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At least 1,171 records · Page 65Linked to original sources
PubMed2005

Promiscuous fatty acyl CoA ligases produce acyl-CoA and acyl-SNAC precursors for polyketide biosynthesis.

The study of bioactive natural products has undergone rapid advancement with the cloning and sequencing of large number of gene clusters and the concurrent progress to manipulate complex biosynthetic systems in heterologous hosts. The genetic reconstitution necessitates that the heterologous hosts possess substrate pools that could be coordinately supplied for biosynthesis. Polyketide synthases (PKS) utilize acyl-coenzyme A (CoA) precursors and synthesize polyketides by repetitive decarboxylative condensations. Here we show that acyl-CoA ligases, which belong to a large family of acyl-activating enzymes, possess potential to produce varied starter CoA precursors that could be utilized in polyketide biosynthesis. Incidentally, such protein domains have been recognized in several PKS and nonribosomal peptide synthetase gene clusters. Our studies with mycobacterial fatty acyl-CoA ligases (FACLs) show remarkable tolerance to activate a variety of fatty acids that contain modifications at alpha, beta, omega, and omega-nu positions. This substrate flexibility extends further such that these proteins also efficiently utilize N-acetyl cysteamine, the shorter acceptor terminal portion of CoASH, to produce acyl-SNACs. We show that the in situ generated acyl-CoAs and acyl-SNACs could be channeled to types I and -III PKS systems to produce new metabolites. Together, the promiscuous activity of FACL and PKSs provides new opportunities to expand the repertoire of natural products.

Acetyl Coenzyme A↗
PubMed1977

Potential inhibitors of collagen biosynthesis. 5,5-Difluoro-DL-lysine and 5,5-dimethyl-DL-lysine and their activation by lysyl-tRNA ligase.

The synthesis of lysine analogues wherein blocking groups are substituted at position 5, the site of hydroxylation by peptidyl lysine hydroxylase, is described. Thus, 5,5-difluorolysine (1) and 5,5-dimethylysine (2) were synthesized via a four- and six-step sequence, respectively, starting from ketone precursors. The propensity for these lysine analogues to be incorporated into procollagen protein in vivo was assessed by their ability to stimulate the lysine-dependent ATP-PPi exchange reaction in the presence of lysyl-tRNA ligase in vitro. The difluoro analogue 1 stimulated exchange, but at a Km (1.3 X 10(-3) M) 1000 times greater than that for lysine itself. The dimethyl analogue 2 did not stimulate exchange, but at high concentrations was a competitive inhibitor of lysine, with an apparent Ki of 1.6 X 10(-2) M. Thus, electronegative and/or bulky substituents at the 5 position of lysine cannot be tolerated by lysyl-tRNA ligase, and this position must be kept free in lysine analogues specifically designed to block collagen biosynthesis.

Amino Acyl-tRNA Synthetases↗
PubMed2004

Stable and specific expression of 4-coumarate:coenzyme A ligase gene (4CL1) driven by the xylem-specific Pto4CL1 promoter in the transgenic tobacco.

The ability of 4-coumarate:coenzyme A ligase promoter from Populus tomentosa (Pto4CL1p) to drive expression of the GUS reporter gene and 4-coumarate:coenzyme A ligase gene in tobacco has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Intense GUS histochemical staining was detected in the xylem of stem in transgenic tobacco plants carrying the 1140 bp Pto4CL1p promoter. To further investigate the regulation function of the tissue-specific expression promoter, Pto4CL1p, a binary vector containing Pto4CL1p promoter fused with 4CL1 gene was transferred into tobacco. The activity of the 4CL1 enzyme doubled in the stems of transgenic tobacco but did not increase in the leaves. The content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.

Base Sequence↗
PubMed2004

The purification and characterisation of 4-chlorobenzoate:CoA ligase and 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain TM-1.

