Disease-associated Mutations Inactivate AMP-Lysine Hydrolase Activity of Aprataxin
2005; Elsevier BV; Volume: 280; Issue: 22 Linguagem: Inglês
10.1074/jbc.m502889200
ISSN1083-351X
AutoresHeather F. Seidle, Paweł Bieganowski, Charles Brenner,
Tópico(s)Mitochondrial Function and Pathology
ResumoAtaxia-oculomotor apraxia syndrome 1 is an early onset cerebellar ataxia that results from loss of function mutations in the APTX gene, encoding Aprataxin, which contains three conserved domains. The forkhead-associated domain of Aprataxin mediates protein-protein interactions with molecules that respond to DNA damage, but the cellular phenotype of the disease does not appear to be consistent with a major loss in DNA damage responses. Disease-associated mutations in Aprataxin target a histidine triad domain that is similar to Hint, a universally conserved AMP-lysine hydrolase, or truncate the protein NH2-terminal to a zinc finger. With novel fluorigenic substrates, we demonstrate that Aprataxin possesses an active-site-dependent AMP-lysine and GMP-lysine hydrolase activity that depends additionally on the zinc finger for protein stability and on the forkhead associated domain for enzymatic activity. Alleles carrying any of eight recessive mutations associated with ataxia and oculomotor apraxia encode proteins with huge losses in protein stability and enzymatic activity, consistent with a null phenotype. The mild presentation allele, APTX-K197Q, associated with ataxia but not oculomotor apraxia, encodes a protein with a mild defect in stability and activity, while enzyme encoded by the atypical presentation allele, APTX-R199H, retained substantial function, consistent with altered and not loss of activity. The data suggest that the essential function of Aprataxin is reversal of nucleotidylylated protein modifications, that all three domains contribute to formation of a stable enzyme, and that the in vitro behavior of cloned APTX alleles can score disease-associated mutations. Ataxia-oculomotor apraxia syndrome 1 is an early onset cerebellar ataxia that results from loss of function mutations in the APTX gene, encoding Aprataxin, which contains three conserved domains. The forkhead-associated domain of Aprataxin mediates protein-protein interactions with molecules that respond to DNA damage, but the cellular phenotype of the disease does not appear to be consistent with a major loss in DNA damage responses. Disease-associated mutations in Aprataxin target a histidine triad domain that is similar to Hint, a universally conserved AMP-lysine hydrolase, or truncate the protein NH2-terminal to a zinc finger. With novel fluorigenic substrates, we demonstrate that Aprataxin possesses an active-site-dependent AMP-lysine and GMP-lysine hydrolase activity that depends additionally on the zinc finger for protein stability and on the forkhead associated domain for enzymatic activity. Alleles carrying any of eight recessive mutations associated with ataxia and oculomotor apraxia encode proteins with huge losses in protein stability and enzymatic activity, consistent with a null phenotype. The mild presentation allele, APTX-K197Q, associated with ataxia but not oculomotor apraxia, encodes a protein with a mild defect in stability and activity, while enzyme encoded by the atypical presentation allele, APTX-R199H, retained substantial function, consistent with altered and not loss of activity. The data suggest that the essential function of Aprataxin is reversal of nucleotidylylated protein modifications, that all three domains contribute to formation of a stable enzyme, and that the in vitro behavior of cloned APTX alleles can score disease-associated mutations. Enzyme's Hydrolase Activity Hints at Root of DiseaseJournal of Biological ChemistryVol. 