Expression, Purification, and Characterization of Human Diacylglycerol Kinase ζ

Takumi Saito, Daisuke Takahashi, Fumio Sakane

研究成果: ジャーナルへの寄稿記事

抄録

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 K m 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.

元の言語英語
ページ(範囲)5540-5546
ページ数7
ジャーナルACS Omega
4
発行部数3
DOI
出版物ステータス出版済み - 3 19 2019

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Diacylglycerol Kinase
Purification
Enzymes
Dichroism
Agglomeration
Monomers
Crystallization
Proteins
Acids
Diglycerides
Phosphatidylserines
Adenosine Triphosphate
Phosphatidic Acids

All Science Journal Classification (ASJC) codes

  • Chemistry(all)
  • Chemical Engineering(all)

これを引用

Expression, Purification, and Characterization of Human Diacylglycerol Kinase ζ. / Saito, Takumi; Takahashi, Daisuke; Sakane, Fumio.

:: ACS Omega, 巻 4, 番号 3, 19.03.2019, p. 5540-5546.

研究成果: ジャーナルへの寄稿記事

Saito, Takumi ; Takahashi, Daisuke ; Sakane, Fumio. / Expression, Purification, and Characterization of Human Diacylglycerol Kinase ζ. :: ACS Omega. 2019 ; 巻 4, 番号 3. pp. 5540-5546.
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abstract = "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 K m 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.",
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