Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “LIGASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,513 records · Page 84Linked to original sources
PubMed1998

Polymorphisms in the human DNA ligase I gene (LIG1) including a complex GT repeat.

Sequencing of a human DNA ligase I cDNA clone derived from HeLa cells revealed two unreported differences with the published sequence: a single base change and a three-base deletion. Both differences are in exon 6, and were analyzed by amplifying a segment containing exon 5, intron 6, and exon 6. The first finding was that intron 6 is approximately 2.6 kb in size, not the 1 kb reported in the literature. By sequence analysis of amplified segments, the single-base difference in exon 6 was shown to be polymorphic, with HeLa cells heterozygous for the A/C difference. Analysis of 60 unrelated individuals found a frequency of 0.5 for each allele. Primer extension reactions across the exon 5/exon 6 boundary were performed on cDNA obtained from HeLa cells and human thymus. The results show that the three-base deletion is due to a variation in splicing. For both HeLa and thymus, two-thirds of the transcripts are like the published cDNA sequence and one-third have the three-base deletion. Finally, sequencing of part of intron 6 revealed the presence of a complex GT repeat consisting of a 48-50 nucleotide polypurine tract followed by a variable number of GT residues. This entire unit of polypurine tract plus GTs is repeated three times. Detection of the repeated sequences required the development of specialized cloning and PCR conditions. Analysis of a pedigree showed that this complex repeat is polymorphic.

Base Sequence↗
PubMed2001

The DSmurf ubiquitin-protein ligase restricts BMP signaling spatially and temporally during Drosophila embryogenesis.

We identified Drosophila Smurf (DSmurf) as a negative regulator of signaling by the BMP2/4 ortholog DPP during embryonic dorsal-ventral patterning. DSmurf encodes a HECT domain ubiquitin-protein ligase, homologous to vertebrate Smurf1 and Smurf2, that binds the Smad1/5 ortholog MAD and likely promotes its proteolysis. The essential function of DSmurf is restricted to its action on the DPP pathway. DSmurf has two distinct, possibly mechanistically separate, functions in controlling DPP signaling. Prior to gastrulation, DSmurf mutations cause a spatial increase in the DPP gradient, as evidenced by ventrolateral expansion in expression domains of target genes representing all known signaling thresholds. After gastrulation, DSmurf mutations cause a temporal delay in downregulation of earlier DPP signals, resulting in a lethal defect in hindgut organogenesis.

Animals↗
PubMed2002

Drosophila Roc1a encodes a RING-H2 protein with a unique function in processing the Hh signal transducer Ci by the SCF E3 ubiquitin ligase.

Substrate specificity of SCF E3 ubiquitin ligases is thought to be determined by the F box protein subunit. Another component of SCF complexes is provided by members of the Roc1/Rbx1/Hrt1 gene family, which encode RING-H2 proteins. Drosophila contains three members of this gene family. We show that Roc1a mutant cells fail to proliferate. Further, while the F box protein Slimb is required for Cubitus interruptus (Ci) and Armadillo/beta-catenin (Arm) proteolysis, Roc1a mutant cells hyperaccumulate Ci but not Arm. This suggests that Slimb and Roc1a function in the same SCF complex to target Ci but that a different RING-H2 protein acts with Slimb to target Arm. Consequently, the identity of the Roc subunit may contribute to the selection of substrates by metazoan SCF complexes.

Animals↗
PubMed2003

Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta.

Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells.

Animals↗
PubMed2003

Expression of the E3 SUMO-1 ligases PIASx and PIAS1 during spermatogenesis in the rat.

