Jennings William, Doshi Sejal, Hota Prasanta Kumar, Prodeus Aaron, Black Stephanie, Epand Richard M
Department of Biochemistry and Biomedical Sciences, McMaster University , 1280 Main Street West, Hamilton, Ontario L8S 4K1, Canada.
Biochemistry. 2017 Mar 7;56(9):1337-1347. doi: 10.1021/acs.biochem.6b01193. Epub 2017 Feb 24.
Diacylglycerol kinase ε (DGKε) catalyzes the phosphorylation of diacylglycerol, producing phosphatidic acid. DGKε demonstrates exquisite specificity for the acyl chains of diacylglycerol. This contributes to the enrichment of particular acyl chains within the lipids of the phosphatidylinositol cycle. Phosphatidylinositol is highly enriched with 1-stearoyl-2-arachidonoyl, which is important for maintaining cellular health. Dysregulation of DGKε perturbs lipid signaling and biosynthesis, which has been linked to epilepsy, Huntington's disease, and heart disease. Recessive loss-of-function mutations in the DGKε gene cause atypical hemolytic uremic syndrome. Because DGKε has never been purified, little is known about its molecular properties. We expressed human DGKε and a truncated version lacking the first 40 residues (DGKεΔ40) and purified both proteins to near homogeneity using nickel affinity chromatography. Kinase activity measurements showed that both purified constructs retained their acyl chain specificity for diacylglycerol with an activity level comparable to that of N-terminally FLAG epitope-tagged forms of these proteins expressed in COS7 cells. Both constructs lost activity upon being stored, particularly upon freezing and thawing, which was minimized by the addition of glycerol. Circular dichroism revealed that DGKε and DGKεΔ40 both contain significant amounts of α-helical and β structure and exhibit biphasic thermal denaturations. The loss of secondary structure upon heating was irreversible for both constructs, with relatively little effect of added dioleoylphosphatidylcholine. The addition of 50% glycerol stabilized both constructs and facilitated refolding of their secondary structures after heating. This is the first successful purification and characterization of DGKε's enzymatic and conformational properties. The purification of DGKε permits detailed analyses of this unique enzyme and will improve our understanding of DGKε-related diseases.
二酰基甘油激酶ε（DGKε）催化二酰基甘油的磷酸化反应，生成磷脂酸。DGKε对二酰基甘油的酰基链表现出极高的特异性。这有助于磷脂酰肌醇循环脂质中特定酰基链的富集。磷脂酰肌醇高度富含1-硬脂酰-2-花生四烯酰，这对维持细胞健康很重要。DGKε的失调会扰乱脂质信号传导和生物合成，这与癫痫、亨廷顿舞蹈症和心脏病有关。DGKε基因的隐性功能丧失突变会导致非典型溶血尿毒综合征。由于DGKε从未被纯化过，对其分子特性知之甚少。我们表达了人DGKε和缺失前40个残基的截短版本（DGKεΔ40），并使用镍亲和色谱法将这两种蛋白质纯化至近乎均一。激酶活性测量表明，两种纯化构建体对二酰基甘油都保留了其酰基链特异性，活性水平与在COS7细胞中表达的这些蛋白质的N端FLAG表位标签形式相当。两种构建体在储存时都会丧失活性，尤其是在冻融后，通过添加甘油可将这种情况最小化。圆二色性显示，DGKε和DGKεΔ40都含有大量的α螺旋和β结构，并表现出双相热变性。加热后二级结构的丧失对两种构建体来说都是不可逆的，添加二油酰磷脂酰胆碱的影响相对较小。添加50%的甘油可稳定两种构建体，并促进加热后其二级结构的重折叠。这是首次成功纯化并表征DGKε的酶学和构象特性。DGKε的纯化使得对这种独特酶的详细分析成为可能，并将增进我们对与DGKε相关疾病的理解。
