Luczkowska Karolina, Stekelenburg Caroline, Sloan-Béna Frédérique, Ranza Emmanuelle, Gastaldi Giacomo, Schwitzgebel Valérie, Maechler Pierre
Department of Cell Physiology and Metabolism, University of Geneva Medical Center, 1206, Geneva, Switzerland.
Faculty Diabetes Center, University of Geneva Medical Center, 1206, Geneva, Switzerland.
Hum Genomics. 2020 Mar 6;14(1):9. doi: 10.1186/s40246-020-00262-8.
Gain-of-function mutations in the GLUD1 gene, encoding for glutamate dehydrogenase (GDH), result in the hyperinsulinism/hyperammonemia HI/HA syndrome. HI/HA patients present with harmful hypoglycemia secondary to protein-induced HI and elevated plasma ammonia levels. These symptoms may be accompanied by seizures and mental retardation. GDH is a mitochondrial enzyme that catalyzes the oxidative deamination of glutamate to α-ketoglutarate, under allosteric regulations mediated by its inhibitor GTP and its activator ADP. The present study investigated the functional properties of the GDH-G446V variant (alias c.1496G > T, p.(Gly499Val) (NM_005271.4)) in patient-derived lymphoblastoid cells.
The calculated energy barrier between the opened and closed state of the enzyme was 41% lower in GDH-G446V compared to wild-type GDH, pointing to altered allosteric regulation. Computational analysis indicated conformational changes of GDH-G446V in the antenna region that is crucial for allosteric regulators. Enzymatic activity measured in patient-derived lymphoblastoid cells showed impaired allosteric responses of GDH-G446V to both regulators GTP and ADP. In particular, as opposed to control lymphoblastoid cells, GDH-G446V cells were not responsive to GTP in the lower range of ADP concentrations. Assessment of the metabolic rate revealed higher mitochondrial respiration in response to GDH-dependent substrates in the GDH-G446V lymphoblastoid cells compared to control cells. This indicates a shift toward glutaminolysis for energy provision in cells carrying the GDH-G446V variant.
Substitution of the small amino acid glycine for the hydrophobic branched-chain valine altered the allosteric sensitivity to both inhibitory action of GTP and activation by ADP, rendering cells metabolically responsive to glutamine.
编码谷氨酸脱氢酶（GDH）的GLUD1基因功能获得性突变会导致高胰岛素血症/高氨血症（HI/HA）综合征。HI/HA患者会出现因蛋白质诱导的高胰岛素血症继发的有害低血糖以及血浆氨水平升高。这些症状可能伴有癫痫发作和智力发育迟缓。GDH是一种线粒体酶，在其抑制剂GTP和激活剂ADP介导的变构调节下，催化谷氨酸氧化脱氨生成α-酮戊二酸。本研究在患者来源的淋巴母细胞中研究了GDH-G446V变体（别名c.1496G>T，p.(Gly499Val) (NM_005271.4)）的功能特性。
与野生型GDH相比，GDH-G446V酶的开放态与关闭态之间计算出的能垒低41%，表明变构调节发生改变。计算分析表明，GDH-G446V在对变构调节剂至关重要的天线区域发生了构象变化。在患者来源的淋巴母细胞中测得的酶活性显示，GDH-G446V对GTP和ADP这两种调节剂的变构反应受损。特别是，与对照淋巴母细胞不同，在较低的ADP浓度范围内，GDH-G446V细胞对GTP无反应。代谢率评估显示，与对照细胞相比，GDH-G446V淋巴母细胞中对GDH依赖性底物的反应导致线粒体呼吸更高。这表明携带GDH-G446V变体的细胞在能量供应方面向谷氨酰胺分解代谢转变。
小氨基酸甘氨酸被疏水性支链缬氨酸取代，改变了对GTP抑制作用和ADP激活作用的变构敏感性，使细胞对谷氨酰胺产生代谢反应。
