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• W2048222765 abstract "Trypanosoma cruzi adenylyl cyclases are encoded by a large polymorphic gene family. Although several genes have been identified in this parasite, little is known about the properties and regulation of these enzymes. Here we report the cloning and characterization of TczAC, a novel member ofT. cruzi adenylyl cyclase family. The TczAC gene is expressed in all of the parasite life forms and encodes a 1,313-amino acid protein that can complement a Saccharomyces cerevisiaemutant deficient in adenylyl cyclase activity. The recombinant enzyme expressed in yeasts is constitutively active, has a low affinity for ATP (Km = 406 μm), and requires a divalent cation for catalysis. TczAC is inhibited by Zn2+and the P-site inhibitor 2′-deoxyadenosine 3′-monophosphate, suggesting some level of conservation in the catalytic mechanism with mammalian adenylyl cyclases. It shows a dose-dependent stimulation by Ca2+ which can be reversed by high concentrations of phenothiazinic calmodulin inhibitors. However, bovine calmodulin fails to stimulate the enzyme. Using a yeast two-hybrid screen it was found that TczAC interacts through its catalytic domain with the paraflagellar rod protein, a component of the flagellar structure. Furthermore, we demonstrate that TczAC can dimerize through the same domain. These results provide novel evidence of the possible localization and regulation of this protein. Trypanosoma cruzi adenylyl cyclases are encoded by a large polymorphic gene family. Although several genes have been identified in this parasite, little is known about the properties and regulation of these enzymes. Here we report the cloning and characterization of TczAC, a novel member ofT. cruzi adenylyl cyclase family. The TczAC gene is expressed in all of the parasite life forms and encodes a 1,313-amino acid protein that can complement a Saccharomyces cerevisiaemutant deficient in adenylyl cyclase activity. The recombinant enzyme expressed in yeasts is constitutively active, has a low affinity for ATP (Km = 406 μm), and requires a divalent cation for catalysis. TczAC is inhibited by Zn2+and the P-site inhibitor 2′-deoxyadenosine 3′-monophosphate, suggesting some level of conservation in the catalytic mechanism with mammalian adenylyl cyclases. It shows a dose-dependent stimulation by Ca2+ which can be reversed by high concentrations of phenothiazinic calmodulin inhibitors. However, bovine calmodulin fails to stimulate the enzyme. Using a yeast two-hybrid screen it was found that TczAC interacts through its catalytic domain with the paraflagellar rod protein, a component of the flagellar structure. Furthermore, we demonstrate that TczAC can dimerize through the same domain. These results provide novel evidence of the possible localization and regulation of this protein. Adenylyl cyclases, the enzymes responsible for cAMP synthesis, have been classified in different families according to their biochemical and structural properties (1Patel T.B., Du, Z. Pierre S. Cartin L. Scholich K. Gene (Amst.). 2001; 269: 13-25Crossref PubMed Scopus (143) Google Scholar, 2Hurley J.H. J. Biol. Chem. 1999; 274: 7599-7602Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). The mammalian adenylyl cyclases that present 12 transmembrane domains (families I–IX) have been the most studied and well characterized members of these families. All of these isoforms share a conserved structure with two domains of six transmembrane helices (MI 1The abbreviations used are: MI, MII, CI, and CII, transmembrane I and II domains and catalytic I and II domains of mammalian adenylyl cyclases, respectively; CD, catalytic domain; AC1, CD fused to LexA DNA binding domain; AC2, CD fused to