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PubMed2004

Characterization of bacteriophage KVP40 and T4 RNA ligase 2.

Bacteriophage T4 RNA ligase 2 (Rnl2) exemplifies a subfamily of RNA strand-joining enzymes that includes the trypanosome RNA editing ligases. A homolog of T4 Rnl2 is encoded in the 244-kbp DNA genome of vibriophage KVP40. We show that the 335-amino acid KVP40 Rnl2 is a monomeric protein that catalyzes RNA end-joining through ligase-adenylate and RNA-adenylate (AppRNA) intermediates. In the absence of ATP, pre-adenylated KVP40 Rnl2 reacts with an 18-mer 5'-PO(4) single-strand RNA (pRNA) to form an 18-mer RNA circle. In the presence of ATP, Rnl2 generates predominantly AppRNA. Isolated AppRNA can be circularized by KVP40 Rnl2 in the absence of ATP. The reactivity of phage Rnl2 and the distribution of the products are affected by the length of the pRNA substrate. Whereas 18-mer and 15-mer pRNAs undergo intramolecular sealing by T4 Rnl2 to form monomer circles, a 12-mer pRNA is ligated intermolecularly to form dimers, and a 9-mer pRNA is unreactive. In the presence of ATP, the 15-mer and 12-mer pRNAs are converted to AppRNAs, but the 9-mer pRNA is not. A single 5' deoxynucleotide substitution of an 18-mer pRNA substrate has no apparent effect on the 5' adenylation or circularization reactions of T4 Rnl2. In contrast, a single deoxyribonucleoside at the 3' terminus strongly and selectively suppresses the sealing step, thereby resulting in accumulation of high levels of AppRNA in the absence of ATP. The ATP-dependent "capping" of RNA with AMP by Rnl2 is reminiscent of the capping of eukaryotic mRNA with GMP by GTP:RNA guanylyltransferase and suggests an evolutionary connection between bacteriophage Rnl2 and eukaryotic RNA capping enzymes.

Adenosine Monophosphate↗
PubMed2006

Enhanced ubiquitination of cytoskeletal proteins in pressure overloaded myocardium is accompanied by changes in specific E3 ligases.

Ubiquitin conjugation of proteins is critical for cell homeostasis and contributes to both cell survival and death. Here we studied ubiquitination of proteins in pressure overloaded (PO) myocardium in the context of cardiomyocyte survival. Analysis using a feline right ventricular pressure overload (RVPO) model revealed a robust and transient increase in ubiquitination of proteins present in the Triton X-100-insoluble fraction in 24 to 48 h PO myocardium, and confocal micrographs indicate this increase in ubiquitination occurs subsarcolemmaly near the intercalated disc area of cardiomyocytes. The ubiquitination was accompanied by changes in E3 ligases including Cbl, E6AP, Mdm2 and cIAP in the same period of PO, although atrophy-related E3 ligases, MuRF1 and MuRF3 were unaltered. Furthermore, Cbl displayed a substantial increase in both levels of expression and tyrosine phosphorylation in 48 h PO myocardium. Confocal studies revealed enrichment of Cbl at the intercalated discs of 48 h PO cardiomyocytes, as evidenced by its colocalization with N-cadherin. Although apoptosis was observed in 48 h PO myocardium by TUNEL staining, cardiomyocytes showing ubiquitin staining were not positive for TUNEL staining. Furthermore, 48 h PO resulted in the phosphorylation of inhibitor of nuclear factor kappa B (IkappaB), suggesting its ubiquitin-mediated degradation and the nuclear localization of NFkappaB for the expression of specific cell survival factors such as cIAPs. Together these data indicate that increased levels of E3 ligases that regulate cell homeostasis and promote cell survival could ubiquitinate multiple cytoskeletal protein targets and that these events that occur during the early phase of PO may contribute to both cardiomyocyte survival and hypertrophy.

Animals↗
PubMed2003

Simultaneous detection of diverse analytes with an aptazyme ligase array.

