Real-time imaging of HIF-1alpha stabilization and degradation.
Real-time imaging of HIF-1alpha stabilization and degradation.
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HIF-1Alpha稳定和降解的实时成像。
DOI：
10.1371/journal.pone.0005077
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发表时间：
2009
期刊：
影响因子：
3.7
通讯作者：
Serganova I
中科院分区：
文献类型：
--
作者：
Moroz E;Carlin S;Dyomina K;Burke S;Thaler HT;Blasberg R;Serganova I
与正常组织相比，HIF-1α在许多人类癌症中过表达，这是由于反映特定遗传改变和细胞外刺激的多种因素和途径的相互作用。本研究建立了HIF-1α/FLuc和HIF-1α（ΔODDD）/FLuc两种报告基因系统，用于研究正常细胞（NIH 3 T3和HEK 293）和胶质瘤细胞（U87）中HIF-1α蛋白的表达水平。这些报告系统提供了一个机会，研究HIF-1α在不同的细胞系，无论是在培养和异种移植的降解。使用免疫荧光显微镜，我们观察到HIF-1α/FLuc融合蛋白在正常细胞和癌细胞之间的不同亚细胞定位模式;在未转导的野生型细胞中观察到HIF-1α的类似差异。在不同条件下（常氧、CoCl 2处理和缺氧），NIH 3 T3和HEK 293细胞中观察到融合蛋白和HIF-1α的动态胞质-核交换。相比之下，U87细胞表现出更持久的核定位模式，受不同生长条件的影响较小。采用蛋白质降解的动力学模型，我们能够区分HIF-1α/FLuc蛋白质降解的两个组分，并定量HIF-1α融合蛋白的半衰期。模拟缺氧的CoCl 2、MG 132处理和ODD结构域的缺失消除了快速清除组分（t1/2 = 4-6 min），并反映了氧/VHL依赖性降解途径。缓慢清除组分（t1/2 ≤ 200 min）与其他未鉴别的非氧/VHL依赖性降解途径一致。总之，HIF-1α/FLuc体外和体内稳定性的连续生物发光读数将促进影响HIF-1α稳定性和蓄积的治疗方法的开发和验证。
HIF-1α is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1α chimeric reporter systems, HIF-1α/FLuc and HIF-1α(ΔODDD)/FLuc, to investigate the tightly controlled level of HIF-1α protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1α in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1α/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1α in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1α was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl2 treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1α/FLuc protein degradation and quantify the half-life of HIF-1α fusion proteins. The rapid clearance component (t1/2 ∼4–6 min) was abolished by the hypoxia-mimetic CoCl2, MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t1/2 ∼200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1α/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1α.
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影响因子：
56.9
作者：
Jaakkola, P;Mole, DR;Ratcliffe, PJ
通讯作者：
Ratcliffe, PJ
影响因子：
4.8
作者：
Isaacs, JS;Jung, YJ;Neckers, LM
通讯作者：
Neckers, LM
影响因子：
11.5
作者：
Creighton-Gutteridge, Mark;Cardellina, John H., II;Melillo, Giovanni
通讯作者：
Melillo, Giovanni
DOI：
10.1093/protein/14.8.529
发表时间：
2001-08-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
Arai, R;Ueda, H;Nagamune, T
通讯作者：
Nagamune, T
影响因子：
5.6
作者：
Di Cristofano, Claudio;Minervini, Andrea;Cavazzana, Andrea
通讯作者：
Cavazzana, Andrea