GAl4 transcription activating domain; AC3, CD fused to VP16 transcription activating domain; 2′deoxy-3′AMP, 2′-deoxyadenosine 3′-monophosphate; GTPγS, guanosine 5′-O-(thiotriphosphate); LAM, lamin protein fused to LexA domain; PAR, paraflagellar rod protein fused to VP16 domain. and MII) and two intracellular catalytic domains (CI and CII), highly homologous but not identical, which are both required to achieve maximal catalytic activity. Trypanosomatid adenylyl cyclases share little sequence homology with their mammalian counterparts and have a completely different structure. So far, these proteins have been described to have a unique intracellular catalytic domain, which is highly conserved, one transmembrane domain, and a large extracellular variable N-terminal domain, a structure that resembles that of guanylyl cyclase receptors (3Lucas K.A. Pitari G.M. Kazerounian S. Ruiz-Stewart I. Park J. Schulz S. Kenneth P. Chepenik K.P. Waldman S.A. Pharmacol. Rev. 2000; 52: 375-414PubMed Google Scholar, 4Seebeck T. Gong K.W. Kunz S. Schaub R. Shalaby T. Zoraghi R. Int. J. Parasitol. 2001; 31: 491-498Crossref PubMed Scopus (44) Google Scholar, 5Naula C. Seebeck T. Parasitol. Today. 2000; 16: 35-38Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 6Taylor M.C. Muhia D.K. Baker D.A. Mondragon A. Schaap P. Kelly J.M. Mol. Biochem. Parasitol. 1999; 104: 205-217Crossref PubMed Scopus (43) Google Scholar). Adenylyl cyclases and cAMP have been involved in the differentiation process of trypanosomatids (7Rangel-Aldao R. Allende O. Triana F. Piras R. Henriquez D. Piras M. Mol. Biochem. Parasitol. 1987; 22: 39-43Crossref PubMed Scopus (42) Google Scholar, 8Flawiá M.M. Tellez-Iñon M.T. Torres H.N. Parasitol. Today. 1997; 13: 30-33Abstract Full Text PDF PubMed Scopus (31) Google Scholar, 9Fraidenraich D. Peña C. Isola E.L. Lammel E.M. Coso O. Diaz Añel A. Sandor P. Baralle F. Torres H.N. Flawiá M.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10140-10144Crossref PubMed Scopus (80) Google Scholar, 10Garcia E.S. Gonzalez M.S. de Azambuja P. Baralle F.E. Fraidenraich D. Torres H.N. Flawiá M.M. Exp. Parasitol. 1995; 81: 255-261Crossref PubMed Scopus (57) Google Scholar, 11Ouaissi A. Cornette J. Schoneck R. Plumas-Marty B. Taibi A. Loyens M. Capron A. Eur. J. Cell Biol. 1992; 59: 68-79PubMed Google Scholar, 12Rangel-Aldao R. Triana F. Fernandez V. Comach G. Abate T. Montoreano R. Biochem. Int. 1988; 17: 337-344PubMed Google Scholar, 13Rolin S. Paindavoine P. Hanocq-Quertier J. Hanocq F. Claes Y., Le Ray D. Overath P. Pays E. Mol. Biochem. Parasitol. 1993; 61: 115-125Crossref PubMed Scopus (66) Google Scholar, 14Vassella E. Reuner B. Yutzy B. Boshart M. J. Cell Sci. 1997; 110: 2661-2671Crossref PubMed Google Scholar). In Trypanosoma cruzi a peptide produced by proteolysis of αd-globin in the hindgut of Triatoma infestans vector can stimulate adenylyl cyclase activity and induce differentiation of epimastigotes to metacyclic trypomastigotes (9Fraidenraich D. Peña C. Isola E.L. Lammel E.M. Coso O. Diaz Añel A. Sandor P. Baralle F. Torres H.N. Flawiá M.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10140-10144Crossref PubMed Scopus (80) Google Scholar, 10Garcia E.S. Gonzalez M.S. de Azambuja P. Baralle F.E. Fraidenraich D. Torres H.N. Flawiá M.M. Exp. Parasitol. 1995; 81: 255-261Crossref PubMed Scopus (57) Google Scholar). In addition, peptides released from proteolysis of fibronectin increase cAMP levels in trypomastigotes (11Ouaissi A. Cornette J. Schoneck R. Plumas-Marty B. Taibi A. Loyens M. Capron A. Eur. J. Cell Biol. 1992; 59: 68-79PubMed Google Scholar) and cAMP analogs and phosphodiesterase inhibitors have also been reported to induce the differentiation of this parasite (12Rangel-Aldao R. Triana F. Fernandez V. Comach G. Abate T. Montoreano R. Biochem. Int. 1988; 17: 337-344PubMed Google Scholar). Similar to this, in Trypanosoma brucei two peaks of cAMP have been observed previous to the differentiation from bloodstream to procyclic forms (13Rolin S. Paindavoine P. Hanocq-Quertier J. Hanocq F. Claes Y., Le Ray D. Overath P. Pays E. Mol. Biochem. Parasitol. 