MutT Motif Research Articles

Emergence of a new lumpy skin disease virus variant in Kurgan Oblast, Russia, in 2018.

In this paper, we report the resurgence of lumpy skin disease (LSD) in Kurgan Oblast, Russia, in 2018. The majority of the outbreaks were silent with no mortality and congregated within an area with a radius of about 30km located 1-50km away from the national border with Kazakhstan. Following primary molecular diagnosis, LSD virus (LSDV) isolates were analyzed using a panel of PCR assays targeting different genetic loci, namely, LSD008 (vaccine), LSDV126 (field), and GPCR (vaccine and field), for differentiation and genotype assignment. All isolates were positive for the vaccine genotype of GPCR and negative for the other field targets tested. A PCR assay with melt curve analysis utilizing LSD008, developed in this work, indicated that the strains melted with a profile similar to those of field strains. Surprisingly, sequence analysis of the RPO30 and GPCR genes aligned the Kurgan/2018 isolate with KSGP O-240 at the GPCR locus, but with Saratov/2017 at the RPO30 locus. The latter cluster forms an association with a sub-cluster of the field strains comprising the South African KSGP O-240 strain and NI-2490 strain. Due to these incongruent phylogenetic patterns, the sequences of three additional loci ORF19 (Kelch-like protein), ORF52 (putative transcriptional elongation factor), and ORF87 (mutT motif protein) were investigated. Phylogenetic analysis of these additional loci placed the strain Kurgan/2018 in either vaccine or field groups, strongly suggesting a novel recombinant profile. This is another piece of evidence exposing the potential for recombination in capripoxviruses and the ignored danger of using live homologous vaccines against LSD. The necessity to revise the PCR-based strategy differentiating infected from vaccinated animals is discussed. The potential scenarios of incursion and the contribution of the KSGP/NI-2490-like strain to the emergence of the recently identified vaccine-like recombinant are discussed.

LTRPC2 Ca 2+-Permeable Channel Activated by Changes in Redox Status Confers Susceptibility to Cell Death

Redox status changes exert critical impacts on necrotic/apoptotic and normal cellular processes. We report here a widely expressed Ca 2+-permeable cation channel, LTRPC2, activated by micromolar levels of H 2O 2 and agents that produce reactive oxygen/nitrogen species. This sensitivity of LTRPC2 to redox state modifiers was attributable to an agonistic binding of nicotinamide adenine dinucleotide (β-NAD +) to the MutT motif. Arachidonic acid and Ca 2+ were important positive regulators for LTRPC2. Heterologous LTRPC2 expression conferred susceptibility to death on HEK cells. Antisense oligonucleotide experiments revealed physiological involvement of “native” LTRPC2 in H 2O 2- and TNFα-induced Ca 2+ influx and cell death. Thus, LTRPC2 represents an important intrinsic mechanism that mediates Ca 2+ and Na + overload in response to disturbance of redox state in cell death.

Studies on Dinucleoside Polyphosphates: Some Intriguing Biochemical, Physiological, and Medical Aspects.

Dinucleoside 5',5'''-P α ',P Ω -polyphosphates have been found in all cells examined. So far, the only known specific enzyme that catalyzes the synthesis of these polyphosphates is GTP:GTP guanylyl-transferase. This enzyme produces Gp 4 G, Gp 3 G, and various Np 3 - 4 Gs. The adenylated counterparts, such as Ap 3 A and Ap 4 A, are synthesized by ligases, at least by some aminoacyl-tRNA synthetases. Ap 4 A phosphorylase, firefly luciferase, acyl-CoA synthetase, and the DNA- and RNA-ligases are also able to produce Np 3 - 6 As. By contrast, in addition to nonspecific enzymes such as phosphodiesterase I and nucleotide pyrophosphatase, there occur in all types of organism specific enzymes that degrade Np 3 - 6 N's. These are the Np 3 N' hydrolases, the asymmetrically and symmetrically acting Np 4 N' hydrolases, the Np n N' phosphorylases, and recently described hydrolases that prefer Ap 6 A and Ap 5 A as substrates. Human Fhit protein, a putative tumor suppressor, behaves as a typical Np 3 N' hydrolase and is a member of the HIT (histidine triad) protein family. The human Np 4 N' hydrolase belongs to the MutT motif or Nudix (nucleoside diphosphate attached to x) hydrolase protein family. The level of Np 3 - 6 N's in the cells increases dramatically under stress. It is suggested that these minor nucleotides act as both intra- and extracellular signalling molecules. Ap n As interact with purine receptors and affect vascular tone. Chemically synthesized analogues of Np n N's help us to understand the mechanism of action of the enzymes mentioned, and some of them are candidates for drugs.

