Greene Amy Styer, Hajduk Stephen L
From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602.
From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602
J Biol Chem. 2016 Feb 5;291(6):3063-75. doi: 10.1074/jbc.M115.680371. Epub 2015 Dec 8.
Human innate immunity against the veterinary pathogen Trypanosoma brucei brucei is conferred by trypanosome lytic factors (TLFs), against which human-infective T. brucei gambiense and T. brucei rhodesiense have evolved resistance. TLF-1 is a subclass of high density lipoprotein particles defined by two primate-specific apolipoproteins: the ion channel-forming toxin ApoL1 (apolipoprotein L1) and the hemoglobin (Hb) scavenger Hpr (haptoglobin-related protein). The role of oxidative stress in the TLF-1 lytic mechanism has been controversial. Here we show that oxidative processes are involved in TLF-1 killing of T. brucei brucei. The lipophilic antioxidant N,N'-diphenyl-p-phenylenediamine protected TLF-1-treated T. brucei brucei from lysis. Conversely, lysis of TLF-1-treated T. brucei brucei was increased by the addition of peroxides or thiol-conjugating agents. Previously, the Hpr-Hb complex was postulated to be a source of free radicals during TLF-1 lysis. However, we found that the iron-containing heme of the Hpr-Hb complex was not involved in TLF-1 lysis. Furthermore, neither high concentrations of transferrin nor knock-out of cytosolic lipid peroxidases prevented TLF-1 lysis. Instead, purified ApoL1 was sufficient to induce lysis, and ApoL1 lysis was inhibited by the antioxidant DPPD. Swelling of TLF-1-treated T. brucei brucei was reminiscent of swelling under hypotonic stress. Moreover, TLF-1-treated T. brucei brucei became rapidly susceptible to hypotonic lysis. T. brucei brucei cells exposed to peroxides or thiol-binding agents were also sensitized to hypotonic lysis in the absence of TLF-1. We postulate that ApoL1 initiates osmotic stress at the plasma membrane, which sensitizes T. brucei brucei to oxidation-stimulated osmotic lysis.
人类针对兽医病原体布氏布氏锥虫的天然免疫是由锥虫溶解因子（TLFs）介导的，而人类感染性冈比亚布氏锥虫和罗德西亚布氏锥虫已对其产生了抗性。TLF-1是高密度脂蛋白颗粒的一个亚类，由两种灵长类动物特有的载脂蛋白定义：形成离子通道的毒素ApoL1（载脂蛋白L1）和血红蛋白（Hb）清除剂Hpr（触珠蛋白相关蛋白）。氧化应激在TLF-1溶解机制中的作用一直存在争议。在此，我们表明氧化过程参与了TLF-1对布氏布氏锥虫的杀伤作用。亲脂性抗氧化剂N,N'-二苯基对苯二胺可保护经TLF-1处理的布氏布氏锥虫不被裂解。相反，添加过氧化物或硫醇结合剂会增加经TLF-1处理的布氏布氏锥虫的裂解。此前，Hpr-Hb复合物被认为是TLF-1裂解过程中自由基的来源。然而，我们发现Hpr-Hb复合物中含铁的血红素并不参与TLF-1的裂解。此外，高浓度的转铁蛋白或胞质脂质过氧化物酶的敲除均不能阻止TLF-1的裂解。相反，纯化的ApoL1足以诱导裂解，且ApoL1的裂解受到抗氧化剂DPPD的抑制。经TLF-1处理的布氏布氏锥虫肿胀类似于低渗应激下的肿胀。此外，经TLF-1处理的布氏布氏锥虫对低渗裂解迅速敏感。暴露于过氧化物或硫醇结合剂的布氏布氏锥虫细胞在没有TLF-1的情况下对低渗裂解也敏感。我们推测ApoL1在质膜上引发渗透应激，使布氏布氏锥虫对氧化刺激的渗透裂解敏感。
