FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2019950056> ?p ?o ?g. }

• W2019950056 endingPage "37706" @default.
• W2019950056 startingPage "37698" @default.
• W2019950056 abstract "Transcriptional signaling from the Ca2+-calmodulin-activated phosphatase calcineurin to its substrate NFAT (nuclear factor of activated T cells, also termed NFATc) is critically dependent on a protein-protein docking interaction between calcineurin and the PXIXIT motif in NFAT. Several inhibitors of NFAT-calcineurin association (INCA compounds) prevent binding of NFAT or the peptide ligand PVIVIT to calcineurin. Here we show that the binding site on calcineurin for INCA1, INCA2, and INCA6 is centered on cysteine 266 of calcineurin Aα and does not coincide with the core PXIXIT-binding site. Although ample evidence indicates that INCA1 and INCA2 react covalently with cysteine 266, covalent derivatization alone is not sufficient for maximal inhibition of the calcineurin-PVIVIT interaction, because the maleimide INCA12 reacts with the same site and produces only very modest inhibition. Thus, inhibition arises through an allosteric change affecting the PXIXIT docking site, which may be assisted by covalent binding but depends on other specific features of the ligand. The spatial arrangement of the binding sites for PVIVIT and INCA makes it probable that the change in conformation involves the β11-β12 loop of calcineurin. The finding that an allosteric site controls NFAT binding opens new alternatives for inhibition of calcineurin-NFAT signaling. Transcriptional signaling from the Ca2+-calmodulin-activated phosphatase calcineurin to its substrate NFAT (nuclear factor of activated T cells, also termed NFATc) is critically dependent on a protein-protein docking interaction between calcineurin and the PXIXIT motif in NFAT. Several inhibitors of NFAT-calcineurin association (INCA compounds) prevent binding of NFAT or the peptide ligand PVIVIT to calcineurin. Here we show that the binding site on calcineurin for INCA1, INCA2, and INCA6 is centered on cysteine 266 of calcineurin Aα and does not coincide with the core PXIXIT-binding site. Although ample evidence indicates that INCA1 and INCA2 react covalently with cysteine 266, covalent derivatization alone is not sufficient for maximal inhibition of the calcineurin-PVIVIT interaction, because the maleimide INCA12 reacts with the same site and produces only very modest inhibition. Thus, inhibition arises through an allosteric change affecting the PXIXIT docking site, which may be assisted by covalent binding but depends on other specific features of the ligand. The spatial arrangement of the binding sites for PVIVIT and INCA makes it probable that the change in conformation involves the β11-β12 loop of calcineurin. The finding that an allosteric site controls NFAT binding opens new alternatives for inhibition of calcineurin-NFAT signaling. Ca2+-calcineurin signaling serves vital purposes in mammalian cells, among them the provision of a direct link between the mobilization of cytoplasmic Ca2+ and gene expression (1Aramburu J. Rao A. Klee C.B. Curr. Top. Cell. Regul. 2000; 36: 237-295Crossref PubMed Scopus (276) Google Scholar, 2Rusnak F. Mertz P. Physiol. Rev. 2000; 80: 1483-1521Crossref PubMed Scopus (1109) Google Scholar). In the physiological activation of T cells, calcineurin acts through NFAT 4The abbreviations used are: NFATnuclear factor of activated T cellsCsAcyclosporin ADTTdithiothreitolIAMiodoacetamideINCAinhibitor of NFAT-calcineurin associationMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightNEMN-ethylmaleimidePVIVIT14-mer peptide MAGPHPVIVITGPHEE-amide family transcription factors and other transcriptional effectors (3Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2223) Google Scholar, 4Serfling E. Berberich-Siebelt F. Chuvpilo S. Jankevics E. Klein-Hessling S. Twardzik T. Avots A. Biochim. Biophys. Acta. 2000; 1498: 1-18Crossref PubMed Scopus (170) Google Scholar, 5Crabtree G.R. Olson E.N. Cell. 2002; 109: S67-S79Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar, 6Hogan P.G. Chen L. Nardone J. Rao A. Genes Dev. 2003; 17: 2205-2232Crossref PubMed Scopus (1557) Google Scholar). Levels of calcineurin in T cells are limiting, and upward or downward modulation of calcineurin enzymatic activity is directly reflected in increased or decreased transcription from cytokine gene promoters (7Fruman D.A. Klee C.B. Bierer B.E. Burakoff S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3686-3690Crossref PubMed Scopus (755) Google Scholar, 8O'Keefe S.J. Tamura J. Kincaid R.L. Tocci M.J. O'Neill E.A. Nature. 