Recent Progress in the Study of the Intracellular Functions of Diadenosine Polyphosphates Alexander G

DRUG DEVELOPMENT RESEARCH 52:249–259 (2001) DDR

Research Overview Recent Progress in the Study of the Intracellular Functions of Diadenosine Polyphosphates Alexander G. McLennan,1* Larry D. Barnes,2 G.Michael Blackburn,3 Charles Brenner,4 Andrzej Guranowski,5 Andrew D. Miller,6 Juan Manuel Rovira,7 Pedro Rotllán,8 Bernat Soria,7 Julian A. Tanner,6 and Antonio Sillero9 1School of Biological Sciences, University of Liverpool, Liverpool, UK 2Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 3Department of Chemistry, University of Sheffield, Sheffield, UK 4Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 5Department of Biochemistry and Biotechnology, Agricultural University, Poznan, Poland 6Imperial College Genetic Therapies Centre, Department of Chemistry, Imperial College of Science, Technology & Medicine, South Kensington, London, UK 7Institute of Bioengineering, Miguel Hernández University, Alicante, Spain 8Departamento de Bioquímica y Biología Molecular, Universidad de La Laguna, Tenerife, Canary Islands, Spain 9Departamento de Bioquímica. Instituto de Investigaciones Biomédicas Alberto Sols, UAM/CSIC, Facultad de Medicina, Madrid, Spain

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ABSTRACT Recent developments in the effort to understand the metabolism and function of the intracellular dinucleoside polyphosphates were described by nine speakers from some of the world’s leading laboratories in this field in a workshop at the Purines 2000 International Symposium on Nucleosides and Nucleotides held in Madrid in July, 2000. Topics were wide-ranging and included phenotypic analyses of yeast mutants defective in enzymes of dinucleoside polyphosphate degradation, virally encoded catabolic enzymes, the structure and function of the Fhit tumor suppressor and Fhit-nitrilase fusion proteins and the relationship of Fhit to human diadenosine triphosphate hydrolase, site-directed mutagenesis of diadenosine tetraphosphate hydrolase, novel nucleotide analogs for studying hydrolase function, the synthesis of dinucleoside polyphosphates by ligases, and the possible roles of diadenosine tri- and tetraphosphates in insulin function and of diadenosine tetraphosphate in the heat-shock response of Escherichia coli. The results presented and the ensuing discussions showed that, while considerable progress is being made in the field, it still has the capacity to tease and frustrate and produce the unexpected result. Drug Dev. Res. 52:249–259, 2001. © 2001 Wiley-Liss, Inc.

Key words: Ap4A; FHIT; diphosphoinositol; stress

Contract grant sponsor: NSF; Contract grant number: MCB- sor: Comunidad de Madrid; Contract grant number: 08.9/0004/98; 9982645; Contract grant sponsor: NIH; Contract grant numbers: Contract grant sponsor: the Wellcome Trust; Contract grant number: CA75954, CA77738; Contract grant sponsor: KBN, the State Com- 053038; Contract grant sponsor: the Leverhulme Trust; Contract grant mittee for Scientific Research; Contract grant number: P04A 06215; number: F25BJ. Contract grant sponsor: the Dirección General de Enseñanza Supe- rior; Contract grant number: PM96-0081; Contract grant sponsor: *Correspondence to: Prof. Alexander G. McLennan, School the Dirección General de Investigación Científica y Técnica; Con- of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, tract grant numbers: PM95/0013, PM 98/0129; Contract grant spon- UK. E-mail: [email protected]

INTRODUCTION phology or growth rate in rich and minimal media. When aps1 and its promoter region were cloned into pUR18, a The functions of the intracellular, mainly cytosolic, multicopy plasmid, and transformed into S. pombe, diadenosine 5′, 5′′′-P1, Pn polyphosphates (Ap A, n = 3– n transformants displayed slow growth in minimal medium 6) are still not clear despite many years of investigation. and showed a subtle change in morphology. The cells Their levels respond to various metabolic stresses, they were slightly enlarged (both width and length) through- bind with relatively high affinity and specificity to a num- out the growth phase and did not shorten upon entering ber of intracellular proteins, and they have been impli- stationary phase. Such transformants exhibited a 2-fold cated in a variety of processes, including the regulation increase in Ap A hydrolase activity in vitro. To intensify of DNA replication and repair, but none of these possi- 6 the change in phenotype, aps1 was expressed using an bilities is supported by unequivocal evidence [McLennan, integrated expression cassette which drives aps1 expres- 2000]. Several new developments were reported at a sion via the cytomegalovirus promoter. These cells dis- workshop devoted to the intracellular dinucleoside played almost no growth in minimal medium and polyphosphates organized by Antonio Sillero (Universi- microcolonies on plates contained multiseptate cells. dad Autonoma de Madrid) and Sandy McLennan (Uni- When aps1 was expressed at an intermediate level via versity of Liverpool) at the Purines 2000 International pREP3X, a plasmid with a thiamine-repressible pro- Symposium on Nucleosides and Nucleotides held in moter, the growth rate was three times slower than wild- Madrid in July, 2000, which encourage the view that the type and the cells exhibited a “round-bottom flask” functions of the Ap As are finally nearing clarification, n morphology. Such transformants exhibited a 5-fold in- although it was also clear that there are still some unex- crease in Ap A hydrolase activity in vitro. To verify that pected obstacles in the way. 6 enzyme activity was required for the change in phenotype, a mutated form of the enzyme, E93Q, which has no Enzymes of ApnA Catabolism detectable Ap6A hydrolase activity in vitro, was also ex- For example, recent research in Larry Barnes’ labo- pressed. Schizosaccharomyces pombe transformed with ratory (University of Texas Health Science Center, San pREP3Xaps1E93Q displayed no changes in growth rate

