Accelerated Transcription of PRPS1 in X-linked Overactivity of Normal Human Phosphoribosylpyrophosphate Synthetase
1999; Elsevier BV; Volume: 274; Issue: 11 Linguagem: Inglês
10.1074/jbc.274.11.7482
ISSN1083-351X
AutoresMaqbool Ahmed, W. Herman Taylor, Patrick Smith, Michael A. Becker,
Tópico(s)Folate and B Vitamins Research
ResumoPhosphoribosylpyrophosphate (PRPP) synthetase (PRS) superactivity is an X-linked disorder characterized by gout with overproduction of purine nucleotides and uric acid. Study of the two X-linked PRS isoforms (PRS1 and PRS2) in cells from certain affected individuals has shown selectively increased concentrations of structurally normal PRS1 transcript and isoform, suggesting that this form of the disorder involves pretranslational dysregulation ofPRPS1 expression and might be more appropriately termed overactivity of normal PRS. We applied Southern and Northern blot analyses and slot blotting of nuclear runoffs to delineate the process underlying aberrant PRPS1 expression in fibroblasts and lymphoblasts from patients with overactivity of normal PRS. NeitherPRPS1 amplification nor altered stability or processing of PRS1 mRNA was identified, but PRPS1 transcription was increased relative to GAPDH (3- to 4-fold normal in fibroblasts; 1.9- to 2.4-fold in lymphoblasts) and PRPS2. Nearly coordinate relative increases in each process mediating transfer of genetic information from PRPS1 transcription to maximal PRS1 isoform expression in patient fibroblasts further supported the idea that accelerated PRPS1 transcription is the major aberration leading to PRS1 overexpression. In addition, modulated relative increases in PRS activities at suboptimal Piconcentration and in rates of PRPP and purine nucleotide synthesis in intact patient fibroblasts indicate that despite an intact allosteric mechanism of regulation of PRS activity, PRPS1transcription is a major determinant of PRPP and purine synthesis. The genetic basis of disordered PRPS1 transcription remains unresolved; normal- and patient-derived PRPS1s share nucleotide sequence identity at least 850 base pairs 5′ to the consensus transcription initiation site. Phosphoribosylpyrophosphate (PRPP) synthetase (PRS) superactivity is an X-linked disorder characterized by gout with overproduction of purine nucleotides and uric acid. Study of the two X-linked PRS isoforms (PRS1 and PRS2) in cells from certain affected individuals has shown selectively increased concentrations of structurally normal PRS1 transcript and isoform, suggesting that this form of the disorder involves pretranslational dysregulation ofPRPS1 expression and might be more appropriately termed overactivity of normal PRS. We applied Southern and Northern blot analyses and slot blotting of nuclear runoffs to delineate the process underlying aberrant PRPS1 expression in fibroblasts and lymphoblasts from patients with overactivity of normal PRS. NeitherPRPS1 amplification nor altered stability or processing of PRS1 mRNA was identified, but PRPS1 transcription was increased relative to GAPDH (3- to 4-fold normal in fibroblasts; 1.9- to 2.4-fold in lymphoblasts) and PRPS2. Nearly coordinate relative increases in each process mediating transfer of genetic information from PRPS1 transcription to maximal PRS1 isoform expression in patient fibroblasts further supported the idea that accelerated PRPS1 transcription is the major aberration leading to PRS1 overexpression. In addition, modulated relative increases in PRS activities at suboptimal Piconcentration and in rates of PRPP and purine nucleotide synthesis in intact patient fibroblasts indicate that despite an intact allosteric mechanism of regulation of PRS activity, PRPS1transcription is a major determinant of PRPP and purine synthesis. The genetic basis of disordered PRPS1 transcription remains unresolved; normal- and patient-derived PRPS1s share nucleotide sequence identity at least 850 base pairs 5′ to the consensus transcription initiation site. Phosphoribosylpyrophosphate (PRPP) 1The abbreviations used are: PRPP, 5-phosphoribosyl 1-pyrophosphate; PRS, phosphoribosylpyrophosphate synthetase; bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase 1The abbreviations used are: PRPP, 5-phosphoribosyl 1-pyrophosphate; PRS, phosphoribosylpyrophosphate synthetase; bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase is a substrate in the synthesis of virtually all nucleotides (1Kornberg A. Lieberman I. Simms E.S. J. Biol. Chem. 1955; 215: 389-402Abstract Full Text PDF PubMed Google Scholar) as well as an important regulator of rates of the de novo pathways of purine and pyrimidine nucleotide synthesis (2Holmes E.W. McDonald J.A. McCord J.M. Wyngaarden J.B. Kelley W.N. J. Biol. Chem. 1973; 248: 144-150Abstract Full Text PDF PubMed Google Scholar, 3Becker M.A. Kim M. J. Biol. Chem. 1987; 262: 14531-14537Abstract Full Text PDF PubMed Google Scholar, 4Tatibana M. Shigesada K. Adv. Enzyme Regul. 1972; 10: 249-271Crossref PubMed Scopus (78) Google Scholar). PRPP synthesis from MgATP and ribose-5-phosphate is catalyzed in mammalian cells by a family of PRPP synthetase (PRS; EC2.7.6.1) isoforms in reactions requiring Mg2+ and Pi as activators and subject to inhibition by purine, pyrimidine, and pyridine nucleotides (5Fox I.H. Kelley W.N. J. Biol. Chem. 1971; 246: 5739-5748Abstract Full Text PDF PubMed Google Scholar, 6Fox I.H. Kelley W.N. J. Biol. Chem. 1972; 247: 2126-2131Abstract Full Text PDF PubMed Google Scholar, 7Ishijima S. Kita K. Ahmad I. Ishizuka T. Taira M. Tatibana M. J. Biol. Chem. 1991; 266: 15693-15697Abstract Full Text PDF PubMed Google Scholar, 8Nosal J.M. Switzer R.L. Becker M.A. J. Biol. Chem. 1993; 268: 10168-10175Abstract Full Text PDF PubMed Google Scholar). Of the three highly homologous human PRS isoforms identified to date, PRS1 and PRS2 are expressed in all tissues (9Taira M. Iizasa T. Yamada K. Shimada H. Tatibana M. Biochim. Biophys. Acta. 1989; 1007: 203-208Crossref PubMed Scopus (49) Google Scholar, 10Becker M.A. Taylor W. Smith P.R. Ahmed M. Adv. Exp. Med. Biol. 1998; 431: 215-220Crossref PubMed Scopus (1) Google Scholar) and are encoded by genes (PRPS1 and PRPS2) that map, respectively, to the long and the short arms of the X chromosome (11Taira M. Kudoh J. Minoshima S. Iizasa T. Shimada H. Shimizu Y. Tatibana M. Shimuzu N. Somatic Cell Mol. Genet. 