JP7366415B2 - phosphatidic acid sensor - Google Patents
phosphatidic acid sensor Download PDFInfo
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- JP7366415B2 JP7366415B2 JP2020019443A JP2020019443A JP7366415B2 JP 7366415 B2 JP7366415 B2 JP 7366415B2 JP 2020019443 A JP2020019443 A JP 2020019443A JP 2020019443 A JP2020019443 A JP 2020019443A JP 7366415 B2 JP7366415 B2 JP 7366415B2
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Description
特許法第３０条第２項適用 Ｔｈｅ ＦＥＢＳ Ｊｏｕｒｎａｌ（２０１９）、Ｆｅｄｅｒａｔｉｏｎ ｏｆ Ｅｕｒｏｐｅａｎ Ｂｉｏｃｈｅｍｉｃａｌ Ｓｏｃｉｅｔｉｅｓのウェブサイト（ｈｔｔｐｓ：／／ｆｅｂｓ．ｏｎｌｉｎｅｌｉｂｒａｒｙ．ｗｉｌｅｙ．ｃｏｍ／ｄｏｉ／ｅｐｄｆ／１０．１１１１／ｆｅｂｓ．１５１３７）において令和元年１１月１３日に公開Article 30, Paragraph 2 of the Patent Act applies The FEBS Journal (2019), Federation of European Biochemical Societies website (https://febs.onlinelibrary.wiley.com/do i/epdf/10.1111/febs.15137) Released on November 13, 2019
本発明は、ホスファチジン酸センサーに関するものである。 The present invention relates to a phosphatidic acid sensor.
ホスファチジン酸（PA）は生体膜中に存在する最も単純なリン脂質であり、量としては全細胞脂質のごく一部に過ぎないが、有糸分裂誘発、遊走、分化などの幅広い生物学的現象に関与している。PAはホスファチジルイノシトール4-リン酸5-キナーゼ（PIP5K）, ラパマイシン標的タンパク質（mTOR）, 非典型プロテインキナーゼC（aPKC）などの多くのシグナルタンパク質を制御することが多くの報告により明らかとなっている。その上、PA量は時空間的にそして厳密に制御されている。例えば、PAは神経芽細胞腫と褐色細胞腫の分化時に非常に増加し、シナプスリボンに富んでいる。他にも、PA量の変動はタンパク質輸送および食作用と相関することが明らかとなっている。 Phosphatidic acid (PA) is the simplest phospholipid present in biological membranes, and although it accounts for only a small portion of total cellular lipids, it is involved in a wide range of biological phenomena such as mitogenesis, migration, and differentiation. is involved in Many reports have revealed that PA regulates many signaling proteins such as phosphatidylinositol 4-phosphate 5-kinase (PIP5K), target of rapamycin (mTOR), and atypical protein kinase C (aPKC). . Moreover, the amount of PA is tightly controlled spatiotemporally. For example, PA is greatly increased during neuroblastoma and pheochromocytoma differentiation and is enriched in synaptic ribbons. It has also been shown that changes in PA levels are correlated with protein transport and phagocytosis.
PAは複数の経路から生成され、シグナル伝達脂質としてのPAはジアシルグリセロールキナーゼ（DGK）によるジアシルグリセロール（DG）のリン酸化、およびホスホリパーゼD（PLD）によるホスファチジルコリン（PC）の加水分解により生成される。DGKには10種のアイソザイム（α,β,γ,δ,η,κ,ε,ζ,ι,θ）が同定されており、ガン、てんかん、強迫性障害、双極性障害、心肥大、高血圧および2型糖尿病などの多種多様な疾患の発症に関与している。 PA is generated from multiple pathways, and PA as a signaling lipid is generated by phosphorylation of diacylglycerol (DG) by diacylglycerol kinase (DGK) and hydrolysis of phosphatidylcholine (PC) by phospholipase D (PLD). . Ten types of DGK isozymes (α, β, γ, δ, η, κ, ε, ζ, ι, θ) have been identified, and they are associated with cancer, epilepsy, obsessive-compulsive disorder, bipolar disorder, cardiac hypertrophy, and hypertension. It is also involved in the development of a wide variety of diseases such as type 2 diabetes.
PLDも2種（1,2）のアイソザイムが存在し、ガンやパーキンソン病やアルツハイマー病を含む神経変性疾患に関与することが明らかとなっている。さらに、細胞膜リン脂質デノボ合成経路の重要な中間体として働くPAは、リゾPA（LPA）アシルトランスフェラーゼ（LPAAT）によって産生され、このLPAATは卵巣ガン、および子宮内膜ガンならびに急性白血病の治療標的でもある。 There are two types of PLD isozymes (1 and 2), and it has been shown that they are involved in neurodegenerative diseases including cancer, Parkinson's disease, and Alzheimer's disease. Furthermore, PA, which serves as a key intermediate in the cell membrane phospholipid de novo synthesis pathway, is produced by lyso-PA (LPA) acyltransferase (LPAAT), which is also a therapeutic target for ovarian and endometrial cancer as well as acute leukemia. be.
ホスファチジン酸センサーに関する文献として、非特許文献１がある。 As a document related to phosphatidic acid sensors, there is Non-Patent Document 1.
非特許文献１に記載のホスファチジン酸結合タンパク質・ドメインは、それ自身の細胞内膜局在性があり、ホスファチジン酸産生時以外でも細胞内の膜領域（形質体やゴルジ体等）に局在してしまう。すなわち、バックグラウンド活性が高すぎて、細胞刺激時またはホスファチジン酸産生酵素発現時に、細胞内膜で産生される細胞内ホスファチジン酸を正確に検出するのが困難であるという課題があった。 The phosphatidic acid-binding protein domain described in Non-Patent Document 1 has its own intracellular membrane localization, and is localized in intracellular membrane regions (plasmosomes, Golgi bodies, etc.) even when phosphatidic acid is not being produced. I end up. That is, the background activity is so high that it is difficult to accurately detect intracellular phosphatidic acid produced in intracellular membranes during cell stimulation or expression of a phosphatidic acid-producing enzyme.
そこで、本発明では、細胞内で産生するホスファチジン酸を高精度で検出することができるホスファチジン酸可視化センサーを提供することを目的とする。 Therefore, an object of the present invention is to provide a phosphatidic acid visualization sensor that can detect phosphatidic acid produced intracellularly with high accuracy.
上記課題を解決するために、本発明の一つの観点によれば、ホスファチジン酸センサーを、α-シヌクレインのN末端領域（α-Syn-N）を含有するものとした。 In order to solve the above problems, according to one aspect of the present invention, a phosphatidic acid sensor contains the N-terminal region of α-synuclein (α-Syn-N).
また、本発明の他の観点によれば、ホスファチジン酸センサーを、下記（１）～（３）のいずれか１つの塩基配列によってコードされるアミノ酸配列を有するペプチドを含有するものとした。
（１）配列番号１で表される塩基配列；
（２）配列番号１で表される塩基配列と90%以上の相同性を有する塩基配列；
（３）配列番号１で表される塩基配列の18個以内の塩基が、欠失、置換及び／又は不可された塩基配列。
また、本発明の他の観点によれば、ホスファチジンセンサーを、下記（１）～（３）のいずれか１つのアミノ酸配列を有するペプチドを含有するものとした。
（１）配列番号２で表されるアミノ酸配列；
（２）配列番号２で表されるアミノ酸配列と90%以上の相同性を有するアミノ酸配列；
（３）配列番号２で表されるアミノ酸配列の6個以内のアミノ酸が、欠失、置換及び／又は付加されたアミノ酸配列。
According to another aspect of the present invention, the phosphatidic acid sensor contains a peptide having an amino acid sequence encoded by any one of the following base sequences (1) to (3).
(1) Base sequence represented by SEQ ID NO: 1;
(2) a nucleotide sequence having 90% or more homology with the nucleotide sequence represented by SEQ ID NO: 1;
(3) A base sequence in which 18 or less bases of the base sequence represented by SEQ ID NO: 1 are deleted, substituted, and/or missing.
According to another aspect of the present invention, the phosphatidine sensor contains a peptide having any one of the following amino acid sequences (1) to (3).
(1) Amino acid sequence represented by SEQ ID NO: 2;
(2) an amino acid sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 2;
(3) An amino acid sequence in which six or fewer amino acids of the amino acid sequence represented by SEQ ID NO: 2 are deleted, substituted, and/or added.
細胞内で産生するホスファチジン酸を検出することができるホスファチジン酸センサーを提供することができる。 A phosphatidic acid sensor capable of detecting phosphatidic acid produced within cells can be provided.
本発明の一つの観点によれば、ホスファチジン酸（可視化）センサーを、α-シヌクレインのN末端領域を含有するものとした。 According to one aspect of the invention, a phosphatidic acid (visualization) sensor contains the N-terminal region of α-synuclein.
