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PubMed2005

Identification of ubiquitin ligase substrates by in vitro expression cloning.

The number of identified E3 ubiquitin ligases has dramatically increased in recent years. However, the substrates targeted for degradation by these particular ligases have not been easily identified. One reason for the inability of matching substrates and ligases is the finding that E3 recognition elements in substrates are often poorly defined. This minimizes the likelihood that bioinformatic approaches will lead to the identification of E3 substrates. For example, the multi-subunit complex the anaphase promoting complex (APC) is an E3 that recognizes destruction boxes (RXXLXXXXD/N/E) or KEN motifs within substrates (Glotzer et al., 1991; Pfleger and Kirschner, 2000). However, many proteins that contain either a potential destruction or a KEN motif are not recognized by the APC in vitro or in vivo, suggesting that there are other, less well-defined characteristics of substrates that contribute to their ability to serve as APC substrates (Ayad, Rankin, and Kirschner, unpublished observations). Aside from bioinformatic approaches of identifying APC substrates, several groups have also attempted to use affinity techniques to discover novel APC substrates. This has not been widely successful, because many APC substrates are not abundant. Also, as is the case with many ligase-substrate interactions, the affinity of substrates for the APC is likely to be very low. All these considerations have motivated a search for other techniques to assist in identifying substrates of this particular E3 ligase. Here, we describe the use of in vitro expression cloning to identify novel APC substrates.

Amino Acid Sequence↗
PubMed2005

Overview of approaches for screening for ubiquitin ligase inhibitors.

E3 ubiquitin ligases are a large family of proteins that, together with ubiquitin-activating enzyme E1 and ubiquitin-conjugating enzyme E2, catalyze the ubiquitination of a variety of protein substrates for targeted degradation by means of the 26S proteasome. Because the turnover of many proteins involves targeted ubiquitination and degradation, E3 ubiquitin ligases, therefore, regulate almost every aspect of eukaryotic cellular functions or biological processes. Accumulated evidence in the past few years has suggested that a subset of E3 ubiquitin ligases that regulates the turnover of tumor suppressors and cell cycle regulators could be promising targets for mechanism-driven cancer drug discovery. Thus, it is highly desirable to optimize the methods of high-throughput screening (HTS) for specific inhibitors of these E3 ubiquitin ligases. Here I will give an overview of several approaches used for HTS for ubiquitin ligase inhibitors with a main focus on assay principles, applications, and the pros and cons of each approach. Experimental details for many of these assays can be found in other chapters in this volume.

Drug Evaluation, Preclinical↗
PubMed1999

DNA double-strand break repair, DNA-PK, and DNA ligases in two human squamous carcinoma cell lines with different radiosensitivity.

PURPOSE: Variation in sensitivity to radiotherapy among tumors has been related to the capacity of cells to repair radiation-induced DNA double-strand breaks (DSBs). DNA-dependent protein kinase (DNA-PK) and DNA ligases may affect DNA dsb rejoining. This study was performed to compare rate of rejoining of radiation-induced DSBs, DNA-PK, and DNA ligase activities in two human squamous carcinoma cell lines with different sensitivity to ionizing radiation. METHODS AND MATERIALS: Cell survival of two human squamous carcinoma cell lines, UM-SCC-1 and UM-SCC-14A, was determined by an in vitro clonogenic assay. DSB rejoining was studied using pulsed field gel electrophoresis (PFGE). DNA-PK activity was determined using BIOTRAK DNA-PK enzyme assay system (Amersham). DNA ligase activity in crude cell extracts was measured using [5'-33P] Poly (dA) x (oligo (dT) as a substrate. Proteolytic degradation of proteins was analyzed by means of Western blotting. RESULTS: Applying the commonly used linear-quadratic equation to describe cell survival, S = e-alphaD-betaD2, the two cell lines roughly have the same alpha value (approximately 0.40 Gy(-1)) whereas the beta value was considerably higher in UM-SCC-14A (0.067 Gy(-2)+/-0.007 Gy(-2) [SEM]) as compared to UM-SCC-1 (0.013 Gy(-2)+/-0.004 Gy(-2) [SEM]). Furthermore, UM-SCC-1 was more proficient in rejoining of X-ray-induced DSBs as compared to UM-SCC-14A as quantified by PFGE. The constitutive level of DNA-PK activity was 1.6 times higher in UM-SCC-1 as compared to UM-SCC-14A ( < 0.05). The constitutive level of DNA ligase activity was similar in the two cell lines. CONCLUSIONS: The results suggest that the proficiency in rejoining of DSBs is associated with DNA-PK activity but not with total DNA ligase activity.

