Fhit Proteins Can Also Recognize Substrates Other Than Dinucleoside Polyphosphates

FEBS Letters 582 (2008) 3152–3158 Fhit proteins can also recognize substrates other than dinucleoside polyphosphates Andrzej Guranowskia,*, Anna M. Wojdyłaa, Małgorzata Pietrowska-Borekb, Paweł Bieganowskic, Elena N. Khursd, Matthew J. Cliffe, G. Michael Blackburnf, Damian Błaziakg, Wojciech J. Stecg a Department of Biochemistry and Biotechnology, The University of Life Sciences, 35 Wołyn´ska Street, 60-637 Poznan´, Poland b Department of Plant Physiology, The University of Life Sciences, 60-637 Poznan´, Poland c International Institute of Molecular and Cell Biology, Warsaw, Poland d Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia e Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield, UK f Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield, UK g Center of Molecular and Macromolecular Studies, Polish Academy of Sciences, Ło´dz´, Poland Received 1 July 2008; revised 17 July 2008; accepted 31 July 2008 Available online 9 August 2008 Edited by Hans Eklund This work is dedicated to Professor Antonio Sillero on the occasion of his 70th birthday and to Professor Maria Antonia Gu¨nther Sillero. 1. Introduction Abstract We show here that Fhit proteins, in addition to their function as dinucleoside triphosphate hydrolases, act similarly to adenylylsulfatases and nucleoside phosphoramidases, liberat- Cells contain various minor nucleotides. Among these are 0 000 1 n 0 ing nucleoside 50-monophosphates from such natural metabolites the dinucleoside 5 ,5 -P ,P -polyphosphates (NpnN s, where as adenosine 50-phosphosulfate and adenosine 50-phosphorami- N and N0 are 50-O-nucleosides and n represents the number date. Moreover, Fhits recognize synthetic nucleotides, such as of phosphate residues in the polyphosphate chain that esterifies 0 0 0 0 0 adenosine 5 -O-phosphorofluoridate and adenosine 5 -O-(c-flu- N and N at their 5 position) [1].NpnN s accumulate as a re- orotriphosphate), and release AMP from them. With respect sult of the activity of certain ligases and transferases that cat- to the former, Fhits behave like a phosphodiesterase I concomi- alyze transfer of a nucleotidyl moiety onto various acceptors tant with cleavage of the P–F bond. Some kinetic parameters containing a pyrophosphate residue, including NTPs (pppNs) and implications of the novel reactions catalyzed by the human and NDPs (ppNs), from a variety of donors. For reviews see and plant (Arabidopsis thaliana) Fhit proteins are presented. 0 Ó 2008 Published by Elsevier B.V. on behalf of the Federation of [2,3].NpnN s play different intracellular and extracellular func- 0 European Biochemical Societies. tions [4–6]. The cellular level of NpnN s can be controlled by various hydrolases or phosphorylases [7]. Among specific Keywords: Fhit protein; Dinucleoside triphosphatase activity; hydrolases is the dinucleoside triphosphatase (EC 3.6.1.29) Nucleoside phosphoramidase activity; Adenylylsulfatase that preferentially hydrolyzes NpppN0 to an NMP and N0DP activity; Phosphodiesterase I activity; P–F bond cleavage (see Reaction 1): This enzyme was first discovered in SillerosÕ laboratory in extracts of rat liver [8] and subsequently in extracts of yellow *Corresponding author. Fax: +48 61 8487146. E-mail address: [email protected] (A. Guranowski). 0 0 Abbreviations: APS or S-pA, adenosine 5 -phosphosulfate; NH2-pA, adenosine 5 -phosphoramidate; ATP-F or F-pppA, adenosine 50-O-(c-fluorotriphosphate); HPLC, high performance liquid chromatography; TLC, thin layer chromatography 0014-5793/$34.00 Ó 2008 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2008.07.060 A. Guranowski et al. / FEBS Letters 582 (2008) 3152–3158 3153 lupin seeds [9], Saccharomyces cerevisiae [10], Artemia [11], and remaining proteins were precipitated with ammonium sulfate added green algae [12]. The intriguing finding that FHIT (from fragile to 70% saturation. Precipitated proteins were resuspended in buffer histidine triad), a putative human tumor suppressor gene, en- A and ammonium sulfate precipitation was repeated. The resulting protein pellet was resuspended in buffer A and loaded onto an codes a typical dinucleoside triphosphatase [13] focused much AMP-agarose column. Unbound proteins were washed out with buffer attention on this particular enzyme. FHIT genes occur in dif- A and Fhit proteins were eluted with buffer A supplemented with ferent eukaryotic organisms [14] and homogeneous Fhit pro- 1 mM adenosine. This procedure yielded proteins that were about teins have been obtained by overexpression of FHIT from 90% pure by SDS–PAGE gel. Molecular mass of the human Fhit monomer is 16800 Da and Arabidopsis Fhit monomer 18120 Da. Both human [15], yeast [16] and Arabidopsis thaliana (present study). Fhit samples were then dialyzed against 20 mM Hepes/NaOH, pH 7.5 Work described here stemmed from the investigations of the containing 100 mM NaCl and 