ABSTRACT
BACKGROUND: Adverse drug reactions (ADRs) are a significant problem for HIV patients, with the risk of developing ADRs increasing as the infection progresses to AIDS. However, the pathophysiology underlying ADRs remains unknown. Sulphamethoxazole (SMX) via its active metabolite SMX-hydroxlyamine, when used prophylactically for pneumocystis pneumonia in HIV-positive individuals, is responsible for a high incidence of ADRs. We previously demonstrated that the HIV infection and, more specifically, that the HIV-1 Tat protein can exacerbate SMX-HA-mediated ADRs. In the current study, Jurkat T cell lines expressing Tat and its deletion mutants were used to determine the effect of Tat on the thiol proteome in the presence and absence of SMX-HA revealing drug-dependent changes in the disulfide proteome in HIV infected cells. Protein lysates from HIV infected Jurkat T cells and Jurkat T cells stably transfected with HIV Tat and Tat deletion mutants were subjected to quantitative slot blot analysis, western blot analysis and redox 2 dimensional (2D) gel electrophoresis to analyze the effects of SMX-HA on the thiol proteome. RESULTS: Redox 2D gel electrophoresis demonstrated that untreated, Tat-expressing cells contain a number of proteins with oxidized thiols. The most prominent of these protein thiols was identified as peroxiredoxin. The untreated, Tat-expressing cell lines had lower levels of peroxiredoxin compared to the parental Jurkat E6.1 T cell line. Conversely, incubation with SMX-HA led to a 2- to 3-fold increase in thiol protein oxidation as well as a significant reduction in the level of peroxiredoxin in all the cell lines, particularly in the Tat-expressing cell lines. CONCLUSION: SMX-HA is an oxidant capable of inducing the oxidation of reactive protein cysteine thiols, the majority of which formed intermolecular protein bonds. The HIV Tat-expressing cell lines showed greater levels of oxidative stress than the Jurkat E6.1 cell line when treated with SMX-HA. Therefore, the combination of HIV Tat and SMX-HA appears to alter the activity of cellular proteins required for redox homeostasis and thereby accentuate the cytopathic effects associated with HIV infection of T cells that sets the stage for the initiation of an ADR.
Subject(s)
Oxidants/pharmacology , Peroxiredoxins/genetics , Sulfamethoxazole/analogs & derivatives , tat Gene Products, Human Immunodeficiency Virus/genetics , Apoptosis/drug effects , Disulfides , Gene Expression/drug effects , HIV-1 , Humans , Jurkat Cells , Mutation , Oxidation-Reduction , Oxidative Stress/drug effects , Peroxiredoxins/antagonists & inhibitors , Peroxiredoxins/metabolism , Plasmids/chemistry , Plasmids/metabolism , Proteome/genetics , Proteome/metabolism , Sulfamethoxazole/pharmacology , Sulfhydryl Compounds/antagonists & inhibitors , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/metabolism , Transgenes , tat Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
HIV infection results in severe immune dysfunction with ensuing sequelae that includes characteristic opportunistic infections. Pneumocystis pneumonia (PCP) is one of the most common of these infections and is routinely treated with sulphamethaxazole (SMX). Although this drug is known to cause hypersensitivity adverse drug reactions (ADRs) in 0.1% of the general population, the incidence of these ADRs increases tenfold in the HIV-positive population. The HIV-1 trans-activator of transcription (HIV-1 Tat) together with the drug metabolite sulphamethaxazole-hydroxylamine (SMX-HA) have both been reported to be factors in these hypersensitivity ADRs. In this study, we use an inducible, Tat-expressing vector system to show that the level of Tat expression contributes to the cellular sensitivity of Jurkat T cells to SMX-HA. We further demonstrated that apoptosis is the likely mechanism by which this occurs. Thus, our data provide insight into the significant increase of SMX-related ADRs during the transition between HIV-1 infection and AIDS.