1993; 61: 115-125Crossref PubMed Scopus (66) Google Scholar). Furthermore, a low mass factor purified from high density cultures of the T. brucei slender form triggers cell cycle arrest and differentiation through a mechanism likely mediated by cAMP (14Vassella E. Reuner B. Yutzy B. Boshart M. J. Cell Sci. 1997; 110: 2661-2671Crossref PubMed Google Scholar). Another second messenger that has been connected to the trypanosomatid differentiation process is calcium (15Stojdl D.F. Clarke M.W. Exp. Parasitol. 1996; 83: 134-146Crossref PubMed Scopus (31) Google Scholar, 16Lammel E.M. Barbieri M.A. Wilkowsky S.E. Bertini F. Isola E.L. Exp. Parasitol. 1996; 83: 240-249Crossref PubMed Scopus (42) Google Scholar). The addition of T. infestans intestinal homogenate, which has been shown to trigger this process, induces an increase in intracellular calcium levels inT. cruzi epimastigotes. Moreover, Ca2+ chelators and calmodulin inhibitors decrease or even block the in vitro differentiation of T. cruzi (16Lammel E.M. Barbieri M.A. Wilkowsky S.E. Bertini F. Isola E.L. Exp. Parasitol. 1996; 83: 240-249Crossref PubMed Scopus (42) Google Scholar). Many of the calcium-binding proteins described in trypanosoma are located in the flagellum, organelle that seems to be crucial for Ca2+ signaling in trypanosomatids (17Engman D.M. Krause K.H. Blumin J.H. Kim K.S. Kirchhoff L.V. Donelson J.E. J. Biol. Chem. 1989; 264: 18627-18631Abstract Full Text PDF PubMed Google Scholar, 18Wu Y. Haghighat N.G. Ruben L. Biochem. J. 1992; 287: 187-193Crossref PubMed Scopus (26) Google Scholar, 19Wu Y. Deford J. Benjamin R. Lee M.G. Ruben L. Biochem. J. 1994; 304: 833-841Crossref PubMed Scopus (43) Google Scholar, 20Maldonado R.A. Linss J. Thomaz N. Olson C.L. Engman D.M. Goldenberg S. Exp. Parasitol. 1997; 86: 200-205Crossref PubMed Scopus (20) Google Scholar, 21Godsel L.M. Engman D.M. EMBO J. 1999; 18: 2057-2065Crossref PubMed Scopus (124) Google Scholar). Interestingly,T. brucei calmodulin is able to interact with the structural flagellar protein paraflagellar rod in a calcium-dependent manner (22Ridgley E. Webster P. Patton C. Ruben L. Mol. Biochem. Parasitol. 2000; 109: 195-201Crossref PubMed Scopus (34) Google Scholar). However, the role of this structural protein in the calcium signaling pathway remains unknown. This article provides novel information on the molecular and biochemical characteristics of the first calcium-activated adenylyl cyclase from T. cruzi. This enzyme can dimerize through its catalytic domain and also interact with the flagellar protein paraflagellar rod, providing new information to the understanding of the regulatory mechanism of the cAMP signaling pathway in this parasite. All radiochemicals used in this work were purchased from PerkinElmer Life Sciences. Restriction endonucleases were from New England Biolabs Inc., Beverly, MA. All other reagents were purchased from Sigma. Bacto-tryptose, yeast nitrogen base, and liver infusion were purchased from Difco Laboratories. T. cruzi epimastigote forms (CL Brenner strain) were cultured for 7 days at 28 °C in LIT medium (5 g/liter liver infusion, 5 g/liter Bacto-tryptose, 68 mmNaCl, 5.3 mm KCl, 22 mmNa2HPO4, 0.2% glucose, 0.002% hemin) supplemented with 10% calf serum, 10 units/ml penicillin, and 10 mg/liter streptomycin. Cell viability was assessed by direct microscopic examination. T. cruzi trypomastigote forms were