Larry D. Barnes

The Diadenosine Hexaphosphate Hydrolases fromSchizosaccharomyces pombe andSaccharomyces 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 fromSaccharomyces 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 (Ap6A and Ap5A). 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 Ap6A and Ap5A, in preference to other diadenosine polyphosphates. The emergence of Ap6A and Ap5A 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.

Adenosine Monophosphoramidase Activity of Hint and Hnt1 Supports Function of Kin28, Ccl1, and Tfb3

The histidine triad superfamily of nucleotide hydrolases and nucleotide transferases consists of a branch of proteins related to Hint and Aprataxin, a branch of Fhit-related hydrolases, and a branch of galactose-1-phosphate uridylyltransferase (GalT)-related transferases. Although substrates of Fhit and GalT are known and consequences of mutations in Aprataxin, Fhit, and GalT are known, good substrates had not been reported for any member of the Hint branch, and mutational consequences were unknown for Hint orthologs, which are the most ancient and widespread proteins in the Hint branch and in the histidine triad superfamily. Here we show that rabbit and yeast Hint hydrolyze the natural product adenosine-5′-monophosphoramidate (AMPNH2) in an active-site-dependent manner at second order rates exceeding 1,000,000m −1 s−1. Yeast strains constructed with specific loss of the Hnt1 active site fail to grow on galactose at elevated temperatures. Loss of Hnt1 enzyme activity also leads to hypersensitivity to mutations in Ccl1, Tfb3, and Kin28, which constitute the TFIIK kinase subcomplex of general transcription factor TFIIH and to mutations in Cak1, which phosphorylates Kin28. The target of Hnt1 regulation in this pathway was shown to be downstream of Cak1 and not to affect stability of Kin28 monomers. Functional complementation of all Hnt1 phenotypes was provided by rabbit Hint, which is only 22% identical to yeast Hnt1 but has very similar adenosine monophosphoramidase activity.

Effect of zwitterionic buffers on measurement of small masses of protein with bicinchoninic acid

Linear standard curves were obtained for 1 to 10 micrograms of bovine serum albumin measured in the presence of 50 mM Mes, Mops, Pipes, Caps, Bes, Hepes, Heppso, and Tes zwitterionic buffers using bicinchoninic acid for detection. The zwitterionic buffers Ada, Tricine, and Bicine that strongly bind Cu2+. Tris, and Bistris, each at 50 mM, inhibited color development to prevent quantitative measurement of protein mass.

THE TUMOR SUPPRESSOR PROTEIN FHIT : A NOVEL INTERACTION WITH TUBULIN

FHIT (fragilehistidine triad) is a candidate human tumor suppressor gene located at chromosome 3p14.2, a location that encompasses the FRA3B chromosomal fragile site. Aberrant transcripts have been detected in a variety of primary tumors, and homozygous deletions in the FHIT locus have been detected in different tumor cell lines. The gene product Fhit in vitro possesses the ability to hydrolyze diadenosine 5′,5′′′-P1,P3-triphosphate (Ap3A). The mechanism of action of Fhit as a tumor suppressor is unknown. Because the tubulin-microtubule system plays an important role in cell division and cell proliferation, we investigated the interaction between wild-type Fhit or mutant Fhit (H96N) and tubulin in vitro. The mutant form of Fhit (H96N) lacks Ap3A hydrolase activity but retains tumor suppressor activity. We found that both wild-type and mutated forms of Fhit bind to tubulin strongly and specifically with K d values of 1.4 and 2.1 μm, respectively. Neither wild-type nor mutant Fhit cause nucleation or formation of microtubules, but in the presence of microtubule-associated proteins, both wild-type and mutant Fhit promote assembly to a greater extent than do microtubule-associated proteins alone, and the microtubules formed appear normal by electron microscopy. Our results suggest the possibility that Fhit may exert its tumor suppressor activity by interacting with microtubules and also indicate that the interaction between Fhit and tubulin is not related to the Ap3A hydrolase activity of Fhit.

