锌指结构的完整对砷剂诱导PML蛋白由核质转移至核基质的必要性及参与蛋白的发现

Arsenic trioxide (As2O3) has been demonstrated to be one of the most effective therapeutic agents for patient with acute promyelocytic leukemia (APL), and generally the possible explanation through which As2O3 exerts its therapeutic effect is degradation of an oncogenic protein PML-RARa by directly binds to cysteine residues of Zinc Fingers in RBCC domain of PML-RARα fusion protein (i.e., Replacement of zinc by As2O3), facilitating the oligomerization of PML proteins and then induces the PML toward the nuclear matrix (i.e., mobility changes), finally degraded by proteasome pathway. However, our preliminary experiment designates extraordinary results; we found that As2O3 completely lost induction of nucleoplasmic PML toward the nuclear matrix if chelating the Zinc ions from Zinc Fingers. In addition, As2O3 also failed to induce PML protein toward the nuclear matrix if we point mutated the cysteine or histidine residues at zinc finger motifs of RBCC in PML proteins, these indicates that the integrity of zinc finger motifs is essential for induction of nucleoplasmic PML toward the nuclear matrix by arsenic. More interestingly, As2O3 is also unable to induce the PML protein toward the matrix at low temperature 4°C, suggesting that only the integrity of zinc finger motifs is not enough condition for induction of nucleoplasmic PML toward the nuclear matrix by arsenic trioxide, it also may be depends on the other factors such as energy or proteins (e.g., proteins or enzymes). Given these discoveries, the interesting new results call into questions what proteins are involved in changes in PML protein mobility based on maintain of zinc finger motifs. In order to reveal the molecular mechanism underneath this, we using three cell lines (i.e., NB4, HEK293T and PML minus MEF cells) or Semi-Intact cells to determine the PML mobility by using the transfection or knock down the target genes after exposure to arsenic trioxide. In fact, we are trying to resolve the following issues; (1) what is the exact molecular mechanism of PML protein degradation following exposure to arsenic? (2) The maintain integrity of zinc finger motifs in PML is enough condition for induction of nucleoplasmic to matrix? (3) What kind of nuclear proteins are involved in this reaction? Our results will be expected to explain the exact mechanism of PML-RARa fusion protein degradation, which will further improve the life span of APL patients by the treatment with arsenic in near future.

三氧化二砷治疗急性早幼粒白血病（APL）成为血液学的研究热点。目前，多数研究者认为砷取代锌离子直接与PML蛋白锌指结构中半胱氨酸残基结合，促使PML发生多聚化、从核质转移至核基质等过程，最终经蛋白酶体途径降解，达到治疗疾病的目的。但我们发现，锌离子被螯合后或破坏PML锌指结构后，砷剂完全丧失了诱导PML由核质转至核基质的能力。因此，保持锌指结构的完整性是砷剂发挥作用的必要条件。在此基础上，我们还发现砷剂诱导PML由核质转至核基质还需要能量以及核内蛋白的参与。本项目通过研究锌指结构在砷剂治疗APL过程中的作用，明确回答以下三个问题：保持PML蛋白的锌指结构是砷剂诱导PML蛋白核质转移至核基质必要条件；确定参与核基质反应的关键核蛋白；揭示该核蛋白的功能及生理学意义，阐明砷剂诱导PML蛋白核质转移至核基质的分子机制。

三氧化二砷（As2O3，又称砷剂、亚砷酸）是目前急性早幼粒细胞白血病（Acute Promyelocytic Leukemia, APL）治疗的一线药物，它主要作用于PML/RARα融合蛋白的PML部分，诱导PML蛋白与PML-RARα融合蛋白的多聚化，并使其发生构象改变（生化表现为该蛋白由可溶转变为不可溶），进而发生一系列的蛋白修饰，最终被蛋白酶体降解。研究表明PML 蛋白的RBCC 结构域包含一个“环指”（Ring Finger）的结构，两个富含半胱氨酸区（B-box1 和B-box2）以及一个卷曲螺旋结构（Coiled-Coil domain），其中Ring、B-box1 和box2 区域中各含有两个锌指结构（共含有6 Zinc Fingers）。通常，锌指结构主要由半胱氨酸和/或组氨酸与锌离子配位组成，锌离子缺乏会破坏锌指蛋白的结构稳定性。因此，在锌指蛋白中，锌的地位是不可替代的，它的存在是锌指蛋白发挥调控作用的关键。目前多数研究者认为As2O3取代锌离子直接与PML 蛋白锌指结构的半胱氨酸残基结合，导致PML-RARα 融合蛋白由可溶性转变为不溶性，最终被蛋白酶体降解。通过本课题的研究，我们发现当锌指结构被破坏时，半胱氨酸和组氨酸残基具有重新组合成新的锌指结构以维持原锌指结构功能的特性。三氧化二砷降解PML蛋白不仅与锌指结构上的半胱氨酸有关，还与锌指结构上的组氨酸有密切联系。而在同一结构域中，锌指结构存在不同氨基酸组合方式，并可发生相互转变以维持原锌指结构功能，并且PML蛋白RING和B-box结构域中锌指结构的完整性不受结构域外的半胱氨酸干扰。在后续研究中，我们将进一步探究RBCC结构与砷剂相互作用的机制，完善砷剂治疗APL的分子机制。