我们要感谢明尼苏达大学的李实验室提供了所用的小鼠脊髓组织样本。这项工作是由国防部卵巢癌研究计划（OCRP）OC093424以MB部的支持下，由兰迪剃须刀癌症研究和社区基金，以MB和明尼苏达卵巢癌联盟（MoCA），以MB。该资助者在研究设计，数据收集和分析，决定发表或准备手稿没有作用。

1.裂解液的制备<ol><li>溶解蔗糖在去离子（DI）水，使2M的储备溶液。过滤蔗糖的使用真空从动0.22μm的聚偏二氟乙烯（PVDF）过滤2M的溶液。 </li><li>溶解二硫苏糖醇（DTT）在去离子水，使厌氧条件下的500mM储备溶液并储存。溶解氯化镁（MgCl 2的）在去离子水，使100mM的储备液。溶解三磷酸腺苷（ATP）的二钠水合物的去离子水，使50mM的储备溶液。 </li><li>弥补在pH7.4 Tris溶液由在去离子水中溶解的Trizma盐酸盐和调节使用氢氧化钠（NaOH）的pH值。 </li><li>结合的储备溶液的部分在去离子水，使裂解缓冲液为250毫米，1毫米，使MgCl 2浓度为5mM，1mM的的ATP浓度和为50mM的Tris浓度的DTT浓度的蔗糖浓度。例如，混合535.3微升三，500微升的MgCl 2，1.25毫升蔗糖，20微升的DTT，200微升ATP和7.4947毫升去离子水，使加入10ml裂解缓冲液。这可以用来大约20实验。 </li></ol>对于实验2培养细胞<ol><li>通过等分30毫升DMEM + 10％胎牛血清（FBS）的到50毫升锥形管中作介质。 </li><li>从液氮储存冷冻转让或M17 HeLa细胞的小烧杯中，用温水。 </li><li>转移细胞到50ml锥形管中。首先，一些媒体加入到细胞小瓶。随后的5秒内解冻转移悬浮细胞。 </li><li>离心细胞悬浮液在450×g离心5分钟以获得细胞沉淀。加20毫升介质成T75烧瓶中。 </li><li>从使用吸细胞取出介质。加入5毫升的新鲜培养基以将细胞沉淀重悬。 </li><li>传送5毫升细胞悬浮液至20毫升介质在T75烧瓶中并在37℃。 CH安格媒体每3天。 </li></ol> 3.细胞收获<ol><li>通过吸取出介质，小心不要碰到细胞。 </li><li>用5ml磷酸盐缓冲盐水（PBS）洗涤细胞。使用抽吸去除PBS。 </li><li>添加3ml胰蛋白酶 - 乙二胺四乙酸（EDTA）的T75烧瓶，并在37℃下进行3分钟。 </li><li>加入6毫升的新鲜培养基的烧瓶中，并悬浮细胞。 </li><li>该悬浮液转移到15毫升锥形管中并离心，在290×g下5分钟。 </li><li>去除上清，加入5毫升的PBS。轻轻拍打管的底部向上突破的沉淀。离心，在290×g离心5分钟。 </li><li>重复步骤3.6三次。 </li><li>除去PBS重悬细胞中加入1ml新鲜的PBS。转移到Eppendorf管中（1.5毫升）。旋转，在290×g离心5分钟。 </li></ol> 4.细胞裂解<ol><li>使用移液管的测量粒料的近似体积ð称出玻璃珠在一团:体积比为1:1。加入裂解缓冲液将沉淀的体积的两倍。 注:添加缓冲添加玻璃珠之前。 </li><li>裂解的细胞在Eppendorf管中以最大搅拌30分钟，在4℃下通过涡旋在冷室中。 </li><li>短暂离心，在200×g离心30秒，将沉淀珠子。回收上清。 </li><li>作为前添加的玻璃珠相同体积的上清液。 </li><li>涡流再次在最大搅拌30分钟，在4℃。 </li><li>短暂离心，在200×g离心30秒以解决玻璃珠。回收上清。 </li><li>离心机在5030×g离心5分钟以除去细胞核，膜和完整细胞。收集上清液，这是细胞裂解物。 </li></ol> 5.组织均匀化<ol><li>使质量:体积的1:9溶液小鼠脊髓组织裂解缓冲液中。 注意:本方法适用于各种不同耳鼻喉科组织样本。 </li><li>均质化使用2级的一个设置在均化30秒的组织，以确保没有剩余的块。 </li><li>降速在5,030×g离心3分钟以除去细胞核，膜和完整细胞。 </li><li>收集上清液，这是溶胞产物。 </li></ol> 6.样品衍生<ol><li>在一个96孔板分析执行二喹啉甲酸（BCA）来确定使用所指定的皮尔斯BCA蛋白测定试剂盒协议比色96孔板读取器的组织裂解液的蛋白质浓度。 </li><li>带来使用去离子（DI）水相当于20微克总蛋白的50微升等分试样。 </li><li>加入2微升1.35微米的HA-UB-乙烯基砜（VS）到溶液中，并培育1小时，在37℃。这将导致20微克裂解物在最终的反应混合物50nM的探针。这是在大的摩尔过量，以确保功能泛素化酶标记。 </li><li>一世ncubate在5分钟的Laemmli的样品缓冲液将样品在95℃。对于52微升反应体积使用26微升2×样品缓冲液，13微升的4倍样品缓冲液，或8.87微升6X样品缓冲液。 </li><li>在装载前凝胶酷冰上。 </li></ol> 7. Western印迹<ol><li>加载一个12孔4-20％tris-甘氨酸凝胶用40微升制备的样品的</li><li>运行在95伏凝胶直到离子前沿到达底部。 </li><li>进行凝胶的过夜转移到聚偏二氟乙烯（PVDF）膜上。 </li><li>孵育在氨基黑染色的膜和扫描membane获得的图像表示蛋白在每个车道的量。 </li><li>脱色，膜，并培育在5％牛奶的PBS中进行1小时到框。 </li><li>孵育在初级抗HA抗体的膜在浓度为1:10,000，在5％牛奶的PBS中任过夜，在4℃或4小时，在室温下进行。 </li><li>执行3次洗涤˚F或每5分钟在PBS与0.1％Tween-20的洗涤剂。 </li><li>孵育在小鼠辣根过氧化物酶的膜在浓度为1:10,000的5％乳的PBS，至少2小时。 </li><li>执行3次洗涤，每次5分钟，在PBS中与0.1％Tween-20的洗涤剂。 </li><li>执行1洗5分钟，在PBS。 </li><li>孵育在化学发光检测试剂的膜10分钟，检测使用化学发光检测器。使用自动曝光的检测器上的设置。 </li></ol>

