ABC蛋白の機能の多様性の分子基盤

• Project/Area Number: 10217205
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Kyoto University
• Principal Investigator: 植田 和光 京都大学, 農学研究科, 助教授 (10151789)
• Co-Investigator(Kenkyū-buntansha): 加藤 博章 京都大学, 薬学研究科, 教授 (90204487) ; 鷹野 誠 京都大学, 医学研究科, 講師 (30236252) ; 天知 輝夫 京都大学, 農学研究科, 教授 (30301245) ; 松尾 道憲 京都大学, 農学研究科, 助手 (00335308) ; 木岡 紀幸 京都大学, 農学研究科, 助手 (90234179)
• Project Period (FY): 1998 – 2002
• Project Status: Completed (Fiscal Year 2002)
• Budget Amount: ¥96,400,000 (Direct Cost: ¥96,400,000); Fiscal Year 2002: ¥28,100,000 (Direct Cost: ¥28,100,000); Fiscal Year 2001: ¥28,200,000 (Direct Cost: ¥28,200,000); Fiscal Year 2000: ¥13,100,000 (Direct Cost: ¥13,100,000); Fiscal Year 1999: ¥13,400,000 (Direct Cost: ¥13,400,000); Fiscal Year 1998: ¥13,600,000 (Direct Cost: ¥13,600,000)
• Keywords: ABCタンパク質 / トランスポーター / チャネル / 動脈硬化 / 膜タンパク質 / コレステロール / 自己免疫疾患 / 生活習慣病 / 糖尿病 / 癌 / 遺伝病 / ATP / レセプター / 残耐射性

Research Abstract

ABCタンパク質は、よく保存されたATP結合領域をもつ膜タンパク質の総称であり、いずれもATPによって駆動あるいは制御されているが、機能はトランスポータ、チャネル、レセプターと多様である。本研究は、ABCタンパク質の機能の多様性の分子基盤を明らかにするために、レセプター型およびトランスポータ型ABCタンパク質のATPとの相互作用の違いを生化学的、分子生物学的、構造生物学的に解明しようとしている。
細胞内および体内のコレステロールなどの脂質のホメオスタシスへのABCA1遺伝子の関与が最近明らかになりつつあるが、その分子メカニズムはいまだ不明である。我々は、血中の高密度リポタンパク質が消失する遺伝病であるタンジール病で見出された変異を導入したABCA1および野生型のGFP融合タンパク質(ABCA1-GFP)を安定的に発現するHEK293細胞株を作製した。蛍光顕微鏡による融合タンパクの蛍光解析とグリコシダーゼによる糖鎖型の解析から、野生型ABCA1とABCA1-W590S変異体は細胞膜に局在し、ABCA1-R587WとABCA1-Q597R変異体は小胞体に残留することが明らかになった。R587WとQ597R2つの変異体は細胞膜に局在できないためタンジール病を引き起こすと考えられる。ATPとの相互作用を検討するため、安定発現株から膜画分を調製し、ATP類似体8-azido-[α-32P]ATPで光親和性標識した。野生型ABCA1とABCA1-W590S変異体はMn存在下8-azido-[α-32P]ATPで標識され、バナジン酸によって標識量が増加した。W590S変異体は野生型と同様にATP結合、加水分解は起こることが明らかになった。

