Fhit-nucleotide Specificity Probed with Novel Fluorescent and Fluorogenic Substrates

Draganescu, Alexandra; Hodawadekar, Santosh; Gee, Kyle R.; Brenner, Charles

doi:10.1074/jbc.275.7.4555

Cited by 71 publications

(84 citation statements)

References 29 publications

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“…As illustrated in Figure 2, Fhit substrate specificity differs from that of Hint with Fhit homologs preferring to hydrolyze substrates such as AppppA (39), ApppA (40), γ-(mNitrobenzyl) adenosine 5′-O-triphosphate (41), and ApppBODIPY (42). This amounts to an altered specificity for the adenylylation step of the reaction because adenylylation of each of these substrates produces an identical Fhit-AMP intermediate that, like Hint-AMP, is enzymatically hydrolyzed.…”

Section: Evolutionary Of Hit Proteins: Bacteria To Birdsmentioning

confidence: 99%

“…In fact, a low barrier hydrogen bond may be so favorable to form that the rate-limiting step in adenylylation may be getting the leaving group to take the proton and go. We have previously observed that substrates with fundamentally unaltered chemical lability such as ApppA, AppppA, ApppBODIPY and GpppBODIPY are cleaved with increasingly poor rates suggesting that slow substrates may prefer to reattack than to exit (42). Ultimately the hydrogen bond network between His35 and His98 is postulated to promote transfer of the His98 Nε proton to the leaving group, rendering the adenylylation reaction not immediately reversible and His98 basic to activate ahydrolytic water.…”

Section: Mechanistic Insights In the Hit Superfamily: Hydrolases Versmentioning

confidence: 99%

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Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

2002

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HIT (histidine triad)1 proteins, named for a motif related to the sequence HφHφHφφ, (φ a hydrophobic amino acid) are a superfamily of nucleotide hydrolases and transferases, which act on the α-phosphate of ribonucleotides, and contain a ∼30 kDa domain that is typically either a homodimer of ∼15 kDa polypeptides with two active-sites or an internally, imperfectly repeated polypeptide that retains a single HIT active site. On the basis of sequence, substrate specificity, structure, evolution and mechanism, HIT proteins can be classified into the Hint branch, which consists of adenosine 5′-monophosphoramide hydrolases, the Fhit branch, which consists of diadenosine polyphosphate hydrolases, and the GalT branch, which consists of specific nucleoside monophosphate transferases including galactose-1-phosphate uridylyltransferase, diadenosine tetraphosphate phosphorylase, and adenylylsulfate:phosphate adenylytransferase. At least one human representative of each branch is lost in human diseases. Aprataxin, a Hint branch hydrolase, is mutated in ataxia-oculomotor apraxia syndrome. Fhit is lost early in development of many epithelially derived tumors. GalT is deficient in galactosemia. Additionally, ASW is an avian Hint family member that has evolved to have unusual gene expression properties and the complete loss of its nucleotide binding-site. The potential roles of ASW and Hint in avian sexual development are discussed in an accompanying manuscript. Here we review what is known about biological activities of HIT proteins, the structural and biochemical bases for their functions, and propose a new enzyme mechanism for Hint and Fhit that may account for the differences between HIT hydrolases and transferases. Mammalian Hint and GalT Structurally Define the HIT SuperfamilyGalactose-1-phosphate uridylyltransferase (GalT), the second enzyme in the Leloir pathway of galactose utilization (1), was crystallized and its structure determined to understand the mechanistic basis for transferring UMP between glucose-1-phosphate and galactose-1-phosphate (2-4). Histidine triad nucleotide-binding protein (Hint), purified as a purine nucleoside/nucleotide-binding protein from rabbit heart cytosol (5) and shown to have a widely conserved sequence (6), was crystallized and its structure determined to establish whether the conserved sequence formed the basis for a new mode of nucleotide-binding (7). GalT (2) and Hint are both dimers (8) but the GalT monomer is more than twice the size of the Hint dimer. Though GalT (9) and Hint (6) homologs could be cloned by similarity and the crystal structures (2,8) co-existed in protein databases, mutual similarity was not noted until inspection of the rabbit Hint crystal structure (7) and computational analysis (10) indicated that a GalT monomer and a Hint dimer can be superimposed and that their nucleotide-binding sites retain some of the same residues for binding a nucleoside monophosphate (7) (Figure 1). The level of sequence similarity of Hint and GalT is difficult to distinguish from n...