1992; 357: 692-694Crossref PubMed Scopus (788) Google Scholar, 9Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1476) Google Scholar, 10Tsuboi A. Masuda E.S. Naito Y. Tokumitsu H. Arai K. Arai N. Mol. Biol. Cell. 1994; 5: 119-128Crossref PubMed Scopus (34) Google Scholar, 11Fruman D.A. Pai S.Y. Burakoff S.J. Bierer B.E. Mol. Cell. Biol. 1995; 15: 3857-3863Crossref PubMed Google Scholar, 12Batiuk T.D. Pazderka F. Enns J. DeCastro L. Halloran P.F. J. Clin. Investig. 1995; 96: 1254-1260Crossref PubMed Scopus (88) Google Scholar, 13Batiuk T.D. Kung L. Halloran P.F. J. Clin. Investig. 1997; 100: 1894-1901Crossref PubMed Scopus (77) Google Scholar, 14Bueno O.F. Brandt E.B. Rothenberg M.E. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9398-9403Crossref PubMed Scopus (158) Google Scholar). The immunosuppressive drugs cyclosporin A (CsA) and FK506 owe their clinical effectiveness to the ability of CsA-cyclophilin or FK506-FKBP12 complexes to inhibit calcineurin in cells of the immune system (15Liu J. Farmer Jr., J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3620) Google Scholar, 16Liu J. Albers M.W. Wandless T.J. Luan S. Alberg D.G. Belshaw P.J. Cohen P. MacKintosh C. Klee C.B. Schreiber S.L. Biochemistry. 1992; 31: 3896-3901Crossref PubMed Scopus (502) Google Scholar). However, the beneficial actions of CsA and FK506 are counterbalanced by serious toxicities attributed at least in part to their interference with calcineurin signaling in other cells and tissues (17Sigal N.H. Dumont F. Durette P. Siekierka J.J. Peterson L. Rich D.H. Dunlap B.E. Staruch M.J. Melino M.R. Koprak S.L. Williams D. Witzel B. Pisano J.M. J. Exp. Med. 1991; 173: 619-628Crossref PubMed Scopus (230) Google Scholar, 18Dumont F.J. Staruch M.J. Koprak S.L. Siekierka J.J. Lin C.S. Harrison R. Sewell T. Kindt V.M. Beattie T.R. Wyvratt M. Sigal N.H. J. Exp. Med. 1992; 176: 751-760Crossref PubMed Scopus (232) Google Scholar). nuclear factor of activated T cells cyclosporin A dithiothreitol iodoacetamide inhibitor of NFAT-calcineurin association matrix-assisted laser desorption ionization time-of-flight N-ethylmaleimide 14-mer peptide MAGPHPVIVITGPHEE-amide The centrality of calcineurin and the calcineurin-NFAT pathway to immune responses has suggested that interrupting signaling at any of several points could provide a therapeutic alternative to current immunosuppressive drugs. Promising target points are the Ca2+ signal that activates calcineurin (19Serafini A.T. Lewis R.S. Clipstone N.A. Bram R.J. Fanger C. Fiering S. Herzenberg L.A. Crabtree G.R. Immunity. 1995; 3: 239-250Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 20Venkatesh N. Feng Y. DeDecker B. Yacono P. Golan D. Mitchison T. McKeon F. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8969-8974Crossref PubMed Scopus (45) Google Scholar), the protein-protein interaction of calcineurin and NFAT (21Loh C. Shaw K.T.-Y. Carew J. Viola J.P.B. Luo C. Perrino B.A. Rao A. J. Biol. Chem. 1996; 271: 10884-10891Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 22Wesselborg S. Fruman D.A. Sagoo J.K. Bierer B.E. Burakoff S.J. J. Biol. Chem. 1996; 271: 1274-1277Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 23Aramburu J. García-Cozár F. Raghavan A. Okamura H. Rao A. Hogan P.G. Mol. Cell. 1998; 1: 627-637Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 24Aramburu J. Yaffe M.B. López-Rodríguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Crossref PubMed Scopus (521) Google Scholar, 25Roehrl M.H.A. Kang S. Aramburu J. Wagner G. Rao A. Hogan P.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7554-7559Crossref PubMed Scopus (139) Google Scholar), and the cooperative binding of NFAT and AP1 on DNA (26Jain J. McCaffrey P.G. Valge-Archer V.E. Rao A. Nature. 1992; 356: 801-804Crossref PubMed Scopus (428) Google Scholar, 27Northrop J.P. Ullman K.S. Crabtree G.R. J. Biol. Chem. 1993; 268: 2917-2923Abstract Full Text PDF PubMed Google Scholar, 28Chen L. Glover J.N. Hogan P.G. Rao A. Harrison S.C. Nature. 