ABSTRACT
Genetic aberrations of the maternal UBE3A allele, which encodes the E3 ubiquitin ligase E6AP, are the cause of Angelman syndrome (AS), an imprinting disorder. In most cases, the maternal UBE3A allele is not expressed. Yet, approximately 10 percent of AS individuals harbor distinct point mutations in the maternal allele resulting in the expression of full-length E6AP variants that frequently display compromised ligase activity. In a high-throughput screen, we identified cyanocobalamin, a vitamin B12-derivative, and several alloxazine derivatives as activators of the AS-linked E6AP-F583S variant. Furthermore, we show by cross-linking coupled to mass spectrometry that cobalamins affect the structural dynamics of E6AP-F583S and apply limited proteolysis coupled to mass spectrometry to obtain information about the regions of E6AP that are involved in, or are affected by binding cobalamins and alloxazine derivatives. Our data suggest that dietary supplementation with vitamin B12 can be beneficial for AS individuals.
Subject(s)
Angelman Syndrome , Ubiquitin-Protein Ligases , Vitamin B 12 , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Angelman Syndrome/genetics , Angelman Syndrome/drug therapy , Angelman Syndrome/metabolism , Humans , Allosteric Regulation/drug effects , Vitamin B 12/metabolism , Vitamin B 12/chemistry , Vitamin B 12/pharmacologyABSTRACT
Ralstonia solanacearum causes bacterial wilt disease in a broad range of plants, primarily through type â ¢ secreted effectors. However, the R. solanacearum effectors promoting susceptibility in host plants remain limited. In this study, we determined that the R. solanacearum effector RipV2 functions as a novel E3 ubiquitin ligase (NEL). RipV2 was observed to be locali in the plasma membrane after translocatio into plant cells. Transient expression of RipV2 in Nicotiana benthamiana could induce cell death and suppress the flg22-induced pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses, mediating such effects as attenuation of the expression of several PTI-related genes and ROS bursts. Furthermore, we demonstrated that the conserved catalytic residue is highly important for RipV2. Transient expression of the E3 ubiquitin ligase catalytic mutant RipV2 C403A alleviated the PTI suppression ability and cell death induction, indicating that RipV2 requires its E3 ubiquitin ligase activity for its role in plant-microbe interactions. More importantly, mutation of RipV2 in R. solanacearum reduces the virulence of R. solanacearum on potato. In conclusion, we identified a NEL effector that is required for full virulence of R. solanacearum by suppressing plant PTI.
Subject(s)
Pathogen-Associated Molecular Pattern Molecules/antagonists & inhibitors , Plant Immunity , Ralstonia solanacearum/enzymology , Solanum tuberosum/immunology , Solanum tuberosum/microbiology , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Virulence , Amino Acid Motifs , Biocatalysis , Cell Death , Cell Membrane/enzymology , Cysteine/metabolism , Flagellin/chemistry , Flagellin/immunology , Pathogen-Associated Molecular Pattern Molecules/immunology , Peptide Fragments/chemistry , Peptide Fragments/immunology , Ralstonia solanacearum/genetics , Ubiquitin-Protein Ligases/chemistry , Virulence/geneticsABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: The dried root of Paeonia lactiflora Pall. (Radix Paeoniae) has been traditionally used to treat various inflammatory diseases in many Asian countries. AIM OF THE STUDY: Cancer cachexia is a catabolic syndrome driven by inflammation and characterised by a loss of skeletal muscle. This study aimed to assess the effects of an ethanolic extract of Radix Paeoniae (RP) on cancer cachexia and elucidate its mechanism of action. MATERIAL AND METHODS: The anti-cachexic effect and mechanism of RP were examined in mouse models of cancer cachexia established in C57BL/6 mice by subcutaneously