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Section: Evolutionary Of Hit Proteins: Bacteria To Birdsmentioning

confidence: 99%

Section: Mechanistic Insights In the Hit Superfamily: Hydrolases Versmentioning

confidence: 99%

Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

2002

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“…methylaminoacetyl and Gppp-S-(4-4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacine-3-yl) methylaminoacetyl [14]. The Abbreviations used: Ap 4 A, diadenosine tetraphosphate; Np n N , dinucleoside 5 ,5 -P 1 ,P n -polyphosphate (where N and N are 5 -O-nucleosides and n is the number of phosphate residues in the polyphosphate chain that links the two 5 -esterified nucleosides).…”

Section: S-(4-4-difluoro-57-dimethyl-4-bora-3a4a-diaza-s-indacine-3mentioning

confidence: 99%

Adenosine-5'-O-phosphorylated and adenosine-5'-O-phosphorothioylated polyols as strong inhibitors of (symmetrical) and (asymmetrical) dinucleoside tetraphosphatases

Guranowski¹,

Starzyńska²,

McLennan

et al. 2003

Biochemical Journal

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Dinucleoside 5 ,5 -P 1 ,P n -polyphosphates, and particularly the diadenosine compounds, have been implicated in extracellular purinergic signalling and in various intracellular processes, including DNA metabolism, tumour suppression and stress responses. If permitted to accumulate, they may also be toxic. One approach to understanding their function is through the various specific degradative enzymes that regulate their levels. Eight adenosine-5 -O-phosphorylated polyols (derivatives of glycerol, erythritol and pentaerythritol) and 11 adenosine-5 -O-phosphorothioylated polyols (derivatives of glycerol, erythritol, pentaerythritol, butanediol and pentanediol) have been tested as inhibitors of specific diadenosine tetraphosphate (Ap 4 A) hydrolases. Of these two groups of novel nucleotides, the adenosine-5 -O-phosphorothioylated polyols were generally stronger inhibitors than their adenosine-5 -O-phosphorylated counterparts. 1,4-Di(adenosine-5 -O-phosphorothio) erythritol appeared to be the strongest inhibitor of (asymmetrical) Ap 4 A hydrolases (EC 3.6.1.17) from both lupin and human, with K i values of 0.15 µM and 1.5 µM respectively. Of eight adenosine-5 -Ophosphorylated polyols, 1,4-di(adenosine-5 -O-phospho) erythritol was the only compound that inhibited the lupin enzyme. Two derivatives of pentaerythritol, di(adenosine-5 -O-phosphorothio)-di(phosphorothio) pentaerythritol and tri(adenosine-5 -Ophosphorothio)-phosphorothio-pentaerythritol, proved to be the strongest inhibitors of the prokaryotic (symmetrical) Ap 4 A hydrolase (EC 3.6.1.41) so far reported. The estimated K i values were 0.04 µM and 0.08 µM respectively. All of these inhibitors were competitive with respect to Ap 4 A. These new selectively acting Ap 4 A analogues should prove to be valuable tools for further studies of Ap 4 A function and of the enzymes involved in its metabolism.

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“…Second, adenoviruses and adeno-associated viruses that express Fhit were used to demonstrate that nitrosomethylbenzamineinduced stomach carcinomas in Fhit-heterozygous mice can be prevented by subsequent infection with FHIT viruses (14). Here, viral reintroduction with quantitative cell-based assays and Fhit enzymology were coupled to perform structure-function analysis on Fhit and to probe the cellular mechanism of its action.A member of branch 2 of the histidine-triad superfamily of nucleotide hydrolases and transferases (15), Fhit encodes a diadenosine polyphosphate (Ap n A) hydrolase (16,17) that cleaves substrates such as diadenosine P 1 ,P 3 -triphosphate (ApppA) and diadenosine 5Ј,5ЈЈЈ-P 1 ,P 4 -tetraphosphate (AppppA) to AMP plus the other nucleotide. Catalysis is mediated via a covalent mechanism involving attack of His-96 on the ␣-phosphate of substrates (18, 19) that bears features in common with the branch 1 hydrolases including Hint (20, 21) and galactose-1-phosphate uridylyltransferase (22), a member of branch 3 of the superfamily (15).…”