1998; 392: 42-48Crossref PubMed Scopus (413) Google Scholar, 29Peterson B.R. Sun L.J. Verdine G.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13671-13676Crossref PubMed Scopus (51) Google Scholar, 30Macián F. García-Rodríguez C. Rao A. EMBO J. 2000; 19: 4783-4795Crossref PubMed Scopus (263) Google Scholar). Each strategy has the potential to be more selective and, hence, less toxic than treatment with CsA or FK506, but none has yet advanced to the stage of small nonpeptide inhibitors that can be tested in vivo. There is considerable evidence that targeting the calcineurin-NFAT protein-protein interaction will produce a selective inhibition. We have demonstrated that the calcineurin-NFAT interaction is based on recognition of a PXIXIT motif in NFAT and that this recognition is essential for efficient signaling (23Aramburu J. García-Cozár F. Raghavan A. Okamura H. Rao A. Hogan P.G. Mol. Cell. 1998; 1: 627-637Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Peptides that compete for binding at the PXIXIT recognition site both inhibit the calcineurin-NFAT interaction in vitro and selectively inhibit calcineurin-NFAT signaling in cells (23Aramburu J. García-Cozár F. Raghavan A. Okamura H. Rao A. Hogan P.G. Mol. Cell. 1998; 1: 627-637Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 24Aramburu J. Yaffe M.B. López-Rodríguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Crossref PubMed Scopus (521) Google Scholar, 31Liu J. Arai K. Arai N. J. Immunol. 2001; 167: 2677-2687Crossref PubMed Scopus (35) Google Scholar). A recently published study (32Noguchi H. Matsushita M. Okitsu T. Moriwaki A. Tomizawa K. Kang S. Li S.-T. Kobayashi N. Matsumoto S. Tanaka K. Tanaka N. Matsui H. Nat. Med. 2004; 10: 305-309Crossref PubMed Scopus (234) Google Scholar) shows that a competitor peptide modified to promote its uptake into cells can prevent heterologous graft rejection in mice. Further, high throughput screening of a library of organic compounds has led to identification of nonpeptide inhibitors of NFAT-calcineurin association (INCA compounds) (25Roehrl M.H.A. Kang S. Aramburu J. Wagner G. Rao A. Hogan P.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7554-7559Crossref PubMed Scopus (139) Google Scholar). These compounds interfere with calcineurin-NFAT signaling in cells, motivating a continued search for inhibitors that have higher affinity and reduced nonspecific toxicity and that are suitable for in vivo administration to animals. The further development of inhibitors can be guided by structural information about the sites of protein-protein interaction and inhibitor binding. To this end, we have determined the structure of the NFAT docking site on calcineurin, in which the PXIXIT recognition peptide of NFAT binds in an extended configuration and each conserved residue of the peptide directly contacts calcineurin (33Li H. Rao A. Hogan P.G. J. Mol. Biol. 2004; 342: 1659-1674Crossref PubMed Scopus (66) Google Scholar). Here we extend the structural studies by identifying a distinct binding site for INCA compounds at a cysteine residue adjacent to the PXIXIT peptide docking site. It is covalent binding or tight noncovalent binding of the bulky INCA compounds at this second site that allosterically inhibits recognition of PXIXIT peptide and NFAT. Our findings serve to identify the approaches that are most likely to be fruitful in developing improved inhibitors. In addition, the findings suggest that calcineurin-substrate recognition, like calcineurin catalytic activity (34Wang X. Culotta V.C. Klee C.B. Nature. 1996; 383: 434-437Crossref PubMed Scopus (241) Google Scholar, 35Rusnak F. Reiter T. Trends Biochem. Sci. 2000; 25: 527-529Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 36Namgaladze D. Hofer H.W. Ullrich V. J. Biol. Chem. 2002; 277: 5962-5969Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 37Sommer D. Coleman S. Swanson S.A. Stemmer P.M. Arch. Biochem. Biophys. 