injecting Lewis lung carcinoma or MC38 colon carcinoma cells. Skeletal muscle tissues were analysed by RNAseq, real-time quantitative reverse transcription PCR, western blotting, and immunofluorescence microscopy. Megestrol acetate, which is recommended for the treatment of cachexia in cancer patients, was used as the comparator treatment in this study. RESULTS: In lung and colon cancer-bearing mice, RP significantly restored food intake and muscle mass, along with muscle function measured by grip strength and treadmill running time. In the skeletal muscle tissue of the cancer-bearing mice, RP suppressed NF-κB signalling and reduced inflammatory cytokines, including TNF-α, IL-6, and IL-1ß; it also down-regulated the muscle-specific E3 ubiquitin ligases MuRF1 and MAFbx. CONCLUSION: RP restored skeletal muscle function and mass in cancer-bearing mice by down-regulating the muscular NF-κB signalling pathway and muscle-specific E3 ubiquitin ligases. Our study indicates that RP is a potential candidate for development as a therapeutic agent against cancer cachexia.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Cachexia/drug therapy , Neoplasms, Experimental/metabolism , Paeonia/chemistry , Plant Extracts/pharmacology , Plant Roots/chemistry , Ubiquitin-Protein Ligases/metabolism , Animals , Antineoplastic Agents, Phytogenic/chemistry , Gene Expression Regulation/drug effects , Mice , Muscle, Skeletal/enzymology , NF-kappa B , Phytotherapy , Plant Extracts/chemistry , Signal Transduction , Ubiquitin-Protein Ligases/chemistryABSTRACT
Modification of proteins with polyubiquitin chains is a key regulatory mechanism to control cellular behavior and alterations in the ubiquitin system are linked to many diseases. Linear (M1-linked) polyubiquitin chains play pivotal roles in several cellular signaling pathways mediating immune and inflammatory responses and apoptotic cell death. These chains are formed by the linear ubiquitin chain assembly complex (LUBAC), a multiprotein E3 ligase that consists of 3 subunits, HOIP, HOIL-1L, and SHARPIN. Herein, we describe the discovery of inhibitors targeting the active site cysteine of the catalytic subunit HOIP using fragment-based covalent ligand screening. We report the synthesis of a diverse library of electrophilic fragments and demonstrate an integrated use of protein LC-MS, biochemical ubiquitination assays, chemical synthesis, and protein crystallography to enable the first structure-based development of covalent inhibitors for an RBR E3 ligase. Furthermore, using cell-based assays and chemoproteomics, we demonstrate that these compounds effectively penetrate mammalian cells to label and inhibit HOIP and NF-κB activation, making them suitable hits for the development of selective probes to study LUBAC biology. Our results illustrate the power of fragment-based covalent ligand screening to discover lead compounds for challenging targets, which holds promise to be a general approach for the development of cell-permeable inhibitors of thioester-forming E3 ubiquitin ligases.
Subject(s)
Enzyme Inhibitors/pharmacology , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/metabolism , Amino Acid Sequence , Drug Evaluation, Preclinical , Enzyme Inhibitors/metabolism , HEK293 Cells , Humans , Ligands , MCF-7 Cells , Models, Molecular , Protein Structure, Secondary , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Ubiquitin-Protein Ligases/chemistryABSTRACT
The ubiquitin/proteasome system is a primary conduit for selective intracellular protein degradation. Since its discovery over 30 years ago, this highly regulated system continues to be an active research area for drug discovery that is exemplified by several approved drugs. Here we review compounds in preclinical testing, clinical trials, and approved drugs, with the aim of highlighting innovative discoveries and breakthrough therapies that target the ubiquitin system.
