Engineering Calpastatin to Develop a Sensor to Detect Active Calpain

Calpains, Ca2+-activated cysteine proteases are essential for early embryonic development and function in signal transduction, cell adhesion, and apoptosis. Calpains also contribute to cataractogenesis, myocardial infarctions, and neurodegenerative diseases such as Alzheimer\u27s. The various methods currently available to demonstrate these roles do not directly identify spatial or temporal activation of calpain in cells. Therefore, a tool to detect active calpain in situ will be useful. Calpastatin is the ubiquitous, endogenous inhibitor that specifically binds the active conformation of the conventional calpains. Calpastatin consists of four homologous domains each containing three subdomains A, B, and C. The crystal structure of a calpastatin C peptide mimic bound to the homodimer of calpain domain VI in the presence of Ca2+ provides a model for the structure of calpastatin subdomains A and C bound to calpain domains IV and VI (56). This structure predicts the calpain domain IV carboxy-terminus is approximately 31 Å from the bound calpastatin domain I amino-terminus and the calpastatin domain I subdomain A aminoterminus is approximately 50 Å from the calpastatin domain I subdomain C carboxy-terminus when bound to calpain. Both are distances that should allow fluorescence resonance energy transfer (FRET) between a pair of fluorophores. These interactions provide a strategy to develop a FRET based sensor to detect active calpain. A strategy to detect endogenous calpain using the binding of fluorophore modified calpastatin subdomain A and C peptides has not yet provided positive results. An alternative strategy utilizing the binding of calpastatin domain I to calpain 2 has successfully demonstrated FRET. Three calpastatin domain I peptides were labeled at a single distinctly located cysteine residue near the amino-terminus with maleimide conjugated tetramethylrhodamine or Alexa Fluor (AF) 546. Lumio Green specifically binds a rare tetra-cysteine motif not found in native calpain 2. Insertion of this motif or eGFP at the carboxy-terminus of calpain 2 domain IV provided two mechanisms for specific labeling of calpain to serve as donors to six acceptor fluorophore labeled calpastatin domain I peptides during in vitro binding assays. All twelve donor-calpain acceptorcalpastatin pairs demonstrated a calcium dependent increase in a ratio of acceptor emission to donor emission (EmA/EmD) indicating the detection of a FRET signal. The increase in EmA/EmD ratio was detected in an E. coli crude cell lysate expressing calpain-eGFP to demonstrate the approach should also work in the complex cellular environment. As expected, calpastatin, calpain 1 and calpain 2 did compete for binding of calpastatin-AF546 to calpain-eGFP. In contrast, two calpain substrates, the cytoplasmic tail of βi integrin and α-casein, did not exhibit competition. These results demonstrate that most substrates should not interfere with the binding signal when applied in situ, however endogenous classical calpains and calpastatin likely will interfere to some extent. Future studies aim to produce a pair of fluorogenic calpastatin subdomain peptides that bind calpain and generate FRET to allow detection of endogenous calpain in cells