The mechanisms of regulation, activation and signal transduction of the angiotensin II (Ang II) type 1 (AT1) receptor have been studied extensively in the decade after its cloning. The AT1 receptor is a major componen...The mechanisms of regulation, activation and signal transduction of the angiotensin II (Ang II) type 1 (AT1) receptor have been studied extensively in the decade after its cloning. The AT1 receptor is a major component of the renin-angiotensin system (RAS). It mediates the classical biological actions of Ang II. Among the structures required for regulation and activation of the receptor, its carboxyl- terminal region plays crucial roles in receptor internalization, desensitization and phosphorylation. The mechanisms involved in heterotrimeric G-protein coupling to the receptor, activation of the downstream signaling pathway by G proteins and the Ang II signal transduction pathways leading to specific cellular responses are discussed. In addition, recent work on the identification and characterization of novel proteins associated with carboxy1-terminus of the AT1 receptor is presented. These novel proteins will advance our understanding of how the receptor is internalized and recycled as they provide molecular mechanisms for the activation and regulation of G-protein-coupled receptors.展开更多
BACKGROUND: Midkine is a heparin-binding growth factor that promotes the proliferation, survival, migration and differentiation of various target cells. Midkine plays an important role in tumorigenesis and tumor progr...BACKGROUND: Midkine is a heparin-binding growth factor that promotes the proliferation, survival, migration and differentiation of various target cells. Midkine plays an important role in tumorigenesis and tumor progression, and is overexpressed in many human malignant tumors. Patients with high tumor midkine expression frequently have a worse prognosis than those with low expression. The present study was designed to investigate the interaction network of midkine in hepatic cancer cells, and to elucidate its role in hepatocellular carcinoma. METHODS: DNA encoding full-length midkine was cloned into pDBLeu vector to serve as bait in yeast two-hybrid screening to identify interacting proteins. Candidate proteins were examined on SC-Leu-Trp-His+3-AT (20 mmol/L) plates and assayed for X-gal activity, then sequenced and classified according to the GenBank. Finally, identified proteins were expressed by the in vitro expression system pCMVTnT, and protein interactions were confirmed by co-immunoprecipitation. RESULTS: Using the yeast two-hybrid system, we found 6 proteins that interacted with midkine: NK-kappa-B inhibitor alpha (I-κ-B-α), Dvl-binding protein naked cuticle 2, granulin, latent active TGF-β binding protein 3, latent active TGF-β binding protein 4, and phospholipid scramblase 1. In vitro co-immunoprecipitation demonstrated that all identified proteins directly interacted with midkine.CONCLUSION: The identification of midkine-interacting proteins in hepatic cancer cells indicates that midkine is a multifunctional factor that may participate in cell migration, differentiation, and proliferation, and is also associated with the multicellular response feedback during the development of hepatocellular carcinoma.展开更多
Research background: The Arabidopsis-resistance protein L5 (AT1G12290) can trigger cell death in Nicotiana benthamiana, which is a characteristic function of an NBS-LRR (Nucleotide-Binding Sites and Leucine-Rich Repea...Research background: The Arabidopsis-resistance protein L5 (AT1G12290) can trigger cell death in Nicotiana benthamiana, which is a characteristic function of an NBS-LRR (Nucleotide-Binding Sites and Leucine-Rich Repeat) protein activation. Purpose: To explore the function and molecular regulatory network of L5. Method: We employed yeast two-hybrid technology to search for interacting proteins of L5, combined with laser confocal microscopy to observe the subcellular localization of these candidate proteins, and analyzed the impact of these proteins on L5 function using an Agrobacterium mediated transient expression system. Results: Seven candidate interacting proteins were identified from the Arabidopsis cDNA library, including PPA1 (AT1G01050), RIN4 (AT3G25070), LSU1 (AT3G49580), BZIP24 (AT3G51960), BOI (AT4G19700), RING/U (AT4G22250) and PPA3 (AT2G46860). Functional analysis of these candidate interacting proteins showed that they participated in multiple pathways, including biological and abiotic stress, programmed cell death, protein degradation, material metabolism and transcriptional regulation. The results of laser confocal microscopy manifested that RIN4 was only localized on the plasma membrane (PM), and RING/U was mainly associated with the PM. PPA1, PPA3, LSU1, BZIP24, and BOI all emerged nuclear and cytoplasmic localization. The results of the transient assay proclaimed that both BOI and RING/U can inhibit cell death caused by L5. Conclusions: These results indicate that L5 immune receptors may participate in various pathways, and their protein levels and activities are strictly regulated at multiple levels, providing a basis for elucidating the mechanism of L5 immune receptors in Arabidopsis resistance.展开更多
