Abstract:
Human PANK1, PANK2, and PANK3 genes encode several pantothenate kinase isoforms that catalyze the phosphorylation of vitamin B5 (pantothenic acid) to phosphopantothenate, a critical step in the biosynthesis of the major cellular cofactor, Coenzyme A (CoA). Mutations in the PANK2 gene, which encodes the mitochondrial pantothenate kinase (PanK) isoform, have been linked to pantothenate-kinase associated neurodegeneration (PKAN), a debilitating and often fatal progressive neurodegeneration of children and young adults. While the biochemical properties of these enzymes have been well-characterized in vitro, their expression in a model organism such as yeast in order to probe their function under cellular conditions have never been achieved. Here we used three yeast mutants carrying missense mutations in the yeast PanK gene, CAB1, which are associated with defective growth at high temperature and iron, mitochondrial dysfunction, increased iron content, and oxidative stress, to assess the cellular function of human PANK genes and functional conservation of the CoA-controlled processes between humans and yeast. Overexpression of human PANK1 and PANK3 in these mutants restored normal cellular activity whereas complementation with PANK2 was partial and could only be achieved with an isoform, PanK2mtmΔ, lacking the mitochondrial transit peptide. These data, which demonstrate functional conservation of PanK activity between humans and yeast, set the stage for the use of yeast as a model system to investigate the impact of PKAN-associated mutations on the metabolic pathways altered in this disease.
摘要:
人PANK1,PANK2和PANK3基因编码几种泛酸激酶同工型,可催化维生素B5(泛酸)磷酸化为磷酸戊酸,主要细胞辅因子生物合成的关键步骤,辅酶A(CoA)。PANK2基因突变,编码线粒体泛酸激酶(PanK)亚型,与泛酸激酶相关的神经变性(PKAN)有关,使儿童和年轻人衰弱且通常致命的进行性神经变性。虽然这些酶的生化特性已经在体外得到了充分的表征,它们在模型生物如酵母中表达以探测它们在细胞条件下的功能从未实现。在这里,我们使用了三个在酵母PanK基因中携带错义突变的酵母突变体,CAB1,与高温和铁的生长缺陷有关,线粒体功能障碍,铁含量增加,和氧化应激,评估人类PANK基因的细胞功能以及人类和酵母之间CoA控制过程的功能保守性。这些突变体中人PANK1和PANK3的过表达恢复正常的细胞活性,而与PANK2的互补是部分的,只能通过同种型实现。PanK2mtmΔ,缺乏线粒体转运肽.这些数据,这证明了PanK活性在人类和酵母之间的功能保守性,为使用酵母作为模型系统研究PKAN相关突变对该疾病中代谢途径改变的影响奠定了基础。