5% glycerol, concentrated by ultrafiltra- 0 regiospecificity of the hydrolysis of NpnN s and in particular tion on Microcone filters from Millipore to 1.3 mg/ml in case of the hu- 0 man Fhit and to 1.5 mg/ml in case of the Arabidopsis Fhit, and stored from a study on the mechanism of action of Fhit/Np3N 0 at À20 °C. hydrolases. Preference for Np3N s and a unique mode of sub- 0 strate cleavage are features that distinguish Np3N hydrolases 0 18 2.2. Enzyme assays from other specific hydrolases acting on NpnN s. Using H2 O Hydrolytic activities of the Fhit proteins were assayed in reaction and mass spectrometry of the isolated reaction products, it was mixture (0.1 ml) containing 50 mM Mes/KOH (pH 6.5), 5 mM MgCl2, 0 first shown that the Np3N hydrolase from yellow lupin exclu- 1 mM substrate and rate-limiting amounts of either human or plant sively hydrolyzes the anhydride bond of its substrates (ApppA, Fhit protein. The reactions were carried out at 30 °C. At time intervals a b (0, 5, 10 and 30 min), 20 ll aliquots were withdrawn and the reaction AppppA, AppCH2pA or AppCHFpA) between P and P , 18 stopped by heating the samples for 5 min at 96 °C. The samples were incorporating O only into AMP [17]. It was subsequently chilled, diluted three-fold with 50 mM TEAB (triethylamine buffer, 0 shown by Abend et al. that human Fhit/Np3N hydrolase acts pH 7.4), filtered through ultrafree-MC filters (from Millipore) and in the same fashion [18]. Moreover, they demonstrated that 10 ll aliquots subjected to high performance liquid chromatography cleavage proceeds with overall retention of configuration at (HPLC) on a Discovery C18 column (4.6 · 250 mm, 5 lm); Supelco at a flow rate 1 ml/min. The column was eluted with a linear gradient phosphorus, implying a double inversion mechanism, and they of 50 mM TEAB (pH 7.4) (solvent A) and solvent A:acetonitrile postulated that all three histidines of the histidine triad (His94, (60:40, v/v) (solvent B); 0–19 min, 40% B. The retention times of the His96 and His98) are involved in the formation of an interme- nucleotides are presented in Table 1. At the aforementioned experi- diate in which His96 first undergoes adenylation and then mental conditions there was a linear dependence between time and deadenylation by water. Pursuing that issue, Frey et al. showed AMP, the reaction product, peak areas. This allowed to calculate and compare the rates of the hydrolysis of investigated substrates. that Fhit and specifically mutated Fhit variants catalyzed the The nucleoside phosphoramidase activity of the Fhits was estimated hydrolysis of adenosine 50-phosphoimidazolide (APS or S- in a reaction mixture containing 50 mM Mes/KOH (pH 6.5) and 3 pA) [19] and, poorly, of p-nitrophenyl-AMP [20]. Thus, Fhits appropriate concentration of NH2-p[8- H]A. When the Km values were behave like a nucleoside phosphoramidase and phosphodies- estimated the radiolabeled substrate concentration varied between 1 and 15 lM. At time intervals, 5 ll aliquots of the reaction mixture were terase, respectively. spotted onto thin layer chromatography (TLC) aluminum plates pre- We demonstrated that two naturally occurring AMP deriv- coated with silica gel containing fluorescent indicator (from Merck), atives: sulfoadenylate (S-pA), a key metabolite on the sulfate standards of NH2-pA and AMP applied at the origin and the plates assimilation pathway, and adenosine 50-phosphoramidate developed for 60 min in dioxane/ammonia/water (6:1:4, by vol.). Spots (NH -pA, aminoadenylate), a less known natural metabolite of the nucleotides were visualized under short-wave UV light and those 2 of the reaction product (AMP) cut out, immersed in a scintillation [21] originating from APS by enzymatic displacement of its sul- cocktail, and radioactivity counted. fate moiety by ammonia [22], are FhitsÕ substrates. We also Ki values of different nucleotides used in competition with NH2- showed that both human and plant (Arabidopsis) Fhits liberate p[8-3H]A in the nucleoside phosphoramidase reaction were determined in a reaction mixture (50 ll) containing 50 mM Mes/KOH (pH 6.5), AMP from such synthetic AMP congeners as fluoro-adenylate 3 5 mM MgCl2, 2.75 lMNH2-p[8- H]A (580000 c.p.m.) and varied con- (F-pA) and fluoro-ATP (ATP-F). centrations of one of the following nucleotides: AMP, ADP, ATP, 0 Ap3A, S-pA, F-pA or adenosine-5 -O-(c-fluoridotriphosphate) (ATP- F or F-pppA). Reaction rates were estimated as described above. Ki values were calculated according [28]. 2. Materials and methods 2.1. Materials Adenosine 50-O-fluorophosphate (F-pA) was synthesized according Table 1 to [23] and adenosine 50-O-(c-fluorophosphate) (Fpp-pA) according Various substrates of human and Arabidopsis Fhit proteins to procedure developed for the synthesis of GTPcF [24]. Other adenine Nucleotide Retention Relative velocities of hydrolysis (di)nucleotides were from Sigma, St.

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