title: Screening and verification of proteins that interact with HSPC238.
abstract: HSPC238 is a recently identified tumor suppressor and demonstrates ubiquitin ligase E3 enzyme activity. HSPC238 was found to be significantly downregulated in human hepatocellular carcinoma (HCC) in vivo and to inhibit the proliferation and invasion of hepatoma cells in vitro; however, the underlying molecular mechanism is largely unknown. In the present study, we screened for and identified proteins that physically interact with HSPC238. A bait vector for yeast two-hybrid was constructed with human HSPC238 gene cDNA. Yeast two-hybrid screening was performed using a human fetal liver cDNA library. Multiple reporter gene assays, DNA sequencing and BLAST comparison analysis were performed on positive clones. Protein interaction of screened candidates with HSPC238 was further validated by confocal microscopy, co-immunoprecipitation and pull-down assays. Yeast two-hybrid screening demonstrated 124 positive clones. Multiple reporter gene assays with LacZ, HIS and ADE2 selective media identified 12 genes. Further co-localization, co-immunoprecipitation and pull-down assays demonstrated that HMOX1, RPS27A, ubiquitinB and MT2A interacted with HSPC238. These four proteins are involved in tumor development and progression, and are associated with the ubiquitin-proteasome pathway. Our results suggest that HSPC238 may play a tumor suppressor role and interact with these proteins via the ubiquitin-proteasome pathway. The identification and validation of proteins interacting with HSP238 may lead to the discovery of novel mechanisms through which HSPC238 suppresses tumorigenesis in human hepatocellular carcinoma.
authors.affiliation: Central Intensive Care Unit, Bo'ai Hospital of Zhongshan City Affiliated with Southern Medical University, Zhongshan 528403, P.R. China.
authors.forename: Jia-Yu
authors.initials: JY
authors.lastname: Tan
authors.affiliation: Prenatal Diagnosis Center, Bo'ai Hospital of Zhongshan City Affiliated with Southern Medical University, Zhongshan 528403, P.R. China.
authors.forename: Jing-Lin
authors.initials: JL
authors.lastname: Chen
authors.affiliation: Prenatal Diagnosis Center, Bo'ai Hospital of Zhongshan City Affiliated with Southern Medical University, Zhongshan 528403, P.R. China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Huang
authors.affiliation: Department of Laboratory Medicine, Bo'ai Hospital of Zhongshan City Affiliated with Southern Medical University, Zhongshan 528403, P.R. China.
authors.forename: Chun-Lei
authors.initials: CL
authors.lastname: Yuan
mesh_terms: D063646:Carcinogenesis / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000378:metabolism / Q000473:pathology; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000378:metabolism
chemical_list: D009363:Neoplasm Proteins; C547603:RNF181 protein, human; D044767:Ubiquitin-Protein Ligases