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Myosin-Va proteolysis by Ca2+/calpain in depolarized nerve endings from rat brain
Luciana Casaletti1, Sinji B F Tauhata, Jorge E Moreira
1Department of Cellular and Molecular Biology, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, 14049-900 Ribeirão Preto, SP, Brazil.
Abstract:
Myosin-Va is a molecular motor that may participate in synaptic vesicle cycling. Calpain cleaves myosin-Va in vitro at methionine 1141 in the tail domain. We show that intracellular proteolysis of myosin-Va occurs in rat cortical synaptosomes depolarized in the presence of calcium, evidenced by the formation of an 80 k polypeptide that co-migrates in SDS-PAGE with the 80 k fragment produced by the in vitro proteolysis of myosin-Va by calpain. Anti-myosin-Va antibody recognized this polypeptide in Western blots and immunoprecipitated it from synaptosome extracts. Calpastatin, a calpain-specific inhibitor, or leupeptin, a general cysteine protease inhibitor, suppressed or blocked formation of the 80 k polypeptide depending on membrane permeability. We conclude that myosin-Va undergoes intracellular proteolysis by endogenous calpain, when synaptosomes are depolarized in the presence of calcium, at the same cleavage site previously identified in vitro, thus, making it a target for calcium signaling during synaptic activation.
Insights
Myosin-Va undergoes calcium-dependent proteolysis in synaptosomes, targeted by calpain at the same site as in vitro. This reveals myosin-Va as a key player in calcium signaling during synaptic activation.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Myosin-Va functions as a molecular motor potentially involved in synaptic vesicle cycling.
- Calpain, a calcium-dependent protease, cleaves myosin-Va in vitro at methionine 1141 within its tail domain.
Purpose of the Study:
- To investigate the intracellular proteolysis of myosin-Va in rat cortical synaptosomes.
- To determine if calpain is responsible for this in situ cleavage and identify the cleavage site.
Main Methods:
- Rat cortical synaptosomes were depolarized in the presence of calcium.
- Proteolysis was analyzed using SDS-PAGE and Western blotting with anti-myosin-Va antibodies.
- Calpain activity was inhibited using calpastatin or leupeptin.
Main Results:
- A specific 80 kDa polypeptide fragment of myosin-Va was formed in depolarized synaptosomes, identical to the in vitro calpain cleavage product.
- This 80 kDa fragment was recognized by anti-myosin-Va antibodies and immunoprecipitated from synaptosome extracts.
- Calpain inhibitors suppressed the formation of the 80 kDa fragment, confirming endogenous calpain activity.
Conclusions:
- Myosin-Va undergoes intracellular proteolysis by endogenous calpain in synaptosomes upon calcium influx during depolarization.
- The cleavage occurs at the same methionine 1141 site identified in vitro.
- This proteolysis positions myosin-Va as a target for calcium signaling pathways regulating synaptic activity.