p94, a skeletal muscle-specific calpain, has attracted much attention because its gene is responsible for limb-girdle muscular dystrophy type 2A. p94, however, has not been characterized at the protein and enzyme levels, owing to its very rapid autolysis. In the present study, a purification procedure for p94 was first established by using a recombinant inactive p94 expressed in COS cells in which the active site cysteine residue was changed to serine [p94(C129S)]. The isolation of native p94 from rabbit skeletal muscle by the established method with conventional procedures was extremely difficult because p94 became highly unstable in a crude extract on the addition of NaCl for separation. Purification of native p94 was possible with an antibody-affinity column but only as an inactive enzyme; p94(C129S) was purified as a homodimer. Characterization of p94, especially autolysis, was performed with partly purified native p94 and p94(C129S). The autolysis of p94, which consisted at least partly of an intermolecular reaction, proceeded in three consecutive steps; 60 and 58 kDa fragments were produced as intermediates before a stable 55 kDa fragment appeared. Autolysis of p94 was regarded as a degradative step rather than for the activation of the enzyme. All the autolysis cleavage sites were located in the p94-specific insertion sequence 1 region, which explains why p94 is unstable compared with the other calpains. The autolysis sites in p94 clearly showed a different specificity relative to the autolytic and proteolytic cleavage sites of the ubiquitous mu- and m-calpains, in its preference for residues at the P3 to P1' sites, indicating a distinct substrate specificity and function for the muscle enzyme.
The 6.5-kilobase mre region at 71 min in the Escherichia coli chromosome map, where genes involved in formation of a rod-shaped cell form a gene cluster, was analyzed by in vivo protein synthesis in a maxicell system and by base sequencing of DNA. An open reading frame that may code for a protein with an Mr of about 37,000 on sodium dodecyl sulfate-polyacrylamide gels was found and was correlated with the mreB gene. N-terminal amino acid sequencing of the hybrid mreB-lacZ protein confirmed the production by mreB of a protein of 347 amino acid residues with a molecular weight of 36,958. The amino acid sequence of this protein deduced from the DNA sequence showed close similarity with that of a protein of theftsA gene which is involved in cell division of E. coli. Three other contiguous genes that formed three proteins with Mrs of about 40,000, 22,000, and 51,000, respectively, were detected downstream of the mreB gene by in vivo protein synthesis. The mreB protein and some of these three proteins may function together in determination of cell shape.Two regions called mrd (15) and mre (11) on the Escherichia coli chromosome involve clusters of genes responsible for determination of the cell shape and the sensitivity of cell growth to an amidinopenicillin, mecillinam. The mrd region located at 15 min on the E. coli chromosome map involves two genes for formation of the rod shape of the cell, mrdA (= pbpA), which codes for penicillin-binding protein 2, a peptidoglycan synthetase, and mrdB (= rodA), which codes for the RodA protein which may also be necessary for functioning of the mrdA protein (5). The mre region located at 71 min on the chromosome map involves genes that probably function together in shape determination and mecillinam sensitivity of the cell (17). Previously, we reported preliminary results of gene analysis of the apparent 7-kilobase (kb) mre region neighboring the fabE gene (17). The gene we called mreB (11), which may be allelic to envB (18) and in which the mutation mre-129 causing a round cell and supersensitivity to mecillinam is located, was cloned in a 2.8-kb DNA fragment. However, we found that another mutation, mre-678, causing a round cell, resistance to mecillinam, and overproduction of penicillin-binding proteins lBs and 3 was due to deletion of a 5.2-kb DNA fragment extending from the mreB gene and could not be complemented by the 2.8-kb fragment (17).The present report describes the in vivo identification of the protein products of the genes located in the mre region (the size of the SaII fragment is corrected to 6.5 kb from that in the previous report) by using a maxicell system (13) MATERIALS AND METHODSBacterial strains. E. coli K-12 strain CSR603 (uvrA6 recAl phr-J thr leu pro his thi arg lac gal ara xyl mtl rpsL) used for protein synthesis in the maxicell system was obtained from B. J. Bachmann, Yale University School of Medicine. Strain JM109 (Alac-proAB recAl endAl gyrA96 thi hsdRJ7 supE44 relAl A-F' traD36 proAB lacIq AlacZMJ5) used for DNA sequencing experiments was obtained...
Emerin is an inner nuclear membrane protein that is involved in X-linked recessive Emery-Dreifuss muscular dystrophy (X-EDMD). Although the function of this protein is still unknown, we revealed that C-terminus transmembrane domain-truncated emerin (amino acid 1-225) binds to lamin A with higher affinity than lamin C. Screening for the emerin binding protein and immunoprecipitation analysis showed that lamin A binds to emerin specifically. We also used the yeast two-hybrid system to clarify that this interaction requires the top half of the tail domain (amino acid 384-566) of lamin A. Lamin A and lamin C are alternative splicing products of the lamin A/C gene that is responsible for autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD). These results indicate that the emerin-lamin interaction requires the tail domains of lamin A and lamin C. The data also suggest that the lamin A-specific region (amino acids 567-664) plays some indirect role in the difference in emerin-binding capacity between lamin A and lamin C. This is the first report that refers the difference between lamin A and lamin C in the interaction with emerin. These data also suggest that lamin A is important for nuclear membrane integrity.
MDC9, also known as meltrin gamma, is a membrane-anchored metalloprotease. MDC9 contains several distinct protein domains: a signal sequence followed by a prodomain and a domain showing sequence similarity to snake venom metalloproteases, a disintegrin-like domain, a cysteine-rich region, an epidermal-growth-factor-like repeat, a transmembrane domain and a cytoplasmic domain. Here we demonstrate that MDC9 expressed in COS cells is cleaved between the prodomain and the metalloprotease domain. Further, when MDC9 was co-expressed in COS cells with amyloid precursor protein (APP695) and treated with phorbol ester, APP695 was digested exclusively at the alpha-secretory site in MDC9-expressing cells. When an artificial alpha-secretory site mutant was also co-expressed with MDC9 and treated with phorbol ester, APP secreted by alpha-secretase was not increased in conditional medium. Inhibition of MDC9 by a hydroxamate-based metalloprotease inhibitor, SI-27, enhanced beta-secretase cleavage. These results suggest that MDC9 has an alpha-secretase-like activity and is activated by phorbol ester.
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