In vitro AMPylation Assays Using Purified, Recombinant Proteins
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Author:
Date:
2017-07-20
[Abstract] Post-translational protein modifications (PTMs) orchestrate the activity of individual proteins and ensure their proper function. While modifications such as phosphorylation or glycosylation are well understood, more unusual modifications, including nitrosylation or AMPylation remain comparatively poorly characterized. Research on protein AMPylation–which refers to the covalent addition of an AMP moiety to the side chains of serine, threonine or tyrosine–has undergone a renaissance (Yarbrough et al., 2009; Engel et al., 2012; Ham et al., 2014; Woolery et al., 2014; Preissler et al., 2015; Sanyal et al., 2015; Truttmann et al., 2016; Truttmann et al., 2017). The identification and characterization of filamentation ...
[摘要] 翻译后蛋白质修饰(PTM)协调各种蛋白质的活性并确保其功能正常。虽然诸如磷酸化或糖基化的修饰被很好地理解,但是更不寻常的修饰,包括亚硝基化或AMP化仍然比较差的表征。关于蛋白质AMP化的研究 - 其是将AMP部分共价加成到丝氨酸,苏氨酸或酪氨酸的侧链,已经经历了复兴(Yarbrough et al。,2009; Engel et al。 2012年; Ham等人,2014年; Woolery等人,2014年; Preissler等人,2015年; ; Sanyal等人,2015; Truttmann等人,2016; Truttmann等人,2017)。鉴定和表征含丝状(fic)结构域的AMPylases引起了对该PTM的新兴趣(Kinch等人,2009; Yarbrough等人,2009)。基于最近的体内和体外研究,我们现在知道分泌的细菌AMPylase共价连接AMP到Rho家族GTP酶的成员,而后生动物AMPylases修饰HSP70家族蛋白在细胞质和内质网(ER)(Itzen等人,2011; Hedberg和Itzen,2015; Truttmann和Ploegh,2017)。认为AMP化学将HSP70置于不能参与蛋白质重折叠反应的引发剂但瞬时失活的状态(Preissler等人,2015)。 ...
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Detection of ASC Oligomerization by Western Blotting
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Author:
Date:
2017-05-20
[Abstract] The apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) adaptor protein bridges inflammasome sensors and caspase-1. Upon inflammasome activation, ASC nucleates in a prion-like manner into a large and single platform responsible for the recruitment and the activation of caspase-1. Active caspase-1 will in turn promote the proteolytic maturation of the pro-inflammatory cytokine IL-1β. ASC oligomerization is direct evidence for inflammasome activation and its detection allows a read-out independent of caspase-1 and IL-1β. This protocol describes how to detect the oligomerization of ASC by Western blot.
[摘要] 具有半胱天冬酶募集区(ASC)衔接蛋白的凋亡相关斑点样蛋白桥联炎症体传感器和半胱天冬酶-1。在炎性体活化后,ASC以类似朊病毒的方式成核,成为负责募集和激活半胱天冬酶-1的大而单一的平台。活性胱天蛋白酶-1将反过来促进促炎细胞因子IL-1β的蛋白水解成熟。 ASC寡聚化是炎性体激活的直接证据,其检测允许读取与caspase-1和IL-1β无关。该方案描述了如何通过蛋白质印迹检测ASC的寡聚化。
背景 Inflammasomes是大量的多蛋白平台,其感测各种微生物,内源和环境胁迫因子,导致促炎IL-1细胞因子家族的成熟(Martinon等人,2002; Sharma和Kanneganti, 2016)。激活后,炎性细胞传感器通过pyrin结构域(PYD)-PYD同型相互作用募集衔接蛋白ASC。 ASC通过胱天蛋白酶激活和募集域(CARD)-CARD相互作用又结合半胱天冬酶-1,并有利于caspase-1的自我蛋白水解切割,导致IL-1β和IL-18的成熟(Hoss等人。,2016)。 Inflammasome激活引发ASC二聚体的超分子寡聚化成称为“ASC-specks”或“pyroptosome”(Fernandes-Alnemri等人,2007)的大交织原纤维。 ASC-speck / ...
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Measurements of Proline and Malondialdehyde Content and Antioxidant Enzyme Activities in Leaves of Drought Stressed Cotton
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Author:
Date:
2016-09-05
[Abstract] Drought stress negatively affects cotton plant growth and induces various biochemical and physiological responses in cotton plants. Proline content and antioxidant enzymes are thought to be associated with maintaining the structure of cellular components or with protecting cellular function. Study of cotton plant responses towards drought stress and investigation of the mechanism of drought tolerance are helpful to develop drought tolerant cotton plants. Here, we describe a protocol to investigate cotton plant response towards drought stress through measurements of biochemical parameters including antioxidant enzyme activities, proline content and malondialdehyde (MDA) content.
[摘要] 干旱胁迫对棉花植物生长有不利影响,并在棉花植物中诱导各种生化和生理反应。 脯氨酸含量和抗氧化酶被认为与保持细胞组分的结构或保护细胞功能相关。 棉花植物对干旱胁迫的反应研究和对干旱耐受机制的研究有助于开发耐旱棉花植物。 在这里,我们描述一个协议调查棉花植物对干旱胁迫的反应,通过测量的生化参数,包括抗氧化酶活性,脯氨酸含量和丙二醛(MDA)含量。
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