2002; 404: 271-278Crossref PubMed Scopus (101) Google Scholar), may be modulated physiologically by redox reactions. cDNA Constructs, Protein Expression, and Synthetic Peptides—The expression construct for GST-calcineurin-(2-347), encoding glutathione S-transferase and the catalytic domain of human calcineurin Aα with the substitutions Y341S, L343A, and M347D, has been described (24Aramburu J. Yaffe M.B. López-Rodríguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Crossref PubMed Scopus (521) Google Scholar). Constructs encoding mutant proteins with individual Cys → Ala or Cys → Val replacements or with the combined Cys → Ala/Cys-266 (all surface Cys residues changed to Ala except for Cys-266) and Cys → Ala/C266V (all surface Cys residues changed to Ala except at position 266, where Cys is changed to Val) replacements were made by PCR mutagenesis with appropriate primers and subcloning. In each case, the sequence of the cDNA insert was verified. GST-calcineurin fusion proteins were expressed in Escherichia coli strain BL21-CodonPlus-RP (Stratagene) by overnight growth at 18 °C after the addition of 1 mm isopropyl-β-d-1-thiogalactopyranoside. Calcineurin was purified from the bacterial lysate by affinity chromatography on glutathione-Sepharose (Amersham Biosciences) and cleavage in 50 mm Tris ·HCl, pH 7.0, 150 mm NaCl, 1 mm EDTA, and 1 mm DTT with PreScission protease (Amersham Biosciences). Purified calcineurin-(2-347) was concentrated by Centricon-30 filtration, aliquoted, flash-frozen, and stored at -80 °C. In most cases, the concentration of DTT in the buffer was reduced to 0.1 mm during concentration of the protein. This step and the further substantial dilution into fluorescence polarization assays minimized any interference of residual DTT with the INCA compounds. In some cases, protein was concentrated in buffer free of DTT immediately before freezing. The PVIVIT 14-mer peptide (24Aramburu J. Yaffe M.B. López-Rodríguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Crossref PubMed Scopus (521) Google Scholar) was labeled overnight at room temperature in a reaction containing 2 mg of peptide, 1.5 mg of Oregon Green (Oregon Green 488 carboxylic acid, succinimidyl ester (5-isomer); Invitrogen), and 5 μl of diisopropylethylamine in 190 μl of anhydrous N,N-dimethylformamide. The labeled peptide was purified by C18 reversed-phase high performance liquid chromatography (HPLC). The calcineurin-(254-273)/C256S peptide, RGSSYFYSYPAVCEFLQHNN, was synthesized and HPLC-purified at Tufts University Core Facility and stored desiccated at -20 °C. Inhibitors—INCA1, INCA2, INCA6, and INCA12 (Fig. 1) were obtained from ChemBridge. Inhibitor stocks were prepared at 10 mm in anhydrous dimethyl sulfoxide (Me2SO) and stored desiccated at -20 °C. Corresponding aliquots of anhydrous Me2SO were stored desiccated at -20 °C for addition to control incubations. Me2SO at concentrations up to 1% had no effect on the parameters studied. Fluorescence Measurements—Fluorescence measurements were made on 10-μl samples in a black 384-well plate (Molecular Devices) using the fluorescein filter set (excitation at 485 nm, emission at 530 nm) in an Analyst plate reader (Molecular Devices). Each well contained 100 mm NaCl, 2 mm magnesium acetate, 20 mm HEPES, pH 7.4, 0.1% (w/v) bovine IgG, calcineurin, 100 nm fluorescent PVIVIT, and other additions as specified. Calcineurin was omitted for measurements of the fluorescence emitted by unbound peptide. In competitive binding assays, calcineurin was typically present at 1 or 1.5 μm; in direct binding titrations, calcineurin was used at 0.15-10 μm. Except in time course experiments, adequate time was allowed for the signal to reach a stable value as verified by repeated readings of the same samples. Pretreatment with iodoacetamide (IAM), N-ethylmaleimide (NEM), or INCA12 was for 30 min to 2 h. Data from direct titrations with calcineurin have been fitted to a model for equilibrium binding to a single class of sites (38Nardone J. Hogan P.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4417-4421Crossref PubMed Scopus (59) Google Scholar). Competition and time course data for INCA compounds have been plotted without attempting to fit a theoretical curve, because the data are not sufficient to specify parameters for covalent binding, noncovalent binding, and side reactions of the INCA compounds. The stability of calcineurin-INCA2 association was examined after equilibration of 6 μm calcineurin, 100 nm labeled PVIVIT, and 20 μm INCA2 to produce an essentially complete blockade of PVIVIT binding. To assess short-term stability of the complex, samples were extracted by vortex mixing with 2 volumes