Subunit interactions of the chloroplast F0F1-ATP synthase were studied using the yeast two-hybrid sys-tem. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid ve...Subunit interactions of the chloroplast F0F1-ATP synthase were studied using the yeast two-hybrid sys-tem. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ subunit with wild type or two truncated mutants of ε sununit, ε△N21 and ε△C45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. coli F0F1-ATP synthase, the δ subunit of chloroplast ATP synthase could interact with β. γ, ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase展开更多
Four human vascular endothelial growth factor receptor Flt-1 cDNA fragments containing extracellular domain loops 2, 1-2, 2-3 and 1-3 respectively were amplified from human placen-tal cDNA library by PCR and used for ...Four human vascular endothelial growth factor receptor Flt-1 cDNA fragments containing extracellular domain loops 2, 1-2, 2-3 and 1-3 respectively were amplified from human placen-tal cDNA library by PCR and used for screening ligand binding domains by yeast two-hybrid system. The result showed that, not only loop 1-3, but also the smaller fragment loop 2-3 could bind to hVEGF165. Recombinant expression plasmids pPIC9K/Flt-1(1-3) and pPIC9K/Flt-1 (2-3) were constructed and transformed to Pichia. pastoris host strain GS115, cultured in flasks, and expressed under the induction of 1 % methanol. The expressed product existed in supernatant in the form of soluble molecules and contained more than 60% of total protein after being induced for 4d. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, its purity reached above 90%. Biological assay in vitro showed that the binding capacity of expressed soluble Flt-1 (2-3) to hVEGF165 and its inhibiting effect on the proliferation of human umbilical veins endothelial cells (HUVEC) stimulated with hVEGF165 were close to those of sFlt-1(1-3). Animal test showed that sFlt-1(2-3) could inhibit the formation of regenerate blood vessels stimulated with hVEGF165 significantly.展开更多
AIM: To identify the pre-X region in hepatitis B virus (HBV)genome and to study the relationship between the genotype and the pre-X region. To investigate the biological function of whole-X (pre-X plus X) protein, we ...AIM: To identify the pre-X region in hepatitis B virus (HBV)genome and to study the relationship between the genotype and the pre-X region. To investigate the biological function of whole-X (pre-X plus X) protein, we performed yeast two-hybrid to screen proteins in liver interacting with whole-X protein.METHODS: The pre-X region of HBV was amplified by polymerase chain reaction (PCR) method, and was cloned to pGEM Teasy vector. After the target region was sequenced, Vector 8.0 software was used to analyze the sequences. The whole-X bait plasmid was constructed by using yeast two-hybrid system 3. Yeast strain AH109 was transformed. After expression of the whole-X protein in AH109 yeast strains was proved, yeast two-hybrid screening was performed by mating AH109 with Y187 containing liver cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between whole-X protein and the protein obtained from positive colonies was further confirmed by repeating yeast two-hybrid. After extracting and sequencing of plasmid from blue colonies, we carried out analysis by bioinformatics. RESULTS: After sequencing, 27 of 45 clones (60%) were found encoding the pre-X peptide. Eighteen of twenty-seven clones (66.7%) of pre-X coding sequences were found from genotype C. Five positive colonies that interacted with whole-X protein were obtained and sequenced; namely, fetuin B, UDP glycosyltransferase 1 family-polypeptide A9, mannose-P-dolichol utilization defect 1, fibrinogen-B beta polypeptide, transmembrane 4 superfamily member 4CD81 (TM4SF4).CONCLUSION: The pre-X gene exists in HBV genome.Genes of proteins interacting with whole-X protein in hepatocytes were successfully cloned. These results brought some new clues for studying the biological functions of whole-X protein.展开更多
Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding a, p, y, 8 and e subunits of Spinach ATP synthase fused ...Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding a, p, y, 8 and e subunits of Spinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding p-galactosidase was detected. Of all the combinations, that of y and e subunit genes showed the highest level of reporter gene expression, while those of a and p, a and e, p and e and p and 8 induced stable and significant reporter gene expression. The combination of 8 and e as well as that of 8 and y induced weak and unstable reporter gene expression. However, combinations of a and y, p and y and a and 8 did not induce reporter gene expression. These results suggested that specific and strong interactions between y and e, a and p, a and e, p and e and p and 8 subunits, and weak and transient interactions between 8 and e and 8 and y subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.展开更多
