Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

<p>Abstract</p> <p>Background</p> <p>Various protein expression systems, such as <it>Escherichia coli </it>(<it>E. coli</it>), <it>Saccharomyces cerevisiae </it>(<it>S. cerevisiae</it>), <it>Pichia pastoris </it>(<it>P. pastoris</it>), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β₂adrenergic receptor, adenosine A_2areceptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, <it>P. pastoris </it>and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies.</p> <p>Results</p> <p>The ideal conditions for the expression of CHRM2 in <it>P. pastoris </it>were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [³H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by <it>P. pastoris </it>was lower than that of Sf9 insect cells, CHRM2 yield in <it>P. pastoris </it>was 2-fold higher than in Sf9 insect cells because <it>P. pastoris </it>was cultured at high cell density. The dissociation constant (Kd) for QNB in <it>P. pastoris </it>was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between <it>P. pastoris </it>and Sf9 insect cells.</p> <p>Conclusion</p> <p>Compared to insect cells, <it>P. pastoris </it>is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, <it>P. pastoris</it>, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in <it>P. pastoris </it>can be applied to the structural and biochemical studies of GPCRs.</p

Structure of the dopamine D2 receptor in complex with the antipsychotic drug spiperone

統合失調症に関わるドパミン受容体の構造解明 --副作用を抑えた薬の迅速な探索・設計が可能に--. 京都大学プレスリリース. 2020-12-24.In addition to the serotonin 5-HT2A receptor (5-HT2AR), the dopamine D2 receptor (D2R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D2R have been described in complex with the inverse agonists risperidone (D2Rris) and haloperidol (D2Rhal). Here we describe the structure of human D2R in complex with spiperone (D2Rspi). In D2Rspi, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D2Rris and D2Rhal, demonstrating that ECL2 in D2R is highly dynamic. Moreover, D2Rspi exhibited an extended binding pocket to accommodate spiperone’s phenyl ring, which probably contributes to the selectivity of spiperone to D2R and 5-HT2AR. Together with D2Rris and D2Rhal, the structural information of D2Rspi should be of value for designing novel antipsychotics with improved safety and efficacy

Coping with Senior Citizens with Dementia in Finnish Communities:Based on interviews with experts on elderly care utilizing fictional cases

本研究の目的は,高齢者ケアの有識者の認識を通してフィンランドの地域における認知症高齢者への典型的な対応を明らかにし,日本の認知症ケアへの示唆を得ることである. フィンランドの高齢者ケアの実際や制度について一定の知識をもつケア専門職の教育機関の教員4名と通訳者1名に対して,模擬事例2ケース(75歳女性,アルツハイマー型認知症,独居,時間の見当識障害のある事例と排泄動作の障害のある事例)を提示し,家族,地域住民,専門職の対応の仕方などについて聞き取った.各質問項目への回答について,意味内容の類似性に従って分類した. 対象者は全員フィンランド人女性であり,年齢は,60歳代が2名,40歳代が2名,50歳代が1名であった.模擬事例への回答結果として,近隣住民は認知機能が低下した高齢者について家族や保健センターのホームケア部門に相談すること,服薬管理においては家族やホームケアナースが責任を持っていることなどが語られた.また,服薬管理において機器が活用されていること,メモリーナースが認知機能のテストや医師への紹介を行うことについても述べられた. 以上より,日本にすでに存在する地域包括支援センター等の機関の地域住民への周知と多様なソーシャル・サポートとの連携のあり方の検討の必要性,認知症ケアに携わるさまざまな専門職の役割の明確化,在宅ケアにおけるICTの活用など,日本における認知症ケアへの示唆が得られた

Identification of neochlorogenic acid as the predominant antioxidant in Polygonum cuspidatum leaves

To identify the predominant antioxidant compound in Polygonum cuspidatum leaves, the methanol extract of fresh samples were separated by liquid–liquid partitioning, octadecylsilyl Sep-pak®cartridge and high-performance liquid chromatography. The main active compound was identified as (1R,3R,4S,5R)-3-{[(2E)-3-(3,4-dihydroxyphenyl)-2-propenoyl]oxy}-1,4,5-trihydroxycyclohexanecarboxylic acid (neochlorogenic acid) by nuclear magnetic resonance and liquid chromatographymassspectroscopic analysis. Its content was found to be 2.31 mg/g of fresh leaves. As shown by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide anion scavenging assays, the contributionsof neochlorogenic acid as an antioxidant were 16.5% and 36.5%, respectively, suggesting that neochlorogenic acid is the predominant antioxidant in P. cuspidatum leaves

Study of the antimicrobial activity of Aminoreductone against pathogenic bacteria in comparison with other antibiotics

Antimicrobial activities of aminoreductone (AR), a product formed in the initial stage of Maillard reaction, were screened against pathogenic bacteria. A significant growth inhibition of AR against all 7 isolates (Staphylococcus aureus ATCC® 25923 TM, Salmonella typhimurium ATCC® 14028 TM, Bacillus cereus ATCC® 13061TM, Bacillus subtilis ATCC® 11774 TM, Escherichia coli ATCC® 25922 TM, Enterococcus faecalis ATCC® 29212 TM, Listeria innocua ATCC® 33090 TM) were observed by the standard disc diffusion methods. The inhibition zone for each isolate by AR (2.5 mg) ranged from 15±0mm to 28.3±0.4mm in diameter. The minimum inhibitory concentration (MIC) of AR ranging from 20mM to 26mM was proven in the 7 isolates tested. AR also showed the similar effect of growth inhibition in comparison with antibiotics frequently used for the treatment of infections bacteria, such as amikacin, ciprofloxacin, meropennem and levofloxacin. The results indicated that foods containing AR are valuable sources of bioactive compounds towards pathogenic bacteria. (Résumé d'auteur