Subject(s)
Enzyme Inhibitors/chemistry , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolism , Drug Evaluation, Preclinical , Enzyme Inhibitors/metabolism , Humans , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/metabolism , Ubiquitin/chemistry , Ubiquitin-Activating Enzymes/chemistry , Ubiquitin-Activating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
An expanded chemical space is essential for improved identification of small molecules for emerging therapeutic targets. However, the identification of targets for novel compounds is biased towards the synthesis of known scaffolds that bind familiar protein families, limiting the exploration of chemical space. To change this paradigm, we validated a new pipeline that identifies small molecule-protein interactions and works even for compounds lacking similarity to known drugs. Based on differential mRNA profiles in multiple cell types exposed to drugs and in which gene knockdowns (KD) were conducted, we showed that drugs induce gene regulatory networks that correlate with those produced after silencing protein-coding genes. Next, we applied supervised machine learning to exploit drug-KD signature correlations and enriched our predictions using an orthogonal structure-based screen. As a proof-of-principle for this regimen, top-10/top-100 target prediction accuracies of 26% and 41%, respectively, were achieved on a validation of set 152 FDA-approved drugs and 3104 potential targets. We then predicted targets for 1680 compounds and validated chemical interactors with four targets that have proven difficult to chemically modulate, including non-covalent inhibitors of HRAS and KRAS. Importantly, drug-target interactions manifest as gene expression correlations between drug treatment and both target gene KD and KD of genes that act up- or down-stream of the target, even for relatively weak binders. These correlations provide new insights on the cellular response of disrupting protein interactions and highlight the complex genetic phenotypes of drug treatment. With further refinement, our pipeline may accelerate the identification and development of novel chemical classes by screening compound-target interactions.
Subject(s)
Drug Discovery/methods , Gene Expression Profiling/methods , Proteins/chemistry , Proteins/drug effects , Cell Line , Computational Biology , Computer Simulation , Databases, Nucleic Acid/statistics & numerical data , Drug Discovery/statistics & numerical data , Drug Evaluation, Preclinical/methods , Drug Evaluation, Preclinical/statistics & numerical data , Gene Expression Profiling/statistics & numerical data , Gene Knockdown Techniques , Gene Ontology , Gene Regulatory Networks/drug effects , Humans , Models, Molecular , Molecular Docking Simulation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Proteins/genetics , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Wortmannin/chemistry , Wortmannin/pharmacology , ras Proteins/antagonists & inhibitors , ras Proteins/chemistry , ras Proteins/geneticsABSTRACT
Beyond the targeting of E3 ubiquitin ligases to inhibit protein homeostasis, E3 ligase binders can be repurposed as targeted protein degraders (PROTACs or molecular glues). We sought to identify new binders of the VHL E3 ligase by biophysical fragment-based screening followed by X-ray crystallographic soaking. We identified fragments binding at the ElonginC:Cullin2 interface and a new cryptic pocket in VHL, along with other potential ligandable sites predicted computationally and found to bind solvent molecules in crystal structures. The elucidated interactions provide starting points for future ligand development.
Subject(s)
Drug Evaluation, Preclinical/methods , Multiprotein Complexes/chemistry , Ubiquitin-Protein Ligases/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Binding Sites , Crystallography, X-Ray , Elongin/chemistry , Elongin/metabolism , Fluorometry/methods , Humans , Ligands , Magnetic Resonance Spectroscopy , Multiprotein Complexes/metabolism , Polycythemia/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Ubiquitin-Protein Ligases/chemistry , Von Hippel-Lindau Tumor Suppressor Protein/chemistryABSTRACT
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.
