高通量荧光素酶法评价单周转蛋白酶 PCSK9 的蛋白质水解
Summary
本协议提出了一种评估细胞内一种内在的低活性、单周转蛋白酶的蛋白水解活性的方法。具体来说, 该方法用于评价 PCSK9 的蛋白水解活性, 这是脂代谢的关键驱动力, 其蛋白水解活性是其最终胆固醇功能所必需的。
Abstract
Proprotein convertase 枯草杆菌9型 (PCSK9) 是一种单周转蛋白酶, 它调节血清低密度脂蛋白 (LDL) 水平, 从而导致心血管疾病。尽管 PCSK9 蛋白水解是其完全胆固醇效果所必需的, 但对其蛋白水解功能的评价是有挑战性的: PCSK9 只知道它自己, 只经历一个单一的营业额, 并在蛋白质水解后, 保留其基底在其活性部位为自动抑制剂。这里提出的方法描述了一种克服这些挑战的试验。该方法的研究重点是细胞间蛋白水解, 并将成功的裂解与分泌的荧光素酶活性联系在一起, 可以很容易地在条件培养基中读出。通过突变、瞬变转染和荧光素酶读数的顺序步骤, 该方法可以在基因或分子摄动条件下, 在高通量的情况下 PCSK9 蛋白质水解。该系统非常适合于临床发现的无义单核苷酸多态性 (SNPs) 的生物化学评价, 以及筛选 PCSK9 蛋白水解物的小分子抑制剂。
Introduction
PCSK9 靶向 ldl 受体 (ldl R) 降解, 提高 ldl 胆固醇 (ldl C) 和驱动动脉粥样硬化性心脏病1,2。治疗靶向 PCSK9 稳健低密度脂蛋白 C 和改善心血管结局的患者, 即使添加到积极的降脂治疗与他汀类药物3,4。然而, 目前批准的疗法仅限于基于抗体的方法, 而且缺乏成本效益5,6。为了解决这个问题, 不那么昂贵的治疗方案, 一种方法来识别患者可能获得更大的利益, 或两者, 是需要的。
小分子方法可以针对细胞内 PCSK9, 提供一个改进的管理路线, 并降低成本, 使它们成为这个领域的 “圣杯”7。然而, PCSK9 已经被小分子所证明是难以用药的。作为蛋白酶, 靶向 PCSK9’s 蛋白水解功能是一个很有吸引力的策略, 因为自我分解是 PCSK9 成熟8的速率限制步骤, 它对 LDL-R9的最大影响是必需的。然而, 到目前为止, 这项战略还没有成功, 可能是由于 PCSK9’s 独特的生物化学: PCSK9 仅10, 执行单翻转反应, 自解裂后, PCSK9 prodomain 仍然绑定在活动站点作为自动抑制剂11, 防止任何进一步的蛋白酶活动的读数。
本文提出了一种评价高通量时尚8PCSK9 蛋白水解功能的方法。通过现场定向诱变, 研究者可以利用这一方法来探讨在临床中发现的编码 snp 的影响, 以评估它们对蛋白质水解的影响, PCSK9 成熟的速率限制步骤。此外, 这种方法将有助于设计高通量的屏幕, 以确定 PCSK9 蛋白降解的调制器, 这将最终扰乱 PCSK9 的表现为 LDL R (和调制 PCSK9’s 胆固醇效应).最后, 该协议可以适应其他具有本质上低活性的蛋白酶, 只要 i) 一个特定的基质蛋白酶对可以找到, 和 ii) 一个合适的细胞内锚可以建立的基质。
Protocol
1. 蛋白酶载体的定点诱变 设计和订购自定义合成的寡核苷酸, 通过修改标准站点定向突变协议来安装感兴趣的变异12。标准淡化底漆 (没有额外的净化) 是完全可以接受的。注: 对底漆设计的一般方法涉及创建部分重叠的底漆, 如表 1所示, 使用的熔融温度 (Tm) 计算器特定的聚合酶的兴趣。 如表 2所示, 在冰上设置聚合酶链反应 (PCRs…
Representative Results
高通量的蛋白质分解测定方法依赖于克服三大挑战。首先, 为了克服单周转 PCSK9 蛋白酶固有的低产量, PCSK9 蛋白酶缺乏抑制 prodomain, 与分裂序列的 prodomain 链接到一个荧光素酶, 可以分泌14。第二, 为了满足蛋白酶在复合体中的折叠与抑制 prodomain 的需要, 这两种多肽在细胞 coexpressed15,16,通过一个 bicistronic ?…
Discussion
上述实验程序提出了一种克服单周转蛋白酶 PCSK9 内在低活性的方法, 并以稳健的方式评价其蛋白水解功能。该检测的关键概念依赖于将单个周转事件转换为酶放大读数。该检测的优点包括相对较短的时间框架和易用性荧光素酶的记者, 以及它的可伸缩性高吞吐量的方法。此外, 该化验结果评估蛋白质水解在其本机, 细胞的上下文。此外, 通过这种检测, 临床鉴定的 snp 可以评估其对 PCSK9 蛋白水解的影…
Declarações
The authors have nothing to disclose.
