| In vitro STING Activation with the cGAMP-STINGΔTM Signaling Complex
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Author:
Date:
2021-02-05
[Abstract] Activating the STING (stimulator of interferon genes) signaling pathway via administration of STING agonist cyclic GMP-AMP (cGAMP) has shown great promise in cancer immunotherapy. While state-of-the-art approaches have predominantly focused on the encapsulation of cGAMP into liposomes or polymersomes for cellular delivery, we discovered that the recombinant STING protein lacking the transmembrane domain (STINGΔTM) could be used as a functional carrier for cGAMP delivery and elicit type I IFN expression in STING-deficient cell lines. Using this approach, we generated anti-tumoral immunity in mouse melanoma and colon cancer models, providing a potential translatable platform for STING agonist-based immunotherapy. Here, we report the detailed in vitro STING activation ...
[摘要] [摘要]通过给予STING激动剂环状GMP-AMP（cGAMP）激活STING（干扰素基因的刺激物）信号通路已显示出在癌症免疫治疗中的广阔前景。尽管目前最先进的方法主要集中在将cGAMP封装进脂质体或聚合物小体中以进行细胞递送，但我们发现缺少跨膜结构域（STINGΔTM）的重组STING蛋白可以用作cGAMP递送的功能载体。在STING缺陷型细胞系中诱导I型IFN表达。使用这种方法，我们在小鼠黑素瘤和结肠癌模型中产生了抗肿瘤免疫力，为基于STING激动剂的免疫疗法提供了潜在的可翻译平台。在这里，我们报告与cGAMP-STINGΔTM复合物的详细体外STING激活方案，以帮助研究人员进一步开发这种方法。该协议还可以轻松扩展到与STING激活相关的其他应用程序，例如控制各种类型的感染。
[背景]在过去的二十年中，STING（干扰素基因的刺激物）信号传导途径已成为免疫系统的关键特征，并有望成为针对病毒和细菌感染，自身免疫性疾病和癌症的治疗靶标。因此，递送STING激动剂以增强免疫应答已经成为学术机构和制药公司的极大兴趣领域（Ohkuri等人，2017）。尽管现有的努力主要集中在开发合成运载工具上（Shae et ...
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| Non-radioactive Assay to Determine Product Profile of Short-chain Isoprenyl Diphosphate Synthases
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Author:
Date:
2021-01-05
[Abstract] Isoprenoids represent the largest class of metabolites with amazing diversities in structure and function. They are involved in protecting plants against pathogens or herbivores or involved in attracting pollinators. Isoprenoids are derived from geranyl diphosphate (GPP; C10), farnesyl diphosphate (FPP; C15), geranylgeranyl diphosphate (GGPP; C20), and geranylfarnesyl diphosphate (GFPP; C25) that are in turn formed by sequential condensations of isopentenyl diphosphate (IPP; C5) with an allylic acceptor such as dimethylallyl diphosphate (DMAPP; C5), GPP, FPP, or GGPP in a reaction catalyzed by isoprenyl diphosphate synthases (IDSs). IDS enzyme assay for determination of prenyl diphosphate products is generally performed ...
[摘要] [Abstrac吨]类异戊二烯代表最大的一类代谢物在结构和功能惊人多样性。它们参与保护植物免受病原体或草食动物侵害，或参与吸引传粉媒介。（;çGPP类异戊二烯是从牻牛儿基二磷酸衍生的10 ），法呢基二磷酸（FPP;Ç 15 ），香叶基香叶基二磷酸（GGPP;Ç 20 ），和geranylfarnesyl二磷酸（GFPP; C ^ 25 ），它们又通过的顺序缩合形成异戊烯基二甲基磷酸酯（IPP; C 5 ）与烯丙基受体，例如二磷酸二甲基烯丙酯（DMAPP; C 5），GPP，FPP或GGPP）由异戊二烯基二磷酸合酶（IDS）催化的反应。用于确定异戊二烯基二磷酸酯产物的IDS酶测定法通常是使用放射性标记的底物进行的，并且所形成的产物是通过使用昂贵的仪器（例如磷光成像仪，radio-GC或radio-HPLC）来鉴定的。尽管已经报道了一种用于测量粗植物提取物中IDS活性的非放射性测定方法，但它需要使用色谱结合串联质谱（LC / MS-MS）的复杂方法。在这里，我们描述了用于确定使用非放射性标记的IPP及其共同烯丙基底物DMAPP，GPP的IDS分析产物非放射性和简单廉价的测定法，和FPP。在测定中生成的异戊二烯基二磷酸产物的检测非常高效，并且在浓度大于40 µM的IPP和DMAPP / GPP / ...
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| Expression and Purification of Recombinant Skd3 (Human ClpB) Protein and Tobacco Etch Virus (TEV) Protease from Escherichia coli
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Author:
Date:
2020-12-05
[Abstract] Skd3 (encoded by human CLPB) is a mitochondrial AAA+ protein comprised of an N-terminal ankyrin-repeat domain and a C-terminal HCLR-clade nucleotide-binding domain. The function of Skd3 has long remained unknown due to challenges in purifying the protein to high quality and near homogeneity. Recently we described Skd3 as a human mitochondrial protein disaggregase that solubilizes proteins in the mitochondrial intermembrane space. This protocol overcomes the challenges associated with purifying Skd3 and allows for in depth in vitro study of Skd3 activity. Tobacco etch virus (TEV) protease is required in the purification of Skd3. Thus, we also describe how to purify high quality TEV protease for use in the purification of Skd3, other purification protocols, and in vitro assays requiring TEV ...
[摘要] [摘要] Skd3（由人类CLPB编码）是一种线粒体AAA +蛋白，由N末端锚蛋白重复域和C末端HCLR分支核苷酸结合域组成。由于在纯化蛋白质达到高质量和接近均质性方面的挑战，Skd3的功能长期未知。最近，我们描述Skd3作为人类线粒体蛋白disaggregase ，在线粒体膜间间隙增溶蛋白。该协议克服了与纯化Skd3相关的挑战，并允许对Skd3活性进行深入的体外研究。Skd3的纯化需要烟草蚀刻病毒（TEV）蛋白酶。因此，我们还描述了如何净化高质量TEV蛋白酶可用于纯化Skd3，其他纯化方案以及需要TEV蛋白酶的体外测定。
[背景] Skd3是一种线粒体AAA +蛋白，与多系统线粒体疾病VII型3-甲基谷氨酸酸尿症（MGCA7）有关（Capo-Chichi等人，2015； Kanabus等人，2015； Saunders等人，， 2015; Wortmann等人，2015 ; Kiykim等人，2016 ）。由于在体外研究Skd3的能力有限，因此对生物学功能和这些突变对Skd3活性的影响的研究仍难以捉摸（Cupo和Shorter，2020； ...
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