Several mutations in human cardiac troponin T (TnT) gene have been reported to cause hypertrophic cardiomyopathy (HCM). To explore the effects of the mutations on cardiac muscle contractile function under physiological conditions, human cardiac TnT mutants, Ile79Asn and Arg92Gln, as well as wild type, were expressed in Escherichia coli and exchanged into permeabilized rabbit cardiac muscle fibers, and Ca2+-activated force was determined. The free Ca2+ concentrations required for tension generation were found to be significantly lower in the mutant TnT-exchanged fibers than in the wild-type TnT-exchanged fibers, whereas no significant differences were found in tension-generating capability under maximal activating conditions and in cooperativity. These results suggest that a heightened Ca2+ sensitivity of cardiac muscle contraction is one of the factors to cause HCM associated with these TnT mutations.
Activation of human platelets by cross-linking of the platelet low-affinity IgG receptor, the Fc gamma receptor IIA (Fc gamma-RIIA), or by collagen is associated with rapid phosphorylation on tyrosine of the non-receptor tyrosine kinase syk. Phosphorylation is still observed, albeit sometimes reduced, in the presence of a combination of a protein kinase C inhibitor, Ro 31-8220, and the intracellular calcium chelator, BAPTA-AM, demonstrating independence from phosphoinositide-specific phospholipase C (PLC) activity. In contrast, the combination of Ro 31-8220 and BAPTA-AM completely inhibits phosphorylation of syk in thrombin-stimulated platelets. Phosphorylation of syk increases its autophosphorylation activity measured in a kinase assay performed on syk immunoprecipitates. Fc gamma-RIIA also undergoes phosphorylation in syk immunoprecipitates from platelets activated by cross-linking of Fc gamma-RIIA but not by collagen, suggesting that it associates with the kinase. Consistent with this, tyrosine-phosphorylated Fc gamma-RIIA is precipitated by a glutathione S-transferase (GST) fusion protein containing the tandem src homology (SH2) domains of syk from Fc gamma-RIIA- but not collagen-activated cells. Two uncharacterized tyrosine-phosphorylated proteins of 40 and 65 kDa are uniquely precipitated by a GST fusion protein containing the tandem syk-SH2 domains in collagen-stimulated platelets. A peptide based on the antigen recognition activation motif (ARAM) of Fc gamma-RIIA, and phosphorylated on the two tyrosine residues found within this region, selectively binds syk from lysates of resting platelets; this interaction is not seen with a non-phosphorylated peptide. Kinase assays on Fc gamma-RIIA immunoprecipitates reveal the constitutive association of an unidentified kinase activity in resting cells which phosphorylates a 67 kDa protein. Syk is not detected in Fc gamma-RIIA immunoprecipitates from resting cells but associates with the receptor following activation and, together with Fc gamma-RIIA, is phosphorylated in the kinase assay in vitro. These results demonstrate that syk is activated by Fc gamma-RIIA cross-linking and collagen, independent of PLC, suggesting that it may have an important role in the early events associated with platelet activation. The association of syk with Fc gamma-RIIA appears to be mediated through the tandem SH2 domains in syk and the ARAM motif of Fc gamma-RIIA. A similar interaction may underlie the response to collagen, suggesting that its signalling receptor contains an ARAM motif.
Human wild-type cardiac troponin T, I, C and five troponin T mutants (I79N, R92Q, F110I, E244D, and R278C) causing familial hypertrophic cardiomyopathy were expressed in Escherichia coli, and then were purified and incorporated into rabbit cardiac myofibrils using a troponin exchange technique. The Ca 2؉ -sensitive ATPase activity of these myofibrillar preparations was measured in order to examine the functional consequences of these troponin mutations. An I79N troponin T mutation was found to cause a definite increase in Ca 2؉ sensitivity of the myofibrillar ATPase activity without inducing any significant change in the maximum level of ATPase activity. A detailed analysis indicated the inhibitory action of troponin I to be impaired by the I79N troponin T mutation. Two more troponin T mutations (R92Q and R278C) were also found to have a Ca 2؉ -sensitizing effect without inducing any change in maximum ATPase activity. Two other troponin T mutations (F110I and E244D) had no Ca 2؉ -sensitizing effects on the ATPase activity, but remarkably potentiated the maximum level of ATPase activity. These findings indicate that hypertrophic cardiomyopathy-linked troponin T mutations have at least two different effects on the Ca 2؉ -sensitive ATPase activity, Ca 2؉-sensitization and potentiation of the maximum level of the ATPase activity.The contraction of striated muscle is regulated by Ca 2ϩ through two specific regulatory proteins, troponin (Tn) 1 and tropomyosin, which are located on the thin filament. Tn is a complex of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnI inhibits myosin-actin interaction and TnC suppresses the inhibitory effect of TnI by binding of Ca 2ϩ . TnT binds to tropomyosin and integrates the whole Tn complex into the thin filament. All three Tn subunits are required for the contraction regulated by Ca 2ϩ (1). Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant cardiac disease associated with a high incidence of sudden death (2-4). This disease has been reported to be caused by mutations in cardiac sarcomeric proteins including TnT (5-7), TnI (8), -myosin heavy chain (9, 10), ␣-tropomyosin (5, 11), myosin-binding protein C (12, 13), and myosin light chains (14). Previous genetic analyses have shown that eight different point mutations in TnT to be associated with HCM. However, it is still not clear as to how these single point mutations are functionally related to and cause this disease.This study was carried out to investigate the functions of five HCM-linked TnT mutants in cardiac myofibrils under physiological conditions. We prepared recombinant human cardiac Tn subunits (wild-type TnC, TnI, and TnT) and five HCM-linked TnT mutants, and these recombinant proteins were then incorporated into rabbit cardiac myofibrils using our previously reported TnT treatment procedure (15-17). The Ca 2ϩ -sensitive ATPase activity of these reconstituted myofibrils was examined. We thus found the HCM-linked TnT mutants to have at least two different effects on ATPase a...
Tumor necrosis ractor a (TNFa) stimulated rapid (seconds) hydrolysis oTsphingomyclin in ML-60 cells, formation of phosphochcline (PCho) and a dmunsc in choline. The response to TNFa was concentration dependent with a muximal et&et at 3-10 nM. The monoclonal antibody (mAb), htr-9, which behaves as an agonist at the 55 kDa subtype of the TNF receptor, also stimulated sphingomyelin hydrolysis in in!act cells. In contrast, the mAb, utr-I, which behaves as an antagonist at the 7.5 kDa receptor subtype, had no et'fixt on sphingomyclin hydrolysis either on its own or in the prcscnce oTTNFa. In addition, htr-9 or TbIFa stimulated hydrolysis of sphingomyelin in a mcmbranc fraction of ML-60 cells. These results are consistent with H role of sphingomyclin hydrolysis as an early event in the sipnalling mechanism of TNFa, and suggest that this pathway is activated through the 55 kDa subtype of the TNF rcccptor.Tumor necrosis factor a; Sphingomyclinase
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