280Issue 22PreviewAtaxia-oculomotor apraxia syndrome 1 is a rare neurological disorder that results from loss of function mutations in the gene for Aprataxin. Normally, Aprataxin mediates protein-protein interactions with molecules that respond to DNA damage. However, the cellular phenotype of the disease does not appear to be associated with a major loss of this ability. Aprataxin contains three conserved domains, a forkhead-associated domain that mediates protein-protein interactions; a histidine triad domain that is similar to Hint, a universally conserved AMP-lysine hydrolase; and a C-terminal zinc finger domain. Full-Text PDF Open Access Ataxia-oculomotor apraxia (AOA) 1The abbreviations used are: AOA, ataxia-oculomotor apraxia; FHA, forkhead-associated; HIT, histidine triad; MCA, methylcoumarin amide. has been characterized as a unique disorder since 1988 (1Aicardi J. Barbosa C. Andermann E. Andermann F. Morcos R. Ghanem Q. Fukuyama Y. Awaya Y. Moe P. Ann. Neurol. 1988; 24: 497-502Crossref PubMed Scopus (109) Google Scholar). Found in many ethnic backgrounds but most frequently diagnosed in Japan and Portugal, AOA is typically an autosomal recessive, early onset cerebellar ataxia with abnormal movements of the head and eye, late neuropathy, long survival, hypercholesterolemia, hypoalbuminemia, and hyperlipidemia. Families with a disease now termed ataxia-oculomotor apraxia 1 (AOA1; MIM 208920) have mutations in the APTX gene (MIM 606350) at 9p13.3, which encodes Aprataxin (2Date H. Onodera O. Tanaka H. Iwabuchi K. Uekawa K. Igarashi S. Koike R. Hiroi T. Yuasa T. Awaya Y. Sakai T. Takahashi T. Nagatomo H. Sekijima Y. Kawachi I. Takiyama Y. Nishizawa M. Fukuhara N. Saito K. Sugano S. Tsuji S. Nat. Genet. 2001; 29: 184-188Crossref PubMed Scopus (333) Google Scholar, 3Moreira M.C. Barbot C. Tachi N. Kozuka N. Uchida E. Gibson T. Mendonca P. Costa M. Barros J. Yanagisawa T. Watanabe M. Ikeda Y. Aoki M. Nagata T. Coutinho P. Sequeiros J. Koenig M. Nat. Genet. 2001; 29: 189-193Crossref PubMed Scopus (376) Google Scholar). Recently, four family members diagnosed with coenzyme Q deficiency and cerebellar ataxia have been shown to harbor Aprataxin mutations (4Quinzii C.M. Kattah A.G. Naini A. Akman H.O. Mootha V.K. DiMauro S. Hirano M. Neurology. 2005; 64: 539-541Crossref PubMed Scopus (153) Google Scholar). AOA2 (MIM 606002) individuals (5Moreira M.C. Klur S. Watanabe M. Nemeth A.H. Le Ber I. Moniz J.C. Tranchant C. Aubourg P. Tazir M. Schols L. Pandolfo M. Schulz J.B. Pouget J. Calvas P. Shizuka-Ikeda M. Shoji M. Tanaka M. Izatt L. Shaw C.E. M'Zahem A. Dunne E. Bomont P. Benhassine T. Bouslam N. Stevanin G. Brice A. Guimaraes J. Mendonca P. Barbot C. Coutinho P. Sequeiros J. Durr A. Warter J.M. Koenig M. Nat. Genet. 2004; 36: 225-227Crossref PubMed Scopus (395) Google Scholar) and children with an early onset form of amyotrophic lateral sclerosis (6Chen Y.Z. Bennett C.L. Huynh H.M. Blair I.P. Puls I. Irobi J. Dierick I. Abel A. Kennerson M.L. Rabin B.A. Nicholson G.A. Auer-Grumbach M. Wagner K. De Jonghe P. Griffin J.W. Fischbeck K.H. Timmerman V. Cornblath D.R. Chance P.F. Am. J. Hum. Genet. 2004; 74: 1128-1135Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar) have mutations in an unrelated gene termed ALS4/SETX (MIM 608465) at 9q34, which is thought to encode a helicase. Aprataxin is a ubiquitously expressed nuclear protein (7Gueven N. Becherel O.J. Kijas A.W. Chen P. Howe O. Rudolph J.H. Gatti R. Date H. Onodera O. Taucher-Scholz G. Lavin M.F. Hum. Mol. Genet. 2004; 13: 1081-1093Crossref PubMed Scopus (140) Google Scholar) translated from several splice variants. The predominant message encodes a primary translation product of 342 amino acids (8Sano Y. Date H. Igarashi S. Onodera O. Oyake M. Takahashi T. Hayashi S. Morimatsu M. Takahashi H. Makifuchi T. Fukuhara N. Tsuji S. Ann. Neurol. 