Structure and Mechanism of Inosine Monophosphate Dehydrogenase in Complex with the Immunosuppressant Mycophenolic Acid

Sintchak, Michael D.; Fleming, Mark; Futer, Olga; Raybuck, Scott A.; Chambers, Stephen P.; Caron, Paul L.; Murcko, Mark A.; Wilson, Keith

doi:10.1016/s0092-8674(00)81275-1

Cited by 402 publications

(459 citation statements)

References 43 publications

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“…A Pfam functional domain search identified similarities of both the ASAP1 BAR and FIP3 coiled-coil domains with IMP dehydrogenase (IMPDH)/GMP reductase domain. IMPDH forms a homotetramer through its core domain (Sintchak et al, 1996). Because both ASAP1 and FIP3 form homodimers (Eathiraj et al, 2006;Nie et al, 2006;Shiba et al, 2006), they might function as heterotetramer.…”

Section: Discussionmentioning

confidence: 99%

Arf GTPase-activating Protein ASAP1 Interacts with Rab11 Effector FIP3 and Regulates Pericentrosomal Localization of Transferrin Receptor–positive Recycling Endosome

Inoue

Prekeris

et al. 2008

MBoC

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ADP-ribosylation factors (Arfs) and Arf GTPase-activating proteins (GAPs) are key regulators of membrane trafficking and the actin cytoskeleton. The Arf GAP ASAP1 contains an N-terminal BAR domain, which can induce membrane tubulation. Here, we report that the BAR domain of ASAP1 can also function as a protein binding site. Two-hybrid screening identified FIP3, which is a putative Arf6-and Rab11-effector, as a candidate ASAP1 BAR domain-binding protein. Both coimmunoprecipitation and in vitro pulldown assays confirmed that ASAP1 directly binds to FIP3 through its BAR domain. ASAP1 formed a ternary complex with Rab11 through FIP3. FIP3 binding to the BAR domain stimulated ASAP1 GAP activity against Arf1, but not Arf6. ASAP1 colocalized with FIP3 in the pericentrosomal endocytic recycling compartment. Depletion of ASAP1 or FIP3 by small interfering RNA changed the localization of transferrin receptor, which is a marker of the recycling endosome, in HeLa cells. The depletion also altered the trafficking of endocytosed transferrin. These results support the conclusion that ASAP1, like FIP3, functions as a component of the endocytic recycling compartment. INTRODUCTIONArf family GTP-binding proteins and their GTPase-activating proteins (Arf GAPs) play crucial roles for membrane traffic and actin remodeling (D'Souza-Schorey and Chavrier, 2006;Gillingham and Munro, 2007). Arf GAPs share the same catalytic domain, the Arf GAP domain, which is responsible for GTP hydrolysis. In humans, 31 genes encoding proteins with Arf GAP domains have been found. They are classified into several subfamilies based on their domain structures Inoue and Randazzo, 2007). Three subfamilies, ArfGAPs, SMAPs, and GITs, have an Arf GAP domain at the N-terminus of the molecules. In contrast, the others, which are called AZAP-family Arf GAPs, contain an Arf GAP domain following a pleckstrin homology (PH) domain in the middle of the protein. They are subdivided into four subfamilies, ASAPs, ACAPs, ARAPs, and AGAPs.ASAP1 is one of the best-characterized Arf GAPs. In addition to PH and Arf GAP domains, ASAP1 contains an N-terminal Bin, Amphiphysin, and Rvs167/Rvs161 (BAR) domain and, in the C-terminal half of the protein, a proline (Pro)-rich domain and Src homology 3 (SH3) domain. Other ASAP isoforms, ASAP2 and ASAP3, have similar domain structures and high homology, especially in the Arf GAP domains, although ASAP3 lacks C-terminal SH3 domain (Ha et al., 2008). Through its Pro-rich and SH3 domains, ASAP1 interacts with a number of proteins, which are mainly adhesion-and actin cytoskeleton-related proteins including Src, focal adhesion kinase (FAK), Crk/CrkL, and cortactin (Brown et al., 1998;Liu et al., 2002;Oda et al., 2003;Onodera et al., 2005;Bharti et al., 2007). ASAP1 is a component of three integrated membrane/actin cytoskeleton structures: focal adhesions, circular dorsal ruffles (CDRs) and invadopodia/podosomes. ASAP1 associates with focal adhesions in fibroblasts . The focal adhesion localization depends on the interaction of ASAP1 with FA...

