钙离子对钙调神经磷酸酶B亚基结构和功能的影响
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摘要
应用原紫外CD光谱、内源荧光光谱以及ANS荧光光谱探究了Ca~(2+)对钙调神经磷酸酶(CN)调节亚基(CNB)结构和功能的影响.结果显示:Ca~(2+)能够激活CN磷酸酶活性提高到约1.5倍;CD谱显示结合Ca~(2+)后CNB出现α-螺旋含量上升、无卷曲含量下降、分子有序性升高等现象;!源荧光光谱显示结合Ca~(2+)导致CNB结构更加紧凑;ANS荧光光谱显示Ca~(2+)结合导致其疏水性增强.结合Ca~(2+)后,CNB分子有序性升高,结构更加紧凑,疏水表面暴露.这些结构变化促进了其与CNA的结合并激活了CNA的磷酸酶活性.
Calcineurin(CN),a Ca~(2+)/calmodulin dependent protein phosphatase,plays a critical role in T cell activation by regulating NF-AT activity,CN is a heterodimer with a catalytic subunit A(CNA)and a Ca~(2+) binding regulatory subunit B(CNB).CNB binding of Ca~(2+) was found to stimulate phosphatase activity of CNA about 1.5 fold.After binding of Ca~(2+) far-UV CD spectra showed an increase ofα-Helix content in CNB.Intrinsic fluorescence spectroscopy also demonstrated increased intrinsic tyrosine fluorescence.Fluorescence spectroscopy of a hydrophobic probe showed CNB hydrophobicity increased after binding Ca~(2+) .These data indicate that calcium binding leads to increasedα-helix content in CNB and exposure of a hydrophobic surface.Hydrophobic surface may be an important structure feature in CNB.
Calcineurin(CN),a Ca~(2+)/calmodulin dependent protein phosphatase,plays a critical role in T cell activation by regulating NF-AT activity,CN is a heterodimer with a catalytic subunit A(CNA)and a Ca~(2+) binding regulatory subunit B(CNB).CNB binding of Ca~(2+) was found to stimulate phosphatase activity of CNA about 1.5 fold.After binding of Ca~(2+) far-UV CD spectra showed an increase ofα-Helix content in CNB.Intrinsic fluorescence spectroscopy also demonstrated increased intrinsic tyrosine fluorescence.Fluorescence spectroscopy of a hydrophobic probe showed CNB hydrophobicity increased after binding Ca~(2+) .These data indicate that calcium binding leads to increasedα-helix content in CNB and exposure of a hydrophobic surface.Hydrophobic surface may be an important structure feature in CNB.
引文
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[26]WATANABE Y,PERRINO B A,SODERLING T R.Activation of calcineurin A subunit phosphatase activity by its calcium-binding B subunit[J].Biochemistry,1996,35(2):562
[2]NORTHRUP J P,ULLMAN K S,CRABTREE G R.Characterization of the nuclear and cytoplasmic components of the lymphoid-specific nuclear factor of activated T cells(NF-AT)complex[J].J Biol chem,1993,268(4):2917
[3]MCCAFFREY P G,PERRINO B A,SOCLERLING TR,et al.NF-ATp,a T lymphocyte DNA-binding protein that is a target for calcineurin and immunosuppressive drugs[J].J Biol chem,1993,268(5):3747
[4]JAIN J,MCCAFFREY P G,MINER Z,et al.The T-cell transcription factor NF-ATp is a substrate for calcineurin and interacts with Fos and Jun[J].Nature,1993,365(6448):721
[5]SHIBASAKI F,PRICE E R,MILAN D,et al.Role of kinases and the phosphatase calcineurin in the nuclear shuttling of transcription factor NF-AT4[J].Nature,1996,382(6589):370
[6]LIU J,FARMER J D,LANE W S,et al.Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506complexes[J].Cell,1991,66(4):807
