The COOH-terminal A168–170 region of the giant sarcomeric protein titin interacts with muscle-specific RING finger-1 (MURF-1). To investigate the functional significance of this interaction, we expressed green fluorescent protein fusion constructs encoding defined fragments of titin's M-line region and MURF-1 in cardiac myocytes. Upon expression of MURF-1 or its central region (containing its titin-binding site), the integrity of titin's M-line region was dramatically disrupted. Disruption of titin's M-line region also resulted in a perturbation of thick filament components, but, surprisingly, not of the NH2-terminal or I-band regions of titin, the Z-lines, or the thin filaments. This specific phenotype also was caused by the expression of titin A168–170. These data suggest that the interaction of titin with MURF-1 is important for the stability of the sarcomeric M-line region.MURF-1 also binds to ubiquitin-conjugating enzyme-9 and isopeptidase T-3, enzymes involved in small ubiquitin-related modifier–mediated nuclear import, and with glucocorticoid modulatory element binding protein-1 (GMEB-1), a transcriptional regulator. Consistent with our in vitro binding data implicating MURF-1 with nuclear functions, endogenous MURF-1 also was detected in the nuclei of some myocytes. The dual interactions of MURF-1 with titin and GMEB-1 may link myofibril signaling pathways (perhaps including titin's kinase domain) with muscle gene expression.

p94 (also called calpain 3) is the skeletal muscle-specific calpain and is considered to be a "modulator protease" in various cellular processes. Analysis of p94 at the protein level is an urgent issue because the loss of p94 protease activity causes limb-girdle muscular dystrophy type 2A. In this study, we enzymatically characterized one alternatively spliced variant of p94, p94:exons 6 ؊ 15 ؊ 16 ؊ (p94⌬), which lacks two of the p94-specific insertion sequences. In contrast to p94, which has hardly been studied enzymatically due to its rapid, thorough, and apparently Ca 2؉ -independent autolytic activity, p94⌬ was stably expressed in COS and insect cells. p94⌬ showed Ca 2؉ -dependent caseinolytic and autolytic activities and an inhibitor spectrum similar to those of the conventional calpains. However, calpastatin did not inhibit p94⌬ and is a substrate for p94⌬, which is consistent with the properties of p94, presenting p94 as a possible regulator of the conventional calpain system. We also established a semi-quantitative fluorescence resonance energy transfer assay using the calpastatin sequence specifically to measure p94 activity. This method detects the activity of COS-expressed p94 and p94⌬, suggesting that it has potential to evaluate p94 activity in vivo and in the diagnosis of limb-girdle muscular dystrophy type 2A.Calpain (EC 3.4.22.17, clan CA, family C2) is a Ca 2ϩ -requiring cysteine protease representing one of the most important families of the cysteine proteases (1-9). To date, various molecules showing significant similarity to the calpain protease domain have been identified in almost all kinds of living organisms and constitute the "calpain superfamily" (6). Two representative members, -and m-calpains, the so-called "conventional" calpains, are ubiquitously expressed and have been well characterized. These two calpains consist of a distinct larger catalytic subunit containing a protease domain (-or m-calpain large subunit, abbreviated as CL 1 or mCL, respectively) and a common smaller regulatory subunit (abbreviated as 30K according to its molecular weight). On the basis of amino acid similarities, the large and small subunits have been described as consisting of four and two domains, respectively, which agrees with the recently resolved three-dimensional structure of m-calpain (10, 11) (Fig. 1A).Conventional calpain has a specific endogenous proteinaceous inhibitor, calpastatin (12). Calpastatin contains four repetitive inhibitory units, each of which inhibits equimolar amounts of conventional calpain. The conserved reactive site interacts with the calpain protease domain, whereas the flanking ␣-helical regions bind to domains IV and VI of the large and small subunits, respectively. Synthetic oligopeptides (see Fig. 4D) corresponding to the calpastatin-reactive site specifically inhibit conventional calpain efficiently, although their inhibitory activity is weaker than that of the full-length inhibitory unit.The primary structure of p94 (also called calpain 3) is very similar to those of CL ...

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