Ca2+ binding to EF hands 1 and 3 is essential for the interaction of apoptosis-linked gene-2 with Alix/AIP1 in ocular melanoma.
Ca2+ binding to EF hands 1 and 3 is essential for the interaction of apoptosis-linked gene-2 with Alix/AIP1 in ocular melanoma.
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DOI：
10.1021/bi048848d
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发表时间：
2004-09
期刊：
影响因子：
2.9
通讯作者：
L. Subramanian;J. Crabb;J. Cox;I. Durussel;T. M. Walker;P. R. Van Ginkel;S. Bhattacharya;Julia M Dellaria;K. Palczewski;A. Polans
中科院分区：
文献类型：
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作者：
L. Subramanian;J. Crabb;J. Cox;I. Durussel;T. M. Walker;P. R. Van Ginkel;S. Bhattacharya;Julia M Dellaria;K. Palczewski;A. Polans
凋亡相关基因-2（ALG-2）编码penta EF-手家族的22 kDa Ca（2+）结合蛋白，其是响应于各种凋亡剂的程序性细胞死亡所需的。在这里，我们证明，ALG-2的mRNA和蛋白质在人类葡萄膜黑色素瘤细胞相比，其祖细胞，正常黑色素细胞下调。ALG-2的下调可能为黑色素瘤细胞提供选择性优势。ALG-2及其推定的靶分子阿利克斯/AIP 1主要定位于黑素细胞和黑素瘤细胞的细胞质中，与细胞内Ca（2+）浓度或凋亡激活无关。交联和分析离心研究支持ALG-2的单种二聚体构象，也不依赖于Ca（2+）浓度。然而，如使用表面等离子体共振光谱所证明的，Ca（2+）与EF-1和EF-3两者的结合对于ALG-2与阿利克斯/AIP 1的相互作用是必需的。EF-5的突变导致靶相互作用降低，而不改变Ca（2+）亲和力。添加N-末端ALG-2肽（残基1-22或残基7-17）不改变ALG-2或ALG-2的N-末端缺失突变体与阿利克斯/AIP 1的相互作用，如从ALG-2的晶体结构衍生的模型所预期的。ALG-2的荧光研究表明，表面疏水性的增加主要是由于Ca（2+）结合EF-3，而Ca（2+）结合EF-1对疏水残基的表面暴露几乎没有影响。总之，这些数据表明总的表面疏水性变化不足以进行靶识别。
Apoptosis-linked gene-2 (ALG-2) encodes a 22 kDa Ca(2+)-binding protein of the penta EF-hand family that is required for programmed cell death in response to various apoptotic agents. Here, we demonstrate that ALG-2 mRNA and protein are down-regulated in human uveal melanoma cells compared to their progenitor cells, normal melanocytes. The down regulation of ALG-2 may provide melanoma cells with a selective advantage. ALG-2 and its putative target molecule, Alix/AIP1, are localized primarily in the cytoplasm of melanocytes and melanoma cells independent of the intracellular Ca(2+) concentration or the activation of apoptosis. Cross-linking and analytical centrifugation studies support a single-species dimer conformation of ALG-2, also independent of Ca(2+) concentration. However, binding of Ca(2+) to both EF-1 and EF-3 is necessary for ALG-2 interaction with Alix/AIP1 as demonstrated using surface plasmon resonance spectroscopy. Mutations in EF-5 result in reduced target interaction without alteration in Ca(2+) affinity. The addition of N-terminal ALG-2 peptides, residues 1-22 or residues 7-17, does not alter the interaction of ALG-2 or an N-terminal deletion mutant of ALG-2 with Alix/AIP1, as might be expected from a model derived from the crystal structure of ALG-2. Fluorescence studies of ALG-2 demonstrate that an increase in surface hydrophobicity is primarily due to Ca(2+) binding to EF-3, while Ca(2+) binding to EF-1 has little effect on surface exposure of hydrophobic residues. Together, these data indicate that gross surface hydrophobicity changes are insufficient for target recognition.