title: Calpain 1 binding capacities of the N1-line region of titin are significantly enhanced by physiological concentrations of calcium.
abstract: Calpain 1, an ubiquitous well-known calcium-dependent intracellular protease, was recently shown to bind tightly to the proximal end of the I-band titin segment in a calcium-dependent manner [Raynaud et al. (2005) FEBS J. 272, 2578-2590]. In the present work we identified the titin Ig-domain of concern by this interaction and the role of calcium in this interaction using a recombinant fragment of titin spanning the I2-I6 region and its subfragments. The heterodimeric form of calpain 1 binds to this titin fragment with a very high affinity ( K d = 5.1 +/- 0.2 x 10 (-7) M) at much lower calcium levels than those saturating the high-affinity binding sites of the peptidase ( K d = 25 microM). Investigation of this interaction with I2-I6 subfragments clearly showed that the dimeric form of calpain 1 binds exclusively to the Ig-domain I4 of titin with an affinity similar to that of the whole I2-I6 segment. As for the I2-I6 fragment, this interaction is calcium regulated. Calcium was shown to bind tightly to titin ( K d = 1.9 x 10 (-7) M), causing an oligomerization of the titin segment. At physiological calcium concentration (10 (-6) to 10 (-8) M), the prevailing form of the titin fragment is a trimer, suggesting that calpain 1 binds to this titin structure. From the present findings, it was concluded that calcium binding to titin increased the amount of bound calpain 1 (up to 40% of the total calpain 1) and that this bound calpain 1 might constitute a reservoir for this peptidase. In this context, we proposed a schematic diagram of this series of calcium-dependent events with the inherent unanswered questions. These events are probably under a complex regulation involving undoubtedly different yet unidentified proteins.
authors.affiliation: INRA, UR370 QuaPA, 63122 Saint Genes Champanelle, France.
authors.forename: Gerald
authors.initials: G
authors.lastname: Coulis
authors.forename: Samira
authors.initials: S
authors.lastname: Becila
authors.forename: Carlos H
authors.initials: CH
authors.lastname: Herrera-Mendez
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Sentandreu
authors.forename: Fabrice
authors.initials: F
authors.lastname: Raynaud
authors.forename: Isabelle
authors.initials: I
authors.lastname: Richard
authors.forename: Yves
authors.initials: Y
authors.lastname: Benyamin
authors.forename: Ahmed
authors.initials: A
authors.lastname: Ouali
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D064211:Connectin; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine
chemical_list: D064211:Connectin; D009124:Muscle Proteins; D011994:Recombinant Proteins; C577285:TTN protein, human; D011494:Protein Kinases; D002154:Calpain; D002118:Calcium