Disruption and overexpression of the Schizosaccharomyces pombe aps1 gene, and effects on growth rate, morphology and intracellular diadenosine 5′,5'-P1,P5-pentaphosphate and diphosphoinositol polyphosphate concentrations

Schizosaccharomyces pombe Aps1 is an enzyme that degrades both diadenosine oligophosphates (Ap(n)A, n =5 or 6) and diphosphoinositol polyphosphates [diphosphoinositol pentakisphosphate (PP-InsP(5)) and bisdiphosphoinositol tetrakisphosphate ([PP](2)-InsP(4))] in vitro. The in vivo substrates of Aps1 are unknown. We report here the identification of Ap(5)A, PP-InsP(5), [PP](2)-InsP(4) and a novel diphosphoinositol polyphosphate ([PP](x)-InsP(x)) in S. pombe using HPLC methods. Ap(5)A was present at 0.06 pmol/mg of protein (approx. 4 nM). PP-InsP(5), [PP](x)-InsP(x) and [PP](2)-InsP(4) were present at 15 pmol/mg (approx. 1.1 microM), 15 pmol/mg (approx. 1.1 microM) and 30 pmol/mg (approx. 2.2 microM) respectively, while the intracellular concentration of InsP(6) was 0.5 nmol/mg of protein (approx. 36 microM). Disruption of aps1 resulted in a 52% decrease in Ap(6)A hydrolase activity in vitro, no detectable change in the intracellular Ap(5)A concentration, and 3-fold increased intracellular concentrations of PP-Ins P(5) and [PP](x)-InsP(x). Disruption of aps1 resulted in no detectable change in morphology or growth rate in minimal or rich media at 30 degrees C. Overexpression of aps1 via two different plasmids that resulted in 60% and 6-fold increases above wild-type enzymic activity in vitro caused no detectable changes in the intracellular concentrations of [PP](2)-InsP(4), [PP](x)-InsP(x) or PP-InsP(5), but paradoxical increases of approx. 2.5- and 55-fold respectively in the intracellular Ap(5)A concentration. Overexpression of aps1 also resulted in a reduced growth rate and in morphological changes, including swollen, rounded and multiseptate cells. No phenotypic changes or changes in intracellular Ap(5)A occurred upon overexpression of aps1 E93Q, which encodes a mutated Aps1 lacking significant enzymic activity. We conclude that Aps1 degrades PP-InsP(5) and [PP](x)-InsP(x) in vivo.

An Adjacent Pair of Human NUDT Genes on Chromosome X Are Preferentially Expressed in Testis and Encode Two New Isoforms of Diphosphoinositol Polyphosphate Phosphohydrolase

Combinatorial expression of the various isoforms of diphosphoinositol synthases and phosphohydrolases determines the rates of phosphorylation/dephosphorylation cycles that have been functionally linked to vesicle trafficking, stress responses, DNA repair, and apoptosis. We now describe two new 19-kDa diphosphoinositol polyphosphate phosphohydrolases (DIPPs), named types 3α and 3β, which possess the canonical Nudix-type catalytic motif flanked on either side by short Gly-rich sequences. The two enzymes differ only in that Pro-89 in the α form is replaced by Arg-89 in the β form, making the latter ∼2-fold more active in vitro. Another Nudix substrate, diadenosine hexaphosphate, was hydrolyzed less efficiently (k cat/K m = 0.2 × 105 m −1s−1) compared with diphosphoinositol polyphosphates (k cat/K m = 2–40 × 105 m −1 s−1). Catalytic activity in vivo was established by individual overexpression of the human (h) DIPP3 isoforms in HEK293 cells, which reduced cellular levels of diphosphoinositol polyphosphates by 40–50%. The hDIPP3 mRNA is preferentially expressed in testis, accompanied by relatively weak expression in the brain, contrasting with hDIPP1 and hDIPP2 which are widely expressed. ThehDIPP3 genes (NUDT10 encodes hDIPP3α;NUDT11 encodes hDIPP3β) are only 152 kbp apart at p11.22 on chromosome X and probably arose by duplication. Transcription of both genes is inactivated on one of the X chromosomes of human females to maintain appropriate gene dosage. The hDIPP3 pair add tissue-specific diversity to the molecular mechanisms regulating diphosphoinositol polyphosphate turnover.

