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PubMed2001

Structure and evolution of 4-coumarate:coenzyme A ligase (4CL) gene families.

The phenylpropanoid enzyme 4-coumarate:coenzyme A ligase (4CL) plays a key role in general phenylpropanoid metabolism. 4CL is related to a larger class of prokaryotic and eukaryotic adenylate-forming enzymes and shares several conserved peptide motifs with these enzymes. In order to better characterize the nature of 4CL gene families in poplar, parsley, and tobacco, we used degenerate primers to amplify 4CL sequences from these species. In each species additional, divergent 4CL genes were found. Complete cDNA clones for the two new poplar 4CL genes were obtained, allowing examination of their expression patterns and determination of the substrate utilization profile of a xylem-specific isoform. Phylogenetic analysis of these genes and gene fragments confirmed previous results showing that 4CL proteins fall into two evolutionarily ancient subgroups . A comparative phylogenetic analysis of enzymes in the adenylate-forming superfamily showed that 4CLs, luciferases, and acetate CoA ligases each form distinct clades within the superfamily. According to this analysis, four Arabidopsis 4CL-like genes identified from the Arabidopsis Genome Project are only distantly related to bona fide 4CLs or are more closely related to fatty acid CoA ligases, suggesting that the three Arabidopsis 4CL genes previously characterized represent the extent of the 4CL gene family in this species.

Adenosine Monophosphate↗
PubMed2000

Xenobiotic-CoA ligases: kinetic and molecular characterization.

This review focuses primarily on the mammalian medium and long-chain fatty acid coenzyme A ligases that have been implicated in the metabolism of xenobiotic carboxylic acids such as pesticides, arylpropionate non steroidal anti-inflammatory drugs and the hypolipidaemic clofibrate and its congeners. Evidence of multiplicity of mitochondrial and microsomal enzymes and their respective substrate/inhibitor profiles are discussed. For completeness, where appropriate, details of non-substrate inhibitors have also been included. Although knowledge is limited at present with respect to the medium-chain enzymes, aspects of regulation particularly the in vivo, in vitro role of peroxisome proliferators and current knowledge of the molecular biology of the long-chain fatty acid CoA ligase superfamily are documented. Additionally, alignment of thirteen cloned mammalian fatty acid CoA ligases using criteria established for the CYP and UGT superfamilies has enabled construction of a phylogenetic tree that clearly defines three families. Catalytic data are still limited and the xenobiotic substrate/inhibitor profiles of the recombinant proteins are incomplete. Finally, with increasing recognition of the importance of fatty acyl-CoA esters as physiological regulators of cell function including gene expression, the review concludes with a discussion of the metabolic fate and toxicity of xenobiotic acyl-CoA esters.

Animals↗
PubMed2006

NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase.

NK lytic-associated molecule (NKLAM) is a protein involved in the cytolytic function of NK cells and CTLs. It has been localized to the cytolytic granules in NK cells and is up-regulated when cells are exposed to cytokines IL-2 or IFN-beta. We report in this study that NKLAM contains a cysteine-rich really interesting new gene (RING) in between RING-RING domain, and that this domain possesses strong homology to the RING domain of the known E3 ubiquitin ligase, Dorfin. To determine whether NKLAM functions as an E3 ligase, we performed coimmunoprecipitation binding assays with ubiquitin conjugates (Ubcs) UbcH7, UbcH8, and UbcH10. We demonstrated that both UbcH7 and UbcH8 bind to full-length NKLAM. We then performed a similar binding assay using endogenous NKLAM and UbcH8 expressed by human NK clone NK3.3 to show that the protein interaction occurs in vivo. Using the yeast two-hybrid system, we identified uridine kinase like-1 (URKL-1) protein as a substrate for NKLAM. We confirmed that NKLAM and URKL-1 interact in mammalian cells by using both immunoprecipitation and confocal microscopy. We demonstrated decreased protein expression and enhanced ubiquitination of URKL-1 in the presence of NKLAM. These data indicate that NKLAM is a RING finger protein that binds Ubcs and has as one of its substrates, URKL-1, thus defining this cytolytic protein as an E3 ubiquitin ligase.

Amino Acid Sequence↗
PubMed1997

Community-based urine screening for Chlamydia trachomatis with a ligase chain reaction assay.