Two DNA fragments encoding PDZ domain （21-110 residues） and BAR domain （ 150-360 residues） from PICK1 （1-416 residues） were amplified by PCR and then introduced into vectors, pET-32M and pMAL-e2X respectively to...Two DNA fragments encoding PDZ domain （21-110 residues） and BAR domain （ 150-360 residues） from PICK1 （1-416 residues） were amplified by PCR and then introduced into vectors, pET-32M and pMAL-e2X respectively to generate recombinant plasmids, pE-pdz and pM-bar. Having been separately transferred into the hosts E. coli BL21 and E. coli JM109, these two strains can express fusion proteins： His-tagged PDZ（PDZ domain） and maltose binding protein-BAR（ MBP-BAR domain） respectively, as confirmed by both SDS-PAGE and Wostem blotting. The interaction between these two domains is dose-dependence, as identified by a pull-down test. Moreover, it has been shown from the ELISA analysis that the actual amount of PDZ bound to MBP-BAR-amylose beads reaches （ 16 ± 0. 5）%, as calculated by the molar ratio of PDZ to MBP-BAR. In addition, the interaction between BAR（bait） and PDZ（prey） in vivo was also examined with a yeast two-hybrid system.展开更多
AIM: To investigate the biological function of 14-3-3σ protein and to look for proteins that interact with 14-3-3σ protein in colon cancer stem cells. METHODS: Reverse transcription polymerase chain reaction was per...AIM: To investigate the biological function of 14-3-3σ protein and to look for proteins that interact with 14-3-3σ protein in colon cancer stem cells. METHODS: Reverse transcription polymerase chain reaction was performed to amplify the 14-3-3σ gene from the mRNA of colon cancer stem cells. The gene was then cloned into the pGEM-T vector. After being sequenced, the target gene 14-3-3σ was cut from the pGEM-T vector and cloned into the pGBKT7 yeast expression plasmid. Then, the bait plasmid pGBKT7-14-3-3σ was transformed into the yeast strain AH109. After the expression of the pGBKT7-14-3-3σ fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening assay was performed by mating AH109 with Y187 that contained a HeLa cDNA library plasmid. The interaction between the 14-3-3σ protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybridscreen. After extracting and sequencing the plasmids from the positive colonies, we performed a bioinformatics analysis. A coimmunoprecipitation assay was performed to confirm the interaction between 14-3-3σ and the proteins obtained from the positive colonies. Finally, we constructed 14-3-3σ and potassium channel modulatory factor 1 (KCMF1) siRNA expression plasmids and transfected them into colon cancer stem cells. RESULTS: The bait plasmid pGBKT7-14-3-3σ was constructed successfully, and the 14-3-3σ protein had no toxic or autonomous activation effect on the yeast. Nineteen true-positive colonies were selected and sequenced, and their full-length sequences were obtained. We searched for homologous DNA sequences for these sequences from GenBank. Among the positive colonies, four coding genes with known functions were obtained, including KCMF1 , quinone oxidore-ductase (NQO2 ), hydroxyisobutyrate dehydrogenase (HIBADH ) and 14-3-3σ . For the subsequent coimmu-noprecipitation assay, the plasmids PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH were successfully construct展开更多
Alzheimer＇s disease（AD） is a neurodegenerative disorder in which amyloid b plaques are a pathological characteristic. Little is known about the physiological functions of amyloid b precursor protein（APP）. Based o...Alzheimer＇s disease（AD） is a neurodegenerative disorder in which amyloid b plaques are a pathological characteristic. Little is known about the physiological functions of amyloid b precursor protein（APP）. Based on its structure as a type I transmembrane protein, it has been proposed that APP might be a receptor, but so far, no ligand has been reported. In the present study, 9 proteins binding to the extracellular domain of APP were identified using a yeast two-hybrid system. After confirming the interactions in the mammalian system, mutated PLP1,members of the FLRT protein family, and KCTD16 were shown to interact with APP. These proteins have been reported to be involved in Pelizaeus-Merzbacher disease（PMD） and axon guidance. Therefore, our results shed light on the mechanisms of physiological function of APP in AD, PMD, and axon guidance.展开更多
