In vitro Reconstitution Assays of Arabidopsis 20S Proteasome
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Author:
Date:
2021-04-05
[Abstract] The majority of cellular proteins are degraded by the 26S proteasome in eukaryotes. However, intrinsically disordered proteins (IDPs), which contain large portions of unstructured regions and are inherently unstable, are degraded via the ubiquitin-independent 20S proteasome. Emerging evidence indicates that plant IDP homeostasis may also be controlled by the 20S proteasome. Relatively little is known about the specific functions of the 20S proteasome and the regulatory mechanisms of IDP degradation in plants compared to other species because there is a lack of systematic protocols for in vitro assembly of this complex to perform in vitro degradation assays. Here, we present a detailed protocol of in vitro reconstitution assay of the 20S proteasome in Arabidopsis by modifying previously ...
[摘要] [摘要]大多数细胞蛋白的s的降解通过26S在真核生物蛋白酶。但是,内在无序的蛋白质(IDPs)包含大量的非结构化区域,并且内在地不稳定,因此很容易通过不依赖泛素的20S蛋白酶体降解。越来越多的证据最近显示ň植物境内流离失所者的平衡也可以通过20S蛋白酶控制。但是,由于缺乏用于体外分离20S蛋白酶体和降解测定的系统协议,因此我们对植物中IDP和20S蛋白酶体降解的功能和调控机制的研究和理解一直处于婴儿期与其他生物。在这里,我们通过采用和修改先前公开的方法,对拟南芥中20S蛋白酶体进行体外重组测定的详细方案。在此获得20S核心蛋白酶体的主要策略是从26S蛋白酶体中去除19S调节亚基。该协议包括两个主要部分:1)的来自表达表位标记的PAG1稳定的转基因品系20S蛋白酶体亲和纯化,的20S蛋白酶(程序AD)的基本组成部分; 2 )体外20S蛋白酶体降解测定法(方法E)。我们预计该协议将提供一种简单有效的方法来研究体外20S蛋白酶体降解,并促进植物中蛋白质代谢的研究。
[背景]蛋白质的降解通常是通过真核生物中的蛋白酶体来实现的。整合的26S蛋白酶体由两个亚颗粒组成:一个或两个末端的19S调节颗粒(RP),用作蛋白酶体激活剂;和20S核心蛋白酶体(CP),执行降解过程。大多数真核蛋白被多聚泛素化并导入26S蛋白酶体进行降解。然而,含有固有蛋白质无序已发现的区域直接通过破坏一个由20S蛋白酶的泛素依赖性降解(本日产等人,2014) ...
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Polyamine Transport Assay Using Reconstituted Yeast Membranes
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Author:
Date:
2021-01-20
[Abstract] ATP13A2/PARK9 is a late endo-/lysosomal P5B transport ATPase that is associated with several neurodegenerative disorders. We recently characterized ATP13A2 as a lysosomal polyamine exporter, which sheds light on the molecular identity of the unknown mammalian polyamine transport system. Here, we describe step by step a protocol to measure radiolabeled polyamine transport in reconstituted vesicles from yeast cells overexpressing human ATP13A2. This protocol was developed as part of our recent publication (van Veen et al., 2020) and will be useful for characterizing the transport function of other putative polyamine transporters, such as isoforms of the P5B transport ATPases.
[摘要] [摘要] ATP13A2 / PARK9是一种晚期内/溶酶体P5B转运ATPase,与多种神经退行性疾病有关。我们最近将ATP13A2表征为溶酶体多胺出口者,这为未知的哺乳动物多胺转运系统的分子身份提供了线索。在这里,我们逐步描述了从过量表达人ATP13A2的酵母细胞中测量重组囊泡中放射性标记的多胺转运的方案。该方案是我们最新出版物的一部分(van Veen等,2020),将有助于表征其他假定的多胺转运蛋白的转运功能,例如P5B转运ATPase的同工型。
[背景] ATP13A2 / PARK9编码一种普遍表达的晚期内-/溶酶体膜蛋白,与一系列神经退行性疾病有关,例如早发性帕金森氏病(Di Fonzo等,2007 ;Lin等,2008)和Kufor -Rakeb综合征(伴痴呆的早期帕金森病)(Ramirez等,2006 ;Park等,2011)。ATP13A2属于P型转运ATPase ,是一类活性转运蛋白,由于ATP水解而暂时形成磷酸中间产物(Kuhlbrandt ,2004年)。ATP13A2是P5亚家族的成员,该家族已在20多年前通过基因组测序鉴定出来(Axelsen和Palmgren ...
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Isolating Multiple Extracellular Vesicles Subsets, Including Exosomes and Membrane Vesicles, from Bovine Milk Using Sodium Citrate and Differential Ultracentrifugation
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Author:
Date:
2020-06-05
[Abstract] Milk is a complex fluid that contains various types of proteins and extracellular vesicles (EVs). Some proteins can mingle with EVs, and interfere with their isolation. Among these proteins, caseins form micelles of a size comparable to milk EVs, and can thus be co-isolated with EVs. Preliminary steps that affect milk are crucial for EV isolation and impact the purity and abundance of isolated EVs. In the course of our previous works on cow’s milk EVs, we found that sodium citrate (1% final), which is a biocompatible reagent capable of breaking down casein micelles into 40-nm monomers, allowed the isolation of high quantities of EVs with low coprecipitation of caseins or other contaminating proteins. Using this protocol, we successfully separated different EV subsets, characterized in ...
[摘要] [摘要 ] 牛奶是一种复杂的流体,其中包含各种类型的蛋白质和细胞外囊泡(EVs),有些蛋白质会与EV混合在一起,并干扰其分离,在这些蛋白质中,酪蛋白形成的胶束大小与牛奶EV相当因此,可以将其与电动汽车共隔离。影响牛奶的初步步骤对于电动汽车的隔离,影响分离电动汽车的纯度和丰度至关重要。在我们以前对牛奶电动汽车的研究过程中,我们发现柠檬酸钠(最终含量为1% )是一种生物相容性试剂,能够将酪蛋白胶束分解为40 nm单体,可分离出大量的EV,而酪蛋白或其他污染蛋白的沉淀率却很低。 EV子集,深入表征其形态,蛋白质含量和小分子RNA富集模式。我们还能够描述其在小鼠肠道炎症模型中的生物学功能。具体来说,是从同一样品中分离出不同乳EV子集的培养基。更具体地说,我们着重介绍了使用柠檬酸钠作为标准化方法来分离和研究乳EV的方法及其在差分超速离心之外的分离技术的潜力。
[背景 ] 在我们以前的出版物(本穆萨等人。,2016 ,2017,2019b和2019c;本穆萨和普罗沃斯特,2019) ,我们强调,牛乳是包含细胞外无数的复杂流体囊泡。(EV)用的子集在这些之中,外泌体是多囊体(MVB)与细胞膜融合时释放的约100 nm囊泡,进行超速离心时,这些沉淀物的离心速度等于或高于100,000 xg (P100K,其中P代表沉淀)(Pieters 等。,2015) ...
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