Antonio) focusing on the deletion and overexpression of or morphology. The levels of Ap5A in the pUR18aps1 and enzymes of ApnA metabolism and subsequent measure- pREP3Xaps1 overexpression strains were, paradoxically, ment of ApnA levels has yielded some surprising results. 3 and 50 times, respectively, the Ap5A level in wild-type Schizosaccharomyces pombe Aps1 is one of three strain. Ap6A was undetectable in the disruption and enzymes, including Saccharomyces cerevisiae Ddp1p overexpression strains. These data indicate that: 1.) Ap5A (YOR163w) and Homo sapiens DIPP, that will hydrolyze and Ap6A probably are not in vivo substrates for Aps1; both ApnA and diphosphorylated inositol polyphosphates 2.) the enzymatic activity of Aps1 is required for the (DIPs) in vitro [Safrany et al., 1999]. Aps1, Ddp1p, and changes in phenotype in the overexpression strains; and DIPP all contain the Nudt sequence motif (also called 3.) either Aps1 or one of its likely DIP substrates may be the MutT motif or nudix box) [Yang et al., 1999]. Aps1 involved in fission yeast morphogenesis and normal pro- prefers Ap6A and Ap5A over all other ApnAs and will de- gression through septation and cytokinesis. Although grade both bisdiphosphoinositol tetrakisphophate ([PP]2- Aps1 was initially characterized as an ApnA hydrolase InsP4) and diphosphoinositol pentakisphosphate [Ingram et al., 1999], previous results [Safrany et al., 1999] ([PP]-InsP5) [Ingram et al., 1999; Safrany et al., 1999]. and the data presented here suggest that the DIPs are Which of these substrates is degraded in vivo by Aps1 is the in vivo substrates. unknown. Ap5A and Ap6A exist in higher eukaryotes and Results similar to the above were also obtained in are proposed to function as both intracellular and extra- Sandy McLennan’s laboratory (University of Liverpool) with cellular signaling molecules. Extracts of S. pombe can the budding yeast S. cerevisiae. Cell extracts were made synthesize DIPs in vitro [Ongusaha et al., 1998] but the from log and stationary phase wild-type and null mutants function of these compounds in S. pombe is unknown. in the DDP1 gene (encoding the Ap6A/DIP hydrolase). Af- To investigate further the in vivo function of Aps1, ter exhaustive alkaline phosphatase treatment of the extracts, the aps1 gene was disrupted and overexpressed in S. ion-exchange chromatography was used to separate the pombe and the endogenous levels of Ap6A and Ap5A mea- remaining dinucleotides into the families Ap4N (where N sured using boronate and anion-exchange chromatogra- = A, G, C, or U), Ap5N and Ap≥6N (presumed to contain phies. Ap5A was present in wild-type yeast at about 5–10% putative Ap6N and possible higher-order polyphosphates) the level of Ap4A, while Ap6A was undetectable. Disrup- on the basis of the known chromatographic properties of tion of aps1 resulted in a significant decrease in Ap6A authentic standards. hydrolase activity, no detectable change in intracellular Four- to eight-fold increases in Ap5N and Ap≥6N Ap5A concentration, and no detectable change in mor- were observed in stationary phase wild-type cells com- INTRACELLULAR DIADENOSINE POLYPHOSPHATES 251 pared to log phase cells, but only a slight increase in Ap4N. because the plant counterpart exhibited the same prop- Similar increases in Ap5N and Ap≥6N were seen in the erty, and finally because the preference of cleavage of DDP1 null mutant as it entered stationary phase; how- (2′-pdA)Ap4A by the human enzyme to the product pairs ever, the differences in levels between the wild-type and ATP + (2′-pdA)AMP (80%) rather than to (2′-pdA)ATP null mutant in both phases were at most 2-fold. The great- + AMP (20%), differed from the preference observed est difference found was in the amount of Ap4N nucle- with the lupin wild-type Ap4A hydrolase. (These mea- otides when comparing stationary phase wild type (4.9 ± surements were achieved using the enzymically synthe- 3 0.6 pmol/mg protein) and DDP1 null mutant (18.5 ± 2.8 sized model substrate, (2′-pdA)[ H]Ap4A, and a thin-layer pmol/mg protein) cells. The corresponding values for log chromatography system which separates [3H]ATP from phase cells were 3.5 ± 0.7 and 7.0 ± 0.6 pmol/mg protein, (2′-pdA)[3H]ATP [Guranowski et al., 2000]). respectively. Thus, despite the fact that Ap4A is barely Some mutants had an altered cleavage pattern for used by Ddp1p as a substrate in vitro [Cartwright and (2′-pdA)Ap4A. Whereas the wild-type enzyme hydrolyzed McLennan, 1999], the greatest effect of disrupting the this model compound to ATP + (2′-pdA]AMP (60%) and

DDP1 gene on ApnN pools is on Ap4N. Ap5N and Ap≥6N to (2′-pdA)ATP + AMP (40%), mutants R54Q, E58D, and show only slight increases. E125Q hydrolyzed this substrate to the same product Corresponding measurements of DIP concentra- pairs in a 70:30 ratio, while E58Q did so in a 55:45 ratio. tions have been made in collaboration with Steve Safrany These results showed that the mutations produced subtle (University of Dundee) and show a 4–8–fold higher level changes in binding of the substrate analog, although they of a [PP]-InsP5 isomer (but no change in [PP]2-InsP4) in changed neither the overall structure of the enzyme, as the DDP1 null mutant compared to wild-type (both log detected by UV circular dichroism, nor the pattern of and stationary phase). Therefore, as with S. pombe, there Ap4A cleavage from asymmetrical to symmetrical. is as yet no strong evidence that the Ddp1p is respon- The other parameter was chosen because high sen- sible for metabolizing the higher-order ApnA compounds sitivity to fluoride is a feature very typical of (asymmetri- in S. cerevisiae but may be of more relevance to DIP cal) Ap4A hydrolases [Guranowski, 1990; Maksel et al., metabolism and function. 1998; Cartwright et al., 1999] and it was expected that a