Subject(s)
Genes, Plant , Salt Tolerance/drug effects , Signal Transduction/drug effects , Sodium Chloride/pharmacology , Solanum/physiology , Stress, Physiological/drug effects , Ubiquitin-Protein Ligases/metabolism , Arabidopsis/genetics , Gene Expression Regulation, Plant/drug effects , Gene Silencing/drug effects , Hydrogen Peroxide/metabolism , Lipid Peroxidation/drug effects , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Shoots/drug effects , Plant Shoots/metabolism , Plants, Genetically Modified , Protein Transport/drug effects , RING Finger Domains , Salt Tolerance/genetics , Seedlings/drug effects , Seedlings/growth & development , Sequence Analysis, DNA , Signal Transduction/genetics , Sodium/metabolism , Solanum/drug effects , Solanum/enzymology , Solanum/genetics , Stress, Physiological/genetics , Subcellular Fractions/metabolism , Ubiquitin-Protein Ligases/chemistryABSTRACT
We investigated the mechanisms of activation and degradation of the E3 ubiquitin ligase Nedd4L combining the available biochemical information with complementary biophysical techniques. Using nuclear magnetic resonance spectroscopy, we identified that the C2 domain binds Ca(2+) and inositol 1,4,5-trisphosphate (IP3) using the same interface that is used to interact with the HECT domain. Thus, we propose that the transition from the closed to the active form is regulated by a competition of IP3 and Ca(2+) with the HECT domain for binding to the C2 domain. We performed relaxation experiments and molecular dynamic simulations to determine the flexibility of the HECT structure and observed that its conserved PY motif can become solvent-exposed when the unfolding process is initiated. The structure of the WW3 domain bound to the HECT-PY site reveals the details of this interaction, suggesting a possible auto-ubquitination mechanism using two molecules, a partially unfolded one and a fully functional Nedd4L counterpart.
Subject(s)
Endosomal Sorting Complexes Required for Transport/chemistry , Endosomal Sorting Complexes Required for Transport/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Amino Acid Sequence , Binding, Competitive , Calcium/metabolism , Enzyme Activation , Humans , Inositol 1,4,5-Trisphosphate/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Nedd4 Ubiquitin Protein Ligases , Protein Conformation , Protein Structure, Tertiary , Signal Transduction , UbiquitinationABSTRACT
In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP251-73, a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundle (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains.
Subject(s)
Protein Structure, Tertiary , Tacrolimus Binding Proteins/chemistry , Ubiquitin-Protein Ligases/chemistry , Amino Acid Sequence , Humans , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Sequence Homology, Amino Acid , Tacrolimus Binding Proteins/metabolism , YY1 Transcription Factor/metabolismABSTRACT
Rheumatoid arthritis (RA) significantly affects quality of life. We recently cloned synoviolin, a RING-type E3 ubiquitin ligase implicated in the endoplasmic reticulum-associated degradation (ERAD) pathway. Synoviolin is highly expressed in rheumatoid synovial cells and may be involved in the pathogenesis of RA. Inhibition of synoviolin activity is a potentially useful therapeutic approach for the treatment of RA. We conducted a high-throughput screen of small molecules to find inhibitors of synoviolin autoubiquitination activity. We identified two classes of small molecules, named LS-101 and LS-102, which inhibited synoviolin activity. LS-102 selectively inhibited synoviolin enzymatic activity, while LS-101 inhibited a broad array of RING-type E3 ligases. Moreover, these inhibitors suppressed the proliferation of rheumatoid synovial cells, and significantly reduced the severity of disease in a mouse model of RA. Our results suggest that inhibition of synoviolin is a potentially useful approach in the treatment of RA.
