Pan Xingxiu, Munan Subrata, Zuckerman Austin L, Pon Andrew, Violante Sara, Cross Justin R, Shah Hardik, Cracan Valentin
Scintillon Institute, Laboratory of Redox Biology and Metabolism, San Diego, CA 92121, USA.
Program in Mathematics and Science Education, University of California San Diego and San Diego State University, San Diego, CA 92120, USA.
bioRxiv. 2025 Jun 5:2025.06.02.657195. doi: 10.1101/2025.06.02.657195.
Dihydroxyacetone phosphate (DHAP), glycerol-3-phosphate (Gro3P) and reduced/oxidized nicotinamide adenine dinucleotide (NADH/NAD) are key metabolites of the Gro3P shuttle system that forms a redox circuit, allowing transfer of reducing equivalents between cytosol and mitochondria. Targeted activation of Gro3P biosynthesis was recently identified as a promising strategy to alleviate reductive stress by promoting NAD recycling, including in cells with an impaired mitochondrial complex I. However, because Gro3P constitutes the backbone of triglycerides under some circumstances, its accumulation can lead to excessive fat deposition. Here, we present the development of a novel genetically encoded tool based on a di-domain glycerol-3-phosphate dehydrogenase from algae (GPDH), which is a bifunctional enzyme that can recycle NAD while converting DHAP to Gro3P. In addition, this enzyme possesses an N-terminal domain which cleaves Gro3P into glycerol and inorganic phosphate (Pi) (in humans and other organisms, this reaction is catalyzed by a separate glycerol-3-phosphate phosphatase, a reaction also known as "glycerol shunt"). When expressed in mammalian cells, GPDH diminished Gro3P levels and boosted the TCA cycle and fatty acid β-oxidation in mitochondria. GPDH expression alone supported proliferation of HeLa cells under conditions of either inhibited activity of the mitochondrial electron transport chain or hypoxia. Moreover, human kidney cancer cells, which exhibit abnormal lipid accumulation, had decreased triglycerides levels when expressing GPDH. Our findings suggest that the coordinated boosting of both Gro3P biosynthesis and glycerol shunt may be a viable strategy to alleviate consequences of redox imbalance and associated impaired lipogenesis in a wide repertoire of conditions, ranging from primary mitochondrial diseases to obesity, type 2 diabetes, and metabolic dysfunction-associated steatotic liver disease (MASLD).
磷酸二羟丙酮（DHAP）、3-磷酸甘油（Gro3P）以及还原型/氧化型烟酰胺腺嘌呤二核苷酸（NADH/NAD）是3-磷酸甘油穿梭系统的关键代谢物，该系统形成一个氧化还原循环，允许还原当量在细胞质和线粒体之间转移。最近，靶向激活3-磷酸甘油生物合成被认为是一种有前景的策略，可通过促进NAD循环来减轻还原应激，包括在具有受损线粒体复合体I的细胞中。然而，由于在某些情况下3-磷酸甘油构成甘油三酯的骨架，其积累会导致脂肪过度沉积。在此，我们展示了一种基于藻类二结构域3-磷酸甘油脱氢酶（GPDH）开发的新型基因编码工具，该酶是一种双功能酶，可在将DHAP转化为Gro3P的同时回收NAD。此外，这种酶具有一个N端结构域，可将Gro3P裂解为甘油和无机磷酸（Pi）（在人类和其他生物体中，此反应由单独的3-磷酸甘油磷酸酶催化，该反应也称为“甘油分流”）。当在哺乳动物细胞中表达时，GPDH降低了Gro3P水平，并增强了线粒体中的三羧酸循环和脂肪酸β氧化。单独的GPDH表达在抑制线粒体电子传递链活性或缺氧条件下支持了HeLa细胞的增殖。此外，表现出异常脂质积累的人肾癌细胞在表达GPDH时甘油三酯水平降低。我们的研究结果表明，协调增强3-磷酸甘油生物合成和甘油分流可能是一种可行的策略，可在从原发性线粒体疾病到肥胖、2型糖尿病和代谢功能障碍相关脂肪性肝病（MASLD）等广泛情况下，减轻氧化还原失衡和相关脂肪生成受损的后果。
