Date of Award

2010-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Manuel Llano

Abstract

The lens epithelium-derived growth factor (LEDGF) proteins p75 and p52 are transcriptional co-activators that protect cells from stresses through modulation of stress and heat shock-related genes. Besides regulating such genes, LEDGF/p75 is also important in the process of HOX gene expression and leukemia transformation driven by the MLL histone methyl transferase complex. By exploiting a similar mechanism of interaction between LEDGF/p75 and MLL, the HIV-1 viral protein Integrase (IN) associates with LEDGF/p75 in order to execute efficient viral DNA integration. This present work has identified that LEDGF proteins are posttranslationally modified by SUMO-1 and -3. SUMOylation was found to target the lysine residues K75, K250 and K254 situated at the shared N-terminal region of these molecules.

Additionally, LEDGF/p75 was demonstrated to be SUMOylated at the residue K364 localized at its C-terminal region. Lysine to arginine mutations of these lysine residues drastically inhibited LEDGF SUMOylation. SUMOylation-deficient LEDGF/p75 and p52 presented chromatin binding abilities similar to the wild type proteins and were both localized in the cell nuclei.

However, inhibition of LEDGF/p75 SUMOylation significantly extended its half-life and increased its transcriptional activity on the Heat shock protein 27 (Hsp27) promoter. Moreover, the HIV-1 co-factor activity of LEDGF/p75 was also affected by SUMO modification. CD4+ T cells expressing SUMOylation-deficient LEDGF/p75 were notably less susceptible to HIV-1 infection when compared to control cells. This impairment in infectivity does not appear to be caused by alterations in the association between LEDGF/p75 and IN since SUMOylation-deficient LEDGF/p75 is able to bind and tether IN to chromatin. Preliminary data suggests that LEDGF/p75 SUMOylation modulates HIV-1 viral DNA integration in a process possibly coupled to mechanisms of DNA repair. This study provides a new perspective for future studies of the molecular mechanisms involved in the cellular and virological functions of LEDGF/p75.

Language

en

Provenance

Received from ProQuest

File Size

79 pages

File Format

application/pdf

Rights Holder

Murilo Tadeu Domingues Bueno

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