The Effects of Interferons on Anthrax Toxicity

描述(申请人提供)：炭疽病是由革兰氏阳性细菌炭疽杆菌引起的一种急性疾病。主要的细胞靶点是单核吞噬细胞，由于暴露于炭疽芽孢杆菌产生的毒素，这些细胞既会发生凋亡，也会发生坏死。致死毒素(LT)由保护性抗原(PA)和致死因子(LF)两个蛋白质亚基组成。PA是一种促进LF进入细胞的膜整合蛋白。Lf是一种金属蛋白水解酶，能特异性地裂解MAPKKs的NH2末端。由此导致的对MAP激酶激活的抑制被认为是炭疽芽孢杆菌逃避天然免疫反应的核心。 我们提出的证据表明，巨噬细胞用干扰素β(IFNbeta)或干扰素伽马(IFNGamma)治疗可促进LT暴露后巨噬细胞的存活。虽然IFNbeta或IFNGamma不能保护巨噬细胞免受LT诱导的MAPKK的切割，但它确实可以保护细胞免受两种重要的蛋白质酪氨酸磷酸酶(SHP-1和SHP-2)的切割，这两种酶以前没有被确定为LT的底物。这些发现表明，干扰素可能提供一种潜在的治疗方法，通过阻止新发现的作为LT靶点的底物的蛋白分解来抑制LT的生物学作用。我们建议： 目的1：鉴定作为LT底物的MAPKK以外的蛋白质，并确定这些底物中的哪些在与IFNbeta或IFNGamma孵育的巨噬细胞中受到LT介导的蛋白分解的保护。目的：确定SHP-1和SHP-2的裂解位点，以及在LT和IFNGamma或IFbeta作用下的细胞中被LT蛋白分解而不被蛋白分解保护的其他蛋白质。

DESCRIPTION (provided by applicant): Anthrax is an acute disease caused by the gram-positive bacterium Bacillus anthracis. Among the primary cellular targets are mononuclear phagocytes, which undergo both apoptosis and necrosis as a result of exposure to toxins produced by Bacillus anthracis. Lethal toxin (LT) is composed of two protein subunits, Protective antigen (PA) and Lethal Factor (LF). PA is a membrane-integrating protein that facilitates LF entry into cells. LF is a metalloprotease that specifically cleaves the NH2 termini of MAP kinase kinases (MAPKKs). The consequential inhibition of the activation of MAP kinases is thought to be central to Bacillus anthracis' evasion of the innate immune response. We present evidence that treatment of macrophages with interferon beta (IFNbeta) or interferon gamma (IFNgamma) promotes survival of macrophages after LT exposure. Although IFNbeta or IFNgamma does not protect macrophages from LT induced cleavage of MAPKKs, it does protect cells from cleavage of two important protein tyrosine phosphatases (SHP-1 and SHP-2), that have not been previously identified as substrates for LT. These findings suggest that interferons may provide a potential therapeutic approach to inhibit the biological actions of LT through prevention of the proteolysis of newly identified substrates that are targets of LT. We propose to: Aim 1: Identify proteins other than MAPKKs that are substrates for LT and determine which of these substrates are protected from LT mediated proteolysis in macrophages incubated with IFNbeta or IFNgamma. Aim 2: Determine the sites of cleavage of SHP-1 and SHP-2 as well as other proteins that are both proteolyzed by LT and are protected from proteolysis in cells exposed to LT and IFNgamma or IFbeta.