Like the Ap6A/DIP hydrolases, the more widely comparison of different mutants would reveal amino studied (asymmetrical) dinucleoside tetraphosphate hy- acid(s) potentially responsible for such strong inhibition drolases (EC 3.6.1.17), commonly referred to as Ap4A of catalysis by this anion—the IC50 values are 2–3 µM hydrolases, are also members of the nudix hydrolase fam- for the plant enzymes [Guranowski, 1990; Maksel et al., ily and possess the Nudt motif [Maksel et al., 1998; 1998]. The E55Q mutant, which had a 20-fold lower Km Cartwright et al., 1999]. Andrzej Guranowski (Agricul- for Ap4A and a 10,000-fold lower kcat than the wild-type tural University of Poznan) reported on site-directed enzyme, was insensitive to fluoride. Mutant R54Q re- mutagenesis studies of the enzyme from the narrow- tained residual susceptibility to fluoride with an IC50 of 1 leafed lupin Lupinus angustifolius recently carried out in mM. Mutants E58D and E58Q retained moderate, and collaboration with Ken Gayler’s group at the Department E59D almost full, sensitivity to fluoride with IC50 values of Biochemistry and Molecular Biology, University of of 50, 20, and 6 µM, respectively. The same values for Melbourne, as part of their effort to determine the im- the mutants generated in another part of the enzyme pro- portance to catalysis of particular amino acid residues tein, E122Q and E125Q, were 4 and 250 µM, respec- acids within this motif and in a short region towards the tively. These data suggest that it is arginine 54, properly carboxyl terminus. oriented to glutamate 55 and to glutamate 125, that in- The sequence encoding the first 39 amino acids was teracts with fluoride. These structural–functional stud- removed from the cloned Ap4A hydrolase cDNA [Maksel ies are of great importance and hopefully will soon be et al., 1998] in order to generate a product equivalent to extended to the animal Ap4A hydrolases, particularly in the native (18.5 kDa) protein (residues previously desig- view of the possibility highlighted by McLennan (Uni- nated M40 to L199 are now 1–160). In addition to Km versity of Liverpool) that the plant and animal Ap4A hy- and kcat, the mutants were characterized by two other drolases may be more distantly related within the nudix measurable parameters—the positional preference of family than might be expected from their very similar cleavage of a model substrate, 2′-deoxyadenylated Ap4A, properties. A phylogenetic analysis of the genes for all (2′-pdA)Ap4A (produced by the action of 2′,5′-oligo- known nudix asymmetrical Ap4A hydrolases shows that adenylate synthetase on Ap4A), and the susceptibility to the animal enzymes cluster with those from thermophilic fluoride inhibition. The former parameter was chosen, bacteria and are in fact more closely related to the Ap6A/ first because it was recently demonstrated that human DIP hydrolases than to the plant Ap4A hydrolases, which Ap4A hydrolase easily hydrolyzes 2′-adenylated- and 2′- cluster with another group of prokaryotic enzymes, those deoxyadenylated Ap4A [Guranowski et al., 2000], second from Gram-negative invasive pathogens. Thus, the plant 252 MCLENNAN ET AL. and animal Ap4A hydrolases may have acquired a com- that hydrolyze a range of dinucleoside polyphosphates mon substrate specificity within the nudix family by con- [Brenner et al., 1999], generating an AMP product plus vergent evolution. the other mononucleotide. The structure of Fhit explains A new member of the nudix hydrolase family with how these enzymes accomplish this specificity: in Fhit specificity for dinucleoside and nucleoside polyphos- bound to nonhydrolyzable Ap3A, one nucleoside is tightly phates and which clusters with the animal Ap4A hydro- bound to each protomer while the other nucleoside moi- lases was also described by McLennan. African swine ety is more solvent-exposed [Pace et al., 1998]. Genetic, fever virus (ASFV) is a double-stranded DNA virus with biochemical, and crystallographic data support the no- a single nudix hydrolase gene (g5R or D250). This gene tion that the active signaling form of Fhit is bound to was cloned and expressed in Escherichia coli and shown ApnA substrates [Pace et al., 1998]. Thus, more slowly to encode an enzyme that degrades a number of cleaved dinucleoside polyphosphates might actually pro- (di)nucleoside polyphosphates. Good substrates (with mote the biological activity of Fhit. relative percentage degradation at 200 µM in brackets) Human and murine genetics have demonstrated were adenosine 5′-pentaphosphate (100), adenosine 5′- that loss of Fhit is associated with cancer in many tissues tetraphosphate (92), GTP (87), Gp4G (76), Gp3G (46), ATP and that re-expression of Fhit induces programmed cell (71), Ap5A (41), Ap4A (29), and Ap3A (13). In the case of death [Huebner et al., 1999; Fong et al., 2000]. If Fhit Gp4G, for example, hydrolysis was mainly symmetrical function depends on complexing with ApnA, then the with GDP as the major product. However, some asym- cellular systems that generate intracellular polyphos- metrical hydrolysis to GTP and GMP was also evident. phates ought to be the upstream regulators of Fhit activ- No interconversion of products was observed. Vaccinia ity. Novel fluorescent and fluorigenic Fhit substrates, virus, another double-stranded DNA virus, has two nudix ApppBODIPY and GpppBODIPY, have been used to test hydrolase genes, one of which (D10) selectively inhibits whether rare ApnA would be able to bind Fhit in the face cap-dependent translation when overexpressed [Shors et of high cellular concentrations of ATP. Indeed, Fhit binds al., 1999]. This suggested that D10 protein might hydro- the ApnA’s 100-fold better than ATP and 10-fold better lyze cap structures. However, when the ASFV g5R pro- than pyrophosphate, potentially indicating that ApnA’ s tein was tested with 7-methylguanine-containing might displace pyrophosphate in a postulated “ground- dinucleotides such as 7-methylguanosine(5′)tetra- state” of Fhit [Draganescu et al., 2000]. phospho(5′)guanosine (m7Gp4G), the activity was greatly The downstream effectors that might transmit Fhit- reduced to the point that cap analog m7Gp3A was inac- substrate complex formation to pro-apoptotic machinery tive as a substrate. Thus, one possible function of the are being hotly pursued. One fascinating molecule to ASFV g5R protein may be to eliminate free guanine di- emerge is Nit, a member of the nitrilase superfamily, that nucleotides such as Gp4G, which may be produced by is fused to Fhit in flies and worms and coordinately ex- viral guanylyltransferases, to prevent them from inter- pressed from separate genes in vertebrates and fungi fering with cap-dependent reactions. Since vaccinia D10 [Pekarsky et al., 1998]. Whether Nit is a Fhit effector is differs considerably from g5R and lacks a conserved ty- not known. Nonetheless, the existence of the NitFhit as rosine residue found in all Ap4A hydrolases, D10 may a fusion protein in invertebrates, the coordinated expres- have a different function from g5R. sion of Nit and Fhit in mouse, and the fact that the Nit branch of the nitrilase superfamily and the Fhit branch FHIT Ap3A Hydrolase and Nucleotide Analogs of the histidine triad superfamily are found in the same Charles Brenner (Thomas Jefferson University, forms of life [Pace et al., 2000] satisfy Eisenberg’s crite- Philadelphia) reported on one of the most exciting de- ria for likely functional significance of NitFhit as a velopments in the field of intracellular ApnA function— “Rosetta Stone” protein fusion event [Marcotte et al., the recent demonstration that the product of the human 1999]. NitFhit turns out to be a tetramer in which a cen-

