Regulation of inosine monophosphate dehydrogenase type I and type II isoforms in human lymphocytes (original) (raw)
Related papers
Clinical Biochemistry, 2001
The immunosuppressive activity of mycophenolate mofetil (MMF) is based on the reversible inhibition of inosine 5Јmonophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). It was the aim of this study to develop a nonradioactive method for specific measurement of IMPDH activity in isolated peripheral mononuclear cells (MNC). Methods: The procedure is based on the incubation of lysed MNC with inosine 5Ј-monophosphate (IMP) followed by direct chromatographic determination of produced xanthosine 5Ј-monophosphate (XMP). IMPDH activity was measured in MNC of MMF-treated patients and nontreated volunteers. Results: The within-run (n ϭ 10) and between-run (n ϭ 20) coefficients of variation (CV) for IMPDH activity were Ͻ 8% and Ͻ 10%, respectively. IMPDH activity in 60 healthy volunteers (19 -63 yr) ranged from 4.72 to 32.92 nmol/h/mg protein (mean ϭ 18.39 Ϯ 6.24). The IC 50 for in vitro inhibition of IMPDH activity was about 2 to 3 g/L. Application of a single dose of 1 g MMF in dialysis patients resulted in a significant inhibition (by 47-95%; p Ͻ 0.05) of IMPDH activity in lysed MNC. Conclusions: The proposed assay specifically and reliably measures IMPDH activity in MNC. The procedure is applicable to evaluate pharmacodynamic activity in MMF-treated patients. The observed interindividual variability of IMPDH activity may reflect pharmacodynamic differences in MMF-treated patients.
Clinical and Experimental Immunology, 2001
The aim of this study was to evaluate the effects of the immunomodulating drug mycophenolic acid (MPA) on splenocytes in an animal model of systemic lupus erythematosus (SLE), using MRLlpr/lpr mice. MPA reversibly inhibits inosine 5 H -monophosphate dehydrogenase, an enzyme involved in the de novo guanosine synthesis. Splenocytes were treated with MPA (at 1 or 10 mm), and stimulated with either lipopolysaccharide (LPS; 10 m g/ml) or concanavalin A (ConA; 1´25 mg/ml). In blocking experiments, guanosine (100 mm) was added to the cultures to inhibit the effects of MPA. Lymphocyte proliferation, enumeration of immunoglobulin producing cells (using ELISPOT) and quantification of anti-double-stranded (ds) DNA antibodies, IFN-g and IL-10 (by ELISA) in supernatants were performed. In addition, cell viability was evaluated using propidium iodide and flow cytometry. We found that MPA-treated splenocytes had dramatically decreased mitogen-induced proliferation and number of immunoglobulin producing cells, down-regulated production of IFN-g, IL-10 and IgM anti-dsDNA antibodies. The viability of MPA-treated cells was also decreased. All of the effect modulated by MPA could be neutralized by the addition of guanosine. We conclude that MPA has potent immunomodulating effects on both B and T lymphocytes, modulating not only proliferation, but also the production of cytokines, immunoglobulins and autoantibodies.