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	<li>Ovaa, H., Kessler, B. M., Rol&#233;n, U., Galardy, P. J., Ploegh, H. L., Masucci, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-+based+ubiquitin-specific+(USP)+profiling+of+virus-infected+and+malignant+human+cells.">Activity- based ubiquitin-specific (USP) profiling of virus-infected and malignant human cells.</a> Proc Natl Acad Sci USA. 101 (8), 2253-2258 (2004).</li><li>Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitination+and+deubiquitination:+Targeting+of+proteins+for+degradation+by+the+proteasome.">Ubiquitination and deubiquitination: Targeting of proteins for degradation by the proteasome.</a> Semin Cell Dev Biol. 11 (3), 141-148 (2000).</li><li>Ziad, M. E., Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+proteolysis+by+human+deubiquitinating+enzymes.">Regulation of proteolysis by human deubiquitinating enzymes.</a> Biochim Biophys Acta. 1843 (1), 114-128 (2014).</li><li>Rol&#233;n, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+Profiling+of+Deubiquitinating+Enzymes+in+Cervical+Carcinoma+Biopsies+and+Cell+Lines.">Activity Profiling of Deubiquitinating Enzymes in Cervical Carcinoma Biopsies and Cell Lines.</a> Mol Carcinog. 45 (4), 260-269 (2006).</li><li>Borodovsky, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry-Based+Functional+Proteomics+Reveals+Novel+Members+of+the+Deubiquitinating+Enzyme+Family.">Chemistry-Based Functional Proteomics Reveals Novel Members of the Deubiquitinating Enzyme Family.</a> Chem Biol. 9 (10), 1149-1159 (2002).</li><li>Andersson, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+Parkinson&#8217;s-disease-associated+mutations+on+the+deubiquitinating+enzyme+UCH-L1.">The effect of Parkinson&#8217;s-disease-associated mutations on the deubiquitinating enzyme UCH-L1.</a> J Mol Biol. 407 (2), 261-272 (2011).</li><li>Poon, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-Amyloid+(A&#946;)+oligomers+impair+brain-derived+neurotrophic+factor+retrograde+trafficking+by+down-regulating+ubiquitin+C-terminal+hydrolase+UCH-L1.">&#946;-Amyloid (A&#946;) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase UCH-L1.</a> J Biol Chem. 288 (23), 16937-16948 (2013).</li><li>Nijman, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deubiquitinating+enzyme+USP1+regulates+the+Fanconi+anemia+pathway.">The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway.</a> Mol Cell. 17 (3), 331-339 (2005).</li><li>Stevenson, L. F., Sparks, A., Allende-Vega, N., Xirodimas, D. P., Lane, D. P., Saville, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deubiquitinating+enzyme+USP2a+regulates+the+p53+pathway+by+targeting+Mdm2.">The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2.</a> EMBO J. 26 (4), 976-986 (2007).</li><li>Coughlin, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+RA-9+inhibits+proteasome-associated+DUBs+and+ovarian+cancer+in+vitro+and+in+vivo+via+exacerbating+unfolded+protein+responses.">Small-molecule RA-9 inhibits proteasome-associated DUBs and ovarian cancer in vitro and in vivo via exacerbating unfolded protein responses.</a> Clin Cancer Res. 20 (12), 3174-3186 (2014).</li><li>Colla, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+Reticulum+Stress+Is+Important+for+the+Manifestations+of+&#945;-Synucleinopathy+In.">Endoplasmic Reticulum Stress Is Important for the Manifestations of &#945;-Synucleinopathy In.</a> Vivo. J Neurosci. 32 (10), 3306-3320 (2012).</li></ol>