Allosteric ribozymes (aptazymes) can transduce the noncovalent recognition of analytes into the catalytic generation of readily observable signals. Aptazymes are easily engineered, can detect diverse classes of biologically relevant molecules, and have high signal-to-noise ratios. These features make aptazymes useful candidates for incorporation into biosensor arrays. Allosteric ribozyme ligases that can recognize a variety of analytes ranging from small organics to proteins have been generated. Upon incorporation into an array format, multiple different aptazyme ligases were able to simultaneously detect their cognate analytes with high specificity. Analyte concentrations could be accurately measured into the nanomolar range. The fact that analytes induced the formation of new covalent bonds in aptazyme ligases (as opposed to noncovalent bonds in antibodies) potentiated stringent washing of the array, leading to improved signal-to-noise ratios and limits of detection.

Biosensing Techniques↗
PubMed2002

Purification and characterization of benzoate:coenzyme A ligase from Clarkia breweri.

Benzoate:CoA ligase (BZL) was partially purified from flowers of the annual California plant Clarkia breweri. BZL catalyzes the formation of benzoyl-CoA and anthraniloyl-CoA, important intermediates for subsequent acyltransferase reactions in plant secondary metabolism. The native enzyme is active as a monomer with a molecular mass of approximately 59-64.5 kDa, and it has K(m) values of 45, 95, and 130 microM for benzoic acid, ATP, and CoA, respectively. BZL is most active in the pH range of 7.2-8.4, and its activity is strictly dependent on certain bivalent cations. BZL is an AMP-forming enzyme. Overall, its properties suggest that it is related to the family of CoA ligase enzymes that includes the plant enzyme 4-hydroxycinnamate:CoA ligase.

Adenosine Monophosphate↗
PubMed2005

Identification, expression, and assay of an oxidation-specific ubiquitin ligase, HOIL-1.

The ubiquitin system plays important roles in the regulation of numerous cellular processes. It is well established that ubiquitin ligases (E3s) are key components in determining the specificity of the system and that the modification of substrates such as phosphorylation often plays a critical role in selective substrate recognition by E3s. Through studies analyzing iron-mediated degradation of iron regulatory protein 2 (IRP2), a central regulator of iron metabolism in mammalian cells, we have identified a RING finger protein, HOIL-1, as an ubiquitin ligase recognizing IRP2 through a signal created by heme-mediated oxidative modification of the protein. We have utilized several types of in vitro ubiquitination assays that detect IRP2 ubiquitination and a differential yeast two-hybrid screen in which yeast cells were cultured either in the presence or in the absence of oxygen to control the oxidation state of the bait in the cells in our studies. This chapter describes the detailed methods used for the identification and functional analysis of the HOIL-1 ligase.

Animals↗
PubMed2005

Purification and assay of the chaperone-dependent ubiquitin ligase of the carboxyl terminus of Hsc70-interacting protein.

It is notable that both chaperone and ubiquitin-proteasome systems are required for the removal of aberrant cellular proteins to ensure protein homeostasis in cells. However, the entity that links the two systems had remained elusive. The carboxyl terminus of Hsc70-interacting protein (CHIP), originally identified as a cochaperone of Hsc70, has both a TPR motif and a U-box domain. The TPR motif associates with Hsp70 and Hsp90, whereas the U-box domain executes ubiquitin ligase activity. Thus, CHIP is an ideal molecule, acting as a protein quality control ubiquitin ligase that selectively leads abnormal proteins recognized by molecular chaperones to degradation by the proteasome. This chapter describes methods of analyzing chaperone-dependent ubiquitin ligase activity of CHIP using firefly luciferase as a model substrate.

Animals↗
PubMed2005

Druggability of SCF ubiquitin ligase-protein interfaces.

The unique mechanism of the SCF ubiquitin ligase poses a challenge to drug discovery. A central enzymatic small molecule-binding active site is not evident in this multisubunit protein enzyme, as is the case with kinases or proteases. Instead, the SCF ligase seems to accomplish ubiquitylation through a series of cooperative movements dependent on the protein interfaces between its components and its substrate. Activity-modulating small molecules, therefore, need to interact with these protein interfaces. The three-dimensional structure of these interfaces may be the key asset in determining their suitability for small molecule binding. Computational tools and a systematic approach described in detail here can assess the "druggability" of an SCF ligase before the investment of effort in high-throughput screening (HTS), structure-based drug design (SBBD), or virtual library screening (VLS).