of buffer-saturated ether for 2 min and briefly centrifuged to separate the phases. Extraction of samples containing only calcineurin and labeled PVIVIT had no effect on peptide binding and did not prevent subsequent displacement of peptide by INCA2. Extraction of samples containing only the INCA compound prior to incubation with calcineurin and PVIVIT demonstrated that removal of unbound INCA2 from the aqueous phase was complete within 10 s. To assess long-term stability of the complex, samples containing PVIVIT and blocked calcineurin were supplemented with 5 mm NEM or 5 mm DTT, and PVIVIT binding was monitored during the following 4 h. Because the calcineurin concentration in fluorescence polarization assays is greater than the Kd of the calcineurin-PVIVIT interaction, each of these procedures is a sensitive test for the unmasking of blocked PVIVIT-binding sites. Synthetic Peptide-INCA Reactions—Freshly dissolved RGSSYFYSYPAVCEFLQHNN peptide, 6 μm in 200 mm NaCl, 4 mm Mg acetate, and 20 mm HEPES, pH 7.35, in 20 μl total volume, was incubated for 60 min at room temperature with 50 μm INCA1, INCA2, or INCA6. At the end of this incubation, samples were either immediately diluted into 240 μl 0.1% (v/v) trifluoroacetic acid and frozen or further incubated for 60 min after the addition of NEM to 1 mm then diluted into 0.1% (v/v) trifluoroacetic acid and frozen. Control samples were reacted first with 1 mm NEM for 60 min and then with 50 μm INCA compound for 60 min. Mass Spectrometry—For MALDI-TOF mass spectrometry, samples were desalted and concentrated by binding the peptides to C18 resin in a ZipTip (Millipore) and eluting with 70% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid. Eluted peptides were diluted 1:10 with α-cyano-4-hydroxycinnamic acid matrix (10 mg/ml in 50% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid) and dried onto the target plate. Mass spectra were obtained using an Applied Biosystems Voyager-DE STR instrument operated in reflector mode with accelerating voltage of 20 kV, grid voltage at 72%, guide wire at 0.1%, and extraction delay time of 175 ns. Calibration was performed using the MSCAL2 mass standard set (Sigma). A minor peak frequently observed in the peptide sample having a mass of ∼1776 Da is apparently the peptide fragment RGSSYFYSYPAVCEF. Corresponding peaks in samples that had been incubated with INCA1, INCA2, or NEM or in samples subjected to prolonged incubation with INCA6 to allow the reaction to approach completion were shifted by the same mass as the main peak in those incubations. A peak of mass ∼1397 Da, matching the calculated mass of the fragment RGSSYFYSYPAV, was present occasionally both in untreated peptide samples and in samples that had been incubated with NEM or INCA compounds. The reaction or lack of reaction observed with these peptide fragments reinforces the conclusion that all of the compounds investigated reacted with the cysteine -SH group. Cellular Assays—Dephosphorylation of NFAT, nuclear import of NFAT, and induction of cytokine mRNAs in D5 T cells were assessed as described in previous publications (23Aramburu J. García-Cozár F. Raghavan A. Okamura H. Rao A. Hogan P.G. Mol. Cell. 1998; 1: 627-637Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 24Aramburu J. Yaffe M.B. López-Rodríguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Crossref PubMed Scopus (521) Google Scholar, 25Roehrl M.H.A. Kang S. Aramburu J. Wagner G. Rao A. Hogan P.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7554-7559Crossref PubMed Scopus (139) Google Scholar). Calcineurin activity in cell lysates was measured using a standard assay (39Fruman D.A. Pai S.Y. Klee C.B. Burakoff S.J. Bierer B.E. Methods. 