FHIT tumor suppressor gene, Fhit, has Ap3A hydrolase tral Nit domain binds two Fhit dimers, presenting them activity. Such an activity, which cleaves Ap3A into ADP on the opposite poles of a 200 kDa complex, now defined and AMP, was first purified from rat liver by Sillero et al. at 2.8 Å resolution. Curiously, the C-terminal elements [1977], but largely ignored until the FHIT gene was po- of Nit polypeptides are extended out of the Nit tetramer sitionally cloned from FRA3B, the most fragile site in and are in intimate contact with Fhit dimer domains [Pace the human genome, by Huebner and co-workers [Ohta et al., 2000]. While the substrates of Nit proteins remain et al., 1996] and shown to be an Ap3A hydrolase by Barnes to be identified and the nature of regulation by Fhit is et al. [1996]. Fhit is the ortholog of the S. pombe Ap4A still unknown, the community of “weird-nucleotide” fan- hydrolase [Huang et al., 1995] and thus it is considered ciers appears to be ready to embrace Nit substrates and that Fhit-related enzymes are a branch of the histidine products if they lead to the normal cell and cancer cell triad superfamily of dimeric nucleotide-binding proteins biology of dinucleoside polyphosphates. INTRACELLULAR DIADENOSINE POLYPHOSPHATES 253 An important aspect of understanding Fhit func- Attempts to achieve further purification of human tion is to assimilate the albeit limited data from much Ap3A hydrolase were performed using affinity chroma- 6 earlier studies of Ap3A hydrolases into the story. In par- tography on N -AMP-agarose. The Ap3A hydrolase ac- ticular, are Fhit and the previously described mamma- tivity present in the cytosolic fractions of human platelets lian Ap3A hydrolase the same enzyme? Pedro Rotllán and and leukocytes and rat brain was adsorbed and specifi- co-workers (University of La Laguna) have addressed this cally eluted as a single peak by 100 µM Ap3A. This pro- problem. The enzyme isolated from various rat tissues cedure yielded highly purified preparations but the Ap3A appears to be a monomeric protein of ∼32 kDa. The hu- hydrolase proved to be extremely unstable at this stage. man Fhit protein, also an Ap3A hydrolase, is a homodimer Electrophoresis of these affinity-purified fractions re- of two 16.8 kDa [Barnes et al., 1996], suggesting that the vealed at least three bands, probably indicating the pres- previously known Ap3A hydrolase and Fhit could be the ence of several Ap3A-binding proteins in addition to Ap3A same protein. While human Fhit protein expressed in E. hydrolase. coli is readily available [Barnes et al., 1996; Pawelczyk et A near homogeneous preparation was obtained from al., 2000], Ap3A hydrolase has not yet been isolated di- the human leukocyte cytosolic fraction after ammonium rectly from human cells. sulfate fractionation, ion-exchange chromatography, and Rotllán and co-workers have undertaken the puri- a final affinity chromatography step. After native PAGE fication of human Ap3A hydrolase and compared its prop- the final preparation showed a single Coomassie blue- erties with those of the human Fhit protein [Asensio et stained band, coincident with the band detected after al., 2000]. Di-ethenoadenosine triphosphate, ε-(Ap3A), a immunoblotting using a Fhit antibody, and with the en- fluorogenic Ap3A analog, was used for fluorimetric mea- zymic activity peak detected after extraction and assay- surement of reaction rates. Human Ap3A hydrolase was ing Ap3A hydrolase activity in parallel sliced lanes. SDS/ purified from platelet, leukocyte, and placental cytosolic PAGE revealed an intense silver-stained band corre- fractions using standard procedures: ammonium sulfate sponding to a polypeptide of about 17 kDa. fractionation, gel-filtration (Sephacryl S-200 HR), and Ap3A hydrolase-containing fractions at several ion-exchange chromatography (DEAE-Sephacel). Spe- stages of purification were subjected to Western blotting cific activity in the cytosol of blood cells was very low, analysis. Results indicated that Ap3A hydrolase activity about 0.1 mU/mg protein. Nonspecific phosphodiesterase and immunoreactivity against Fhit antibodies copurified. activity was absent from platelet and leukocyte extracts After SDS/PAGE and immunoblotting of purified human but clearly detected in placenta. Partially purified Ap3A Ap3A hydrolase (gel filtration, ion-exchange, and affinity hydrolase after the ion-exchange chromatography step chromatography fractions) and rat brain Ap3A hydrolase was used for biochemical studies. (affinity chromatography), only bands close to 17 kDa,

