Fiddler Joanna L, Blum Jamie E, Heyden Katarina E, Castillo Luisa F, Thalacker-Mercer Anna E, Field Martha S
Division of Nutritional Sciences, Cornell University, Ithaca, NY, USA.
Department of Food, Nutrition, and Packaging Sciences, Clemson University, Clemson, SC, 29634, USA.
Genes Nutr. 2023 Mar 24;18(1):5. doi: 10.1186/s12263-023-00724-3.
Serine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine-producing THF-conjugated one-carbon units and glycine in the mitochondria. Biallelic SHMT2 variants were identified in humans and suggested to alter the protein's active site, potentially disrupting enzymatic function. SHMT2 expression has also been shown to decrease with aging in human fibroblasts. Immortalized cell models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels, suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. This study examined the role of SHMT2 and folate availability in regulating mitochondrial function, energy metabolism, and cellular proliferative capacity in both heterozygous and homozygous cell models of reduced SHMT2 expression. In this study, primary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6J dam crossed with a heterozygous Shmt2 male to generate Shmt2 (wild-type) or Shmt2 (HET) MEF cells. In addition, haploid chronic myeloid leukemia cells (HAP1, wild-type) or HAP1 cells lacking SHMT2 expression (ΔSHMT2) were cultured for 4 doublings in either low-folate or folate-sufficient culture media. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1α, pyruvate kinase enzyme activity, mitochondrial membrane potential, and mitochondrial function.
Homozygous loss of SHMT2 in HAP1 cells impaired cellular folate accumulation and altered mitochondrial DNA content, formate production, membrane potential, and basal respiration. Formate rescued proliferation in HAP1, but not ΔSHMT2, cells cultured in low-folate medium. Pyruvate kinase activity and protein levels were impaired in ΔSHMT2 cells and in MEF cells exposed to low-folate medium. Mitochondrial biogenesis protein levels were elevated in Shmt2 MEF cells, while mitochondrial mass was increased in both homozygous and heterozygous models of SHMT2 loss.
The results from this study indicate disrupted mitochondrial FOCM impairs mitochondrial folate accumulation and respiration, mitochondrial formate production, glycolytic activity, and cellular proliferation. These changes persist even after a potentially compensatory increase in mitochondrial biogenesis as a result of decreased SHMT2 levels.
丝氨酸羟甲基转移酶2（SHMT2）催化四氢叶酸（THF）与丝氨酸之间的可逆转化，在线粒体中生成与THF结合的一碳单位和甘氨酸。在人类中已鉴定出双等位基因SHMT2变体，提示其改变了该蛋白的活性位点，可能破坏酶功能。在人类成纤维细胞中，SHMT2表达也随衰老而降低。完全缺失SHMT2或叶酸缺乏的永生化细胞模型表现出氧化能力下降、线粒体复合物I组装受损及蛋白水平降低，提示叶酸介导的一碳代谢（FOCM）与氧化磷酸化系统在功能上相互协调。本研究在SHMT2表达降低的杂合和纯合细胞模型中，研究了SHMT2和叶酸可用性在调节线粒体功能、能量代谢及细胞增殖能力中的作用。在本研究中，从与杂合Shmt2雄性小鼠交配的C57Bl/6J母鼠中分离出原代小鼠胚胎成纤维细胞（MEF），以产生Shmt2（野生型）或Shmt2（杂合型）MEF细胞。此外，将单倍体慢性髓性白血病细胞（HAP1，野生型）或缺乏SHMT2表达的HAP1细胞（ΔSHMT2）在低叶酸或叶酸充足的培养基中培养4代。检测细胞的增殖、总叶酸水平、线粒体DNA含量、丙酮酸激酶和PGC1α的蛋白水平、丙酮酸激酶酶活性、线粒体膜电位及线粒体功能。
HAP1细胞中SHMT2的纯合缺失损害了细胞叶酸积累，并改变了线粒体DNA含量、甲酸生成、膜电位和基础呼吸。甲酸挽救了在低叶酸培养基中培养的HAP1细胞而非ΔSHMT2细胞的增殖。ΔSHMT2细胞及暴露于低叶酸培养基的MEF细胞中丙酮酸激酶活性和蛋白水平受损。Shmt2 MEF细胞中线粒体生物发生蛋白水平升高，而在SHMT2缺失的纯合和杂合模型中，线粒体质量均增加。
本研究结果表明，线粒体FOCM紊乱会损害线粒体叶酸积累和呼吸、线粒体甲酸生成、糖酵解活性及细胞增殖。即使由于SHMT2水平降低导致线粒体生物发生可能出现代偿性增加后，这些变化仍持续存在。