Carcinoma, Squamous Cell↗
PubMed2001

Identification of candidate mitochondrial RNA editing ligases from Trypanosoma brucei.

Most mitochondrial genes of Trypanosoma brucei do not contain the necessary information to make translatable mRNAs. These transcripts must undergo RNA editing, a posttranscriptional process by which uridine residues are added and deleted from mitochondrial mRNAs. RNA editing is believed to be catalyzed by a ribonucleoprotein complex containing endonucleolytic, terminal uridylyl transferase (TUTase), 3' uridine-specific exonucleolytic (U-exo), and ligase activities. None of the catalytic enzymes for RNA editing have been identified. Here we describe the identification of two candidate RNA ligases (48 and 52 kDa) that are core catalytic components of the T. brucei ribonucleoprotein editing complex. Both enzymes share homology to the covalent nucleotidyl transferase superfamily and contain five key signature motifs, including the active site KXXG. In this report, we present data on the proposed 48 kDa RNA editing ligase. We have prepared polyclonal antibodies against recombinant 48 kDa ligase that specifically recognize the trypanosome enzyme. When expressed in trypanosomes as an epitope-tagged fusion protein, the recombinant ligase localizes to the mitochondrion, associates with RNA editing complexes, and adenylates with ATP. These findings provide strong support for the enzymatic cascade model for kinetoplastid RNA editing.

Adenosine Triphosphate↗
PubMed1988

Binding interactions between yeast tRNA ligase and a precursor transfer ribonucleic acid containing two photoreactive uridine analogues.

Yeast tRNA ligase, from Saccharomyces cerevisiae, is one of the protein components that is involved in the splicing reaction of intron-containing yeast precursor tRNAs. It is an unusual protein because it has three distinct catalytic activities. It functions as a polynucleotide kinase, as a cyclic phosphodiesterase, and as an RNA ligase. We have studied the binding interactions between ligase and precursor tRNAs containing two photoreactive uridine analogues, 4-thiouridine and 5-bromouridine. When irradiated with long ultraviolet light, RNA containing these analogues can form specific covalent bonds with associated proteins. In this paper, we show that 4-thiouridine triphosphate and 5-bromouridine triphosphate were readily incorporated into a precursor tRNA(Phe) that was synthesized, in vitro, with bacteriophage T7 RNA polymerase. The analogue-containing precursor tRNAs were authentic substrates for the two splicing enzymes that were tested (endonuclease and ligase), and they formed specific covalent bonds with ligase when they were irradiated with long-wavelength ultraviolet light. We have determined the position of three major cross-links and one minor cross-link on precursor tRNA(Phe) that were located within the intron and near the 3' splice site. On the basis of these data, we present a model for the in vivo splicing reaction of yeast precursor tRNAs.

Base Sequence↗
PubMed2006

The Prp19 U-box crystal structure suggests a common dimeric architecture for a class of oligomeric E3 ubiquitin ligases.

Prp19 is an essential splicing factor and a member of the U-box family of E3 ubiquitin ligases. Prp19 forms a tetramer via a central coiled-coil domain. Here, we show the U-box domain of Prp19 exists as a dimer within the context of the Prp19 tetramer. A high-resolution structure of the homodimeric state of the Prp19 U-box was determined by X-ray crystallography. Mutation of the U-box dimer interface abrogates U-box dimer formation and is lethal in vivo. The structure of the U-box dimer enables construction of a complete model of Prp19 providing insights into how the tetrameric protein functions as an E3 ligase. Finally, comparison of the Prp19 U-box homodimer with the heterodimeric complex of BRCA1/BARD1 RING-finger domains uncovers a common architecture for a family of oligomeric U-box and RING-finger E3 ubiquitin ligases, which has mechanistic implications for E3 ligase-mediated polyubiquitination and E4 polyubiquitin ligases.