Research Products

• [Publications] Tanaka, A.R.: "Effects of mutations of ABCA1 in the first extracellular domain on subcellular trafficking and ATP binding/hydrolysis"J Biol Chem.. 278. 8815-8819 (2003)
• [Publications] Shitan, N.: "Involvement of CjMDR1, a plant MDR-type ABC protein, in alkaloid transport in Coptis japonica"Proc. Natl. Acad. Sci. USA. 100. 751-756 (2003)
• [Publications] Sugimoto, Y.: "Reversal of breast cancer resistance protein-mediated drug resistence by estrogen antagonists and agonists"Mol. Cancer Ther.. 2. 105-112 (2003)
• [Publications] Tanaka, A.R.: "ATP binding/hydrolysis by and phosphorylation of peroxisomal ABC proteins PMP70 (ABCD3) and ALDP (ABCD1)"J Biol Chem.. 277. 40142-40147 (2002)
• [Publications] Matsuo, M.: "Mutations in the linker domain of NBD2 of SUR inhibit transduction but not nucleotide binding"EMBO J. 21. 4250-4258 (2002)
• [Publications] Hashimoto, K.: "Trafficking-defect and functional-defect by mutations of the ATP-binding domains in multidrug resistance protein 2 (MRP2, ABCC2) in patients with Dubin-Johnson syndrome"Hepatology. 36. 1246-1252 (2002)
• [Publications] 稲垣暢也: "ABCトランスポーター-生体防御のABC-遺伝子から疾患まで"診断と治療社. 82 (2002)
• [Publications] Tanaka, A.R.: "Human ABCA1 Contains a Large Amino-Terminal Extracellular Domain Homologous to an Epitope of Sjogren's Syndrome"Biochem. Biophys. Res. Commun.. 283. 1019-1025 (2001)
• [Publications] Chen, Z.: "Reversal of drug resistance mediated by multidrug resistance protein (MRP) 1 by dual effects of agosterol a on MRP1 function"Int. J. Cancer. 93. 107-113 (2001)
• [Publications] Yazaki, K.: "A novel Coptis japonica multidrug resistant protein preferentially expressed in the alkaloid-accumulating rhizome"J. Exp. Bot.. 357. 877-879 (2001)
• [Publications] 植田和光: "多剤排出トランスポーターMDR1の分子機構?生体防御のABC."蛋白質 核酸 酵素. 46. 588-595 (2001)
• [Publications] 植田和光: "ABCA1蛋白質の構造と機能."The Lipid. 13. 42-48 (2002)
• [Publications] 稲垣暢也: "ABCトランスポーター-生体防御のABC-遺伝子から疾患まで"診断と治療社. 82 (2002)
• [Publications] Matsuo,M.: "Different binding properties and affinities for ATP and ADP among sulfonylurea receptor subtypes. SUR1,SUR2A, and SUR2B."J.Biol.Chem. 275. 28757-28763 (2000)
• [Publications] Matsuo,M.: "Functional analysis of a mutant sulfonylurea receptor, SUR1-R1420C, that is responsible for persistent hyperinsulinemic hypoglycemia of infancy."J.Biol.Chem.. 275. 41184-41191 (2000)
• [Publications] Nagata,K: "Nonequivalent Nucleotide Trapping in the Two Nucleotide Binding Folds of the Human Multidrug Resistance Protein MRP1"J.Biol.Chem.. 275. 17626-17630 (2000)
• [Publications] Tanizawa,Y.: "Genetic analysis of Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy : nucleotide-binding fold-2 mutation impairs cooperative binding of adenine nucleotides to SUR1"Diabetes. 49. 114-20 (2000)
• [Publications] Tanabe,K: "Direct Photoaffinity Labeling of Kir6.2 by [γ-32P]ATP-[γ]4-Azidoanilide."Biochem.Biophys.Res.Commun.. 272. 316-319 (2000)
• [Publications] Zhao,L.-X.,: "Cloning, characterization, and tissue distribution of the ABC transporter ABC2."Biochem,J.,. 350. 865-872 (2000)
• [Publications] Ueda,K.: "How do P-glycoprotein and other ABC proteins recognize their substrates? In The first International Conference on Cotrol and Diseases of Sodium Dependent Transportation Proteins and Ion Channels."Amsterdam : Elsevier. 425-428 (2000)
• [Publications] Ueda, K.: "Cooperative binding of ATP and MgADP in the sulfonylurea receptor is modulated by glibenclamide"Proc. Natl. Acad. Sci. USA. 96. 1268-1272 (1999)
• [Publications] Matsuo, M.: "ATP binding properties of the nucleotide binding folds of SUR1"J. Biol. Chem.. 274. 37479-37482 (1999)
• [Publications] Tanabe, K.: "Direct Photoaffinity Labelling of the Kir6.2 subunit of the ATP-sensitive K+ channel by 8-Azido-ATP"J. Biol. Chem.. 274. 3931-3933 (1999)
• [Publications] Akamatsu, M.: "Vinexin Forms a Signaling Complex with Sos and Modulates Epidermal Growth Factor induced c-Jun N-terminal Kinase/Stress-activated Protein Kinase Activities"J. Biol. Chem.. 274. 35933-35937 (1999)
• [Publications] Ueda, K.: "Comparative aspects of the function and mechanism of SUR1 and MDR1 proteins"Biochim. Biophys. Acta. 1461. 305-313 (1999)
• [Publications] Tanizawa, Y.: "Genetic analysis of Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy : nucleotide binding fold-2 mutation impairs cooperative binding of adenine nucleotides to sulfonylurea receptor 1"Diabetes. 49. 114-120 (2000)
• [Publications] Ashizawa, T.: "Antitumor activity of KF22678, a novel thioester derivative of leinamycin"Anticancer Drugs. 10. 829-836 (1999)
• [Publications] Chen, Z.: "ATP-Dependent efflux of CPT-11 and SN-38 by the multidrug resistance protein (MRP) and its inhibition by PAK-104P"Mol Pharmacol. 55. 921-928 (1999)
• [Publications] Matsuo, M.: "NEM modification prevents high-affinity ATP-binding to the first nucleotide binding fold of the sulphonylurea receptor, SUR1"FEBS Lett. 458. 292-294 (1999)
• [Publications] Tsuruoka, S.: "Removal of digoxin and doxorubicin by multidrug resistance protein-overexpressed cell culture in hollow fiber"Kidney Int. 56. 154-163 (1999)
• [Publications] Ueda, K.: "Recent progress in P-glycoprotein research"Anti-cancer drug design. 14. 115-121 (1999)
• [Publications] Nishii, Y.: "CROP/Luc7A, a novel serine/Arginine-rich nuclear protein, isolated from cisplatin resistant cell line"FEBS Lett. 465. 153-156 (2000)
• [Publications] Takada,Y.: "Non-equivalent cooperation between the two nucleotide-binding folds of P-glycoprotein." Biochim.Biophys.Acta.1373. 131-136 (1998)
• [Publications] Kusunoki,N.: "Inhibitory effects of a cyclosporin derivative,SDZ PSC 833,on transport of doxorubicin and vinblastine via human P-glycoprotein." Jpn.J.Cancer Res.89. 1220-1228 (1998)
• [Publications] Tanabe,K.: "Direct Photoaffinity Labelling of the Kir6.2 subunit of the ATP-sensitive K+ channel by 8-Azido-ATP." J.Biol.Chem.274. 3931-3933 (1999)
• [Publications] Ueda,K.: "Cooperative binding of ATP and MgADP in the sulfonylurea receptor is modulated by glibenclamide." Proc.Natl.Acad.Sci.USA. 96. 1268-1272 (1999)
• [Publications] Ueda,K.: "Recent progress in P-glycoprotein research." Anti-cancer drug design. 14. 印刷中 (1999)