Department of Pathology and Translational Genomics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, 06351, Republic of Korea.
Division of Translational Science, National Cancer Center, Ilsan-ro 323, Ilsan-gu, Goyang, 10408, Republic of Korea.
Sci Rep. 2019 Oct 31;9(1):15771. doi: 10.1038/s41598-019-52364-6.
Angiogenesis is involved in both normal physiological and pathological conditions. Vascular endothelial growth factor (VEGF) is a major factor for promoting angiogenesis. The current anti-VEGF therapies have limited efficacy and significant adverse effects. To find novel targets of VEGFA for angiogenesis inhibition, we performed yeast two-hybrid screening and identified calpain-6 as a novel VEGFA-interaction partner and confirmed the endogenous VEGFA-calpain-6 interaction in mammalian placenta. A domain mapping study revealed that the Gly321-Asp500 domain in calpain-6 is required for the interaction with the C-terminus of the VEGFA protein. The functional significance of the VEGFA-calpain-6 interaction was explored by assessing its effect on angiogenesis in vitro. Whereas forced overexpression of calpain-6 increased the secretion of the VEGF protein and tube formation, knockdown of calpain-6 expression abrogated the calpain-6-mediated VEGF secretion and tube formation in HUVECs. Consistent with the domain mapping result, overexpressing calpain-6 without the VEGFA-interacting domain III (Gly321-Asp500) failed to increase the secretion of VEGF protein. Our results identify calpain-6, an unconventional non-proteolytic calpain, as a novel VEGFA-interacting protein and demonstrate that their interaction is necessary to enhance VEGF secretion. Thus, calpain-6 might be a potential molecular target for angiogenesis inhibition in many diseases.
血管生成参与正常生理和病理状态。血管内皮生长因子 (VEGF) 是促进血管生成的主要因素。目前的抗 VEGF 治疗方法疗效有限，且存在显著的不良反应。为了寻找抑制血管生成的 VEGFA 的新靶点，我们进行了酵母双杂交筛选，发现钙蛋白酶-6 是 VEGFA 的一个新的相互作用伙伴，并在哺乳动物胎盘内证实了内源性 VEGFA-钙蛋白酶-6 相互作用。结构域映射研究表明，钙蛋白酶-6 的 Gly321-Asp500 结构域是与 VEGFA 蛋白 C 端相互作用所必需的。通过评估其对体外血管生成的影响，研究了 VEGFA-钙蛋白酶-6 相互作用的功能意义。尽管钙蛋白酶-6 的强制过表达增加了 VEGF 蛋白的分泌和管形成，但钙蛋白酶-6 表达的下调消除了 HUVEC 中钙蛋白酶-6 介导的 VEGF 分泌和管形成。与结构域映射结果一致，过表达没有与 VEGFA 相互作用结构域 III（Gly321-Asp500）的钙蛋白酶-6 不能增加 VEGF 蛋白的分泌。我们的研究结果将非常规非蛋白水解钙蛋白酶-6 鉴定为 VEGFA 的一个新的相互作用蛋白，并证明它们的相互作用对于增强 VEGF 分泌是必需的。因此，钙蛋白酶-6 可能成为许多疾病中抑制血管生成的潜在分子靶点。