To further research the regulatory network of pyruvate dehydrogenase kinase （designated as TaPDK） in physiological male-sterility （PHYMS） of wheat induced by chemical hybridizing agent （CHA） SQ-1, an anther cDNA...To further research the regulatory network of pyruvate dehydrogenase kinase （designated as TaPDK） in physiological male-sterility （PHYMS） of wheat induced by chemical hybridizing agent （CHA） SQ-1, an anther cDNA library was constructed, and the proteins interacting with TaPDK were screened via yeast two-hybrid technique. Subsequently, a few candidate proteins in nucleotide expression levels were detected by real-time quantitative PCR. Yeast-two hybrid screening was performed by mating yeast strain Y2HGold containing BD-TaPDK bait plasmid with yeast strain Y187 including anther cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium （SD/-Ade/-His/-Leu/-Trp） （QDO）, and further were incubated on QDO medium containing AbA and X-α-Gal. The interactions between TaPDK and the proteins obtained from positive colonies were further confirmed by co-transformation validation. After plasmids DNA were extracted from blue colonies and sequenced, the sequences results were analyzed by bioinformatic methods. Finally, 24 colonies were obtained, including eight genes, namely non-specific lipid-transfer protein precursor （TanLTP）, polyubiquitin （TaPUbi）, glyceraldehyde-3-phosphate dehydrogenase, proliferating cell nuclear antigen （TaPCNA）, CBS domain containing protein （TaCBS）, actin, guanine nucleotide-binding protein beta subunit, chalcone synthase, and three new genes with unknown function. The results of quantitative RT-PCR showed that the expression levels of TanLTP, TaPUbi, and TaPCNA were obviously up-regulated in PHYMS anther, and TaCBS expression was only increased at the tricellular stage in PHYMS anther compared with in fertile lines. Whereas, the expression of TaPDK was obviously down-regulated in PHYMS lines. Collectively, these datas indicated that the majority of candidate proteins might be related to pollen abortion in PHYMS lines, which further suggested that TaPDK plays multiple roles in pollen development, besides participating in regulating p展开更多
Restin,a member of melanoma-associated antigen superfamily gene,was first cloned from differentiated leukemia cell induced by all trans-retinoic acid,and was able to inhibit cell proliferation,but the molecular mechan...Restin,a member of melanoma-associated antigen superfamily gene,was first cloned from differentiated leukemia cell induced by all trans-retinoic acid,and was able to inhibit cell proliferation,but the molecular mechanism was not clear.Since Restin was localized in cell nucleus,and its homolog member,Necdin(neuronal growth suppressor factor),could interact with transcription factors p53 and E2F1,we proposed that Restin might also function as Necdin through interacting with some transcription factors.In this study,transcription factors p53,AP1,ATFs and E2Fs were cloned and used in the mammalian two-hybrid system to identify their in-teraction with Restin.The results showed that only ATF3 had a strong interaction with Restin.It is interesting to know that ATF3 was an important transcription factor for G1 cell cycle initiation in physiological stress response.It was possible that the inhibition of cell proliferation by Restin might be related with the inhibition of ATF3 activity.展开更多
asy gene is a novel apoptosis-inducing gene, but its mechanism is unclear. To investigate the mechanism of asy inducing apoptosis, a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell...asy gene is a novel apoptosis-inducing gene, but its mechanism is unclear. To investigate the mechanism of asy inducing apoptosis, a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system. The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels. Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5' end and different 3' end, and share a common open reading frame encoding a polypeptide of 236 amino acids. Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence. Two highly hydrophobic regions encoding potentially two transmembrane domains are present. The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu). Immunoprecipitation assay confirmed the interaction of ASY and ASYIP in mammalian cells. Compared with asy gene, overexpression of asyip gene can inhibit growth of tumor cell Saos2 and induce cell apoptosis with a low efficiency.展开更多
Gram-negative bacteria have become the main pathogens and cause serious clinical problems with increased morbidity and mortality. However, the slow discovery of new antimicrobial agents is unable to meet the need for ...Gram-negative bacteria have become the main pathogens and cause serious clinical problems with increased morbidity and mortality. However, the slow discovery of new antimicrobial agents is unable to meet the need for the treatment of bacterial infections caused by drug-resistant strains. The interaction of L12 and L10 is essential for ribosomal function and protein synthesis. In this study, a yeast two-hybrid system was established to successfully detect the interaction between L12 and L10 proteins from gram-negative bacteria Escherichia coli, which allows us to screen compounds that specifically disrupt this interaction. With this system, we identified two compounds IMB-84 and IMB-87 that block L12-L10 interaction and show bactericidal activity against E. coli. We used glutathione-S-transferase(GST) pull-down and surface plasmon resonance(SPR) assays to demonstrate that these compounds disrupt L12-L10 interaction in vitro and the target of compounds was further confirmed by the overexpression of target proteins. Moreover, protein synthesis and elongation factor G-dependent GTPase activities are inhibited by two compounds. Therefore, we have identified two antibacterial agents that disrupt L12-L10 interaction by using yeast two-hybrid system.展开更多
