Degradation Mechanism Of FBXO31 And Its Expression Profile Analysis

F-box protein FBXO31 is a senescence gene and a candidate breast cancer tumor suppressor, which associates with Skpl and Cullin to form a SCF complex and involves in ubiquitin-proteasome pathway. FBXO31 fulfill its cellular function such as proliferation,senescence,cell cycle surveillance by mediating its substrate degradation. One of the most important functions of FBXO31 had been unveiled. It can mediate cyclin D1 degradation to induce G1 arrest after DNA damage in melanoma cell line SK-MEL-28.FBXO31 located in human chromosome 16q24.2 between nt85974895-85920445. FBXO31 mRNA contains a 1,620-bp ORF that is transcribed from the genomic DNA with nine exons spanning～52kb. The FBXO31 transcript has an exceptionally short 23-base 5'-untranslated region. In silico analysis of the EST database and 5'rapid amplification of cDNA ends failed to identify any additional 5' transcript sequence. In addition, the presence of a 2.48-kb CpG island (66% G+C and with 9% CpG) encompassing the first 350-bp exon of FBXO31 also supports the 5'start of the transcript. FBXO31 encodes a 539-amino-acid protein with a predicted molecular mass of 61 kDa. The predicted amino acid sequence of FBXO31 does not have significant homology to known proteins, except for a consensus F-box domain at the amino terminus (Fig.1C). The 40-amino-acid F-box domain is involved in protein-protein interactions and is present in a large family of proteins (16). FBXO31 also contains six minimal (RxxL) destruction box (D-box) motifs that are hallmark of proteins degraded via the anaphase-promoting complex/cyclosome (APC/C).To identify degradation mechanism of FBXO31, the first thing to do is to identify the degradation pathway. Accumulated FBXO31 levels were found in Proteasome inhibitor MG132 treated Hella cells, which suggests that FBXO31 protein may degraded via ubiquitin-proteasome pathway. FBXO31 contains six minimal (RxxL) destruction box (D-box) motifs that are hallmark of proteins degraded via the anaphase-promoting complex/cyclosome (APC/C). FBXO31 endogenous expression fluctuated through cell cycle and at a maximum at late G2 to early G1 phase. The timing of FBXO31 destruction is consistent with APC-mediated degradation. All these findings suggest that FBXO31 degradation mediate by APC/C complex.To investigate whether FBXO31 degradation mediate by APC/C complex, Myc-FBX031,Flag-CDH1 and Flag-CDC20 expression plasmids were constructed. Coinmmunoprecipitation and western blot were used to identify interaction between FBXO31 and CDC20 or CDH1. Results showed that FBXO31 could bind CDH1. Decreased endogenous expression of FBXO31 were found when increase CDH1 expression in Hella cells. In vivo ubiquitination showed that CDH1 could mediate ubiquitination of FBXO31. All these findings suggest that CDH1 mediated FBXO31 degradation.To investigate which specific D-box motif could mediate FBXO31-CDH1 binding,6 D-box mutants were constructed using PCR. Coinmmunoprecipitation and western blot were used to identify interaction between D-box mutants and CDH1. Results showed that 5 D-box mutants could bind CDH1 except D-box mutant 5, which indicated that the fifth D-box motif may play an important role in FBXO31-CDH1 binding.We also investigated D-box mutation's effect on FBXO31- Skp1 binding. Results showed that D-box mutation did not abrogate FBXO31- Skp1 except the first D-box motif mutation, which suggest that the first D-box motif may play an important role in formation of SCFFBXO31 complex.Real-time reverse transcription-PCR and western blot were used to detect FBXO31 mRNA and protein expression levels in HCC cell lines and specimens. Results showed that FBXO31 expression levels were down-regulated in Hep3B, MHCC97L, MHCC97H, HCCLM3, SNU-449 cell lines, up-regulated in HepG2 and SNU-449 cell lines, compare to normal liver cell line H17. In 30/32 detected specimens, FBXO31 expression levels were down-regulated, compare to corresponding healthy tissues.Hep3B cell line was used to investigate the possibility that FBXO31 might function as a tumor suppressor in HCC. Ectopic expression of FBXO31 inhibited cell proliferation and induced G1 arrest in Hep3B cell line. Increased expression of FBXO31 did not induce apoptosis in Hep3B. Senescence did not detect in FBXO31 transfected cells. Decreased endogenous expression of cyclin D1 were found when increase FBXO31 expression in Hep3B cells. In conclusion, FBXO31 might function as a tumor suppressor in HCC by regulating cyclin D1 expression to induce G1 arrest.