PIASx and PIAS1, two members of the conserved protein inhibitor of activated STAT (PIAS) family, are able to interact with and modulate activities of several distinct nuclear proteins, including androgen receptor (AR). PIASx and PIAS1 also function as E3-type ligases in small ubiquitin-related modifier 1 modifications. To gain better insight into the physiological roles of PIASx and PIAS1 in vivo, their cell-type specific expression and regulation were analyzed during testicular development and spermatogenesis in the rat. The expression of both PIASx and PIAS1 was low or undetectable in newborn rats. PIASx mRNA started to accumulate after day 20 of postnatal life, whereas expression of PIAS1 mRNA increased around day 30 after birth. In the adult rat testis, both PIASx and PIAS1 mRNA were present in Sertoli cells and in germ cells in the seminiferous epithelium at all stages. However, PIASx mRNA was more abundant in spermatocytes than in other cell types, whereas higher levels of PIAS1 mRNA were detected in late spermatocytes and round spermatids than in early spermatocytes. Since PIASx and PIAS1 accumulate in developing male germ cells, their regulatory functions are not only restricted to AR in Sertoli cells, but they also participate in molecular processes during meiosis.

Animals↗
PubMed1984

T4 RNA ligase mediated preparation of novel "chemically misacylated" tRNAPheS.

T4 RNA ligase was employed for the condensation of Escherichia coli tRNAPhe missing cytidine-75 and adenosine-76 (tRNAPhe-COH; the acceptor "oligomer") with each of several chemically acylated derivatives of pCpA (the donor "oligomer"). The resulting "chemically misacylated " tRNAPheS were obtained in 20-65% yields following chromatographic workup on DEAE-cellulose and benzoylated DEAE-cellulose. Characterization of the chemically misacylated tRNAs was accomplished by (i) enzymatic reaminoacylation of chemically misacylated tRNAPhe with phenylalanine by E. coli phenylalanyl-tRNA synthetase following chemical deacylation of the "incorrect" amino acid, (ii) comparison of the hydrolytic effects of Cu2+ solutions on chemically and enzymatically prepared samples of N-acetyl-L-phenylalanyl- tRNAPheS , and (iii) measurement of the chromatographic behavior of the tRNA species derived from chemical misacylation .

Escherichia coli↗
PubMed1985

A stereochemical study of the mechanism of activation of donor oligonucleotides by RNA ligase from bacteriophage T4 infected Escherichia coli.

RNA ligase from bacteriophage T4 infected Escherichia coli catalyzes the activation of adenosine 3',5'-bisphosphate (representing the donor oligonucleotide) by adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate with retention of configuration at P alpha. Since single-step enzyme-catalyzed nucleotidyl transfer reactions proceed with inversion, this stereochemical result provides support for a double displacement mechanism involving an adenylyl-enzyme intermediate as proposed previously from isotope exchange experiments.

Adenine Nucleotides↗
PubMed1986

Multiple forms of ubiquitin-protein ligase. Binding of activated ubiquitin to protein substrates.

Enzyme activities that catalyzed the covalent attachment of ubiquitin to protein substrates (ubiquitin-protein ligase, UbL) were purified from the extracts of human red blood cells. These activities required the presence of ubiquitin-activating enzyme and ATP for activity. Four fractions (UbL A, B1, B2, and C) were resolved and showed different specificities toward added substrates [carboxymethylated bovine serum albumin (CM-BSA), G-actin, lysozyme, and alpha-lactalbumin]. The enzyme fractions gave different products with a given substrate. UbL A and UbL B1 were exclusively active with CM-BSA and alpha-lactalbumin, respectively. UbL B2 was most active toward CM-BSA with substantial activities to G-actin and alpha-lactalbumin and with no activity to lysozyme. UbL C showed significant activities with all four substrates, having a highest activity toward CM-BSA. There were many endogenous proteins present in the erythrocyte extract which were efficient substrates for ubiquitin conjugation. In particular, a pair of substrates were identified from erythrocyte extracts which were far more efficient substrates than the denatured proteins exogenously added.

Chromatography↗
PubMed1978

T4 RNA Ligase joins 2'-deoxyribonucleoside 3',5'-bisphosphates to oligodeoxyribonucleotides.

