Multi-sites cleavage of leukemogenic AML1-ETO fusion protein by caspase-3 and its contribution to increased apoptotic sensitivity
Multi-sites cleavage of leukemogenic AML1-ETO fusion protein by caspase-3 and its contribution to increased apoptotic sensitivity
复制标题
Caspase-3 对白血病 AML1-ETO 融合蛋白的多位点切割及其对提高细胞凋亡敏感性的贡献
DOI：
10.1038/sj.leu.2405020
复制
发表时间：
2008-02-01
期刊：
影响因子：
11.4
通讯作者：
Chen, G-Q
中科院分区：
文献类型：
--
作者：
Lu, Y.;Peng, Z-G;Chen, G-Q
白血病相关融合蛋白AML 1-ETO是急性髓细胞白血病（AML）中常见的染色体易位（8; 21）的产物。融合癌蛋白阻断白血病细胞分化，并且它还诱导生长停滞，增加对凋亡诱导的敏感性。这种二分功能使得很难阐明AML 1-ETO在白血病发生中的作用。在这里，我们系统地表明，组成性和过表达的AML 1-ETO蛋白裂解为四个片段的70，49，40和25 kDa的激活caspase-3在细胞凋亡诱导的外源性线粒体和死亡受体信号通路。体外蛋白水解系统结合MALDI-TOF/TOF质谱仪证实AML 1-ETO和野生型ETO蛋白是凋亡执行蛋白caspase-3的直接底物，而RUNX 1（AML 1）蛋白不是。定点突变分析确定了两个非经典的突变位点（TMPD 188和LLLD 368）作为AML 1-ETO序列中的caspase-3靶向位点。当这两种谷氨酸突变为丙氨酸时，更有趣的是，AML 1-ETO诱导的凋亡放大作用完全消失，而在四环素去除后，AML 1-ETO的caspase-3切割的70 kDa片段的诱导表达足以增强凋亡敏感性。对这种切割的潜在体内效应及其在白血病发生中的可能作用的进一步研究将为理解AML 1-ETO相关白血病的生物学和治疗提供新的见解。
Leukemia-associated fusion protein AML1-ETO is a product of the chromosome translocation (8; 21) frequently occurred in acute myeloid leukemia (AML). The fusion oncoprotein blocks leukemic cell differentiation, and it also induces growth arrest with increased sensitivity to apoptosis induction. Such dichotomous functions make it difficult to clarify the role of AML1-ETO in leukemogenesis. Here, we systematically showed that constitutively and overexpressed AML1-ETO protein was cleaved to four fragments of 70, 49, 40 and 25 kDa by activated caspase-3 during apoptosis induction by extrinsic mitochondrial and death receptor signaling pathways. The in vitro proteolytic system combined with MALDI-TOF/TOF mass spectrometer confirmed that AML1-ETO and wild-type ETO but not RUNX1 (AML1) proteins were direct substrates of apoptosis executioner caspase-3. Site-directed mutagenesis analyses identified two nonclassical aspartates (TMPD 188 and LLLD 368) as caspase-3-targeted sites in the AML1-ETO sequence. When these two aspartates were mutated into alanines, more intriguingly, the apoptosis-amplified action of AML1-ETO induction completely disappeared, while inducible expression of the caspase-3-cleaved 70 kDa fragment of AML1-ETO after tetracycline removal is sufficient to enhance apoptotic sensitivity. Further investigations on the potential in vivo effects of such a cleavage and its possible role in leukemogenesis would provide new insights for understanding the biology and treatment of AML1-ETO-associated leukemia.