また、本発明の一つの観点によれば、ホスファチジン酸センサーを、下記（１）～（３）のいずれか１つの塩基配列によってコードされるアミノ酸配列を有するペプチドを含有するものとした。
（１）配列番号１で表される塩基配列；
（２）配列番号１で表される塩基配列と90%以上の相同性を有する塩基配列；
（３）配列番号１で表される塩基配列の18個以内の塩基が、欠失、置換及び／又は不可された塩基配列。
また、本発明の他の観点によれば、ホスファチジンセンサーを、下記（１）～（３）のいずれか１つのアミノ酸配列を有するペプチドを含有するものとした。
（１）配列番号２で表されるアミノ酸配列；
（２）配列番号２で表されるアミノ酸配列と90%以上の相同性を有するアミノ酸配列；
（３）配列番号２で表されるアミノ酸配列の6個以内のアミノ酸が、欠失、置換及び／又は付加されたアミノ酸配列。
Further, according to one aspect of the present invention, the phosphatidic acid sensor contains a peptide having an amino acid sequence encoded by any one of the following base sequences (1) to (3).
(1) Base sequence represented by SEQ ID NO: 1;
(2) a nucleotide sequence having 90% or more homology with the nucleotide sequence represented by SEQ ID NO: 1;
(3) A base sequence in which 18 or less bases of the base sequence represented by SEQ ID NO: 1 are deleted, substituted, and/or missing.
According to another aspect of the present invention, the phosphatidine sensor contains a peptide having any one of the following amino acid sequences (1) to (3).
(1) Amino acid sequence represented by SEQ ID NO: 2;
(2) an amino acid sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 2;
(3) An amino acid sequence in which six or fewer amino acids of the amino acid sequence represented by SEQ ID NO: 2 are deleted, substituted, and/or added.
１．序論
前述のとおり、細胞内PAの局在および動態の追跡はPAによって制御される生物学的現象を理解するために必須である。胞子形成特異的タンパク質（Spo20p）やcAMPホスホジエステラーゼ4A1（PDE4A1）といったタンパク質のPA結合ドメイン（PABD）がしばしばPAセンサーとして用いられているが、Spo20pは細胞膜に、PDE4A1はゴルジ体に細胞刺激非依存的に局在を示す。これらの細胞刺激非依存的な局在はPAセンサーとしての機能を妨害し、それらの適用を困難とさせる。したがって、あらゆる細胞刺激、細胞種に広く適用可能なPAセンサーは未だ開発されていない。
1. Introduction As mentioned above, tracking the localization and dynamics of intracellular PA is essential for understanding the biological phenomena regulated by PA. PA-binding domains (PABD) of proteins such as sporulation-specific protein (Spo20p) and cAMP phosphodiesterase 4A1 (PDE4A1) are often used as PA sensors, but Spo20p is attached to the cell membrane and PDE4A1 is attached to the Golgi apparatus in a cell stimulus-independent manner. shows localization. Their cellular stimulus-independent localization interferes with their function as PA sensors, making their application difficult. Therefore, a PA sensor that is widely applicable to all cell stimuli and cell types has not yet been developed.
最近、発明者らの検討によりαシヌクレイン（α-Syn）が強く、そして選択的にPAに結合することが分かった。そこで発明者は、α-Synの有用なPAセンサーとしての可能性について探索を行った。in vitroにてα-SynのN末端領域（α-Syn-N）がPAに結合し、細胞内においてα-Syn-Nは膜局在性を示さないことが明らかとなった。なお、α-SynのN末端領域の配列は、配列番号１に示すものである。また、配列番号１に示す塩基配列によりコードされるアミノ酸配列は、配列番号２に示すものである。また、α-Syn-NはDGKやPLDなどのPA産生酵素とは活性依存的に共局在する一方で、PI（4,5）P2産生酵素であるPIP5Kとは共局在せず、α-Syn-Nが細胞内PAに選択的に結合することが示唆された。以上より、α-Syn-Nは細胞内にて信頼性が高く、幅広く適用可能なPAセンサーとして利用可能である。 Recently, the inventors' studies revealed that α-synuclein (α-Syn) binds strongly and selectively to PA. Therefore, the inventor investigated the possibility of α-Syn as a useful PA sensor. It was revealed that the N-terminal region of α-Syn (α-Syn-N) binds to PA in vitro, and that α-Syn-N does not show membrane localization within cells. The sequence of the N-terminal region of α-Syn is shown in SEQ ID NO: 1. Furthermore, the amino acid sequence encoded by the base sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2. In addition, while α-Syn-N colocalizes with PA-producing enzymes such as DGK and PLD in an activity-dependent manner, it does not colocalize with PIP5K, a PI(4,5)P2-producing enzyme, and α It was suggested that -Syn-N selectively binds to intracellular PA. From the above, α-Syn-N is highly reliable in cells and can be used as a widely applicable PA sensor.
２．結果
（１）α-Synとその領域欠損変異体のPA結合活性
まず初めに、His×6-SUMOタグ融合α-Synとそのドメイン欠損変異体（α-Syn-N, non-amyloid-b-component; α-Syn-NAC, C末端領域; α-Syn-C, NAC and C; α-Syn-NAC-C）のリポソーム共沈降法を、全長のα-Synが強く結合することが明らかとなっている18:1/18:1-PAを用いて行った。図２に示すように、α-Syn-Nが最も強く18:1/18:1-PAに結合し、一方でα-Syn-NAC, α-Syn-C, α-Syn-NAC-CはPA結合活性を示さなかった。また、α-Syn-NのPA結合活性は全長α-Synと同等であった。
2. Results (1) PA-binding activity of α-Syn and its domain-deleted mutant component; α-Syn-NAC, C-terminal region; α-Syn-C, NAC and C; α-Syn-NAC-C), it is clear that full-length α-Syn binds strongly. This was done using 18:1/18:1-PA. As shown in Figure 2, α-Syn-N binds most strongly to 18:1/18:1-PA, while α-Syn-NAC, α-Syn-C, α-Syn-NAC-C It showed no PA binding activity. Furthermore, the PA binding activity of α-Syn-N was equivalent to that of full-length α-Syn.
次に、16:0/16:0-, 16:1/18:1-, 18:1/18:1-, 18:0/18:0-PAなどの様々なPA分子種を用いたα-Syn-Nのリポソーム沈降法を行ったところα-Syn-Nは全長α-Synと同様に18:1/18:1-PAに最も強く結合し、16:0/16:0-, 16:1/18:1-,とも結合した（図３、４）。 Next, α using various PA molecular species such as 16:0/16:0-, 16:1/18:1-, 18:1/18:1-, 18:0/18:0-PA When -Syn-N was subjected to liposome precipitation, α-Syn-N bound most strongly to 18:1/18:1-PA, similar to full-length α-Syn, and 16:0/16:0-, 16 :1/18:1-, also combined (Figs. 3 and 4).
α-Syn-Nの18:1/18:1-PAに対する親和性を、様々な18:1/18:1-PA濃度でリポソーム沈降法を行う事で決定した。α-Syn-NのPA濃度依存的共沈降が観察され、解離定数（Kd）は6.6μMと求められた。これはSpo20p-PABD（2.2μM）や、PDE4A1-PABD（6.8μM）と同程度であった（図５、６）。 The affinity of α-Syn-N for 18:1/18:1-PA was determined by liposome precipitation at various 18:1/18:1-PA concentrations. PA concentration-dependent coprecipitation of α-Syn-N was observed, and the dissociation constant (Kd) was determined to be 6.6 μM. This was comparable to Spo20p-PABD (2.2 μM) and PDE4A1-PABD (6.8 μM) (Figs. 5 and 6).
α-Syn-Nの酸性グリセロリン脂質に対する親和性を比較するため、18:1/18:1-PA, ホスファチジルセリン（PS）, PI（4,5）P2を用いてリポソーム沈降法を行った。図６、７に示すようにα-Syn-Nは18:1/18:1-PAに最も強く結合した一方で、弱いシグナルのみがPI（4,5）P2では検出され、PSでは検出されなかった。 To compare the affinity of α-Syn-N for acidic glycerophospholipids, liposome precipitation was performed using 18:1/18:1-PA, phosphatidylserine (PS), and PI(4,5)P2. As shown in Figures 6 and 7, α-Syn-N bound most strongly to 18:1/18:1-PA, while only weak signals were detected with PI(4,5)P2 and not with PS. There wasn't.
PA、ホスファチジルエタノールアミン（PE）、セラミド-1-リン酸のような親水性の頭部が小さい円錐形脂質は、負の膜曲率を生じさせる。そこで、PEとC1Pを用いたリポソーム沈降法を行ったところ、α-Syn-NはほぼPEやC1Pと相互作用を示さなかった（図８）。よって、α-Syn-Nは膜曲率選択的ではなく、PA選択的であると考えられる。 Conical lipids with small hydrophilic heads, such as PA, phosphatidylethanolamine (PE), and ceramide-1-phosphate, give rise to negative membrane curvature. Therefore, when we performed a liposome sedimentation method using PE and C1P, α-Syn-N showed almost no interaction with PE or C1P (Figure 8). Therefore, it is considered that α-Syn-N is not membrane curvature selective but PA selective.