1996; 9: 146-154Crossref PubMed Scopus (64) Google Scholar) with phosphorylated RII peptide as substrate. D5 T cells were chilled on ice, collected by centrifugation at 4 °C, and resuspended in 50 mm Tris ·HCl, pH 7.5, 1 mm EDTA, 0.1 mm EGTA, 0.2% (v/v) Nonidet P-40, 50 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and, where specified, 30 mm sodium pyrophosphate. After 5 min on ice, lysates were centrifuged, and supernatants were transferred to fresh tubes containing an equal volume of 50 mm Tris ·HCl, pH 7.5, 100 mm NaCl, 2.5 mm CaCl2, 100 μg/ml bovine serum albumin, and 1.5 μm okadaic acid. Blank samples were prepared with resuspension buffer in place of cell lysate. The reaction was initiated by the addition of 32P-labeled RII peptide, each tube was incubated for 15 min at 30 °C, and the reaction was terminated by the addition of 100 mm potassium phosphate buffer containing 5% (w/v) trichloroacetic acid. Each sample was applied to a Dowex AG 50W-X8 cation exchange column (Bio-Rad), and the effluent was collected and its content of radiolabel determined by liquid scintillation counting. Structural Modeling—Structural modeling was based on the coordinates of calcineurin Aα from Protein Data Bank entries 1AUI and 1TCO (40Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (772) Google Scholar, 41Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E Moomaw E.W. Gastinel L.N. Habuka N. Chen X Maldonado F. Barker J.E. Bacquet R. Villafranca J.E. Nature. 1995; 378: 641-644Crossref PubMed Scopus (697) Google Scholar). The coordinates of docked PVIVIT peptide were from Li et al. (33Li H. Rao A. Hogan P.G. J. Mol. Biol. 2004; 342: 1659-1674Crossref PubMed Scopus (66) Google Scholar). Fig. 9 was prepared using RasMol. The compounds INCA1, INCA2, INCA6, and INCA12 (Fig. 1) have been shown to inhibit recognition of NFAT by calcineurin as assessed quantitatively by the binding of the fluorescent PVIVIT peptide to calcineurin. Several observations have raised the possibility that a covalent INCA-protein complex is involved in the inhibition of PVIVIT peptide binding. First, the most effective inhibitors were quinones or quinoneimines, compounds that are chemically reactive and that are known to form protein adducts. Second, the kinetics of binding were slow at intermediate concentrations of an INCA compound, as illustrated for INCA1 (Fig. 2A), with a plateau level of inhibition reached only after tens of minutes at room temperature. This behavior is often indicative of the formation of a covalent ligand-protein complex. Finally, INCA1, INCA2, and INCA6 were inactivated as inhibitors in the competitive binding assay by preincubation with DTT (Fig. 2B and not shown). Inactivation could reflect either reduction of the compounds by DTT to a less reactive form or covalent reaction with DTT. Effect of Sulfhydryl-modifying Reagents—Protein sulfhydryl groups are likely sites of covalent modification by quinones. Consistent with this possibility, preincubation of calcineurin with the sulfhydryl-modifying reagent IAM impaired INCA2 competition, whereas preincubation of calcineurin by itself had no effect (Fig. 3A). In more extensive studies, preincubation of calcineurin with the maleimide INCA12 impaired competition by INCA1, INCA2, and INCA6 (Fig. 3B and not shown). Pretreatment of calcineurin with the sulfhydryl-modifying reagents IAM, NEM, and INCA12 reduced the calcineurin-PVIVIT polarization signal even in the absence of an INCA competitor (Fig. 3 and not shown). Very low concentrations of INCA12 were sufficient to reduce the calcineurin-PVIVIT polarization signal, but even high concentrations did not fully eliminate the signal from bound PVIVIT (Fig. 4A). Similarly, for high concentrations of IAM and NEM, the calcineurin-PVIVIT signal settled to a plateau that was well above the signal of free peptide (not shown). The explanation for this behavior, documented below, is that pretreated calcineurin retains the ability to bind PVIVIT with reduced affinity. In principle, the effect of the nonspecific alkylating reagents IAM and NEM could be due either to true impairment of calcineurin-peptide binding or to impaired immobilization of the fluorescent label when the PVIVIT peptide is bound. The second case would be evident in a reduction in the maximal polarization signal obtained by titrating fluorescent PVIVIT with IAM-pretreated or NEM-pretreated calcineurin and a corresponding reduction in the slope of the central portion of the binding curve. In fact, the curves for pretreated calcineurin showed no change in slope but instead were shifted rightward to an extent compatible with a 1.5-2-fold loss in affinity of the calcineurin-peptide interaction (Fig. 4B). Thus IAM and NEM are themselves partial inhibitors of the calcineurin-PVIVIT interaction, and experiments described below indicate that they act at the same site as the INCA compounds. Identification