T4 RNA ligase catalyzes the ATP-dependent addition of a single 2'-deoxyribonucleoside 3',5'-bisphosphate to the 3'-hydroxyl of an oligodeoxyribonucleotide. The bisphosphate is joined to the deoxyoligomer by a 3' leads to 5' phosphodiester bond and the product, which is terminated by a 3'-phosphate, is one nucleotide longer than the substrate. Bisphosphates of dAdo, dCyd, dGuo, dThd, and dUrd are donors and oligodeoxyribonucleotides with dA, dC, dG, dT, or dU 3' termini act as acceptors. The preferred residue for both donor and acceptor is dCyd. Deoxyoligomers from 3 to 12 residues in length are active as acceptors. To obtain good yields, high concentration of enzyme, long incubation time at low temperature, and manganous rather than Mg(II) ion are required. Under optimal conditions, yields calculated with respect to deoxyoligomer converted to product vary from 40 to greater than 95%. The turnover number of the enzyme for DNA joining is extremely low but, because the preparation is nearly free of DNases, there is less than 3% degradation of substrate or product after 6 days of reaction. We anticipate that this reaction will serve as the basis for a method for the stepwise enzymatic synthesis of DNA of defined sequence.

Adenosine Triphosphate↗
PubMed2000

The dual function steroid receptor coactivator/ubiquitin protein-ligase integrator E6-AP is overexpressed in mouse mammary tumorigenesis.

Steroid receptor coactivator and corepressor proteins are important mediators of steroid receptor function. Changes in the expression or activity of these limiting cofactors can contribute to the etiology of steroidal cancers. Using a mouse mammary model of multistage tumorigenesis we have examined whether the expression of select steroid receptor coactivators is altered. The 10 kb transcript of the novel dual function steroid receptor coactivator/ubiquitin protein-ligase integrator E6-AP is overexpressed 2.5-4.5 fold in the mammary tumors but not in the precursor steps of tumorigenesis; that is, immortal ductal and alveolar hyperplastic outgrowths. The over expression is striking because the 10 kb transcript is expressed to variable levels in other wild type tissues like the uterus, ovary, testis, kidney and brain but is undetectable in normal virgin mammary gland and the prostate gland. The E6-AP overexpression in the mammary tumors is substantiated by western blot analysis and immunohistochemical analysis. Absence of ER and PR in these tumors in the presence of high levels of E6-AP could contribute to steroid receptor-independent function and tumorigenesis. There is no obvious correlation between p53 (a well-characterized substrate of E6-AP) status (wt vs. mutant) and levels of E6-AP in the mouse mammary tumors.

Animals↗
PubMed1999

A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation.

The TGF-beta superfamily of proteins regulates many different biological processes, including cell growth, differentiation and embryonic pattern formation. TGF-beta-like factors signal across cell membranes through complexes of transmembrane receptors known as type I and type II serine/threonine-kinase receptors, which in turn activate the SMAD signalling pathway. On the inside of the cell membrane, a receptor-regulated class of SMADs are phosphorylated by the type-I-receptor kinase. In this way, receptors for different factors are able to pass on specific signals along the pathway: for example, receptors for bone morphogenetic protein (BMP) target SMADs 1, 5 and 8, whereas receptors for activin and TGF-beta target SMADs 2 and 3. Phosphorylation of receptor-regulated SMADs induces their association with Smad4, the 'common-partner' SMAD, and stimulates accumulation of this complex in the nucleus, where it regulates transcriptional responses. Here we describe Smurf1, a new member of the Hect family of E3 ubiquitin ligases. Smurf1 selectively interacts with receptor-regulated SMADs specific for the BMP pathway in order to trigger their ubiquitination and degradation, and hence their inactivation. In the amphibian Xenopus laevis, Smurf1 messenger RNA is localized to the animal pole of the egg; in Xenopus embryos, ectopic Smurf1 inhibits the transmission of BMP signals and thereby affects pattern formation. Smurf1 also enhances cellular responsiveness to the Smad2 (activin/TGF-beta) pathway. Thus, targeted ubiquitination of SMADs may serve to control both embryonic development and a wide variety of cellular responses to TGF-beta signals.