Subject(s)
Antirheumatic Agents/pharmacology , Arthritis, Rheumatoid/drug therapy , Benzodiazepinones/pharmacology , Benzoxazoles/pharmacology , Enzyme Inhibitors/pharmacology , Triazines/pharmacology , Ubiquitin-Protein Ligases/antagonists & inhibitors , Animals , Antirheumatic Agents/chemistry , Arthritis, Rheumatoid/pathology , Benzodiazepinones/chemistry , Benzoxazoles/chemistry , Cell Proliferation/drug effects , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Forelimb/drug effects , Forelimb/pathology , HeLa Cells , High-Throughput Screening Assays , Hindlimb/drug effects , Hindlimb/pathology , Humans , Joints/drug effects , Joints/pathology , Male , Mice , Mice, Inbred DBA , Triazines/chemistry , Ubiquitin-Protein Ligases/chemistryABSTRACT
Ubiquitination of proteins is one of the critical regulatory mechanisms in eukaryotes. In higher plants, protein ubiquitination plays an essential role in many biological processes, including hormone signaling, photomorphogenesis, and pathogen defense. However, the roles of protein ubiquitination in the reproductive process are not clear. In this study, we identified four plant-specific RING-finger genes designated Aberrant Pollen Development 1 (APD1) to APD4, as regulators of pollen mitosis II (PMII) in Arabidopsis thaliana (L.). The apd1 apd2 double mutant showed a significantly increased percentage of bicellular-like pollen at the mature pollen stage. Further downregulation of the APD3 and APD4 transcripts in apd1 apd2 by RNA interference (RNAi) resulted in more severe abnormal bicellular-like pollen phenotypes than in apd1 apd2, suggesting that cell division was defective in male gametogenesis. All of the four genes were expressed in multiple stages at different levels during male gametophyte development. Confocal analysis using green florescence fusion proteins (GFP) GFP-APD1 and GFP-APD2 showed that APDs are associated with intracellular membranes. Furthermore, APD2 had E2-dependent E3 ligase activity in vitro, and five APD2-interacting proteins were identified. Our results suggest that these four genes may be involved, redundantly, in regulating the PMII process during male gametogenesis.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Arabidopsis/enzymology , Mitosis/physiology , Pollen/cytology , Pollen/enzymology , Ubiquitin-Protein Ligases/metabolism , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Molecular Sequence Data , Pollen/genetics , Sequence Homology, Amino Acid , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/geneticsABSTRACT
PDZ (Post-synaptic density, 95 kDa, Discs large, Zona Occludens-1) domains are protein interaction domains that bind to the carboxy-terminal amino acids of binding partners, heterodimerize with other PDZ domains, and also bind phosphoinositides. PDZ domain containing proteins are frequently involved in the assembly of multi-protein complexes and clustering of transmembrane proteins. LNX1 (Ligand of Numb, protein X 1) is a RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligase that also includes four PDZ domains suggesting it functions as a scaffold for a multi-protein complex. Here we use a human protein array to identify direct LNX1 PDZ domain binding partners. Screening of 8,000 human proteins with isolated PDZ domains identified 53 potential LNX1 binding partners. We combined this set with LNX1 interacting proteins identified by other methods to assemble a list of 220 LNX1 interacting proteins. Bioinformatic analysis of this protein list was used to select interactions of interest for future studies. Using this approach we identify and confirm six novel LNX1 binding partners: KCNA4, PAK6, PLEKHG5, PKC-alpha1, TYK2 and PBK, and suggest that LNX1 functions as a signalling scaffold.
Subject(s)
Ubiquitin-Protein Ligases/chemistry , Computational Biology , Drug Evaluation, Preclinical , Humans , PDZ Domains , Protein Binding , Protein Interaction Mapping , Signal Transduction , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/physiologyABSTRACT
Enzyme-linked immunosorbent assays (ELISAs) are a long established and widely used assay format for drug discovery and diagnostics. They offer many advantages over homogeneous assay formats, including high sensitivity and separation (wash) steps that remove detection-interfering compounds. Many high-throughput screening assays are now performed in miniaturized formats (1,536- and 3,456-well plates) for higher throughput and lower reagent consumption. With miniaturization, separation steps in assays such as ELISA can become difficult to implement. Here we report on the implementation of the Kalypsys, Inc. (San Diego, CA) 1,536-well plate washer to enable the successful miniaturization and full automation of an ELISA that monitors ubiquitin ligase activity. The 1,536-well plate ELISA was robust and used for the high-throughput screening of a large screening collection (>1 million compounds).