さらにα-Syn-Nの脂質選択性について検証するため、トリアシルグリセロール（TG）, DG, PA, PS, PE, PC, ホスファチジルグリセロール（PG）, カルジオリピン（CL）, PI, PI（4）P, PI（4, 5）P2, PI（3, 4, 5）P3, コレステロール（chol）, スフィンゴミエリン （SM）, スルファチド（SGC）らをスポットしたメンブレンを用いて脂質オーバーレイアッセイを行った。図９、１０に示すように、α-Syn-NはPAに最も強く結合し、他の脂質に対しては非常に弱い結合能しか示さなかった。これらの実験は16:0/16:0-グリセロ脂質を用いて行ったが、18:1/18:1-PA, PC, PE, PG, PS, PI, PI（4, 5）P2,を用いても同様の結果が得られた（図２７、２８）。また、PAはLPA, C1Pと比較しても非常に強くα-Syn-Nと結合した（図１１、１２）。以上より、α-Syn-NはPAに選択的に結合する事が示された。 In order to further verify the lipid selectivity of α-Syn-N, triacylglycerol (TG), DG, PA, PS, PE, PC, phosphatidylglycerol (PG), cardiolipin (CL), PI, PI(4)P Lipid overlay assays were performed using membranes spotted with , PI(4,5)P2, PI(3,4,5)P3, cholesterol (chol), sphingomyelin (SM), and sulfatide (SGC). As shown in Figures 9 and 10, α-Syn-N bound most strongly to PA, and showed very weak binding ability to other lipids. These experiments were performed using 16:0/16:0-glycerolipids, but 18:1/18:1-PA, PC, PE, PG, PS, PI, PI(4,5)P2, Similar results were obtained using the same method (FIGS. 27 and 28). Furthermore, PA bound to α-Syn-N very strongly compared to LPA and C1P (FIGS. 11 and 12). From the above, it was shown that α-Syn-N selectively binds to PA.
（２）COS-7細胞にてα-Syn-NはDGKβと細胞膜にて共局在する
次に、α-Syn-NがPA産生酵素であるDGKとPLDと細胞内にて共局在するか検証を行った。まず初めに我々はDsRed融合α-Syn-NがDsRedタグ単独と同様に、細胞質および核に広く分布することを確認し、これはα-Syn-Nが細胞膜やゴルジ体のような生体膜に局在化しないことを示している。
(2) α-Syn-N co-localizes with DGKβ in the cell membrane in COS-7 cells Next, α-Syn-N co-localizes with PA-producing enzymes DGK and PLD in the cell. We conducted a verification. First, we confirmed that DsRed-fused α-Syn-N is widely distributed in the cytoplasm and nucleus, similar to the DsRed tag alone, which suggests that α-Syn-N is distributed in biological membranes such as the plasma membrane and the Golgi apparatus. It shows that it is not localized.
DGKβは基質であるDGが存在する細胞膜に局在する事が知られている。発明者は、EGFPタグ融合DGKβがCOS-7細胞において細胞膜に局在する事を確認し（図１３）、そしてDsRed-α-Syn-NがEGFP-DGKβと共局在する事を明らかとした（図１３、１４）。一方で、不活性（KD）型変異EGFP-DGKβ-KDとはDsRed-α-Syn-Nは共局在しなかった。また、1型DGK（α,β,γ）阻害剤である化合物A（IC50:0.02μM）がDsRed-α-Syn-NとEGFP-DGKβの共局在を打ち消したことから、共局在化がPA依存的に生じ、α-Syn-NはDGKβタンパク質自体を認識しない事が示され、α-Syn-Nが細胞膜にてDGKβが産生するPAを認識できる事が示された。 DGKβ is known to be localized in the cell membrane where the substrate DG is present. The inventors confirmed that EGFP-tagged DGKβ was localized to the cell membrane in COS-7 cells (Figure 13), and clarified that DsRed-α-Syn-N was colocalized with EGFP-DGKβ. (Figures 13, 14). On the other hand, DsRed-α-Syn-N did not colocalize with the inactive (KD) mutant EGFP-DGKβ-KD. In addition, compound A (IC50: 0.02 μM), a type 1 DGK (α,β,γ) inhibitor, canceled the colocalization of DsRed-α-Syn-N and EGFP-DGKβ, indicating that colocalization It was shown that α-Syn-N occurs in a PA-dependent manner and that α-Syn-N does not recognize the DGKβ protein itself, indicating that α-Syn-N can recognize PA produced by DGKβ at the cell membrane.
同様の検証をSpo20p-PABDと、PDE4A1-PABDを用いて行ったところ、DsRed-Spo20p-PABDは細胞膜にてEGFP-DGKβと共局在したが、DsRed-Spo20p-PABDはEGFP-DGKβ非存在下においても細胞膜へと局在した。次に、報告されているようにDsRed-PDE4A1-PABDがゴルジ体へと局在する事を確認し、次にEGFP-DGKβと共発現させたものの、DsRed-PDE4A1-PABDはゴルジ体に留まりEGFP-DGKβとの共局在を示さなかった（図２８）。 When similar verification was performed using Spo20p-PABD and PDE4A1-PABD, DsRed-Spo20p-PABD colocalized with EGFP-DGKβ at the cell membrane, but DsRed-Spo20p-PABD colocalized with EGFP-DGKβ in the absence of EGFP-DGKβ. It was also localized to the cell membrane. Next, we confirmed that DsRed-PDE4A1-PABD localized to the Golgi apparatus as reported, and then coexpressed it with EGFP-DGKβ, but DsRed-PDE4A1-PABD remained in the Golgi apparatus and EGFP - It did not show colocalization with DGKβ (Figure 28).
（３）COS-7細胞にてα-Syn-Nはミリストイル化DGKζと細胞膜およびゴルジ体にて共局在する
DGKζは細胞質および核に広く局在する事が報告されており、PAを産生するためにDGKζは基質が存在する生体膜へと移行する必要がある。膜局在化を促進する修飾としてミリストイル化が知られており、我々はDGKζの膜局在化を誘導するため、N末端ミリストイル化として機能するc-Srcのコンセンサス配列を有するAcGFP-DGKζのcDNA （Myr-AcGFP-DGKζ）を作成した。まず、Myr-AcGFP-DGKζが核周辺領域と細胞膜に共局在する事を確認した（図１７、１８）。Myr-AcGFP-DGKζはトランスゴルジマーカーであるTGN46やゴルジ体局在タンパク質であるDsRed-PDE4A1-PABDと局在が一致したことから、Myr-AcGFP-DGKζがゴルジ体へと局在している事が示された。DsRed-α-Syn-Nは細胞膜およびトランスゴルジにてMyr-AcGFP-DGKζと著しく共局在したが、不活性型変異体Myr-AcGFP-DGKζ-KDとは共局在しなかった（図１７、１８）ことから、共局在が、DGKζが産生したPA由来である事が示された。さらに、これらの結果は、DsRed-α-Syn-Nは細胞膜だけでなくゴルジ体においてもPAを標識できる事を示している。
(3) In COS-7 cells, α-Syn-N colocalizes with myristoylated DGKζ at the plasma membrane and Golgi apparatus.
DGKζ has been reported to be widely localized in the cytoplasm and nucleus, and in order to produce PA, DGKζ must be translocated to biological membranes where substrates are present. Myristoylation is known as a modification that promotes membrane localization, and we used the cDNA of AcGFP-DGKζ with a c-Src consensus sequence that functions as an N-terminal myristoylation to induce membrane localization of DGKζ. (Myr-AcGFP-DGKζ) was created. First, we confirmed that Myr-AcGFP-DGKζ was colocalized in the perinuclear region and the cell membrane (Figures 17 and 18). The localization of Myr-AcGFP-DGKζ coincided with the trans-Golgi marker TGN46 and the Golgi-localized protein DsRed-PDE4A1-PABD, indicating that Myr-AcGFP-DGKζ is localized to the Golgi apparatus. It has been shown. DsRed-α-Syn-N significantly colocalized with Myr-AcGFP-DGKζ at the plasma membrane and trans-Golgi, but not with the inactive mutant Myr-AcGFP-DGKζ-KD (Figure 17 , 18), indicating that the colocalization was derived from PA produced by DGKζ. Furthermore, these results indicate that DsRed-α-Syn-N can label PA not only at the plasma membrane but also at the Golgi apparatus.
Spo20p-PABDもゴルジ体においてMyr-AcGFP-DGKζとは共局在したが、Myr-AcGFP-DGKζ-KDとは共局在しなかった（図３２、３３）ことから、Spo20p-PABDもゴルジ体におけるPAを標識できる事が示された。しかしながら、Spo20p-PABDは細胞膜においてはMyr-AcGFP-DGKζ-KDと共局在を示したことから、Spo20p-PABDはCOS-7細胞内で細胞膜におけるPA量の変動を検出することが困難である。 Spo20p-PABD also colocalized with Myr-AcGFP-DGKζ in the Golgi apparatus, but not with Myr-AcGFP-DGKζ-KD (Figures 32 and 33). It was shown that it is possible to label PA in However, Spo20p-PABD colocalized with Myr-AcGFP-DGKζ-KD at the cell membrane, indicating that it is difficult to detect changes in the amount of PA at the cell membrane in COS-7 cells. .
