Mechanism of Proteotoxicity and Experimental Therapeutic Approaches in Porphyria

项目摘要 卟啉症是由涉及八种顺序生物合成的酶的突变引起的遗传性疾病。 在第一个酶促步骤中结合甘氨酸和琥珀酰辅酶-A的转化最终生成 亚铁血红素。在“继发性卟啉症”中也会出现卟啉蓄积，并与其他疾病有关，如 丙型肝炎病毒感染。目前尚未满足的有关卟啉症的主要需求包括：(A)我们的 目前对细胞和组织损伤的生化机制了解有限，(B)分子触发因素 (C)一些人出现严重器官并发症的原因，例如 作为终末期肝病，需要肝脏或骨髓移植，而其他人不需要，以及(D) 治疗不同的门静脉症的药物供应有限。我们的中心假设是肝脏易受影响 导致细胞和组织损伤的不依赖光的卟啉介导的蛋白毒性损伤，以及 可以确定导致卟啉蓄积增加或减少的药物。这一假设将是 通过追求三个相互关联的特定目标进行测试：(I)确定与光无关的卟啉的机制- 诱导蛋白质在内脏中聚集，主要集中在肝脏；(Ii)阐明 利用体外和体内模型对卟啉诱导的蛋白毒性损伤的解毒作用；以及(Iii)表征 减少或增加组织中的卟啉蓄积和卟啉介导的蛋白毒性的小分子。 我们已经收集了广泛的初步结果，以支持我们的目标可能取得的成功，包括 卟啉介导的蛋白质聚集是光不依赖的证据，蛋白质的可逆性 可能参与逆转蛋白质氧化的聚集和酶，以及斑马鱼的使用 高通量筛选，以确定减少或增加卟啉在肝脏中蓄积的已知药物。这个 减少卟啉蓄积的药物将被测试其作用机制，并在 临床前的卟啉症实验模型，可作为潜在的新治疗方法。在……里面 同时，增加卟啉蓄积的药物将被定性为潜在的候选药物，以避免 在患有卟啉症的患者中。 我们提出的目标的完成提供了关于哪些蛋白质容易 卟啉介导的氧化和聚集，定义这种聚集的分子特征， 聚集周转和解聚的机制，我们表征的化合物是否是候选的 对门静脉症患者进行测试，以及目前用于非门静脉症的药物是否需要 避免或监测有卟啉症的患者。该提案使用了最先进的技术，有多个 生化和卟啉症动物模型工具，包括斑马鱼和小鼠，并引入了新的概念 蛋白毒性作为卟啉症恶化和进展的替代机制可能会带来曙光 在一些患者中解释肝病进展的遗传修饰物，而不是其他患者。

Project Summary Porphyrias are genetic disorders caused by mutations in enzymes involved in eight sequential biosynthetic conversions that combine glycine and succinyl coenzyme-A in the first enzymatic step to ultimately generate heme. Porphyrin accumulation also occurs in ‘secondary porphyrias’ in association with other diseases such as hepatitis C virus infection. Current major unmet needs with regard to the porphyria disorders include: (a) our present limited understanding of the biochemical mechanism of cell and tissue injury, (b) the molecular triggers of porphyria acute attacks, (c) the reasons why some individuals develop significant organ complications such as end-stage liver disease that requires liver or bone marrow transplantation, while others do not, and (d) the limited availability of drugs to treat the different porphyrias. Our central hypothesis is that the liver is susceptible to light-independent porphyrin-mediated proteotoxic damage that leads to cell and tissue injury in porphyria, and that drugs can be identified that lead to increased or decreased porphyrin accumulation. This hypothesis will be tested by pursuing three interconnected specific aims: (i) Define the mechanism of light-independent porphyrin- induced protein aggregation in internal organs, with a focus on the liver; (ii) Elucidate the mechanism of detoxification of porphyrin-induced proteotoxic damage using in vitro and in vivo models; and (iii) Characterize small molecules that decrease or increase tissue porphyrin accumulation and porphyrin-mediated proteotoxicity. We have assembled extensive preliminary results to support the likely success of our aims, including substantial evidence for porphyrin-mediated protein aggregation that is light-independent, the reversibility of protein aggregation and enzymes that are likely to be involved in reversing protein oxidation, and the use of zebrafish high-throughput screening to identify known drugs that decrease or increase porphyrin accumulation in liver. The drugs that decrease porphyrin accumulation will be tested for their mechanism of action and examined in preclinical porphyria experimental models as drugs that may be repurposed as potential new therapies. In parallel, drugs that increase porphyrin accumulation will be characterized as potential candidate drugs to avoid in patients with porphyria. Completion of our proposed aims provides fundamental knowledge regarding which proteins are prone to porphyrin-mediated oxidation and aggregation, the molecular signatures that define such aggregation, the mechanism of aggregate turnover and disaggregation, whether compounds we characterize are candidates for testing in patients with porphyria, and whether currently used drugs in non-porphyria disorders might need to be avoided or monitored in patients with porphyria. This proposal uses state-of-the-art technologies, multiple biochemical and porphyria animal model tools including zebrafish and mice, and introduces the novel concept of proteotoxicity as an alternative mechanism for porphyria exacerbations and progression that may shed light on genetic modifiers that account for liver disease progression in some patients but not others.