Molecular Mechanisms of Fibrinolysis

描述(申请人提供)：全球每年有50多万人死于严重的链球菌感染。多重耐药细菌病原体的迅速出现，以及在制药开发中缺乏新的抗生素，代表着一个巨大的公共卫生问题。链激酶(SK)是一种主要的链球菌毒力因子，通过激活纤溶酶原(PG)作用于蛋白水解酶(Pm)来靶向纤溶作用。通过尚不清楚的机制，PM降解了由宿主反应产生的保护性纤维蛋白(FBN)屏障。与另一种毒力因子PAM一起，SK以一种未知的机制产生与细菌表面结合的PM，使涂有PM的细菌能够在组织中传播。背景-我们提出了一个统一的机制，即通过SK结合启动SK-PG激活，并通过NH2-末端插入机制诱导PG酶原的构象激活。第二个PG分子作为特定的底物与SK7Pg*催化复合体结合，在最初的催化循环中被裂解成PM。Pg被产生的Pm的高亲和力结合从SK7Pg*取代，产生作为底物结合Pg的SK7Pm催化络合物，启动第二个繁殖周期，将剩余的Pg转化为Pm广泛的目标--SK激活PG的生化和生物物理研究，目的是了解SK颠覆人的纤溶作用以促进严重链球菌疾病的分子机制，最终目的是开发基于机制的新药物。这项调查提出了一些假说，以填补在了解感染机制和评估新的治疗方法方面的主要空白。实验方法包括酶动力学、快速反应动力学、平衡结合、诱变、协同电子显微镜、结晶学和体内感染模型。目的1：确定[Glu]PG在催化和底物模式下与SK结合的构象变化的作用，以及[Glu]PG、[Lys]PG和PM的底物/产物识别机制目的：在快速反应动力学研究中明确SK诱导的[Glu]PG和[Lys]PG构象活化机制中的分子事件途径，以及SK7Pg*/Pm催化络合物识别[Glu]PG/[Lys]PG底物的机制。目的：确定结合Pg和Pm的SK结构域间的灵活性在调节SK功能中的作用，并评估连接SK结构域的柔性片段的抗体在抗链球菌疾病中的潜在用途。目的：阐明FBG、FBN和PAM在侵袭性链球菌感染中调节SK诱导的PG激活的机制，并评价一种抗底物PG识别抗体抑制剂。目的：明确Skizzle(SkzL)作为一种新的tPA激活PG的辅因子的作用机制，并评价其在无乳链球菌感染中的潜在致病作用。 公共卫生相关性：全球每年有50万人死于严重链球菌感染，至少有1810万人受到影响。抗生素抗药性细菌的迅速出现和正在开发的新抗生素的缺乏是一个巨大的公共卫生问题。该项目建议确定链球菌致病因子链激酶颠覆人类血液凝块溶解系统以传播危及生命的感染的分子机制，最终目标是开发针对链激酶的新药来对抗这些细菌感染。

DESCRIPTION (provided by applicant): Severe streptococcal infections account for more than 500,000 deaths globally per year. The rapid emergence of multi-drug resistant bacterial pathogens, and the lack of new antibiotics in pharmaceutical development, represents a huge public health problem. Streptokinase (SK), a major streptococcal virulence factor, targets human fibrinolysis by activating plasminogen (Pg) to the proteinase, plasmin (Pm). By mechanisms that are not understood, Pm degrades protective fibrin (Fbn) barriers generated by the host response. Along with another virulence factor, PAM, SK produces, by an unknown mechanism, Pm bound to the bacterial surface, allowing Pm-coated bacteria to spread through tissues. Background - We proposed a unified mechanism of SK-Pg activation initiated by SK binding and inducing conformational activation of the Pg zymogen by the NH2-terminal insertion mechanism. A second Pg molecule binds to the SK7Pg* catalytic complex as a specific substrate, which is cleaved to Pm in an initial, triggering catalytic cycle. Pg is displaced from SK7Pg* by the high affinity binding of Pm produced, yielding the SK7Pm catalytic complex that binds Pg as a substrate, initiating the second, propagation cycle that converts the remaining Pg to Pm. Broad Goals - Biochemical and biophysical studies of Pg activation by SK are proposed with the goal of understanding the molecular mechanism by which SK subverts human fibrinolysis to promote severe streptococcal diseases, with the ultimate purpose of developing new mechanism-based drugs. The investigation addresses hypotheses to fill major gaps in the understanding of the infection mechanisms, and evaluation of novel therapeutic approaches. Experimental approaches include enzyme kinetics, rapid-reaction kinetics, equilibrium binding, mutagenesis, and collaborative electron microscopy, crystallography, and in vivo models of infection. Aim 1: To define the role of [Glu]Pg conformational changes in its binding to SK in the catalytic and substrate modes, and the mechanism of substrate/product recognition for [Glu]Pg, [Lys]Pg, and Pm. Aim 2: To define in rapid-reaction kinetic studies the pathway of molecular events in the mechanism of SK-induced conformational activation of [Glu]Pg and [Lys]Pg, and the mechanism of [Glu]Pg/[Lys]Pg substrate recognition by the SK7Pg*/Pm catalytic complexes. Aim 3: To determine the role of inter-domain flexibility of SK bound to Pg and Pm in dictating SK function, and to evaluate antibodies against the flexible segments linking SK domains as inhibitors for potential use in combating streptococcal diseases. Aim 4: To elucidate the mechanisms by which Fbg, Fbn, and PAM regulate SK-initiated Pg activation that underlie their roles in invasive streptococcal infections, and to evaluate an anti-substrate Pg recognition antibody inhibitor. Aim 5: To define the mechanism of skizzle (SkzL) as a novel cofactor of tPA-catalyzed Pg activation, and to evaluate its potential role as pathogenicity factor in Streptococcus agalactiae infections. PUBLIC HEALTH RELEVANCE: Severe streptococcal infections account for 500,000 deaths per year globally, and there are at least 18.1 million people affected. The rapid emergence of antibiotic-resistant bacteria and the lack of new antibiotics under development represent a huge public health problem. This project proposes to define the molecular mechanisms by which streptokinase, a streptococcal pathogenicity factor, subverts the human blood clot- dissolving system to propagate life-threatening infections, with the ultimate goal of developing new drugs for targeting streptokinase to combat these bacterial infections.