（４）COS-7細胞にてα-Syn-Nはホルボールエステル刺激条件においてDGKγと共局在する
次に我々はDsRed-が短期間のみ生成されるPAを標識できるか検証を行った。DGKγはホルボールエステル（PMA）刺激により細胞質から基質が存在する細胞膜へと移行する事が知られている。そこで、EGFP-DGKγを発現させたCOS-7細胞をホルボールエステルで30分処理を行ったところEGFP-DGKγの細胞膜への移行が観測された（図１９）。特にこの刺激時にDsRed-α-Syn-Nは細胞膜にてEGFP-DGKγと共局在し、一方でEGFP-DGKγ-KDとはPMA刺激時にも共局在しなかった（図１９、２０）。これらよりDGK活性が共局在に必須である事が示され、α-Syn-Nが短期間に生成されるPAを標識できる事が示された。
(4) α-Syn-N colocalizes with DGKγ under phorbol ester stimulation conditions in COS-7 cells Next, we verified whether DsRed- could label PA that is produced only for a short period of time. It is known that DGKγ is translocated from the cytoplasm to the cell membrane where the substrate is present upon stimulation with phorbol ester (PMA). Therefore, when COS-7 cells expressing EGFP-DGKγ were treated with phorbol ester for 30 minutes, translocation of EGFP-DGKγ to the cell membrane was observed (FIG. 19). In particular, DsRed-α-Syn-N colocalized with EGFP-DGKγ at the cell membrane during this stimulation, while it did not colocalize with EGFP-DGKγ-KD even during PMA stimulation (FIGS. 19 and 20). These results showed that DGK activity is essential for colocalization, and that α-Syn-N can label PA generated in a short period of time.
（５）COS-7細胞にてα-Syn-NはPLD2と共局在する
次に、DGK以外のPA産生酵素で検証を行うためPLD2を用いた。EGFP-PLD2は細胞膜およびゴルジ体への局在を示した（図２１、３４）。DsRed-α-Syn-NはEGFP-PLD2と共局在を示したが、DsRed単体はしなかった。さらに、PLD阻害剤であるFIPIにより共局在が減弱されたことから、共局在はPLD活性による事が示された。これらの結果は、α-Syn-Nが様々な膜環境において今回検証された全てのPA産生酵素により産生されたPAを標識できる事を示している。
(5) α-Syn-N colocalizes with PLD2 in COS-7 cells Next, we used PLD2 to perform verification with PA-producing enzymes other than DGK. EGFP-PLD2 was localized to the plasma membrane and Golgi apparatus (Figures 21, 34). DsRed-α-Syn-N showed colocalization with EGFP-PLD2, but DsRed alone did not. Furthermore, the colocalization was attenuated by the PLD inhibitor FIPI, indicating that the colocalization was due to PLD activity. These results indicate that α-Syn-N can label PA produced by all PA-producing enzymes tested in various membrane environments.
（６）COS-7細胞にてα-Syn-NはPIP5K1Aと共局在しない
PI（4,5）P2のスポットやリポソームに対して、α-Syn-Nの弱いシグナルが検出されたため（図７から図１２）、α-Syn-NがPI（4,5）P2産生酵素であるPIP5K1Aと共局在するかどうか検証を行ったところ、DsRed-α-Syn-NはEGFP-PIP5K1Aと共局在しなかった（図２３、２４）。また、PI（4,5）P2センサーとして確立されているDsRed-PLCTM1-pleckstrin homology domain（PHD）はPIP5K1Aと共局在することを確認した（図２３、２４）。これらの結果は、α-Syn-Nは細胞内にてPI（4,5）P2に結合せず、PA特異的である事を示している。
(6) α-Syn-N does not colocalize with PIP5K1A in COS-7 cells
Because weak signals of α-Syn-N were detected for PI(4,5)P 2 spots and liposomes (Figures 7 to 12), α-Syn-N was detected as PI(4,5)P 2 When we verified whether DsRed-α-Syn-N colocalized with the produced enzyme PIP5K1A, DsRed-α-Syn-N did not colocalize with EGFP-PIP5K1A (Figures 23 and 24). Furthermore, it was confirmed that DsRed-PLC TM 1-pleckstrin homology domain (PHD), which has been established as a PI(4,5)P 2 sensor, colocalizes with PIP5K1A (FIGS. 23 and 24). These results indicate that α-Syn-N does not bind to PI(4,5)P 2 in cells and is PA-specific.
（７）考察
現在、信頼性と汎用性の高いPAセンサーは存在していない。Spo20p-PABDとPDE4A1-PABDはしばしば細胞内PAセンサーとして用いられているが、それらは細胞膜（Spo20p-PABD）やゴルジ体（PDE4A1-PABD）のように、細胞刺激誘発性PA産生に非依存的にそれら自身の局在を示す。発明者はα-Syn-Nが新規の信頼性があり、広く適用可能なPAセンサーであると明らかにした。特にDsRed-α-Syn-Nはその局在がDsRed単体と同様であるため、PA産生非依存的な非特異的細胞内局在を示さず、これは優れた脂質センサーとして必須の条件である。そして、この結果はα-Syn-NがPI（4,5）P2（図10）に加えて、in vitroで有意な結合を示さなかった（図７から図１２）主要なリン脂質であるPC, PS, PEを細胞内で標識しない事を示している。さらに、α-Syn-Nは活性PA産生酵素であるDGKβ, Myr-DGKζ, PMA刺激DGKγ,PLD2が局在する生体膜（細胞膜、ゴルジ体）へと移行した一方で、それらの阻害剤存在下および不活性型変異体とは共局在しなかった（図１３から図２２）。これらの結果はα-Syn-Nはそこで生成されたPAを標識できた事を表している。さらに、α-Syn-Nは24時間以上のPA産生酵素による定常状態のPA産生に加え、PMAにより移行したDGKγが短時間（30分）に生成したPAも標識できた事が示された（図１９、２０）。
(7) Discussion Currently, there is no highly reliable and versatile PA sensor. Although Spo20p-PABD and PDE4A1-PABD are often used as intracellular PA sensors, they are independent of cellular stimulation-induced PA production, such as the plasma membrane (Spo20p-PABD) or Golgi apparatus (PDE4A1-PABD). show their own localization. The inventors have demonstrated that α-Syn-N is a novel reliable and widely applicable PA sensor. In particular, DsRed-α-Syn-N has the same localization as DsRed alone, so it does not exhibit nonspecific intracellular localization independent of PA production, which is an essential condition for an excellent lipid sensor. . And this result shows that α-Syn-N is a major phospholipid that did not show significant binding in vitro (Figures 7 to 12), in addition to PI(4,5)P 2 (Figure 10). This shows that PC, PS, and PE are not labeled within the cells. Furthermore, α-Syn-N was transferred to biological membranes (cell membranes, Golgi apparatus) where active PA-producing enzymes DGKβ, Myr-DGKζ, PMA-stimulated DGKγ, and PLD2 are localized, whereas in the presence of their inhibitors, and did not colocalize with the inactive mutant (Figures 13 to 22). These results indicate that α-Syn-N was able to label the PA generated there. Furthermore, it was shown that α-Syn-N was able to label PA produced in a short period of time (30 minutes) by DGKγ transferred by PMA, in addition to steady-state PA production by the PA-producing enzyme over 24 hours ( Figures 19, 20).
Spo20p-PABDとPDE4A1-PABDはそれぞれ細胞膜および核、ゴルジ体に局在する事が報告されており、本研究ではこれを再確認した（図２９から図３４）。α-Syn-NのPAに対する解離定数6.6μMはSpo20p-PABD（2.2μM）やPDE4A1-PABD（6.8μM）と同等であり、α-Syn-NはPAに対しSpo20p-PABDやPDE4A1-PABDと同等に高感度であった。しかし、細胞内における非特異的（PA産生酵素非依存的）局在の観点から、α-Syn-NはSpo20p-PABDとPDE4A1-PABDよりも優れている可能性がある。例えば、Spo20p-PABDはDGKβ非存在下においても細胞膜に強く局在するため、DGKβが細胞膜で産生するPAを標識できなかった。（図２９）。さらに、PDE4A1-PABDはMyr-DGKzζ非存在下においてもゴルジ体に強く局在するため、Myr-DGKζがゴルジ体にて産生するPAを標識できなかった（図２９から図３１）。一方、α-Syn-Nは細胞膜やゴルジ体への局在能を示さない（図１３から図１６）ため様々な生体膜におけるPA産生を容易に標識する事ができ、これは大きな利点であると言える。 It has been reported that Spo20p-PABD and PDE4A1-PABD are localized in the cell membrane, nucleus, and Golgi apparatus, respectively, and this study reconfirmed this (Figures 29 to 34). The dissociation constant of α-Syn-N to PA, 6.6 μM, is equivalent to that of Spo20p-PABD (2.2 μM) and PDE4A1-PABD (6.8 μM), and It was equally sensitive. However, α-Syn-N may be superior to Spo20p-PABD and PDE4A1-PABD in terms of nonspecific (PA-producing enzyme-independent) localization within cells. For example, Spo20p-PABD was strongly localized to the cell membrane even in the absence of DGKβ, and therefore could not label PA produced by DGKβ at the cell membrane. (Figure 29). Furthermore, since PDE4A1-PABD is strongly localized in the Golgi apparatus even in the absence of Myr-DGKzζ, it was not possible to label PA produced by Myr-DGKzζ in the Golgi apparatus (Figures 29 to 31). On the other hand, α-Syn-N does not show the ability to localize to the cell membrane or Golgi apparatus (Figures 13 to 16), so it can easily label PA production in various biological membranes, which is a major advantage. I can say that.
