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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21GM057502-02
Application #
6180553
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Shapiro, Bert I
Project Start
1999-09-01
Project End
2001-08-31
Budget Start
2000-09-01
Budget End
2001-08-31
Support Year
2
Fiscal Year
2000
Total Cost
$104,650
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
800771594
City
Houston
State
TX
Country
United States
Zip Code
77225