Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, 117543, Singapore.
School of Biological Sciences, Nanyang Technological University, Singapore, 637551, Singapore.
Cell Death Dis. 2022 Feb 23;13(2):172. doi: 10.1038/s41419-022-04618-x.
A Disintegrin and Metalloproteinase with ThromboSpondin motif (ADAMTS) 5 functions as an anti-angiogenic and anti-cancer protein independent of its metalloproteinase activity. Both full-length ADAMTS5 and TS5-p45, the autocatalytically cleaved C-terminal 45 kDa truncate of ADAMTS5, inhibits angiogenesis, and induces endothelial cell (EC) apoptosis. However, how ADAMTS5 triggers EC apoptosis remains unclear. This work shows that caspase-8 (Cas-8) and caspase-9 (Cas-9) are involved in TS5-p45-induced EC apoptosis. We identify cell surface nucleolin (NCL) as a novel high-affinity receptor for TS5-p45 in ECs, mediating TS5-p45's cell surface binding and pro-apoptotic function. We show that the central RNA-binding domain (RBD) of NCL is essential and sufficient for its binding to TS5-p45. Upon interacting with EC surface NCL, TS5-p45 is internalized through clathrin- and caveolin-dependent endocytosis and trafficked to the nucleus via late endosomes (LEs). We demonstrate that the nuclear trafficking of TS5-p45 is important for its pro-apoptotic activity as disruption of LE membrane integrity with an endosomolytic peptide suppressed both nuclear trafficking and pro-apoptotic activity of TS5-p45. Through cell surface biotinylation, we revealed that cell surface NCL shuttles extracellular TS5-p45 to the nucleus to mediate apoptosis. Furthermore, blocking the importin α1/ß1 receptor hindered the nuclear trafficking of TS5-p45, suggesting the involvement of the nuclear importing machinery for this nuclear translocation. RNA-seq identified many apoptosis-related genes that are differentially expressed at least two-fold in TS5-p45-treated ECs, with 10 of them qRT-PCR-validated and at least 5 of these genes potentially contributing to TS5-p45-NCL-induced apoptosis. Altogether, our work identifies NCL as a novel cell surface receptor for ADAMTS5 and demonstrates the critical role of NCL-mediated internalization and nuclear trafficking for ADAMTS5-induced EC apoptosis. These findings reveal novel mechanistic insights of the secreted metalloproteinase ADAMTS5 in angiogenesis inhibition.
整合素和金属蛋白酶与血栓反应蛋白基序（ADAMTS）5 作为一种抗血管生成和抗癌蛋白，其功能独立于其金属蛋白酶活性。全长 ADAMTS5 和 TS5-p45，即 ADAMTS5 的自动催化切割的 C 端 45 kDa 截断物，均可抑制血管生成，并诱导内皮细胞（EC）凋亡。然而，ADAMTS5 如何引发 EC 凋亡尚不清楚。本工作表明半胱天冬酶-8（Cas-8）和半胱天冬酶-9（Cas-9）参与了 TS5-p45 诱导的 EC 凋亡。我们鉴定出细胞表面核仁素（NCL）是 EC 中 TS5-p45 的新型高亲和力受体，介导 TS5-p45 的细胞表面结合和促凋亡功能。我们表明，NCL 的中央 RNA 结合域（RBD）对于其与 TS5-p45 的结合是必需和充分的。与 EC 表面 NCL 相互作用后，TS5-p45 通过网格蛋白和小窝蛋白依赖性内吞作用内化，并通过晚期内体（LE）转运到核内。我们证明，TS5-p45 的核内转运对其促凋亡活性很重要，因为用内体溶酶体肽破坏 LE 膜完整性会抑制 TS5-p45 的核内转运和促凋亡活性。通过细胞表面生物素化，我们揭示了细胞表面 NCL 将细胞外 TS5-p45 穿梭到核内以介导凋亡。此外，阻断导入蛋白 α1/β1 受体阻碍了 TS5-p45 的核内转运，这表明核内转运涉及核导入机制。RNA-seq 鉴定了许多在 TS5-p45 处理的 EC 中差异表达至少两倍的凋亡相关基因，其中 10 个经 qRT-PCR 验证，至少 5 个基因可能对 TS5-p45-NCL 诱导的凋亡有贡献。总之，我们的工作确定 NCL 是 ADAMTS5 的一种新型细胞表面受体，并证明了 NCL 介导的内化和核内转运在 ADAMTS5 诱导的 EC 凋亡中的关键作用。这些发现揭示了分泌型金属蛋白酶 ADAMTS5 在血管生成抑制中的新的机制见解。