2004; 55: 241-249Crossref PubMed Scopus (72) Google Scholar), although transcripts encoding forms of Aprataxin of 168 (2Date H. Onodera O. Tanaka H. Iwabuchi K. Uekawa K. Igarashi S. Koike R. Hiroi T. Yuasa T. Awaya Y. Sakai T. Takahashi T. Nagatomo H. Sekijima Y. Kawachi I. Takiyama Y. Nishizawa M. Fukuhara N. Saito K. Sugano S. Tsuji S. Nat. Genet. 2001; 29: 184-188Crossref PubMed Scopus (333) Google Scholar) and 356 amino acids (9Habeck M. Zuhlke C. Bentele K.H. Unkelbach S. Kress W. Burk K. Schwinger E. Hellenbroich Y. J. Neurol. 2004; 251: 591-594Crossref PubMed Scopus (24) Google Scholar) have been reported. As shown in Fig. 1, Aprataxin contains three domains: a forkhead-associated (FHA) domain that resembles that of the vertebrate polynucleotide kinase, a histidine triad (HIT) domain, and a COOH-terminal zinc finger domain (2Date H. Onodera O. Tanaka H. Iwabuchi K. Uekawa K. Igarashi S. Koike R. Hiroi T. Yuasa T. Awaya Y. Sakai T. Takahashi T. Nagatomo H. Sekijima Y. Kawachi I. Takiyama Y. Nishizawa M. Fukuhara N. Saito K. Sugano S. Tsuji S. Nat. Genet. 2001; 29: 184-188Crossref PubMed Scopus (333) Google Scholar, 3Moreira M.C. Barbot C. Tachi N. Kozuka N. Uchida E. Gibson T. Mendonca P. Costa M. Barros J. Yanagisawa T. Watanabe M. Ikeda Y. Aoki M. Nagata T. Coutinho P. Sequeiros J. Koenig M. Nat. Genet. 2001; 29: 189-193Crossref PubMed Scopus (376) Google Scholar). The FHA domain is a phosphoprotein binding motif found in many proteins involved in DNA damage responses (10Durocher D. Taylor I.A. Sarbassova D. Haire L.F. Westcott S.L. Jackson S.P. Smerdon S.J. Yaffe M.B. Mol. Cell. 2000; 6: 1169-1182Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). Indeed, consistent with a possible role in single-strand break repair, Aprataxin has been reported to associate with Xrcc1, PARP-1, p53 (7Gueven N. Becherel O.J. Kijas A.W. Chen P. Howe O. Rudolph J.H. Gatti R. Date H. Onodera O. Taucher-Scholz G. Lavin M.F. Hum. Mol. Genet. 2004; 13: 1081-1093Crossref PubMed Scopus (140) Google Scholar), and Xrcc4 (11Clements P.M. Breslin C. Deeks E.D. Byrd P.J. Ju L. Bieganowski P. Brenner C. Moreira M.C. Taylor A.M. Caldecott K.W. DNA Repair (Amst.). 2004; 3: 1493-1502Crossref PubMed Scopus (164) Google Scholar) and lead to mild sensitivity to hydrogen peroxide (7Gueven N. Becherel O.J. Kijas A.W. Chen P. Howe O. Rudolph J.H. Gatti R. Date H. Onodera O. Taucher-Scholz G. Lavin M.F. Hum. Mol. Genet. 2004; 13: 1081-1093Crossref PubMed Scopus (140) Google Scholar, 11Clements P.M. Breslin C. Deeks E.D. Byrd P.J. Ju L. Bieganowski P. Brenner C. Moreira M.C. Taylor A.M. Caldecott K.W. DNA Repair (Amst.). 2004; 3: 1493-1502Crossref PubMed Scopus (164) Google Scholar). However, in contrast to the striking defects in DNA damage responses of cultured cells with atm mutations (for a recent review, see Chun and Gatti (12Chun H.H. Gatti R.A. DNA Repair (Amst.). 2004; 3: 1187-1196Crossref PubMed Scopus (343) Google Scholar)), cultured aptx-mutant cells do not exhibit defects in ATM signaling or radioresistant DNA synthesis (11Clements P.M. Breslin C. Deeks E.D. Byrd P.J. Ju L. Bieganowski P. Brenner C. Moreira M.C. Taylor A.M. Caldecott K.W. DNA Repair (Amst.). 2004; 3: 1493-1502Crossref PubMed Scopus (164) Google Scholar). The potential relationships between other biochemical pathways, such as coenzyme Q deficiency (13Shults C.W. Curr. Med. Chem. 2003; 10: 1917-1921Crossref PubMed Scopus (95) Google Scholar) or glycogen synthase 3 signaling (14Kaytor M.D. Orr H.T. Curr. Opin. Neurobiol. 