Acknowledgements
提交人感谢 NHLBI/nih (K08 HL124068 和 LRP HMOT1243)、NCATS/nih 通过 UCSF 临床和转化科学研究所催化剂方案 (UL1 TR000004)、UCSF 学术参议院、赫尔曼基金会提供的慷慨资助,基列科学研究学者奖, 辉瑞渴望心血管奖 (全部对 John s Chorba) 和霍华德休斯医学研究所 (阿德里 m. 盖文和凯万 m. Shokat)。
Materials
| PCR Tubes | USA Scientific | 1402-2900 | For PCR |
| Q5 Hot Start | New England Biolabs | M0493L | High-fidelity DNA Polymerase |
| Deoxynucleotide Solution Mix | New England Biolabs | N0447L | dNTPs (for PCR) |
| pPCSK9-NLucProteaseAssay-WT | Autores | n/a | Available from authors |
| pPCSK9-NLucProteaseAssay-S386A | Autores | n/a | Available from authors |
| Agarose LE | Gold Biotechnology | A-201-100 | For DNA gels |
| E-Gel Imager System with Blue Light Base | ThermoFisher Scientific | 4466612 | For imaging DNA gels |
| SYBR Safe DNA Gel Stain | ThermoFisher Scientific | S33102 | For DNA gels |
| Tris Base | ThermoFisher Scientific | BP152-1 | For DNA gel running buffer |
| Glacial acetic acid | ThermoFisher Scientific | A38-500 | For DNA gel running buffer |
| Ethylenediaminetetraacetic acid solution | Millipore Sigma | 3690 | EDTA, for DNA gel running buffer |
| 1 kb DNA ladder | Gold Biotechnology | D010 | DNA ladder |
| DpnI | New England Biolabs | R0176S | Restriction enzyme |
| LB Agar plates with 100 µg/mL carbenicillin | Teknova | L1010 | LB-Carb plates |
| One Shot Mach1 T Phage-Resistent Chemically Competent E. coli | ThermoFisher Scientific | C862003 | Chemically competent cells |
| LB Broth, Miller | ThermoFisher Scientific | BP1426-2 | LB |
| Carbenicillin | Gold Biotechnology | C-103-5 | Selective antibiotic |
| E.Z.N.A. Plasmid Mini Kit I | Omega BioTek | D6942-02 | DNA Purification Miniprep kit |
| NanoDrop 2000 Spectrophotomer | ThermoFisher Scientific | ND-2000C | Spectrophotometer |
| 293T Cells | American Tissue Culture Collection (ATCC) | CRL-3216 | HEK 293T cells |
| DMEM, high glucose, pyruvate | ThermoFisher Scientific | 11995065 | DMEM, mammalian cell media |
| Fetal Bovine Sera | Axenia Biologix | F001 | FBS |
| Trypsin-EDTA (0.05%), phenol red | ThermoFisher Scientific | 25300062 | Trypsin, for cell dissociation |
| Phosphate buffered saline (PBS) | ThermoFisher Scientific | 10010023 | PBS |
| Countess automated cell counter | ThermoFisher Scientific | C10227 | Automated cell counting |
| Countess cell counting chamber slides | ThermoFisher Scientific | C10228 | Slides for cell counting |
| CELLSTAR Tissue Culture Plates, White, White-Bottom, with Lid | Grenier Bio-One | 655083 | White, white-bottom 96 well plate |
| TempPlate non-skirted 96-well PCR plate, natural | USA Scientific | 1402-9596 | 96 well plate for master plasmid plate |
| Nunc 2.0mL DeepWell Plates | ThermoFisher Scientific | 278743 | 96 well deep well plate |
| Lipofectamine 3000 | ThermoFisher Scientific | L3000008 | Lipid transfection reagent, Lf3K |
| P3000 Reagent | ThermoFisher Scientific | L3000008 | DNA pre-complexation reagent, provided with Lf3K |
| OptiMEM I Reduced Serum Medium | ThermoFisher Scientific | 31985062 | Reduced serum medium for transfection |
| (+)-Sodium L-ascorbate | Millipore Sigma | A4034 | Sodium ascorbate |
| Sodium chloride | Millipore Sigma | S9888 | NaCl |
| Albumin, Bovine Serum, Fraction V, Low Heavy Metals | Millipore Sigma | 12659 | BSA |
| Methanol (HPLC) | ThermoFisher Scientific | A4524 | MeOH |
| Hydrochloric acid | VWR | JT9535-2 | Concentrated HCl |
| Coelenterazine | Gold Biotechnology | CZ2.5 | Luciferase substrate |
| Syringe Filter, Sterile | ThermoFisher Scientific | 09-720-3 | Sterile filter, PVDF, 0.22 µm pore |
| Pipet-Lite Multi Pipette L12-200XLS+ | Rainin | 17013810 | Multichannel pipette |
| Pipet-Lite Multi Pipette L12-20XLS+ | Rainin | 17013808 | Multichannel pipette |
| Pipet-Lite Multi Pipette L12-10XLS+ | Rainin | 17013807 | Multichannel pipette |
| Reagent reservoir | Corning | 4870 | Trough for reagents |
| Centrifuge tubes, 15 mL | ThermoFisher Scientific | 05-539-12 | 15 mL tubes |
| Centrifuge tubes, 50 mL | Corning | 430829 | 50 mL tubes |
| Spark Microplate Reader | Tecan | N/a | Plate Reader |
| Excel | Microsoft | 2016 for Mac | Spreadsheet software |
| Prism | GraphPad Software | v7 | Scientific data analysis software |
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Citar este artigo
Chorba, J. S., Galvan, A. M., Shokat, K. M. A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9. J. Vis. Exp. (138), e58265, doi:10.3791/58265 (2018).