作者什么都没有透露。

由于泛素化是一个基本的细胞活性，了解监管机制可能是关键发掘疾病和病理过程。使用医管局标记这里报告泛素衍生的活性部位定向探头提供了一种简单，但非常适用的方法成为研究泛素介导的蛋白质降解。这种方法更快，更便宜不是单独研究泛素化酶中的每一个。 在该方法中，细胞的裂解是通过机械装置来实现 - 利用玻璃珠。这种温和的裂解方法保留代谢细胞内的画面?...

泛素 - 蛋白酶体系统（UPS），作为在哺乳动物细胞的主要降解途径之一。基板在蛋白酶体结合的降解共价标记的泛素（泛素）1的聚合物。前的目标衬底进入蛋白酶体进行降解，聚遍在蛋白标记必须被去除。一类称为酶去泛素（泛素化酶）的酶负责泛素分子2的去除和回收。据预测，从人类基因组中，有近百名在泛素化酶小区3工作。有了这样一个大量的泛素化酶泛素控制介导的细胞过程，研究这些酶是一种挑战，因为基因技术不给上的活动和免疫印迹信息，仅给出了表达水平的信息。 使用流感血凝素（HA）的标记，泛素衍生的活性部位定向探头允许的共价国防部官能泛素化酶并且因此ification给出了这些酶的活性对Western印迹4直接可视化。探针具有用作活性位点半胱氨酸残基5一自杀底物的C-末端硫醇反应性基团。用这些探针，能够研究细胞的两个病理和生理状态下许多泛素化酶的活性和潜在的表达。 变化DUB活性已牵涉多种病理状况，如帕金森氏，阿尔茨海默氏症，贫血和多种癌症6-10。这种技术提供了一个强大的工具，疾病的研究。在本文中，我们将展示这项技术在HeLa和M17细胞已使用玻璃珠裂解的应用。此外，我们概述了如何使用此方法在小鼠脊髓组织样本。从这种技术获得的信息可以被用来作为一个起点，用于识别治疗目标以及用于不同疾病状况的研究建立的模型。这种技术的真正的实用在于它能够提供在多个泛素化酶信息在单个测定能力。

<table><tbody><tr><td>PowerGen 125 (homogenizer)</td><td>Fischer Scientific</td><td>14-261-02P</td><td></td></tr><tr><td>Vacuum-driven filters 0.22 &amp;micro;m</td><td></td><td>BPV2210</td><td></td></tr><tr><td>Glass beads, acid washed &amp;le;106 &amp;micro;m (~140 U.S. sieve)</td><td>Sigma-aldrich</td><td>G4649-10G</td><td></td></tr><tr><td>Sucrose</td><td>Fischer Scientific</td><td>S6-212</td><td></td></tr><tr><td>DL-dithiothreitol</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Adenosine 5&#039;-triphosphate disodium salt hydrate</td><td>Sigma-Aldrich</td><td>A26209</td><td></td></tr><tr><td>Trizma hydrochloride</td><td>Sigma-Aldrich</td><td>T3253</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Life Technologies</td><td>11965-092</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Tissue culture flask 75 cm&lt;sup&gt;2&lt;/sup&gt; w/ filter cup 250 ml 120/cs</td><td>Cellstar</td><td>T-3001-2</td><td></td></tr><tr><td>0.05% Trypsin-EDTA (1x)</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>HI FBS</td><td>Life Technologies</td><td>16140-071</td><td></td></tr><tr><td>Monoclonal anti-HA antibody produced in mouse</td><td>Sigma-Aldrich</td><td>H9658</td><td></td></tr><tr><td>Ubiquitin vinyl sulfone (HA-tag)</td><td>Enzo Life Sciences</td><td>BML-UW0155-0025</td><td></td></tr><tr><td>Laemmli&#039;s SDS-Sample Buffer (4x, reducing)</td><td>Boston BioProducts</td><td>BP-110R</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td></td></tr></tbody></table>

method for measuring the activity of deubiquitinating enzymes in cell lines and tissue samples

