Screening The Interaction Proteins Of CITED1by Yeast Two-hybrid System

BACKGROUNDOsteoporosis (OP) is a systemic bone disease, which is characterized by bone mass reduced and bone micro-structural damaged. Osteoporosis increased bone fragility and was more vulnerable to bone fractures at the same time (World Health Organization, WHO). In2001, the concept was presented by U.S. National Institutes of Health (NIH):osteoporosis is a skeletal system disease featured by decreased bone strength and increased fracture risk. The key concept includes two aspects:bone mineral density and bone quality.A recent U.S. survey showed that10million individuals over the age of50years-8million women and2million men-are estimated to already have the disease. As the population ages, numbers suffered from osteoporosis are expected to increase to14million by2020. Economic losses caused by osteoporosis in the United States were estimated to be betweens13.7billion and$20.3billion in2005. Also, it is projected that the expenditure will up to$25.3billion per year by2025. An epidemiological survey in China found that the rate of person suffering from osteoporosis were16.1%over the age of40, the incidence of osteoporosis is gradually increased with age, the rate in people over the age of60was22.6%, the rate is up to50%in those greater than80. As the population ages, osteoporosis disease and its complications have become a hot topic, which poses a serious threat on the health and life of the elderly population.Currently, drugs used in the treatment of osteoporosis mainly included:bone resorption inhibitor, bone formation accelerator and increasing bone mineralization drugs and Tranditional China Medicine drugs. Calcium and vitamin D is the basis of anti-osteoporosis therapy. The main treatment of osteoporosis is antiresorptive drugs, such as bisphosphonates, calcitonin, estrogen and estrogen receptor modifier. PTH (parathyroid hormone) is an important drug which increased bone mass by increasing bone mass. PTH increases bone mass when administered intermittently at appropriate doses, whereas continuous exposure to high concentrations of PTH accelerates bone turnover and sustains bone resorption and bone loss, More detailed understanding of the molecular mechanisms of PTH action on bone metabolism is of great interest, and it would be particularly advantageous to learn whether it is possible to maximize the anabolic and minimize catabolic effects of the PTH.Our results indicate that expression of the transcriptional cofactor CITED1(Cbp/p300-interacting transactivator with Glutamic Acid/Aspartic Acid-rich carboxy-terminal domain-1) is significantly increased in osteoblastic cells during brief exposure to PTH, and showed a dose and time dependent relationship. Further research suggests that CITED1inhibits osteoblast mineralization. PTH appears to be mediated mainly via cAMP/PKA signaling, while CITED1may be one component of an autoregulatory feedback loop that serves to restrain the extent of PTH regulation of osteoblastic function. CITED1as a potential pharmacologic have potential in the treatment of bone disease. However, the molecular mechanisms of CITED1and it’s role on PTH are still unclear.To understand the role of CITED1in osteoblasts and in their functional response to PTH, the yeast two-hybrid system was used to screening protein interacted with the transcriptionalcofactor CITED1in this study. On one hand, it is helpful to research the mechanism of its action. On the other hand, CITED1inhibits cAMP/PKA sig-naling and may serve as one component of an autoregulatory feedback loop for cAMP/PKAsignaling. The antagonistic peptide of CITED1can serve as adjuvant to PTH, which will optimize osteogenesis produced by PTH.CITED1, formerly MSG1(melanocyte-specific gene, melanocyte-specific gene1), was originally found in mouse melanoma cell lines, and identified to participate in the formation of melanin. CITED1binds CBP/p300to form a complex, enhancing transcription induced by TGF-β.The response is suppressed by heat-shock cognate protein70, possibly via a competition mechanism. CITED1also binds the function domain of the liganded estrogen receptors-a and-β and enhances estrogen-dependent gene transcription. CITED1functions as a cellcycle-dependent transcriptional cofac-tor whose activity is regulated by phosphorylation.The absence of CITED1gene expression was associated with increased osteoblastic differentiation. CITED1knockout osteoblasts can expressed higher bone marker gene relative to wild-type osteoblasts. G1R19(1-28) is a cAMP/PKA signaling pathway selective PTH analog material, it significantly enhanced ability to promote osteoblast differentiation after CITED1knockout. In vivo results showed CITED1KO mice have higher bone density in5weeks, but have lower bone mass in12weeks. CITED1may play a role in regulating the metabolism of bone.The system of yeast two-hybrid based on the regulation of eukaryotic transcription initiation process. Transcriptional activator is the componentin structure (MODULAR), including two (or more) relatively independent domain:the DNA binding domain (binding domain, BD) and transcriptional activation domain (activition domain, AD). The connection between two domains is Key to transcriptional activation, butany of the two domains alone is unable to activate the transcription process. Prey (prey) protein and the gene encoding the sequence of the AD combined bait (bait) protein and the BD formed fusion gene separately. When the two fusion plasmids, prey-AD and bait-BDfusion protein, were transfected into Y187and Y2H yeast cells respectively. If the bait protein interacted with the target protein in the nucleus, AD and BD will be closed to form a