Expression, Purification, and Characterization of Human Diacylglycerol Kinase ζ
Expression, Purification, and Characterization of Human Diacylglycerol Kinase ζ
复制标题
人二酰甘油激酶的表达、纯化和表征 ❖
作者：
Takumi Saito;D. Takahashi;F. Sakane
甘油二酯激酶β（DGK β）磷酸化甘油二酯（DG）产生磷脂酸。DGK β的功能障碍与几种疾病有关，如心脏肥大、缺血和癫痫发作。此外，DGK β和DGKα作为肿瘤免疫治疗的潜在靶点受到了广泛关注。然而，DGK β从来没有被纯化，因此，它的酶性质和它的结构尚未被报道，阻碍了我们对DGK β的催化机制的理解和开发合理的基于结构的药物设计。在本研究中，我们使用杆状病毒-昆虫细胞表达系统产生了全长DGK酶，用于酶学和结构研究。全长DGK β保持可溶性，并作为单体纯化至接近均一性，产率适合于蛋白质结晶（0.63 mg/1 L培养物）。酶促鉴定表明，纯化的DGK酶处于完全功能状态。对三磷酸腺苷（ATP）和DG的Km值分别为0.05 mM和1.5 mol %，对激活剂磷脂酰丝氨酸的EC 50为8.6 mol %，表明其对ATP的亲和力略高于DGKα和DGKε，对DG和磷脂酰丝氨酸的亲和力与DGKα/DGKε相当。我们进一步证实，纯化的酶可以被浓缩而没有任何显著的聚集。圆二色性分析表明DGK β链由25%的α-螺旋和18%的β-链组成。这是第一次成功的纯化和表征的酶和构象特性的DGK酶。DGK β的纯化允许对这种重要的酶进行详细分析，并将促进我们对DGK β相关疾病和治疗的理解。
Diacylglycerol kinase ζ (DGKζ) phosphorylates diacylglycerol (DG) to generate phosphatidic acid. The dysfunction of DGKζ has been linked to several diseases, such as cardiac hypertrophy, ischemia, and seizures. Moreover, much attention has been paid to DGKζ, together with DGKα, as a potential target for cancer immunotherapy. However, DGKζ has never been purified and, thus, neither its enzymatic properties nor its structure has yet been reported, hindering our understanding of the catalytic mechanism of DGKζ and the development of a reasonable structure-based drug design. In the present study, we generated a full-length DGKζ using a baculovirus–insect cell expression system for enzymological and structural studies. Full-length DGKζ remained soluble and was purified to near homogeneity as a monomer with yields suitable for protein crystallization (0.63 mg/1 L culture). Enzymatic characterization showed that the purified DGKζ is in a fully functional state. The Km values for adenosine triphosphate (ATP) and DG were 0.05 mM and 1.5 mol %, respectively, and the EC50 for the activator phosphatidylserine was 8.6 mol %, indicating that its affinity for ATP is moderately higher than those of DGKα and DGKε, and its affinities for DG and phosphatidylserine are comparable to those of DGKα/DGKε. We further confirmed that the purified enzyme could be concentrated without any significant aggregation. Circular dichroism revealed that DGKζ is comprised of 25% α-helices and 18% β-strands. This is the first successful purification and characterization of the enzymatic and conformational properties of DGKζ. The purification of DGKζ allows detailed analyses of this important enzyme and will advance our understanding of DGKζ-related diseases and therapies.
影响因子：
2.9
作者：
Li, Junan;Mahajan, Anjali;Tsai, Ming-Daw
通讯作者：
Tsai, Ming-Daw
影响因子：
3.3
作者：
Abramovici, Hanan;Mojtabaie, Parmiss;Gee, Stephen H.
通讯作者：
Gee, Stephen H.