BioTherapeutics Research Laboratory, Molecular Medicine Research Laboratories, Robarts Research Institute, Rm 2214, 1151 Richmond Street North, London, Ontario, Canada.
Department of Microbiology and Immunology, University of Western Ontario, 1151 Richmond Street North, London, ON, N6A 5B7, Canada.
Virol J. 2018 May 9;15(1):82. doi: 10.1186/s12985-018-0991-x.
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.
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.
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.
药物不良反应（ADR）是 HIV 患者的一个重大问题，随着感染进展为艾滋病，发生 ADR 的风险增加。然而，ADR 的病理生理学机制尚不清楚。磺胺甲恶唑（SMX）通过其活性代谢物 SMX-羟胺，在 HIV 阳性个体中预防性用于肺囊虫肺炎时，会导致 ADR 的发生率很高。我们之前的研究表明，HIV 感染，更具体地说，HIV-1 Tat 蛋白会加剧 SMX-HA 介导的 ADR。在目前的研究中，使用表达 Tat 及其缺失突变体的 Jurkat T 细胞系来确定 Tat 在存在和不存在 SMX-HA 的情况下对硫醇蛋白质组的影响，揭示了 HIV 感染细胞中二硫键蛋白质组的药物依赖性变化。从 HIV 感染的 Jurkat T 细胞和稳定转染 HIV Tat 和 Tat 缺失突变体的 Jurkat T 细胞的蛋白裂解物进行定量插槽印迹分析、western blot 分析和氧化还原 2 维（2D）凝胶电泳，以分析 SMX-HA 对硫醇蛋白质组的影响。
氧化还原 2D 凝胶电泳表明，未经处理的表达 Tat 的细胞含有许多具有氧化硫醇的蛋白质。其中最明显的蛋白巯基是过氧化物酶。未经处理的表达 Tat 的细胞系与亲本 Jurkat E6.1 T 细胞系相比，过氧化物酶的水平较低。相反，用 SMX-HA 孵育会导致硫醇蛋白氧化增加 2-3 倍，并且所有细胞系，特别是表达 Tat 的细胞系中过氧化物酶的水平显著降低。
SMX-HA 是一种能够诱导反应性蛋白半胱氨酸巯基氧化的氧化剂，其中大多数形成分子间蛋白质键。用 SMX-HA 处理时，表达 HIV Tat 的细胞系比 Jurkat E6.1 细胞系显示出更高水平的氧化应激。因此，HIV Tat 和 SMX-HA 的组合似乎改变了细胞蛋白质的活性，这些蛋白质是氧化还原稳态所必需的，从而加剧了与 HIV 感染 T 细胞相关的细胞病变效应，为 ADR 的发生奠定了基础。
