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Proteasome Assay in Cell Lysates
细胞匀浆中的蛋白酶体分析   

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Abstract

The ubiquitin-proteasome system (UPS) mediates the majority of the proteolysis seen in the cytoplasm and nucleus of mammalian cells. As such it plays an important role in the regulation of a variety of physiological and pathophysiological processes including tumorigenesis, inflammation and cell death (Ciechanover, 2005; Kisselev and Goldberg, 2001). A number of recent studies have shown that proteasome activity is decreased in a variety of neurological disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and stroke as well as during normal aging (Chung et al., 2001; Ciechanover and Brundin, 2003; Betarbet et al., 2005). This decrease in proteasome activity is thought to play a critical role in the accumulation of abnormal and oxidized proteins. Protein clearance by the UPS involves two sequential reactions. The first is the tagging of protein lysine residues with ubiquitin (Ub) and the second is the subsequent degradation of the tagged proteins by the proteasome. We herein describe an assay for the second of these two reactions (Valera et al., 2013). This assay uses fluorogenic substrates for each of the three activities of the proteasome: chymotrypsin-like activity, trypsin-like activity and caspase-like activity. Cleavage of the fluorophore from the substrate by the proteasome results in fluorescence that can be detected with a fluorescent plate reader.

Keywords: Nerve cells(神经细胞), Fluorescence(荧光), Protein degradation(蛋白质的降解)

Materials and Reagents

  1. Cells
  2. HEPES (Sigma-Aldrich, catalog number: H3375 )
  3. MgCl2 (Sigma-Aldrich, catalog number: M2670 )
  4. EDTA (Sigma-Aldrich, catalog number: E5134 )
  5. EGTA (Sigma-Aldrich, catalog number: E4378 )
  6. Sucrose (MP Biomedicals, catalog number: 821713 )
  7. DTT (Life Technologies, catalog number: 15508013 )
  8. Proteasome substrates [dissolved in DMSO (Sigma-Aldrich, catalog number: D8418 ) to a final concentration of 10 mM and stored frozen at -20 °C]
    1. Suc-LLVY-AMC (chymotrypsin-like activity substrate) (Enzo Life Sciences, catalog number: P802 )
    2. Z-ARR-AMC (trypsin-like activity substrate) (EMD Millipore, catalog number: 539149 )
    3. Z-LLE-AMC (caspase-like activity substrate) (EMD Millipore, catalog number: 539141 )
  9. PBS without Ca2+ and Mg2+ (Sigma-Aldrich, catalog number: P802)
  10. Coomassie Protein Assay Reagent (Thermo Fisher Scientific, catalog number: 1856209 )
  11. BSA protein standard (Thermo Scientific, catalog number: 23209 )
  12. ATP (Sigma-Aldrich, catalog number: A3377 )
  13. Proteasome Lysis/Assay Buffer (see Recipes)   

Equipment

  1. 60 mm tissue culture dishes
  2. Rubber policeman or other type of cell scraper
  3. 1.7 ml microcentrifuge tube
  4. Black walled 96 well plates (Corning, Costar®, catalog number: 3603 )
  5. Clear 96 well plates (Greiner Bio-One GmbH, catalog number: 655101 )
  6. Sonicator (GlobalSpec, model: W-380 )
  7. Microcentrifuge
  8. Fluorescent plate reader
  9. Visible plate reader

Procedure

  1. Sample Preparation
    1. Rinse cells in 60 mm dishes 2 times with ice cold PBS.
    2. Scrape cells using a rubber policeman or other type of cell scraper into 400 µl lysis buffer, transfer to a 1.7 ml microcentrifuge tube and place on ice.
    3. Sonicate for 10 sec using microtip set on ~2.
    4. Centrifuge at 16,000 x g for 10 min at 4 °C.
    5. Transfer supernatant to a fresh 1.7 ml microcentrifuge tube (can store at -70 °C until use in assay).

  2. Assay
    1. Prepare assay buffer (make up more than you need; e.g. if you need 3 ml, make 3.5 ml).
    2. Add proteasome substrates to assay buffer (2.5 µl/sample; 100 µM final concentration) (make up slightly more than you need; e.g. if you have 12 samples, make up enough for 13).
    3. Put 2 x 200 µl aliquots of assay buffer with substrate into 2 wells of a black walled 96 well plate for each sample.
    4. Add 50 µl cell lysate to each well (include 2 wells with lysis buffer with no cells as assay blanks).
    5. Incubate 60 min at 37 °C in order to allow the cleavage of the fluorophore from the proteasome substrate.
    6. Read A360ex/A460em on fluorescent plate reader.
    7. Normalize to protein determined with the Coomassie Protein Assay following the manufacturer’s instructions for a microplate reader and using 5 µl of lysate.