領域欠損変異体を用いた実験は、N末端のみがPAに強く結合し、NACやC末端は結合しない事を示した（図１、２）。全長α-Synを用いた以前の報告と同様に、α-Syn-Nは18:1/18:1-PAや16:0/18:1-PA, 16:0/16:0-PAを含む幅広いPA分子種に対し比較的広い選択性を示した（図３、４）。最近、我々はグルコース刺激条件下C2C12細胞にて、DGKδが14:0/16:0-, 14:0/16:1-, 16:0/16:0-, 16:0/16:1-, 16:0/18:0-, 16:0/18:1-PAを産生する事を明らかとした。また、16:0/16:0-と16:0/18:0-PAの産生がDGKα選択的阻害剤によって減弱されることを示した。さらに、DGKζは神経芽細胞腫分化の初期段階において16:0/16:0-PAを選択的に産生し、PLDはPCを加水分解し多量の16:0/18:1-, 18:1/18:1-PAを産生する。また、LPAATは主要リン脂質のde novo合成に関与しており幅広いPA分子種を生成する。したがって、PA分子種に対する比較的広い選択性により、α-Syn-Nは多くの細胞種における細胞刺激に応答するPAセンサーとして用いる事ができる。 Experiments using region-deleted mutants showed that only the N-terminus strongly binds to PA, while NAC and the C-terminus do not bind (Figures 1 and 2). Similar to previous reports using full-length α-Syn, α-Syn-N can be used to synthesize 18:1/18:1-PA, 16:0/18:1-PA, and 16:0/16:0-PA. It showed relatively wide selectivity for a wide range of PA molecular species, including (Figures 3 and 4). Recently, we showed that DGKδ is 14:0/16:0-, 14:0/16:1-, 16:0/16:0-, 16:0/16:1- in C2C12 cells under glucose-stimulated conditions. , 16:0/18:0-, 16:0/18:1-PA. We also showed that the production of 16:0/16:0- and 16:0/18:0-PA was attenuated by a DGKα selective inhibitor. Furthermore, DGKζ selectively produces 16:0/16:0-PA during the early stages of neuroblastoma differentiation, and PLD hydrolyzes PC to produce large amounts of 16:0/18:1-, 18:1. Produces /18:1-PA. Additionally, LPAAT is involved in the de novo synthesis of major phospholipids and produces a wide range of PA molecular species. Therefore, due to its relatively broad selectivity towards PA molecular species, α-Syn-N can be used as a PA sensor in response to cellular stimulation in many cell types.
一般的に、細胞内における酵素を標的とした化合物ライブラリーの阻害剤または活性化剤のスクリーニングは、in vitroよりも困難である。いくつかのDGKアイソザイムはガン、てんかん、強迫性障害、双極性障害、自己免疫、ガン免疫、心肥大、高血圧、および2型糖尿病の薬物標的となりうると考えられている。また、PLDはガンやパーキンソン病、アルツハイマー病などの神経変性疾患の薬物標的であり、LPAATも婦人性およびリンパ性悪性腫瘍における治療標的であると報告されている。PAセンサーとしてα-Syn-Nは、PA産生酵素の阻害剤および活性化剤のスクリーニングによる上記の疾患に対する薬物開発のための有用なツールとなりうると言える。実際、我々はCOS-7細胞において、DGKβ阻害剤である化合物AとPLD阻害剤であるFIPIの効果を検出することに成功している（図１３から図１６、図２１、２２）。 In general, screening compound libraries targeting enzymes in cells for inhibitors or activators is more difficult than in vitro. Several DGK isozymes are thought to be potential drug targets for cancer, epilepsy, obsessive-compulsive disorder, bipolar disorder, autoimmunity, cancer immunity, cardiac hypertrophy, hypertension, and type 2 diabetes. Additionally, PLD is a drug target for cancer and neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, and LPAAT is also reported to be a therapeutic target for gynecological and lymphoid malignancies. As a PA sensor, α-Syn-N can be a useful tool for drug development for the above-mentioned diseases by screening for inhibitors and activators of PA-producing enzymes. In fact, we succeeded in detecting the effects of Compound A, a DGKβ inhibitor, and FIPI, a PLD inhibitor, in COS-7 cells (Figures 13 to 16, Figures 21 and 22).
まとめると、発明者はα-Syn-Nが多種多様な生理学的現象に適用可能な信頼性ある有望な細胞内PAセンサーである事を明らかとした。PAセンサーの1種としてα-Syn-Nが追加されたが、PAに複数のセンサーを利用することは、PA標識の適用範囲と信頼性が向上するといえる。さらにα-Syn-Nは、多種多様な疾患の有望な薬物標的であるDGK, PLD,そしてLPAATなどのPA酸性酵素の阻害剤および活性化剤のスクリーニングツールとして有用である。 In summary, the inventors have demonstrated that α-Syn-N is a reliable and promising intracellular PA sensor that can be applied to a wide variety of physiological phenomena. Although α-Syn-N has been added as one type of PA sensor, the use of multiple sensors for PA can improve the coverage and reliability of PA labeling. Furthermore, α-Syn-N is useful as a screening tool for inhibitors and activators of PA acid enzymes such as DGK, PLD, and LPAAT, which are promising drug targets for a wide variety of diseases.
（８）図の説明
図１は、α-Synとその領域欠損変異体の概略図、図２は、精製したSUMO-α-Synとその領域欠損変異体（2.5μM）を18:1/18:1-PAまたは18:1/18:1-PSリポソーム（PAまたはPS150μM）をインキュベート後、超遠心により分離、SDS-PAGE（15%）によって分離したタンパク質をクマシーブリリアントブルーにより染色したものを示す図である。α-Synとその領域欠損変異体の位置を矢印で表した。上清（S）および沈殿（P）画分のタンパク質量をImageJソフトウェアでデンシトメトリーにより定量し、結合活性は全バンド強度に対する沈殿画分のバンド強度の百分率として表した。値は4回の独立した実験の平均値±標準偏差, 18:1/18:1-PSリポソームに対し**P < 0.01。
(8) Explanation of figures Figure 1 is a schematic diagram of α-Syn and its region-deficient mutant, and Figure 2 shows purified SUMO-α-Syn and its region-deficient mutant (2.5 μM) at 18:1/18 :1-PA or 18:1/18:1-PS liposomes (PA or PS 150 μM) were incubated, separated by ultracentrifugation, and proteins separated by SDS-PAGE (15%) stained with Coomassie brilliant blue are shown. It is a diagram. The positions of α-Syn and its region deletion mutants are indicated by arrows. The protein content of the supernatant (S) and precipitate (P) fractions was quantified by densitometry with ImageJ software, and the binding activity was expressed as the percentage of the band intensity of the precipitate fraction relative to the total band intensity. Values are means ± standard deviations of four independent experiments, ** P < 0.01 versus 18:1/18:1-PS liposomes.
図３は、精製したSUMO-α-Syn-N（2.5μM）とコントロール（PCとcholのみ）, 16:0/16:0-PA, 16:0/18:1-PA, 18:1/18:1-PA, 18:0/18:0-PAリポソーム（PA150μM）をインキュベート後、超遠心により分離、SDS-PAGE（15%）によって分離したタンパク質をクマシーブリリアントブルーにより染色したものを示す図である。α-Syn-Nの位置を矢印で表した。 Figure 3 shows purified SUMO-α-Syn-N (2.5 μM) and control (PC and chol only), 16:0/16:0-PA, 16:0/18:1-PA, 18:1/ 18:1-PA, 18:0/18:0-PA liposomes (PA 150 μM) were incubated, separated by ultracentrifugation, and proteins separated by SDS-PAGE (15%) were stained with Coomassie brilliant blue. It is. The position of α-Syn-N is indicated by an arrow.
図４は、上清（S）および沈殿（P）画分のタンパク質量をImageJソフトウェアでデンシトメトリーにより定量し、結合活性は全バンド強度に対する沈殿画分のバンド強度の百分率として表した図である。値は3回の独立した実験の平均値±標準偏差, 18:1/18:1-PAリポソームに対し、#P < 0.05, ##P < 0.01, ###P < 0.005, コントロールリポソームに対し*P < 0.05, ***P < 0.005。 Figure 4 shows the amount of protein in the supernatant (S) and precipitate (P) fractions determined by densitometry using ImageJ software, and the binding activity expressed as a percentage of the band intensity in the precipitate fraction relative to the total band intensity. be. Values are mean ± standard deviation of three independent experiments, # P < 0.05, ## P < 0.01, ### P < 0.005, vs. control liposomes for 18:1/18:1-PA liposomes. * P < 0.05, *** P < 0.005.
図５は、精製したSUMO-α-Syn-N（0.1μM）と18:1/18:1-PAリポソーム（0-20μM）をインキュベート後、超遠心により分離、SDS-PAGE（15%）によって分離したタンパク質を銀染色により染色したものを示す図である。α-Syn-Nの位置を矢印で表した。 Figure 5 shows the results of incubation of purified SUMO-α-Syn-N (0.1 μM) and 18:1/18:1-PA liposomes (0-20 μM), followed by separation by ultracentrifugation and SDS-PAGE (15%). FIG. 2 is a diagram showing separated proteins stained with silver staining. The position of α-Syn-N is indicated by an arrow.