2002; 12: 275-278Crossref PubMed Scopus (185) Google Scholar), and Aprataxin function has not been investigated. HIT domains, named for a motif related to His-ϕ-His-ϕ-His-ϕ-ϕ, where ϕ is a hydrophobic amino acid, are found in a superfamily of nucleotide hydrolases and nucleotide transferases that includes the universally conserved Hint enzyme, the Fhit tumor suppressor protein, and galactose-1-phosphate uridylyltransferase (15Brenner C. Biochemistry. 2002; 41: 9003-9014Crossref PubMed Scopus (243) Google Scholar). Contrary to assertion (2Date H. Onodera O. Tanaka H. Iwabuchi K. Uekawa K. Igarashi S. Koike R. Hiroi T. Yuasa T. Awaya Y. Sakai T. Takahashi T. Nagatomo H. Sekijima Y. Kawachi I. Takiyama Y. Nishizawa M. Fukuhara N. Saito K. Sugano S. Tsuji S. Nat. Genet. 2001; 29: 184-188Crossref PubMed Scopus (333) Google Scholar, 16Hirano M. Furiya Y. Kariya S. Nishiwaki T. Ueno S. Biochem. Biophys. Res. Commun. 2004; 322: 380-386Crossref PubMed Scopus (13) Google Scholar), the HIT domain of Aprataxin is more similar to the Hint branch of the HIT superfamily than to Fhit (15Brenner C. Biochemistry. 2002; 41: 9003-9014Crossref PubMed Scopus (243) Google Scholar, 17Kwasnicka D.A. Krakowiak A. Thacker C. Brenner C. Vincent S.R. J. Biol. Chem. 2003; 278: 39051-39058Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Whereas Fhit-homologous enzymes hydrolyze diadenosine polyphosphates such as ApppA and AppppA into mononucleotides (18Barnes L.D. Garrison P.N. Siprashvili Z. Guranowski A. Robinson A.K. Ingram S.W. Croce C.M. Ohta M. Huebner K. Biochemistry. 1996; 35: 11529-11535Crossref PubMed Scopus (371) Google Scholar, 19Draganescu A. Hodawadekar S.C. Gee K.R. Brenner C. J. Biol. Chem. 2000; 275: 4555-4560Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), Hint-homologous enzymes have extremely low activity on such substrates and instead hydrolyze model substrates in which AMP is joined by a phosphoramidate linkage to the ϵ amino group of lysine (20Bieganowski P. Garrison P.N. Hodawadekar S.C. Faye G. Barnes L.D. Brenner C. J. Biol. Chem. 2002; 277: 10852-10860Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 21Krakowiak A. Pace H.C. Blackburn G.M. Adams M. Mekhalfia A. Kaczmarek R. Baraniak J. Stec W.J. Brenner C. J. Biol. Chem. 2004; 279: 18711-18716Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Recently, in work to characterize a potential role for Hint hydrolases in regulating sexual differentiation in birds (22Pace H.C. Brenner C. Genome Biol. 2003; 4: R18Crossref PubMed Google Scholar), we developed an adenylylated lysine substrate linked to aminomethylcoumarin that allows Hint enzymatic activity, coupled to trypsin cleavage, to produce a bright fluorescent product (23Parks K.P. Seidle H. Wright N. Sperry J.B. Bieganowski P. Howitz K. Wright D.L. Brenner C. Physiol. Genomics. 2004; 20: 12-14Crossref PubMed Scopus (23) Google Scholar). In yeast, the enzymatic activity of the Hint homolog, Hnt1, functions as a positive regulator of components of general transcription factor TFIIH (20Bieganowski P. Garrison P.N. Hodawadekar S.C. Faye G. Barnes L.D. Brenner C. J. Biol. Chem. 2002; 277: 10852-10860Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In Escherichia coli, the Hint-homologous hinT gene is required for resistance to elevated levels of certain salts (24Chou T.F. Bieganowski P. Shilinski K. Cheng J. Brenner C. Wagner C.R. J. Biol. Chem. 2005; 280: 15356-15361Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Although specific protein targets remain to be identified, it has been hypothesized that Hint hydrolases may reverse nucleotidylylated protein modifications of lysine (15Brenner C. Biochemistry. 2002; 41: 9003-9014Crossref PubMed Scopus (243) Google Scholar). Two specific models linking Aprataxin inactivation to disease have been proposed. According to the first model, diadenosine polyphosphate hydrolysis is required for DNA repair (2Date H. Onodera O. Tanaka H. Iwabuchi K. Uekawa K. Igarashi S. Koike R. Hiroi T. Yuasa T. Awaya Y. Sakai T. Takahashi T. Nagatomo H. Sekijima Y. Kawachi I. Takiyama Y. Nishizawa M. Fukuhara N. Saito K. Sugano S. Tsuji S. Nat. Genet. 