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Ubiquitin-Protein Ligases/chemistry , Automation , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Enzyme-Linked Immunosorbent Assay/instrumentation , Miniaturization , RoboticsABSTRACT
LIM Mineralization Protein-1 (LMP-1) has been cloned and shown to be osteoinductive. Our efforts to understand the mode of action of LMP-1 led to the determination that LMP-1 interacts with Smad Ubiquitin Regulatory Factor-1 (Smurf1). Smurf1 targets osteogenic Smads, Smad1/5, for ubiquitin-mediated proteasomal degradation. Smurf1 interaction with LMP-1 or Smads is based on the presence of unique WW-domain interacting motif in these target molecules. By performing site-directed mutagenesis and binding studies in vitro on purified recombinant proteins, we identified a specific motif within the osteogenic region of several LMP isoforms that is necessary for Smurf1 interaction. Similarly, we have identified that the WW2 domain of Smurf1 is necessary for target protein interaction. Here, we present a homology-based modeling of the Smurf1 WW2 domain and its interacting motif of LMP-1. We performed computational docking of the interacting domains in Smurf1 and LMPs to identify the key amino acid residues involved in their binding regions. In support of the computational predictions, we also present biochemical evidence supporting the hypothesis that the physical interaction of Smurf1 and osteoinductive forms of LMP may prevent Smurf1 from targeting osteogenic Smads by ubiquitin-mediated proteasomal degradation.
Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Protein Isoforms/metabolism , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Base Sequence , Binding Sites , Blotting, Western , Cloning, Molecular , Cytoskeletal Proteins , DNA Primers , DNA, Complementary , Electrophoresis, Polyacrylamide Gel , Genetic Vectors , Intracellular Signaling Peptides and Proteins/isolation & purification , LIM Domain Proteins , Ligands , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Sequence Homology, Amino Acid , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/isolation & purificationABSTRACT
The E3 ubiquitin ligase neuregulin receptor degrading protein 1 (Nrdp1) mediates the ligand-independent degradation of the epidermal growth factor receptor family member ErbB3/HER3. By regulating cellular levels of ErbB3, Nrdp1 influences ErbB3-mediated signaling, which is essential for normal vertebrate development. Nrdp1 belongs to the tripartite or RBCC (RING, B-box, coiled-coil) family of ubiquitin ligases in which the RING domain is responsible for ubiquitin ligation and a variable C-terminal region mediates substrate recognition. We report here the 1.95 A crystal structure of the C-terminal domain of Nrdp1 and show that this domain is sufficient to mediate ErbB3 binding. Furthermore, we have used site-directed mutagenesis to map regions of the Nrdp1 surface that are important for interacting with ErbB3 and mediating its degradation in transfected cells. The ErbB3-binding site localizes to a region of Nrdp1 that is conserved from invertebrates to vertebrates, in contrast to ErbB3, which is only found in vertebrates. This observation suggests that Nrdp1 uses a common binding site to recognize its targets in different species.
Subject(s)
Receptor Protein-Tyrosine Kinases/metabolism , Ubiquitin-Protein Ligases/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cloning, Molecular , Crystallization , DNA, Complementary , Humans , Molecular Sequence Data , Mutagenesis , Protein Binding , Sequence Homology, Amino Acid , Ubiquitin-Protein Ligases/chemistryABSTRACT
The interface of chemistry and biology offers many opportunities to explore different aspects of cell biology. The emerging field of chemical genetics is providing the chemical means to understand biological systems not easily accessible using classical genetic manipulations. In this article, we will discuss how natural product mode of action studies and novel bio-organic manipulation of intracellular protein levels are proving useful in the exploration of cell biology.
