含硫氨基酸代谢关键酶CBS的活性调控生化分子基础和新型激活剂研究

Cystathionine β-synthase (CBS) is the key enzyme for the transsulfuration pathway of sulfur amino acids. The pathogenic missense mutations in the allosteric domain of the C-terminus of CBS are found to cause homocystinuria. To date, the biochemical and molecular basis of the allosteric activation of CBS and the deregulation by CBS pathogenic mutations have not been elucidated. Moreover, no specific allosteric modulator for the CBS allosteric domain has been identified yet. These have become bottlenecks on studying the molecular basis of the allosteric regulation of CBS and the pathogenic mechanism of homocystinuria, as well as for developing novel treatments for the disease. ..In this study, we would like to utilize the multidisciplinary approaches, our established high-throughput assay for CBS and the basis of preliminary studies for the CBS activator, i) to further discover new specific activators for CBS that function for purified CBS, in cells and in vivo; ii) to study the regulatory effects of them on the key metabolites of homocystinuria and reveal mode of actions of these modulators. Meanwhile, the underlying molecular mechanisms of the allosteric activation of CBS, the dysfunction of CBS pathogenic mutants and the interactions between these activators and CBS wild-type or mutants, will be explored...This study could not only increase our understanding of the biochemical and molecular basis for the activity regulation of CBS and the pathogenic mechanism of homocystinuria, but also offer us the probe tool for the mechanism study of the CBS protein or the allosteric domain, and drug leads for developing new treatments of homocystinuria. Thus, the study has theoretical and potential application significance.

胱硫醚β合酶（cystathionine β-synthase，CBS）是含硫氨基酸代谢的关键酶，其羧基端别构结构域的突变能引发高胱氨酸尿症。但至今，CBS的别构激活调控和其病理性失调的生化分子机制尚不明确，且靶向该结构域的特异激活剂也无报道，这已成为研究CBS的活性调控机制、揭示高胱氨酸尿症的致病机理和发展该疾病治疗新方法的瓶颈。.本项目将采用多学科交叉结合的方法，基于我们已建立的CBS高通量测活特色技术，在前期工作基础上，去得到在分子、细胞和动物水平上有效的CBS新型特异激活剂，阐述其对疾病关键代谢物的调控作用，并揭示其作用机制；同时，探究CBS的别构激活、致病突变体活性失调以及其与激活剂相互作用的生化分子新机制。.这一研究，能加速我们对CBS的活性调控机制和高胱氨酸尿症致病机制的认知，并可为CBS的分子机制研究及该疾病的治疗提供新的探针工具和药物先导物,具有理论和潜在的应用意义。

CBS酶的羧基端含有一个受内源性代谢物SAM别构激活的结构域。该CBS别构结构域的家族性突变被发现可导致内源性激活剂SAM的激活能力下降和CBS酶活下降，并引发代谢疾病高胱氨酸尿症。目前针对CBS别构结构域的特异激活剂还未见报道，CBS结构域的遗传突变造成该酶活性失调的分子机制也未解析。这些都已成为发展高胱氨酸尿症新型治疗方法的瓶颈。..本项目中，申请者团队通过生物化学结合药物筛选等方法，发现了多个小分子化合物能在体外激活CBS野生型或D444N突变体，其中化合物1为特异的CBS D444N激活剂。通过解析化合物1和CBS结构域的共晶晶体结构，结合分子突变等实验，揭示了化合物1在CBS中结合位点以及互作残基。通过细胞热迁移试验、靶向代谢物定量分析等试验，发现化合物1处理可提高细胞内CBS D444N的稳定性并增加CBS产物胱硫醚的含量，提示化合物1具有较好的生物活性。本项目还创建了human CBS D444N人源化小鼠模型以及mouse CBS D440N点突变小鼠模型，并发现其具有高胱氨酸尿症的病理特征。化合物1在这些小鼠疾病模型上的有效性正在研究之中。本项目的完成，对阐明CBS别构调控机制和发展治疗高胱氨酸尿症的新型治疗方法具有重要意义。..受本项目支持，申请人团队已在本领域知名杂志上发表多篇论文，培养了多位具有相关经历的研究生。

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