ABSTRACT
Adult T-cell leukemia (ATL) is an often fatal malignancy caused by infection with the complex retrovirus, human T-cell Leukemia Virus, type 1 (HTLV-1). In ATL patient samples, the tumor suppressor, p53, is infrequently mutated; however, it has been shown to be inactivated by the viral protein, Tax. Here, we show that another HTLV-1 protein, HBZ, represses p53 activity. In HCT116 p53+/+ cells treated with the DNA-damaging agent, etoposide, HBZ reduced p53-mediated activation of p21/CDKN1A and GADD45A expression, which was associated with a delay in G2 phase-arrest. These effects were attributed to direct inhibition of the histone acetyltransferase (HAT) activity of p300/CBP by HBZ, causing a reduction in p53 acetylation, which has be linked to decreased p53 activity. In addition, HBZ bound to, and inhibited the HAT activity of HBO1. Although HBO1 did not acetylate p53, it acted as a coactivator for p53 at the p21/CDKN1A promoter. Therefore, through interactions with two separate HAT proteins, HBZ impairs the ability of p53 to activate transcription. This mechanism may explain how p53 activity is restricted in ATL cells that do not express Tax due to modifications of the HTLV-1 provirus, which accounts for a majority of patient samples.
Subject(s)
Basic-Leucine Zipper Transcription Factors/metabolism , Histone Acetyltransferases/metabolism , Retroviridae Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , p300-CBP Transcription Factors/metabolism , Acetylation/drug effects , Animals , Basic-Leucine Zipper Transcription Factors/genetics , Blotting, Western , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Etoposide/pharmacology , G2 Phase Cell Cycle Checkpoints/drug effects , G2 Phase Cell Cycle Checkpoints/genetics , Gene Expression Regulation, Neoplastic/drug effects , HCT116 Cells , HEK293 Cells , HeLa Cells , Histone Acetyltransferases/antagonists & inhibitors , Histone Acetyltransferases/genetics , Human T-lymphotropic virus 1/genetics , Human T-lymphotropic virus 1/metabolism , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Retroviridae Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Protein p53/genetics , p300-CBP Transcription Factors/antagonists & inhibitors , p300-CBP Transcription Factors/geneticsABSTRACT
Bla g 1 is a major allergen from Blatella germanica and one of the primary allergens used to assess cockroach allergen exposure. The epitope of an anti-Bla g 1 scFv was mapped in order to better understand cross reactivity with other group 1 cockroach allergens and patient IgE epitopes. X-ray crystallography was used to determine the structure of the scFv. The scFv epitope on Bla g 1 was located by alanine scanning site-directed mutagenesis and ELISA. Twenty-six rBla g 1-GST alanine mutants were evaluated for variations in binding to the scFv compared to the wild type allergen. Six mutants showed a significant difference in scFv binding affinity. These mutations clustered to form a discontinuous epitope mainly comprising two helices of Bla g 1. The allergen-scFv complex was modeled based on the results, and the epitope region was found to have low sequence similarity with Per a 1, especially among the residues identified as functionally important for the scFv binding to Bla g 1. Indeed, the scFv failed to bind Per a 1 in American cockroach extract. The scFv was unable to inhibit the binding of IgE antibodies from a highly cockroach allergic patient to Bla g 1. Based on the surface area of Bla g 1 occluded by the scFv, putative regions of patient IgE-Bla g 1 interactions can be inferred. This scFv could be best utilized as a capture antibody in an IgE detection ELISA, or to differentiate Bla g 1 from Per a 1 in environmental exposure assays.