Binding Sites↗
PubMed1991

Identification of vancomycin resistance protein VanA as a D-alanine:D-alanine ligase of altered substrate specificity.

High-level glycopeptide resistance in Enterococcus faecium BM4147 is mediated by a 38-kDa protein VanA, whose amino acid sequence is related to Gram-negative D-alanine:D-alanine (D-Ala-D-Ala) ligases [Dutka-Malen, S., Molinas, C., Arthur, M., & Courvalin, P. (1990) Mol. Gen. Genet. 224, 364-372]. We report purification of VanA and demonstrate that it has D-Ala-D-Ala ligase activity but has substantially modified substrate specificity, compared with Gram-negative D-Ala-D-Ala ligases. VanA preferentially condenses D-Ala with D-Met or D-Phe, raising the possibility that its cellular role is to synthesize a modified cell-wall component, which is subsequently not recognized by vancomycin.

Amino Acid Sequence↗
PubMed1979

Photocross-linking analysis of the contact surface of tRNA Met in complexes with Escherichia coli methionine:tRNA ligase.

Photoinduced covalent cross-linking has been used to identify a common surface of four methionine-accepting tRNAs which interact specifically with the Escherichia coli methionine:tRNA ligase (EC 6.1.1.10). tRNA--ligase mixtures were irradiated, and the covalently linked complexes were isolated and digested with T1 RNase (Schimmel & Budzik, 1977). The fragments lost from the elution profile of the T1 RNase digest were considered to have been cross-linked to the protein and therefore in intimate contact with the enzyme. Only specific cognate tRNA--ligase pairs produce covalently linked complexes. The four substrate tRNAs used in this study have substantially different sequences, but all showed a common cross-linking pattern, supporting the view that the sites cross-linked to the enzyme reflect the functionally common contact surface rather than particularly photoreactivity regions of tRNA. The cross-linked contact surface is comprised of three regions: (1) the narrow groove of the anticodon stem and its extension into the anticodon loop; (2) the 3' terminal residues; and (3) the 3' side of the "T arm". Unlike previous studies with other tRNAs, the D arm is not involved and significant radiation damage is suffered by the tRNA which must be taken into account in the analysis. The results are consistent with and complement chemical modification studies [Schulman, L. H., & Pelka, H. (1977) Biochemistry 16, 4256].

Amino Acyl-tRNA Synthetases↗
PubMed2002

ATP-dependent L-cysteine:1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase, mycothiol biosynthesis enzyme MshC, is related to class I cysteinyl-tRNA synthetases.

Mycothiol is a novel thiol produced only by actinomycetes and is the major low molecular weight thiol in mycobacteria. The mycothiol biosynthetic pathway has been postulated to involve ATP-dependent ligation of L-cysteine (Cys) with 1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside; GlcN-Ins) catalyzed by MshC to produce Cys-GlcN-Ins. The ligase activity was purified approximately 2400-fold from Mycobacterium smegmatis and two proteins of slightly different M(r) approximately 47000 were identified with MshC activity. The N-terminal sequence of the smaller protein revealed that it was coded by a gene in the databases for M. smegmatis and M. tuberculosis previously designated as cysS2. The larger protein was coded by the same gene in M. smegmatis but included an eight amino acid N-terminal extension involving a different start codon. The ligase was found to have K(m) values of 40 +/- 3 and 72 +/- 9 microM for Cys and GlcN-Ins, respectively. The cysS2 gene was thought to encode a second cysteinyl-tRNA synthetase in addition to cysS but the present results indicate that cysS2 is actually the mshC gene encoding ATP-dependent Cys:GlcN-Ins ligase.

Adenosine Triphosphate↗
PubMed1997

Acyl-adenylate motif of the acyl-adenylate/thioester-forming enzyme superfamily: a site-directed mutagenesis study with the Pseudomonas sp. strain CBS3 4-chlorobenzoate:coenzyme A ligase.

