砷剂诱导核内LC3参与蛋白酶体途径降解PML-RARa融合蛋白新机制研究

Arsenic trioxide (As2O3) has been demonstrated to be one of the most effective therapeutic agents for patient with acute promyelocytic leukemia (APL), and the possible explanations for arsenic trioxide induced the cells differentiation is that As2O3 directly targeted PML-RARa fusion protein, resulted in initiating PML-RARa oncoprotein degradation. Regarding the mechanism of PML-RARa fusion protein degradation, two different pathways has been identified; one is a lysosomal degradation pathway (autophagy) and another is a proteasomal degradation pathway. In our preliminary experiment, we have clearly found that degradation of PML-RARa fusion protein in NB4 cells is not dependent on a lysosomal degradation pathway, but dependent on the proteasomal degradation pathway. Surprisingly, we first time found that nuclear LC3 is necessary protein for proteasomal degradation, because LC3 is normally thought to be a key factor for lysosomal degradation. Given these discovery, the interesting new results call into questions why nuclear LC3 is involved in proteasomal degradation. In order to reveal the molecular mechanism, we using three cell lines to determine the PML protein degradation by using the transfection or knock down the target genes by exposure to arsenic compounds. In fact, we trying to answer the following issues; (1) what is the role of nuclear LC3 for PML-RARa fusion protein degradation, (2) which steps is necessary for nuclear LC3 involvement during the PML protein degradation, (3) degradation of PML-RARa protein is not resulted by induction of autophagy (but proteasomal degradation). Our results are expected to explain the exact mechanism of PML-RARa fusion protein degradation, and it will improve the life span of APL patients after treatment of arsenic in near future.

目前利用三氧化二砷的毒性治疗急性早幼粒白血病（APL）已成为血液学的研究热点，研究发现三氧化二砷可通过降解APL特征性PML/RARa融合蛋白起到治疗作用。关于此融合蛋白的降解，现存在两种不同的学说，即自噬溶酶体和蛋白酶体途径。本课题组前期结果显示，PML/RARa融合蛋白与自噬溶酶降解途径无因果无关，但发现核内LC3参与泛素-蛋白酶体途径来降解PML/RARa融合蛋白。本项目拟进一步深入展开砷和核内LC3对PML蛋白的降解和调控机理，明确回答以下三个问题：细胞核内LC3在PML/RARa融合蛋白降解过程中的具体机理；核内LC3与PML蛋白的相互作用的位点；自噬发生与PML蛋白降解无因果关系。本课题组已取得切实可靠的研究数据，突破LC3仅参与溶酶体降解途径的理论束缚，率先提出核内LC3可能参与蛋白酶体的降解过程，阐明三氧化二砷治疗急性早幼粒白血病过程中PML/RARa 蛋白降解的确切机理。

三氧化二砷（As2O3）是目前治疗APL的有效药物之一，可通过靶向性降解急性早幼粒细胞白血病（Acute Promyelocytic Leukemia, APL）的肿瘤特异性致病癌蛋白（PML-RARα融合蛋白）而达到治疗目的。目前已经发现的PML蛋白主要有七种亚型，它们具有相同的N端(包含RBCC区域)和不同的C端。本课题首次发现，内源性PML V对砷剂具有比其他亚型高很多的灵敏性，其对加速PML-RARα融合蛋白的降解起到关键的作用。除蛋白酶体外，PML-RARα融合蛋白还可以通过自噬溶酶体途径降解。有文献报告，PML-RARα融合蛋白可以与SQSTM-1/p62蛋白在胞浆中共定位，SQSTM-1/p62可携带PML-RARα融合蛋白进入溶酶体中降解。本课题研究显示，SQSTM-1/p62特异性地与PML V蛋白相互作用，而不与PML其他亚型蛋白及PML-RARα融合蛋白直接相互作用。该结果表明，PML V可同时分别与PML-RARα融合蛋白、SQSTM-1/p62蛋白相互作用，进而形成一个复合体，使PML-RARα与SQSTM-1/p62共定位。SQSTM-1/p62蛋白可特异性地促进PML V蛋白及其复合体的泛素化修饰，进一步经蛋白酶体途径降解。除此之外，PML V蛋白还可以竞争性地与TRAF6蛋白结合SQSTM-1/p62蛋白，从而抑制NF-kB通路的激活。由此可见，PML V蛋白为一个潜在的肿瘤抑制因子。本课题拟阐明PML V蛋白的功能，完善As2O3治疗APL的分子机制，并希望以此为基础，结合临床，为治疗白血病提供理论基础。