2001; 29: 184-188Crossref PubMed Scopus (333) Google Scholar). However, because intact, purified Aprataxin was reported to be devoid of diadenosine polyphosphate hydrolase activity (16Hirano M. Furiya Y. Kariya S. Nishiwaki T. Ueno S. Biochem. Biophys. Res. Commun. 2004; 322: 380-386Crossref PubMed Scopus (13) Google Scholar), and there is no known connection between diadenosine polyphosphate hydrolysis and DNA repair, this model appears to be unlikely. According to the second model, Aprataxin stabilizes Xrcc1 by a physical association between phosphorylated Xrcc1 and the FHA domain of Aprataxin (25Luo H. Chan D.W. Yang T. Rodriguez M. Chen B.P. Leng M. Mu J.J. Chen D. Songyang Z. Wang Y. Qin J. Mol. Cell. Biol. 2004; 24: 8356-8365Crossref PubMed Scopus (122) Google Scholar). This model appears to be undermined by the lack of x-ray sensitivity of aptx fibroblasts (11Clements P.M. Breslin C. Deeks E.D. Byrd P.J. Ju L. Bieganowski P. Brenner C. Moreira M.C. Taylor A.M. Caldecott K.W. DNA Repair (Amst.). 2004; 3: 1493-1502Crossref PubMed Scopus (164) Google Scholar) and the fact that disease-associated mutations in APTX do not alter the ability of Aprataxin to associate with Xrcc1 (25Luo H. Chan D.W. Yang T. Rodriguez M. Chen B.P. Leng M. Mu J.J. Chen D. Songyang Z. Wang Y. Qin J. Mol. Cell. Biol. 2004; 24: 8356-8365Crossref PubMed Scopus (122) Google Scholar). Here we demonstrate that Aprataxin possesses an intrinsic, active site-dependent AMP-lysine and GMP-lysine hydrolase activity. AMP-lysine hydrolase activity on a model substrate was also greatly reduced by expression of Aprataxin without the NH2-terminal FHA domain and eliminated by truncation of the zinc finger domain, indicating that all three domains contribute to formation of a stable enzyme. Finally, biochemical analysis indicated that eight reported disease-associated alleles are null or nearly null for the Hint active site, while a reported mild presentation allele and an atypical presentation allele can be functionally diagnosed by in vitro stability and activity. Plasmid Construction and Mutagenesis—E. coli strain BL21* and DH10B were used for cloning. Plasmid pB352 and derivatives were used for expression of human APTX (11Clements P.M. Breslin C. Deeks E.D. Byrd P.J. Ju L. Bieganowski P. Brenner C. Moreira M.C. Taylor A.M. Caldecott K.W. DNA Repair (Amst.). 2004; 3: 1493-1502Crossref PubMed Scopus (164) Google Scholar). Site-directed mutagenesis of plasmid pB352 was used to create plasmids to express APTX alleles K197Q, A198V, R199H, P206L, H260A, V263G, D267G, W279R, W279X, 689insT, and 840delT using primers listed in Supplemental Table I. The 168-amino acid Aprataxin coding sequence was produced by adding an NdeI site by site-directed mutagenesis using primer 7779 and amplification by PCR with primers 0720 and 0721. The construct was recovered from ligation of an NdeI and XhoI digestion of the resulting PCR product. All constructs were confirmed by DNA sequencing. Enzyme Expression and Purification—E. coli strain BB2-1 was used for protein expression. BB2-1 was produced by disrupting the E. coli hinT gene in strain BL21* as described (26Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11205) Google Scholar). Primers 7024 and 7025 were used for PCR amplification of the chloramphenicol resistance marker of plasmid pKD3. Stable chloramphenicol-resistant transformants of BL21* were tested by PCR with primers 7026 and 7027 to confirm correct recombination of the chloramphenicol resistance marker into the hinT locus. E. coli transfomants carrying APTX expression plasmids were