Background The hepatitis B virus （HBV） genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 （human gene 5 transactivated by pre-S1 protein o...Background The hepatitis B virus （HBV） genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 （human gene 5 transactivated by pre-S1 protein of HBV） is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory （GenBank accession： AY427953）. In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS1TP5 protein. Methods The reverse transcription polymerase chain reaction （RT-PCR） was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then ＂bait＂ plasmid pGBKT7-PS1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blotting hybridization. After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH109 with Y187 containing a leukocyte cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis. Results Forty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes with known functions were 展开更多
Tuberculosis(TB)is one of the deadly diseases caused by Mycobacterium tuberculosis(Mtb),which presents a significant public health challenge.Treatment of TB relies on the combination of several anti-TB drugs to create...Tuberculosis(TB)is one of the deadly diseases caused by Mycobacterium tuberculosis(Mtb),which presents a significant public health challenge.Treatment of TB relies on the combination of several anti-TB drugs to create shorter and safer regimens.Therefore,new anti-TB agents working by different mechanisms are urgently needed.FtsZ,a tubulin-like protein with GTPase activity,forms a dynamic Z-ring in cell division.Most of FtsZ inhibitors are designed to inhibit GTPase activity.In Mtb,the function of Z-ring is modulated by SepF,a FtsZ binding protein.The FtsZ/SepF interaction is essential for FtsZ bundling and localization at the site of division.Here,we established a yeast twohybrid based screening system to identify inhibitors of FtsZ/SepF interaction in M.tuberculosis.Using this system,we found compound T0349 showing strong anti-Mtb activity but with low toxicity to other bacteria strains and mice.Moreover,we have demonstrated that T0349 binds specifically to SepF to block FtsZ/SepF interaction by GST pull-down,fluorescence polarization(FP),surface plasmon resonance(SPR)and CRISPRi knockdown assays.Furthermore,T0349 can inhibit bacterial cell division by inducing filamentation and abnormal septum.Our data demonstrated that FtsZ/SepF interaction is a promising anti-TB drug target for identifying agents with novel mechanisms.展开更多
Mitogen-activated protein kinases(MAPKs,also known as MPKs)regulate diverse cellular and physiological functions,and dual-specificity MAPK phosphatases(MKPs)modulate MAPK signalling through MAPK dephosphorylation and ...Mitogen-activated protein kinases(MAPKs,also known as MPKs)regulate diverse cellular and physiological functions,and dual-specificity MAPK phosphatases(MKPs)modulate MAPK signalling through MAPK dephosphorylation and inactivation.Due to lacking of overall understanding for the regulatory networks between Chrysanthemum morifolium MKPs(CmMKPs)and C.morifolium MAPKs(CmMPKs),we systematically studied the interactions between four groups of CmMPKs and eight identified CmMKPs in chrysanthemum and found that the interaction between the specific CmMKP and the specific CmMPK differed from those in other plants.Furthermore,the expression of CmMKP1 and CmMKP1-LIKE1showed opposite trends during the development of chrysanthemum flower buds under salt treatment and Alternaria alternata inoculation,but these genes could interact with the same CmMPKs,providing insight into the subfunctionalization of paralogues.Amino acid variations(M87V,T277P and V6L)in dual-specificity protein phosphatases(DsPTP1)-LIKE1/2/3 changed the interactions of these proteins with the four CmMPK groups in chrysanthemum,providing evidence for the de/neofunctionalization of paralogues in polyploids,suggesting that we can identify the key functional sites of proteins by studying polyploid paralogues.展开更多
文摘The mechanisms of regulation, activation and signal transduction of the angiotensin II (Ang II) type 1 (AT1) receptor have been studied extensively in the decade after its cloning. The AT1 receptor is a major component of the renin-angiotensin system (RAS). It mediates the classical biological actions of Ang II. Among the structures required for regulation and activation of the receptor, its carboxyl- terminal region plays crucial roles in receptor internalization, desensitization and phosphorylation. The mechanisms involved in heterotrimeric G-protein coupling to the receptor, activation of the downstream signaling pathway by G proteins and the Ang II signal transduction pathways leading to specific cellular responses are discussed. In addition, recent work on the identification and characterization of novel proteins associated with carboxy1-terminus of the AT1 receptor is presented. These novel proteins will advance our understanding of how the receptor is internalized and recycled as they provide molecular mechanisms for the activation and regulation of G-protein-coupled receptors.
