Immunization with cardiac myosin induces T cell-mediated myocarditis in genetically predisposed mice and serves as a model for autoimmune heart disease. This study was undertaken to identify pathogenic epitopes on the myosin molecule. Our approach was based on the comparison of the pathogenicity between cardiac ( ␣ -) myosin and soleus muscle ( ␤ -) myosin. We show that ␣ -myosin is the immunodominant isoform and induces myocarditis at high severity and prevalence whereas ␤ -myosin induces little disease. Therefore the immunodominant epitopes of ␣ -myosin must reside in regions of different amino acid sequence between ␣ -and ␤ -myosin isoforms. Cardiac myosin peptides corresponding to these regions of difference were synthesized and tested for their ability to induce inflammatory heart disease. Three pathogenic peptides were identified. One peptide that is located in the head portion of the molecule induced severe myocarditis, whereas two others that reside in the rod portion possessed only minor pathogenicity. The identification of pathogenic epitopes on the cardiac myosin molecule will allow detailed studies on the recognition of this antigen by the immune system and might be used to downmodulate ongoing heart disease. (

The specificity of SH3 domain complex formation plays an important role in determining signal transduction events. We have previously identified a highly specific interaction between the first CrkSH3 domain [CrkSH3(1)] and proline-rich sequences in the guanine nucleotide exchange factor C3G. A 10 amino acid peptide derived from the first proline-rich sequence (p3P4P5A6L7P8P9K'0K11R12) bound with a Kd of 1.89 ± 0.06,uM and fully retained the high affinity and unique selectivity for the CrkSH3(1) domain. Mutational analysis showed that P5 P8, L7 and K'0 are critical for high affinity binding. A conservative mutation, KiOR, significantly decreased the affinity for the CrkSH3(1) domain while increasing the affinity for Grb2. Comparative binding studies with the KlOR and K1OA mutant peptides to c-Crk and v-Crk further suggested that K'0 binds via a charge-dependent and a chargeindependent interaction to the RT loop of the CrkSH3(1) domain. Besides determining important structural features necessary for high affinity and specificity binding to the CrkSH3(1) domain, our results also demonstrate that a conservative mutation in a single amino acid can significantly alter the specificity of an SH3 binding peptide.

Many Src Homology 3 (SH3) domains function as molecular adhesives in intracellular signal transduction. Based on previous ultrastructural studies, short motifs which bind to the ®rst SH3 domains of the adapters Crk and CRKL were selectively mutagenised to generate Crk/ CRKL SH3-binding peptides of very high a nity and selectivity. A nities were increased up to 20-fold compared to the best wildtype sequences, while the selectivity against a similar SH3 domain [Grb2SH3(N)] was not only retained, but sometimes increased. Blot techniques with GST-fusion peptides and in solution precipitation assays with biotinylated high a nity Crk binding peptides (HACBPs) were subsequently used to analyse the binding of these sequences to a large panel of SH3 domain-containing fusion proteins. Only those proteins which contained the CrkSH3(1) or CRKLSH3(1) domains bound e ciently to the HACBPs. A GST-HACBP fusion protein precipitated Crk and CRKL proteins out of 35 S-labelled and unlabelled cell lysates. Very little binding of other cellular proteins to HACBP was detectable, indicative of a great preference for Crk and CRKL when compared to the wide variety of other endogenous cellular proteins. Moreover, HACBP disrupted in vitro preexisting Crk-complexes with DOCK180 and the exchange factors SoS and C3G, which are known targets of Crk adapters, in a concentration dependent manner. HACBP-based molecules should therefore be useful as highly selective inhibitors of intracellular signalling processes involving Crk and CRKL.

The 72-kDa nuclear lamina protein lamin A is synthesized as a 74-kDa farnesylated precursor. Conversion of this precursor to mature lamin A appears to be mediated by a specific endoprotease. Prior studies of overexpressed wild-type and mutant lamin A proteins in cultured cells have indicated that the precursor possesses the typical carboxyl-terminal S-farnesylated, cysteine methyl ester and that farnesylation is required for endoproteolysis to occur. In this report, we describe the synthesis of an S-farnesyl, cysteinyl methyl ester peptide corresponding to the carboxyl-terminal 18 amino acid residues of human prelamin A. This peptide acts as a substrate for the prelamin A endoprotease in vitro, with cleavage of the synthetic peptide at the expected site between Tyr 657 and Leu 658 . Endoproteolytic cleavage requires the S-prenylated cysteine methyl ester and, in agreement with transfection studies, is more active with the farnesylated than geranylgeranylated cysteinyl substrate. N-Acetyl farnesyl methyl cysteine is shown to be a noncompetitive inhibitor of the enzyme. Taken together, these observations suggest that there is a specific farnesyl binding site on the enzyme which is not at the active site.Proteins with a CAAX consensus sequence at their carboxyl terminus undergo serial post-translational modifications of the cysteinyl residue (1, 2). These modifications include derivitization of the cysteine sulfhydryl with an isoprenoid moiety followed by the endoproteolytic removal of the -AAX tripeptide and methylation of the cysteine ␣-carboxyl group. When the X amino acid is S, C, Q, or M, a 15-carbon farnesyl residue is attached in thioether linkage to the cysteine (3), whereas when X is a leucine, a 20-carbon geranylgeranyl residue is found instead (4).The nuclear lamina is a thin, fibrous structure that lines the inner nuclear membrane and is believed to function in maintaining nuclear shape and volume (5) and may also be involved in the organization of chromatin in the interphase nucleus (6). In most mammalian cells, it consists of three class V intermediate filament proteins, lamins A, B, and C (5, 6). Prelamin A is the 74-kDa precursor of the 72-kDa nuclear lamin A protein (7).It possesses a CAAX box sequence (CSIM) (8, 9) and has been shown to be farnesylated in vitro (10) and in vivo (11). Despite the loss of the carboxyl-terminal 18 amino acids of prelamin A in its proteolytic conversion to lamin A, it nevertheless undergoes all of the reactions characteristic of other CAAX proteins (11). Experiments with mutants, in which the cysteine of the CAAX box is replaced by another amino acid, demonstrate that farnesylation is required for the maturation of prelamin A (12). These nonprenylated CAAX box mutants of prelamin A enter the nucleus, yet are not proteolytically processed and are not incorporated into the nuclear lamina. Similar results have been obtained with nonprenylated prelamin A produced by treating cultured mammalian cells with mevinolin (13,14) or inhibitors of protein farnesylation (15).Prela...