aerated at 24 °C. At an A600 of 0.5, expression was induced with 100 μm isopropyl β-d-thiogalactopyranoside. Cultures were grown 16 h and harvested by centrifugation. Frozen (–80 °C) and thawed cell pellets (∼5 g wet weight) were lysed using Bugbuster (Novagen) with DNase I and EDTA-free complete protease inhibitor mixture (Roche Applied Science). Clarified lysates were loaded on 1.5 ml Talon columns (Clontech). Aprataxin proteins were eluted with 10 ml of 250 mm imidazole, 10 mm HEPES, pH 7, and concentrated into 100 mm NaCl, 10 mm HEPES, pH 7.2, with 10-kDa cutoff centrifugal concentrators (Amicon). Proteins were transferred to nitrocellulose membranes and detected using an anti-penta-His antibody conjugated to horseradish peroxidase (Qiagen) and SuperSignal West Pico chemiluminescent substrate (Pierce). Enzyme Assays—The fluorigenic substrate t-Boc-LysAMP-MCA was synthesized as described (23Parks K.P. Seidle H. Wright N. Sperry J.B. Bieganowski P. Howitz K. Wright D.L. Brenner C. Physiol. Genomics. 2004; 20: 12-14Crossref PubMed Scopus (23) Google Scholar). Synthesis of t-Boc-LysGMP-MCA is reported in the supplemental material. t-Boc-LysAMP-MCA and t-Boc-LysGMP-MCA hydrolytic activities were assayed in 25-μl volumes containing 2.5–250 μm substrate, 12 nmol of wild-type enzyme or 12–60 nmol of mutant enzyme, 100 mm NaCl, 10 mm HEPES, pH 7.2, for 30–60 min at room temperature. Reactions were initiated by the addition of substrate and stopped by addition of 75 μl of 80 mg/ml trypsin. After a 10-min incubation with trypsin, fluorescence (excitation 355 nm, emission 460 nm) was measured with a Wallac 1420 multilabel counter. pH and divalent cation analyses (supplemental material) were completed in 10 mm HEPES, with pH from 6.8 to 8.2. GpppBODIPY hydrolysis was assayed as described previously (19Draganescu A. Hodawadekar S.C. Gee K.R. Brenner C. J. Biol. Chem. 2000; 275: 4555-4560Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Assays were initated by addition of 12 nmol of enzyme and stopped after 30–60 min by the addition of sodium citrate, pH 3. GpppBODIPY concentration ranged between 0 and 25 μm. Dinucleoside polyphosphate hydrolysis assays were performed using 60 nmol of wild-type enzyme with 5–200 μm substrate in a 50-μl volume. Assays were incubated at room temperature for 30 min and stopped by addition of 50 μl of Na2CO3, pH 11.6. 50 μl of the reaction mixture was injected onto a strong anion exchange column (Princeton Chromatography) equilibrated with 10 mm K2HPO4, pH 2.58 (Buffer A) on a Varian Prostar HPLC. The elution program was 100% Buffer A for 10 min, a 2-min gradient to 100% 750 mm K2HPO4, pH 2.58 (Buffer B), and 100% Buffer B for 6 min. Elution times for AMP, ADP, ATP, ApppA, and AppppA were 6.7, 15.3, 16.2, 15.7, and 16.3 min, respectively. Product amounts were determined from the peak areas and a standard curve of AMP using Varian Galaxie Software. Assays to determine divalent cation effects (supplemental material) were completed as described above with the addition of 0.5 mm MgCl2. All assays were performed at least in triplicate. Aprataxin Exhibits AMP- and GMP-Lysine Hydrolase Activity—The 342-amino acid APTX cDNA was fused to an aminoterminal His-tag, and the resulting protein was purified to homogeneity by metal chelate affinity chromatography. The resulting enzyme was assayed for activity with t-Boc-AMPLys-MCA, a model substrate containing an adenylylated lysine (23Parks K.P. Seidle H. Wright N. Sperry J.B. Bieganowski P. Howitz K. Wright D.L. Brenner C. Physiol. Genomics. 