Cloning, expression and characterization of YSA1H, a human adenosine 5′-diphosphosugar pyrophosphatase possessing a MutT motif

The human homologue of the Saccharomyces cerevisiae YSA1 protein, YSA1H, has been expressed as a thioredoxin fusion protein in Escherichia coli. It is an ADP-sugar pyrophosphatase with similar activities towards ADP-ribose and ADP-mannose. Its activities with ADP-glucose and diadenosine diphosphate were 56% and 20% of that with ADP-ribose respectively, whereas its activity towards other nucleoside 5′-diphosphosugars was typically 2-10%. cADP-ribose was not a substrate. The products of ADP-ribose hydrolysis were AMP and ribose 5-phosphate. Km and kcat values with ADP-ribose were 60 μM and 5.5 s-1 respectively. The optimal activity was at alkaline pH (7.4-9.0) with 2.5-5 mM Mg2+ or 100-250 μM Mn2+ ions; fluoride was inhibitory, with an IC50 of 20 μM. The YSA1H gene, which maps to 10p13-p14, is widely expressed in all human tissues examined, giving a 1.4 kb transcript. The 41.6 kDa fusion protein behaved as an 85 kDa dimer on gel filtration. After cleavage with enterokinase, the 24.4 kDa native protein fragment ran on SDS/PAGE with an apparent molecular mass of 33 kDa. Immunoblot analysis with a polyclonal antibody raised against the recombinant YSA1H revealed the presence of a protein of apparent molecular mass 33 kDa in various human cells, including erythrocytes. The sequence of YSA1H contains a MutT sequence signature motif. A major proposed function of the MutT motif proteins is to eliminate toxic nucleotide metabolites from the cell. Hence the function of YSA1H might be to remove free ADP-ribose arising from NAD+ and protein-bound poly- and mono-(ADP-ribose) turnover to prevent the occurrence of non-enzymic protein glycation.

Cloning, expression and characterization of YSA1H, a human adenosine 5′-diphosphosugar pyrophosphatase possessing a MutT motif

The human homologue of the Saccharomyces cerevisiae YSA1 protein, YSA1H, has been expressed as a thioredoxin fusion protein in Escherichia coli. It is an ADP-sugar pyrophosphatase with similar activities towards ADP-ribose and ADP-mannose. Its activities with ADP-glucose and diadenosine diphosphate were 56% and 20% of that with ADP-ribose respectively, whereas its activity towards other nucleoside 5'-diphosphosugars was typically 2-10%. cADP-ribose was not a substrate. The products of ADP-ribose hydrolysis were AMP and ribose 5-phosphate. K(m) and k(cat) values with ADP-ribose were 60 microM and 5.5 s(-1) respectively. The optimal activity was at alkaline pH (7.4-9.0) with 2.5-5 mM Mg(2+) or 100-250 microM Mn(2+) ions; fluoride was inhibitory, with an IC(50) of 20 microM. The YSA1H gene, which maps to 10p13-p14, is widely expressed in all human tissues examined, giving a 1.4 kb transcript. The 41.6 kDa fusion protein behaved as an 85 kDa dimer on gel filtration. After cleavage with enterokinase, the 24.4 kDa native protein fragment ran on SDS/PAGE with an apparent molecular mass of 33 kDa. Immunoblot analysis with a polyclonal antibody raised against the recombinant YSA1H revealed the presence of a protein of apparent molecular mass 33 kDa in various human cells, including erythrocytes. The sequence of YSA1H contains a MutT sequence signature motif. A major proposed function of the MutT motif proteins is to eliminate toxic nucleotide metabolites from the cell. Hence the function of YSA1H might be to remove free ADP-ribose arising from NAD(+) and protein-bound poly- and mono-(ADP-ribose) turnover to prevent the occurrence of non-enzymic protein glycation.

The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif.