of a Target Cysteine Residue—The inhibitory activity common to IAM, maleimides, and quinones/quinoneimines strongly supported the notion that these compounds derivatize a cysteine sulfhydryl of calcineurin. Pilot experiments examining the effect of several single Cys → Ala substitutions in calcineurin on the inhibitory activity of INCA2 pointed to Cys-266 as the likely reactive residue, and a more detailed study confirmed that the point mutant C266A was insensitive to the inhibitory effect of INCA2 (Fig. 5). Similar experiments demonstrated that this substitution also compromised the effectiveness of INCA1, INCA6, IAM, and maleimides (not shown). Conversely, the other surface-exposed cysteine sulfhydryl groups were not required for the action of INCA compounds. The crystal structure of calcineurin shows six exposed cysteine residues in the catalytic domain, Cys-166, Cys-184, Cys-228, Cys-256, Cys-266, and Cys-336. We compared calcineurin in which all exposed cysteine side chains except Cys-266 were changed to alanine (Cys → Ala/Cys-266) to calcineurin with the same substitutions plus a C266V substitution (Cys → Ala/C266V). Binding of fluorescent PVIVIT to the two proteins was comparable (Fig. 6A). However, whereas replacement of the cysteines other than Cys-266 did not compromise the effectiveness of INCA1, INCA2, or INCA6, the C266V substitution in the context of these other Cys → Ala replacements completely blocked the effects of the inhibitors (Fig. 6B and not shown). The C266V substitution in this context also blocked the effect of NEM (not shown). These observations showed that the presence of Cys-266 is necessary and sufficient for a complete block of PVIVIT binding by INCA compounds. Formation of a Peptide-INCA Adduct—We tested the ability of the three INCA compounds to react with the synthetic calcineurin peptide RGSSYFYSYPAVCEFLQHNN. The peptide is calcineurin-(254-273) with a C256S substitution to eliminate the possibility of a covalent reaction at that position. MALDI-TOF mass spectrometry demonstrated that INCA1 and INCA2, at micromolar concentrations, react covalently with the synthetic calcineurin peptide (Fig. 7, A-C). Peptide-INCA adduct formation was blocked in each case by prior incubation with excess NEM under conditions that derivatized the -SH group (Fig. 7A and not shown). Conversely, NEM failed to react with the peptide-INCA adducts present after a first incubation with INCA1 and INCA2 (Fig. 7, B and C), confirming that the INCA compounds block the sulfhydryl group. Because the peptide is unlikely to have a preferred conformation in solution, the experiments show that INCA1 and INCA2 are sufficiently reactive to modify accessible cysteine sulfhydryl groups without necessarily forming an initial noncovalent complex. INCA6 reacted more slowly (Fig. 7D), with full labeling of the peptide requiring several hours. Reaction was again blocked by pretreatment with excess NEM. The sluggish reaction suggests that covalent reaction with Cys-266 in calcineurin would require higher concentrations of INCA6, a local environment that increases the nucleophilicity of the Cys-266 thiol or a noncovalent interaction that assists in targeting INCA6 to the site. Direct examination of tryptic digests of INCA6-treated calcineurin did not provide evidence of an INCA-protein adduct. Rather, the same tryptic peptide containing Cys-266, identified by comparison with digests of C266V calcineurin, was detected in the digests of untreated and treated calcineurin. However, it has been difficult to demonstrate adducts in tryptic digests of other proteins that are known to be covalently modified by quinones, probably because the sulfu" @default.
• W2019950056 created "2016-06-24" @default.
• W2019950056 creator A5002370058 @default.
• W2019950056 creator A5040839623 @default.
• W2019950056 creator A5080472610 @default.
• W2019950056 creator A5083052216 @default.
• W2019950056 date "2005-11-01" @default.
• W2019950056 modified "2023-10-17" @default.
• W2019950056 title "Inhibition of the Calcineurin-NFAT Interaction by Small Organic Molecules Reflects Binding at an Allosteric Site" @default.
• W2019950056 cites W1484729021 @default.
• W2019950056 cites W1489389999 @default.
• W2019950056 cites W1493538154 @default.
• W2019950056 cites W1545126526 @default.