Animals↗
PubMed2001

Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation.

In eukaryotes, endoplasmic reticulum-associated degradation (ERAD) functions in cellular quality control and regulation of normal ER-resident proteins. ERAD proceeds by the ubiquitin-proteasome pathway, in which the covalent attachment of ubiquitin to proteins targets them for proteasomal degradation. Ubiquitin-protein ligases (E3s) play a crucial role in this process by recognizing target proteins and initiating their ubiquitination. Here we show that Hrd1p, which is identical to Der3p, is an E3 for ERAD. Hrd1p is required for the degradation and ubiquitination of several ERAD substrates and physically associates with relevant ubiquitin-conjugating enzymes (E2s). A soluble Hrd1 fusion protein shows E3 activity in vitro - catalysing the ubiquitination of itself and test proteins. In this capacity, Hrd1p has an apparent preference for misfolded proteins. We also show that Hrd1p functions as an E3 in vivo, using only Ubc7p or Ubc1p to specifically program the ubiquitination of ERAD substrates.

Catalysis↗
PubMed2002

E3 ubiquitin ligase that recognizes sugar chains.

N-glycosylation of proteins in the endoplasmic reticulum (ER) has a central role in protein quality control. Here we report that N-glycan serves as a signal for degradation by the Skp1-Cullin1-Fbx2-Roc1 (SCF(Fbx2)) ubiquitin ligase complex. The F-box protein Fbx2 (ref. 4) binds specifically to proteins attached to N-linked high-mannose oligosaccharides and subsequently contributes to ubiquitination of N-glycosylated proteins. Pre-integrin beta 1 is a target of Fbx2; these two proteins interact in the cytosol after inhibition of the proteasome. In addition, expression of the mutant Fbx2 Delta F, which lacks the F-box domain that is essential for forming the SCF complex, appreciably blocks degradation of typical substrates of the ER-associated degradation pathway. Our results indicate that SCF(Fbx2) ubiquitinates N-glycosylated proteins that are translocated from the ER to the cytosol by the quality control mechanism.

Animals↗
PubMed2003

CUL-4 ubiquitin ligase maintains genome stability by restraining DNA-replication licensing.

To maintain genome stability, DNA replication is strictly regulated to occur only once per cell cycle. In eukaryotes, the presence of 'licensing proteins' at replication origins during the G1 cell-cycle phase allows the formation of the pre-replicative complex. The removal of licensing proteins from chromatin during the S phase ensures that origins fire only once per cell cycle. Here we show that the CUL-4 ubiquitin ligase temporally restricts DNA-replication licensing in Caenorhabditis elegans. Inactivation of CUL-4 causes massive DNA re-replication, producing cells with up to 100C DNA content. The C. elegans orthologue of the replication-licensing factor Cdt1 (refs 2, 3) is required for DNA replication. C. elegans CDT-1 is present in G1-phase nuclei but disappears as cells enter S phase. In cells lacking CUL-4, CDT-1 levels fail to decrease during S phase and instead remain constant in the re-replicating cells. Removal of one genomic copy of cdt-1 suppresses the cul-4 re-replication phenotype. We propose that CUL-4 prevents aberrant re-initiation of DNA replication, at least in part, by facilitating the degradation of CDT-1.

Animals↗
PubMed2002

A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies.

Membrane proteins with transmembrane domains (TMDs) that contain polar residues exposed to the lipid bilayer are selectively sorted into multivesicular bodies (MVBs) and delivered to the yeast vacuole. Sorting of some, although not all, proteins into these structures is mediated by ubiquitination. We have identified a transmembrane ubiquitin ligase, Tul1, that is resident in the Golgi apparatus and is required for the ubiquitination of proteins with polar TMDs, including vacuolar proteins such as carboxypeptidase S. We suggest that Tul1 provides quality control, identifying misfolded membrane proteins and marking them for transport to endosomes and degradation in the vacuole.