基金supported by grants from the National Natural Science Foundation of China (30772534)the Natural Science Foundation of Huzhou (2010YS05)
文摘BACKGROUND: Midkine is a heparin-binding growth factor that promotes the proliferation, survival, migration and differentiation of various target cells. Midkine plays an important role in tumorigenesis and tumor progression, and is overexpressed in many human malignant tumors. Patients with high tumor midkine expression frequently have a worse prognosis than those with low expression. The present study was designed to investigate the interaction network of midkine in hepatic cancer cells, and to elucidate its role in hepatocellular carcinoma. METHODS: DNA encoding full-length midkine was cloned into pDBLeu vector to serve as bait in yeast two-hybrid screening to identify interacting proteins. Candidate proteins were examined on SC-Leu-Trp-His+3-AT (20 mmol/L) plates and assayed for X-gal activity, then sequenced and classified according to the GenBank. Finally, identified proteins were expressed by the in vitro expression system pCMVTnT, and protein interactions were confirmed by co-immunoprecipitation. RESULTS: Using the yeast two-hybrid system, we found 6 proteins that interacted with midkine: NK-kappa-B inhibitor alpha (I-κ-B-α), Dvl-binding protein naked cuticle 2, granulin, latent active TGF-β binding protein 3, latent active TGF-β binding protein 4, and phospholipid scramblase 1. In vitro co-immunoprecipitation demonstrated that all identified proteins directly interacted with midkine.CONCLUSION: The identification of midkine-interacting proteins in hepatic cancer cells indicates that midkine is a multifunctional factor that may participate in cell migration, differentiation, and proliferation, and is also associated with the multicellular response feedback during the development of hepatocellular carcinoma.
文摘Research background: The Arabidopsis-resistance protein L5 (AT1G12290) can trigger cell death in Nicotiana benthamiana, which is a characteristic function of an NBS-LRR (Nucleotide-Binding Sites and Leucine-Rich Repeat) protein activation. Purpose: To explore the function and molecular regulatory network of L5. Method: We employed yeast two-hybrid technology to search for interacting proteins of L5, combined with laser confocal microscopy to observe the subcellular localization of these candidate proteins, and analyzed the impact of these proteins on L5 function using an Agrobacterium mediated transient expression system. Results: Seven candidate interacting proteins were identified from the Arabidopsis cDNA library, including PPA1 (AT1G01050), RIN4 (AT3G25070), LSU1 (AT3G49580), BZIP24 (AT3G51960), BOI (AT4G19700), RING/U (AT4G22250) and PPA3 (AT2G46860). Functional analysis of these candidate interacting proteins showed that they participated in multiple pathways, including biological and abiotic stress, programmed cell death, protein degradation, material metabolism and transcriptional regulation. The results of laser confocal microscopy manifested that RIN4 was only localized on the plasma membrane (PM), and RING/U was mainly associated with the PM. PPA1, PPA3, LSU1, BZIP24, and BOI all emerged nuclear and cytoplasmic localization. The results of the transient assay proclaimed that both BOI and RING/U can inhibit cell death caused by L5. Conclusions: These results indicate that L5 immune receptors may participate in various pathways, and their protein levels and activities are strictly regulated at multiple levels, providing a basis for elucidating the mechanism of L5 immune receptors in Arabidopsis resistance.
基金This work was supported by theState Key Basic Research and Development Plan (Grant No. G1998010100) the National Natural Science Foundation of China (Grant No. 39730040).
文摘Subunit interactions of the chloroplast F0F1-ATP synthase were studied using the yeast two-hybrid sys-tem. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ subunit with wild type or two truncated mutants of ε sununit, ε△N21 and ε△C45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. coli F0F1-ATP synthase, the δ subunit of chloroplast ATP synthase could interact with β. γ, ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase
基金National High Technology Programs of China (Grant No. 102-08-01-03) and Natural Science Fund Program of Guangdong Province (Grant No.001098).
文摘Four human vascular endothelial growth factor receptor Flt-1 cDNA fragments containing extracellular domain loops 2, 1-2, 2-3 and 1-3 respectively were amplified from human placen-tal cDNA library by PCR and used for screening ligand binding domains by yeast two-hybrid system. The result showed that, not only loop 1-3, but also the smaller fragment loop 2-3 could bind to hVEGF165. Recombinant expression plasmids pPIC9K/Flt-1(1-3) and pPIC9K/Flt-1 (2-3) were constructed and transformed to Pichia. pastoris host strain GS115, cultured in flasks, and expressed under the induction of 1 % methanol. The expressed product existed in supernatant in the form of soluble molecules and contained more than 60% of total protein after being induced for 4d. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, its purity reached above 90%. Biological assay in vitro showed that the binding capacity of expressed soluble Flt-1 (2-3) to hVEGF165 and its inhibiting effect on the proliferation of human umbilical veins endothelial cells (HUVEC) stimulated with hVEGF165 were close to those of sFlt-1(1-3). Animal test showed that sFlt-1(2-3) could inhibit the formation of regenerate blood vessels stimulated with hVEGF165 significantly.