4-Chlorobenzoate:CoA ligase, the first enzyme in the pathway for 4-chlorobenzoate dissimilation, has been partially purified from Arthrobacter sp. strain TM-1, by sequential ammonium sulphate precipitation and chromatography on DEAE-Sepharose and Sephacryl S-200. The enzyme, a homodimer of subunit molecular mass approximately 56 kD, is dependent on Mg2+-ATP and coenzyme A, and produces 4-chlorobenzoyl CoA and AMP. Besides Mg2+, Mn2+, Co2+, Fe2+ and Zn2+ are also stimulatory, but not Ca2+. Maximal activity is exhibited at pH 7.0 and 25 degrees C. The ligase demonstrates broad specificity towards other halobenzoates, with 4-chlorobenzoate as best substrate. The apparent Michaelis constants (Km) of the enzyme for 4-chlorobenzoate, CoA and ATP were determined as 3.5, 30 and 238 microM respectively. 4-Chlorobenzoyl CoA dehalogenase, the second enzyme, has been purified to homogeneity by sequential column chromatography on hydroxyapatite, DEAE-Sepharose and Sephacryl S-200. It is a homotetramer of 33 kD subunits with an isoelectric point of 6.4. At pH 7.5 and 30 degrees C, Km and kcat for 4-CBCoA are 9 microM and 1 s(-1) respectively. The optimum pH is 7.5, and maximal enzymic activity occurs at 45 degrees C. The properties of this enzyme are compared with those of the 4-chlorobenzoyl CoA dehalogenases from Arthrobacter sp. strain 4-CB1 and Pseudomonas sp. strain CBS-3, which differ variously in their N-terminal amino acid sequences, optimal pH values, pI values and/or temperatures of maximal activity.

Arthrobacter↗
PubMed2003

BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3.

Programmed destruction of regulatory proteins through the ubiquitin-proteasome system is a widely used mechanism for controlling signalling pathways. Cullins are proteins that function as scaffolds for modular ubiquitin ligases typified by the SCF (Skp1-Cul1-F-box) complex. The substrate selectivity of these E3 ligases is dictated by a specificity module that binds cullins. In the SCF complex, this module is composed of Skp1, which binds directly to Cul1, and a member of the F-box family of proteins. F-box proteins bind Skp1 through the F-box motif, and substrates by means of carboxy-terminal protein interaction domains. Similarly, Cul2 and Cul5 interact with BC-box-containing specificity factors through the Skp1-like protein elongin C. Cul3 is required for embryonic development in mammals and Caenorhabditis elegans but its specificity module is unknown. Here we report the identification of a large family of BTB-domain proteins as substrate-specific adaptors for C. elegans CUL-3. Biochemical studies using the BTB protein MEL-26 and its genetic target MEI-1 (refs 12, 13) indicate that BTB proteins merge the functional properties of Skp1 and F-box proteins into a single polypeptide.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex.

SUMO-1 (for small ubiquitin-related modifier) belongs to the ubiquitin (Ub) and ubiquitin-like (Ubl) protein family. SUMO conjugation occurs on specific lysine residues within protein targets, regulating pathways involved in differentiation, apoptosis, the cell cycle and responses to stress by altering protein function through changes in activity or cellular localization or by protecting substrates from ubiquitination. Ub/Ubl conjugation occurs in sequential steps and requires the concerted action of E2 conjugating proteins and E3 ligases. In addition to being a SUMO E3, the nucleoporin Nup358/RanBP2 localizes SUMO-conjugated RanGAP1 to the cytoplasmic face of the nuclear pore complex by means of interactions in a complex that also includes Ubc9, the SUMO E2 conjugating protein. Here we describe the 3.0-A crystal structure of a four-protein complex of Ubc9, a Nup358/RanBP2 E3 ligase domain (IR1-M) and SUMO-1 conjugated to the carboxy-terminal domain of RanGAP1. Structural insights, combined with biochemical and kinetic data obtained with additional substrates, support a model in which Nup358/RanBP2 acts as an E3 by binding both SUMO and Ubc9 to position the SUMO-E2-thioester in an optimal orientation to enhance conjugation.

Amino Acid Sequence↗
PubMed2006

Spatially regulated ubiquitin ligation by an ER/nuclear membrane ligase.

The ubiquitin system targets many cellular proteins. Doa10 (also known as Ssm4), a yeast transmembrane ubiquitin ligase (E3), resides in the endoplasmic reticulum (ER), but it attaches ubiquitin to soluble proteins that concentrate in the nucleus. A central question is how nuclear substrates gain access to an enzyme in the ER. Here we show that Doa10 reaches the inner nuclear membrane. A subcomplex of nuclear pore subunits is important for this transport. Notably, another ER transmembrane E3, Hrd1 (also known as Der3), cannot localize efficiently to the inner nuclear membrane. Tethering Doa10 at the cell periphery inhibits degradation of soluble nuclear substrates but not cytoplasmic ones. If Doa10 is released from these peripheral sites, localization of Doa10 to the nuclear envelope and degradation of its nuclear substrates are restored in parallel. Thus, localization of Doa10 to the inner nuclear membrane is necessary for nuclear substrate degradation. These data indicate that different membrane ubiquitin ligases are spatially sorted within the ER-nuclear envelope membrane system and that this differential localization contributes to their specificity.

Endoplasmic Reticulum↗
PubMed2006

Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery.

