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对细胞中的FIH活性进行了基于氧体和报告基因的分析

Yulia L Volkova¹, Agnieszka E Jucht¹, Carsten C Scholz²

¹Institute of Physiology, University of Zurich, Zurich, Switzerland.

Methods in molecular biology (Clifton, N.J.)

|February 6, 2024

概括

这项研究引入了测量细胞中抑制因子HIF (FIH) 酶活性的新方法. 这些测试可以帮助开发新的治疗方法,用于与低氧相关的疾病,而不会影响红细胞的生产.

科学领域:

生物化学生化学
细胞生物学细胞生物学
身体生理学身体生理学

背景情况:

细胞对缺氧 (缺氧) 的反应对正常生理和疾病至关重要.
低氧诱导因子 (HIF) 是由氧气传感器调节的,包括prolyl-4-hydroxylase域 (PHD) 蛋白质和抑制因子HIF (FIH).
FIH调节HIF和其他蛋白质,如OTUB1,但其更广泛的生理作用尚未完全理解.

研究的目的:

开发和介绍评估FIH细胞内酶活性的协议.
为了使FIH活动在HIF依赖和独立的路径中进行调查.
促进在各种 (病理) 生理条件下研究FIH以及对新型抑制剂的评估.

主要方法:

开发一种依赖于HIF的火虫 luciferase-reporter基因试验,以测量FIH活动.
建立一种依赖氧基的测定方法,使用由FIH形成的依赖氧基的蛋白质寡合体.
验证这些方法分析FIH在纤维素中的酶活性.

主要成果:

描述了评估细胞FIH酶活性的两种不同的可靠方法.
这些分析允许对FIH活动对HIF和非HIF目标进行分析.
氧合物分析提供了FIH活动的直接和易于使用的读数.

结论:

开发的测试为研究FIH (病理) 生理学提供了有价值的工具.
了解FIH活动对于开发针对性治疗缺氧相关疾病至关重要.
这些方法可以帮助发现新的治疗策略,避免有害的副作用,如增加红细胞的生产.

关键词:

阿斯巴拉金的基化.第五百零五章没有了在FIH-1中,FIH-1被认为是FIH-1.抑制HIF的因素可以抑制HIF.氧化酶是一种氧化酶.氧气过低是因为缺氧.抑制剂的抑制剂路西法拉斯就是路西法拉斯.氧化物的氧化物.

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Reporter Genes

Reporter Genes

Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.

FISH - Fluorescent In-situ Hybridization

FISH - Fluorescent In-situ Hybridization

Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in cytogenetics. Labeled probes are used to bind complementary DNA or RNA sequences on a chromosome or in a region within a cell. Earlier, the probes could only be obtained by cloning or reverse transcription of a DNA template. Currently, the probe oligonucleotides can be synthesized synthetically. Additionally, with the advancement of optical techniques,...