基金Supported by the grants from the National Natural Science Foundation, No. C03011402, No. C30070690the 9.5 Research and Technique Foundation of PLA, No. 98D063+1 种基金 the Launching Foundation for Student Studying Abroad of PLA, No. 98H038 the 10.5 Youth Research and Technique Foundation of PLA, No. 01Q138andNo. 01MB135
文摘AIM: To identify the pre-X region in hepatitis B virus (HBV)genome and to study the relationship between the genotype and the pre-X region. To investigate the biological function of whole-X (pre-X plus X) protein, we performed yeast two-hybrid to screen proteins in liver interacting with whole-X protein.METHODS: The pre-X region of HBV was amplified by polymerase chain reaction (PCR) method, and was cloned to pGEM Teasy vector. After the target region was sequenced, Vector 8.0 software was used to analyze the sequences. The whole-X bait plasmid was constructed by using yeast two-hybrid system 3. Yeast strain AH109 was transformed. After expression of the whole-X protein in AH109 yeast strains was proved, yeast two-hybrid screening was performed by mating AH109 with Y187 containing liver cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between whole-X protein and the protein obtained from positive colonies was further confirmed by repeating yeast two-hybrid. After extracting and sequencing of plasmid from blue colonies, we carried out analysis by bioinformatics. RESULTS: After sequencing, 27 of 45 clones (60%) were found encoding the pre-X peptide. Eighteen of twenty-seven clones (66.7%) of pre-X coding sequences were found from genotype C. Five positive colonies that interacted with whole-X protein were obtained and sequenced; namely, fetuin B, UDP glycosyltransferase 1 family-polypeptide A9, mannose-P-dolichol utilization defect 1, fibrinogen-B beta polypeptide, transmembrane 4 superfamily member 4CD81 (TM4SF4).CONCLUSION: The pre-X gene exists in HBV genome.Genes of proteins interacting with whole-X protein in hepatocytes were successfully cloned. These results brought some new clues for studying the biological functions of whole-X protein.
文摘Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding a, p, y, 8 and e subunits of Spinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding p-galactosidase was detected. Of all the combinations, that of y and e subunit genes showed the highest level of reporter gene expression, while those of a and p, a and e, p and e and p and 8 induced stable and significant reporter gene expression. The combination of 8 and e as well as that of 8 and y induced weak and unstable reporter gene expression. However, combinations of a and y, p and y and a and 8 did not induce reporter gene expression. These results suggested that specific and strong interactions between y and e, a and p, a and e, p and e and p and 8 subunits, and weak and transient interactions between 8 and e and 8 and y subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.
基金the National Natural Science Foundation of China(No 30400065)
文摘Two DNA fragments encoding PDZ domain （21-110 residues） and BAR domain （ 150-360 residues） from PICK1 （1-416 residues） were amplified by PCR and then introduced into vectors, pET-32M and pMAL-e2X respectively to generate recombinant plasmids, pE-pdz and pM-bar. Having been separately transferred into the hosts E. coli BL21 and E. coli JM109, these two strains can express fusion proteins： His-tagged PDZ（PDZ domain） and maltose binding protein-BAR（ MBP-BAR domain） respectively, as confirmed by both SDS-PAGE and Wostem blotting. The interaction between these two domains is dose-dependence, as identified by a pull-down test. Moreover, it has been shown from the ELISA analysis that the actual amount of PDZ bound to MBP-BAR-amylose beads reaches （ 16 ± 0. 5）%, as calculated by the molar ratio of PDZ to MBP-BAR. In addition, the interaction between BAR（bait） and PDZ（prey） in vivo was also examined with a yeast two-hybrid system.
基金Supported by The Medical Guidance Projects of Shanghai Science Committee,No.10411961800National Natural Science Foundation of China,No.81101617
文摘AIM: To investigate the biological function of 14-3-3σ protein and to look for proteins that interact with 14-3-3σ protein in colon cancer stem cells. METHODS: Reverse transcription polymerase chain reaction was performed to amplify the 14-3-3σ gene from the mRNA of colon cancer stem cells. The gene was then cloned into the pGEM-T vector. After being sequenced, the target gene 14-3-3σ was cut from the pGEM-T vector and cloned into the pGBKT7 yeast expression plasmid. Then, the bait plasmid pGBKT7-14-3-3σ was transformed into the yeast strain AH109. After the expression of the pGBKT7-14-3-3σ fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening assay was performed by mating AH109 with Y187 that contained a HeLa cDNA library plasmid. The interaction between the 14-3-3σ protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybridscreen. After extracting and sequencing the plasmids from the positive colonies, we performed a bioinformatics analysis. A coimmunoprecipitation assay was performed to confirm the interaction between 14-3-3σ and the proteins obtained from the positive colonies. Finally, we constructed 14-3-3σ and potassium channel modulatory factor 1 (KCMF1) siRNA expression plasmids and transfected them into colon cancer stem cells. RESULTS: The bait plasmid pGBKT7-14-3-3σ was constructed successfully, and the 14-3-3σ protein had no toxic or autonomous activation effect on the yeast. Nineteen true-positive colonies were selected and sequenced, and their full-length sequences were obtained. We searched for homologous DNA sequences for these sequences from GenBank. Among the positive colonies, four coding genes with known functions were obtained, including KCMF1 , quinone oxidore-ductase (NQO2 ), hydroxyisobutyrate dehydrogenase (HIBADH ) and 14-3-3σ . For the subsequent coimmu-noprecipitation assay, the plasmids PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH were successfully construct