1989; 15: 29-37Crossref PubMed Scopus (41) Google Scholar, 12Becker M.A. Heidler S.A. Bell G.I. Seino S. LeBeau M.M Westbrook C.A. Neuman W. Shapiro L.J. Mohandas T.K. Roessler B.J. Palella T.D. Genomics. 1990; 8: 550-561Crossref Scopus (40) Google Scholar). PRS3 expression is detectable only in the testes and is encoded autosomally (9Taira M. Iizasa T. Yamada K. Shimada H. Tatibana M. Biochim. Biophys. Acta. 1989; 1007: 203-208Crossref PubMed Scopus (49) Google Scholar, 13Taira M. Iizasa T. Shimada H. Kudoh J. Shimizu N. Tatibana M. J. Biol. Chem. 1990; 265: 16491-16497Abstract Full Text PDF PubMed Google Scholar).Superactivity of PRS is an X chromosome-linked human disorder (14Yen R.C.K. Adams W.B. Lazar C. Becker M.A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 482-485Crossref PubMed Scopus (28) Google Scholar) characterized by PRPP, purine nucleotide, and uric acid overproduction (15Sperling O. Boer P. Persky-Brosh S. Kanarek E. deVries A. Rev. Eur. Etudes Clin. Biol. 1972; 17: 703-706PubMed Google Scholar, 16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar, 17Zoref E. deVries A. Sperling O. J. Clin. Invest. 1975; 56: 1093-1099Crossref PubMed Scopus (91) Google Scholar), gout (15Sperling O. Boer P. Persky-Brosh S. Kanarek E. deVries A. Rev. Eur. Etudes Clin. Biol. 1972; 17: 703-706PubMed Google Scholar, 18Becker M.A. Puig J.G. Mateos F.A. Jimenez M.L. Kim M. Simmonds H.A. Am. J. Med. 1988; 85: 383-390Abstract Full Text PDF PubMed Scopus (68) Google Scholar), and, in some affected families, neurodevelopmental impairment (18Becker M.A. Puig J.G. Mateos F.A. Jimenez M.L. Kim M. Simmonds H.A. Am. J. Med. 1988; 85: 383-390Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 19Becker M.A. Raivio K.O. Bakay B. Adams W.B. Nyhan W.L. J. Clin. Invest. 1980; 65: 109-120Crossref PubMed Scopus (65) Google Scholar, 20Simmonds H.A. Webster D.R. Lingham S. Wilson J. Neuropediatrics. 1985; 16: 106-108Crossref PubMed Scopus (20) Google Scholar, 21Becker M.S. Smith P.R. Taylor W. Mustafi R. Switzer R.L. J. Clin. Invest. 1995; 96: 2133-2141Crossref PubMed Scopus (75) Google Scholar). The kinetic mechanisms underlying inherited PRS superactivity are diverse and include defective allosteric regulation of PRS1 activity (regulatory defects) (15Sperling O. Boer P. Persky-Brosh S. Kanarek E. deVries A. Rev. Eur. Etudes Clin. Biol. 1972; 17: 703-706PubMed Google Scholar, 16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar, 17Zoref E. deVries A. Sperling O. J. Clin. Invest. 1975; 56: 1093-1099Crossref PubMed Scopus (91) Google Scholar, 18Becker M.A. Puig J.G. Mateos F.A. Jimenez M.L. Kim M. Simmonds H.A. Am. J. Med. 1988; 85: 383-390Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 19Becker M.A. Raivio K.O. Bakay B. Adams W.B. Nyhan W.L. J. Clin. Invest. 1980; 65: 109-120Crossref PubMed Scopus (65) Google Scholar, 21Becker M.S. Smith P.R. Taylor W. Mustafi R. Switzer R.L. J. Clin. Invest. 1995; 96: 2133-2141Crossref PubMed Scopus (75) Google Scholar, 22Becker M.A. Losman M.J. Wilson J. Simmonds H.A. Biochim. Biophys. Acta. 1986; 882: 168-176Crossref PubMed Scopus (23) Google Scholar), increased apparent affinity of PRS for the substrate ribose-5-phosphate (23Becker M.A. J. Clin. Invest. 1976; 59: 308-318Crossref Scopus (61) Google Scholar), and increased activity of the normal PRS1 isoform (formerly called catalytic superactivity) (24Becker M.A. Taylor W. Smith P.R. Ahmed M. J. Biol. Chem. 1996; 271: 19894-19899Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 25Becker M.A. Losman M.J. Rosenberg A.L. Mehlman I. Levinson D.J. Holmes E.W. Arthritis Rheum. 1986; 29: 880-888Crossref PubMed Scopus (28) Google Scholar, 26Becker M.A. Meyer L.J. Wood A.W. Seegmiller J.E. Science. 1973; 179: 1123-1126Crossref PubMed Scopus (60) Google Scholar, 27Becker M.A. Losman M.J. Itkin P. Simkin P.A. J. Lab. Clin. Med. 1982; 99: 485-511Google Scholar). Study of the genetic and mechanistic bases of the heterogeneous kinetic alterations associated with PRS superactivity has shed light on the manner in which the synthesis of PRPP is regulated (3Becker M.A. Kim M. J. Biol. Chem. 1987; 262: 14531-14537Abstract Full Text PDF PubMed Google Scholar, 16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar, 21Becker M.S. Smith P.R. Taylor W. Mustafi R. Switzer R.L. J. Clin. Invest. 1995; 96: 2133-2141Crossref PubMed Scopus (75) Google Scholar). In the case of regulatory defects, for example, patient-derived PRS1 cDNAs bear point mutations encoding recombinant mutant PRS1s with altered allosteric properties (resistance to noncompetitive purine nucleotide inhibition and increased sensitivity to Pi activation) characteristic of those of PRS in cells from the respective affected individual (21Becker M.S. Smith P.R. Taylor W. Mustafi R. Switzer R.L. J. Clin. Invest. 1995; 96: 2133-2141Crossref PubMed Scopus (75) Google Scholar). This finding provides evidence that allosteric control of PRS1 activity is important in regulating PRPP synthesis in human cells.In contrast, overexpression of normal PRS1 transcript as well as PRS1 isoform has been demonstrated in cells from patients with overactivity of normal PRS (24Becker M.A. Taylor W. Smith P.R. Ahmed M. J. Biol. Chem. 1996; 271: 19894-19899Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The association of increased PRS1 transcript level with increased PRS1 isoform content and enzyme activity suggests a pretranslational defect in the expression of PRPS1 in this type of inherited PRS superactivity (24Becker M.A. Taylor W. Smith P.R. Ahmed M. J. Biol. Chem. 1996; 271: 19894-19899Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In studies aimed at further defining the process responsible for overexpression of