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Section: Discussionmentioning

confidence: 99%

Arf GTPase-activating Protein ASAP1 Interacts with Rab11 Effector FIP3 and Regulates Pericentrosomal Localization of Transferrin Receptor–positive Recycling Endosome

Inoue

Prekeris

et al. 2008

MBoC

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“…The molecular mechanisms underlying the antiproliferative e ect of MM on lymphocytes have been partially characterized . MPA speci®cally inhibits inosine monophosphate dehydrogenase (Sintchak et al, 1996), thus inhibiting de novo GTP formation and depleting cells of guanosine and deoxyguanosine nucleotides required for lymphocyte proliferation. The GTP depleting e ect is less pronounced in other cell types, such as EC, that have a salvage pathway for purine synthesis .…”

Section: Discussionmentioning

confidence: 99%

“…MPA is a potent uncompetitive and reversible inhibitor of inosine-5'-monophosphate dehydrogenase (IMPDH) (Sintchak et al, 1996), and thus interferes with the biosynthesis of guanosine and deoxyguanosine nucleotides. Since proliferating B and T lymphocytes are more dependent on this de novo pathway for purine biosynthesis rather than on the salvage pathway, MPA more e ectively inhibits lymphocyte proliferation than proliferation of other cell types (Allison et al, 1977).…”

Section: Introductionmentioning

confidence: 99%

Effect of mycophenolic acid on TNFα‐induced expression of cell adhesion molecules in human venous endothelial cells in vitro

Hauser

Johnson

Thévenod

et al. 1997

British J Pharmacology

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Mycophenolic acid (MPA) is an inhibitor of inosine‐5′‐monophosphate dehydrogenase and therefore interferes with cellular GTP biosynthesis. Recently, MPA has been used as an antiproliferative and immunosuppressive agent. In the present study, the effect of MPA on the expression of the endothelial cell adhesion molecules (CAMs), intercellular (I) CAM‐1, vascular (V) CAM‐1 and endothelial (E)‐selectin, was investigated in tumour necrosis factor‐α (TNFα)‐activated cultured human venous endothelial cells (EC). Surface expression of CAMs was measured by flow cytometry and mRNA expression by Northern blot analysis. Transcriptional activation of CAMs by the nuclear factor NF‐κB was determined by an electromobility shift assay. The function of CAMs was studied by a static adhesion assay with human monocyte‐like undifferentiated U937 cells. Pretreatment of TNFα‐ (5 ng ml−1, 12 h) activated EC with MPA (10 μM, 24 h) increased the binding of U937 cells, which had not been treated with MPA, by ∼amp;2 fold. MPA‐pretreatment of EC did not affect TNFα‐induced surface expression of ICAM‐1. However, VCAM‐1 and E‐selectin were increased 2–3 fold and remained elevated up to 24 h, by which time TNFα‐activated control EC had returned to baseline levels of expression. The effect of MPA on the surface expression of CAMs was half‐maximal at ∼amp;1 μM and required 12 h of pretreatment. Guanosine (0.3 mM), a precursor of GTP, did not prevent the effect of MPA on the expression of CAMs in TNFα‐activated EC. Kinetics of mRNA expression of CAMs mirrored protein expression: mRNA for ICAM‐1 was unaffected, whereas TNFα‐induced mRNA expression for E‐selectin and VCAM‐1 was prolonged and increased by MPA. This effect was not due to increased transcription mediated by the nuclear transcription factor NF‐κB. However, half‐life for E‐selectin mRNA was increased 10 fold by MPA, whereas ICAM‐1 mRNA half‐life was unchanged. The data demonstrate that apart from its antiproliferative effects on lymphocytes, MPA enhances TNFα‐induced VCAM‐1 and E‐selectin surface expression on EC by selectively increasing the mRNA‐stability of these cell adhesion molecules. This effect of MPA on EC appears to be independent from inhibition of inosine‐5′‐monophosphate dehydrogenase.

