Regulation Of EMT Factor Twist1 By HUWE1 And Role Of The HUWE1 Homolog CG8184 In Drosophila Melanogaster Development

Ubiquitin-proteasome system(UPS)is a major pathway for selective degradation of the most of cellular proteins.HUWE1,an conserved HECT E3 ligase,ubiqutinates many vital proteins such as p53,Mcl-1,Histones,N-Myc and HDAC2,and so on.HUWE1 takes part in series of important physiological functions,including apoptosis,spermatogenesis,hemidesmosome maturation and neuron precursor differentiation.Epithelial-mesenchymal transition(EMT)takes place as epithelial cells lose celluar tight junctions,transform into fibroblast phenotype and metastasize.Cancer cells utilize EMT to metastasize,which is the main cause of cancer lethality.The bHLH transcription factor Twist1 plays a key role in EMT induction,and overexpression of Twistl often leads to cell malicious transforming.Twist1 also functions during organism development as Twist is indispensible for mesodermal induction and somites and muscle formation in Drosophila melanogaste.It was reported that Twist1 degraded by F-box E3 ligase complex SCFPPA and SCF?-TRCP.In here,we found E3 liagse HU WE1 could bind and ubiquitinate Twistl to promote its degradation,regulating its transcriptional actvities.We found overexpression of HUWE1 prompted ectopic Twistl degradation and proved that HUWE1 could ubiquitinate Twistl.We detected HUWE1 interacted with both of Twistl and Twist2,HU WEI bound to Twistl through its C-trminal fragment.At the same time,the C-terninal fragment of CG8184,the Drosophila homolog of HUWE1,is proved to interact with Drosophila Twist.Gene silence of huwe1 enhanced the mobility of HeLa cells and increased Twistl protein level.Quantative RT-PCR analysis revealed that E-Cad mRNA levels decreased as mRNA levels of genes including N-Cad increased,Twist1 mRNA levels were not affected.It was indicated that HUWE1 modulated transcriptional activity of Twistl via Twistl degradation.Immunofluorescent imaging illustrated that ectopic Twistl located in nucleus,however,ectopic Twistl was redistributed to plasma upon HU WE1 coexpression and colocated with HUWE1,it was that HUWE1 probably arrested Twist1 in plasma and degraded it.In order to investigate the effects of CG8184 expression levels on Twist transcriptional activity and Drosophila development,we constructed CG8184 gene knock-out Drosophila melanogaster strains.We observed abnormal eyes,or bristles,ovaries and imaginal discs in these Drosophilas,and the Drosophilas had low viability.We proceeded to quantative RT-PCR analysis of CG8184 gene knock-out Drosophila and found that E-Cad mRNA levels decreased,mRNA levels of genes including N-Cad increased.In addition,quantative RT-PCR analysis of CG8184 gene overxpressing Drosophila also approved the conclusion.Ubiquitin ligase HUWE1 and EMT factor Twist1 are both evolutionarily conserved and play vital roles in cancer signaling pathways and organism developmental progress.This research proved that HUWE1 regulated Twistl transcriptional activity through affecting Twist1 proein levels,and it would uncover a new research perspect of cancer metastasis and organism development.