normal PRS1 transcript and isoform, we have found selective acceleration ofPRPS1 transcription in this disorder as well as evidence that in fibroblasts from affected individuals the rate of transcription of PRPS1 serves as a major determinant of PRPP and purine nucleotide production rates despite intact allosteric regulation of PRS activity.DISCUSSIONThe current studies provide evidence for a selectively increased rate of transcription of PRPS1 as the pretranslational aberration underlying increased expression of the normal PRS1 isoform in inherited PRS overactivity. In each of the fibroblast strains and lymphoblast lines cultured from affected individuals, transcription ofPRPS1 was consistently greater relative to that ofGAPDH than was the case in corresponding normal cells. Although the relative increases in PRPS1 transcription rates were greater in patient fibroblasts than lymphoblasts, study of the latter cell type permitted a more accurate demonstration of the selectivity of accelerated PRPS1 transcription. That is, relative rates of PRPS2 transcription in normal cells were substantially greater in lymphoblasts than in fibroblasts, but for both lymphoblasts and fibroblasts, relative rates of PRPS2transcription in normal and patient cells were indistinguishable. Consistency was also found in the relative differences between normal and patient cells in each of the processes mediating flow of genetic information from PRPS1 transcription to maximal PRS1 isoform expression (enzyme activity at 32 mm Pi). This finding, in conjunction with the demonstration that patient and normal cells did not differ in PRS1 mRNA or isoform structure or in alternative pretranslational mechanisms that might otherwise explain PRS1 isoform overexpression, supports the contentions that PRS1 isoform concentrations are determined, at least in major part, at the level of transcription and that an inherited increase in PRPS1transcription rate provides the basis for the increase in the concentration of the normal PRS1 isoform in cells of affected individuals.The genetic basis of inherited acceleration of PRPS1transcription remains to be determined. Sequence identity of patient and normal PRPS1 DNAs in the 850-bp region 5′ to the consensus transcription initiation site (32Ishizuka T. Iizasa T. Taira M. Ishijima S. Sonoda T. Shimada H. Nagatake N. Tatibana M. Biochim. Biophys. Acta. 1992; 1130: 139-148Crossref PubMed Scopus (20) Google Scholar) excludes mutation in the gene promoter and immediate 5′ PRPS1 flanking sequence, for which examples of transcriptional dysregulation have been established, as in the thalassemias (37Orkin S.H. Antonarakis S.E Kazazian Jr., H.H. J. Biol. Chem. 1984; 259: 8679-8681Abstract Full Text PDF PubMed Google Scholar, 38Antonarakis S.E. Orkin S.H. Cheng T.-C. Scott A.F. Sexton J.P. Trusko S.P. Charache S. Kazazian Jr., H.H. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1154-1158Crossref PubMed Scopus (138) Google Scholar, 39Matsuda M. Sakamoto N. Fukinaki Y. Blood. 1992; 80: 1347-1351Crossref PubMed Google Scholar) and hereditary persistence of fetal hemoglobin (40Collins F.S Stoeckert Jr., C.J. Serjeant G.R. Forget B.G. Weissman S.M. Proc Natl. Acad. Sci. U. S. A. 1984; 81: 4894-4898Crossref PubMed Scopus (78) Google Scholar, 41Martin D.I.K. Tsai S.-F. Orkin S.H. Nature. 1989; 338: 435-438Crossref PubMed Scopus (181) Google Scholar). Among alternative possibilities to explain accelerated PRPS1 transcription are mutations in a more remote promoter element, either in contiguity with the immediate 5′-flanking sequence (42Berg P.I. Mittelman M. Elion J. Labie D. Schechter A.N. Am. J. Hematol. 1991; 36: 42-47Crossref PubMed Scopus (52) Google Scholar) or even substantially distant (43Grosveld F. van Assendelft G.B. Greaves D.R. Kollias G. Cell. 1987; 51: 975-985Abstract Full Text PDF PubMed Scopus (1430) Google Scholar); in a cis-acting element within or adjacent to the PRPS1 gene, such as an intronic enhancer or suppressor (44Aranow B. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (92) Google Scholar) or a 3′-flanking DNA sequence (45Moi P. Loudianos G. Lavinha J. Murru S. Cossu P. Casu R. Oggiano L. Longinotti M. Cao A. Pirastu M. Blood. 1992; 79: 512-516Crossref PubMed Google Scholar); or in a trans-acting gene influencing the regulation of PRPS1 transcription. In any case, X chromosome-linked transmission of PRS catalytic superactivity (14Yen R.C.K. Adams W.B. Lazar C. Becker M.A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 482-485Crossref PubMed Scopus (28) Google Scholar) favors the view that the primary defect altering PRPS1 transcription is itself X-linked. In addition to extended PRPS1 5′-flanking region sequencing, functional analysis of the PRPS1 promoter and adjacent 5′-flanking DNA, comparing PRPS1 promoter-plasmid construct expression in normal and patient cells, should prove helpful in distinguishing among these possibilities.Prior studies (3Becker M.A. Kim M. J. Biol. Chem. 1987; 262: 14531-14537Abstract Full Text PDF PubMed Google Scholar, 16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar) comparing mechanisms of purine nucleotide overproduction in fibroblasts from individuals with PRS superactivity (either catalytic or regulatory defects in PRS1) and severe deficiency of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) have confirmed that the rate of the pathway of purine synthesis de novo is controlled at the sequential PRS and amidophosphoribosyltransferase (EC 2.4.2.14) reactions, the latter the first committed step in the pathway. Within this regulatory domain, purine nucleotides inhibit both reactions (3Becker M.A. Kim M. J. Biol. Chem. 1987; 262: 14531-14537Abstract Full Text PDF PubMed Google Scholar, 16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar, 36Yen R.C.K. Raivio K.O. Becker M.A. J. Biol. Chem. 1981; 256: 1839-1845Abstract Full Text PDF PubMed Google Scholar), exerting more potent inhibition of amidophosphoribosyltransferase by antagonizing PRPP activation of this reaction (3Becker M.A. Kim M. J. Biol. Chem. 1987; 262: 14531-14537Abstract Full Text PDF PubMed Google Scholar, 46Holmes E.W. Wyngaarden J.B. Kelley W.N. J. Biol. Chem. 1973; 248: 6035-6040Abstract Full Text PDF PubMed Google Scholar). Although fibroblasts with either regulatory defects in PRS or overactivity of normal PRS share the biochemical hallmarks of PRS superactivity, increased rates of PRPP and purine synthesis and increased intracellular purine nucleotide concentrations, defect-specific differences in the intracellular control of PRPP and purine nucleotide synthesis are apparent (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar).In the case of PRS regulatory superactivity, where PRS activity in cell extracts or purified enzyme preparations are resistant to purine nucleotide inhibition, accelerated rates of intracellular PRPP and purine nucleotide synthesis are refractory to inhibition by exogenous purine base precursors of purine nucleotides or by endogenous increases in purine nucleotide concentrations (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar). Thus, in cells bearing PRS1s with any of an array of point mutations (21Becker M.S. Smith P.R. Taylor W. Mustafi R. Switzer R.L. J. Clin. Invest. 1995; 96: 2133-2141Crossref PubMed Scopus (75) Google Scholar), in vitrodefects in allosteric regulation of PRS1 activities are paralleled by dysregulation of PRPP and purine synthesis. In contrast, allosteric regulation of PRS activity is normal in enzyme preparations from fibroblasts with overactivity of normal PRS, and suppression of PRPP and purine nucleotide synthesis in response to purine base addition is intact in the corresponding cells (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar). Nevertheless, these cells express increased rates of PRPP and purine nucleotide synthesis.The apparent paradox of increased rates of PRPP and purine nucleotide synthesis in fibroblasts with overactivity of normal PRS catalytic superactivity despite increased purine nucleotide inhibitor pools and normal allosteric regulation of PRS activity (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar) is best resolved by the view that the increased concentration of the normal PRS1 isoform (24Becker M.A. Taylor W. Smith P.R. Ahmed M. J. Biol. Chem. 1996; 271: 19894-19899Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) results in a rate of PRPP synthesis sufficient to activate amidophosphoribosyltransferase despite coexisting increased levels of inhibitory purine nucleotides (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar). Consistent with this formulation are the substantially more modest increases in rates of PRPP and purine nucleotide synthesis in fibroblasts with intact allosteric regulation than in cells in which mutations in PRPS1 impair this control mechanism (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar). Thus, although intact allosteric inhibition apparently modulates expression of PRPP and purine overproduction in fibroblasts with excessive PRS1 isoform, this regulatory mechanism is insufficient to overcome the excessive expression of enzyme activity resulting from acceleration of PRPS1 transcription. The current studies provide, then, an example of a circumstance in which transcription of PRPS1 is a major determinant of PRPP and purine nucleotide synthetic rates. Phosphoribosylpyrophosphate (PRPP) 1The abbreviations used are: PRPP, 5-phosphoribosyl 1-pyrophosphate; PRS, phosphoribosylpyrophosphate synthetase; bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase 1The abbreviations used are: PRPP, 5-phosphoribosyl 1-pyrophosphate; PRS, phosphoribosylpyrophosphate synthetase; bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase is a substrate in the synthesis of virtually all nucleotides (1Kornberg A. Lieberman I. Simms E.S. J. Biol. Chem. 1955; 215: 389-402Abstract Full Text PDF PubMed Google Scholar) as well as an important regulator of rates of the de novo pathways of purine and pyrimidine nucleotide synthesis (2Holmes E.W. McDonald J.A. McCord J.M. Wyngaarden J.B. Kelley W.N. J. Biol. Chem. 1973; 248: 144-150Abstract Full Text PDF PubMed Google Scholar, 3Becker M.A. Kim M. J. Biol. Chem. 1987; 262: 14531-14537Abstract Full Text PDF PubMed Google Scholar, 4Tatibana M. Shigesada K. Adv. Enzyme Regul. 1972; 10: 249-271Crossref PubMed Scopus (78) Google Scholar). PRPP synthesis from MgATP and ribose-5-phosphate is catalyzed in mammalian cells by a family of PRPP synthetase (PRS; EC2.7.6.1) isoforms in reactions requiring Mg2+ and Pi as activators and subject to inhibition by purine, pyrimidine, and pyridine nucleotides (5Fox I.H. Kelley W.N. J. Biol. Chem. 1971; 246: 5739-5748Abstract Full Text PDF PubMed Google Scholar, 6Fox I.H. Kelley W.N. J. Biol. Chem. 1972; 247: 2126-2131Abstract Full Text PDF PubMed Google Scholar, 7Ishijima S. Kita K. Ahmad I. Ishizuka T. Taira M. Tatibana M. J. Biol. Chem. 1991; 266: 15693-15697Abstract Full Text PDF PubMed Google Scholar, 8Nosal J.M. Switzer R.L. Becker M.A. J. Biol. Chem. 1993; 268: 10168-10175Abstract Full Text PDF PubMed Google Scholar). Of the three highly homologous human PRS isoforms identified to date, PRS1 and PRS2 are expressed in all tissues (9Taira M. Iizasa T. Yamada K. Shimada H. Tatibana M. Biochim. Biophys. Acta. 1989; 1007: 203-208Crossref PubMed Scopus (49) Google Scholar, 10Becker M.A. Taylor W. Smith P.R. Ahmed M. Adv. Exp. Med. Biol. 1998; 431: 215-220Crossref PubMed Scopus (1) Google Scholar) and are encoded by genes (PRPS1 and PRPS2) that map, respectively, to the long and the short arms of the X chromosome (11Taira M. Kudoh J. Minoshima S. Iizasa T. Shimada H. Shimizu Y. Tatibana M. Shimuzu N. Somatic Cell Mol. Genet. 1989; 15: 29-37Crossref PubMed Scopus (41) Google Scholar, 12Becker M.A. Heidler S.A. Bell G.I. Seino S. LeBeau M.M Westbrook C.A. Neuman W. Shapiro L.J. Mohandas T.K. Roessler B.J. Palella T.D. Genomics. 