mentioning

confidence: 99%

“…A member of branch 2 of the histidine-triad superfamily of nucleotide hydrolases and transferases (15), Fhit encodes a diadenosine polyphosphate (Ap n A) hydrolase (16,17) that cleaves substrates such as diadenosine P 1 ,P 3 -triphosphate (ApppA) and diadenosine 5Ј,5ЈЈЈ-P 1 ,P 4 -tetraphosphate (AppppA) to AMP plus the other nucleotide. Catalysis is mediated via a covalent mechanism involving attack of His-96 on the ␣-phosphate of substrates (18, 19) that bears features in common with the branch 1 hydrolases including Hint (20, 21) and galactose-1-phosphate uridylyltransferase (22), a member of branch 3 of the superfamily (15).…”

mentioning

confidence: 99%

Designed FHIT alleles establish that Fhit-induced apoptosis in cancer cells is limited by substrate binding

Trapasso

Krakowiak

Cesari

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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The FHIT gene is inactivated early in the development of many human tumors, and Fhit-deficient mice have increased cancer incidence. Viral reexpression of Fhit kills Fhit-deficient cells by induction of apoptosis. Fhit, a member of branch 2 of the histidinetriad superfamily of nucleoside monophosphate hydrolases and transferases, is a diadenosine polyphosphate hydrolase, the activesite histidine of which is not required for tumor suppression. To provide a rigorous test of the hypothesis that Fhit function depends on forming a complex with substrates, we designed a series of alleles of Fhit intended to reduce substrate-binding and͞or hydrolytic rates, characterized these mutants biochemically, and then performed quantitative cell-death assays on cancer cells virally infected with each allele. The allele series covered defects as great as 100,000-fold in k cat and increases as large as 30-fold in KM. Nonetheless, when mutant FHIT genes were expressed in two human cancer cell lines containing FHIT deletions, reductions in apoptotic activity correlated exclusively with K M. Mutants with 2-and 7-fold increases in K M significantly reduced apoptotic indices, whereas the mutant with a 30-fold increase in K M retained little cellular function. These data indicate that the proapoptotic function of Fhit is limited by substrate binding and is unrelated to substrate hydrolysis.T he FHIT gene spans the most active common fragile site in the human genome, FRA3B, and is inactivated by deletion in a large proportion of human malignancies (1-3). Fhitheterozygous mice suffer high rates of stomach and sebaceous tumors when exposed to levels of nitrosomethylbenzamine that do not induce carcinomas in wild-type mice (4). In addition, untreated Fhit-deficient and Fhit-heterozygous mice suffer high rates of spontaneous tumors (5), and the ability of human Fhit-deficient cells to form tumors in nude mice is blocked by reintroduction and expression of Fhit (6-8). Beyond the demonstration that Fhit is an authentic tumor suppressor via these types of assays, two additional issues have been explored. First, viral and transfection-mediated induction of Fhit indicated that tumor suppression by Fhit is accompanied by induction of apoptosis (9, 10), and subsequent studies have begun to dissect the downstream players in Fhit-dependent cell death (11-13). Second, adenoviruses and adeno-associated viruses that express Fhit were used to demonstrate that nitrosomethylbenzamineinduced stomach carcinomas in Fhit-heterozygous mice can be prevented by subsequent infection with FHIT viruses (14). Here, viral reintroduction with quantitative cell-based assays and Fhit enzymology were coupled to perform structure-function analysis on Fhit and to probe the cellular mechanism of its action.A member of branch 2 of the histidine-triad superfamily of nucleotide hydrolases and transferases (15), Fhit encodes a diadenosine polyphosphate (Ap n A) hydrolase (16,17) that cleaves substrates such as diadenosine P 1 ,P 3 -triphosphate (ApppA) and diadenosine 5Ј,5ЈЈЈ-P ...

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Fhit-nucleotide Specificity Probed with Novel Fluorescent and Fluorogenic Substrates

Cited by 71 publications

References 29 publications

Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

Adenosine-5'-O-phosphorylated and adenosine-5'-O-phosphorothioylated polyols as strong inhibitors of (symmetrical) and (asymmetrical) dinucleoside tetraphosphatases

Designed FHIT alleles establish that Fhit-induced apoptosis in cancer cells is limited by substrate binding

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