Institute of Biochemistry & Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada.
Institute of Biochemistry & Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada.
Biochim Biophys Acta Proteins Proteom. 2020 Sep;1868(9):140448. doi: 10.1016/j.bbapap.2020.140448. Epub 2020 May 20.
The citric acid cycle (CAC) is a central metabolic pathway that links carbohydrate, lipid, and amino acid metabolism in the mitochondria and, hence, is a crucial target for metabolic regulation. The α-ketoglutarate dehydrogenase complex (KGDC) is the rate-limiting step of the CAC, the three enzymes of the complex catalyzing the transformation of α-ketoglutarate to succinyl-CoA with the release of CO and reduction of NAD to NADH. During hibernation, the metabolic rate of small mammals is suppressed, in part due to reduced body temperature but also active controls that suppress aerobic metabolism. The present study examined KGDC regulation during hibernation in skeletal muscle of the Richardson's ground squirrel (Urocitellus richardsonii). The KGDC was partially purified from skeletal muscle of euthermic and hibernating ground squirrels and kinetic properties were evaluated at 5°, 22°, and 37 °C. KGDC from hibernator muscle at all temperatures compared with euthermic controls exhibited a decreased affinity for CoA as well as reduced activation by Ca ions at 5 °C from both euthermic and hibernating conditions. Co-immunoprecipitation was employed to isolate the E1, E2 and E3 enzymes of the complex (OGDH, DLST, DLD) to allow immunoblot analysis of post-translational modifications (PTMs) of each enzyme. The results showed elevated phospho-tyrosine content on all three enzymes during hibernation as well as increased ADP-ribosylation and succinylation of hibernator OGDH. Taken together these results show that the KGDC is regulated by posttranslational modifications and temperature effects to reorganize enzyme activity and mitochondrial function to aid suppression of mitochondrial activity during hibernation.
柠檬酸循环（CAC）是一种核心代谢途径，它将碳水化合物、脂质和氨基酸代谢联系在一起，因此是代谢调节的关键靶点。α-酮戊二酸脱氢酶复合物（KGDC）是 CAC 的限速步骤，该复合物的三种酶催化α-酮戊二酸转化为琥珀酰辅酶 A，同时释放 CO 和将 NAD 还原为 NADH。在冬眠期间，小型哺乳动物的代谢率受到抑制，部分原因是体温降低，但也有主动控制因素抑制有氧代谢。本研究检查了冬眠期间在 Richardson's 地松鼠（Urocitellus richardsonii）骨骼肌中的 KGDC 调节。从恒温动物和冬眠地松鼠的骨骼肌中部分纯化了 KGDC，并在 5°C、22°C 和 37°C 下评估了其动力学特性。与恒温对照组相比，来自冬眠动物肌肉的 KGDC 在所有温度下对 CoA 的亲和力降低，并且在 5°C 时对 Ca 离子的激活作用降低，无论是来自恒温还是冬眠条件。采用共免疫沉淀法分离复合物的 E1、E2 和 E3 酶（OGDH、DLST、DLD），以允许对每个酶的翻译后修饰（PTMs）进行免疫印迹分析。结果表明，在冬眠期间，所有三种酶的磷酸酪氨酸含量升高，同时冬眠动物 OGDH 的 ADP-ribosylation 和 succinylation 增加。综上所述，这些结果表明，KGDC 通过翻译后修饰和温度效应进行调节，以重新组织酶活性和线粒体功能，从而有助于在冬眠期间抑制线粒体活性。