Both the human Ap3A hydrolase and Fhit proteins the size of Fhit monomers, were detected. However, af- had Km values close to 2 µM for ε-(Ap3A), exhibited simi- ter native PAGE and immunoblotting, evident differences lar pH optima of 7.0–7.2, and were similarly activated by of mobility between the bands detected in human and Mg2+, Ca2+, and Mn2+ and inhibited by Zn2+. Suramin, rat fractions and Fhit were observed. Isoelectric focus- the most potent inhibitor of rat brain Ap3A hydrolase ing followed by immunoblotting allowed the detection [Rotllán et al., 1998], displayed a competitive pattern of of bands with pI values of about 6.3 for platelet and leu- inhibition for human Ap3A hydrolase but a rather mixed kocyte Ap3A hydrolase and around 6.5 for Fhit. Human pattern for Fhit, while Ki values for both enzymes were Ap3A hydrolase is therefore a slightly more acidic pro- about 25 nM. Fluoride, heparin, and heparin disaccha- tein than Fhit. rides were not inhibitors. The histidine-specific reagent Taken together, these results indicate that human Ap3A diethyl pyrocarbonate rapidly inactivated both human hydrolase and human Fhit protein are closely related pro-

Ap3A hydrolase and Fhit. Thus, kinetically, both hydro- teins kinetically and immunochemically. They present some lases are barely distinguishable. slight structural differences accounting for some distinc- Fhit antibodies differentially inhibited the enzymic tions in their observed behavior. Their unequal pI values activity of Fhit and Ap3A hydrolase, Fhit protein being the suggest minor posttranslational or purification-generated more sensitive. Purified human Ap3A hydrolase eluted dur- differences involving charged amino acids. ing gel filtration as a single peak with an apparent molecu- The areas of extra- and intracellular ApnA function lar mass of ~32 kDa. However, Fhit protein eluted as a and metabolism come together in the work of Mike broad peak, suggesting the existence of several enzymically Blackburn (University of Sheffield). The design and active Fhit oligomers in addition to the homodimer. This preparation of successful agonists/antagonists for nucle- point was confirmed by native PAGE and immunodetection otide receptors has to meet three requirements. First, with Fhit antibodies. SDS/PAGE, however, proved that the the analog has to have high affinity for the receptor pro- Fhit monomer is a 17 kDa polypeptide. tein. Second, it has to resist hydrolysis by specific and 254 MCLENNAN ET AL. nonspecific nucleotide phosphatases and pyrophos- otic enzyme hydrolysis, both of these features were phohydrolases. Third, it has to possess good bioavail- introduced into species such as (P1,P2-methylene-P3- ability. Over the last 15 years the work of Blackburn and thio)-diadenosine triphosphate, AdoPCH2POPSAdo collaborators on ApnA analogs has primarily focused on [Blackburn et al., 1990b]. This has a useful affinity for the rational design of the first two of these features, with the Fhit protein with a Kd of 40 µM and effectively re- particular attention to nucleotide analog stability to eu- sists hydrolysis. X-ray crystal structures of two diaste- karyotic and prokaryotic degradative enzymes. Many of reoisomers of this analog complexed to wild-type and these enzymes show high specificities with Km values in to an active-site mutant of Fhit show one adenosine the micromolar range for both natural and synthetic sub- bound in a deep cleft and with opposite stereospecifici- strates. ties for the stereochemistry of a P(S) ligand in the P1 Two devices have been prominent in delivering and P3 sites [Pace et al., 1998]. This explains the gen- resistance of analogs to hydrolysis in vitro and in vivo. eral observation that one adenosine is recognized with The first employed a range of carbon bridges to replace high specificity while there is ample scope for modifi- the P-O-P pyrophosphate by an alkylenebisphosphonate, cation of the second adenosine either to other nucleo-

P-(C)n-P. The virtue of this construct is the stability of the sides or to non-nucleoside ligands. Some indication of C-P(O) bond to enzymic hydrolysis and its close stere- weakened binding of thiophosphate relative to phos- ochemical relationship to the parent O-P(O) entity. Its phate analogs was also observed for Fhit, although the disadvantage is the inevitable reduction of electronega- reverse is the case for the P2T receptor. tivity resulting from the exchange of an oxygen ligand for Studies were next directed to the question of the a carbon ligand at phosphorus. This deficiency was cor- contribution of anionic charge on nucleotide binding to rected in two ways to restore isoelectronic character to Fhit by the synthesis of “supercharged” analogs of pyro- the phosphonate moieties: the use of the CF2 group and phosphoric acid—specifically, methylenetrisphosphonic the insertion of an acetylenic linkage adjacent to phos- acid, HC(PO3H2)3, and its halo-derivatives. The corre- phorus, as in P(O)CF2P(O) and P(O)-C≡C-P(O) bisphos- sponding supercharged ATP analog, AdoPOPC(P)2, has phonates, respectively [Blackburn et al., 1984]. X-ray increased anionic charge (≥5 –ve) relative to ATP (4 –ve). analysis of nucleotides containing the former show that Nucleotides incorporating such trisphosphonates show they are isosteric and isopolar with the parent pyrophos- differential competitive inhibition between two specific phate linkage. By contrast, the acetylenic bridge is hydrolases: the human Fhit protein and the Ap3A hydro- strongly electronegative for phosphorus but is clearly lase from yellow lupin seeds [Liu et al., 1999]. The nonisosteric. In particular, it means that an analog of ATP adenylated polyphosph(on)ates are not hydrolyzed by such as AdoP-O-P-C≡C-P will not bind magnesium be- these enzymes; however, they strongly and competitively tween the β- and γ-phosphoryl groups. The single inser- inhibit Fhit with Ki values similar to Km for Ap3A (1.9 tion of CX2 bisphosphonates into ApnA analogs can µM), while they are significantly less effective as inhibi- maintain nucleotide binding in the micromolar range. tors of the lupin enzyme. The competitive inhibition of However, the insertion of two such moieties, as in Fhit shows a rather good LFE (linear free energy) rela-