4-Chlorobenzoate:coenzyme A (4-CBA:CoA) ligase catalyzes 4-chlorobenzoyl-coenzyme A formation in a two-step reaction consisting of the adenylation of 4-chlorobenzoate with adenosine 5'-triphosphate followed by acyl transfer from the 4-chlorobenzoyl adenosine 5'-monophosphate diester intermediate to coenzyme A. In this study, two core motifs present in the Pseudomonas sp. strain CBS3 4-CBA:CoA ligase (motif I, 161T-S-G-T-T-G-L-P-K-G170, and motif II, 302Y-G-T-T-E306) and conserved among the sequences representing the acyl-adenylate/thioester-forming enzyme family (to which the ligase belongs) were tested for their possible role in substrate binding and/or catalysis. The site-directed mutants G163I, G166I, P168A, K169M, and E306Q were prepared and then subjected to steady-state and transient kinetic studies. The results, which indicate reduced catalysis of the adenylation of 4-chlorobenzoate in the mutant enzymes, are interpreted within the context of the three-dimensional structure of the acyl-adenylate/thioester-forming enzyme family member, firefly luciferase.

Adenosine Triphosphate↗
PubMed2000

Kinetic framework for ligation by an efficient RNA ligase ribozyme.

The class I RNA ligase ribozyme, isolated previously from random sequences, performs an efficient RNA ligation reaction. It ligates two substrate RNAs, promoting the attack of the 3'-hydroxyl of one substrate upon the 5'-triphosphate of the other substrate with release of pyrophosphate. This ligation reaction has similarities to the reaction catalyzed by RNA polymerases. Using data from steady-state kinetic measurements and pulse-chase/pH-jump experiments, we have constructed minimal kinetic frameworks for two versions of the class I ligase, named 207t and 210t. For both ligases, as well as for the self-ligating parent ribozyme, the rate constant for the chemical step (k(c)) is log-linear with pH in the range 5.7-8.0. At physiological pH, the k(c) is 100 min(-1), a value similar to those reported for the fastest naturally occurring ribozymes. At higher pH, product release is limiting for both 207t and 210t. The 210t ribozyme, with its faster product release, attains multiple-turnover rates (k(cat) = 360 min(-1), pH 9.0) exceeding those of 207t and other reported ribozyme reactions. The kinetic framework for the 210t ribozyme describes the limits of this catalysis and suggests how key steps can be targeted for improvement using design or combinatorial approaches.

Binding Sites↗
PubMed2001

A de novo designed peptide ligase: a mechanistic investigation.

A 33-residue de novo designed peptide ligase is reported which catalyzes the template-directed condensation of suitably activated short peptides with catalytic efficiencies in excess of 10(5) ([k(cat)/K(m)]/k(uncat)). The ligase peptide, derived from natural and designed alpha-helical coiled-coil proteins, presents a surface for substrate assembly via formation of a hydrophobic core at the peptide interface. Charged residues flanking the core provide additional binding specificity through electrostatic complementarity. Addition of the template to an equimolar fragment solution results in up to 4100-fold increases in initial reaction rates. Dramatic decreases in efficiency upon mutation of charged residues or increase in ionic strength establishes the importance of electrostatic recognition to ligase efficiency. Although most of the increase in reaction efficiency is due to entropic gain from binding of substrates in close proximity, mechanistic studies with altered substrates demonstrate that the system is highly sensitive to precise ordering at the point of ligation. Taken together these results represent the first example of a peptide catalyst with designed substrate binding sites which can significantly accelerate a bimolecular reaction and support the general viability of alpha-helical protein assemblies in artificial enzyme design.

Amino Acid Sequence↗
PubMed2004

De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling.

NF-kappaB transcription factors mediate the effects of pro-inflammatory cytokines such as tumour necrosis factor-alpha and interleukin-1beta. Failure to downregulate NF-kappaB transcriptional activity results in chronic inflammation and cell death, as observed in A20-deficient mice. A20 is a potent inhibitor of NF-kappaB signalling, but its mechanism of action is unknown. Here we show that A20 downregulates NF-kappaB signalling through the cooperative activity of its two ubiquitin-editing domains. The amino-terminal domain of A20, which is a de-ubiquitinating (DUB) enzyme of the OTU (ovarian tumour) family, removes lysine-63 (K63)-linked ubiquitin chains from receptor interacting protein (RIP), an essential mediator of the proximal TNF receptor 1 (TNFR1) signalling complex. The carboxy-terminal domain of A20, composed of seven C2/C2 zinc fingers, then functions as a ubiquitin ligase by polyubiquitinating RIP with K48-linked ubiquitin chains, thereby targeting RIP for proteasomal degradation. Here we define a novel ubiquitin ligase domain and identify two sequential mechanisms by which A20 downregulates NF-kappaB signalling. We also provide an example of a protein containing separate ubiquitin ligase and DUB domains, both of which participate in mediating a distinct regulatory effect.

