Gu J, Dong R P, Zhang C, McLaughlin D F, Wu M X, Schlossman S F
Division of Tumor Immunology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1999 Jul 23;274(30):20759-62. doi: 10.1074/jbc.274.30.20759.
DNA fragmentation factor (DFF) functions downstream of caspase-3 and directly triggers DNA fragmentation during apoptosis. Here we described the identification and characterization of DFF35, an isoform of DFF45 comprised of 268 amino acids. Functional assays have shown that only DFF45, not DFF35, can assist in the synthesis of highly active DFF40. Using the deletion mutants, we mapped the function domains of DFF35/45 and demonstrated that the intact structure/conformation of DFF45 is essential for it to function as a chaperone and assist in the synthesis of active DFF40. Whereas the amino acid residues 101-180 of DFF35/45 mediate its binding to DFF40, the amino acid residues 23-100, which is homologous between DFF35/45 and DFF40, may function to inhibit the activity of DFF40. In contrast to DFF45, DFF35 cannot work as a chaperone, but it can bind to DFF40 more strongly than DFF45 and can inhibit its nuclease activity. These findings suggest that DFF35 may function in vivo as an important alternative mechanism to inhibit the activity of DFF40 and further, that the inhibitory effects of both DFF35 and DFF45 on DFF40 can put the death machinery under strict control.
DNA片段化因子(DFF)在半胱天冬酶-3下游发挥作用,并在细胞凋亡过程中直接触发DNA片段化。在此,我们描述了DFF35的鉴定和特性,它是由268个氨基酸组成的DFF45的一种同工型。功能分析表明,只有DFF45而非DFF35能够协助合成高活性的DFF40。利用缺失突变体,我们绘制了DFF35/45的功能结构域,并证明DFF45完整的结构/构象对于其作为伴侣蛋白发挥作用并协助合成活性DFF40至关重要。DFF35/45的氨基酸残基101 - 180介导其与DFF40的结合,而DFF35/45与DFF40之间同源的氨基酸残基23 - 100可能具有抑制DFF40活性的作用。与DFF45不同,DFF35不能作为伴侣蛋白发挥作用,但它比DFF45更能强烈地结合DFF40并能抑制其核酸酶活性。这些发现表明,DFF35可能在体内作为一种重要的替代机制来抑制DFF40的活性,此外,DFF35和DFF45对DFF40的抑制作用能够使死亡机制受到严格控制。