Amino Acid Sequence↗
PubMed1996

Determination of the chemical pathway for 4-chlorobenzoate:coenzyme A ligase catalysis.

4-Chlorobenzoate:coenzyme A ligase (4-CBA:CoA ligase) catalyzes the first step of the 4-CBA degradation pathway of Pseudomonas sp. strain CBS3. In this reaction, 4-CBA-CoA thioester synthesis is coupled to ATP cleavage. The studies described in this paper examine the intermediacy of 4-chlorobenzoyl-adenosine 5'phosphate diester (4-CBA-AMP) in the ligase reaction. The 4-CBA-AMP adduct was isolated from the ligase reaction mixture generated from magnesium adenosine 5-triphosphate (MgATP) and 4-CBA in the absence of CoA. The structure of the 4-CBA-AMP was verified by 1H- 13C-, and 31P-nuclear magnetic resonance analysis. Single-turnover reactions carried out with 14C-labeled 4-CBA in a rapid quench apparatus demonstrated formation of the enzyme. 4-CBA-AMP.MgPPi complex from the enzyme.4-CBA.MgATP complex at a rate of 135 s-1. The rate of ligand release from the enzyme.4-CBA-AMP.MgPPi complex was measured at 0.013 s-1. Single-turnover reactions of [14C]-4-CBA, MgATP, and CoA catalyzed by the ligase revealed that the 4-CBA-AMP intermediate formed reaches a maximum level of 25% of the starting 4-CBA within 10 ms and then declines with the formation of the 4-CBA-CoA. The rates of the adenylation and thioesterification partial reactions, determined by kinetic simulation of the rate data, are nearly equal (135 and 100 s-1). Substitution of CoA with the slow substrate pantetheine did not significantly alter the rate of the adenylation step but did reduce the rate of the thioesterification step to 2 s-1. The maximum level of 4-CBA-AMP reached during the single-turnover reaction of 4-CBA, MgATP, and pantetheine corresponded to one-half of the starting 4-CBA.

Adenosine Monophosphate↗
PubMed2001

Isolation and preliminary characterization of the medium-chain fatty acid:CoA ligase responsible for activation of short- and medium-chain fatty acids in colonic mucosa from swine.

The ATP-dependent activation of short- and medium-chain fatty acids to their respective CoA thioester adducts was investigated in the colonic mucosa from swine. Subcellular fractionation of a homogenate of the mucosa from the entire length of the colon revealed a predominantly mitochondrial localization for activity toward fatty acids ranging from propionate through laurate. These activities could be released from mitochondria in soluble form by freeze-thaw lysis. Purification of these activities revealed that they all appeared to reside with a single enzyme. This suggests that the entire colon contains a single form of medium-chain fatty acid:CoA ligase (MCFA:CoA ligase). The ligase also had activity toward benzoate and salicylate, although this activity was significantly lower than activity toward medium-chain fatty acids. The enzyme had the highest activity at Vmax with butyrate as substrate and had the lowest Km for octanoate. Butyrate and octanoate were mutually inhibitory. Activity toward both substrates was also efficiently inhibited by cyclohexane carboxylate. The molecular weight of the enzyme was estimated by gel filtration chromatography to be ca. 46,500. These data indicate that the colonic MCFA:CoA ligase is significantly different from the hepatic and kidney MCFA:CoA ligases.

Acetate-CoA Ligase↗
PubMed2001

Characterization of an ATP-dependent type I DNA ligase from Arabidopsis thaliana.

Here we report the purification and biochemical characterization of recombinant Arabidopsis thaliana DNA ligase I. We show that this ligase requires ATP as a source for adenylation. The calculated Km [ATP] for ligation is 3 microM. This enzyme is able to ligate nicks in oligo(dT)/poly(dA) and oligo(rA)/poly(dT) substrates, but not in oligo(dT)/poly(rA) substrates. Double-stranded DNAs with cohesive or blunt ends are also good substrates for the ligase. These biochemical features of the purified enzyme show the characteristics typical of a type I DNA ligase. Furthermore, this DNA ligase is able to perform the reverse reaction (relaxation of supercoiled DNA) in an AMP-dependent and PPi-stimulated manner.