Cell, 1996
is also found in bacteria and protozoa, and these forms share 30%-40% sequence identity with the human en-Scott A. Raybuck, Stephen P. Chambers, Paul R. Caron, Mark A. Murcko, zyme (Natsumeda and Carr, 1993). Regardless of species, native IMPDHs exist as homotetramers with sub-and Keith P. Wilson Vertex Pharmaceuticals Incorporated unit molecular masses in the 56-58 kDa range and follow an ordered Bi-Bi reaction sequence, 40 Allston Street Cambridge, Massachusetts 02139-4211
Journal of Medicinal Chemistry, 2002
Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme that is involved in the de novo synthesis of purine nucleotides. Novel 2-aminooxazoles were synthesized and tested for inhibition of IMPDH catalytic activity. Multiple analogues based on this chemotype were found to inhibit IMPDH with low nanomolar potency. One of the analogues (compound 23) showed excellent in vivo activity in the inhibition of antibody production in mice and in the adjuvant induced arthritis model in rats. Introduction. Inosine monophosphate dehydrogenase (IMPDH) is an enzyme that catalyzes the nicotinamide adenosine dinucleotide (NAD) dependent conversion of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP). 1 The reaction is irreversible and is the first step in the de novo synthesis of guanine nucleotides. Rapidly proliferating cells such as lymphocytes are dependent on the availability of the nucleotide pool to meet their metabolic requirement, and it is known that the activity of IMPDH is higher in proliferating cells. 2 Because of these cell requirements, IM-PDH is an attractive target for immunosuppressive, anticancer, and antiviral therapies. 3 Two isoforms of the IMPDH enzyme are known to exist: type I and type II. Human types I and II IMPDH cDNAs encode the same-size proteins (514 amino acids) and show 84% sequence identity. 4 It was initially thought that because type II expression is upregulated in neoplastic and replicating cells, it is this isoform that is responsible for cell differentiation and neoplastic transformation. 5 However, more recent work has indicated that induction of types I and II isoforms contributes significantly to the T-cell proliferation response. 6 The mechanism of IMPDH reaction has been studied in detail (Figure 1). 7 The oxidation of IMP to XMP is an irreversible reaction and utilizes NAD as the cofac
Inosine monophosphate dehydrogenase activity in acute leukaemia
Leukemia Research, 1987
Inosine monophosphate dehydrogenase (IMPD) is an important enzyme in de-novo purine synthesis. The level of IMPD activity has been suggested to determine whether acute leukaemia cells proliferate (if the activity is high) or differentiate (if IMPD activity is low). IMPD activity measured by the conversion of inosine monophosphate to xanthine monophosphate ranged from 12.5 to 87.0 (mean 49.4) pmol/h/106 cells in normal bone marrow. The levels were significantly raised in AML (range 14-374, mean 184 pmol/h/106 cells) and ALL (range 65-228, 6 mean 172 pmol/h/10 cells). Normal tonsillar (B) lymphocytes showed higher levels (range 78-159, mean 110 pmol/h/106 ceils) than resting peripheral blood T lymphocytes (range 8.8-51.2, 6 6 mean 28.1 pmol/h/10 cells). In CLL, the results (range 19-173, mean 64.3 pmol/h/10 cells) were comparable to those of normal tonsillar B lymphocytes. IMPD levels could be related to cell cycle in PHA-stimulated lymphocytes, since IMPD activity increased in parallel with increase in DNA synthesis measured by labelled thymidine incorporation. On the other hand, IMPD activity did not correlate with the proportion of proliferating cells measured on a FACS sorter in either AML or ALL or in normal tonsillar B cells. We conclude that IMPD levels are higher in B than T lymphocytes and in acute leukaemia blasts compared to more differentiated mixed bone marrow cells. The results do not suggest, however, that IMPD assay will be of value in differentiation of the various subtypes of acute leukaemia or of malignant haemopoietic cells from the equivalent normal cell at the same level of differentiation.
Expression of IMPDH1 and IMPDH2 After Transplantation and Initiation of Immunosuppression
Transplantation, 2008
Background. Mycophenolic acid (MPA) mediates immunosuppressive effects by inhibiting inosine monophosphate dehydrogenase (IMPDH). Induction of IMPDH activity has been observed in whole blood and erythrocyte samples during immunosuppressive therapy. Information concerning the mechanisms for increased IMPDH activity is limited and the potential implications of induction have been debated. Methods. Whole blood, CD4ϩ cell, and reticulocyte samples were collected from 30 renal transplant patients pre-and posttransplantation. The expressions of two IMPDH isoforms, type 1 and 2, were analyzed by real-time reversetranscription polymerase chain reaction and quantified using a housekeeping gene index. The IMPDH activity was determined by ultraviolet high-performance liquid chromatography. Results. Transplantation and the initiation of immunosuppressive therapy was associated with increased IMPDH1 (50-88%, PϽ0.0005) and decreased IMPDH2 (42-56%, PϽ0.0005) expression. In CD4ϩ cells, however, IMPDH2 increased (15%, Pϭ0.009). These changes are probably related to glucocorticoid effects. Two weeks posttransplant, MPA-treated patients displayed elevated IMPDH 1 and 2 in reticulocytes, suggesting enzyme induction in these cells during prolonged MPA therapy. Patients with acute rejection during follow-up demonstrated higher IMPDH2 expression in CD4ϩ cells pretransplant than nonrejecting patients (median expression 1.26 vs. 0.87 respectively, Pϭ0.017). Conclusions. Knowledge of changes in IMPDH 1 and 2 expression after transplantation and initiation of immunosuppression is important considering the action of MPA on IMPDH and the potential for pharmacodynamic monitoring of MPA by measuring IMPDH activity. The expression of IMPDH2 in CD4ϩ cells pretransplant may be an indicator of immune activation.