a gift from Dr. M. Maria Julia Manso-Alvez. Saccharomyces cerevisiae strains, T503A (Mat-α, leu2, his3, trp1, ura3, cyr 1–2) and L40 (Mat-α, his3D200, trp1–901, leu2–3,112 ade2 LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lacZ) were grown in YPD medium at 30 °C before transformation. Transformants were selected in minimal medium containing 0.17% yeast nitrogen base (without amino acids and ammonium sulfate), 0.5% ammonium sulfate, and 2% glucose, supplemented with the corresponding amino acid mixture. The minimal medium used for two-hybrid assays also contained 1% succinic acid and 0.6% NaOH. Genomic DNA was purified as described by Pereira et al. (23Pereira C.A. Alonso G.D. Paveto M.C. Iribarren A. Cabanas M.L. Torres H.N. Flawiá M.M. J. Biol. Chem. 2000; 275: 1495-1501Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Total RNA was prepared from 4×1010 epimastigotes using the total RNA isolation (TRIzol) reagent (Invitrogen) as described by the manufacturer. Two degenerate oligonucleotides were designed from previously cloned adenylyl cyclases fromLeishmania donovani and T. brucei(5′-AC(G/T/C)CT(G/T/C)AT(C/T)TT(C/T)AC(G/T/C)GA(C/T)AT-3′ L1 primer and 5′-GA(A/G)GT(G/T/C)AAGAC(G/T/C)GT(G/T/C)GG(G/T/C)GA-3′ L2 primer). PCR amplifications were carried out using 600–800 ng of T. cruzi genomic DNA, 100 ng of each primer, 2.5 mmMgCl2, 0.2 mm dNTPs, and 1–2 units ofTaq polymerase (Promega, Madison, WI). After sequencing, the 642-bp product showed homology to trypanosomatid adenylyl cyclases. This fragment was used as a probe to screen a λ FIX II (Stratagene, La Jolla, CA) genomic library from T. cruzi (24Zingales B. Rondinelli E. Degrave W. da Silevira F. Levin M., Le Paslier D. Modabber F. Dobrokhotov B.B. Swindle J. Kelly J.M. Åslung L. Hoheisel J.D. Ruiz A.M. Cazzulo J.J. Petterson U. Frash A.C. Parasitol. Today. 1997; 13: 16-22PubMed Google Scholar). DNA from one phage clone was purified using the Qiagen Lambda midi kit (Qiagen, Valencia, CA) following the manufacturer's instructions. Oligonucleotides designed from the sequence previously obtained were used for sequencing. For Northern blot analysis, 15 μg of total RNA was electrophoresed on a 1.5% formaldehyde-agarose gel, transferred to a Hybond N+ nylon membrane (AmershamBiosciences), and hybridized at 65 °C in Church's buffer (1% bovine serum albumin, 7% SDS, 1 mm EDTA, pH 8, 0.5% Na2PO4) with the specific TczAC probe. This probe was obtained by PCR amplification of phage DNA with the primers 5′-CAGCCGTCGGGTGGTGACCGTGTC-3′ and 5′-TTAACCACTGGCACAGTCACTATA-3′. Blots were subjected to sequential stringent washes at 65 °C and exposed to AGFA CP-BU NEW films (AGFA-Gevaert N.V., Belgium). Southern blot analysis were performed with 5 μg of genomic DNA previously digested with the indicated restriction endonucleases. The products were resolved on 0.8% agarose gels, transferred, and hybridized as described for Northern blots. All probes were labeled with [α-32P]dCTP using the Prime-a-Gene kit (Promega) following the manufacturer's instructions. cDNA for PCR amplification was obtained from total RNA of epimastigote, trypomastigote, and amastigote forms of T. cruzi using the ThermoScriptTM reverse transcription-PCR system (Invitrogen) following the manufacturer's instructions. Amplifications were performed using a specific oligonucleotide from the 5′-untranslated region of TczAC and an oligonucleotide within the coding region which were not present in previously described TczAC sequences. TczAC full-length or catalytic domains (CD; amino acids 854–1105) were amplified using the following oligonucleotides: 5′-ATGGCGGTGGGATGGGTGGCTGTG-3′ (forward Mx6) plus 5′-TTATTGAGGAATAGACGGAGATCC-3′ (reverse Mx7) and 5′-TACTTCAGCCACAGCTCGCGTGAC-3′ (forward Mx1) plus reverse Mx7, respectively. The products were subcloned in the pYES2 yeast expression vector (Invitrogen). The T503A yeast strain was transformed with this constructions using the lithium acetate procedure (25Gietz R.D. Schiestl R.H. Yeast. 