Expression, purification and characterization of human diacylglycerol kinase zeta
Expression, purification and characterization of human diacylglycerol kinase zeta
复制标题
人二酰甘油激酶 zeta 的表达、纯化和表征
作者：
Saito;T.;Takahashi;D. and Sakane;F.
甘油二酯激酶ζ(DGKζ)使甘油二酯(DG)磷酸化，生成磷脂酸。DGKζ功能障碍与多种疾病有关，如心肌肥大、缺血和癫痫发作。此外，DGKζ和DGKα作为肿瘤免疫治疗的潜在靶点也受到了广泛的关注。然而，DGKζ一直没有得到纯化，因此，其酶学性质和结构尚未见报道，这阻碍了我们对DGKζ催化机理的了解，也阻碍了基于结构的合理药物设计的发展。在本研究中，我们利用杆状病毒-昆虫细胞表达系统获得了全长DGKζ，用于酶学和结构研究。全长DGKζ保持可溶状态，经纯化后为接近均一的单体，产量适合蛋白质结晶(L培养0.63 mg/l)。酶学鉴定表明，纯化的DGKζ处于完全功能状态。其对三磷酸腺苷和DG的Km值分别为0.0 5 mM和1.5mol%，对激活剂磷脂酰丝氨酸的EC50为8.6mol%，表明其对三磷酸腺苷的亲和力略高于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，纯化的酶可以在没有任何明显聚集的情况下被浓缩。圆二色谱显示DGKζ由25%的α-螺旋和18%的β-链组成。这是首次成功纯化和鉴定DGKζ的酶和构象性质。DGKζ的纯化可以对这一重要的酶进行详细的分析，并将促进我们对DGKζ相关疾病和治疗的了解。
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. TheKmvalues 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.