Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2001. / Vita. Bibliography: 139-169.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 86-98.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19612号 / 医博第4119号 / 新制||医||1015(附属図書館) / 32648 / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 渡邉 大, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development, and nervous system function. Previous studies have suggested that the regulatory domain (RD) of CaN binds Calmodulin (CaM) towards the N-terminal end of CaN. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the regulatory domain, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto inhibitory domain (AID) from the active site of CaN, which activates phosphatase activity. In the absence of calcium-loaded CaM, the RD is at least partially disordered, and binding of CaM induces folding in the RD. Previous studies have shown that an ?-helical structure forms in the N-terminal half of the RD, but organization may occur in the C-terminal region as well. Here, we are presenting a model for the structural transition of the full length RD as it binds to CaM. Using nuclear magnetic resonance (NMR) spectroscopy, we have successfully assigned >85% of the 15N, 13C?, 13C? and HN chemical shifts of the unbound, regulatory domain of CaN. Secondary chemical shifts support a model where the RD is highly disordered. Our study of the CaM and CaN interaction supports the formation of a distal helix in the region between the AID and calmodulin-binding region. Heat capacity changes upon binding predict that 43 residues fold when CaM binds to CaN, consistent with the formation of this distal helix. Paramagnetic relaxation enhancement (PRE) studies of this interaction suggest a potential binding mode where the distal helix binds to CaM near residues I10-A11. Mutagenesis in the distal helix disrupts PREs, further supporting this hypothesis. Together, these data suggest that the interactions between CaM and the distal helix of CaN can be important in regulation of phosphatase activity.

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Herzinsuffizienz mit konsekutivem Herzversagen ist ein zentrales kardiovaskuläres Problem der heutigen Bevölkerung.Ursächlich ist insbesondere eine progrediente Herzhypertrophie. Die Calcium-Calmodulin abhängige Phosphatase Calcineurin (CnR) spielt hierbei in der Pathogenese eine entscheidende Rolle. CnR wird über seine Calciumbindungsstellen an der regulatorischen Untereinheit Calcineurin B (CnB) aktiviert.Um zu untersuchen, inwieweit CnB bei der Hypertrophie verschiedener Ätiologien reguliert wird, wurde in linksventrikulären Myokardbiopsien von Patienten mit Aortenstenose (AS= 14) bzw. aus explantierten Herzen mit Dilatativer Kardiomyopathie (DCM=27) und Koronarer Herzerkrankung (KHK=7) der mRNA-und Proteingehalt von CnB bestimmt und mit der Expression von ANP und BNP korreliert. Als Kontrollgruppe dienten 15 abgelehnte Spenderherzen mit normaler systolischer Funktion und gesunder Morphologie. In den Herzen der Kontroll-, DCM-, und KHK-Gruppen wurde der linksventrikuläre Fibrosegehalt bestimmt. Hierzu wurden eine extern standardisierte Real-Time-PCR-Technik und ein etabliertes Western Blot Verfahren angewandt. Die Ergebnisse werden im Median ± 25%/75%-Perzentile angegeben und mit dem Mann-Whitney-Test bzw. Korrelationsanalysen nach Spearman berechnet. In den Herzen mit DCM zeigte sich eine signifikante Erhöhung der CnB mRNA auf ca. das Dreifache der Kontrollen (293% der Ko, p / Heart failure is a central cardiovascular problem for the current population. Cardiac hypertrophy is a central factor. The calcium-Calmodulin dependent phosphatase Calcineurin (CnR) plays a crucial role in the pathogenesis. CnR is activated via its calcium-binding site in the regulatory subunit Calcineurin B (CnB). In order to examine, to what extent CnB is regulated in different aetiologies of hypertrophy, we analysed CnB´s mRNA and protein in left ventricular samples from patients with aortic valve stenosis (AS = 14) and from explanted hearts with dilated (DCM=27) and ischemic (ICM=7) cardiomyopathy and correlated them with the expression of ANP and BNP. As a control, 15 rejected donor hearts with normal systolic function and non-pathologic changed morphology were used. Fibrosis of the left ventricle was determined in three groups: control , DCM and ICM. Therefore, we used an externally standardized real-time PCR and an established Western Blot. Data are given as median ± 25%/75%- percentiles; Mann Whitney test and Spearman´s correlation-analyses were used. CnB mRNA was significantly raised in DCM (293% of control, p

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