1991; 7: 253-263Crossref PubMed Scopus (368) Google Scholar), and the transformants were selected in minimal medium lacking uracil (ura− medium) at 30 °C. For complementation assays, transformants were plated at 34 °C in minimal medium lacking uracil and glucose and supplemented with 2% raffinose, 3% galactose, and 2% glycerol (gal+ medium) to induce the vector promoter. Controls were grown at 30 and 34 °C in the presence of cAMP. No differences were observed under these conditions. Yeast transformants were grown for 2 days in ura− medium, centrifuged at 4,000 × g for 5 min, resuspended in gal+ medium, and grown for 16 h more. Cells were harvested, washed twice with cold TE buffer, resuspended in lysis buffer (20 mm Tris-HCl, pH 7.5, 20% glycerol, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 25 units/ml aprotinin, 0.5 mm N α-p-tosyl-l-lysine chloromethyl ketone), and lysed by 10 cycles of 1 min of vortexing in the presence of glass beads (425–600 μm) and cooling on ice. Unbroken cells were discarded by centrifugation at 2,500 ×g at 4 °C. The supernatants were centrifuged further for 1 h at 100,000 × g. The pellet was resuspended in 20 mm Tris-HCl, pH 7.5, 5% glycerol plus antiproteases and used as a membrane fraction. T. cruzi cultures were centrifuged at 4,000 ×g for 5 min at 4 °C, resuspended in lysis buffer, and broken by 10 cycles of freezing in liquid N2 and thawing at 4 °C. Soluble extracts and membranes were then prepared as described for yeast. For endogenous calmodulin displacement, membranes were washed with 20 mm Tris-HCl, pH 7.5, 5% glycerol, 5 mm EGTA, and 0.1 mm NaCl with stirring for 30 min at 4 °C and then washed twice with resuspension buffer to eliminate all EGTA. Adenylyl cyclase activity was determined as described by Flawiá et al.(26Flawiá M.M. Kornblihtt A.R. Reig J.A. Torruella M. Torres H.N. J. Biol. Chem. 1983; 258: 8255-8259Abstract Full Text PDF PubMed Google Scholar). Unless indicated otherwise, assays were performed in triplicate with 30–50 μg of protein from the corresponding yeast fractions or 10–15-μg T. cruzi extracts, in the presence of 50 mm Tris-HCl, pH 7.5, 2.5 mm Mg2+ or Mn2+, 5 mm 3-isobutylmethylxanthine, 1 mm cAMP, and 1 mm [α-32P]ATP (200 cpm/pmol). Incubations were carried out for 20 min at 37 °C in a total volume of 200 μl. The CD of TczAC used for complementation assays was subcloned in the pBTM116 vector (trp+) in fusion with the LexA DNA binding domain (27Bartel P. Chien C. Sternglanz R. Fields S. Hartley D.A. Cellular Interactions in Development: A Practical Approach. Oxford University Press, Oxford, UK1993: 153-179Google Scholar). The construct was used to transform the L40 strain. Expression of the fusion protein was confirmed by Western blot analysis using anti-LexA antibodies (CLONTECH), and correct folding of the catalytic site was confirmed by adenylyl cyclase assays. L40 cells expressing the LexA-TczAC fusion protein were transformed with 300 μg of a T. cruzi cDNA library subcloned in the pVP16 vector (leu+ (28Gomez E.B. Santori M.I. Ları́a S. Engel J.C. Swindle J. Eisen H. Szankasi P. Téllez-Iñon M.T. Mol. Biochem. Parasitol. 