The major pathway of mRNA degradation in yeast occurs through deadenylation, decapping and subsequent 5' to 3' exonucleolytic decay of the transcript body. To identify proteins that control the activity of the decapping enzyme, which is encoded by the DCP1 gene, we isolated a high-copy suppressor of the temperature-sensitive dcp1-2 allele, termed DCP2. Overexpression of Dcp2p partially suppressed the dcp1-2 decapping defect. Moreover, the Dcp2 protein was required for the decapping of both normal mRNAs and aberrant transcripts that are degraded by the mRNA surveillance pathway. The Dcp2 protein contains a MutT motif, which is found in a class of pyrophosphatases. Mutational analyses indicated that the region of Dcp2p containing the MutT motif is necessary and sufficient for Dcp2p's function in mRNA decapping. The Dcp2p also coimmunoprecipitates with the DCP1 decapping enzyme and is required for the production of enzymatically active decapping enzyme. These results suggest that direct or indirect interaction of Dcp1p with Dcp2p is required for the production of active decapping enzyme, perhaps in a process requiring the hydrolysis of a pyrophosphate bond.

The MutT motif family of nucleotide phosphohydrolases in man and human pathogens (review).

Human cells express at least eight members of the MutT motif protein (or nudix hydrolase) family. These enzymes are believed to eliminate toxic nucleotide derivatives from the cell and regulate the levels of important signalling nucleotides and their metabolites. Six have been fully or partially characterized: i) hMTH1 is a nucleoside triphosphatase which restricts AT-->CG transversions by specifically degrading the oxidized nucleotide 8-oxo-dGTP; ii) hAPAH1 preferentially degrades the signalling dinucleotide Ap4A; iii) DIPP is unusual in hydrolysing two seemingly unrelated signalling substrate groups - the dinucleotides Ap6A and Ap5A, and the diphosphoinositol polyphosphates; iv) DIPP2 is closely related to DIPP; v) hYSAH1 is an NDP-sugar hydrolase which prefers ADP-ribose, and vi) hGFG is a protein of unknown function encoded by the antisense transcript of the basic fibroblast growth factor gene. Although not yet associated with known hereditary or acquired disorders, the functional loss of any one of these hydrolases would be expected to be detrimental to cellular function. Furthermore, the ialA invasion gene of Bartonella bacilliformis and other invasive pathogens encodes a MutT motif Ap4A hydrolase while poxviruses express two MutT motif proteins, at least one of which is essential for infectivity. This protein family, therefore, occupies a position of some importance in controlling human health and disease.

The Diadenosine Hexaphosphate Hydrolases fromSchizosaccharomyces pombe and Saccharomyces cerevisiae Are Homologues of the Human Diphosphoinositol Polyphosphate Phosphohydrolase: OVERLAPPING SUBSTRATE SPECIFICITIES IN A MutT-TYPE PROTEIN

Aps1 from Schizosaccharomyces pombe (Ingram, S. W., Stratemann, S. A. , and Barnes, L. D. (1999) Biochemistry 38, 3649-3655) and YOR163w from Saccharomyces cerevisiae (Cartwright, J. L., and McLennan, A. G. (1999) J. Biol. Chem. 274, 8604-8610) have both previously been characterized as MutT family hydrolases with high specificity for diadenosine hexa- and pentaphosphates (Ap(6)A and Ap(5)A). Using purified recombinant preparations of these enzymes, we have now discovered that they have an important additional function, namely, the efficient hydrolysis of diphosphorylated inositol polyphosphates. This overlapping specificity of an enzyme for two completely different classes of substrate is not only of enzymological significance, but in addition, this finding provides important new information pertinent to the structure, function, and evolution of the MutT motif. Moreover, we report that the human protein previously characterized as a diphosphorylated inositol phosphate phosphohydrolase represents the first example, in any animal, of an enzyme that degrades Ap(6)A and Ap(5)A, in preference to other diadenosine polyphosphates. The emergence of Ap(6)A and Ap(5)A as extracellular effectors and intracellular ion-channel ligands points not only to diphosphorylated inositol phosphate phosphohydrolase as a candidate for regulating signaling by diadenosine polyphosphates, but also suggests that diphosphorylated inositol phosphates may competitively inhibit this process.