transcription factor, and then combined with the upstream activating sequence (upstream activating sequence, UAS) to activate the corresponding report gene (report gene) transcription. Conversely, it can be judged whether the interaction between the target protein and the bait proteins by the expression of a reporter gene or not. Analysis of protein-protein interactions can be realized by detecting the expression of reporter gene.Currently, the technology of yeast two-hybrid have increasingly improved. It would be particularly helpful to learn function of CITED1and the molecular mechanisms of PTH action on bone metabolism.Purpose1.To construct a cDNA library of neonatal rat osteoblasts by homologous recombination method.2.Construct PGBKT7-CITED1△CR2plasmid and verify its toxicity and self-activated, to lay the foundation for screening interaction protein of CITED1.3.To screening protein interacted with PGBKT7-CITED1△CR2plasmid from neonatal rat osteoblast cDNA library using yeast two-hybrid system, it will be helpful for researching function of CITED1.Materials and Methods1.Neonatal rat osteoblasts RNA was extracted using TRIzol method. Synthesis double-stranded cDNA by reverse transcription according to the SMART cDNA Library Construction Kit (CLONTECH) manual, then remove a short segment using Spin Column, and finally double-stranded cDNA and PGADT7-rec carrier were co-transformed into yeast cells Y187by homologous recombination method.2.Digested the recombinant plasmid CITED1△CR2and the expression vector pGBKT7with EcoR I and BamH I, then electrophoresis, agarose gel extraction, connection were done, and then plasmid was transformed into E. coli DH5a, screened positive bacterial clones and extracted plasmid. PCR was used to identified if plasmid construct was right. Recombinant pGBKT7-CITED1△CR2and empty vector pGBKT7were transformed into competent yeast Y2H separately, then observed its growth on SD/-Trp plates; the recombinant pGBKT7-CITED1△CR2was transformed to competent yeast Y2H, observed its color change and colonies on SD/-Trp, SD/-Trp/X-a-Gal/AbA plates.3.The pGBKT7-CITED1△CR2bait vector was transformed into Y2H yeast cells, and then the neonatal rat osteoblast cDNA library was transformed into Y187yeast cells, screening if there exists protein interacted with pGBKT7-CITED1△CR2by hybridization. The interaction between PIK3C2A and CITED1was verified by Co-transformation experiments and co-immunoprecipitation experiments.Result1.Total RNA absorptiometry ratio A260/A280was1.99, indicating that the purity of the extracted RNA was fine; Gel electrophoresis results suggest that the band luminance of28S is as higher as two times of18S and the concentration of total RNA was780ng/ul. The results show that the quality of total RNA is fine. The long-range PCR product was identified by1.0%agarose gel electrophoresis, cDNA fragment elongated waterfall-like, mainly ranged from500bp to4500bp. cDNA fragments is concentrated in the500-4500bp after removal of short segments, mainly ranged from1500bp to4500bp. The conversion efficiency of library was3.27*107, the titer was1.68x107cfu/mL.16colonies were identified with1.0%agarose gel electrophoresis, the size of inserted fragments were range from1.5kb to4.01Kb and the average length was approximately2.5kb, the rate of recombinant was100%.2.pGBKT7-CITED1△CR2plasmid was transformed into competent yeast Y2H by the lithium acetate method and screened positive clones on SD/-Trp agar plates. The positive clones picked were identified with the method of PCR using the of pGBKT7vector primers. The result showed that pGBKT7-CITED1△CR2plasmid was constructed successfully. Then plasmid and vector were transformed into yeast separately and cultured3days, the colony of the plasmid is mainly as same as the vector in size and number, indicating that the bait protein does not affect the normal growth of yeast, and is non-toxic to the yeast. The recombinant pGBKT7-CITED1△CR2was transformed into competent yeast Y2H, the result showed that the pGBKT7-CITED1△CR2plasmid was no self-activation, and can be used to further screen protein interacted with CITED1in the yeast two-hybrid system.3.Clover-shaped diploid cells were observed under an inverted microscopeafter culturing20h, indicating that the yeast mating is successful.46positive clones were screened three times, eventually36positive clones were obtained. cDNA library fragments of different sizes were verified by PCR using Matchmaker Insert Check PCR Mix2kit. The positive clones were sequenced and the BLAST nucleotide similarity was analysed. Finally, five open reading frames were obtained, including: the phosphatidylinositol3-kinase (PIK3C2a), flavin electronic the transfer protein (Etfa), voltage-dependent anion channel1(Vdac1), the copper poisoning metabolic gene region5(Commd5), diphosphate urine nucleotides glucose pyrophosphorylase2(Ugp2). Co-transformation and co-immunoprecipitate experiments showed the interaction between CITED1and PIK3C2a were positive.Conclusion1. Yeast two-hybrid cDNA library of Neonatal rat osteoblasts was constructed successfully.2.PGBKT7-CITED1△CR2plasmid was successfully constructed and expressed in yeast cells, pGBKT7-CITED1△CR2plasmid was proven no yeast toxic and self-activated.3.Five kinds of candidate proteins may interact with CITED1by yeast two-hybrid technology, including:phosphatidylinositol3-kinase (PIK3C2a), flavin electron transfer protein (Etfa), voltage-dependent anion channel (Vdac1) copper poisoning metabolic genes area5(Commd5), diphosphate urine the nucleotide glucose pyrophosphorylase enzyme2(Ugp2). The interaction between CITED1and PIK3C2a were positive. The result is helpful for further researching function of CITED1.