2001; 113: 97-108Crossref PubMed Scopus (33) Google Scholar)). Transformants were selected by growing in minimal medium lacking tryptophan and leucine for 16 h to obtain an efficient expression of the HIS3 gene. Positive clones were then selected for histidine protrotrophy and assayed for β-galactosidase activity. cDNA fragments subcloned in the pVP16 vector from positive clones were amplified by PCR from yeast plasmid extracts using primers matching the vector sequences. The amplified fragments were subcloned in the pGEM-T-easy vector (Promega) and sequenced. L40 yeasts expressing CD-LexA were transformed with the plasmid pGAD (leu+) also containing the CD region of the TczAC gene but in fusion with the Gal4 transcription-activating domain (CLONTECH). Positive clones were selected as described previously, and β-galactosidase assays were performed to assess dimerization of the catalytic domain. For plate assays of β-galactosidase activity, yeast cells were grown in the corresponding minimal medium and transferred to a nitrocellulose membrane. Cells were permeabilized by floating for 30 s and then submerging for 5 s more in liquid N2. Membranes were suspended in Whatman 3MM filters soaked in Z buffer containing 0.06% 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal; Amresco Inc., Solon, OH). Liquid assays were performed as described by Pestka et al. (29Pestka S. Daugherty B.L. Jung V. Hotta K. Pestka R.K. Genetics. 1984; 81: 7525-7528Google Scholar). Experiments were performed in triplicate. Sequence identity was analyzed using the BLAST program (www.ncbi.nlm.nih.gov/blast/index.html). Signal peptide and transmembrane regions were determined using the programs SMART (smart.embl-heidelberg.de/), DAS (www.sbc.su.se/∼miklos/DAS/), SPLIT (pref.etfos.hr/cgi-bin/split/), TMPRED (www.ch.embnet.org/software/TMPRED_Form.html), and PRED-TMT (o2.db.uoa.gr/ PRED-TMR/). Two degenerate oligonucleotides designed from L. donovani andT. brucei adenylyl cyclase gene sequences were used to amplify a DNA fragment from T. cruzigenomic DNA. A 642-bp fragment was obtained and sequenced, showing a significant homology with previously cloned trypanosomatid adenylyl cyclases. The fragment was used to screen a λ FIX II genomic library from T. cruzi (24Zingales B. Rondinelli E. Degrave W. da Silevira F. Levin M., Le Paslier D. Modabber F. Dobrokhotov B.B. Swindle J. Kelly J.M. Åslung L. Hoheisel J.D. Ruiz A.M. Cazzulo J.J. Petterson U. Frash A.C. Parasitol. Today. 1997; 13: 16-22PubMed Google Scholar). After three rounds of screening, six clones with 10–15-kbp inserts were isolated. One of these clones was sequenced and revealed an open reading frame coding a 1,313-amino acid polypeptide (Fig.1 A). This sequence was designated TczAC and deposited in the GenBank data base under the accession number AAC61849. Transmembrane regions predicted using the DAS software showed that TczAC has five putative transmembrane domains, corresponding to amino acid positions 1–38, 608–628, 658–679, 830–852, and 1281–1307 (Fig. 1 B). The first domain corresponds to a predicted signal peptide with a putative cleavage site between amino acids 37 and 38. If only the domain with higher score (amino acids 830–852) is considered, the structure of TczAC would be identical to those assigned previously to trypanosomatid adenylyl cyclases having a large extracellular N-terminal domain, a unique transmembrane domain, and one intracellular catalytic domain (5Naula C. Seebeck T. Parasitol. Today. 2000; 16: 35-38Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 6Taylor M.C. Muhia D.K. Baker D.A. Mondragon A. Schaap P. Kelly J.M. Mol. Biochem. Parasitol. 1999; 104: 205-217Crossref PubMed Scopus (43) Google Scholar, 7Rangel-Aldao R. Allende O. Triana F. Piras R. Henriquez D. Piras M. Mol. Biochem. Parasitol. 