In the thymus, positive and negative selection shape the T cell repertoire. It has previously been shown that positive selection, like negative selection, is the result of the interaction of the TCR with self-peptides bound to MHC. However, little is known about the number or nature of the self-peptide ligands that mediate positive selection in vivo. We devised a novel assay with enhanced sensitivity for low affinity TCR ligands to identify self-peptides that may be biologically relevant. At least eight K(b)-bound self-peptides were detected by this assay using thymocytes bearing the OT-I TCR (specific for OVAp/K(b)). The sequence of one of these peptides was determined using the recently developed technique of membrane preconcentration-capillary electrophoresis-tandem mass spectrometry. This peptide, CP alpha1, has limited sequence similarity to OVAp, yet was found to induce positive selection of OT-I thymocytes in fetal thymic organ culture.
Actin capping protein (CP) binds barbed ends of actin filaments to regulate actin assembly. CP is an α/β heterodimer. Vertebrates have conserved isoforms of each subunit. Muscle cells contain two β isoforms. β1 is at the Z-line; β2 is at the intercalated disc and cell periphery in general. To investigate the functions of the isoforms, we replaced one isoform with another using expression in hearts of transgenic mice.Mice expressing β2 had a severe phenotype with juvenile lethality. Myofibril architecture was severely disrupted. The β2 did not localize to the Z-line. Therefore, β1 has a distinct function that includes interactions at the Z-line. Mice expressing β1 showed altered morphology of the intercalated disc, without the lethality or myofibril disruption of the β2-expressing mice.The in vivo function of CP is presumed to involve binding barbed ends of actin filaments. To test this hypothesis, we expressed a β1 mutant that poorly binds actin. These mice showed both myofibril disruption and intercalated disc remodeling, as predicted.Therefore, CPβ1 and CPβ2 each have a distinct function that cannot be provided by the other isoform. CPβ1 attaches actin filaments to the Z-line, and CPβ2 organizes the actin at the intercalated discs.
Abstract-Actin capping protein (CapZ) binds the barbed ends of actin at sarcomeric Z-lines. In addition to anchoring actin, Z-discs bind protein kinase C (PKC). Although CapZ is crucial for myofibrillogenesis, its role in muscle function and intracellular signaling is unknown. We hypothesized that CapZ downregulation would impair myocardial function and disrupt PKC-myofilament signaling by impairing PKC-Z-disc interaction. To test these hypotheses, we examined transgenic (TG) mice in which cardiac CapZ protein is reduced. Fiber bundles were dissected from papillary muscles and detergent extracted. Some fiber bundles were treated with PKC activators phenylephrine (PHE) or endothelin (ET) before detergent extraction. We simultaneously measured Ca 2ϩ -dependent tension and actomyosin MgATPase activity. CapZ downregulation increased myofilament Ca 2ϩ sensitivity without affecting maximum tension or actomyosin MgATPase activity. Maximum tension and actomyosin MgATPase activity were decreased after PHE or ET treatment of wild-type (WT) muscle. Fiber bundles from TG hearts did not respond to PHE or ET. Immunoblot analysis revealed an increase in myofilament-associated PKC-⑀ after PHE or ET exposure of WT preparations. In contrast, myofilamentassociated PKC-⑀ was decreased after PHE or ET treatment in TG myocardium. Protein levels of myofilamentassociated PKC- were decreased in TG ventricle. C-protein and troponin I phosphorylation was increased after PHE or ET treatment in WT and TG hearts. Basal phosphorylation levels of C-protein and troponin I were higher in TG myocardium. These results indicate that downregulation of CapZ, or other changes associated with CapZ downregulation, increases cardiac myofilament Ca 2ϩ sensitivity, inhibits PKC-mediated control of myofilament activation, and decreases myofilament-associated PKC-.
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