1990; 8: 550-561Crossref Scopus (40) Google Scholar). PRS3 expression is detectable only in the testes and is encoded autosomally (9Taira M. Iizasa T. Yamada K. Shimada H. Tatibana M. Biochim. Biophys. Acta. 1989; 1007: 203-208Crossref PubMed Scopus (49) Google Scholar, 13Taira M. Iizasa T. Shimada H. Kudoh J. Shimizu N. Tatibana M. J. Biol. Chem. 1990; 265: 16491-16497Abstract Full Text PDF PubMed Google Scholar). Superactivity of PRS is an X chromosome-linked human disorder (14Yen R.C.K. Adams W.B. Lazar C. Becker M.A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 482-485Crossref PubMed Scopus (28) Google Scholar) characterized by PRPP, purine nucleotide, and uric acid overproduction (15Sperling O. Boer P. Persky-Brosh S. Kanarek E. deVries A. Rev. Eur. Etudes Clin. Biol. 1972; 17: 703-706PubMed Google Scholar, 16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar, 17Zoref E. deVries A. Sperling O. J. Clin. Invest. 1975; 56: 1093-1099Crossref PubMed Scopus (91) Google Scholar), gout (15Sperling O. Boer P. Persky-Brosh S. Kanarek E. deVries A. Rev. Eur. Etudes Clin. Biol. 1972; 17: 703-706PubMed Google Scholar, 18Becker M.A. Puig J.G. Mateos F.A. Jimenez M.L. Kim M. Simmonds H.A. Am. J. Med. 1988; 85: 383-390Abstract Full Text PDF PubMed Scopus (68) Google Scholar), and, in some affected families, neurodevelopmental impairment (18Becker M.A. Puig J.G. Mateos F.A. Jimenez M.L. Kim M. Simmonds H.A. Am. J. Med. 1988; 85: 383-390Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 19Becker M.A. Raivio K.O. Bakay B. Adams W.B. Nyhan W.L. J. Clin. Invest. 1980; 65: 109-120Crossref PubMed Scopus (65) Google Scholar, 20Simmonds H.A. Webster D.R. Lingham S. Wilson J. Neuropediatrics. 1985; 16: 106-108Crossref PubMed Scopus (20) Google Scholar, 21Becker M.S. Smith P.R. Taylor W. Mustafi R. Switzer R.L. J. Clin. Invest. 1995; 96: 2133-2141Crossref PubMed Scopus (75) Google Scholar). The kinetic mechanisms underlying inherited PRS superactivity are diverse and include defective allosteric regulation of PRS1 activity (regulatory defects) (15Sperling O. Boer P. Persky-Brosh S. Kanarek E. deVries A. Rev. Eur. Etudes Clin. Biol. 1972; 17: 703-706PubMed Google Scholar, 16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar, 17Zoref E. deVries A. Sperling O. J. Clin. Invest. 1975; 56: 1093-1099Crossref PubMed Scopus (91) Google Scholar, 18Becker M.A. Puig J.G. Mateos F.A. Jimenez M.L. Kim M. Simmonds H.A. Am. J. Med. 1988; 85: 383-390Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 19Becker M.A. Raivio K.O. Bakay B. Adams W.B. Nyhan W.L. J. Clin. Invest. 1980; 65: 109-120Crossref PubMed Scopus (65) Google Scholar, 21Becker M.S. Smith P.R. Taylor W. Mustafi R. Switzer R.L. J. Clin. Invest. 1995; 96: 2133-2141Crossref PubMed Scopus (75) Google Scholar, 22Becker M.A. Losman M.J. Wilson J. Simmonds H.A. Biochim. Biophys. Acta. 1986; 882: 168-176Crossref PubMed Scopus (23) Google Scholar), increased apparent affinity of PRS for the substrate ribose-5-phosphate (23Becker M.A. J. Clin. Invest. 1976; 59: 308-318Crossref Scopus (61) Google Scholar), and increased activity of the normal PRS1 isoform (formerly called catalytic superactivity) (24Becker M.A. Taylor W. Smith P.R. Ahmed M. J. Biol. Chem. 1996; 271: 19894-19899Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 25Becker M.A. Losman M.J. Rosenberg A.L. Mehlman I. Levinson D.J. Holmes E.W. Arthritis Rheum. 1986; 29: 880-888Crossref PubMed Scopus (28) Google Scholar, 26Becker M.A. Meyer L.J. Wood A.W. Seegmiller J.E. Science. 1973; 179: 1123-1126Crossref PubMed Scopus (60) Google Scholar, 27Becker M.A. Losman M.J. Itkin P. Simkin P.A. J. Lab. Clin. Med. 1982; 99: 485-511Google Scholar). Study of the genetic and mechanistic bases of the heterogeneous kinetic alterations associated with PRS superactivity has shed light on the manner in which the synthesis of PRPP is regulated (3Becker M.A. Kim M. J. Biol. Chem. 1987; 262: 14531-14537Abstract Full Text PDF PubMed Google Scholar, 16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar, 21Becker M.S. Smith P.R. Taylor W. Mustafi R. Switzer R.L. J. Clin. Invest. 1995; 96: 2133-2141Crossref PubMed Scopus (75) Google Scholar). In the case of regulatory defects, for example, patient-derived PRS1 cDNAs bear point mutations encoding recombinant mutant PRS1s with altered allosteric properties (resistance to noncompetitive purine nucleotide inhibition and increased sensitivity to Pi activation) characteristic of those of PRS in cells from the respective affected individual (21Becker M.S. Smith P.R. Taylor W. Mustafi R. Switzer R.L. J. Clin. Invest. 1995; 96: 2133-2141Crossref PubMed Scopus (75) Google Scholar). This finding provides evidence that allosteric control of PRS1 activity is important in regulating PRPP synthesis in human cells. In contrast, overexpression of normal PRS1 transcript as well as PRS1 isoform has been demonstrated in cells from patients with overactivity of normal PRS (24Becker M.A. Taylor W. Smith P.R. Ahmed M. J. Biol. Chem. 1996; 271: 19894-19899Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The association of increased PRS1 transcript level with increased PRS1 isoform content and enzyme activity suggests a pretranslational defect in the expression of PRPS1 in this type of inherited PRS superactivity (24Becker M.A. Taylor W. Smith P.R. Ahmed M. J. Biol. Chem. 1996; 271: 19894-19899Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In studies aimed at further defining the process responsible for overexpression of normal PRS1 transcript and isoform, we have found selective acceleration ofPRPS1 transcription in this disorder as well as evidence that in fibroblasts from affected individuals the rate of transcription of PRPS1 serves as a major determinant of PRPP and purine nucleotide production rates despite intact allosteric regulation of PRS activity. DISCUSSIONThe current studies provide evidence for a selectively increased rate of transcription of PRPS1 as the pretranslational aberration underlying increased expression of the normal PRS1 isoform in inherited PRS overactivity. In each of the fibroblast strains and lymphoblast lines cultured