The importance of conserved amino acid residues in p94 protease sub-domain IIb and the IS2 region for constitutive autolysis.
p94/calpain 3, a skeletal muscle-specific member of calpain protease family, is characterized by apparent Ca(2+)-independence during exhaustive autolysis and concomitant proteolysis of non-self substrates. The purpose of our study was to comprehensively profile the structural basis of p94 enabling activation in the cytosol without an extra Ca(2+). Ca(2+)-dependent p94 mutants were screened using "p94-trapping", which is an application of yeast genetic reporter system called "proteinase-trapping". Several amino acids were revealed as critical for apparent Ca(2+)-independent p94 activity. These results highlight the importance of conserved amino acids in domain IIb as well as in the p94-specific IS2 region.
Ono
Yasuko
Y
Department of Enzymatic Regulation for Cell Functions (Calpain Project), The Tokyo Metropolitan Institute of Medical Science (Rinshoken), Tokyo, Japan. yakoono@rinshoken.or.jp
Hayashi
Chikako
C
Doi
Naoko
N
Tagami
Mai
M
Sorimachi
Hiroyuki
H
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000494:pharmacology; D002154:Calpain / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017434:Protein Structure, Tertiary; D012964:Sodium / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects
D009124:Muscle Proteins; D050505:Mutant Proteins; D012964:Sodium; D010447:Peptide Hydrolases; C105884:CAPN3 protein, human; D002154:Calpain; D002118:Calcium