αβ:α′β′-disubstituted phosphonate analogs of Ap4A, has tionship to anionic charge for six adenosine phosphonate generally resulted in weakened affinity for the enzyme analogs, requiring at least one adenosine moiety for bind- [McLennan et al., 1989]. ing and with the best inhibitor having six negative It has therefore proved desirable to introduce a charges. However, the apparently beneficial effect of ad- second feature to endow analogs with resistance to hy- ditional negative charge will certainly have adverse ef- drolysis: the substitution of sulfur for oxygen as a fects on bioavailability and makes a search for non-ionic nonbridging phosphorus ligand. This device, introduced bioisosteres of phosphates a key feature of future work. for nucleotides in the 1960s by Fritz Eckstein, can be By contrast, the use of a very sensitive fluorescence-based employed readily in complementation with alkylene- assay [Draganescu et al., 2000] for nucleotide binding to bisphosphonates. It has to be noted that the introduction Fhit has revealed that incorporation of sulfur at the phos- of each P(S) moiety creates a new stereochemical center phoryl centers adjacent to the adenosine moiety can pro- in the nucleotide analog, for which chirality can be con- mote protein affinity by up to two orders of magnitude trolled either by stereospecific synthesis or by chromato- and provide ligands with nanomolar affinities for Fhit. graphic separation of diastereoisomers. Some early results Clearly, the stereochemical preferences for such modifi- showed a preference for one or the other chirality at the cation will be the focus of future work.

P(S) center for resistance to specific enzyme cleavage, Assays with two recombinant asymmetrical Ap4A while good overall affinity for the protein was retained hydrolases (lupin and human) has shown that none of the [Blackburn et al., 1990a]. above nucleotide analogs acts as a substrate, while they

In designing analogs of Ap3A resistant to eukary- are poor to moderately effective inhibitors of these en- INTRACELLULAR DIADENOSINE POLYPHOSPHATES 255 zymes. For example, the derivative with two adenylates Because a dinucleoside polyphosphate resembles and one adenosine did not inhibit the human enzyme the 3′-OH end of an RNA, it could conceivably behave and was a poor inhibitor of the lupin counterpart (I50 = as an RNA acceptor analog in reaction 3. This possibility 650 µM). Only the three-adenylated compound moder- was explored using cytidine-3′,5′-bisphosphate (pCp) as ately and competitively inhibited both enzymes with I50 an RNA donor in reaction 2 and several NpnN as RNA values (estimated at 50 µM Ap4A) of 80 µM and 40 µM acceptors in reaction 3. for the human and lupin enzymes, respectively. Reaction mixtures were set up containing ATP,

These results raise the question of improved intra- pCp, NpnN and T4 RNA ligase and analyzed by HPLC cellular availability of nucleotide analogs. The inherent at different times of incubation. In this way, it could be liability to ready hydrolysis and alkylating potential of demonstrated that the 5′-adenylated cytidine 5′,3′-bis- fully esterified polyphosphate species (e.g., tetraethyl phosphate (AppCp) synthesized from ATP and pCp in pyrophosphate) disfavors the pursuit of fully esterified reaction 5 is used as a substrate to transfer the pCp resi- analogs of ApnAs as delivery vehicles and pro-ligands for due to the 3′-OH end of a dinucleoside polyphosphate receptor proteins in vivo. Moreover, the potential ben- (reaction 6) and to a lesser extent to both 3′-OH ends efit of thiophosphates for cell availability remains largely (reaction 7): unexplored. Consequently, a search for non-ionic bioisosteres of phosphate will dominate the next phase 5.) E-AMP + pCp ↔ E-AppCp of work on nucleotide ligand design and synthesis. A few 6.) E-AppCp + NpnN → NpnNpnCp + E + AMP leads are available, as from the design of stable mimics 7.) E-AppCp + NpnNpCp → pCpNpnNpCp + E for anionic intermediates in the hydrolysis of esters by + AMP catalytic antibodies of peptides by aspartyl proteases. In particular, the potential of tetrahedral moieties such as Using pCp and Gp4G as substrates, in the presence secondary hydroxyl functions (CH(OH) as in statin) and of ATP, formation of Gp4GpCp and pCpGp4GpCp was ob- fluoroketones (as CF2(OH)2 covalent hydrates) are two tained in a ratio of 10:1, respectively. Reactions 6 and 7 promising candidates for further exploration. can also be followed indirectly by measuring the forma-

tion of AMP. The Gp4GpCp synthesized was isolated and Synthesis of ApnAs characterized by treatment with snake venom phosphodi- The ability of a variety of ligases to synthesize esterase and alkaline phosphatase and analysis (chromato- dinucleoside polyphosphates has been investigated in de- graphic position and UV spectra) of the reaction products tail in recent years by Antonio Sillero’s group. The follow- by HPLC. The apparent Km values measured for Gp4G ing enzymes belonging to this group were shown to catalyze and Ap2Cp were around 4 and 0.4 mM, respectively. In the synthesis of nucleoside polyphosphates (pnN) and the presence of 0.5 mM ATP and 0.5 mM pCp, the rela- dinucleoside polyphosphates (NpnN): aminoacyl-tRNA syn- tive efficiencies of the following NpnN as acceptors of pCp thetases; firefly luciferase (EC 1.13.12.); acetyl-CoA syn- from Ap2Cp are indicated in parentheses: Gp4G (100); thetase (EC 6.2.1.1); acyl-CoA synthetase (EC 6.2.1.8); DNA Gp5G (101); Ap4G (47); Ap4A (39). Gp2G, Gp3G and Xp4X ligase (AMP forming) (EC 6.5.1.1); RNA ligase (EC 6.5.1.3) were not substrates for the reaction [Atencia et al., 2000].