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“…Potent MPA-inhibition of the ratelimiting enzyme, inosine monophosphate dehydrogenase (IMPDH) [7], is highly selective toward type II IMPDH isozyme over type I because of its specific role in de-novo synthesis of guanosine monophosphate in activated mononuclear cell proliferation. Selective inhibition of type II IMPDH by MPA prevents proliferation of activated T-and B-lymphocytes and allows the agent to exert a potent immunosuppressant effect, as well as it induces apoptosis of activated T-lymphocytes [8].…”

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confidence: 99%

Targeted inhibition of glucuronidation markedly improves drug efficacy in mice—A model

Basu

Kole

Basu

et al. 2007

Biochemical and Biophysical Research Communications

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Finding UDP-glucuronosyltransferases (UGT) require protein kinase C-mediated phosphorylation is important information that allows manipulation of this critical system. UGTs glucuronidate numerous aromatic-like chemicals derived from metabolites, diet, environment and, inadvertently, therapeutics to reduce toxicities. As UGTs are inactivated by downregulating PKCs with reversiblyacting dietary curcumin, we determined the impact of gastro-intestinal glucuronidation on free-drug uptake and efficacy using immunosuppressant, mycophenolic acid (MPA), in mice. Expressed in COS-1 cells, mouse GI-distributed Ugt1a1 glucuronidates curcumin and MPA and undergoes irreversibly and reversibly dephosphorylation by PKC-specific inhibitor calphostin-C and generalkinase inhibitor curcumin, respectively, with parallel effects on activity. Moreover, oral curcuminadministration to mice reversibly inhibited glucuronidation in GI-tissues. Finally, successive oraladministration of curcumin and MPA to antigen-treated mice increased serum free-MPA and immunosuppression up to 9-fold. Results indicate targeted inhibition of GI-glucuronidation in mice markedly improved free-chemical uptake and efficacy using MPA as a model.Whereas the primary role of UDP-glucurononsyltransferases (UGT) is to detoxify numerous structurally diverse lipophilic chemicals, including metabolites, dietary constituents, environmental toxicants, carcinogens and, inadvertently, therapeutic chemicals, there is a limited capacity to manipulate glucuronidation to improve protection or reduce premature ‡ Corresponding authors at: National Institutes of Health, Building 10, Room 8D-42, Bethesda, MD 20892-1830, E-mail addresses, telephone and fax numbers: owensi@mail.nih.gov; 301-496-6091, and 301-480-8042; basun@mail.nih.gov, 301-496-8825, 301-451-4288. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Whereas MPA readily forms ether-linked-(MPAG) or acyl-glucuronides (AcMPAG) [9-11], we studied MPA glucuronidation in vitro with human UGTs and found 4 isozymes are avid metabolizers [12] that reach saturation kinetics between 1.6 and 2.4 mM with Km values between 0.25 and 0.55 mM. These UGT isozymes, encoded at the UGT1 complex locus, were found strategically and differentially expressed in the gastrointestinal (GI) mucosa [1]. In this report, we established under both in-vitro and in-vivo conditions that mouse Ugt1a1 also requires phosphorylation, which can be transiently downregulated by nontoxic kinase inhibitor, curcumin [2,3,13], and irreversibly by calphostin-C, similar to that for human UGTs [2,3]. Moreover, effects of...

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Structure and Mechanism of Inosine Monophosphate Dehydrogenase in Complex with the Immunosuppressant Mycophenolic Acid

Cited by 402 publications

References 43 publications

Arf GTPase-activating Protein ASAP1 Interacts with Rab11 Effector FIP3 and Regulates Pericentrosomal Localization of Transferrin Receptor–positive Recycling Endosome

Arf GTPase-activating Protein ASAP1 Interacts with Rab11 Effector FIP3 and Regulates Pericentrosomal Localization of Transferrin Receptor–positive Recycling Endosome

Effect of mycophenolic acid on TNFα‐induced expression of cell adhesion molecules in human venous endothelial cells in vitro

Targeted inhibition of glucuronidation markedly improves drug efficacy in mice—A model

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