E2-EPF UCP regulates stability and functions of missense mutant pVHL via ubiquitin mediated proteolysis.
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.


METHODS
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. Anticancer 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.


CONCLUSIONS
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.
Gene Therapy Research Unit, KRIBB, Daejeon, Republic of Korea. Park-pksu@equisnzaroo.com.|EQUISnZAROO R&D center, Gyeonggi-do, Republic of Korea. Park-pksu@equisnzaroo.com.
Kyeong-Su
KS
Park
Gene Therapy Research Unit, KRIBB, Daejeon, Republic of Korea. Kim-wooahaha@kribb.re.kr.|University of Science and Technology, Daejeon, Republic of Korea. Kim-wooahaha@kribb.re.kr.
Ju Hee
JH
Kim
Gene Therapy Research Unit, KRIBB, Daejeon, Republic of Korea. Shin-shinhw@kribb.re.kr.|University of Science and Technology, Daejeon, Republic of Korea. Shin-shinhw@kribb.re.kr.
Hee Won
HW
Shin
Genome research center, KRIBB, Daejeon, Republic of Korea. Chung-kschung@kribb.re.kr.|University of Science and Technology, Daejeon, Republic of Korea. Chung-kschung@kribb.re.kr.
Kyung-Sook
KS
Chung
Gene Therapy Research Unit, KRIBB, Daejeon, Republic of Korea. Im-imdongsu@kribb.re.kr.
Dong-Soo
DS
Im
Gene Therapy Research Unit, KRIBB, Daejeon, Republic of Korea. jhwa@kribb.re.kr.
Jung Hwa
JH
Lim
Gene Therapy Research Unit, KRIBB, Daejeon, Republic of Korea. crjung@kribb.re.kr.|University of Science and Technology, Daejeon, Republic of Korea. crjung@kribb.re.kr.
Cho-Rok
CR
Jung
D000818:Animals; D005260:Female; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D020125:Mutation, Missense / Q000502:physiology*; D055550:Protein Stability; D059748:Proteolysis; D025801:Ubiquitin / Q000502:physiology*; D044763:Ubiquitin-Conjugating Enzymes / Q000502:physiology*; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics* / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays / Q000379:methods
D025801:Ubiquitin; C544962:Ube2S protein, human; D044763:Ubiquitin-Conjugating Enzymes; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human