1. E2-EPF UCP regulates stability and functions of missense mutant pVHL via ubiquitin mediated proteolysis
- Author
-
Hee Won Shin, Cho Rok Jung, Kyeong Su Park, Kyung-Sook Chung, Dong-Soo Im, Ju Hee Kim, and Jung Hwa Lim
- Subjects
Cancer Research ,VHL disease ,Mutant ,Mutation, Missense ,Mice, Nude ,pVHL missense mutation ,E2-EPF UCP ,Ubiquitination ,Proteininstability ,Ubiquitin-conjugating enzyme ,Protein instability ,Mice ,Ubiquitin ,Genetics ,Animals ,Humans ,Missense mutation ,Mice, Inbred BALB C ,biology ,Protein Stability ,Cell growth ,HEK 293 cells ,Wild type ,Xenograft Model Antitumor Assays ,Molecular biology ,Ubiquitin ligase ,HEK293 Cells ,Oncology ,Von Hippel-Lindau Tumor Suppressor Protein ,Proteolysis ,Ubiquitin-Conjugating Enzymes ,biology.protein ,Female ,HeLa Cells ,Research Article - Abstract
Background 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. 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. Results 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. Conclusion 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. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1786-8) contains supplementary material, which is available to authorized users.
- Full Text
- View/download PDF