[see Sillero and Sillero, 2000 for a review]. Thus, T4 RNA ligase: 1.) discriminates between NpnN T4 RNA ligase catalyzes the joining of RNA mol- containing the same number of internal phosphates (Gp4G, ecules via the following reversible steps: Ap4G, Ap4A, and Xp4X) and between homodinucleotides containing chains of internal phosphates of differing lengths

1.) E + ATP ↔ E-AMP + PPi (Enzyme ad- (Gp2G, Gp3G, Gp4G, and Gp5G); 2.) requires a minimum enylylation) of four internal phosphates; and 3.) prefers guanine to ad- 2.) E-AMP + 5′-P-RNA ↔ E-AppRNA (RNA ad- enine or xanthine dinucleotides. The nucleotide specific- enylylation) ity of T4 RNA ligase is different from that exhibited by 3.) E-AppRNA + 3′-OH-RNA ↔ RNA-p-RNA + two enzymes that specifically cleave dinucleoside

AMP + E (RNA joining) polyphosphates: Ap3A hydrolase and Ap4A hydrolase [Guranowski and Sillero, 1992]. The 5′-P and the 3′-OH ends correspond to that of Apart from its utility for dinucleotide modification the RNA donor and RNA acceptor, respectively. T4 RNA in vitro, it is difficult to grasp the possible physiological ligase also transfers the adenylyl moiety of the E-AMP meaning of the reactions described here. Very little is complex to a nucleoside triphosphate (NTP) giving rise known about the function of the enzyme itself in vivo. to a dinucleoside tetraphosphate [Atencia et al., 1999]: The only phenotype attributed to mutations in T4 gene 63, coding for T4 RNA ligase, is the failure of the

4.) E-AMP + NTP → Ap4N + E noncovalent attachment of the tail fibers, leading to 256 MCLENNAN ET AL. fiberless T4 particles. In the process of T4 phage mor- there were no published data on the existence or con- phogenesis, the enzyme could recognize certain motifs centrations of ApnA in pancreatic β-cells, measurements of the phage proteins which resemble the structures of of Ap3A and Ap4A were first made using high-performance Gp4G and Gp5G and which have been revealed by chance liquid chromatography. The concentrations of both nucle- using this experimental approach. otides were estimated under two different sets of conditions: in nonstimulated cells, i.e., cells incubated in basal ApnA and Insulin Function glucose (3 mM), and in cells incubated in high glucose

Insulin secretion from pancreatic β-cells is essen- (22 mM). The increase in glucose augmented Ap4A and tial in glucose homeostasis. This hormone is released by Ap3A levels from submicromolar to tens of micromolar. exocytosis in response to the presence of glucose in Stimulatory glucose concentrations were found to in- plasma. Glucose enters the cell, generating metabolites crease cytosolic ApnA to concentrations sufficient to block that selectively inhibit ATP-sensitive potassium (KATP) KATP channels. Furthermore, other nutrients such as leu- channels, depolarizing the cell and triggering insulin se- cine and 2-ketoisocaproate raised Ap4A and Ap3A as effi- cretion. KATP channels are involved in a variety of signal- ciently as glucose, while non-nutrient secretagogues, such ing pathways that transduce cellular metabolic events into as high extracellular potassium or tolbutamide, had no membrane potential changes. Due to their potential clini- effect. In conclusion, β-cell KATP channels can be effec- cal implications, KATP has generated most interest in two tively blocked by ApnA at concentrations induced by nu- particular processes: glucose-induced insulin secretion trient (glucose, amino acids) stimulation [Martin et al., of pancreatic β-cells and ischemia-associated action po- 1998]. However, unlike ATP, Ap4A does not restore KATP tential in heart muscle. Although the defining property channel activity after rundown [Ripoll et al., 1996; Mar- of KATP channels is their inhibition by intracellular ATP, tin et al., 1998]. it is now widely accepted that other intracellular modu- In the light of these results, Soria’s group proposed lators of KATP channels may also be involved in the regu- ApnA as a new KATP channel regulator in pancreatic β- lation of insulin secretion in response to glucose cells, and so provide a physiological meaning to the ex- stimulation. Particularly, the role of several nucleotides istence of two metabolic pathways in the early steps that in KATP channel regulation, namely ADP, GTP, GDP, or lead from glucose sensing to insulin secretion that con- UTP, has been extensively analyzed. verge in KATP channel regulation. These data also high- Recent studies have demonstrated that ApnA modu- light the fact that the list of messengers derived from the late the gating of KATP channels in isolated membrane metabolism of glucose may still be incomplete. patches of ventricular cells [Jovanovic and Terzic, 1995]. Given the similarities found in the physiological behav- ApnA In Prokaryotic Systems ior of KATP channel activity in cardiac and β-cells, it seems Although most current investigations of ApnA in- feasible that ApnA could also be pharmacologically ac- volve eukaryotic systems, it must not be forgotten that tive on β-cells. Current work from Bernat Soria’s labora- many of the early studies describing their response to tory (Miguel Hernández University) was outlined by Juan metabolic stresses were carried out in prokaryotes and

Manuel Rovira. These studies are directed toward de- some recent studies on the possible involvement of Ap4A termining the effects of ApnA on pancreatic KATP chan- in the heat-shock response in E. coli, in particular the nels and then analyzing their suitability as potential binding of Ap4A to the molecular chaperone GroEL and mediators in β-cell signaling. the posttranslational modification of the LysU lysyl-tRNA

To investigate the effect of ApnA on pancreatic KATP synthetase, were described by Julian Tanner from An- channels, the patch-clamp technique was used. Ap4A was drew Miller’s lab (Imperial College, London). Ap4A is applied at micromolar concentrations to the intracellu- present in submicromolar concentrations in prokaryotes, lar side of β-cell membrane patches and found to pro- but is induced to much higher levels (>100 µM) under a voke a rapid and fully reversible decrease of KATP channel variety of stress conditions [Lee et al., 1983]. Ap4A may activity. The effect was comparable to that observed for act as a signal molecule whose function is to regulate or ATP, both in time-course and effective concentration modify stress responses on the basis that it binds to sev- range. The estimated half-maximal inhibition (18 µM) for eral E. coli heat-shock proteins, including molecular

