Journal
BIOCHEMISTRY
Volume 47, Issue 35, Pages 9174-9183Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi800315v
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Funding
- INRA
- Assiociation Francaise contre les Myopathies (AFM)
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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 mu M). Investigation of this interaction with I2-I6 subfragments clearly showed that the dimeric form of calpain 1 binds exclusively to the Ig-domain 14 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 (Kd = 1.9 x 10(-1) 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.
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