Hlaing T, Guo R F, Dilley K A, Loussia J M, Morrish T A, Shi M M, Vincenz C, Ward P A
Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 2001 Mar 23;276(12):9230-8. doi: 10.1074/jbc.M009853200. Epub 2000 Nov 13.
We report the deduced amino acid sequences of two alternately spliced isoforms, designated DEFCAP-L and -S, that differ in 44 amino acids and encode a novel member of the mammalian Ced-4 family of apoptosis proteins. Similar to the other mammalian Ced-4 proteins (Apaf-1 and Nod1), DEFCAP contains a caspase recruitment domain (CARD) and a putative nucleotide binding domain, signified by a consensus Walker's A box (P-loop) and B box (Mg(2+)-binding site). Like Nod1, but different from Apaf-1, DEFCAP contains a putative regulatory domain containing multiple leucine-rich repeats (LRR). However, a distinguishing feature of the primary sequence of DEFCAP is that DEFCAP contains at its NH(2) terminus a pyrin-like motif and a proline-rich sequence, possibly involved in protein-protein interactions with Src homology domain 3-containing proteins. By using in vitro coimmunoprecipitation experiments, both long and short isoforms were capable of strongly interacting with caspase-2 and exhibited a weaker interaction with caspase-9. Transient overexpression of full-length DEFCAP-L, but not DEFCAP-S, in breast adenocarcinoma cells MCF7 resulted in significant levels of apoptosis. In vitro death assays with transient overexpression of deletion constructs of both isoforms using beta-galactosidase as a reporter gene in MCF7 cells suggest the following: 1) the nucleotide binding domain may act as a negative regulator of the killing activity of DEFCAP; 2) the LRR/CARD represents a putative constitutively active inducer of apoptosis; 3) the killing activity of LRR/CARD is inhibitable by benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone and to a lesser extent by Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone; and 4) the CARD is critical for killing activity of DEFCAP. These results suggest that DEFCAP is a novel member of the mammalian Ced-4 family of proteins capable of inducing apoptosis, and understanding its regulation may elucidate the complex nature of the mammalian apoptosis-promoting machinery.
我们报道了两种交替剪接异构体（命名为DEFCAP-L和-S）的推导氨基酸序列，它们在44个氨基酸上存在差异，编码哺乳动物凋亡蛋白Ced-4家族的一个新成员。与其他哺乳动物Ced-4蛋白（凋亡蛋白酶激活因子-1（Apaf-1）和核苷酸结合寡聚化结构域1（Nod1））相似，DEFCAP含有一个半胱天冬酶募集结构域（CARD）和一个假定的核苷酸结合结构域，由保守的沃克A框（P环）和B框（镁离子结合位点）表示。与Nod1一样，但与Apaf-1不同，DEFCAP含有一个假定的调节结构域，其中包含多个富含亮氨酸的重复序列（LRR）。然而，DEFCAP一级序列的一个显著特征是，DEFCAP在其氨基末端含有一个类吡啉基序和一个富含脯氨酸的序列，可能参与与含Src同源结构域3的蛋白的蛋白质-蛋白质相互作用。通过体外共免疫沉淀实验，长、短异构体均能与半胱天冬酶-2强烈相互作用，并与半胱天冬酶-9表现出较弱的相互作用。在乳腺腺癌细胞MCF7中瞬时过表达全长DEFCAP-L而非DEFCAP-S，导致显著水平的细胞凋亡。在MCF7细胞中使用β-半乳糖苷酶作为报告基因对两种异构体的缺失构建体进行瞬时过表达的体外死亡分析表明：1）核苷酸结合结构域可能作为DEFCAP杀伤活性的负调节因子；2）LRR/CARD代表一种假定的组成型活性凋亡诱导剂；3）LRR/CARD的杀伤活性可被苄氧羰基-Val-Ala-Asp（OMe）-氟甲基酮抑制，在较小程度上可被Asp-Glu-Val-Asp（OMe）-氟甲基酮抑制；4）CARD对DEFCAP的杀伤活性至关重要。这些结果表明，DEFCAP是哺乳动物Ced-4蛋白家族中能够诱导细胞凋亡的一个新成员，了解其调控机制可能有助于阐明哺乳动物凋亡促进机制的复杂本质。