Cell Line↗
PubMed2003

Targeting of protein ubiquitination by BTB-Cullin 3-Roc1 ubiquitin ligases.

The concentrations and functions of many cellular proteins are regulated by the ubiquitin pathway. Cullin family proteins bind with the RING-finger protein Roc1 to recruit the ubiquitin-conjugating enzyme (E2) to the ubiquitin ligase complex (E3). Cul1 and Cul7, but not other cullins, bind to an adaptor protein, Skp1. Cul1 associates with one of many F-box proteins through Skp1 to assemble various SCF-Roc1 E3 ligases that each selectively ubiquitinate one or more specific substrates. Here, we show that Cul3, but not other cullins, binds directly to multiple BTB domains through a conserved amino-terminal domain. In vitro, Cul3 promoted ubiquitination of Caenorhabditis elegans MEI-1, a katanin-like protein whose degradation requires the function of both Cul3 and BTB protein MEL-26. We suggest that in vivo there exists a potentially large number of BCR3 (BTB-Cul3-Roc1) E3 ubiquitin ligases.

Animals↗
PubMed2006

CUL4-DDB1 ubiquitin ligase interacts with multiple WD40-repeat proteins and regulates histone methylation.

The CUL4-DDB1-ROC1 ubiquitin E3 ligase regulates cell-cycle progression, replication and DNA damage response. However, the substrate-specific adaptors of this ligase remain uncharacterized. Here, we show that CUL4-DDB1 complexes interact with multiple WD40-repeat proteins (WDRs) including TLE1-3, WDR5, L2DTL (also known as CDT2) and the Polycomb-group protein EED (also known as ESC). WDR5 and EED are core components of histone methylation complexes that are essential for histone H3 methylation and epigenetic control at K4 or K9 and K27, respectively, whereas L2DTL regulates CDT1 proteolysis after DNA damage through CUL4-DDB1 (ref. 8). We found that CUL4A-DDB1 interacts with H3 methylated mononucleosomes and peptides. Inactivation of either CUL4 or DDB1 impairs these histone modifications. However, loss of WDR5 specifically affects histone H3 methylation at K4 but not CDT1 degradation, whereas inactivation of L2DTL prevents CDT1 degradation but not histone methylation. Our studies suggest that CUL4-DDB1 ligases use WDR proteins as molecular adaptors for substrate recognition, and modulate multiple biological processes through ubiquitin-dependent proteolysis.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Function and regulation of cullin-RING ubiquitin ligases.

Cullin-RING complexes comprise the largest known class of ubiquitin ligases. Owing to the great diversity of their substrate-receptor subunits, it is possible that there are hundreds of distinct cullin-RING ubiquitin ligases in eukaryotic cells, which establishes these enzymes as key mediators of post-translational protein regulation. In this review, we focus on the composition, regulation and function of cullin-RING ligases, and describe how these enzymes can be characterized by a set of general principles.

Animals↗
PubMed2001

A covalent crosslink converts the hammerhead ribozyme from a ribonuclease to an RNA ligase.

The hammerhead ribozyme is a more efficient ribonuclease than an RNA ligase. Under typical reaction conditions, the rate of RNA chain cleavage is approximately 100-fold faster than the rate of the reverse ligation reaction such that virtually all of the hammerhead is in its cleaved form at equilibrium. Here we show that the introduction of a crosslink away from the catalytic core of the hammerhead has little effect on the cleavage rate but dramatically increases the ligation rate, thereby making the hammerhead an efficient RNA ligase. This experiment emphasizes the role of molecular flexibility in defining the properties of a macromolecular catalyst and suggests why other small ribozymes are more efficient ligases than ribonucleases.

Base Sequence↗