Adenosine Triphosphate↗
PubMed2004

Cullin-based ubiquitin ligases: Cul3-BTB complexes join the family.

Cullin-based E3 ligases target substrates for ubiquitin-dependent degradation by the 26S proteasome. The SCF (Skp1-Cul1-F-box) and ECS (ElonginC-Cul2-SOCS box) complexes are so far the best-characterized cullin-based ligases. Their atomic structure has been solved recently, and several substrates have been described in different organisms. In addition to Cul1 and Cul2, higher eucaryotic genomes encode for three other cullins: Cul3, Cul4, and Cul5. Recent results have shed light on the molecular composition and function of Cul3-based E3 ligases. In these complexes, BTB-domain-containing proteins may bridge the cullin to the substrate in a single polypeptide, while Skp1/F-box or ElonginC/SOCS heterodimers fulfill this function in the SCF and ECS complexes. BTB-containing proteins are evolutionary conserved and involved in diverse biological processes, but their function has not previously been linked to ubiquitin-dependent degradation. In this review, we present these new findings and compare the composition of Cul3-based ligases to the well-defined SCF and ECS ligases.

Animals↗
PubMed2006

Regulation of p53 localization and transcription by the HECT domain E3 ligase WWP1.

As a key cellular regulatory protein p53 is subject to tight regulation by several E3 ligases. Here, we demonstrate the role of HECT domain E3 ligase, WWP1, in regulating p53 localization and activity. WWP1 associates with p53 and induces p53 ubiquitylation. Unlike other E3 ligases, WWP1 increases p53 stability; inhibition of WWP1 expression or expression of a ligase-mutant form results in decreased p53 expression. WWP1-mediated stabilization of p53 is associated with increased accumulation of p53 in cytoplasm with a concomitant decrease in its transcriptional activities. WWP1 effects are independent of Mdm2 as they are seen in cells lacking Mdm2 expression. Whereas WWP1 limits p53 activity, p53 reduces expression of WWP1, pointing to a possible feedback loop mechanism. Taken together, these findings identify the first instance of a ubiquitin ligase that causes stabilization of p53 while inactivating its transcriptional activities.

Amino Acid Motifs↗
PubMed1998

Genes of succinyl-CoA ligase from Saccharomyces cerevisiae.

Succinyl-CoA ligase (succinyl-CoA synthetase) catalyzes the nucleotide-dependent conversion of succinyl-CoA to succinate. This enzyme functions in the tricarboxylic acid (TCA) cycle and is also involved in ketone-body breakdown in animals. The enzyme is composed of alpha and beta subunits that are required for catalytic activity. Two genes, LSC1 (YOR142W) and LSC2 (YGR244C), with high similarity to succinyl-CoA ligase subunits from other species were isolated from Saccharomyces cerevisiae. The expression of these genes was repressed by growth on glucose and was induced threefold to sixfold during growth on nonfermentable carbon sources. The LSC genes were deleted singly and in combination. Unlike other yeast strains with defects in TCA cycle genes, strains lacking either or both LSC genes were able to grow with acetate as a carbon source. However, growth on glycerol or pyruvate was impaired. An antiserum against both subunits of the Escherichia coli enzyme was capable of recognizing the yeast succinyl-CoA ligase alpha subunit, and this band was absent in delta lsc1 deletion strains. Succinyl-CoA ligase activity was absent in mitochondria isolated from strains deleted for one or both LSC genes, but activity was restored by the presence of the appropriate LSC gene on a plasmid. The yeast succinyl-CoA ligase was shown to utilize ATP but not GTP for succinyl-CoA synthesis.

Acetates↗
PubMed2005

Primate lentiviral virion infectivity factors are substrate receptors that assemble with cullin 5-E3 ligase through a HCCH motif to suppress APOBEC3G.