Recipes

  1. Proteasome Lysis/Assay Buffer
    50 mM HEPES (pH 7.8)
    10 mM NaCl
    1.5 mM MgCl2
    1 mM EDTA
    1 mM EGTA
    250 mM sucrose
    Sterile filter and stored at 4 °C
    For lysis buffer: add DTT to 5 mM final concentration (use 1 M stock)
    For assay buffer: add DTT to 5 mM final concentration and ATP to 2 mM final concentration

Acknowledgments

This protocol was adapted from Valera et al. (2013). The research was supported by grants from NIH (RO1AG035055), the Fritz B. Burns Foundation and the Alzheimer’s Association.

References

  1. Betarbet, R., Sherer, T. B. and Greenamyre, J. T. (2005). Ubiquitin-proteasome system and Parkinson's diseases. Exp Neurol 191 Suppl 1: S17-27. 
  2. Chung, K. K., Dawson, V. L. and Dawson, T. M. (2001). The role of the ubiquitin-proteasomal pathway in Parkinson's disease and other neurodegenerative disorders. Trends Neurosci 24(11 Suppl): S7-14. 
  3. Ciechanover, A. (2005). Proteolysis: from the lysosome to ubiquitin and the proteasome. Nat Rev Mol Cell Biol 6(1): 79-87.
  4. Ciechanover, A. and Brundin, P. (2003). The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg. Neuron 40(2): 427-446. 
  5. Kisselev, A. F. and Goldberg, A. L. (2001). Proteasome inhibitors: from research tools to drug candidates. Chem Biol 8(8): 739-758. 
  6. Valera, E., Dargusch, R., Maher, P. A. and Schubert, D. (2013). Modulation of 5-lipoxygenase in proteotoxicity and Alzheimer's disease. J Neurosci 33(25): 10512-10525.

简介

泛素 - 蛋白酶体系统(UPS)介导哺乳动物细胞的细胞质和细胞核中大部分蛋白水解。因此,它在调节各种生理和病理生理过程中起重要作用,包括肿瘤发生,炎症和细胞死亡(Ciechanover,2005; Kisselev和Goldberg,2001)。许多最近的研究表明,在多种神经障碍(包括帕金森病,阿尔茨海默病,肌萎缩性侧索硬化和中风)以及正常衰老期间,蛋白酶体活性降低(Chung等,2001; Ciechanover和Brundin,2003; Betarbet等,2005)。蛋白酶体活性的降低被认为在异常和氧化蛋白质的积累中发挥关键作用。 UPS的蛋白质清除涉及两个连续的反应。第一个是用泛素(Ub)标记蛋白质赖氨酸残基,第二个是蛋白酶体随后降解标记的蛋白质。我们本文描述了这两个反应中第二个的测定(Valera等,2013)。该测定法使用荧光底物作为蛋白酶体的三种活性:糜蛋白酶样活性,胰蛋白酶样活性和半胱天冬酶样活性。通过蛋白酶体从底物中切割荧光团可导致可用荧光板读数器检测的荧光。

关键字:神经细胞, 荧光, 蛋白质的降解

材料和试剂

  1. 单元格
  2. HEPES(Sigma-Aldrich,目录号:H3375)
  3. MgCl 2(Sigma-Aldrich,目录号:M2670)
  4. EDTA(Sigma-Aldrich,目录号:E5134)
  5. EGTA(Sigma-Aldrich,目录号:E4378)
  6. 蔗糖(MP Biomedicals,目录号:821713)
  7. DTT(Life Technologies,目录号:15508013)
  8. 将蛋白酶体底物[溶于DMSO(Sigma-Aldrich,目录号:D8418)至终浓度为10mM,并冷冻保存在-20℃下]
    1. Suc-LLVY-AMC(胰凝乳蛋白酶样活性底物)(Enzo Life Sciences,目录号:P802)
    2. Z-ARR-AMC(胰蛋白酶样活性底物)(EMD Millipore,目录号:539149)
    3. Z-LLE-AMC(胱天蛋白酶样活性底物)(EMD Millipore,目录号:539141)
  9. PBS(不含Ca 2+和Mg 2+)(Sigma-Aldrich,目录号:P802)。
  10. 考马斯蛋白测定试剂(Thermo Fisher Scientific,目录号:1856209)
  11. BSA蛋白标准品(Thermo Scientific,目录号:23209)
  12. ATP(Sigma-Aldrich,目录号:A3377)
  13. 蛋白酶体裂解/测定缓冲液(见配方)  