TheIalAInvasion Gene ofBartonella bacilliformisEncodes a (Di)Nucleoside Polyphosphate Hydrolase of the MutT Motif Family and Has Homologs in Other Invasive Bacteria

The product of theialAinvasion gene ofBartonella bacilliformishas been expressed as a thioredoxin fusion protein. It is a (di)nucleoside polyphosphate hydrolase of the MutT motif protein family with strong sequence similarity to plant diadenosine tetraphosphate hydrolases. It hydrolyses nucleoside and dinucleoside polyphosphates with four or more phosphate groups, always producing an NTP as one product. Diadenosine tetraphosphate (Ap4A) is the preferred substrate with aKmof 10 μM and akcatof 3.0 s−1. It is inhibited by Ca2+and F−(Ki= 30 μM). Hydrolysis of Ap4A in H218O yielded [18O]AMP as the only labelled product. In terms of sequence, reaction mechanism and properties, IalA is very similar to eukaryotic Ap4A hydrolases and unlike previously described bacterial Ap4A hydrolases. Homologs are present in the genomes of other invasive pathogens. They may function to reduce stress-induced dinucleotide levels during invasion and so enhance pathogen survival.

The Saccharomyces cerevisiae YOR163w Gene Encodes a Diadenosine 5′,5‴-P1,P6-Hexaphosphate (Ap6A) Hydrolase Member of the MutT Motif (Nudix Hydrolase) Family

The YOR163w open reading frame on chromosome XV of the Saccharomyces cerevisiae genome encodes a member of the MutT motif (nudix hydrolase) family of enzymes of Mr 21,443. By cloning and expressing this gene in Escherichia coli and S. cerevisiae, we have shown the product to be a (di)adenosine polyphosphate hydrolase with a previously undescribed substrate specificity. Diadenosine 5',5"'-P1, P6-hexaphosphate is the preferred substrate, and hydrolysis in H218O shows that ADP and adenosine 5'-tetraphosphate are produced by attack at Pbeta and AMP and adenosine 5'-pentaphosphate are produced by attack at Palpha with a Km of 56 microM and kcat of 0.4 s-1. Diadenosine 5',5"'-P1,P5-pentaphosphate, adenosine 5'-pentaphosphate, and adenosine 5'-tetraphosphate are also substrates, but not diadenosine 5',5"'-P1,P4-tetraphosphate or other dinucleotides, mononucleotides, nucleotide sugars, or nucleotide alcohols. The enzyme, which was shown to be expressed in log phase yeast cells by immunoblotting, displays optimal activity at pH 6.9, 50 degrees C, and 4-10 mM Mg2+ (or 200 microM Mn2+). It has an absolute requirement for a reducing agent, such as dithiothreitol (1 mM), and is inhibited by Ca2+ with an IC50 of 3.3 mM and F- (noncompetitively) with a Ki of 80 microM. Its function may be to eliminate potentially toxic dinucleoside polyphosphates during sporulation.

Recovery of soluble, active recombinant protein from inclusion bodies.

Recovery of soluble, active recombinant protein from inclusion bodies.

Molecular cloning of diadenosine tetraphosphatase from pig small intestinal mucosa and identification of sequence blocks common to diadenosine polyphosphate hydrolases and phosphorylases

Diadenosine 5′,5‴- P 1, P 4-tetraphosphate (Ap 4A) pyrophosphohydrolase is the enzyme responsible for reducing intracellular levels of the stress-responsive nucleotide diadenosine 5′,5‴- P 1, P 4-tetraphosphate. In order to gain more information on the relationships between the enzymes hydrolysing diadenosine polyphosphates in different eukaryotes, the Ap 4A hydrolase and a corresponding cDNA have been isolated from pig small intestinal mucosa by standard procedures. The enzyme is a typical mammalian Ap 4A hydrolase ( K m = 0.8 μM), being sensitive to inhibition by fluoride ( K i = 24 μM) and adenosine 5′-tetraphosphate ( K i = 10 nM) and yielding ATP and AMP as products. A low K m Ap 4A hydrolase ( K m = 0.3 μM) was also isolated from rabbit small intestinal mucosa. These enzymes differ from the rat intestinal mucosal hydrolase, which has much higher values of K m for Ap 4A and K i for adenosine 5′-tetraphosphate. A cDNA encoding the pig enzyme was isolated from a pig ileum cDNA library. The derived amino acid sequence of the 16.8 kDa gene product shows 88% identity and 96% similarity to that of the human enzyme. The sequence has the same modification of the MutT motif found in the human enzyme in which a threonine residue replaces a hydrophobic amino acid. Sequence comparisons among eukaryotic diadenosine polyphosphate hydrolases and phosphorylases reveal two blocks of amino acid similarity, including a motif, Z[AD]Gx[ED]AGQ, which may be involved in polyphosphate binding by the hydrolases, and an invariant histidine residue that may be involved in catalysis. These sequence similarities may have arisen by convergent evolution.