from affected individuals, transcription ofPRPS1 was consistently greater relative to that ofGAPDH than was the case in corresponding normal cells. Although the relative increases in PRPS1 transcription rates were greater in patient fibroblasts than lymphoblasts, study of the latter cell type permitted a more accurate demonstration of the selectivity of accelerated PRPS1 transcription. That is, relative rates of PRPS2 transcription in normal cells were substantially greater in lymphoblasts than in fibroblasts, but for both lymphoblasts and fibroblasts, relative rates of PRPS2transcription in normal and patient cells were indistinguishable. Consistency was also found in the relative differences between normal and patient cells in each of the processes mediating flow of genetic information from PRPS1 transcription to maximal PRS1 isoform expression (enzyme activity at 32 mm Pi). This finding, in conjunction with the demonstration that patient and normal cells did not differ in PRS1 mRNA or isoform structure or in alternative pretranslational mechanisms that might otherwise explain PRS1 isoform overexpression, supports the contentions that PRS1 isoform concentrations are determined, at least in major part, at the level of transcription and that an inherited increase in PRPS1transcription rate provides the basis for the increase in the concentration of the normal PRS1 isoform in cells of affected individuals.The genetic basis of inherited acceleration of PRPS1transcription remains to be determined. Sequence identity of patient and normal PRPS1 DNAs in the 850-bp region 5′ to the consensus transcription initiation site (32Ishizuka T. Iizasa T. Taira M. Ishijima S. Sonoda T. Shimada H. Nagatake N. Tatibana M. Biochim. Biophys. Acta. 1992; 1130: 139-148Crossref PubMed Scopus (20) Google Scholar) excludes mutation in the gene promoter and immediate 5′ PRPS1 flanking sequence, for which examples of transcriptional dysregulation have been established, as in the thalassemias (37Orkin S.H. Antonarakis S.E Kazazian Jr., H.H. J. Biol. Chem. 1984; 259: 8679-8681Abstract Full Text PDF PubMed Google Scholar, 38Antonarakis S.E. Orkin S.H. Cheng T.-C. Scott A.F. Sexton J.P. Trusko S.P. Charache S. Kazazian Jr., H.H. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1154-1158Crossref PubMed Scopus (138) Google Scholar, 39Matsuda M. Sakamoto N. Fukinaki Y. Blood. 1992; 80: 1347-1351Crossref PubMed Google Scholar) and hereditary persistence of fetal hemoglobin (40Collins F.S Stoeckert Jr., C.J. Serjeant G.R. Forget B.G. Weissman S.M. Proc Natl. Acad. Sci. U. S. A. 1984; 81: 4894-4898Crossref PubMed Scopus (78) Google Scholar, 41Martin D.I.K. Tsai S.-F. Orkin S.H. Nature. 1989; 338: 435-438Crossref PubMed Scopus (181) Google Scholar). Among alternative possibilities to explain accelerated PRPS1 transcription are mutations in a more remote promoter element, either in contiguity with the immediate 5′-flanking sequence (42Berg P.I. Mittelman M. Elion J. Labie D. Schechter A.N. Am. J. Hematol. 1991; 36: 42-47Crossref PubMed Scopus (52) Google Scholar) or even substantially distant (43Grosveld F. van Assendelft G.B. Greaves D.R. Kollias G. Cell. 1987; 51: 975-985Abstract Full Text PDF PubMed Scopus (1430) Google Scholar); in a cis-acting element within or adjacent to the PRPS1 gene, such as an intronic enhancer or suppressor (44Aranow B. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (92) Google Scholar) or a 3′-flanking DNA sequence (45Moi P. Loudianos G. Lavinha J. Murru S. Cossu P. Casu R. Oggiano L. Longinotti M. Cao A. Pirastu M. Blood. 1992; 79: 512-516Crossref PubMed Google Scholar); or in a trans-acting gene influencing the regulation of PRPS1 transcription. In any case, X chromosome-linked transmission of PRS catalytic superactivity (14Yen R.C.K. Adams W.B. Lazar C. Becker M.A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 482-485Crossref PubMed Scopus (28) Google Scholar) favors the view that the primary defect altering PRPS1 transcription is itself X-linked. In addition to extended PRPS1 5′-flanking region sequencing, functional analysis of the PRPS1 promoter and adjacent 5′-flanking DNA, comparing PRPS1 promoter-plasmid construct expression in normal and patient cells, should prove helpful in distinguishing among these possibilities.Prior studies (3Becker M.A. Kim M. J. Biol. Chem. 1987; 262: 14531-14537Abstract Full Text PDF PubMed Google Scholar, 16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar) comparing mechanisms of purine nucleotide overproduction in fibroblasts from individuals with PRS superactivity (either catalytic or regulatory defects in PRS1) and severe deficiency of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) have confirmed that the rate of the pathway of purine synthesis de novo is controlled at the sequential PRS and amidophosphoribosyltransferase (EC 2.4.2.14) reactions, the latter the first committed step in the pathway. Within this regulatory domain, purine nucleotides inhibit both reactions (3Becker M.A. Kim M. J. Biol. Chem. 1987; 262: 14531-14537Abstract Full Text PDF PubMed Google Scholar, 16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar, 36Yen R.C.K. Raivio K.O. Becker M.A. J. Biol. Chem. 1981; 256: 1839-1845Abstract Full Text PDF PubMed Google Scholar), exerting more potent inhibition of amidophosphoribosyltransferase by antagonizing PRPP activation of this reaction (3Becker M.A. Kim M. J. Biol. Chem. 1987; 262: 14531-14537Abstract Full Text PDF PubMed Google Scholar, 46Holmes E.W. Wyngaarden J.B. Kelley W.N. J. Biol. Chem. 1973; 248: 6035-6040Abstract Full Text PDF PubMed Google Scholar). Although fibroblasts with either regulatory defects in PRS or overactivity of normal PRS share the biochemical hallmarks of PRS superactivity, increased rates of PRPP and purine synthesis and increased intracellular purine nucleotide concentrations, defect-specific differences in the intracellular control of PRPP and purine nucleotide synthesis are apparent (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar).In the case of PRS regulatory superactivity, where PRS activity in cell extracts or purified enzyme preparations are resistant to purine nucleotide inhibition, accelerated rates of intracellular PRPP and purine nucleotide synthesis are refractory to inhibition by exogenous purine base precursors of purine nucleotides or by endogenous increases in purine nucleotide concentrations (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar). Thus, in cells bearing PRS1s with any of an array of point mutations (21Becker M.S. Smith P.R. Taylor W. Mustafi R. Switzer R.L. J. Clin. Invest. 