E2-EPF UCP regulates stability and functions of missense mutant pVHL via ubiquitin mediated proteolysis.
E2-EPF UCP regulates stability and functions of missense mutant pVHL via ubiquitin mediated proteolysis.
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DOI：
10.1186/s12885-015-1786-8
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发表时间：
2015-10-26
期刊：
影响因子：
3.8
通讯作者：
Jung CR
中科院分区：
文献类型：
--
作者：
Park KS;Kim JH;Shin HW;Chung KS;Im DS;Lim JH;Jung CR
VHL基因的错义突变在2型VHL疾病中是常见的，并与pVHL广泛的功能和稳定性有关。某些突变的pVHL保留了调节HIF的能力，但由于不稳定而失去了功能。在这种情况下，可以假定调节突变pVHL的降解为治疗方法。在HEK 293 T瞬时表达细胞和786-O稳定细胞系中测定错义突变pVHL的稳定性和细胞功能。在体外系统中进行突变VHL蛋白的泛素化测定。采用小鼠异种移植试验评价腺病毒介导的shUCP表达的抗痤疮效果。三个VHL错义突变体（V155 A、L158 Q和Q164 R）在体外被E2-EPF UCP（UCP）直接泛素化。突变型pVHL在细胞中比野生型更不稳定。错义突变体pVHL在体外和细胞系统中与UCP直接相互作用。缺乏pVHL的所有赖氨酸残基导致对泛素化的抗性，从而增加其稳定性。错义突变体pVHLs在体外保持了E3连接酶泛素化HIF-1α的功能。与表达野生型的细胞中的水平相比，在表达突变型pVHL的细胞中，Glut-1和VEGF相对上调。UCP的耗尽恢复错义突变体pVHL水平并抑制细胞生长。腺病毒介导的shUCP RNA递送在离体小鼠异种移植模型中抑制肿瘤生长。这些数据表明，靶向UCP可以成为治疗由不稳定但功能性错义突变pVHL引起的2型VHL疾病的方法之一。本文的在线版本（doi：10.1186/s12885-015-1786-8）包含补充材料，可供授权用户使用。
Missense mutation of VHL gene is frequently detected in type 2 VHL diseases and linked to a wide range of pVHL functions and stability. Certain mutant pVHLs retain ability to regulate HIFs but lose their function by instability. In this case, regulating of degradation of mutant pVHLs, can be postulated as therapeutic method. The stability and cellular function of missense mutant pVHLs were determine in HEK293T transient expressing cell and 786-O stable cell line. Ubiquitination assay of mutant VHL proteins was performed in vitro system. Anticacner effect of adenovirus mediated shUCP expressing was evaluated using ex vivo mouse xenograft assay. Three VHL missense mutants (V155A, L158Q, and Q164R) are directly ubiquitinated by E2-EPF UCP (UCP) in vitro. Mutant pVHLs are more unstable than wild type in cell. Missense mutant pVHLs interact with UCP directly in both in vitro and cellular systems. Lacking all of lysine residues of pVHL result in resistance to ubiquitination thereby increase its stability. Missense mutant pVHLs maintained the function of E3 ligase to ubiquitinate HIF-1α in vitro. In cells expressing mutant pVHLs, Glut-1 and VEGF were relatively upregulated compared to their levels in cells expressing wild-type. Depletion of UCP restored missense mutant pVHLs levels and inhibited cell growth. Adenovirus-mediated shUCP RNA delivery inhibited tumor growth in ex vivo mouse xenograft model. These data suggest that targeting of UCP can be one of therapeutic method in type 2 VHL disease caused by unstable but functional missense mutant pVHL. The online version of this article (doi:10.1186/s12885-015-1786-8) contains supplementary material, which is available to authorized users.