Protein ubiquitination is a common form of post-translational modification that regulates a broad spectrum of protein substrates in diverse cellular pathways. Through a three-enzyme (E1-E2-E3) cascade, the attachment of ubiquitin to proteins is catalysed by the E3 ubiquitin ligase, which is best represented by the superfamily of the cullin-RING complexes. Conserved from yeast to human, the DDB1-CUL4-ROC1 complex is a recently identified cullin-RING ubiquitin ligase, which regulates DNA repair, DNA replication and transcription, and can also be subverted by pathogenic viruses to benefit viral infection. Lacking a canonical SKP1-like cullin adaptor and a defined substrate recruitment module, how the DDB1-CUL4-ROC1 E3 apparatus is assembled for ubiquitinating various substrates remains unclear. Here we present crystallographic analyses of the virally hijacked form of the human DDB1-CUL4A-ROC1 machinery, which show that DDB1 uses one beta-propeller domain for cullin scaffold binding and a variably attached separate double-beta-propeller fold for substrate presentation. Through tandem-affinity purification of human DDB1 and CUL4A complexes followed by mass spectrometry analysis, we then identify a novel family of WD40-repeat proteins, which directly bind to the double-propeller fold of DDB1 and serve as the substrate-recruiting module of the E3. Together, our structural and proteomic results reveal the structural mechanisms and molecular logic underlying the assembly and versatility of a new family of cullin-RING E3 complexes.

Amino Acid Motifs↗
PubMed2004

Targeted ubiquitination of CDT1 by the DDB1-CUL4A-ROC1 ligase in response to DNA damage.

Cullins assemble a potentially large number of ubiquitin ligases by binding to the RING protein ROC1 to catalyse polyubiquitination, as well as binding to various specificity factors to recruit substrates. The Cul4A gene is amplified in human breast and liver cancers, and loss-of-function of Cul4 results in the accumulation of the replication licensing factor CDT1 in Caenorhabditis elegans embryos and ultraviolet (UV)-irradiated human cells. Here, we report that human UV-damaged DNA-binding protein DDB1 associates stoichiometrically with CUL4A in vivo, and binds to an amino-terminal region in CUL4A in a manner analogous to SKP1, SOCS and BTB binding to CUL1, CUL2 and CUL3, respectively. As with SKP1-CUL1, the DDB1-CUL4A association is negatively regulated by the cullin-associated and neddylation-dissociated protein, CAND1. Recombinant DDB1 and CDT1 bind directly to each other in vitro, and ectopically expressed DDB1 bridges CDT1 to CUL4A in vivo. Silencing DDB1 prevented UV-induced rapid CDT1 degradation in vivo and CUL4A-mediated CDT1 ubiquitination in vitro. We suggest that DDB1 targets CDT1 for ubiquitination by a CUL4A-dependent ubiquitin ligase, CDL4A(DDB1), in response to UV irradiation.

Amino Acid Sequence↗
PubMed2005

Membrane-bound Ubx2 recruits Cdc48 to ubiquitin ligases and their substrates to ensure efficient ER-associated protein degradation.

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is a quality control system that removes misfolded proteins from the ER. ERAD substrates are channelled from the ER via a proteinacious pore to the cytosolic ubiquitin-proteasome system - a process involving dedicated ubiquitin ligases and the chaperone-like AAA ATPase Cdc48 (also known as p97). How the activities of these proteins are coupled remains unclear. Here we show that the UBX domain protein Ubx2 is an integral ER membrane protein that recruits Cdc48 to the ER. Moreover, Ubx2 mediates binding of Cdc48 to the ubiquitin ligases Hrd1 and Doa10, and to ERAD substrates. In addition, Ubx2 and Cdc48 interact with Der1 and Dfm1, yeast homologues of the putative dislocation pore protein Derlin-1 (refs 11-13). Lack of Ubx2 causes defects in ERAD that are exacerbated under stress conditions. These findings are consistent with a model in which Ubx2 coordinates the assembly of a highly efficient ERAD machinery at the ER membrane.

Adenosine Triphosphatases↗
PubMed2005

Ubiquitin ligase component Cul4 associates with Clr4 histone methyltransferase to assemble heterochromatin.

