The tail-specific protease (Tsp) of Chlamydiae spp.: Mechanistic and structural characterization of its interactions with host-cell proteins

衣原体是一种专性细胞内病原体，它利用分泌的效应蛋白来改变真核宿主细胞的信号通路。在人类病原体沙眼衣原体中，丝氨酸蛋白酶CT441被报道通过裂解p65来干扰宿主炎症反应的核因子-kappaB途径。此外，CT441还专门针对雌激素受体α的类固醇受体RNA激活剂1(SRA1)的蛋白质成分SRAP1。有研究表明，这种相互作用将SRAP1保留在宿主细胞的细胞质中，从而阻止雌激素诱导的许多靶基因的表达，这些基因对衣原体在宿主细胞内的生存和发育至关重要。CT441在很大程度上没有特征性，显示出与大肠杆菌中参与细菌蛋白质质量控制的尾部特异性蛋白酶(TSP)的序列相似之处。该项目的目标是帮助理解衣原体对宿主细胞通路的调控。我们将重点阐明衣原体TSP与宿主细胞蛋白相互作用的详细机制。我们的目标是确定CT441及其近缘物种的同源物的三维结构。基于结构信息，将使用定点突变来进行详细的机理分析。此外，还将在体外研究CT441与SRAP1的相互作用。利用CT441和SRAP1的全长蛋白质或单个结构域进行生化相互作用分析、蛋白酶分析和X射线结晶学分析将有助于阐明相互作用的模式。最终，我们的目标是确定SRAP1和衣原体TSP蛋白之间的复合体的分子结构。利用人输卵管组织上生殖道衣原体感染模型，研究CT441在生理环境中对宿主细胞SRAP1水平的影响。该项目有望揭示衣原体调节宿主细胞代谢和雌激素感染性的分子基础。

Chlamydiae are obligate intracellular pathogens that use secreted effector proteins to modify signaling pathways of eukaryotic host cells. In the human pathogen Chlamydia trachomatis, the serine protease CT441 has been reported to interfere with the NF-kappaB pathway of the host inflammatory response by cleaving p65. In addition, CT441 specifically targets SRAP1, the protein component of steroid-receptor RNA activator 1 (SRA1) of estrogen receptor alpha. It has been suggested that this interaction retains SRAP1 in the host-cell cytoplasm, thereby preventing the expression of many estradiol-induced target genes which is important for chlamydial survival and development within the host cell. The largely uncharacterized CT441 shows sequence similarities to tail-specific proteases (Tsps) involved in bacterial protein-quality control in E. coli. The goal of this project is to contribute to the understanding of the modulation of host-cell pathways by Chlamydiae. We will focus on elucidating the detailed mechanisms of interactions between chlamydial Tsps with host-cell proteins. We aim to determine three-dimensional structures for CT441 and homologues from related species. Based on structural information, site-directed mutagenesis will be used to conduct a detailed mechanistic analysis. In addition, the interaction between CT441 and SRAP1 will be studied in vitro. Using full-length proteins or individual domains of CT441 and SRAP1 in biochemical interaction analyses, protease assays and X-ray crystallography will help to clarify the mode of the interaction. Ultimately, we aim to determine the molecular structure of a complex between SRAP1 and a chlamydial Tsp protein. A human fallopian-tube tissue model of chlamydial infections of the upper genital tract will be used to study the influence of CT441 on host cell SRAP1 levels in a physiological environment. The project is expected to reveal the molecular basis for chlamydial modulation of host-cell metabolism and infectivity by estrogen.