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01.12.2018 | Research | Ausgabe 1/2018 Open Access

Virology Journal 1/2018

HIV-1 tat expression and sulphamethoxazole hydroxylamine mediated oxidative stress alter the disulfide proteome in Jurkat T cells

Zeitschrift:
Virology Journal > Ausgabe 1/2018
Autoren:
Kemi Adeyanju, John R. Bend, Michael J. Rieder, Gregory A. Dekaban
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12985-018-0991-x) contains supplementary material, which is available to authorized users.

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.
Zusatzmaterial
Additional file 1: Figure S1. R2D SDS-PAGE of thiol proteins, formed by reduction of cellular protein-protein mixed disulphides, in lysates of (A) Jurkat E6.1, (B) Jurkat-HIV, (C) Tat101, (D) Tat101GFP, (E) Tat72GFP and (F) Tat∆GFP at 0 ng/ml Dox. The R2D gels in panels A and B are replicas of those presented in Fig. 2. Cells from each of the various lines were treated with 0.05% DMSO for 2 h, collected and the protein was isolated. Protein lysate (85 μg) was loaded onto the first dimension gel and run for 3 h followed by an overnight run of the second dimension gel. On the left side of the diagonal on each gel are molecular weight protein standards that are enumerated to the left of panels A, C and E. (TIF 4102 kb)
Additional file 2: Figure S2. R2D SDS-PAGE of thiol proteins, formed by reduction of cellular protein-protein mixed disulphides, in lysates of (A) Jurkat E6.1, (B) Jurkat-HIV, (C) Tat101, (D) Tat101GFP, (E) Tat72GFP, (F) Tat∆GFP, with panels C-F induced for 40 h with 400 ng/ml Dox (C and F) and 200 ng/ml Dox (D and E) prior to DMSO treatment. The redox 2D gels in panels A and B are replicas of those represented in Fig. 2. Cells from each of the various lines were treated with 0.05% DMSO for 2 h, collected and the protein was isolated. Protein lysate (85 μg) was loaded onto the first dimension gel and run for 3 h followed by an overnight run of the second dimension gel. On the left side of the diagonal on each gel are molecular weight protein standards that are enumerated the left of panels A, C and E . (TIF 4107 kb)
Additional file 3: Figure S3. R2D SDS-PAGE of thiol proteins formed by reduction of cellular protein-protein mixed disulphides, in lysates of (A) Jurkat E6.1, (B) Jurkat-HIV, (C) Tat101, (D) Tat101GFP, (E) Tat72GFP and (F) Tat∆GFP at 0 ng/ml Dox. The R2D gels in panels A and B are replicas of those represented in Fig. 4. Cells from each of the various lines were treated with 200 μM SMX-HA for 2 h, collected and the protein was isolated. Protein lysate (85 μg) was loaded onto the first dimension gel and run for 3 h followed by an overnight run of the second dimension gel. On the left side of the diagonal on each gel are molecular weight protein standards that are enumerated to the left of panels A, C and E. (TIF 3759 kb)
Additional file 4: Figure S4. R2D SDS-PAGE of thiol proteins formed by reduction of cellular protein-protein mixed disulphides, in lysates of (A) Jurkat E6.1, (B) Jurkat-HIV, (C) Tat101, (D) Tat101GFP, (E) Tat72GFP and (F) Tat∆GFP, induced for 40 h with 400 ng/ml Dox (C and F) and 200 ng/ml Dox (D and E) prior to drug treatment. The R2D gels in panels A and B are replicas of those represented in Fig. 4. Cells from each of the various lines were then treated with 200 μM SMX-HA for 2 h, collected and the protein was isolated. Protein lysate (85 μg) was loaded onto the first dimension gel and run for 3 h followed by an overnight run of the second dimension gel. On the left side of the diagonal on each gel are molecular weight protein standards that are enumerated to the left of panels A, C and E. (TIF 3762 kb)
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