PATHOBIOCHEMISTRY OF ACUTE PROMYELOCYTIC LEUKEMIA

PML是一种环指蛋白，通常位于称为核体的特定亚核区室中。 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。 我们已经表明，野生型PML，但不是PML-RAR α融合蛋白，是共价修饰的sentrin蛋白。Sentrin是一个泛素样分子家族，可以共价连接到其他细胞蛋白。 在哺乳动物细胞中，有三种sentrin蛋白在所有组织中表达，并且似乎具有重叠的功能。 sentrin对蛋白质的共价修饰（sentrin化）通过一系列酶促步骤进行，使用专门的活化酶复合物（Aos 1/Uba 2）和独特的缀合酶（Ubc 9）。 虽然sentrinization和泛素化之间的酶促原理非常相似，但sentrinization不靶向蛋白质进行蛋白酶体降解，甚至可能抑制泛素化。 本研究旨在阐明PML中性化的机制及其功能意义。我们最近发现，赖氨酸-65在环指结构域，赖氨酸-160在B1盒，赖氨酸-490在核定位信号的PML作为三个主要的受体sentrin-1。 一个三重赖氨酸到精氨酸取代PML突变体正常表达，但不能sentrinized。 这一重要发现将为研究PML中性化的机制和功能提供必要的试剂。 其目标是：1）确定sentrin是否可以形成多聚体，2）确定Ubc 9是否是PML修饰的缀合酶，3）研究PML是否在细胞核中被正常sentrin化，4）评估sentrin化的PML是否靶向核体，以及5）评估sentrin化是否是PML的生物活性所需的。这些研究将为深入了解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认识。

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.