| Manganese Superoxide Dismutase Activity Assay in the Yeast Saccharomyces cerevisiae
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Author:
Date:
2020-03-05
[Abstract] Superoxide dismutases (SODs) act as a primary defence against reactive oxygen species (ROS) by converting superoxide anion radicals (O2-) into molecular oxygen (O2) and hydrogen peroxide (H2O2). Members of this enzyme family include CuZnSODs, MnSODs, FeSODs, and NiSODs, depending on the nature of the cofactor that is required for proper activity. Most eukaryotes, including yeast, possess CuZnSOD and MnSOD. This protocol aims at assessing the activity of the yeast Saccharomyces cerevisiae MnSOD Sod2p from cellular extracts using nitroblue tetrazolium staining. This method can be used to estimate the cellular bioavailability of Mn2+ as well as to evaluate the redox state of the cell.
[摘要] [摘要 ] 超氧化物歧化酶（SOD能）充当主防御针对反应性氧物质（ROS）通过转换的超氧阴离子自由基（O 2 - ）为分子氧（O 2 ）和过氧化氢（H 2 ? 2 ）。这种酶的家庭成员包括CuZnSODs ，MnSODs ，FeSODs 和NiSODs ，这取决于是需要适当的活动辅助因子的性质。大多数真核生物，包括酵母，都具有CuZnSOD 和MnSOD 。该协议旨在评估酵母的活性 使用硝基蓝四唑染色法从细胞提取物中提取酿酒酵母MnSOD Sod2p 。该方法可用于估计Mn 2+ 的细胞生物利用度以及评估细胞的氧化还原状态。
[背景 ] 的SODs被定义为减少正常有氧代谢为氧气和过氧化氢期间形成的氧的有害自由基含金属的抗氧化剂酶。：这些酶是基于需要作为辅因子进行适当的酶活性的金属分类CuZnSODs ，MnSODs ，FeSODs ，和NiSODs 。在酿酒酵母中，有两个S OD ：CuZn-Sod1p和Mn-Sod2p（Abreu和Cabelli ...
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| Measurement of CD74 N-terminal Fragment Accumulation in Cells Treated with SPPL2a Inhibitor
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Author:
Date:
2019-06-05
[Abstract] The recent discovery of human signal peptide peptidase-like 2a (SPPL2a) deficiency in humans revealed the toxicity associated with the accumulation of one of its substrates, CD74 N-terminal fragment (CD74-NTF), for certain type of dendritic cells (cDC2). We developed a two-step protocol for monitoring the accumulation of this molecule in different subsets of PBMCs and immortalized B cells, in which SPPL2a is chemically inhibited and CD74-NTF levels are then assessed by flow cytometry or western blotting. The chemical inhibition of SPPL2a has been described elsewhere, but this is the first time that this inhibition has been reported as a protocol.
[摘要] 最近发现的人类信号肽类肽2a (SPPL2a)缺失揭示了其底物之一CD74 n端片段(CD74- ntf)对某些树突状细胞(cDC2)积累的毒性。我们开发了一个两步方案来监测该分子在不同亚群的PBMCs和永生B细胞中的积累，其中SPPL2a被化学抑制，然后通过流式细胞术或western blotting评估CD74-NTF水平。SPPL2a的化学抑制已经在其他地方被描述过，但这是第一次将这种抑制作为一种方案被报道。
【背景】信号肽肽样2A (SPPL2a)是来自GxGD家族的跨膜细胞内蛋白酶(Voss et al.， 2013)。在人类和小鼠中，该酶的缺失导致其底物之一CD74 n端片段(CD74- ntf)在MHC ii类阳性细胞中积累(Beisner et al.， 2013;Bergmann 等。， 2013;Schneppenheim et al.， 2013;孔等。， 2018)。这种累积的CD74-NTF对小鼠B细胞具有选择性毒性(Beisner et al.， 2013;Bergmann 等。， ...
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| Protein Expression Protocol for an Adenylate Cyclase Anchored by a Vibrio Quorum Sensing Receptor
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Author:
Date:
2017-01-20
[Abstract] The direct regulation of a mycobacterial adenylate cyclase (Rv1625c) via exchange of its membrane anchor by the quorum sensing receptor CqsS (Vibrio harveyi) has recently been reported (Beltz et al., 2016). This protocol describes the expression and membrane preparation for these chimeric proteins.
[摘要] 最近报道了通过其群岛感应受体CqsS（Vibrio harveyi）交换其膜锚定点直接调节分枝杆菌腺苷酸环化酶（Rv1625c）（Beltz等，2016）。 该方案描述了这些嵌合蛋白的表达和膜制备。
【背景】膜分隔的哺乳动物腺苷酸环化酶（AC）是IIIa类AC。调节是间接通过第一信使细胞外刺激G蛋白偶联受体（GPCR）而在细胞内释放的刺激（或抑制性）Gα-蛋白。 AC产生使用ATP作为底物的通用第二信使cAMP。脊椎动物AC中两个六角膜结构域的大小远远超过了对简单膜锚固的要求。然而，这种内在的膜锚定/受体结构域的调节特征是未知的。为了研究潜在的功能，选择了可以容易地在细菌中表达的分类细菌的典型类IIIa AC Rv1625c（Guo等人，2001和2005）与哺乳动物AC同工型相反。我们用来自哈维氏弧菌CqsS的六价体积感知（QS）受体的受体结构域取代了Rv1625c AC的六角膜锚，以检查我们是否可以通过QS配体的霍乱自动诱导剂直接调节AC -1'，CAI-1。 QS受体和IIIa类膜锚的设计是非常相似的，即最小的跨膜α-螺旋和极短的连接环。
我们已经证明了通过细胞外信号直接调节IIIa ...
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