STUDIES OF THE SENTRIN FAMILY OF UBIQUITIN-LIKE PROTEINS

前丝蛋白是一类泛素类分子，可以共价连接到其他细胞蛋白上。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。利用一种特殊的活化酶复合体(Aos1/Uba2)和一种独特的结合酶(Ubc9)，通过一系列的酶步骤，对蛋白质进行Sentrin的共价修饰。尽管Sentrin化和泛素化之间的酶原理非常相似，Sentrin化并不以蛋白酶体降解为目标，甚至可能抑制泛素化。这一建议旨在阐明以PML为模型底物的句子化的机制和功能。PML是一种环指蛋白，通常存在于被称为核体的特定亚核室中。在急性早幼粒细胞白血病中，PML基因与维甲酸受体α(RARpha)基因融合，导致PML-RARpha融合蛋白的产生。我们已经证明，野生型PML，而不是PML-RARpha融合蛋白，是由Sentrin蛋白共价修饰的。突变分析表明，位于PML环指结构域的Lys-65、位于B1盒的Lys-160和位于核定位信号中的Lys-490是Sent rin-1的三个主要受体。本文旨在验证关于PML句子化机制和功能的五个假说。其目的是：1)确定Sentrin是否可以形成多聚体；2)确定Ubc9是否是PML修饰的结合酶；3)研究PML是否正常地在细胞核内定位；4)评价定位后的PML是否针对核体；5)评估PML的生物学活性是否需要定位。这些研究将为前哨化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。

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 and function of sentrinization using PML as a model substrate. 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. Mutational analyses showed 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. The following aims are proposed to test five hypotheses concerning 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.