BACKGROUND: Urine tests for Chlamydia trachomatis permit expansion of screening beyond traditional clinic environments. Prevention of infection in teenagers is a high priority. OBJECTIVE: To define the prevalence of C. trachomatis among teenagers by using the ligase chain reaction assay on urine specimens and to evaluate leukocyte esterase testing of urine specimens as an indicator of infection. DESIGN: Cross-sectional study. SETTING: An adolescent clinic, a juvenile detention facility, seven school-based clinics, and three community-based youth organizations in Seattle, Washington. PARTICIPANTS: 10,118 sexually active teenagers and young adults. MEASUREMENTS: Chlamydia trachomatis infection detected in urine specimens by ligase chain reaction assay and leukocyturia detected by leukocyte esterase testing. RESULTS: The prevalence of chlamydial infection among female participants was 8.6% and declined with increasing age; among male participants, it was 5.4% and increased with increasing age. In female participants, independent predictors of infection were being 17 years of age or younger (odds ratio [OR], 1.49), having had two or more sex partners in the previous 2 months (OR, 1.61), and having genitourinary symptoms (OR, 1.46). In male participants, independent predictors were being of nonwhite race or ethnicity (OR, 2.00 to 3.08), having had two or more sex partners in the previous 2 months (OR, 1.57), and having used a condom during the most recent sexual encounter (OR, 0.67). For identifying infection in male participants, the sensitivity of leukocyte esterase testing was 58.9%, the specificity was 94.9%, the positive predictive value was 38.4%, and the negative predictive value was 97.7%. CONCLUSIONS: Chlamydial infection is common in teenagers and young adults in community settings. The urine ligase chain reaction assay will permit widespread screening for C. trachomatis, but leukocyte esterase testing had low sensitivity for selecting persons for screening with this assay. Indicators of chlamydial infection differed substantially in male and female participants.

Adolescent↗
PubMed1975

Millipore filter assay for long-chain fatty acid:CoASH ligase activity using 3H-labeled coenzyme A.

A novel radiochemical assay for long-chain fatty acid:CoASH ligase activity (AMP) (EC 6.2.1.3) has been developed based on the conversion of [3H]CoASH to long-chain fatty acyl CoA. Fatty acyl [3H]CoA was quantitatively retained on Millipore filters upon filtration of the acidified reaction mixture under conditions where the [3H]CoASH was not retained. The assay was developed using microsomes derived from isolated fat cells as the source of fatty acid:CoASH ligase activity. The assay performed at 25 degrees C for 10 min was linear with added microsomal protein up to 7 mug. The assay was linear with time up to 24 min when 1 mug of protein was employed. Fatty acid:CoASH ligase activity was strongly dependent on ATP and magnesium, was stimulated by Triton WR-1339, and was two- to fivefold dependent on added fatty acid. The filter assay is easier than existing assays based on incorporation of labeled fatty acid and is equally sensitive.

Animals↗
PubMed1992

Consequences of 6-thioguanine incorporation into DNA on polymerase, ligase, and endonuclease reactions.

The incorporation of 6-thioguanine (S6G) in place of guanine proceeds readily in DNA synthesis reactions catalyzed by mammalian and bacterial polymerases. This report summarizes the consequences of such incorporation studied to date. S6G was incorporated into one strand of a defined M13mp18 phage sequence in a (+)reaction catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. After denaturation of the newly synthesized strand (containing S6G) and annealing with a reverse (-) 32P-labeled primer, polymerization catalyzed by the Klenow enzyme as well as by human DNA polymerases alpha, gamma, and delta was slowed considerably, compared with that across the corresponding guanine-containing template. To evaluate S6G-containing DNA as a substrate for DNA ligases, two oligodeoxynucleotides (19- and 20-mers) antisense to a 40-mer were synthesized so that the 40-mer coded for guanine at the 3' terminus of the 19-mer. After annealing of the synthetic oligonucleotides to form a duplex DNA containing a one-nucleotide gap (opposite cytosine in the 40-mer), the 19-mer was extended with 2'-deoxythioguanosine 5'-triphosphate using DNA polymerase, forming a nicked duplex DNA. The abilities of T4 DNA ligase and HeLa and calf thymus DNA ligase I to join the 5'-phosphate with the 3'-S6G-OH were severely inhibited, compared with the 3'-guanine-extended control. This finding suggests that incorporation of S6G at the 3' terminus of Okazaki fragments would inhibit lagging strand DNA synthesis. In other experiments, cleavage of S6G-containing DNA by some but not all restriction endonucleases progressed poorly, compared with the control guanine-containing DNA, independently of the location of S6G at recognition or cleavage sites, as previously observed by Iwaniec et al. [Mol. Pharmacol. 39:299-306 (1991)] with a different spectrum of enzymes. These findings indicate altered DNA-protein interactions due to S6G incorporation. The poor template function of S6G-containing DNA is consistent with the known delayed cytotoxicity and DNA damage previously reported to occur in S6G-treated cells.

