Profiling lethal factor interacting proteins from human stomach using T7 phage display screening.
Profiling lethal factor interacting proteins from human stomach using T7 phage display screening.
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DOI：
10.3892/mmr.2016.5031
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发表时间：
2016-05
影响因子：
3.4
通讯作者：
Rios-Velazquez C
中科院分区：
文献类型：
--
作者：
Cardona-Correa A;Rios-Velazquez C
炭疽致死因子（LF）是一种锌依赖性金属蛋白酶，其裂解大多数丝裂原活化蛋白激酶激酶和NOD样受体蛋白的成员，诱导细胞凋亡。尽管努力充分了解炭疽杆菌毒素成分，胃肠道（GI）炭疽机制尚未完全阐明。以前的研究表明胃溃疡，并在派尔集合淋巴结的大量细菌生长率。然而，LF蛋白水解活性导致组织损伤的疾病的完整分子途径仍不清楚。在本研究中，以确定潜在参与GI炭疽的蛋白质的配置文件，蛋白质-蛋白质的相互作用进行了研究，使用人胃T7噬菌体展示（T7 PD）cDNA文库。T7 PD是一种高通量技术，其允许克隆的DNA序列在噬菌体表面上表达为肽，从而能够选择和鉴定蛋白质配体。野生型和突变型LF（E687 A）用于区分相互作用位点。通过计算机分析，从194个相互作用位点中鉴定出124个克隆，包括DNA和蛋白质水平。数据库显示，所选择的候选人是来自不同家族的蛋白质，包括脂肪酶，肽酶-A1和阳离子转运家族等。此外，针对LF测试个别T7 PD候选物以检测其对靶分子的特异性，从而产生10种LF相互作用肽。当LF的最小相互作用浓度为1 μg/ml时，T7 PD分离的胃蛋白酶A3前蛋白（PAP）表现出对两种类型LF的亲和力。此外，PAP被分离在不同长度的相同的蛋白质，表现出共同的区域后，PRALINE比对。这些发现将有助于阐明和提高对GI炭疽分子发病机制的理解，并有助于开发潜在的治疗药物。
The anthrax lethal factor (LF) is a zinc dependent metalloproteinase that cleaves the majority of mitogen-activated protein kinase kinases and a member of NOD-like receptor proteins, inducing cell apoptosis. Despite efforts to fully understand the Bacillus anthracis toxin components, the gastrointestinal (GI) anthrax mechanisms have not been fully elucidated. Previous studies demonstrated gastric ulceration, and a substantial bacterial growth rate in Peyer's patches. However, the complete molecular pathways of the disease that results in tissue damage by LF proteolytic activity remains unclear. In the present study, to identify the profile of the proteins potentially involved in GI anthrax, protein-protein interactions were investigated using human stomach T7 phage display (T7PD) cDNA libraries. T7PD is a high throughput technique that allows the expression of cloned DNA sequences as peptides on the phage surface, enabling the selection and identification of protein ligands. A wild type and mutant LF (E687A) were used to differentiate interaction sites. A total of 124 clones were identified from 194 interacting-phages, at both the DNA and protein level, by in silico analysis. Databases revealed that the selected candidates were proteins from different families including lipase, peptidase-A1 and cation transport families, among others. Furthermore, individual T7PD candidates were tested against LF in order to detect their specificity to the target molecule, resulting in 10 LF-interacting peptides. With a minimum concentration of LF for interaction at 1 μg/ml, the T7PD isolated pepsin A3 pre-protein (PAP) demonstrated affinity to both types of LF. In addition, PAP was isolated in various lengths for the same protein, exhibiting common regions following PRALINE alignment. These findings will help elucidate and improve the understanding of the molecular pathogenesis of GI anthrax, and aid in the development of potential therapeutic agents.