[7]MOLKENTIN J D,LU J R,ANTOS C,et al.Acalcineurin-dependent transcriptional pathway for cardiac hypertrophy[J].Cell,1998,93(2):215
[8]SUSSMAN M A,LIM H W,GUDE N,et al.Prevention of cardiac hypertrophy in mice by calcineurin inhibition[J].Science,1998,281(5383):1690
[9]SEMSARIN C,WU M J,JU K,et al.Skeletal muscle hypertrophy is mediated by Ca2+-dependent calcineurin signaling pathway[J].Nature,1999,400(6744):576
[10]GRIFFITH J P,KIM E E,SINTCHAK M D,et al.X-ray structure of calcineurin inhibited the immunophilinimmunosupperessant FKBP12-FK506 complex[J].Cell,1995,82(3):507
[11]KISSINGER C R,PARGE H E,KNIGHTON D R,et al.Crystal structures of human Calcineurin and the human FKBP12-FK506calcineurin complex[J].Nature,1995,378(6557):641
[12]FENG B,STEMMER P M.Interactions of calcineurin A,calcineurin B,and Ca2+[J].Biochemistry,1999,38(38):12481
[13]PERRINO B A,FONG Y L,BRICKEY D A,et al.Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform[J].J Biol Chem,1992(22),267:15965
[14]HADDY A,SWANSON S K H,BOM T L,et al.Inhibition of calcineurin by cyclosporin A-cyclophilin requires calcineurin B[J].FEBS Letters,1992,314(1):37
[15]LI W,HANDSCHUMACHER E R.Identification of two calcineurin B-binding protei:tublin and heat shock protein 60[J].Biochim Biophys Acta,2002,1599(1/2):72
[16]LI N,ZHANG Z,WEI Q,et al.Calcineurin B subunit interacts with proteasome subunit alpha type 7 and represses hypoxia-inducible factor-1αactivity via the proteasome pathway[J].Biochim Biophys Res Commun,2011,405(3):468
[17]WU X,NGUYEN B C,DZIUNYEZ P,et al.Opposing roles for calcineurin and ATF3in squamous skin cancer[J].Nature,2010,465(7296):368
[18]WEI Q,LIAN M L,JIN F Z,et al.Studies of calcineurin B subunit from genetic engineering for use in medicine[J].Drug Dev Res,2002,56(1):40
[19]WEI Q,LEE E Y C.Expression and reconstitution of calcineurin A and B subunits[J].Biochem Mol Biol Int,1997,41(1):169
[20]WEI Q,LEE E Y C.Mutagenesis of the L7loop connecting beta strands 12 and 13 of calcineurin:evidence for a structural role in activity changes[J].Biochemistry,1997,96(8):7418
[21]CHEN Y H,YANG J T,CHAU K H.Determination of the helix and beta form of proteins in aqueous solution by circular dichroism[J].Biochemistry,1974,13(16):3350
[22]STEMMER P M,KLEE C B.Dual calcium ion regulation of calcineurin by calmodulin and calcineurin B[J].Biochemistry,1994,33(22):6859
[23]GUERINI D,KRINKS M H,SIKELA J M,et al.Isolation and sequence of a cDNA clone for human calcineurin B,the Ca2+-binding subunit of the Ca2+/calmodulin-stimulated protein phosphatase.[J].DNA,1989,8:675
[24]JEAN-PAUL V E,KAWASAKI Y.Ca2+induced conformational change in Ca2+binding component of Troponin[J].Biochem Biophys Res Commun,1972,47(4):859
[25]YAO Y H,SCHONEICH C,SQUIER T C.Resolution of structural changes associated with calcium activation of calmodulin using frequency domain fluorescence spectroscopy[J].Biochemistry,1994,33(25):7797
[26]WATANABE Y,PERRINO B A,SODERLING T R.Activation of calcineurin A subunit phosphatase activity by its calcium-binding B subunit[J].Biochemistry,1996,35(2):562