Discrete proteolysis of neuronal calcium sensor-1 (NCS-1) by μ-calpain disrupts calcium binding

Abstract

SummaryNeuronal Calcium Sensor-1 (NCS-1) is a high-affinity, low-capacity Ca 2+ -binding protein expressed in many cell types. We previously showed that NCS-1 interacts with inositol 1,4,5-trisphosphate receptor (InsP3R) and modulates Ca 2+ -signaling by enhancing InsP3-dependent InsP3R channel activity and intracellular Ca 2+ transients. Recently we reported that the chemotherapeutic agent, paclitaxel (taxol) triggers μ-calpain dependent proteolysis of NCS-1, leading to reduced Ca 2+ -signaling within the cell. Degradation of NCS-1 may be critical in the induction of peripheral neuropathy associated with taxol treatment for breast and ovarian cancer. To begin to design strategies to protect NCS-1, we treated NCS-1 with μ-calpain in vitro and identified the cleavage site by N-terminal sequencing and MALDI-mass spectroscopy. μ-calpain cleavage of NCS-1 occurs within an N-terminal pseudoEF-hand domain, which by sequence analysis appears to be unable to bind Ca 2+ . Our results suggest a role for this pseudoEF-hand in stabilizing the three functional EFhands within NCS-1. Using isothermal titration calorimetry (ITC) we found that loss of the pseudoEF-hand markedly decreased NCS-1's affinity for Ca 2+ . Physiologically, this significant decrease in Ca 2+ affinity may render NCS-1 incabable of responding to changes in Ca 2+ levels in vivo. The reduced ability of μ-calpain treated NCS-1 to bind Ca 2+ may explain the altered Ca 2+ signaling in the presence of taxol and suggests a strategy for therapeutic intervention of peripheral neuropathy in cancer patients undergoing taxol treatment.

Protection of Neuronal Calcium Sensor 1 Protein in Cells Treated with Paclitaxel

“…Digestion of NCS-1 by -Calpain-Digestion of NCS-1 WT and mutants was performed using a modification of a previously published protocol (14). Briefly, -calpain was purchased from Sigma (calpain-1, active from human plasma; Sigma C-6108).…”

“…and mutants, I35H and I35A, were expressed as previously described (14). Mutations were made at amino acid 35 of NCS-1 to either a histidine or an alanine using the QuikChange multiple site-directed mutagenesis kit (Stratagene).…”

“…Isothermal Titration Calorimetry (ITC)-Calcium titrations were performed as described previously (14). Briefly, purified NCS-1 WT and mutants were stripped of calcium using a modification of a previously published protocol (22).…”

“…Paclitaxel increases the interaction between NCS-1 and the InsP 3 R (11,13). Prolonged treatment with paclitaxel activates calpain, a calcium-dependent enzyme with numerous targets, including NCS-1 (13,14). The loss in functional NCS-1 results in a decrease in the activity of the InsP 3 R, which in turn decreases ability of the cell to produce intracellular calcium signals (6,11,13,15).…”

Inhibition of Paclitaxel-induced Decreases in Calcium Signaling

“…Interestingly, Taxol increases the interaction between NCS-1 and the InsP 3 R (11) which enhances intracellular calcium signaling. Treatment with Taxol also activates calpain, a calciumdependent enzyme, which cleaves a number of substrates, including NCS-1 (11,16) and the InsP 3 R (32). This cleavage of NCS-1 results in a decrease in the activity of the InsP 3 R, resulting in a decrease in the generation of intracellular calcium signals (10,11,15,17).…”