量化龙脑中的亚细胞泛基蛋白-蛋白酶体活性
Summary
该协议旨在有效地量化啮齿动物大脑不同细胞舱中的泛泛蛋白-蛋白酶体系统(UPS)活性。用户能够检查UPS在同一动物中核、细胞质和突触分数的功能,从而减少执行这些复杂分析所需的时间和动物数量。
Abstract
泛素蛋白体系统是真核生物中蛋白质降解和各种其他细胞过程的关键调节器。在大脑中,泛英蛋白酶体活性的增加对突触可塑性至关重要,记忆形成和本系统中的异常变化与各种神经、神经退行性和精神障碍有关。研究大脑中泛泛蛋白蛋白酶功能的一个问题是,它存在于所有细胞隔间中,其中蛋白质靶点、功能作用和调节机制可能有很大差异。因此,能够直接比较同一动物内不同亚细胞室中大脑泛性蛋白靶向和蛋白酶体催化活性的能力,对于充分理解UPS如何促进突触可塑性至关重要,记忆和疾病。此处描述的方法允许从同一啮齿动物(大鼠)大脑中收集核、细胞质和粗突触分数,然后同时定量蛋白酶体催化活性(间接提供蛋白酶体核心的活性)仅)和连杆特异性泛基蛋白标记。因此,该方法可用于直接比较同一动物在突触可塑性、记忆形成和不同疾病状态期间不同大脑区域泛性蛋白酶体活性的亚细胞变化。该方法还可用于评估同一动物中其他蛋白质的亚细胞分布和功能。
Introduction
泛性蛋白-蛋白酶体系统(UPS)是一个复杂的网络,由相互连接的蛋白质结构和气体组成,控制着细胞1中大多数短寿命蛋白质的降解。在这个系统中,蛋白质被小修饰剂泛性蛋白标记为降解或其他细胞过程/命运。靶蛋白可以获得1-7种泛基质修饰,它可以在7个流氨酸(K11、K27、K29、K33、K48和K63)或N端蛋氨酸(M1;称为线性)中的七个基氨酸(K)位点之一连接在一起。其中一些多维他金标记是降解特异性(K48)3,而其他基本上与蛋白质降解过程(M1)4,5,6无关。因此,蛋白质泛化过程极其复杂,量化特定多聚素标记的变化的能力对于最终理解这种给定修饰在细胞功能中的作用至关重要。进一步复杂化的研究这个系统,蛋白酶体,这是UPS7的催化结构,既降解蛋白质,但也可以参与其他非蛋白酶过程8,9。毫不奇怪,自最初发现以来,正常和异常的泛性蛋白-蛋白酶体活动一直与长期记忆形成和各种疾病状态有关,包括许多神经、神经退行性和精神病紊乱10,11。因此,能够有效和高效地量化UPS在大脑中的活动的方法对于最终理解这个系统在疾病状态下是如何调节不良以及最终开发针对泛基丁和/或的治疗方案至关重要。蛋白酶体功能。
在量化大鼠和小鼠脑组织中泛基蛋白酶体活性方面存在许多问题,这是用于研究UPS功能的最常见模型系统,包括1)泛基蛋白修饰的多样性,以及2)分布和UPS在亚细胞舱12、13、14之间运作的差分调节。例如,在记忆形成过程中大脑中泛素蛋白酶体功能的许多早期演示使用全细胞赖沙,并表明蛋白质泛化和蛋白酶体活性15的时间依赖性增加,16,17,18,19,20.然而,我们最近发现,泛基蛋白-蛋白酶体活性在亚细胞间中因学习而差异很大,有些区域同时增加,另一些区域减少,这一模式差异很大从之前报告在整个细胞莱沙21。这与整个细胞方法的限制是一致的,因为它不能分离不同亚细胞隔间UPS活动的变化。虽然最近的研究已经使用突触分数协议,以研究UPS专门在突触响应学习22,23,24,使用的方法遮挡了测量的能力同一动物的核质和细胞质泛素蛋白体变化。这导致不必要的需要重复实验多次,收集不同的亚细胞分数在每个。这不仅会导致动物生命损失,而且消除了直接比较不同亚细胞间的UPS活动以响应给定事件或特定疾病状态的能力。考虑到泛素蛋白和蛋白酶体的蛋白质靶点在整个细胞中差异很大,了解泛素蛋白-蛋白酶体信号在不同亚细胞室中如何不同,对于确定UPS在细胞中的功能作用至关重要。大脑在记忆形成和神经,神经退行性和精神障碍。
为了满足这一需求,我们最近开发了一个程序,其中核,细胞质和突触分数可以收集从同一动物21的给定大脑区域。此外,为了考虑从同一样品中收集多个亚细胞分数可以获得的有限蛋白质量,我们优化了先前建立的协议,以检测体外蛋白酶体活性和联动特异性从啮齿动物脑组织收集的分片细胞中的蛋白质普遍化。使用该协议,我们能够收集和直接比较蛋白酶体活性、K48、K63、M1和细胞质中整体蛋白多聚体化水平以及大鼠侧杏仁核的突合体中学习相关变化。在这里,我们详细介绍了我们的程序(图1),这可以显著提高我们对UPS如何参与长期记忆形成和各种疾病状态的理解。然而,应该指出的是,我们的协议中讨论的体外蛋白酶体活性虽然被广泛使用,但并不能直接测量完整的26S蛋白酶体复合物的活性。相反,此检测测量 20S 内核的活动,这意味着它只能作为代理来了解内核本身的活动,而不是整个 26S 蛋白酶体复合体。
Protocol
Representative Results
Discussion
在这里,我们演示了一种有效的方法,用于量化同一动物中不同亚细胞腔中泛基蛋白酶体活性的变化。目前,测量泛基蛋白蛋白酶体系统活性亚细胞变化的大多数尝试被限制在每个样品的单个隔间内,因此需要重复实验。这会导致动物生命的巨大成本和损失。我们的协议通过分裂半球来缓解这个问题,允许从同一动物的每个半球收集不同的细胞分数。使用这个协议,我们能够首次证明,在同一动物泛素-蛋?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了农业和生命科学学院和弗吉尼亚理工大学科学学院的启动基金的支持。
Materials
| 0.5M EDTA | Fisher | 15575020 | Various other vendors |
| 20S Proteasome Activity Kit | Millipore Sigma | APT280 | Other vendors carry different versions |
| ATP | Fisher | FERR1441 | Various other vendors |