2004; 20: 12-14Crossref PubMed Scopus (23) Google Scholar), as well as a newly synthesized t-Boc-GMPLys-MCA substrate, and the Fhit substrates GpppBODIPY (19Draganescu A. Hodawadekar S.C. Gee K.R. Brenner C. J. Biol. Chem. 2000; 275: 4555-4560Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), ApppA, and AppppA (Table I). To avoid contamination by the E. coli Hint hydrolase, wild-type and all mutant APTX constructs were expressed in and purified from E. coli strain BB2, which contains a deletion for the Hint-homologous hinT gene (24Chou T.F. Bieganowski P. Shilinski K. Cheng J. Brenner C. Wagner C.R. J. Biol. Chem. 2005; 280: 15356-15361Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar).Table ISubstrate specificity of AprataxinSubstratekcatKmkcat/Kms-1μms-1 m-1t-Boc-LysAMP-MCA0.0028 ± 0.000247 ± 960 ± 22t-Boc-LysGMP-MCA0.003 ± 0.0004116 ± 2426 ± 17GpppBODIPY0.0004 ± 0.0000513.1 ± 4.731 ± 11ApppA0.00008 ± 0.0000221 ± 1.63.8 ± 1.3AppppA0.0009 ± 0.0000539 ± 623 ± 8.3 Open table in a new tab Aprataxin exhibits strong kcat discrimination against hydrolysis of the dinucleoside polyphosphates ApppA and AppppA and the dinucleoside polyphosphate analog GpppBODIPY, with 30-, 3-, and 7-fold lower turnover rates with these compounds, respectively, than the turnover rates against substrates with nucleotidylylated lysine residues. The enzyme exhibited relatively less Km discrimination between the nucleotide substrates presented to it and, in fact, displayed a preference for the dinucleoside polyphosphates and their analogs (13 to 39 μm) versus substrates containing nucleotidylylated lysines (47 and 116 μm). The relatively high Km values for t-Boc-LysAMP-MCA and t-Boc-LysGMP-MCA did not offset their advantages in the kcat term. As shown in Table I, on the basis of the specificity constant, kcat/Km, Aprataxin is an AMP-lysine hydrolase whose activity is limited by the relatively high Km values of the model peptide substrates presented to it. Metal Independence, Active Site Dependence, FHA Domain Dependence, and pH Dependence of the AMP-Lysine Hydrolase Activity of Aprataxin—Fhit enzymatic activity on dinucleoside polyphosphate substrates is magnesium-dependent (27Huang K. Arabshahi A. Wei Y. Frey P.A. Biochemistry. 2004; 43: 7637-7642Crossref PubMed Scopus (22) Google Scholar), but Hint enzymatic activity is metal-independent (23Parks K.P. Seidle H. Wright N. Sperry J.B. Bieganowski P. Howitz K. Wright D.L. Brenner C. Physiol. Genomics. 2004; 20: 12-14Crossref PubMed Scopus (23) Google Scholar). Consistent with biochemical similarity to Hint, none of the observed activities on nucleotidylylated lysine substrates or dinucleoside polyphosphate substrates were inhibited by EDTA or stimulated by magnesium (supplemental Fig. 1). We constructed a H260A allele, targeted to the middle His residue of the HIT motif, to determine whether all of the AMP-lysine hydrolase activity is due to the Hint-homologous active site of Aprataxin. As shown in Table II, the H260A substitution eliminates all detectable activity, demonstrating that the AMP-lysine hydrolase activity of Aprataxin depends on the Hint-homologous active site.Table IIEnzymatic activity of expressed APTX transcripts and alleles with t-Boc-LysAMP-MCAEnzymekcatKmkcat/KmTranscript or allele references-1μms-1 m-1Aptx-3420.003 ± 0.000247 ± 964 ± 222Date H. Onodera O. Tanaka H. Iwabuchi K. Uekawa K. Igarashi S. Koike R. Hiroi T. Yuasa T. Awaya Y. Sakai T. Takahashi T. Nagatomo H. Sekijima Y. Kawachi I. Takiyama Y. Nishizawa M. Fukuhara N. Saito K. Sugano S. Tsuji S. Nat. Genet. 2001; 29: 184-188Crossref PubMed Scopus (333) Google Scholar, 3Moreira M.C. Barbot C. Tachi N. Kozuka N. Uchida E. Gibson T. Mendonca P. Costa M. Barros J. Yanagisawa T. Watanabe M. Ikeda Y. Aoki M. Nagata T. Coutinho P. Sequeiros J. Koenig M. Nat. Genet. 2001; 29: 189-193Crossref PubMed Scopus (376) Google ScholarAptx-1680.00006 ± 0.000005187 ± 520.3 ± 0.12Date H. Onodera O. Tanaka H. 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