図６は、沈殿画分のタンパク質量をImageJソフトウェアでデンシトメトリーにより定量し、結合活性は全バンド強度（インプット）に対する沈殿画分のバンド強度の百分率として表した図である。値は4回の独立した実験の平均値±標準偏差, KdはGraphPad Prism 8（Dissociation-One phase exponential decay）により求めた。 FIG. 6 is a diagram in which the amount of protein in the precipitate fraction was quantified by densitometry using ImageJ software, and the binding activity was expressed as a percentage of the band intensity in the precipitate fraction relative to the total band intensity (input). Values are the average ± standard deviation of four independent experiments, and K d was determined using GraphPad Prism 8 (Dissociation-One phase exponential decay).
図７は、精製したSUMO-α-Syn-N（0.1μM）と18:1/18:1-PA, 18:1/18:1-PS, 18:1/18:1-PI（4,5）P2, 18:1/18:1-PE, d18:1/18:1-C1Pリポソーム（20μM）をインキュベート後、超遠心により分離、SDS-PAGE（15%）によって分離したタンパク質を銀染色により染色したものを示す図である。α-Syn-Nの位置を矢印で表した。 Figure 7 shows purified SUMO-α-Syn-N (0.1 μM) and 18:1/18:1-PA, 18:1/18:1-PS, 18:1/18:1-PI (4, 5) After incubating P2, 18:1/18:1-PE, d18:1/18:1-C1P liposomes (20 μM), separate by ultracentrifugation, and silver stain the separated proteins by SDS-PAGE (15%). FIG. The position of α-Syn-N is indicated by an arrow.
図８は、沈殿画分のタンパク質量をImageJソフトウェアでデンシトメトリーにより定量し、α-Syn-NのPAに対する結合活性を100%とした図である。値は3回の独立した実験の平均値±標準偏差, 18:1/18:1-PAリポソームに対し***P < 0.005。 FIG. 8 is a diagram in which the amount of protein in the precipitate fraction was quantified by densitometry using ImageJ software, and the binding activity of α-Syn-N to PA was set as 100%. Values are mean ± standard deviation of three independent experiments, *** P < 0.005 for 18:1/18:1-PA liposomes.
図９は、等モル（100pmol）の様々な脂質をニトロセルロース膜上にスポットしたものを示す図である。グリセロ脂質のアシル鎖は16:0である（Echelon Biosciences）。 FIG. 9 shows equimolar (100 pmol) of various lipids spotted on a nitrocellulose membrane. The acyl chain of glycerolipids is 16:0 (Echelon Biosciences).
図１１は、等モル（300pmol）の18:1/18:1-PA, 18:1-LPA, d18:1/18:1-C1Pをニトロセルロースメンブレン上にスポットしたものを示す図である。メンブレンを精製したGST-α-Syn-N（20μM）とインキュベート後、脂質結合タンパク質を抗GST抗体にて検出した。 FIG. 11 is a diagram showing equimolar (300 pmol) of 18:1/18:1-PA, 18:1-LPA, and d18:1/18:1-C1P spotted on a nitrocellulose membrane. After incubating the membrane with purified GST-α-Syn-N (20 μM), lipid-bound proteins were detected using an anti-GST antibody.
図１０、１２は、ブロットをImageJソフトウェアで定量し、α-Syn-NのPAに対する結合活性（スポット強度）を100%とした図である。値は3回の独立した実験の平均値±標準偏差, PAに対し***P < 0.005。 Figures 10 and 12 are diagrams in which the blots were quantified using ImageJ software, and the binding activity (spot intensity) of α-Syn-N to PA was set as 100%. Values are means ± standard deviation of three independent experiments, *** P < 0.005 versus PA.
図１３から図１６の画像は、EGFP, EGFP-DGKβ-WTまたはEGFP-DGKβ-KDをDsRed, DsRed-α-Syn-NとCOS-7細胞にて共発現させ、トランスフェクションの24時間後に細胞を固定し、画像化したものである。3回の独立した実験の代表的なデータを示し、スケールバーは20μmである。 The images in Figures 13 to 16 were obtained by coexpressing EGFP, EGFP-DGKβ-WT, or EGFP-DGKβ-KD with DsRed, DsRed-α-Syn-N in COS-7 cells, and 24 hours after transfection. was fixed and imaged. Representative data of three independent experiments are shown, scale bar is 20 μm.
図１４は、EGFP-DGKβ-WTまたはEGFP-DGKβ-KD, とDsRed-α-Syn-Nの局在をImageJソフトウェアで定量したものである。図１５は、EGFP-DGKβ-WTをDsRed-α-Syn-NとCOS-7細胞にて共発現させ、トランスフェクションの24時間後に1μMの化合物Aと成長培地中で1時間インキュベートし、その後細胞を固定し、画像化したものである。3回の独立した実験の代表的なデータを示し、スケールバーは20μmである。 Figure 14 shows the localization of EGFP-DGKβ-WT or EGFP-DGKβ-KD, and DsRed-α-Syn-N quantified using ImageJ software. Figure 15 shows that EGFP-DGKβ-WT was coexpressed with DsRed-α-Syn-N in COS-7 cells, and 24 hours after transfection, the cells were incubated with 1 μM compound A for 1 hour in growth medium, and then the cells was fixed and imaged. Representative data of three independent experiments are shown, scale bar is 20 μm.
図１６の下のグラフは、EGFP-DGKβ-WTとDsRed-α-Syn-Nの局在をImageJソフトウェアで定量したものである。 The lower graph in FIG. 16 shows the localization of EGFP-DGKβ-WT and DsRed-α-Syn-N quantified using ImageJ software.
図１７は、Myr-AcGFP-DGKζ-WTまたはMyr-AcGFP-DGKζ-KDをDsRed, DsRed-α-Syn-NとCOS-7細胞にて共発現させ、トランスフェクションの24時間後に細胞を固定し、画像化したものである。3回の独立した実験の代表的なデータを示し、スケールバーは20μmである。 Figure 17 shows that Myr-AcGFP-DGKζ-WT or Myr-AcGFP-DGKζ-KD was coexpressed with DsRed, DsRed-α-Syn-N in COS-7 cells, and the cells were fixed 24 hours after transfection. , is an image. Representative data of three independent experiments are shown, scale bar is 20 μm.
図１８は、Myr-AcGFP-DGKζ-WTまたはMyr-AcGFP-DGKζ-KD,とDsRed-α-Syn-Nの局在をImageJソフトウェアで定量したものである。 Figure 18 shows the localization of Myr-AcGFP-DGKζ-WT or Myr-AcGFP-DGKζ-KD and DsRed-α-Syn-N quantified using ImageJ software.
図１９、図２０は、EGFP, EGFP-DGKγ-WTまたはEGFP-DGKγ-KDをDsRedまたはDsRed-α-Syn-NとCOS-7細胞にて共発現させ、トランスフェクションの24時間後に1μMのPMAまたはDMSOと成長培地中で30分間インキュベートし、その後細胞を固定し、画像化したものである。3回の独立した実験の代表的なデータを示し、スケールバーは20μmである。図２０は、EGFP-DGKγ-WTまたはEGFP-DGKγ-KDとDsRed-α-Syn-Nの局在をImageJソフトウェアで定量したものである。 Figures 19 and 20 show that EGFP, EGFP-DGKγ-WT, or EGFP-DGKγ-KD was coexpressed with DsRed or DsRed-α-Syn-N in COS-7 cells, and 1 μM PMA was added 24 hours after transfection. or incubated for 30 min in growth medium with DMSO, after which cells were fixed and imaged. Representative data of three independent experiments are shown, scale bar is 20 μm. FIG. 20 shows the localization of EGFP-DGKγ-WT or EGFP-DGKγ-KD and DsRed-α-Syn-N quantified using ImageJ software.
図２１、２２は、EGFP-PLDをDsRedまたはDsRed-α-Syn-NとCOS-7細胞にて共発現させ、トランスフェクションの24時間後に750nMのFIPIと成長培地中で4時間インキュベートし、その後細胞を固定し、画像化したものである。3回の独立した実験の代表的なデータを示し、スケールバーは20μmである。 Figures 21 and 22 show that EGFP-PLD was coexpressed with DsRed or DsRed-α-Syn-N in COS-7 cells, incubated with 750 nM FIPI for 4 hours in growth medium 24 hours after transfection, and then Cells were fixed and imaged. Representative data of three independent experiments are shown, scale bar is 20 μm.
図２２は、EGFP-PLD2とDsRed-α-Syn-Nの局在をImageJソフトウェアで定量したものである。 Figure 22 shows the localization of EGFP-PLD2 and DsRed-α-Syn-N quantified using ImageJ software.
図２３は、EGFPまたはEGFP-PIP5K1AをDsRed, DsRed-α-Syn-NまたはDsRed-PLCTM1-PHDとCOS-7細胞にて共発現させ、トランスフェクションの24時間後に細胞を固定し、画像化したものである。3回の独立した実験の代表的なデータを示し、スケールバーは20μmである。 Figure 23 shows that EGFP or EGFP-PIP5K1A was coexpressed with DsRed, DsRed-α-Syn-N, or DsRed-PLC TM 1-PHD in COS-7 cells, and the cells were fixed 24 hours after transfection. It has become. Representative data of three independent experiments are shown, scale bar is 20 μm.