Amino Acid Sequence↗
PubMed1997

De novo truncating mutations in E6-AP ubiquitin-protein ligase gene (UBE3A) in Angelman syndrome.

Angelman syndrome (AS) is associated with maternal deletions of human chromosome 15q11-q13 and with paternal uniparental disomy for this region indicating that deficiency of an imprinted, maternally expressed gene within the critical interval is the likely cause of the syndrome. Although the gene for E6-AP ubiquitin-protein ligase (UBE3A) was mapped to the critical region for AS, evidence of expression from both parental alleles initially suggested that it was an unlikely candidate gene for this disorder. Because attempts to identify any novel maternally expressed transcripts were unsuccessful and because the UBE3A gene remained within a narrowed AS critical region, we searched for mutations in UBE3A in 11 AS patients without known molecular defects (large deletion, uniparental disomy, or imprinting mutation). This analysis tested the possibility that deficiency of an undefined, maternally expressed transcript or isoform of the UBE3A gene could cause AS. Four mutations were identified including a de novo frameshift mutation and a de novo nonsense mutation in exon 3 and two missense mutations of less certain significance. The de novo truncating mutations indicate that UBE3A is the AS gene and suggest the possibility of a maternally expressed gene product in addition to the biallelically expressed transcript. Intragenic mutation of UBE3A in AS is the first example of a genetic disorder of the ubiquitin-dependent proteolytic pathway in mammals. It may represent an example of a human genetic disorder associated with a locus producing functionally distinct imprinted and biallelically expressed gene products.

Angelman Syndrome↗
PubMed1998

The itchy locus encodes a novel ubiquitin protein ligase that is disrupted in a18H mice.

Non-agouti-lethal 18H (a18H) mice are dark agouti with black pinna hairs. What makes these mice unique is that they develop a spectrum of immunological diseases not seen in other agouti mutant mice. On the JU/Ct background, a18H mice develop an inflammatory disease of the large intestine. On the C57BL/6J background, they develop a fatal disease characterized by pulmonary chronic interstitial inflammation and alveolar proteinosis, inflammation of the glandular stomach and skin resulting in scarring due to constant itching, and hyperplasia of lymphoid cells, haematopoietic cells and the forestomach epithelium. Previous studies suggested that the a18H mutation results from a paracentric inversion that affects two loci: agouti and another, as yet unidentified locus designated itchy (the provisional gene symbol is Itch), that is responsible for the immunological phenotype of a18H mice. Here we confirm that a18H results from an inversion and show that Itch encodes a novel E3 ubiquitin protein ligase, a protein involved in ubiquitin-mediated protein degradation. Our results indicate that ubiquitin-dependent proteolysis is an important mediator of the immune response in vivo and provide evidence for Itch's role in inflammation and the regulation of epithelial and haematopoietic cell growth.

Amino Acid Sequence↗
PubMed2001

MID1, mutated in Opitz syndrome, encodes an ubiquitin ligase that targets phosphatase 2A for degradation.

The gene MID1, the mutation of which causes X-linked Opitz G/BBB syndrome (OS, MIM 300000), encodes a microtubule-associated protein (MAP). We show that mutation of MID1 leads to a marked accumulation of the catalytic subunit of protein phosphatase 2A (PP2Ac), a central cellular regulator. PP2Ac accumulation is caused by an impairment of a newly identified E3 ubiquitin ligase activity of the MID1 protein that normally targets PP2Ac for degradation through binding to its alpha4 regulatory subunit in an embryonic fibroblast line derived from a fetus with OS. Elevated PP2Ac causes hypophosphorylation of MAPs, a pathological mechanism that is consistent with the OS phenotype.

Animals↗