Subject(s)
Proteins/chemistry , Animals , Biotinylation , Combinatorial Chemistry Techniques , Drug Design , Drug Evaluation, Preclinical , Genomics , Green Fluorescent Proteins/metabolism , Humans , Ketones/chemistry , Models, Chemical , Molecular Probe Techniques , Nanotechnology , Oligopeptides/chemistry , Phosphorylation , Protein Binding , Receptors, Androgen/metabolism , Recombinant Fusion Proteins/chemistry , Serine/analogs & derivatives , Serine/chemistry , Sesquiterpenes/chemistry , Signal Transduction , Ubiquitin-Protein Ligases/chemistryABSTRACT
Exhibiting rapid polarized growth, the pollen tube delivers the male gametes into the ovule for fertilization in higher plants. To get an overall picture of gene expression during pollen germination and pollen tube growth, we profiled the transcription patterns of 1,536 pollen cDNAs from lily (Lilium longiflorum) by microarray. Among those that exhibited significant differential expression, a cDNA named lily ankyrin repeat-containing protein (LlANK) was thoroughly studied. The full-length LlANK cDNA sequence predicts a protein containing five tandem ankyrin repeats and a RING zinc-finger domain. The LlANK protein possesses ubiquitin ligase activity in vitro. RNA blots demonstrated that LlANK transcript is present in mature pollen and its level, interestingly contrary to most pollen mRNAs, up-regulated significantly during pollen germination and pollen tube growth. When fused with green fluorescent protein and transiently expressed in pollen, LlANK was found dominantly associated with membrane-enclosed organelles as well as the generative cell. Overexpression of LlANK, however, led to abnormal growth of the pollen tube. On the other hand, transient silencing of LlANK impaired pollen germination and tube growth. Taken together, these results showed that LlANK is a ubiquitin ligase associated with membrane-enclosed organelles and required for polarized pollen tube growth.
Subject(s)
Lilium/enzymology , Lilium/growth & development , Plant Proteins/physiology , Pollen/enzymology , Pollen/growth & development , Ubiquitin-Protein Ligases/physiology , Ankyrin Repeat , Gene Expression Profiling , Gene Silencing , Green Fluorescent Proteins/analysis , Intracellular Membranes/enzymology , Lilium/genetics , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Organelles/enzymology , Plant Proteins/chemistry , Plant Proteins/genetics , Pollen/cytology , Recombinant Fusion Proteins/analysis , Sequence Homology, Amino Acid , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Up-Regulation , Zinc FingersABSTRACT
Individual ubiquitin (Ub)-protein ligases (E3s) cooperate with specific Ub-conjugating enzymes (E2s) to modify cognate substrates with polyubiquitin chains. E3s belonging to the Really Interesting New Gene (RING) and Homologous to E6-Associated Protein (E6AP) C-Terminus (HECT) domain families utilize distinct molecular mechanisms. In particular, HECT E3s, but not RING E3s, form a thiol ester with Ub before transferring Ub to the substrate lysine. Here we report that different HECT domain E3s can employ distinct mechanisms of polyubiquitin chain synthesis. We show that E6AP builds up a K48-linked chain on its HECT cysteine residue, while KIAA10 builds up K48- and K29-linked chains as free entities. A small region near the N-terminus of the conserved HECT domain helps to bring about this functional distinction. Thus, a given HECT domain can specify both the linkage of a polyubiquitin chain and the mechanism of its assembly.
Subject(s)
Polyubiquitin/chemistry , Ubiquitin-Protein Ligases/chemistry , Binding Sites , Blotting, Western , Cloning, Molecular , Cysteine/chemistry , DNA, Complementary/metabolism , Escherichia coli/metabolism , Glutathione Transferase/metabolism , Humans , Lysine/chemistry , Multienzyme Complexes/chemistry , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Time Factors , Ubiquitin/chemistry , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Protein Ligase Complexes/chemistryABSTRACT
In the present study, the human TEB4 is identified as a novel ER (endoplasmic reticulum)-resident ubiquitin ligase. TEB4 has homologues in many species and has a number of remarkable properties. TEB4 contains a conserved RING (really interesting new gene) finger and 13 predicted transmembrane domains. The RING finger of TEB4 and its homologues is situated at the N-terminus and has the unconventional C4HC3 configuration. The N-terminus of TEB4 is located in the cytosol. We show that the isolated TEB4 RING domain catalyses ubiquitin ligation in vitro in a reaction that is ubiquitin Lys48-specific and involves UBC7 (ubiquitin-conjugating enzyme 7). These properties are reminiscent of E3 enzymes, which are involved in ER-associated protein degradation. TEB4 is an ER degradation substrate itself, promoting its own degradation in a RING finger- and proteasome-dependent manner.