図２４は、EGFP-PIP5K1AとDsRed-α-Syn-NまたはDsRed-PLCδ1-PHDの局在をImageJソフトウェアで定量したものである。 Figure 24 shows the localization of EGFP-PIP5K1A and DsRed-α-Syn-N or DsRed-PLCδ1-PHD quantified using ImageJ software.
図２５、２６は、大腸菌にて発現させ、アフィニティクロマトグラフィーにより精製したGSTまたはSUMO融合α-Synとその領域欠損変異体をSDS-PAGE（15%）で分離したものである。分離したタンパク質をクマシーブリリアントブルーにより染色し、イムノブロットにより決定したタンパク質の位置を矢印で表記した（図２６）。予想分子量: GST alone, 28.4kDa; GST-α-Syn-N, 31.7kDa; SUMO alone, 12.2kDa, SUMO-α-Syn-N, 18.4 kDa; SUMO-α-Syn-NAC, 15.5kDa; SUMO-α-Syn-C, 17.3kDa; SUMO-α-Syn-（NAC-C）, 20.5kDa; SUMO-α-Syn, 26.7kDa。 Figures 25 and 26 show GST- or SUMO-fused α-Syn expressed in Escherichia coli and purified by affinity chromatography, and its region deletion mutant, separated by SDS-PAGE (15%). The separated proteins were stained with Coomassie brilliant blue, and the positions of the proteins determined by immunoblotting were indicated with arrows (FIG. 26). Expected molecular weight: GST alone, 28.4kDa; GST-α-Syn-N, 31.7kDa; SUMO alone, 12.2kDa, SUMO-α-Syn-N, 18.4 kDa; SUMO-α-Syn-NAC, 15.5kDa; SUMO- α-Syn-C, 17.3kDa; SUMO-α-Syn-(NAC-C), 20.5kDa; SUMO-α-Syn, 26.7kDa.
図２７は、等モル（500pmol）の18:1/18:1-PA, 18:1/18:1-PC, 18:1/18:1-PE, 18:1/18:1-PG, 18:1/18:1-PS, 18:1/18:1-PI, 18:1/18:1-PI（4,5）P2をニトロセルロースメンブレン上にスポットし、精製したGST-α-Syn-N （20μM）とインキュベート後、脂質結合タンパク質を抗GST抗体にて検出したものである。 Figure 27 shows equimolar (500 pmol) of 18:1/18:1-PA, 18:1/18:1-PC, 18:1/18:1-PE, 18:1/18:1-PG, 18:1/18:1-PS, 18:1/18:1-PI, 18:1/18:1-PI(4,5) P2 were spotted on nitrocellulose membrane and purified GST-α After incubation with -Syn-N (20 μM), lipid-bound proteins were detected using an anti-GST antibody.
図２８は、ブロットをImageJソフトウェアで定量し、α-Syn-NのPAに対する結合活性（スポット強度）を100%としたものである。値は3回の独立した実験の平均値±標準偏差, 18:1/18:1-PAに対し***P < 0.005。 In FIG. 28, the blot was quantified using ImageJ software, and the binding activity (spot intensity) of α-Syn-N to PA was set as 100%. Values are means ± standard deviation of three independent experiments, *** P < 0.005 for 18:1/18:1-PA.
図２９は、EGFPまたはEGFP-DGKβをDsRed, DsRed-Spo20p-PABDまたはDsRed-PDE4A1-PABDとCOS-7細胞にて共発現させ、トランスフェクションの24時間後に細胞を固定し、画像化したものである。3回の独立した実験の代表的なデータを示し、スケールバーは20μmである。 Figure 29 shows that EGFP or EGFP-DGKβ was coexpressed with DsRed, DsRed-Spo20p-PABD, or DsRed-PDE4A1-PABD in COS-7 cells, and the cells were fixed and imaged 24 hours after transfection. be. Representative data of three independent experiments are shown, scale bar is 20 μm.
図３０、３１は、Myr-AcGFP-DGKζをDsRed-PDE4A1-PABDとCOS-7細胞にて共発現させ、トランスフェクションの24時間後、Myr-AcGFP-DGKζのみを発現させた細胞は抗TGN46抗体とAlexa Fluor 594-conjugated二次抗体を用いて免疫染色し、画像化したものである。3回の独立した実験の代表的なデータを示し、スケールバーは20μmである。 Figures 30 and 31 show that Myr-AcGFP-DGKζ was coexpressed with DsRed-PDE4A1-PABD in COS-7 cells, and 24 hours after transfection, cells expressing only Myr-AcGFP-DGKζ were treated with anti-TGN46 antibody. and Alexa Fluor 594-conjugated secondary antibody. Representative data of three independent experiments are shown, scale bar is 20 μm.
図３２、３３は、Myr-AcGFP-DGKζ-WTまたはMyr-AcGFP-DGKζ-KDをDsRed-Spo20pとCOS-7細胞にて共発現させ、トランスフェクションの24時間後に細胞を固定し、画像化したものである。3回の独立した実験の代表的なデータを示し、スケールバーは20μmである。 Figures 32 and 33 show that Myr-AcGFP-DGKζ-WT or Myr-AcGFP-DGKζ-KD was coexpressed with DsRed-Spo20p in COS-7 cells, and the cells were fixed and imaged 24 hours after transfection. It is something. Representative data of three independent experiments are shown, scale bar is 20 μm.
図３４、３５は、EGFP-PLD2をDsRed-PDE4A1-PABDとCOS-7細胞にて共発現させ、トランスフェクションの24時間後、EGFP-PLD2のみを発現させた細胞は抗TGN46抗体とAlexa Fluor 594-conjugated二次抗体を用いて免疫染色し、画像化した。3回の独立した実験の代表的なデータを示し、スケールバーは20μmである。 Figures 34 and 35 show that EGFP-PLD2 was coexpressed with DsRed-PDE4A1-PABD in COS-7 cells, and 24 hours after transfection, cells expressing only EGFP-PLD2 were treated with anti-TGN46 antibody and Alexa Fluor 594. -conjugated secondary antibody was used for immunostaining and imaging. Representative data of three independent experiments are shown, scale bar is 20 μm.
（９）実験手順
（a）SUMOとGSTタグ融合α-Synとその領域欠損変異体の発現と精製
His×6-SUMO融合コンストラクト作成のため、Nde1とXho1制限酵素サイトを融合したα-Syn（Met1‐Ala140）, α-Syn-N（Met1‐Lys60）, α-Syn-NAC（Glu61‐Val95）, α-Syn-C（Lys96‐Ala140）, and α-Syn-（NAC-C） （Glu61‐Ala140） cDNAをpET-14b-マウスα-SynベクターからPCRにより増幅し、pSUMOベクターに挿入した。このプラスミドで大腸菌 Rosetta-gami 2 （DE3）を形質転換した。菌体をLB培地にて OD600=0.6~0.8まで37℃で培養し、0.1 mM IPTGによりタンパク質発現を誘導し37℃で3時間培養した。遠心により回収した菌体を溶解バッファー （50 mM sodium phosphate, pH 8.0, containing 300 mM NaCl, 10 mM imidazole, 20 μg/mL aprotinin, 20 μg/mL leupeptin, and 20 μg/mL pepstatin）で溶解し、氷上で超音波破砕を行った。遠心後、His×6-SUMO融合タンパク質を精製するため、Ni-NTAアガロースを用いてNi-アフィニティクロマトグラフィーを行なった。カラムは洗浄バッファー（50 mM sodium phosphate, pH 8.0, 300 mM NaCl, and 10 and 50 mM imidazole）で洗浄し、300mM imidazole含有バッファーにて溶出させた。精製タンパク質はHEPESバッファー（25 mM HEPES, pH 7.4, 100 mM NaCl）に透析し、タンパク質濃度はbicinchoninic acid protein assay kit （Thermo Fisher Scientific）により決定した。
(9) Experimental procedure (a) Expression and purification of SUMO and GST tag-fused α-Syn and its region deletion mutant
α-Syn (Met1-Ala140), α-Syn-N (Met1-Lys60), α-Syn-NAC (Glu61-Val95) fused with Nde1 and Xho1 restriction enzyme sites to create a His × 6-SUMO fusion construct. , α-Syn-C (Lys96-Ala140), and α-Syn-(NAC-C) (Glu61-Ala140) cDNA was amplified by PCR from pET-14b-mouse α-Syn vector and inserted into pSUMO vector. E. coli Rosetta-gami 2 (DE3) was transformed with this plasmid. The bacterial cells were cultured in LB medium at 37°C until OD 600 =0.6 to 0.8, protein expression was induced with 0.1 mM IPTG, and cultured at 37°C for 3 hours. Cells collected by centrifugation were lysed with lysis buffer (50 mM sodium phosphate, pH 8.0, containing 300 mM NaCl, 10 mM imidazole, 20 μg/mL aprotinin, 20 μg/mL leupeptin, and 20 μg/mL pepstatin). Ultrasonic disruption was performed on ice. After centrifugation, Ni-affinity chromatography was performed using Ni-NTA agarose to purify the His×6-SUMO fusion protein. The column was washed with a washing buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, and 10 and 50 mM imidazole) and eluted with a buffer containing 300 mM imidazole. The purified protein was dialyzed into HEPES buffer (25 mM HEPES, pH 7.4, 100 mM NaCl), and the protein concentration was determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific).