Current Medicinal Chemistry, 2004
IMP dehydrogenase, the key enzyme in de novo synthesis of purine nucleotides, is an important therapeutic target. Three inhibitors of IMP dehydrogenase reached the market; ribavirin (Rebetol) a broadspectrum antiviral agent, which in combination with interferon-α is now used for treatment of hepatitis C virus infections, mizoribine (Bredinin) and mycophenolic mofetil (CellCept) have been introduced as immunosuppressants. Numerous novel inhibitors are under development. This review describes recent progress in the development of new drugs based on inhibition of IMP dehydrogenase.
Transplantation Proceedings, 2004
Mycophenolate mofetil (MMF) is an immunosuppressant used for the prophylaxis of rejection in renal, pancreas, and liver transplantation. It inhibits the inducible isoform of the enzyme inosine-monophosphate dehydrogenase (IMPDH II) via its active metabolite mycophenolic acid (MPA). IMPDH II is necessary for de novo purine synthesis in activated lymphocytes. The aims of the present study were to evaluate the feasibility of a real-time polymerase chain reaction (PCR) quantitative assessment of IMPDH II gene expression in liver transplant recipients as well as to provide a preliminary evaluation of possible correlations with drug tolerability. RNA was extracted from peripheral blood mononuclear cells of liver recipients after at least 6 months of MMF administration. IMPDH II gene expression was assessed using quantitative, real-time PCR and normalized using glyceraldheyde-3-phosphate dehydrogenase (GAPDH). Finally, adverse events associated with MMF administration were recorded. Real-time PCR quantitation of IMPDH II gene expression was reliable, sensitive, and specific. The intrapatient variability for both IMPDH II and GAPDH assays was lower than 0.6% in all patients. The results demonstrated a wide interpatient variability, with the mean value Ϯ standard deviation of 0.949 Ϯ 0.525 (95% confidence interval, 0.669 -1.229) and a median value of 0.797. Patients with treatment-related toxicities displayed a trend to a higher level of IMPDH II expression than those without toxicity (mean, 1.126 vs 0.771). In conclusion, pharmacogenetic analysis of IMPDH II may represent a novel approach to MMF therapeutic monitoring.
Human IMP dehydrogenase:: Kinetics and regulatory properties
Biochimica et Biophysica Acta (BBA) …, 1974
Human IMP dehydrogenase has been partially purified from placenta and characterized. The enzyme activity is located in the cytosol. The Michaelis constants for IMP and NAD are 14 and 46/~M, respectively. In addition, the enzyme requires a monovalent cation for maximal activity and the apparent Km for K + is 17 mM. The purine ribonucleotides XMP, GMP, and AMP inhibit the enzyme in a manner which is competitive with respect to IMP. The Ki values are 30/~M, 100 #M, and 530 #M, respectively. The enzyme is also sensitive to inhibition to a lesser degree by pyrimidine ribonucleotides. The pharmacologic agents, 6-mercaptopurine ribonucleotide, allopurinol ribonucleotide and mycophenolic acid are also inhibitors of human IMP dehydrogenase. A kinetic analysis indicates an ordered sequential reaction mechanism in which IMP binds first and XMP is released last.
Two distinct cDNAs for human IMP dehydrogenase
The Journal of biological chemistry, 1990
IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, is a promising target in antileukemic chemotherapy. We have isolated two distinct cDNA clones (types I and II) encoding IMP dehydrogenase from a human spleen cDNA library. Both clones encode closely related proteins of 514 residues showing 84% sequence identity. Northern hybridization analyses of poly(A)+ RNA from human normal leukocytes and human ovarian tumors demonstrated a striking contrast in mRNA expression in that type I mRNA is the main species in normal leukocytes and type II predominates over type I in the tumor. This is the first report suggesting the existence of two distinct types of human IMP dehydrogenase molecular species which may have different sensitivities to the drugs targeted against IMP dehydrogenase.