Ap4A closely resembled that found for ATP channel inhi- chaperones GroEL, DnaK, and ClpB that assist protein bition (17 µM). These results demonstrated that Ap4A folding/refolding in vivo [Johnstone and Farr, 1991]. This acts as an effective inhibitor of β-cell KATP channel activ- view is supported by experiments showing that Ap4A ity, as expected from results previously obtained in car- overproduction causes cells to be oversensitive to heat diac muscle [Ripoll et al., 1996]. This first observation shock [Farr et al., 1989]. Aminoacyl-tRNA synthetases are led Soria and co-workers to investigate the potential sig- a major source of Ap4A in cells and in E. coli the heat- nificance of ApnA as a modulator of β-cell function. As inducible lysyl-tRNA synthetase (LysU) is the most ef- INTRACELLULAR DIADENOSINE POLYPHOSPHATES 257 fective Ap4A synthetase—80% of total Ap4A synthesis in release of over five times more refolded MDH from E. coli cell extracts has been ascribed to lysyl-tRNA syn- GroEL than was released in the absence Ap4A. This ob- thetase catalysis [Charlier and Sanchez, 1987]. servation was corroborated by differential scanning calo-

Escherichia coli lysyl-tRNA synthetase (heat-induc- rimetry studies that showed Ap4A to significantly ible LysU and constitutively expressed LysS) can be re- destabilize a complex formed between GroEL and un- solved into several peaks of enzymic activity during the folded α-lactalbumin, giving rise to a two-step unfolding last stage of purification by hydroxyapatite chromatogra- transition consistent with differing interactions between phy [Charlier and Sanchez, 1987]. Recombinant LysU cis and trans rings of GroEL. Taken together, these re- was further resolved in the Miller laboratory into two sults suggest that Ap4A is able to bind to GroEL at a site main peaks of activity by hydroxyapatite chromatogra- distinct from the well-established ADP/ATP binding site. phy. Using anti-phosphothreonine Western blots and The functional purpose of this allosteric binding interac- Ames malachite green assays, it was shown that the first tion then appears to be destabilization of the high-tem- peak eluting at 70 mM potassium phosphate corre- perature protective complex formed between an unfolded sponded with unphosphorylated LysU, while that elut- protein substrate and GroEL under heat-shock condi- ing at 100 mM potassium phosphate corresponded with tions [Llorca et al., 1998], forcing the release of refolded LysU phosphorylated on threonine. Under normal growth protein back into solution. conditions (37°C), approximately 30% of the recombinant In conclusion, phosphorylation of LysU resulted in LysU was found to be phosphorylated. This result is con- some significant changes to the rate and thermal persis- sistent with some of the earliest studies on the lysyl-tRNA tence of Ap4A synthesis catalyzed by LysU. Therefore, synthetase of E. coli, which suggested that the enzyme phosphorylation of LysU may represent an important way was phosphorylated, but at less than 1 mole of phosphate to control the synthesis Ap4A in E. coli under stress con- per mole of enzyme [Stern and Peterkofsky, 1969]. An ditions. There may also be a link between Ap4A, the E. analysis of phosphorylated and unphosphorylated LysU coli hsp70 DnaK, and the aminoacyl-tRNA synthetases. by circular dichroism (CD) spectroscopy revealed only DnaK has been shown to participate in the phosphoryla- very slight changes in tertiary structure as a result of tion of the glutaminyl-tRNA synthetase and threonyl- phosphorylation. However, phosphorylation did induce tRNA synthetase of E. coli [Wada et al., 1986]. Therefore, some changes in the kinetics of Ap4A synthesis. The over- DnaK may well be the source of LysU phosphorylation all rate of Ap4A synthesis was slightly increased as a con- as well, a possibility that is under active investigation. –1 sequence of phosphorylation (134 ± 10 mol min for The effects of Ap4A on GroEL point towards a cellular –1 unphosphorylated LysU, 150 ± 12 mol min for phos- role in which Ap4A assists the return of cells to normal phorylated LysU). However, the specificity constant kcat/ growth conditions following stress, and promotes release –1 –1 Km for lysine of phosphorylated LysU (290 sec .mol ) of molecular chaperone-bound cellular proteins under was found to be over three times that of the unphos- prolonged stress to maintain cellular viability. These ef- –1 –1 phorylated enzyme (80 sec mol ), suggesting that phos- fects could also account for the heat-sensitivity of Ap4A phorylation had significantly increased the velocity of hydrolase-deficient apaH mutants, which have elevated aminoacylation. Phosphorylated LysU also appeared to intracellular levels of Ap4A [Farr et al., 1989]. be significantly more thermostable than the unphosphorylated enzyme. ACKNOWLEDGMENTS In order to characterize a molecular role for Ap4A This work was supported in part by grants and awards: in the heat-shock response, the interaction between Ap4A NSF grant MCB-9982645 (LDB); NIH grants CA75954 and the molecular chaperone GroEL was also studied. and CA77738 (CB); KBN Project Grant P04A 06215 from µ Ap4A was found to bind tightly to GroEL (Kd < 10 M), the State Committee for Scientific Research (AG); grant both in the presence and absence of ADP, according to PM96-0081 from the Dirección General de Enseñanza the results of a CD binding assay [Holler, 1984]. Using Superior (PR), grants from the Dirección General de isothermal calorimetry, Ap4A was found not to inhibit the Investigación Científica y Técnica (PM95/0013; PM 98/ binding of ADP to GroEL. Moreover, no hydrolysis of 0129) and Comunidad de Madrid (08.9/0004/98) (AS), and Ap4A could be detected as a result of binding to GroEL. project grants 053038 from the Wellcome Trust and F25BJ The potential significance of this tight binding interac- from the Leverhulme Trust (AGM). tion was investigated using two model protein refolding ° assay systems. At low temperatures (<30 C), Ap4A was REFERENCES found to have no effect on the refolding of unfolded malate Asensio AC, Rodriguez-Ferrer CR, Oaknin S, Rotllán P. 2000. Human dehydrogenase (MDH) mediated by GroEL (and co- diadenosine triphosphate hydrolase: preliminary characterisation chaperone GroES). However, at higher heat-shock tem- and comparison with the Fhit protein, a human tumour suppressor. peratures (>40°C), Ap4A was found to promote the Acta Biochim Polon 47:435–441. 258 MCLENNAN ET AL.

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