PATHOBIOCHEMISTRY OF ACUTE PROMYELOCYTIC LEUKEMIA

PML是一种环指蛋白，通常存在于称为核体的特定亚核室中。在急性早幼粒细胞白血病中，PML基因与视黄酸受体α (rar α)基因融合，导致PML- rar α融合蛋白的产生。我们已经证明野生型PML，而不是PML- rar α融合蛋白，被前哨蛋白共价修饰。Sentrin是一类泛素类分子，可与其他细胞蛋白共价结合。在哺乳动物细胞中，有三种sentrin蛋白在所有组织中表达，似乎具有重叠的功能。哨兵蛋白的共价修饰（哨兵化）是通过一系列酶的步骤发生的，这些步骤使用一个特殊的激活酶复合物（Aos1/Uba2）和一个独特的偶联酶（Ubc9）。尽管sentrinization和泛素化之间的酶学原理非常相似，但sentrinization并不针对蛋白酶体降解的蛋白质，甚至可能抑制泛素化。本文旨在阐明PML sentrinization的机制及其功能意义。我们最近发现环指结构域中的Lys-65、B1盒中的Lys-160和PML核定位信号中的Lys-490是sentrin-1的三个主要受体。三赖氨酸到精氨酸置换的PML突变体正常表达，但不能被诱导。这一关键的见解将为研究PML sentrinization的机制和功能提供必要的试剂。目的是：1)确定sentrin是否能形成多聚体，2)确定Ubc9是否为PML修饰的偶联酶，3)研究PML在细胞核中是否正常sentrin化，4)评价sentrin化后的PML是否靶向核体，5)评估PML的生物活性是否需要sentrin化。这些研究将为sentrinization的机制和功能提供新的见解，并增加我们对急性早幼粒细胞白血病发病机制的理解。

PML is a RING finger protein that normally resides within a specific subnuclear compartment termed the nuclear body. In acute promyelocytic leukemia, the PML gene is fused to the retinoic acid receptor alpha (RARalpha) gene, resulting in the production of PML-RARalpha fusion proteins. We have shown that wild type PML, but not the PML-RARalpha fusion proteins, are covalently modified by the sentrin proteins. Sentrin is a family of ubiquitin-like molecules which can be covalently attached to other cellular proteins. In mammalian cells, there are three sentrin proteins that are expressed in all tissues and appear to have overlapping function. Covalent modification of proteins by sentrin (sentrinization) occurs through a series of enzymatic steps using a specialized activating-enzyme complex (Aos1/Uba2) and a unique conjugating enzyme (Ubc9). Although the enzymatic principles between sentrinization and ubiquitination are remarkably similar, sentrinization does not target proteins for proteasomal degradation and may even inhibit ubiquitination. This proposal is designed to elucidate the mechanism of PML sentrinization and its functional significance. We have recently shown that Lys-65 in the RING finger domain, Lys-160 in the B1 box, and Lys-490 in the nuclear localization signal of PML serve as three major acceptors for sentrin-1. A triple Lys to Arg substitution PML mutant is expressed normally, but cannot be sentrinized. This critical insight will provide an essential reagent to examine the mechanism and function of PML sentrinization. The aims are: 1) to determine whether sentrin can form multimers, 2) to define whether Ubc9 is the conjugating enzyme for PML modification, 3) to study whether PML is normally sentrinized in the nucleus, 4) to evaluate whether sentrinized PML is targeted to the nuclear body, and 5) to assess whether sentrinization is required for PML's biological activities. These studies should provide novel insights into the mechanism and function of sentrinization and increase our understanding of the pathogenesis of acute promyelocytic leukemia.