| Beta-actin antibody | Cell signaling | 4967S | Various other vendors |
| Beta-tubulin antibody | Cell signaling | 2128T | Various other vendors |
| BioTek Synergy H1 plate reader | BioTek | VATECHH1MT3 | Other vendors carry different versions |
| B-mercaptoethanol | Fisher | ICN19024280 | Various other vendors |
| clasto lactacystin b-lactone | Millipore Sigma | L7035 | Various other vendors |
| Cryogenic cup | Fisher | 033377B | Various other vendors |
| DMSO | DMSO | D8418 | Varous other vendors |
| DTT | Millipore Sigma | D0632 | Various other vendors |
| Glycerol | Millipore Sigma | G5516 | Various other vendors |
| H3 antibody | Abcam | ab1791 | Various other vendors |
| HEPES | Millipore Sigma | H3375 | Various other vendors |
| Hydrochloric acid | Fisher | SA48 | Various other vendors |
| IGEPAL (NP-40) | Millipore Sigma | I3021 | Various other vendors |
| K48 Ubiquitin Antibody | Abcam | ab140601 | Various other vendors |
| K63 Ubiquitin Antibody | Abcam | ab179434 | Various other vendors |
| KCl | Millipore Sigma | P9541 | Various other vendors |
| KONTES tissue grinder | VWR | KT885300-0002 | Various other vendors |
| Laemmli sample buffer | Bio-rad | 161-0737 | Various other vendors |
| Linear Ubiquitin Antibody | Life Sensors | AB-0130-0100 | Only M1 antibody |
| MgCl | Millipore Sigma | 442611 | Various other vendors |
| Microcentrifuge | Eppendorf | 2231000213 | Various other manufacturers/models |
| myr-AIP | Enzo Life Sciences | BML-P212-0500 | Carried by Millipore-Sigma |
| NaCl | Millipore Sigma | S3014 | Various other vendors |
| Odyssey Fc Imaging System | LiCor | 2800-02 | Other vendors carry different versions |
| Phosphatase Inhibitor | Millipore Sigma | 524625 | Various other vendors |
| Precision Plus Protein Standard | Bio-rad | 161-0373 | Various other vendors |
| Protease Inhibitor | Millipore Sigma | P8340 | Various other vendors |
| PSD95 antibody | Cell signaling | 3450T | Various other vendors |
| SDS | Millipore Sigma | L3771 | Various other vendors |
| Sodium hydroxide | Fisher | SS255 | Various other vendors |
| Sucrose | Millipore Sigma | S0389 | Various other vendors |
| TBS | Alfa Aesar | J62938 | Varous other vendors |
| Tris | Millipore Sigma | T1503 | Various other vendors |
| Tween-20 | Fisher | BP337-100 | Various other vendors |
| Ubiquitin Antibody | Enzo Life Sciences | BML-PW8810 | Various other vendors |
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