目的:癌细胞中的代谢重编程与线粒体功能障碍有关。线粒体2-酮戊二酸/苹果酸载体(OGC)已被认为是预防癌症进展的潜在靶标。尽管OGC参与了苹果酸/天冬氨酸穿梭,其在癌症代谢中的确切作用尚不清楚。我们旨在研究OGC是否可能通过运输质子来改变线粒体内膜电位。
方法:通过PCR和Westernblot分析研究OGC在小鼠组织和癌细胞中的表达。通过测量平面脂质双层的膜电导(Gm)来评估重组鼠OGC的质子转运功能。使用14C-苹果酸在蛋白脂质体中测量OGC介导的底物转运。
结果:OGC仅在天然(长链脂肪酸,FA)或化学(2,4-二硝基苯酚)质子载体。OGC活性的增加与FA的不饱和键数量的增加直接相关。OGC底物和抑制剂与FA竞争相同的蛋白质结合位点。精氨酸90被确定为与FA结合的关键氨基酸,ATP,2-氧戊二酸,和苹果酸,这是了解OGC介导的质子传输机制的第一步。
结论:OGC扩展了线粒体转运蛋白家族,具有双重功能:(i)代谢物转运和(ii)质子转运在质子存在下促进。阐明OGC对解偶联的贡献可能对于设计用于治疗癌症和其他代谢疾病的靶向药物是必不可少的。
Metabolic reprogramming in cancer cells has been linked to mitochondrial dysfunction. The mitochondrial 2-oxoglutarate/malate carrier (OGC) has been suggested as a potential target for preventing cancer progression. Although OGC is involved in the malate/aspartate shuttle, its exact role in cancer metabolism remains unclear. We aimed to investigate whether OGC may contribute to the alteration of mitochondrial inner membrane potential by transporting protons.
The expression of OGC in mouse tissues and cancer cells was investigated by PCR and Western blot analysis. The proton transport function of recombinant murine OGC was evaluated by measuring the membrane conductance (Gm) of planar lipid bilayers. OGC-mediated substrate transport was measured in proteoliposomes using 14C-malate.
OGC increases proton Gm only in the presence of natural (long-chain fatty acids, FA) or chemical (2,4-dinitrophenol) protonophores. The increase in OGC activity directly correlates with the increase in the number of unsaturated bonds of the FA. OGC substrates and inhibitors compete with FA for the same protein binding site. Arginine 90 was identified as a critical amino acid for the binding of FA, ATP, 2-oxoglutarate, and malate, which is a first step towards understanding the OGC-mediated proton transport mechanism.
OGC extends the family of mitochondrial transporters with dual function: (i) metabolite transport and (ii) proton transport facilitated in the presence of protonophores. Elucidating the contribution of OGC to uncoupling may be essential for the design of targeted drugs for the treatment of cancer and other metabolic diseases.