Assay of diadenosine tetraphosphate hydrolytic enzymes by boronate chromatography.

A new procedure was described for assay of diadenosine tetraphosphate (Ap4A) hydrolases based on boronate chromatography. Potential reaction products, AMP, ADP, and ATP, of the hydrolysis of Ap4A were separated from residual substrate by chromatography on a boronate-derivatized cation-exchange resin, Bio-Rex 70. Separation was achieved by changing the concentrations of ethanol and ammonium acetate in the elution buffers. Picomole masses of products were detectable, blank dpm values were less than 0.5% of the total dpm, and auxiliary enzymes were not required. The procedure was specifically described for Ap4A pyrophosphohydrolase from Physarum polycephalum. The assay is generally applicable for dinucleoside polyphosphate hydrolases which hydrolyze other substrates such as Ap3A, Ap5A, Ap6A, and Gp4G. Dinucleotide polyphosphates are readily purified by chromatography on this boronate resin in a volatile buffer. Tes, Tricine, and Tris buffers significantly interfered with the chromatography of ATP.

High-performance liquid chromatographic application of the hummel and dreyer method for the determination of colchicine—tubulin binding parameters

An application of the Hummel and Dreyer gel chromatography procedure modified for high-performance liquid chromatography has been used to determine the dissociation constant for the colchicine-tubulin interaction at 25 degrees C. The results obtained are compared with results of other equilibrium and non-equilibrium techniques and demonstrate that the initial interaction of colchicine with tubulin must be rapid and probably reversible. This rapid and sensitive technique, which does not require radioisotopes for measurement of the binding parameters, will be extremely useful for characterization of tubulin-ligand interactions.

Correlation of Fragile Histidine Triad (Fhit) Protein Structural Features with Effector Interactions and Biological Functions

We have previously shown that Fhit tumor suppressor protein interacts with Hsp60 chaperone machinery and ferredoxin reductase (Fdxr) protein. Fhit-effector interactions are associated with a Fhit-dependent increase in Fdxr stability, followed by generation of reactive oxygen species and apoptosis induction under conditions of oxidative stress. To define Fhit structural features that affect interactions, downstream signaling, and biological outcomes, we used cancer cells expressing Fhit mutants with amino acid substitutions that alter enzymatic activity, enzyme substrate binding, or phosphorylation at tyrosine 114. Gastric cancer cell clones stably expressing mutants that do not bind substrate or cannot be phosphorylated showed decreased binding to Hsp60 and Fdxr and reduced mitochondrial localization. Expression of Fhit or mutants that bind interactor proteins results in oxidative damage and accumulation of cells in G2/M or sub-G1 fractions after peroxide treatment; noninteracting mutants are defective in these biological effects. Gastric cancer clones expressing noncomplexing Fhit mutants show reduction of Fhit tumor suppressor activity, confirming that substrate binding, interaction with heat shock proteins, mitochondrial localization, and interaction with Fdxr are important for Fhit tumor suppressor function.

Analysis of angiotensins I, II, III, and iodinated derivatives by high-performance liquid chromatography

Abstract Angiotensins I, II, and III were separated by reversed-phase high-performance liquid chromatography on an octadecylsilyl column. The peptides were isocratically eluted with 50 m m NaH2PO4-25% ( v v ) acetonitrile, pH 6.0. The retention times were 3.3, 6.0, and 9.6 min for angiotensin II, III, and I, respectively. 125I-Angiotensins II, III, and I eluted with retention times of 5.4, 16.8, and 19.9 min, respectively, under the same chromatographic conditions used for the unlabeled angiotensins. The effect of iodination of the tyrosine residue on the retention time was also demonstrated by chromatographic comparison of tyrosine and diiodotyrosine. Saralasin (Sar1, Ala8-angiotensin II), a partial agonist of angiotensin II, and des-Asp1, Ile8-angiotensin II, an inhibitor of angiotensin III, eluted with retention times of 2.5 and 3.9 min, respectively.