A muscle-specific calpain, CAPN3, forms a homotrimer
A muscle-specific calpain, CAPN3, forms a homotrimer
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DOI：
10.1016/j.bbapap.2020.140411
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发表时间：
2020-07-01
影响因子：
3.2
通讯作者：
Ono, Yasuko
中科院分区：
文献类型：
--
作者：
Hata, Shoji;Doi, Naoko;Ono, Yasuko
钙蛋白酶-3（CAPN 3）是钙蛋白酶蛋白酶家族的一个94-kDa成员，在骨骼肌中含量丰富。CAPN 3基因突变导致肢带型肌营养不良2A型，表明CAPN 3在肌肉生理学中起重要作用。CAPN 3有几个独特的功能。晶体学研究显示，其C-末端五EF-手结构域形成同源二聚体，表明CAPN 3作为同源二聚体蛋白酶起作用。为了以更方便的方式分析CAPN 3的复合物形成，我们进行了蓝色非变性聚丙烯酰胺凝胶电泳，发现观察到的非变性CAPN 3以及重组CAPN 3的分子量大于240 kDa。通过交联和连续免疫沉淀的进一步分析揭示，CAPN 3实际上形成同源三聚体。三聚体的形成被废除的PEF结构域的删除，但不是CAPN 3特异性插入序列NS，IS 1，和IS 2。如所报道的，单独的PEF结构域形成同二聚体，但是将相邻的CBSW结构域添加到其N-末端增强了三聚体形成性质。总的来说，这些结果表明CAPN 3形成同源三聚体，其中PEF结构域的二聚体形成能力受到其他结构域的影响。
Calpain-3 (CAPN3), a 94-kDa member of the calpain protease family, is abundant in skeletal muscle. Mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A, indicating that CAPN3 plays important roles in muscle physiology. CAPN3 has several unique features. A crystallographic study revealed that its C-terminal penta-EF-hand domains form a homodimer, suggesting that CAPN3 functions as a homodimeric protease. To analyze complex formation of CAPN3 in a more convenient manner, we performed blue native polyacrylamide gel electrophoresis and found that the observed molecular weight of native CAPN3, as well as recombinant CAPN3, was larger than 240 kDa. Further analysis by cross-linking and sequential immunoprecipitation revealed that CAPN3 in fact forms a homotrimer. Trimer formation was abolished by the deletion of the PEF domain, but not the CAPN3-specific insertion sequences NS, IS1, and IS2. The PEF domain alone formed a homodimer, as reported, but addition of the adjacent CBSW domain to its N-terminus reinforced the trimer-forming property. Collectively, these results suggest that CAPN3 forms a homotrimer in which the PEF domain's dimer-forming ability is influenced by other domains.