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Protein Ligase (protein + ligase)

Distribution by Scientific Domains

Chemistry	66%

Selected Abstracts

Rnf19a, a ubiquitin protein ligase, and Psmc3, a component of the 26S proteasome, Tether to the acrosome membranes and the head,tail coupling apparatus during rat spermatid development

DEVELOPMENTAL DYNAMICS, Issue 7 2009
Eugene Rivkin
Abstract We report the cDNA cloning of rat testis Rnf19a, a ubiquitin protein ligase, and show 98% and 93% protein sequence identity of testicular mouse and human Rnf19a, respectively. Rnf19a interacts with Psmc3, a protein component of the 19S regulatory cap of the 26S proteasome. During spermatid development, Rnf19a and Psmc3 are initially found in Golgi-derived proacrosomal vesicles. Later on, Rnf19a, Psmc3, and ubiquitin are seen along the cytosolic side of the acrosomal membranes and the acroplaxome, a cytoskeletal plate linking the acrosome to the spermatid nuclear envelope. Rnf19a and Psmc3 accumulate at the acroplaxome marginal ring,manchette perinuclear ring region during spermatid head shaping and in the developing sperm head,tail coupling apparatus and tail. Rnf19a and Psmc3 may interact directly or indirectly with each other, presumably pointing to the participation of the ubiquitin,proteasome system in acrosome biogenesis, spermatid head shaping, and development of the head-tail coupling apparatus and tail. Developmental Dynamics 238:1851,1861, 2009. © 2009 Wiley-Liss, Inc. [source]

Inhibition of the D -alanine:D -alanyl carrier protein ligase from Bacillus subtilis increases the bacterium's susceptibility to antibiotics that target the cell wall

FEBS JOURNAL, Issue 12 2005
Juergen J. May
The surface charge as well as the electrochemical properties and ligand binding abilities of the Gram-positive cell wall is controlled by the d -alanylation of the lipoteichoic acid. The incorporation of d -Ala into lipoteichoic acid requires the d -alanine:d -alanyl carrier protein ligase (DltA) and the carrier protein (DltC). We have heterologously expressed, purified, and assayed the substrate selectivity of the recombinant proteins DltA with its substrate DltC. We found that apo-DltC is recognized by both endogenous 4,-phosphopantetheinyl transferases AcpS and Sfp. After the biochemical characterization of DltA and DltC, we designed an inhibitor (d -alanylacyl-sulfamoyl-adenosine), which is able to block the d -Ala adenylation by DltA at a Ki value of 232 nmin vitro. We also performed in vivo studies and determined a significant inhibition of growth for different Bacillus subtilis strains when the inhibitor is used in combination with vancomycin. [source]

Biotinylation in the hyperthermophile Aquifex aeolicus

FEBS JOURNAL, Issue 6 2003
Isolation of a cross-linked BPL:BCCP complex
Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl CoA carboxylase and this post-translational modification of a single lysine residue is exceptionally specific. The exact details of the protein,protein interactions involved are unclear as a BPL:BCCP complex has not yet been isolated. Moreover, detailed information is lacking on the composition, biosynthesis and role of fatty acids in hyperthermophilic organisms. We have cloned, overexpressed and purified recombinant BPL and the biotinyl domain of BCCP (BCCP,67) from the extreme hyperthermophile Aquifex aeolicus. In vitro assays have demonstrated that BPL catalyses biotinylation of lysine 117 on BCCP,67 at temperatures of up to 70 °C. Limited proteolysis of BPL with trypsin and chymotrypsin revealed a single protease-sensitive site located 44 residues from the N-terminus. This site is adjacent to the predicted substrate-binding site and proteolysis of BPL is significantly reduced in the presence of MgATP and biotin. Chemical crosslinking with 1-ethyl-3-(dimethylamino-propyl)-carbodiimide (EDC) allowed the isolation of a BPL:apo-BCCP,67 complex. Furthermore, this complex was also formed between BPL and a BCCP,67 mutant lacking the lysine residue (BCCP,67 K117L) however, complex formation was considerably reduced using holo-BCCP,67. These observations provide evidence that addition of the biotin prosthetic group reduces the ability of BCCP,67 to heterodimerize with BPL, and emphasizes that a network of interactions between residues on both proteins mediates protein recognition. [source]

The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activity

PROTEIN SCIENCE, Issue 12 2001
Anne Chapman-Smith
BCCP, biotin carboxyl carrier protein; IPTG, isopropyl-1-thio-,-D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; S.D., standard deviation Abstract Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5,-AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA. No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP. Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain. [source]

Anaesthesia for an adult with Angelman syndrome

ANAESTHESIA, Issue 11 2009
M. Maguire
Summary Angelman syndrome is a complex genetic condition involving abnormalities of chromosome 15 in the majority of cases. These defects involve a gene encoding an ubiquitin protein ligase and may be associated with abnormal ,-aminobutyric acid (GABA)A receptor subunits. Angelman syndrome may have profound implications for anaesthesia: potential exists for airway difficulties; refractory bradyarrythmias; and pharmacodynamic unpredictability. A case of an adult with Angelman syndrome undergoing dental work under general anaesthesia is presented. Induction and maintenance of anaesthesia was unremarkable but emergence was complicated by generalised muscular hypertonia and temporary respiratory embarrassment which resolved spontaneously. [source]

Remodeling of the SCF complex-mediated ubiquitination system by compositional alteration of incorporated F-box proteins

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2010
Mitsunori Kato
Abstract Ubiquitination regulates not only the stability but the localization and activity of substrate proteins involved in a plethora of cellular processes. The Skp1,Cullin,F-box protein (SCF) complexes constitute a major family of ubiquitin protein ligases, in each member of which an F-box protein serves as the variable component responsible for substrate recognition, thereby defining the function of each complex. Here we studied whether the composition of F-box proteins in the SCF complexes is remodeled under different conditions. We exploited stable isotope labeling and MS for relative quantification of F-box proteins in the SCF complexes affinity-purified en masse from budding yeast cells at log and post-diauxic phases, and revealed an increment of Saf1, an F-box protein involved in entry into quiescence, during the diauxic shift. Similarly, we found that Met4 overexpression induces a specific increment of Met30, the F-box protein responsible for ubiquitination of Met4. These results illustrate a cellular response to environmental and genetic perturbations through remodeling of the SCF complex-mediated ubiquitination system. Compositional alteration of incorporated F-box proteins may redirect the activity of this system toward appropriate substrates to be ubiquitinated under individual conditions for the maintenance of cellular homeostasis. [source]