Cullin-Ring E3 ubiquitin ligases target substrates for ubiquitin-dependent, proteasome-mediated degradation and regulate critical cellular processes. These cullins assemble with cellular substrate receptor proteins through specific adaptor molecules. F-box- and BC-box-containing receptors use Skp1, ElonginB, and ElonginC as adaptors to recruit Cul1/Cul7 and Cul2/Cul5, respectively. At present, the determinants of Cul2 vs. Cul5 specificity for the BC-box-containing receptors are poorly defined. Here, we demonstrate that primate lentiviral Vif (virion infectivity factor) proteins represent previously uncharacterized substrate receptor proteins that contain divergent BC-box motifs. These molecules selectively assemble with a Cul5-E3 ligase to suppress the antiviral activity of autologous cytidine deaminase APOBEC3G. A previously unrecognized Hx5Cx(17-18)Cx(3-5)H motif that is highly conserved among all primate lentiviral Vif proteins was found to be critical for the selective assembly and activity of Vif-Cul5-E3 ligase. Non-primate lentiviral Vif proteins, which lack this HCCH motif, displayed reduced interaction with Cul5. These data suggest that in addition to target protein specificity, substrate receptor proteins play important roles in cullin selection and functional assembly of cullin-Ring E3 ligases. The discovery of these viral substrate receptor molecules that recruit Cul5 through distinct mechanisms from cellular proteins may facilitate the identification of additional cellular factors that regulate cellular functions through Cul5-E3 ligase. Motifs in Vif that are absent from cellular proteins could also be targets for the development of innovative therapeutics.

APOBEC-3G Deaminase↗
PubMed1998

The action of DNA ligase at abasic sites in DNA.

Apurinic/apyrimidinic (AP) sites occur frequently in DNA as a result of spontaneous base loss or following removal of a damaged base by a DNA glycosylase. The action of many AP endonuclease enzymes at abasic sites in DNA leaves a 5'-deoxyribose phosphate (dRP) residue that must be removed during the base excision repair process. This 5'-dRP group may be removed by AP lyase enzymes that employ a beta-elimination mechanism. This beta-elimination reaction typically involves a transient Schiff base intermediate that can react with sodium borohydride to trap the DNA-enzyme complex. With the use of this assay as well as direct 5'-dRP group release assays, we show that T4 DNA ligase, a representative ATP-dependent DNA ligase, contains AP lyase activity. The AP lyase activity of T4 DNA ligase is inhibited in the presence of ATP, suggesting that the adenylated lysine residue is part of the active site for both the ligase and lyase activities. A model is proposed whereby the AP lyase activity of DNA ligase may contribute to the repair of abasic sites in DNA.

Animals↗
PubMed2003

Genetic and biochemical analysis of the functional domains of yeast tRNA ligase.

Yeast tRNA ligase (Trl1) converts cleaved tRNA half-molecules into spliced tRNAs containing a 2'-PO4, 3'-5' phosphodiester at the splice junction. Trl1 performs three reactions: (i) the 2',3'-cyclic phosphate of the proximal fragment is hydrolyzed to a 3'-OH, 2'-PO4 by a cyclic phosphodiesterase (CPD); (ii) the 5'-OH of the distal fragment is phosphorylated by an NTP-dependent polynucleotide kinase; and (iii) the 3'-OH, 2'-PO4, and 5'-PO4 ends are sealed by an ATP-dependent RNA ligase. Trl1 consists of an N-terminal adenylyltransferase domain that resembles T4 RNA ligase 1, a central domain that resembles T4 polynucleotide kinase, and a C-terminal CPD domain that resembles the 2H phosphotransferase enzyme superfamily. Here we show that all three domains are essential in vivo, although they need not be linked in the same polypeptide. We identify five amino acids in the adenylyltransferase domain (Lys114, Glu266, Gly267, Lys284, and Lys286) that are essential for Trl1 activity and are located within motifs I (114KANG117), IV (266EGFVI270), and V (282FFKIK286) that comprise the active sites of DNA ligases, RNA capping enzymes, and T4 RNA ligases 1 and 2. Mutations K404A and T405A in the P-loop (401GXGKT405) of the central kinase-like domain had no effect on Trl1 function in vivo. The K404A and T405A mutations eliminated ATP-dependent kinase activity but preserved GTP-dependent kinase activity. A double alanine mutant in the P-loop was lethal in vivo and abolished GTP-dependent kinase activity. These results suggest that GTP is the physiological substrate and that the Trl1 kinase has a single NTP binding site of which the P-loop is a component. Two other mutations in the central domain were lethal in vivo and either abolished (D425A) or severely reduced (R511A) GTP-dependent RNA kinase activity in vitro. Mutations of the signature histidines of the CPD domain were either lethal (H777A) or conferred a ts growth phenotype (H673A).