• W2019950056 cites W1569503463 @default.
• W2019950056 cites W1601157315 @default.
• W2019950056 cites W1847275787 @default.
• W2019950056 cites W1968949754 @default.
• W2019950056 cites W1970842110 @default.
• W2019950056 cites W1975107946 @default.
• W2019950056 cites W1981956565 @default.
• W2019950056 cites W1982323880 @default.
• W2019950056 cites W1983118151 @default.
• W2019950056 cites W1984754431 @default.
• W2019950056 cites W1989229394 @default.
• W2019950056 cites W1990851531 @default.
• W2019950056 cites W1991210089 @default.
• W2019950056 cites W2003460765 @default.
• W2019950056 cites W2003807987 @default.
• W2019950056 cites W2012124354 @default.
• W2019950056 cites W2014093955 @default.
• W2019950056 cites W2014223927 @default.
• W2019950056 cites W2018160971 @default.
• W2019950056 cites W2018365938 @default.
• W2019950056 cites W2020955202 @default.
• W2019950056 cites W2021903164 @default.
• W2019950056 cites W2027640067 @default.
• W2019950056 cites W2028211858 @default.
• W2019950056 cites W2033938885 @default.
• W2019950056 cites W2034891977 @default.
• W2019950056 cites W2041512157 @default.
• W2019950056 cites W2046459625 @default.
• W2019950056 cites W2047198875 @default.
• W2019950056 cites W2047973654 @default.
• W2019950056 cites W2048127934 @default.
• W2019950056 cites W2054730594 @default.
• W2019950056 cites W2062005756 @default.
• W2019950056 cites W2065567658 @default.
• W2019950056 cites W2066661679 @default.
• W2019950056 cites W2068716929 @default.
• W2019950056 cites W2069585297 @default.
• W2019950056 cites W2074593407 @default.
• W2019950056 cites W2077499686 @default.
• W2019950056 cites W2080169144 @default.
• W2019950056 cites W2080334198 @default.
• W2019950056 cites W2081790221 @default.
• W2019950056 cites W2087700080 @default.
• W2019950056 cites W2089988801 @default.
• W2019950056 cites W2091538386 @default.
• W2019950056 cites W2109734459 @default.
• W2019950056 cites W2125977083 @default.
• W2019950056 cites W2137304324 @default.
• W2019950056 cites W2138958920 @default.
• W2019950056 cites W2140707292 @default.
• W2019950056 cites W2141589798 @default.
• W2019950056 cites W2157973267 @default.
• W2019950056 cites W2169523708 @default.
• W2019950056 cites W2172196619 @default.
• W2019950056 doi "https://doi.org/10.1074/jbc.m502247200" @default.
• W2019950056 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16148011" @default.
• W2019950056 hasPublicationYear "2005" @default.
• W2019950056 type Work @default.
• W2019950056 sameAs 2019950056 @default.
• W2019950056 citedByCount "38" @default.
• W2019950056 countsByYear W20199500562012 @default.
• W2019950056 countsByYear W20199500562013 @default.
• W2019950056 countsByYear W20199500562014 @default.
• W2019950056 countsByYear W20199500562015 @default.
• W2019950056 countsByYear W20199500562016 @default.
• W2019950056 countsByYear W20199500562017 @default.
• W2019950056 countsByYear W20199500562018 @default.
• W2019950056 countsByYear W20199500562019 @default.
• W2019950056 countsByYear W20199500562020 @default.
• W2019950056 countsByYear W20199500562021 @default.
• W2019950056 crossrefType "journal-article" @default.
• W2019950056 hasAuthorship W2019950056A5002370058 @default.
• W2019950056 hasAuthorship W2019950056A5040839623 @default.
• W2019950056 hasAuthorship W2019950056A5080472610 @default.
• W2019950056 hasAuthorship W2019950056A5083052216 @default.
• W2019950056 hasBestOaLocation W20199500561 @default.
• W2019950056 hasConcept C104317684 @default.
• W2019950056 hasConcept C107824862 @default.
• W2019950056 hasConcept C12554922 @default.
• W2019950056 hasConcept C126322002 @default.
• W2019950056 hasConcept C128057223 @default.
• W2019950056 hasConcept C161624437 @default.
• W2019950056 hasConcept C166342909 @default.
• W2019950056 hasConcept C181199279 @default.
• W2019950056 hasConcept C185592680 @default.

• next