1995; 96: 2133-2141Crossref PubMed Scopus (75) Google Scholar), in vitrodefects in allosteric regulation of PRS1 activities are paralleled by dysregulation of PRPP and purine synthesis. In contrast, allosteric regulation of PRS activity is normal in enzyme preparations from fibroblasts with overactivity of normal PRS, and suppression of PRPP and purine nucleotide synthesis in response to purine base addition is intact in the corresponding cells (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar). Nevertheless, these cells express increased rates of PRPP and purine nucleotide synthesis.The apparent paradox of increased rates of PRPP and purine nucleotide synthesis in fibroblasts with overactivity of normal PRS catalytic superactivity despite increased purine nucleotide inhibitor pools and normal allosteric regulation of PRS activity (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar) is best resolved by the view that the increased concentration of the normal PRS1 isoform (24Becker M.A. Taylor W. Smith P.R. Ahmed M. J. Biol. Chem. 1996; 271: 19894-19899Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) results in a rate of PRPP synthesis sufficient to activate amidophosphoribosyltransferase despite coexisting increased levels of inhibitory purine nucleotides (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar). Consistent with this formulation are the substantially more modest increases in rates of PRPP and purine nucleotide synthesis in fibroblasts with intact allosteric regulation than in cells in which mutations in PRPS1 impair this control mechanism (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar). Thus, although intact allosteric inhibition apparently modulates expression of PRPP and purine overproduction in fibroblasts with excessive PRS1 isoform, this regulatory mechanism is insufficient to overcome the excessive expression of enzyme activity resulting from acceleration of PRPS1 transcription. The current studies provide, then, an example of a circumstance in which transcription of PRPS1 is a major determinant of PRPP and purine nucleotide synthetic rates. The current studies provide evidence for a selectively increased rate of transcription of PRPS1 as the pretranslational aberration underlying increased expression of the normal PRS1 isoform in inherited PRS overactivity. In each of the fibroblast strains and lymphoblast lines cultured from affected individuals, transcription ofPRPS1 was consistently greater relative to that ofGAPDH than was the case in corresponding normal cells. Although the relative increases in PRPS1 transcription rates were greater in patient fibroblasts than lymphoblasts, study of the latter cell type permitted a more accurate demonstration of the selectivity of accelerated PRPS1 transcription. That is, relative rates of PRPS2 transcription in normal cells were substantially greater in lymphoblasts than in fibroblasts, but for both lymphoblasts and fibroblasts, relative rates of PRPS2transcription in normal and patient cells were indistinguishable. Consistency was also found in the relative differences between normal and patient cells in each of the processes mediating flow of genetic information from PRPS1 transcription to maximal PRS1 isoform expression (enzyme activity at 32 mm Pi). This finding, in conjunction with the demonstration that patient and normal cells did not differ in PRS1 mRNA or isoform structure or in alternative pretranslational mechanisms that might otherwise explain PRS1 isoform overexpression, supports the contentions that PRS1 isoform concentrations are determined, at least in major part, at the level of transcription and that an inherited increase in PRPS1transcription rate provides the basis for the increase in the concentration of the normal PRS1 isoform in cells of affected individuals. The genetic basis of inherited acceleration of PRPS1transcription remains to be determined. Sequence identity of patient and normal PRPS1 DNAs in the 850-bp region 5′ to the consensus transcription initiation site (32Ishizuka T. Iizasa T. Taira M. Ishijima S. Sonoda T. Shimada H. Nagatake N. Tatibana M. Biochim. Biophys. Acta. 1992; 1130: 139-148Crossref PubMed Scopus (20) Google Scholar) excludes mutation in the gene promoter and immediate 5′ PRPS1 flanking sequence, for which examples of transcriptional dysregulation have been established, as in the thalassemias (37Orkin S.H. Antonarakis S.E Kazazian Jr., H.H. J. Biol. Chem. 1984; 259: 8679-8681Abstract Full Text PDF PubMed Google Scholar, 38Antonarakis S.E. Orkin S.H. Cheng T.-C. Scott A.F. Sexton J.P. Trusko S.P. Charache S. Kazazian Jr., H.H. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1154-1158Crossref PubMed Scopus (138) Google Scholar, 39Matsuda M. Sakamoto N. Fukinaki Y. Blood. 1992; 80: 1347-1351Crossref PubMed Google Scholar) and hereditary persistence of fetal hemoglobin (40Collins F.S Stoeckert Jr., C.J. Serjeant G.R. Forget B.G. Weissman S.M. Proc Natl. Acad. Sci. U. S. A. 1984; 81: 4894-4898Crossref PubMed Scopus (78) Google Scholar, 41Martin D.I.K. Tsai S.-F. Orkin S.H. Nature. 1989; 338: 435-438Crossref PubMed Scopus (181) Google Scholar). Among alternative possibilities to explain accelerated PRPS1 transcription are mutations in a more remote promoter element, either in contiguity with the immediate 5′-flanking sequence (42Berg P.I. Mittelman M. Elion J. Labie D. Schechter A.N. Am. J. Hematol. 