Quantification and structure-function analysis of calpain-1 and calpain-2 protease subunit interactions.

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Calpains (calpain-1 and calpain-2) are proteases crucial in cell signaling, cancer progression, and drug resistance.
• Current therapeutic strategies lack effective, specific calpain inhibitors.
• Calpain structure suggests susceptibility to allosteric inhibition by disrupting subunit heterodimerization.

Purpose of the Study:

• To develop and utilize split-Nanoluciferase biosensors to quantify calpain-1 and calpain-2 heterodimerization.
• To determine dissociation constants (K_D) and half-maximal calcium concentrations for calpain heterodimerization.
• To identify key residues involved in calpain heterodimerization using molecular modeling and mutagenesis.

Main Methods:

• Split-Nanoluciferase biosensor development for protein-protein interaction quantification.
• Measurement of heterodimer dissociation constants (K_D) in the presence of Ca²⁺ and Mg²⁺.
• Molecular modeling based on calpain-2 crystal structure and site-directed mutagenesis (CAPNS1 Q263).

Main Results:

• Calpain-1 and calpain-2 heterodimer K_D values were determined under varying ionic conditions.
• Half-maximal Ca²⁺ concentrations for interactions were established for both calpains.
• Molecular modeling identified 20 key residues in penta-EF-hand domains; CAPNS1 Q263 mutation significantly reduced calpain-2 activity in cells.

Conclusions:

• Split-Nanoluciferase biosensors provide a quantitative method to study calpain heterodimerization.
• Understanding calpain subunit interactions is vital for developing allosteric inhibitors.
• Identification of critical residues offers targets for novel therapeutic strategies against calpain-mediated diseases.