In eukaryotes, heterochromatin mediates diverse processes including gene silencing and regulation of long-range chromatin interactions. The formation of heterochromatin involves a conserved array of histone modifications; in particular, methylation of histone H3 at Lys 9 (H3K9me) is essential for recruiting HP1/Swi6 proteins. In fission yeast, the Clr4 methyltransferase is responsible for H3K9me across all heterochromatic domains. However, the mechanism of Clr4 recruitment to these loci is poorly understood. We show that Clr4 associates with Cul4, a cullin family protein that serves as a scaffold for assembling ubiquitin ligases. Mutations in Cul4 result in defective localization of Clr4 and loss of silencing at heterochromatic loci. This is accompanied by a severe reduction in H3K9me and Swi6 levels, and accumulation of transcripts corresponding to naturally silenced repeat elements within heterochromatic domains. Moreover, heterochromatin defects in Cul4 mutants could not be rescued by expression of Cul4 protein lacking Nedd8 modification, which is essential for its ubiquitin ligase activity. Rik1, a protein related to DNA damage binding protein DDB1 and required for H3K9me, also interacts with Cul4, the association of which might serve to target Clr4 to heterochromatic loci. These analyses uncover a role for Cul4-based protein ubiquitination in regulating H3K9me and heterochromatin formation.

Cell Cycle Proteins↗
PubMed2003

A novel ubiquitin ligase is deficient in Fanconi anemia.

Fanconi anemia is a recessively inherited disease characterized by congenital defects, bone marrow failure and cancer susceptibility. Cells from individuals with Fanconi anemia are highly sensitive to DNA-crosslinking drugs, such as mitomycin C (MMC). Fanconi anemia proteins function in a DNA damage response pathway involving breast cancer susceptibility gene products, BRCA1 and BRCA2 (refs. 1,2). A key step in this pathway is monoubiquitination of FANCD2, resulting in the redistribution of FANCD2 to nuclear foci containing BRCA1 (ref. 3). The underlying mechanism is unclear because the five Fanconi anemia proteins known to be required for this ubiquitination have no recognizable ubiquitin ligase motifs. Here we report a new component of a Fanconi anemia protein complex, called PHF9, which possesses E3 ubiquitin ligase activity in vitro and is essential for FANCD2 monoubiquitination in vivo. Because PHF9 is defective in a cell line derived from an individual with Fanconi anemia, we conclude that PHF9 (also called FANCL) represents a novel Fanconi anemia complementation group (FA-L). Our data suggest that PHF9 has a crucial role in the Fanconi anemia pathway as the likely catalytic subunit required for monoubiquitination of FANCD2.

Amino Acid Sequence↗
PubMed2004

E3 ubiquitin ligases as T cell anergy factors.

E3 ubiquitin ligases have emerged as key molecular regulators of immune cell function. Three families of proteins with ubiquitin ligase activity have been described (the HECT, RING and U-box proteins), and each may be involved in the regulation of immune responses during infection by targeting specific inhibitory molecules for proteolytic destruction. Several HECT and RING E3 proteins have now also been linked to the induction and maintenance of immune self-tolerance: c-Cbl, Cbl-b, GRAIL, Itch and Nedd4 each negatively regulate T cell growth factor production and proliferation. This review will discuss the relationship between the ubiquitination of select components of the antigen-sensing signaling apparatus in T cells and the development and maintenance of the clonal anergy state.

Animals↗
PubMed2004

The SCF ubiquitin ligase: insights into a molecular machine.

Ubiquitin ligases are well suited to regulate molecular networks that operate on a post-translational timescale. The F-box family of proteins - which are the substrate-recognition components of the Skp1-Cul1-F-box-protein (SCF) ubiquitin ligase - are important players in many mammalian functions. Here we explore a unifying and structurally detailed view of SCF-mediated proteolytic control of cellular processes that has been revealed by recent studies.

Animals↗
PubMed2007

Regulation of the Drosophila ubiquitin ligase DIAP1 is mediated via several distinct ubiquitin system pathways.

Inhibitors of apoptosis proteins (IAPs) suppress cell death by inactivating proapoptotic regulators, and therefore play important roles in controlling apoptosis in normal and malignant cells. Many IAPs are ubiquitin ligases, and their activity is mediated via ubiquitination and subsequent degradation of their targets. Here we corroborate a previous observation that DIAP1 (Drosophila IAP1) can be degraded via a two-step mechanism: (i) limited caspase-mediated cleavage and (ii) degradation of the released fragment via the ubiquitin N-end rule pathway. Yet, we demonstrate that this pathway is not the only one involved in DIAP1 degradation, and the intact protein can be degraded independent of prior caspase cleavage. Importantly, this mode of degradation does not require the RING-finger-mediated autoubiquitinating activity of DIAP1, believed to target many RING-finger E3s for self-destruction. Our preliminary data suggest that DIAP2 mediates DIAP1 degradation, suggesting a novel regulatory loop within the apoptotic pathway. Studying the role of the autoubiquitinating activity of DIAP1, we demonstrate that it does not involve formation of Lys48-based polyubiquitin chains, but probably chains linked via Lys63. Our preliminary data suggest that the autoubiquitination serves to attenuate the ligase activity of DIAP1 towards its exogenous substrates.