Autoradiography↗
PubMed2003

ZNRF proteins constitute a family of presynaptic E3 ubiquitin ligases.

Protein ubiquitination has been implicated recently in neural development, plasticity, and degeneration. We previously identified ZNRF1/nin283, a protein with a unique, evolutionarily conserved C-terminal domain containing a juxtaposed zinc finger/RING finger combination. Here we describe the identification of a closely related protein, ZNRF2, thus defining a novel family of ZNRF E3 ubiquitin ligases. Both ZNRF1 and ZNRF2 have E3 ubiquitin ligase activity and are highly expressed in the nervous system, particularly during development. In neurons, ZNRF proteins are located in different compartments within the presynaptic terminal: ZNRF1 is associated with synaptic vesicle membranes, whereas ZNRF2 is present in presynaptic plasma membranes. Mutant ZNRF proteins with a disrupted RING finger, a domain necessary for their E3 function, can each inhibit Ca2+-dependent exocytosis in PC12 cells. These data suggest that ZNRF proteins play a role in the establishment and maintenance of neuronal transmission and plasticity via their ubiquitin ligase activity.

Amino Acid Sequence↗
PubMed2004

FANCL replaces BRCA1 as the likely ubiquitin ligase responsible for FANCD2 monoubiquitination.

Monoubiquitination of FANCD2 is a key step in the DNA damage response pathway involving Fanconi anemia proteins and the breast cancer susceptibility gene products, BRCA1 and BRCA2. One critical unresolved issue is the identity of the ubiquitin ligase responsible for this reaction. Two proteins, BRCA1 and FANCL(PHF9), have been suggested to be this ligase. Here we found that FANCL, but not BRCA1, evolutionarily co-exists with FANCD2 in several species. Moreover, the proportion of FANCD2 in chromatin and nuclear matrix is drastically reduced in a cell line mutated in FANCL, but not in that mutated in BRCA1. This defective distribution of FANCD2 in the FANCL-mutant cell line is likely due to its defective monoubiquitination, because the monoubiquitinated FANCD2 preferentially associates with chromatin and nuclear matrix, whereas non-ubiquitinated FANCD2 largely resides in the soluble fraction. Our data support the notion that FANCL, but not BRCA1, is the likely ligase for FANCD2 monoubiquitination.

Aged↗
PubMed2005

Down-regulation of BRCA1-BARD1 ubiquitin ligase by CDK2.

BRCA1, a breast and ovarian tumor suppressor, is a phosphoprotein whose cellular expression level is regulated in a cell cycle-dependent manner. BRCA1 interacts with BARD1 to generate significant ubiquitin ligase activity which catalyzes nontraditional Lys-6-linked polyubiquitin chains. However, it is not clear how the activity is regulated and how this affects BRCA1's multiple cellular functions. Here we show that the ubiquitin ligase activity of BRCA1-BARD1 is down-regulated by CDK2. During the cell cycle, BARD1 expression can largely be categorized into three patterns: moderately expressed in a predominantly unphosphorylated form in early G(1) phase, expressed at low levels in both phosphorylated and unphosphorylated forms during late G(1) and S phases, and highly expressed in its phosphorylated form during mitosis coinciding with BRCA1 expression. CDK2-cyclin A1/E1 and CDK1-cyclin B1 phosphorylate BARD1 on its NH(2) terminus in vivo and in vitro. Intriguingly, the BRCA1-BARD1-mediated in vivo ubiquitination of nucleophosmin/B23 (NPM) and autoubiquitination of BRCA1 are dramatically disrupted by coexpression of CDK2-cyclin A1/E1, but not by CDK1-cyclin B1. The inhibition of ubiquitin ligase activity is not due to the direct effect of the kinases on BARD1 because an unphosphorylatable mutant of BARD1, S148A/S251A/S288A/T299A, is still inhibited by CDK2-cyclin E1. Alternatively, BRCA1 and BARD1 are likely exported to the cytoplasm and their expressions are remarkably reduced by CDK2-cyclin E1 coexpression. Recognizing the importance of cyclin E1 overexpression in breast cancer development, these results suggest a CDK2-BRCA1-NPM pathway that coordinately functions in cell growth and tumor progression pathways.

BRCA1 Protein↗
PubMed1976

Modification of Escherichia coli DNA ligase by cleavage with trypsin.