Amino Acid Sequence↗
PubMed2004

Characterization of a baculovirus enzyme with RNA ligase, polynucleotide 5'-kinase, and polynucleotide 3'-phosphatase activities.

The end-healing and end-sealing steps of the phage T4-induced RNA restriction-repair pathway are performed by two separate enzymes, a bifunctional polynucleotide 5'-kinase/3'-phosphatase and an ATP-dependent RNA ligase. Here we show that a single trifunctional baculovirus enzyme, RNA ligase 1 (Rnl1), catalyzes the identical set of RNA repair reactions. Three enzymatic activities of baculovirus Rnl1 are organized in a modular fashion within a 694-amino acid polypeptide consisting of an autonomous N-terminal RNA-specific ligase domain, Rnl1-(1-385), and a C-terminal kinase-phosphatase domain, Rnl1-(394-694). The ligase domain is itself composed of two functional units. The N-terminal module Rnl1-(1-270) contains essential nucleotidyltransferase motifs I, IV, and V and suffices for both enzyme adenylylation (step 1 of the ligation pathway) and phosphodiester bond formation at a preactivated RNA-adenylate end (step 3). The downstream module extending to residue 385 is required for ligation of a phosphorylated RNA substrate, suggesting that it is involved specifically in the second step of the end-joining pathway, the transfer of AMP from the ligase to the 5'-PO(4) end to form RNA-adenylate. The end-healing domain Rnl1-(394-694) consists of a proximal 5'-kinase module with an essential P-loop motif ((404)GSGKS(408)) and a distal 3'-phosphatase module with an essential acylphosphatase motif ((560)DLDGT(564)). Our findings have implications for the evolution of RNA repair systems and their potential roles in virus-host dynamics.

Adenosine Triphosphate↗
PubMed2004

Smac/Diablo antagonizes ubiquitin ligase activity of inhibitor of apoptosis proteins.

Inhibitor of apoptosis proteins (IAPs) can block apoptosis through binding to active caspases and antagonizing their function. IAP function can be neutralized by Smac/Diablo, an IAP-binding protein that is released from mitochondria during apoptosis. In addition to their ability to interact with caspases, certain IAPs also display ubiquitin-protein isopeptide ligase activity because of the presence of a RING domain. However, it is not known whether the ubiquitin-protein isopeptide ligase activities of human IAPs contribute to their apoptosis inhibitory activity or whether this IAP property can be modulated through association with Smac/Diablo. Here we demonstrate that the ubiquitin ligase activities of XIAP, and to a lesser extent c-IAP-1 and c-IAP2, are potently repressed through binding to Smac/Diablo. We also show that mutation of the XIAP RING domain rendered this IAP a less effective inhibitor of apoptosis, suggesting that the ubiquitin ligase activity of XIAP contributes to its anti-apoptotic function. These data suggest that Smac/Diablo potentiates apoptosis by simultaneously antagonizing caspase-IAP interactions and repressing IAP ubiquitin ligase activities.

Amino Acid Motifs↗
PubMed2005

Molecular architecture and ligand recognition determinants for T4 RNA ligase.

RNA ligase type 1 from bacteriophage T4 (Rnl1) is involved in countering a host defense mechanism by repairing 5'-PO4 and 3'-OH groups in tRNA(Lys). Rnl1 is widely used as a reagent in molecular biology. Although many structures for DNA ligases are available, only fragments of RNA ligases such as Rnl2 are known. We report the first crystal structure of a complete RNA ligase, Rnl1, in complex with adenosine 5'-(alpha,beta-methylenetriphosphate) (AMPcPP). The N-terminal domain is related to the equivalent region of DNA ligases and Rnl2 and binds AMPcPP but with further interactions from the additional N-terminal 70 amino acids in Rnl1 (via Tyr37 and Arg54) and the C-terminal domain (Gly269 and Asp272). The active site contains two metal ions, consistent with the two-magnesium ion catalytic mechanism. The C-terminal domain represents a new all alpha-helical fold and has a charge distribution and architecture for helix-nucleic acid groove interaction compatible with tRNA binding.

Bacteriophage T4↗