Shapovalov Ivan, Rimal Prawin, Poudel Pitambar, Lewtas Victoria, Bell Mathias, Panday Shailesh Kumar, Laight Brian J, Harper Danielle, Grieve Stacy, Baillie George S, Nouri Kazem, Davies Peter L, Alexov Emil, Greer Peter A
Department Pathology and Molecular Medicine, School of Medicine, Queen's University, Kingston, Ontario, Canada; Division of Cancer Biology and Genetics, Sinclair Cancer Research Institute, Queen's University, Kingston, Ontario, Canada.
Department of Physics, College of Science, Clemson University, Clemson, South Carolina, USA.
J Biol Chem. 2025 May 16;301(6):110243. doi: 10.1016/j.jbc.2025.110243.
Calpain-1 and calpain-2 are heterodimeric proteases consisting of a common small regulatory subunit CAPNS1 and a large catalytic subunit, CAPN1 or CAPN2, respectively. These calpains have emerged as potential therapeutic targets in cancer and other diseases through their roles in cell signaling pathways affecting sensitivity to chemotherapeutic and targeted drugs and in promoting metastasis. While inhibition of calpains has the potential to provide therapeutic benefit to cancer patients, there are currently no clinically approved active site-directed drugs that specifically and effectively inhibit them. However, the structures of calpain-1 and calpain-2 make them susceptible to allosteric inhibition aimed at interfering with heterodimerization of the catalytic and regulatory subunits, which is necessary for stability and proteolytic activity. Split-Nanoluciferase biosensors were generated to quantify the protein-protein interactions between the calcium-binding penta-EF-hand domains of CAPN1 or CAPN2 and CAPNS1. These biosensors were used to quantify the heterodimer dissociation constants (K) of calpain-1 and calpain-2, estimated at 185 nM and 509 nM, respectively, in the presence of 5 mM Ca; and 362 nM and 1651 nM, respectively, in the presence of Mg. The half-maximal Ca concentrations supporting these protein-protein interactions for calpain-1 and calpain-2 were 59.9 μM and 940.8 μM, respectively. Molecular modeling, based on the crystal structure of calpain-2, was used to predict 20 residues of the penta-EF-hand domains that contribute to heterodimerization. Individual point mutation of CAPNS1 at Q263 reduced the catalytic activity of calpain-2 to 51.0 ± 6.4% in live cells.
钙蛋白酶-1和钙蛋白酶-2是异二聚体蛋白酶，分别由一个共同的小调节亚基CAPNS1和一个大催化亚基CAPN1或CAPN2组成。这些钙蛋白酶通过在影响对化疗药物和靶向药物敏感性的细胞信号通路以及促进转移中发挥作用，已成为癌症和其他疾病潜在的治疗靶点。虽然抑制钙蛋白酶有可能为癌症患者带来治疗益处，但目前尚无临床批准的能特异性有效抑制其活性位点的药物。然而，钙蛋白酶-1和钙蛋白酶-2的结构使其易受变构抑制，这种抑制旨在干扰催化亚基和调节亚基的异二聚化，而异二聚化对稳定性和蛋白水解活性至关重要。构建了分裂纳米荧光素酶生物传感器来定量CAPN-1或CAPN-2的钙结合五聚EF手结构域与CAPNS1之间的蛋白质-蛋白质相互作用。这些生物传感器用于定量钙蛋白酶-1和钙蛋白酶-2的异二聚体解离常数（K），在存在5 mM Ca的情况下，估计分别为185 nM和509 nM；在存在Mg的情况下，分别为362 nM和1651 nM。支持钙蛋白酶-1和钙蛋白酶-2这些蛋白质-蛋白质相互作用的半数最大Ca浓度分别为59.9 μM和940.8 μM。基于钙蛋白酶-2的晶体结构进行分子建模，以预测对异二聚化有贡献的五聚EF手结构域的20个残基。在活细胞中，CAPNS1的Q263位点单个点突变使钙蛋白酶-2的催化活性降低至51.0±6.4%。