设备

  1. 60 mm组织培养皿
  2. 橡胶警察或其他类型的细胞刮刀
  3. 1.7 ml微量离心管
  4. 黑色的96孔板(Corning,Costar ,目录号:3603)
  5. 清除96孔板(Greiner Bio-One GmbH,目录号:655101)
  6. 超声波仪(GlobalSpec,型号:W-380)
  7. 微量离心机
  8. 荧光平板读取器
  9. 可见读板机

程序

  1. 样品制备
    1. 用冰冷的PBS冲洗60 mm培养皿中的细胞2次
    2. 使用橡胶警察或其他类型的细胞刮刀将细胞刮入400μl裂解缓冲液中,转移至1.7ml微量离心管中并置于冰上。
    3. 使用微尖设置在〜2上超声处理10秒。
    4. 在4℃下以16,000xg离心10分钟。
    5. 将上清转移到新鲜的1.7ml微量离心管(可储存在-70℃,直到用于测定)
  2. 测定
    1. 准备测定缓冲液(补足超过你需要的; 如如果你需要3毫升,使3.5毫升)。
    2. 将蛋白酶体底物添加到测定缓冲液(2.5μl/样品;100μM最终浓度)(补偿略多于你需要;例如如果你有12个样品,补足13) >
    3. 将2×200μl含底物的测定缓冲液的等分试样加入到每个样品的黑色壁96孔板的2个孔中。
    4. 向每个孔中加入50μl细胞裂解液(包括2个具有无细胞裂解缓冲液的孔作为测定空白)
    5. 在37℃孵育60分钟,以允许荧光团从蛋白酶体底物裂解。
    6. 在荧光板读取器上读取A 360 ex/A
    7. 使用考马斯蛋白质测定法根据制造商关于酶标仪的说明并使用5μl裂解物来确定蛋白质的规格。

食谱

  1. 蛋白酶体裂解/测定缓冲液
    50mM HEPES(pH7.8)
    10mM NaCl 1.5mM MgCl 2·h/v 1mM EDTA
    1 mM EGTA
    250mM蔗糖 无菌过滤并在4℃下保存
    对于裂解缓冲液:加入DTT至终浓度为5mM(使用1M储备液) 对于测定缓冲液:加入DTT至终浓度为5mM,ATP至终浓度为2mM

致谢

此协议改编自Valera等人(2013)。 研究得到NIH(RO1AG035055),Fritz B. Burns基金会和阿尔茨海默病协会的资助。

参考文献

  1. Betarbet,R.,Sherer,T.B.and Greenamyre,J.T。(2005)。 泛素 - 蛋白酶体系统和帕金森病。 Exp Neurol 191 Suppl 1:S17-27。
  2. Chung,K.K.,Dawson,V.L。和Dawson,T.M。(2001)。 泛素 - 蛋白酶体途径在帕金森病和其他神经变性疾病中的作用。 em> Trends Neurosci 24(11 Suppl):S7-14。 
  3. Ciechanover,A。(2005)。 蛋白水解:从溶酶体到泛素和蛋白酶体。 Cell Biol 6(1):79-87。
  4. Ciechanover,A。和Brundin,P。(2003)。 神经退行性疾病中的泛素蛋白酶体系统:有时是鸡,有时是鸡蛋。 em> Neuron 40(2):427-446。 
  5. Kisselev,A.F。和Goldberg,A.L。(2001)。 蛋白酶体抑制剂:从研究工具到候选药物。 em> 8(8):739-758。
  6. Valera,E.,Dargusch,R.,Maher,P.A。和Schubert,D。(2013)。 在蛋白毒性和阿尔茨海默氏病中调节5-脂氧合酶 J Neurosci 33(25):10512-10525。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Maher, P. (2014). Proteasome Assay in Cell Lysates. Bio-protocol 4(2): e1028. DOI: 10.21769/BioProtoc.1028.
  2. Valera, E., Dargusch, R., Maher, P. A. and Schubert, D. (2013). Modulation of 5-lipoxygenase in proteotoxicity and Alzheimer's disease. J Neurosci 33(25): 10512-10525.
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