Mechanism of Proteotoxicity and Experimental Therapeutic Approaches in Porphyria

摘要 卟啉病是由参与八种连续生物合成的酶突变引起的遗传性疾病。 结合联合收割机甘氨酸和琥珀酰辅酶-A生成血红素的转化。卟啉积累可以 导致与其他疾病如丙型肝炎病毒感染相关的“继发性卟啉症”。电流 关于卟啉病的主要未满足的需求是我们目前对卟啉病的有限理解。 卟啉症急性发作的分子触发因素，除了与发烧或药物的间接关联;原因 为什么有些人会出现严重的终末器官并发症，如需要肝脏或骨髓 移植，而其他人没有;重要的是，有限的药物供应，以治疗不同的 卟啉症我们的中心假设是蛋白毒性卟啉介导的损伤，加上修饰剂 影响，有助于与卟啉症相关的细胞损伤和发病率。这一假设 将通过追求三个具体目标进行测试：（i）定义卟啉诱导蛋白的机制 （ii）表征促进或保护免受卟啉诱导的细胞损伤的潜在修饰剂 文化和动物模型;和（iii）使用斑马鱼卟啉症模型筛选和表征小- 防止组织卟啉积累的分子化合物。我们已经收集了大量的初步资料 结果支持我们的目标可能取得成功，包括大量证据表明卟啉介导的蛋白质 聚集，这是依赖于卟啉积累的模式，脂肪酸结合蛋白的证据- 1作为卟啉介导的细胞和肝损伤的调节剂，以及两种新的急性斑马鱼卟啉病模型 我们产生的，是适合高通量药物筛选与潜在的积极化合物， 建议验证和表征。 完成我们提出的目标应该提供有关卟啉如何导致 蛋白质聚集、所涉及的亚细胞区室、这种聚集是否是由于 直接共价卟啉-蛋白质加合物的形成，以及聚集体周转的机制。我们也 预期能够表征特定的卟啉结合蛋白，调节的倾向， 在细胞和组织中积累卟啉。这可能会提供潜在的线索，为什么一些人与 卟啉症在没有伴随的潜在肝病的情况下发展为终末期肝病， 其他病人则避免了严重的肝脏相关并发症。重要的是，我们预计我们的提案将 允许我们使用我们开发的两种强大的新型急性卟啉症斑马鱼模型来识别潜在的 最终可能具有治疗效用的化合物。这项提案使用了最先进的技术， 多种生物化学和动物模型工具，包括斑马鱼和小鼠，并介绍了新的概念， 蛋白毒性作为卟啉病恶化和进展的替代机制。

Abstract Porphyrias are genetic disorders caused by mutations in enzymes involved in eight sequential biosynthetic conversions that combine glycine and succinyl coenzyme-A to generate heme. Porphyrin accumulation can result in `secondary porphyrias' in association with other diseases such as hepatitis C virus infection. Current major unmet needs with regard to the porphyria disorders are our present limited understanding of the molecular triggers of porphyria acute attacks, aside from indirect association with fever or drugs; the reasons why some individuals develop significant end-organ complications such as the need for liver or bone marrow transplantation while others do not; and importantly the limited availability of drugs to treat the different porphyrias. Our central hypothesis is that proteotoxic porphyrin-mediated damage, coupled with modifier effects, contributes to the cellular damage and morbidity that is associated with porphyrias. This hypothesis will be tested by pursuing three specific aims: (i) Define the mechanism of porphyria-induced protein aggregation; (ii) Characterize potential modifiers that promote or protect from porphyria-induced injury in cell culture and animal models; and (iii) Use zebrafish porphyria models to screen and characterize small- molecule compounds that prevent tissue porphyrin accumulation. We have assembled extensive preliminary results to support the likely success of our aims, including substantial evidence for porphyrin-mediated protein aggregation that is dependent on the mode of porphyrin accumulation, evidence for fatty acid binding protein- 1 as a modulator of porphyrin-mediated cell and liver injury, and two novel acute zebrafish porphyria models we generated that are amenable to high-throughput drug screening with potential positive compounds that we propose to validate and characterize. Completion of our proposed aims should provide fundamental knowledge regarding how porphyrins cause protein aggregation, the subcellular compartments that are involved, whether such aggregation is due to direct covalent porphyrin-protein adduct formation, and the mechanism of aggregate turnover. We also anticipate being able to characterize specific porphyrin-binding proteins that regulate the propensity to accumulate porphyrins in cells and tissues. This could provide potential clues as to why some individuals with porphyria develop end-stage liver disease in the absence of concomitant underlying liver disorders, while other patients are spared serious liver-related complications. Importantly, we anticipate that our proposal will allow us to use two powerful novel acute porphyria zebrafish models we have developed to identify potential compounds that may ultimately have therapeutic utility. This proposal uses state-of-the-art technologies, multiple biochemical and animal model tools including zebrafish and mice, and introduces the novel concept of proteotoxicity as an alternative mechanism for porphyria exacerbations and progression.