Animals↗
PubMed2004

Bsd2 binds the ubiquitin ligase Rsp5 and mediates the ubiquitination of transmembrane proteins.

Membrane proteins destined for the vacuolar or lysosomal lumen are typically ubiquitinated, the ubiquitin serving as a targeting signal for the multivesicular body pathway. The RING-domain ubiquitin ligase Tul1 is an integral membrane protein that modifies the yeast vacuolar enzyme carboxypeptidase S (Cps1), the polyphosphatase Ppn1/Phm5 and other proteins containing exposed hydrophilic residues within their transmembrane domains (TMDs). Here we show that Bsd2 provides an alternative ubiquitination mechanism for Cps1, Phm5 and other proteins. Bsd2 is a three-TMD protein with a PPXY motif that binds the HECT domain ubiquitin ligase Rsp5. It can thus act as a specific adaptor linking Rsp5 to its substrates. Like Tul1, the Bsd2 system recognises polar TMDs. Bsd2 also controls the vacuolar targeting of a manganese transporter and a mutant plasma membrane ATPase, and together with the ER retrieval receptor Rer1, it protects cells from stress. We suggest that Bsd2 has a wide role in the quality control of membrane proteins. Bsd2 is the yeast homologue of human NEDD4 binding protein N4WBP5, which may therefore have similar functions.

Amino Acid Sequence↗
PubMed2005

The Rsp5 ubiquitin ligase is coupled to and antagonized by the Ubp2 deubiquitinating enzyme.

Saccharomyces cerevisiae Rsp5 is an essential HECT ubiquitin ligase involved in several biological processes. To gain further insight into regulation of this enzyme, we identified proteins that copurified with epitope-tagged Rsp5. Ubp2, a deubiquitinating enzyme, was a prominent copurifying protein. Rup1, a previously uncharacterized UBA domain protein, was required for binding of Rsp5 to Ubp2 both in vitro and in vivo. Overexpression of Ubp2 or Rup1 in the rsp5-1 mutant elicited a strong growth defect, while overexpression of a catalytically inactive Ubp2 mutant or Rup1 deleted of the UBA domain did not, suggesting an antagonistic relationship between Rsp5 and the Ubp2/Rup1 complex. Consistent with this model, rsp5-1 temperature sensitivity was suppressed by either ubp2Delta or rup1Delta mutations. Ubp2 reversed Rsp5-catalyzed substrate ubiquitination in vitro, and Rsp5 and Ubp2 preferentially assembled and disassembled, respectively, K63-linked polyubiquitin chains. Together, these results indicate that Rsp5 activity is modulated by being physically coupled to the Rup1/Ubp2 deubiquitinating enzyme complex, representing a novel mode of regulation for an HECT ubiquitin ligase.

Endopeptidases↗
PubMed2006

PDGF regulates the actin cytoskeleton through hnRNP-K-mediated activation of the ubiquitin E3-ligase MIR.

PDGF is a potent chemotactic mitogen and a strong inductor of fibroblast motility. In Swiss 3T3 fibroblasts, exposure to PDGF but not EGF or IGF-1 causes a rapid loss of actin stress fibers (SFs) and focal adhesions (FAs), which is followed by the development of retractile dendritic protrusions and induction of motility. The PDGF-specific actin reorganization was blocked by inhibition of Src-kinase and the 26S proteasome. PDGF induced Src-dependent association between the multifunctional transcription/translation regulator hnRNP-K and the mRNA-encoding myosin regulatory light-chain (MRLC)-interacting protein (MIR), a E(3)-ubiquitin ligase that is MRLC specific. This in turn rapidly increased MIR expression, and led to ubiquitination and proteasome-mediated degradation of MRLC. Downregulation of MIR by RNA muting prevented the reorganization of actin structures and severely reduced the migratory and wound-healing potential of PDGF-treated cells. The results show that activation of MIR and the resulting removal of diphosphorylated MRLC are essential for PDGF to instigate and maintain control over the actin-myosin-based contractile system in Swiss 3T3 fibroblasts. The PDGF induced protein destabilization through the regulation of hnRNP-K controlled ubiquitin -ligase translation identifies a novel pathway by which external stimuli can regulate phenotypic development through rapid, organelle-specific changes in the activity and stability of cytoskeletal regulators.

Actin Cytoskeleton↗