The Effects of Interferons on Anthrax Toxicity

描述（由申请人提供）：炭疽是一种由革兰氏阳性细菌炭疽杆菌引起的急性疾病。主要的细胞靶标是单核吞噬细胞，其由于暴露于炭疽杆菌产生的毒素而经历凋亡和坏死。致死毒素（LT）由保护性抗原（PA）和致死因子（LF）两个蛋白亚基组成。PA是促进LF进入细胞的膜整合蛋白。LF是一种特异性切割MAP激酶（MAPKK）的NH 2末端的金属蛋白酶。MAP激酶活化的相应抑制被认为是炭疽杆菌逃避先天免疫应答的核心。 我们目前的证据表明，治疗巨噬细胞与干扰素β（IFN β）或干扰素γ（IFN γ）促进巨噬细胞的生存LT曝光后。虽然IFN β或IFN γ不能保护巨噬细胞免受LT诱导的MAPKK裂解，但它确实保护细胞免受两种重要蛋白酪氨酸磷酸酶的裂解（SHP-1和SHP-2），这些发现表明，干扰素可能提供了一种潜在的治疗方法，通过阻止新发现的底物的蛋白水解来抑制LT的生物学作用我们建议： 目标1：鉴定作为LT底物的MAPKK以外的蛋白质，并确定在与IFN β或IFN γ孵育的巨噬细胞中，这些底物中的哪一种被保护免于LT介导的蛋白水解。目标二：确定SHP-1和SHP-2以及其他蛋白质的切割位点，这些蛋白质均被LT蛋白水解，并且在暴露于LT和IFN γ或IFN β的细胞中受到保护而不被蛋白水解。

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.