泛素 - 蛋白酶体系统最近已经牵连在各种病症，包括神经变性疾病和癌症。鉴于此，技术研究本系统的调控机制是必不可少的阐明上述疾病的细胞和分子过程。使用本文中概述血凝素衍生的泛素探针作为本系统的研究的宝贵工具。本文详述的方法，使用户能够执行检测，让去泛素酶活性的直接可视化。去泛素化酶控制蛋白酶体降解，并在它们的活性位点，其允许用户进行调查多种酶的活性在一个测定共享功能同源性。裂解液是通过温和的化学细胞破碎，并获得与培养活性部位定向探头。功能酶标记探针，同时不活跃的酶保持绑定。通过运行宁该测定中，用户获得上的活性和多个去泛素酶在一个快速和容易的方式潜在表达信息。当前的方法是显著比使用单个抗体对于预测百在人类细胞去泛素酶更有效。

使用获得的协议（3细胞收集）的详细方法和小鼠脊髓组织培养的M17和HeLa细胞收获。将细胞沉淀/脊髓组织置于与在试剂制备部分所述裂解缓冲液的管中。细胞沉淀物用玻璃珠（ 图1A）和小鼠脊髓组织样品使用均化器（ 图1B）进行匀浆裂解。裂解或均化后，将样品然后在5030×g离心离心除去玻璃珠和/或完整的膜和细胞器（ 图2）。这两种方法是裂解的机械装置以...

Watch this Scientific Journal Video about Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples at JoVE.com

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

目前的协议细节，用于测量功能性同源去泛素酶的活性的方法。专门探针共价修饰的酶，并允许进行检测。这种方法保存，以确定新的治疗靶点的潜力。

测量去泛素酶的活性细胞系和组织样品的方法

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

method-for-measuring-activity-deubiquitinating-enzymes-cell-lines

Research

JoVE Journal

Biology

9.6K 次观看.  University of Minnesota. 目前的协议细节，用于测量功能性同源去泛素酶的活性的方法。专门探针共价修饰的酶，并允许进行检测。这种方法保存，以确定新的治疗靶点的潜力。 

视频: Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

科研

生物化学

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

核糖体组蛋白的体外泛化和二次基质测定

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

蛋白泛素化的检测

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

生物学

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

This method describes the assessment of global changes in ubiquitin chain topology. The assessment is performed by the application of a mass spectrometry-based targeted proteomics approach.

通过并行反应监测进行无处不在的链分析

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

测量泛素化、SUMO 化和 ADP 核糖基化 DNA-蛋白质交联

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

作者聚焦: DNA 蛋白交联及其翻译后修饰的定量检测  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

<meta charset="utf8"></head><body>使用切割测定评估神经发育障碍中的 DUB 变异

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. Discrete disorders caused by single genes provide models to investigate mechanisms driving atypical neurodevelopment. Variants of genes encoding deubiquitylating enzyme (DUB) family proteins are associated with several NDDs, but there is a need to determine the pathogenic mechanisms of disorders driven by these gene variants. The impact of gene variants on DUB activity can be experimentally determined using a substrate-independent in vitro ubiquitin cleavage assay. This assay does not require knowledge of downstream substrates to directly measure catalytic activity. Here, the protocol for determining the impact of gene variants on enzymatic activity is modeled using the DUB Ubiquitin Specific Protease 27, X-linked (USP27X), which is mutated in X-linked intellectual disability 105 (XLID105). This experimental pipeline can be used to clarify the mechanisms underlying neurodevelopmental disorders driven by variants in DUB genes.

<meta charset="utf8"></head><body>通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性

This protocol explains how to measure the effect of genetic variation associated with neurodevelopmental disorders on deubiquitylating enzyme activity by combining recombinant protein purification with ubiquitin chain cleavage assays.

通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. ...

测量去泛素酶的活性细胞系和组织样品的方法

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此视频中的章节

通过肽浓缩和质谱检测蛋白质泛化位点检测

核糖体组蛋白的体外泛化和二次基质测定

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

蛋白泛素化的检测

通过并行反应监测进行无处不在的链分析

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性

通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性