Limited treatment of Escherichia coli DNA ligase with trypsin results in rapid loss of DNA joining activity. However, the ability to react with DPN to form the covalent enzyme-AMP intermediate is unaffected. The cleaved enzyme is also unable to catalyze the formation of DNA-adenylate, the second covalent intermediate in the ligase-catalyzed reaction. These findings demonstrate that portions of the DNA ligase molecule that are required for phosphodiester bond formation are not required for at least one of the partial reactions catalyzed by this enzyme.

Escherichia coli↗
PubMed1979

Evidence that cholic acid CoA ligase is located asymmetrically on the cytoplasmic surface of hepatic microsomal vesicles.

The cholic acid CoA ligase activity of rat liver was quantitatively inactivated by proteolysis with pronase, chymotrypsin, subtilisin, or proteinase K in intact microsomal vesicles. Under the conditions employed, less than 14% of the lumenal mannose-6-phosphate phosphatase activity was lost, and the mannose-6-phosphate phosphatase activity remained highly latent. After microsomal integrity was disrupted with sodium deoxycholate, protease treatment resulted in a loss of greater than 74% of the mannose-6-phosphate phosphatase activity. Cholic acid CoA ligase activity was unaffected by preincubation of microsomes with sodium taurocholate under conditions that led to the complete expression of latent mannose-6-phosphate phosphatase activity. The data suggest that cholic acid CoA ligase activity is located on the cytoplasmic surface of hepatic microsomal vesicles.

Animals↗
PubMed1979

Kinetic characterization of long chain fatty acyl coenzyme A ligase from rat liver mitochondria.

Long chain fatty acyl coenzyme A ligase (EC 6.2.1.3) purified from rat liver mitochondria has been characterized with respect to several kinetic parameters. Many of the kinetic properties of the mitochondrial enzyme are similar to those of the purified microsomal enzyme with respect to palmitoyl-CoA formation, but there are distinct differences. The fatty acid and nucleotide specificities of the mitochondrial enzyme are similar to those of the microsomal enzyme, as are the apparent Km values for ATP and coenzyme A. On the other hand, the mitochondrial enzyme differs from the microsomal enzyme in that it has a lower pH optimum, is different in molecular weight, and does not show simple saturation kinetics with palmitate as substrate. Of particular interest is the evidence presented which indicates that the mitochondrial long chain fatty acyl-CoA ligase, unlike short and medium chain ligases, does not utilize an acyladenylate as an intermediate in the formation of fatty acyl-CoA.

Animals↗
PubMed1981

Coordinated regulation of 4-coumarate:CoA ligase and phenylalanine ammonia-lyase mRNAs in cultured plant cells.

4-Coumarate:CoA ligase (EC 6.2.1.12) from cell suspension cultures of parsley (Petroselinum hortense) was extensively purified. The enzyme behaved as a monomer with a molecular weight of approximately 60,000. A rabbit antiserum to the purified enzyme was obtained and used to determine the rates of 4-coumarate:CoA ligase synthesis under various conditions of induction, such as short term or continuous irradiation and treatment of the cells with an "elicitor" preparation from a fungal pathogen. In all cases, the time course of changes in the rate of synthesis, measured both in vivo and in vitro, was very similar for 4-coumarate:CoA ligase and a closely related enzyme, phenylalanine ammonia-lyase (EC 4.3.1.5). The results suggest that the mRNA activities encoding the two enzymes are regulated in a coordinated manner.

Ammonia-Lyases↗
PubMed1997

[Development of novel clinical tests and their contribution to laboratory diagnosis--ligase chain reaction].

There are several methods that have been developed for the detection of a small amount of nucleic acids. Ligase chain reaction(LCR) is a highly sensitive assay for the detection of a specific DNA sequence. LCR utilizes four oligodeoxynucleotides(probes) complementary to each of target DNA strands and repeats a thermalcycling amplification step. Two pairs of oligodeoxynucleotides renature adjacent to one another on each of the separated target DNA strands, resulting in nick formation. A thermostable DNA ligase joins the nick covalently. Each ligated product can serve as a template sequence in subsequent rounds of thermalcycling (denaturation, annealing/ligation). In this manner LCR accumulates the ligated products exponentially by repeating thermalcycling. One of the distinctive features of LCR is to be able to detect a known point-mutation easily. Gap-LCR, a modified LCR, has been developed to improve the sensitivity and the specificity of the standard LCR by setting up gaps (3 < or = nucleotides) at the ligatable 3' and 5' termini of the probes. The gap-LCR probes form a short gap after annealing of the probes to the target DNA. The gap is filled in by a thermostable DNA polymerase and the resultant nick is sealed by a thermostable DNA ligase to generate the ligated probes. Gap-LCR repeats this step on thermalcycling and accumulates the LCR products in an exponential fashion.