1987; 22: 39-43Crossref PubMed Scopus (42) Google Scholar). However, compared with the 12 transmembrane domains of the mammalian adenylyl cyclase Cya1 (Fig.1 B), the possibility that the other predicted regions could represent transmembrane domains should not be discarded. If all of these elements are taking into account, TczAC would differ from the previously described structure of trypanosomatid proteins in that the extracellular N-terminal domain would be connected by three transmembrane spans to the catalytic domain, and this one would be anchored to the membrane by its C-terminal hydrophobic region resembling mammalian adenylyl cyclase catalytic domains. Similar results were obtained using the programs TMPRED, SPLIT, and PRED-TMR. Using the Blast program, it was found that TczAC presents an identity with trypanosomatid adenylyl cyclases which varies from 60 to 67% forT. cruzi enzymes to 35 to 48% for L. donovani and T. brucei enzymes (TableI). In contrast, the identity observed with mammalian adenylyl cyclase was less than 25%. Dyctiostelium discoideum-specific germination adenylyl cyclase (ACG) was also compared because it has a trypanosoma-like structure. The sequence identity with this protein was very low as well. Further analysis showed that the sequence corresponding to the catalytic domain was highly conserved, up to 80%, whereas the N-terminal domain was less conserved (<57%) and showed no significant similarity with mammalian counterparts (Table I).Table IComparison of amino acid identity of TczAC entire protein, catalytic domain or N-terminal domainOrganism (gene)Entire proteinCatalytic domainN-terminal domain%%%T. cruzi (ADC1)677557T. cruzi (ADC4)607349T. equiperdum(eESAG4c)5861T. brucei (GRESAG 4.3)435834T. congolense(TcADCYC1)4857L. donovani(RacA)355334H. sapiens(NPGCR)2427NSaNS, not significant.D. discodeum (ACG)2525NSB. taurus (AC1 cya1)23<20NSSequences were compared using the BLAST program. Except the guanylyl cyclase NPGCR, the other sequences correspond to adenylyl cyclases. GenBank accession numbers: AJ012096, T30876,P26338, Q99280, Z67964, U17042, NP_000897, M87278, and P26338, respectively.a NS, not significant. Open table in a new tab Sequences were compared using the BLAST program. Except the guanylyl cyclase NPGCR, the other sequences correspond to adenylyl cyclases. GenBank accession numbers: AJ012096, T30876,P26338, Q99280, Z67964, U17042, NP_000897, M87278, and P26338, respectively. Despite the poor conservation between TczAC and mammalian adenylyl cyclases, most of the amino acids required for catalytic activity and ATP specificity were strongly conserved (Fig. 1 C). In contrast, amino acids involved in the interaction with the diterpene activator forskolin and Gα or βγ subunits of G proteins were absent, suggesting a different regulatory mechanism for this enzyme (Fig. 1 C). It is worth mentioning that the putative signal peptide of TczAC presented no homology, even compared with trypanosomatids proteins. This may suggest that this sequence could act as a specific localization signal for TczAC. Southern blot analysis using a specific probe representing most of TczAC gene (nucleotides 112–3292) showed that even when T. cruzi genomic DNA was digested with restriction endonucleases that do not cut within the TczAC gene, more than one band was recognized. This confirms that TczAC is part of a large multiple gene family ofT. cruzi (Fig. 2 A(6Taylor M.C. Muhia D.K. Baker D.A. Mondragon A. Schaap P. Kelly J.M. Mol. Biochem. Parasitol. 1999; 104: 205-217Crossref PubMed Scopus (43) Google Scholar)). Northern blot analysis of T. cruzi epimastigote RNA reveals at least three bands, of ∼5.8, 4.7, and 3.9 kbp, indicating that more than one isoform of this protein are expressed in this stage of the parasite (Fig. 2 B). DNA fragments corresponding to the TczAC full-length sequence or the CD were cloned in the yeast expression vector pYES2. The T503A yeast strain was transformed with these constructions. This strain has a thermosensitive adenylyl cyclase inserted in thecyr1-2 allele and cannot grow at 34 °C