Treatment of CBS deficiency with proteostasis modulators

项目摘要/摘要 先天性新陈代谢障碍包括一大类涉及细胞紊乱的遗传性疾病 新陈代谢。同型半胱氨酸是一种由蛋氨酸衍生的中间代谢物，可以循环使用 回到蛋氨酸，或在胱硫醚β-合成酶的作用下顺着硫化途径分流 (CBS)，一种主要在肝脏和肾脏表达的酶。CBS基因突变的个体患有CBS 缺乏，以血浆总同型半胱氨酸(THcy)和表型极端升高为特征，包括 血栓、骨质疏松症、晶状体脱位和智力低下的发生率增加。在健康的成年人中， 血浆总同型半胱氨酸浓度从5到15M不等，但未经治疗的CBS缺乏症患者通常有 THcy超过200M目前的治疗策略包括饮食限制和维生素治疗，但 这些方法只有部分有效，并不是对所有患者都有效。 在CBS缺乏的患者中，超过85%的所描述的突变是错义突变，其中单个 错误的氨基酸被替换到CBS多肽中。在过去的十年里，我们的实验室已经开发出六种 不同的人源化小鼠模型，每个模型都表达不同的患者来源的突变人CBS蛋白 使用在肝脏中高水平表达的可诱导启动子。在所有这些模型中，肝脏CBS活性是 大大降低(野生型的5%)，血清tHcy至少升高20倍。然而，这种行为 每个突变的CBS蛋白都不完全相同，突变会影响蛋白质的稳定性，蛋白质的聚集， 酶功能，或三者的组合。突变导致的蛋白质稳定性降低的影响是 通过泛素/蛋白酶体系统介导。令人惊讶的是，对突变表达小鼠的治疗 蛋白酶体抑制物(PI)可以在功能上逆转大多数错义突变的影响， 导致肝脏中CBS活性的大幅增加，在某些情况下tHcy降低到接近野生型 级别。PIS是FDA批准的治疗某些类型癌症的药物，但从未被研究过 在新陈代谢先天缺陷的背景下。我们的数据表明，小剂量PI治疗可能能够 恢复足够的CBS酶活性以表现出有益的表现，但不会产生相关的毒性 癌症治疗中使用的高水平药物。此外，我们有初步数据表明， PI与其他调节细胞蛋白平衡网络的药物相结合，可能在 提高绩效指标的有效性。因此，当前提案的总体目标是延长这些研究 并确定PI和蛋白抑制调节剂在恢复患者来源的CBS功能方面是否有效 等位基因。如果成功，这里描述的实验可能会带来治疗CBS缺乏症和 可能还有其他与错义突变相关的遗传病。

PROJECT SUMMARY/ABSTRACT Inborn errors of metabolism comprise a large class of genetic diseases involving disorders of cellular metabolism. Homocysteine is an intermediary metabolite derived from methionine that can either be recycled back to methionine, or shunted down the transsulfuration pathway by the action of cystathionine beta-synthase (CBS), an enzyme primarily expressed in the liver and kidney. Individuals with mutations in CBS have CBS deficiency, characterized by extreme elevations in plasma total homocysteine (tHcy) and phenotypes including increased incidence of thrombosis, osteoporosis, dislocated lenses, and mental retardation. In healthy adults, tHcy concentration in plasma ranges from 5 to 15 M, but untreated patients with CBS deficiency often have tHcy in excess of 200 M. Current treatment strategies involve dietary restriction and vitamin therapy, but these are only partially effective and do not work in all patients. Over 85% of the described mutations in CBS deficient patients are missense mutations in which a single incorrect amino acid is substituted into the CBS polypeptide. Over the past decade, our lab has developed six different humanized mouse models that each express a different patient-derived mutant human CBS protein using an inducible promoter expressed at high levels in the liver. In all of these models, liver CBS activity is greatly diminished (<5% of wild-type), and serum tHcy is elevated by at least 20-fold. However, the behavior of each mutant CBS protein is not identical, with mutations affecting protein stability, protein aggregation, enzymatic function, or a combination of all three. The decreased protein stability effects of the mutations are mediated via the ubiquitin/proteasome system. Amazingly, treatment of mutant-expressing mice with proteasome inhibitors (PIs) can functionally reverse the effects of a majority of the missense mutations, resulting in large increases in CBS activity in the liver, and in some cases lowering of tHcy to near wild-type levels. PIs are FDA approved drugs for the treatments of certain types of cancer, but have never been studied in the context of inborn errors of metabolism. Our data suggests that low-dose PI treatment may be able to restore sufficient CBS enzyme activity to be phenotypically beneficial, but not have the toxicities associated with the high levels used in cancer therapy. In addition, we have preliminary data that suggests the combination of PIs with other drugs that modulate the cellular proteostasis network, may be useful in increasing the effectiveness of PIs. Thus, the overall goal of the current proposal is to extend these studies and determine if PIs and proteostasis modulators are effective in restoring function to patient-derived CBS alleles. If successful, the experiments described here could lead to novel treatments of CBS deficiency and potentially other genetic diseases associated with missense mutations.