Clinical Laboratory Techniques↗
PubMed1998

Protection provided by exogenous DNA ligase in G0 human lymphocytes treated with restriction enzyme MspI or bleomycin as shown by the comet assay.

DNA double-strand breaks (DSB) may arise either spontaneously during cellular processes or as a result of exposure to DNA-damaging agents such as ionizing radiation, or radiomimetic agents such as restriction endonucleases or bleomycin. It is widely accepted that nonrepaired or misrepaired DSB are the main lesions leading to the production of chromosomal aberrations, mutagenesis, oncogenic transformation, and cell killing. Studies focusing on this relationship, as well as the possible modulation of DNA repair mechanisms, are currently of major interest. A wide variety of test systems are available to study DNA damage. In the last few years, single-cell gel electrophoresis, commonly known as "comet assay," has been considered a rapid, sensitive, and visual method for quantifying DNA strand breaks and alkali-labile damage in individual cells. In this study, making use of the comet assay, we tried to find out if under conditions that maintain chromatin structure the DNA ligase from T4 phage is able to facilitate the rejoining of strand breaks with different end structures, induced by the restriction endonuclease MspI or bleomycin in living human lymphocytes in a nonproliferating state. T4 DNA ligase, as well as the restriction endonuclease or bleomycin, were introduced together by electroporation into human lymphocytes. Our results support the idea that it is possible to modulate the DSB-rejoining of different DNA strand-breaking agents by exogenous T4 DNA ligase.

Bleomycin↗
PubMed1999

ROC1, a homolog of APC11, represents a family of cullin partners with an associated ubiquitin ligase activity.

We have identified two highly conserved RING finger proteins, ROC1 and ROC2, that are homologous to APC11, a subunit of the anaphase-promoting complex. ROC1 and ROC2 commonly interact with all cullins while APC11 specifically interacts with APC2, a cullin-related APC subunit. YeastROC1 encodes an essential gene whose reduced expression resulted in multiple, elongated buds and accumulation of Sic1p and Cln2p. ROC1 and APC11 immunocomplexes can catalyze isopeptide ligations to form polyubiquitin chains in an E1- and E2-dependent manner. ROC1 mutations completely abolished their ligase activity without noticeable changes in associated proteins. Ubiquitination of phosphorylated I kappa B alpha can be catalyzed by the ROC1 immunocomplex in vitro. Hence, combinations of ROC/APC11 and cullin proteins proteins potentially constitute a wide variety of ubiquitin ligases.

Amino Acid Sequence↗
PubMed2001

Ligand-dependent degradation of Smad3 by a ubiquitin ligase complex of ROC1 and associated proteins.

Smads are signal mediators for the members of the transforming growth factor-beta (TGF-beta) superfamily. Upon phosphorylation by the TGF-beta receptors, Smad3 translocates into the nucleus, recruits transcriptional coactivators and corepressors, and regulates transcription of target genes. Here, we show that Smad3 activated by TGF-beta is degraded by the ubiquitin-proteasome pathway. Smad3 interacts with a RING finger protein, ROC1, through its C-terminal MH2 domain in a ligand-dependent manner. An E3 ubiquitin ligase complex ROC1-SCF(Fbw1a) consisting of ROC1, Skp1, Cullin1, and Fbw1a (also termed betaTrCP1) induces ubiquitination of Smad3. Recruitment of a transcriptional coactivator, p300, to nuclear Smad3 facilitates the interaction with the E3 ligase complex and triggers the degradation process of Smad3. Smad3 bound to ROC1-SCF(Fbw1a) is then exported from the nucleus to the cytoplasm for proteasomal degradation. TGF-beta/Smad3 signaling is thus irreversibly terminated by the ubiquitin-proteasome pathway.

Active Transport, Cell Nucleus↗
PubMed1999

Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34.

SCFCdc4 (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development. Mass spectrometric analysis of proteins copurifying with Cdc53 identified the RING-H2 finger protein Hrt1 as a subunit of SCF. Hrt1 shows striking similarity to the Apc11 subunit of anaphase-promoting complex. Conditional inactivation of hrt1(ts) results in stabilization of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G1/S. Hrt1 assembles into recombinant SCF complexes and individually binds Cdc4, Cdc53 and Cdc34, but not Skp1. Hrt1 stimulates the E3 activity of recombinant SCF potently and enables the reconstitution of Cln2 ubiquitination by recombinant SCFGrr1. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality of hrt1Delta, and human HRT2 binds CUL-1. We conclude that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases.

Amino Acid Sequence↗