GST融合α-Syn-N作成のため、α-Syn-N（Met1‐Lys60）cDNAをpET-14b-マウスα-SynベクターからPCRにより増幅し、pGEX6P-1ベクターのBamH1とSal1制限酵素サイトに挿入し、このプラスミドで大腸菌 Rosetta 2 （DE3）を形質転換した。菌体をLB培地にてOD600=0.6~0.8まで37℃で培養し、0.1mM IPTGによりタンパク質発現を誘導し37℃で3時間培養し、遠心により回収した菌体を溶解バッファー（1.47mM KH2PO4, 8.09mM Na2HPO4, pH7.2, 2.67mM KCl, 137mM NaCl, 20μg/mL aprotinin, 20μg/mL leupeptin, and 20μg/mL pepstatin） で溶解し、氷上で超音波破砕を行なった。遠心後、GST融合タンパク質を精製するため、glutathione-Sepharose 4Bを用いてアフィニティクロマトグラフィーを行なった。カラムは溶解バッファーで洗浄し、溶出バッファー（50mM Tris-HCl, 150mM NaCl, and 10mM reduced glutathione）でタンパク質を溶出させた。脂質オーバーレイアッセイのため、精製タンパク質をHEPESバッファーに透析し、タンパク質濃度はbicinchoninic acid protein assay kit （Thermo Fisher Scientific） により決定した。 To create GST-fused α-Syn-N, α-Syn-N (Met1-Lys60) cDNA was amplified by PCR from pET-14b-mouse α-Syn vector and inserted into the BamH1 and Sal1 restriction enzyme sites of pGEX6P-1 vector. E. coli Rosetta 2 (DE3) was transformed with this plasmid. The cells were cultured in LB medium at 37℃ until OD 600 =0.6~0.8, protein expression was induced with 0.1mM IPTG, cultured at 37℃ for 3 hours, and the cells recovered by centrifugation were added to lysis buffer (1.47mM KH 2 PO 4 , 8.09mM Na 2 HPO 4 , pH7.2, 2.67mM KCl, 137mM NaCl, 20μg/mL aprotinin, 20μg/mL leupeptin, and 20μg/mL pepstatin) and sonicated on ice. . After centrifugation, affinity chromatography was performed using glutathione-Sepharose 4B to purify the GST fusion protein. The column was washed with lysis buffer, and the protein was eluted with elution buffer (50mM Tris-HCl, 150mM NaCl, and 10mM reduced glutathione). For lipid overlay assays, purified protein was dialyzed into HEPES buffer, and protein concentration was determined by a bicinchoninic acid protein assay kit (Thermo Fisher Scientific).
（b）リポソーム沈降法
コントロールリポソーム（Chol （50mol%） and 16:0/18:1-PC （50mol%））、PAリポソーム Chol （50 mol%）, 16:0/18:1-PC （35 mol%） and each PA species （15 mol%）の組成でリポソームを作成した。乾燥脂質混合物をHEPESバッファーに懸濁し、1分間ボルテックス後に75℃で超音波破砕し、リポソーム形成を誘導した。タンパク質はリポソームと1時間インキュベートし、200.000g, 4℃で1時間遠心した。ペレットをHEPESバッファーに溶解後、SDS-PAGEにより分離しクマシーブリリアントブルーまたは銀染色にて検出した。定量はImageJソフトウェアを用いて行った。脂質は2重層を形成するため、実際の半分の濃度を考慮した。
(b) Liposome sedimentation method Control liposomes (Chol (50 mol%) and 16:0/18:1-PC (50 mol%)), PA liposomes Chol (50 mol%), 16:0/18:1-PC (35 Liposomes were prepared with a composition of (15 mol%) and each PA species (15 mol%). The dry lipid mixture was suspended in HEPES buffer, vortexed for 1 minute, and then sonicated at 75°C to induce liposome formation. Proteins were incubated with liposomes for 1 hour and centrifuged at 200.000g, 4°C for 1 hour. After dissolving the pellet in HEPES buffer, it was separated by SDS-PAGE and detected by Coomassie brilliant blue or silver staining. Quantification was performed using ImageJ software. Since lipids form a double layer, half the actual concentration was considered.
（c）脂質オーバーレイアッセイ
脂質をニトロセルロースメンブレンにスポットし作成し、またMembrane Lipid Stripを用いた。メンブレンは1% skim milk in Tris-buffered saline, pH 7.4,で1時間、室温でブロッキングし、GSTタグ融合タンパク質（20 nM）を溶解した10 ml of 3% bovine serum albumin in Tris-buffered saline, pH 7.4で1時間、室温でインキュベート後、抗GST抗体peroxidase-conjugated goat anti-rabbit IgG antibodyによりタンパク質を検出した。定量はImageJソフトウェアを用いて行った。
(c) Lipid overlay assay Lipids were spotted onto a nitrocellulose membrane and a Membrane Lipid Strip was used. The membrane was blocked with 1% skim milk in Tris-buffered saline, pH 7.4, for 1 hour at room temperature, and then treated with 10 ml of 3% bovine serum albumin in Tris-buffered saline, pH 7.4, containing dissolved GST-tagged protein (20 nM). After incubation with 7.4 for 1 hour at room temperature, proteins were detected with anti-GST peroxidase-conjugated goat anti-rabbit IgG antibody. Quantification was performed using ImageJ software.
（d）細胞培養
COS-7細胞は10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin含有Dulbecco’s modified Eagle’s mediumにて37℃, 5% CO2中で培養した。細胞は製造者のプロトコル通りにポリフェクトを用いて行なった。
(d) Cell culture
COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37° C. in 5% CO 2 . Cell culture was performed using Polyfect according to the manufacturer's protocol.
（e）共焦点レーザー顕微鏡
COS-7細胞は以上の手順でトランスフェクションした。pEGFP-DGKβをトランスフェクションした細胞は、24時間後に1 μM化合物A / DMEMにて1時間インキュベートし、DGKβ活性を阻害した。pEGFP-PLD2をトランスフェクションした細胞は、750 nM FIPI / growth medium にて4時間インキュベートし、PLD2活性を阻害した。細胞は4%パラホルムアルデヒドで固定し、Vectashieldにてスライドガラスにマウントした。蛍光画像はOlympus FV1000-D （IX81） confocal laser scanning microscopeを用いて観察し、GFPは488 nm、DsRedは543 nmにて励起させた。画像取得にはFV-10 ASWソフトウェアを用いた。
(e) Confocal laser microscope
COS-7 cells were transfected using the above procedure. After 24 hours, cells transfected with pEGFP-DGKβ were incubated with 1 μM compound A/DMEM for 1 hour to inhibit DGKβ activity. Cells transfected with pEGFP-PLD2 were incubated with 750 nM FIPI/growth medium for 4 hours to inhibit PLD2 activity. Cells were fixed with 4% paraformaldehyde and mounted on glass slides with Vectashield. Fluorescent images were observed using an Olympus FV1000-D (IX81) confocal laser scanning microscope, and GFP was excited at 488 nm and DsRed was excited at 543 nm. FV-10 ASW software was used for image acquisition.
（１０）統計解析
データは平均値±標準偏差で表し、GraphPad Prism 8ソフトウェアにより、Student’s t-testまたはone-way ANOVA followed by Tukey’s post hoc testにより解析した。
(10) Statistical analysis Data were expressed as mean value ± standard deviation and analyzed by Student's t-test or one-way ANOVA followed by Tukey's post hoc test using GraphPad Prism 8 software.
本発明は、細胞内におけるホスファチジン酸センサーとして利用可能である。 The present invention can be used as an intracellular phosphatidic acid sensor.
Claims (3)
（１）配列番号１で表される塩基配列；
（２）配列番号１で表される塩基配列と90%以上の相同性を有する塩基配列；
（３）配列番号１で表される塩基配列の18個以内の塩基が、欠失、置換及び／又は付加された塩基配列。 A phosphatidic acid sensor containing a peptide having an amino acid sequence encoded by any one of the following base sequences (1) to (3):
(1) Base sequence represented by SEQ ID NO: 1;
(2) a nucleotide sequence having 90% or more homology with the nucleotide sequence represented by SEQ ID NO: 1;
(3) A base sequence in which within 18 bases of the base sequence represented by SEQ ID NO: 1 are deleted, substituted, and/or added.
（１）配列番号２で表されるアミノ酸配列；
（２）配列番号２で表されるアミノ酸配列と90%以上の相同性を有するアミノ酸配列；
（３）配列番号２で表されるアミノ酸配列の6個以内のアミノ酸が、欠失、置換及び／又は付加されたアミノ酸配列。 A phosphatidic acid sensor containing a peptide having an amino acid sequence of any one of the following (1) to (3):
(1) Amino acid sequence represented by SEQ ID NO: 2;
(2) an amino acid sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 2;
(3) An amino acid sequence in which six or fewer amino acids of the amino acid sequence represented by SEQ ID NO: 2 are deleted, substituted, and/or added.
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| US20100105078A1 (en) | 2008-07-22 | 2010-04-29 | Board Of Trustees Of Michigan State University | Compositions And Methods Of A Phosphatidic Acid Binding Protein |
| US20150024419A1 (en) | 2013-02-26 | 2015-01-22 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Method for the assay of synucleins |
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