyses support the inference that MARCH6 and Doa10 are functionally similar. Moreover, our results with the yeast enzyme suggest that the CTE is involved in the recognition and/or ubiquitylation of specific protein substrates. PMID:27068744