in the absence of exogenous cAMP. Full-length TczAC and a control plasmid containing the yeast wild type cyr1 gene, but not the empty pYES2 vector, were able to rescue the mutation, allowing yeast growth at the restrictive temperature (Fig.3 A). The TczAC CD showed less growth, suggesting a partial complementation. At 30 °C no differences were observed among all transformants (Fig.3 A). Adenylyl cyclase activity was measured in soluble and membrane extracts of the complemented yeast cells. The full-length protein TczAC showed the highest specific activity (93.7 ± 1.5 pmol of cAMP/min/mg) associated with the membrane fraction (Fig. 3 B), but the CD showed significant lower activity associated with both particulate (7.2 ± 2.7 pmol of cAMP/min/mg) and soluble extracts (4.2 ± 0.5 pmol of cAMP/min/mg). The membrane association of the CD may indicate its anchor to the plasma membrane through the hydrophobic residues in the C-terminal region of TczAC. The lower activity observed for this domain would explain the partial complementation phenotype. It is important to emphasize that the transformants expressing the cyr1 gene showed very low activity. This did not correlate with the growth observed at 34 °C. It is possible to speculate that because this enzyme would be correctly regulated, small amounts of it would be enough to generate the cAMP levels required for growth. TczAC activity was characterized in membrane preparations of yeast cells expressing the entire protein. In these cells, TczAC is constitutively active and shows 8–10-fold less activity when assayed in the presence of Mg2+ than Mn2+ as a divalent cation (Fig.4 A). It has a low affinity for ATP with a Km of 406 ± 55 μm, a value within the range reported for trypanosomatid enzymes (Fig. 4 B). The enzyme is specific for ATP because a 20-fold excess of GTP did not affect its activity (data not shown). Zn2+ is known to inhibit mammalian adenylyl cyclases by binding to the A metal binding site (30Tesmer J.J. Sprang S.R. Curr. Opin. Struct. Biol. 1998; 8: 713-719Crossref PubMed Scopus (100) Google Scholar). In TczAC the metal-binding residues are conserved with mammalian enzymes (Fig. 1 B) so the effect of this divalent cation was tested. When added to the reaction mixture, Zn2+ inhibited the enzyme with an IC50 of 7 ± 2 μm (Fig.4 C). To characterize further the catalytic mechanism of TczAC, the effect of the P-site inhibitor 2′-deoxyadenosine 3′-monophosphate (2′deoxy-3′AMP) was also analyzed. P-site inhibitors are adenosine or adenine derivatives that specifically inhibit mammalian adenylyl cyclases by binding to the purine ring binding site (31Dessauer C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 32Tesmer J.J. Dessauer C.W. Sunahara R.K. Murray L.D. Johnson R.A. Gilman A.G. Sprang S.R. Biochemistry. 2000; 39: 14464-14471Crossref PubMed Scopus (95) Google Scholar). 2′Deoxy-3′AMP inhibited TczAC with an IC50 of ∼100 μm. This value is between those reported for mammalian (<1 μm (33Désaubry L. Shoshani I. Johnson R.A. J. Biol. Chem. 1996; 271: 2380-2382Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar)) and bacterial (>1 mm (34Johnson R.A. Shoshani I. J. Biol. Chem. 1990; 265: 19035-19039Abstract Full Text PDF PubMed Google Scholar)) adenylyl cyclases. As expected from the sequence analysis, the recombinant enzyme responded neither to the diterpene forskolin nor to the G protein regulators GTPγS, AlF3−, cholera toxin, or pertussis toxin (data not shown)" @default.
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• W2048222765 title "A Novel Calcium-stimulated Adenylyl Cyclase fromTrypanosoma cruzi, Which Interacts with the Structural Flagellar Protein Paraflagellar Rod" @default.
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