1991; 36: 42-47Crossref PubMed Scopus (52) Google Scholar) or even substantially distant (43Grosveld F. van Assendelft G.B. Greaves D.R. Kollias G. Cell. 1987; 51: 975-985Abstract Full Text PDF PubMed Scopus (1430) Google Scholar); in a cis-acting element within or adjacent to the PRPS1 gene, such as an intronic enhancer or suppressor (44Aranow B. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (92) Google Scholar) or a 3′-flanking DNA sequence (45Moi P. Loudianos G. Lavinha J. Murru S. Cossu P. Casu R. Oggiano L. Longinotti M. Cao A. Pirastu M. Blood. 1992; 79: 512-516Crossref PubMed Google Scholar); or in a trans-acting gene influencing the regulation of PRPS1 transcription. In any case, X chromosome-linked transmission of PRS catalytic superactivity (14Yen R.C.K. Adams W.B. Lazar C. Becker M.A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 482-485Crossref PubMed Scopus (28) Google Scholar) favors the view that the primary defect altering PRPS1 transcription is itself X-linked. In addition to extended PRPS1 5′-flanking region sequencing, functional analysis of the PRPS1 promoter and adjacent 5′-flanking DNA, comparing PRPS1 promoter-plasmid construct expression in normal and patient cells, should prove helpful in distinguishing among these possibilities. Prior studies (3Becker M.A. Kim M. J. Biol. Chem. 1987; 262: 14531-14537Abstract Full Text PDF PubMed Google Scholar, 16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar) comparing mechanisms of purine nucleotide overproduction in fibroblasts from individuals with PRS superactivity (either catalytic or regulatory defects in PRS1) and severe deficiency of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) have confirmed that the rate of the pathway of purine synthesis de novo is controlled at the sequential PRS and amidophosphoribosyltransferase (EC 2.4.2.14) reactions, the latter the first committed step in the pathway. Within this regulatory domain, purine nucleotides inhibit both reactions (3Becker M.A. Kim M. J. Biol. Chem. 1987; 262: 14531-14537Abstract Full Text PDF PubMed Google Scholar, 16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar, 36Yen R.C.K. Raivio K.O. Becker M.A. J. Biol. Chem. 1981; 256: 1839-1845Abstract Full Text PDF PubMed Google Scholar), exerting more potent inhibition of amidophosphoribosyltransferase by antagonizing PRPP activation of this reaction (3Becker M.A. Kim M. J. Biol. Chem. 1987; 262: 14531-14537Abstract Full Text PDF PubMed Google Scholar, 46Holmes E.W. Wyngaarden J.B. Kelley W.N. J. Biol. Chem. 1973; 248: 6035-6040Abstract Full Text PDF PubMed Google Scholar). Although fibroblasts with either regulatory defects in PRS or overactivity of normal PRS share the biochemical hallmarks of PRS superactivity, increased rates of PRPP and purine synthesis and increased intracellular purine nucleotide concentrations, defect-specific differences in the intracellular control of PRPP and purine nucleotide synthesis are apparent (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar). In the case of PRS regulatory superactivity, where PRS activity in cell extracts or purified enzyme preparations are resistant to purine nucleotide inhibition, accelerated rates of intracellular PRPP and purine nucleotide synthesis are refractory to inhibition by exogenous purine base precursors of purine nucleotides or by endogenous increases in purine nucleotide concentrations (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar). Thus, in cells bearing PRS1s with any of an array of point mutations (21Becker M.S. Smith P.R. Taylor W. Mustafi R. Switzer R.L. J. Clin. Invest. 1995; 96: 2133-2141Crossref PubMed Scopus (75) Google Scholar), in vitrodefects in allosteric regulation of PRS1 activities are paralleled by dysregulation of PRPP and purine synthesis. In contrast, allosteric regulation of PRS activity is normal in enzyme preparations from fibroblasts with overactivity of normal PRS, and suppression of PRPP and purine nucleotide synthesis in response to purine base addition is intact in the corresponding cells (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar). Nevertheless, these cells express increased rates of PRPP and purine nucleotide synthesis. The apparent paradox of increased rates of PRPP and purine nucleotide synthesis in fibroblasts with overactivity of normal PRS catalytic superactivity despite increased purine nucleotide inhibitor pools and normal allosteric regulation of PRS activity (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar) is best resolved by the view that the increased concentration of the normal PRS1 isoform (24Becker M.A. Taylor W. Smith P.R. Ahmed M. J. Biol. Chem. 1996; 271: 19894-19899Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) results in a rate of PRPP synthesis sufficient to activate amidophosphoribosyltransferase despite coexisting increased levels of inhibitory purine nucleotides (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar). Consistent with this formulation are the substantially more modest increases in rates of PRPP and purine nucleotide synthesis in fibroblasts with intact allosteric regulation than in cells in which mutations in PRPS1 impair this control mechanism (16Becker M.A. Losman M.J. Kim M. J. Biol. Chem. 1987; 262: 5596-5602Abstract Full Text PDF PubMed Google Scholar). Thus, although intact allosteric inhibition apparently modulates expression of PRPP and purine overproduction in fibroblasts with excessive PRS1 isoform, this regulatory mechanism is insufficient to overcome the excessive expression of enzyme activity resulting from acceleration of PRPS1 transcription. The current studies provide, then, an example of a circumstance in which transcription of PRPS1 is a major determinant of PRPP and purine nucleotide synthetic rates. We thank Danette Shine for excellent manuscript preparation. We appreciate the helpful comments of Drs. Craig B. Thompson, Tullia Lindsten, and Harinder Singh (University of Chicago) during the course of this work.
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