基金supported by the National Natural Science Foundation of China for Distinguished Young Scholars(81425009)the Beijing Natural Science Foundation,China(7142085)the Peking University Collaborative Fund,China(464-10606-00416)
文摘Alzheimer＇s disease（AD） is a neurodegenerative disorder in which amyloid b plaques are a pathological characteristic. Little is known about the physiological functions of amyloid b precursor protein（APP）. Based on its structure as a type I transmembrane protein, it has been proposed that APP might be a receptor, but so far, no ligand has been reported. In the present study, 9 proteins binding to the extracellular domain of APP were identified using a yeast two-hybrid system. After confirming the interactions in the mammalian system, mutated PLP1,members of the FLRT protein family, and KCTD16 were shown to interact with APP. These proteins have been reported to be involved in Pelizaeus-Merzbacher disease（PMD） and axon guidance. Therefore, our results shed light on the mechanisms of physiological function of APP in AD, PMD, and axon guidance.
基金supported by the National High-Tech R&D Program of China(2011AA10A106)the National Natural Science Foundation of China(31071477,31171611)the Key Scientific and Technological Innovation Special Projects of Shaanxi"13115",China(2010ZDKG-68,2011KTZB02-01-01)
文摘To further research the regulatory network of pyruvate dehydrogenase kinase （designated as TaPDK） in physiological male-sterility （PHYMS） of wheat induced by chemical hybridizing agent （CHA） SQ-1, an anther cDNA library was constructed, and the proteins interacting with TaPDK were screened via yeast two-hybrid technique. Subsequently, a few candidate proteins in nucleotide expression levels were detected by real-time quantitative PCR. Yeast-two hybrid screening was performed by mating yeast strain Y2HGold containing BD-TaPDK bait plasmid with yeast strain Y187 including anther cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium （SD/-Ade/-His/-Leu/-Trp） （QDO）, and further were incubated on QDO medium containing AbA and X-α-Gal. The interactions between TaPDK and the proteins obtained from positive colonies were further confirmed by co-transformation validation. After plasmids DNA were extracted from blue colonies and sequenced, the sequences results were analyzed by bioinformatic methods. Finally, 24 colonies were obtained, including eight genes, namely non-specific lipid-transfer protein precursor （TanLTP）, polyubiquitin （TaPUbi）, glyceraldehyde-3-phosphate dehydrogenase, proliferating cell nuclear antigen （TaPCNA）, CBS domain containing protein （TaCBS）, actin, guanine nucleotide-binding protein beta subunit, chalcone synthase, and three new genes with unknown function. The results of quantitative RT-PCR showed that the expression levels of TanLTP, TaPUbi, and TaPCNA were obviously up-regulated in PHYMS anther, and TaCBS expression was only increased at the tricellular stage in PHYMS anther compared with in fertile lines. Whereas, the expression of TaPDK was obviously down-regulated in PHYMS lines. Collectively, these datas indicated that the majority of candidate proteins might be related to pollen abortion in PHYMS lines, which further suggested that TaPDK plays multiple roles in pollen development, besides participating in regulating p
文摘Restin,a member of melanoma-associated antigen superfamily gene,was first cloned from differentiated leukemia cell induced by all trans-retinoic acid,and was able to inhibit cell proliferation,but the molecular mechanism was not clear.Since Restin was localized in cell nucleus,and its homolog member,Necdin(neuronal growth suppressor factor),could interact with transcription factors p53 and E2F1,we proposed that Restin might also function as Necdin through interacting with some transcription factors.In this study,transcription factors p53,AP1,ATFs and E2Fs were cloned and used in the mammalian two-hybrid system to identify their in-teraction with Restin.The results showed that only ATF3 had a strong interaction with Restin.It is interesting to know that ATF3 was an important transcription factor for G1 cell cycle initiation in physiological stress response.It was possible that the inhibition of cell proliferation by Restin might be related with the inhibition of ATF3 activity.
文摘asy gene is a novel apoptosis-inducing gene, but its mechanism is unclear. To investigate the mechanism of asy inducing apoptosis, a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system. The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels. Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5' end and different 3' end, and share a common open reading frame encoding a polypeptide of 236 amino acids. Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence. Two highly hydrophobic regions encoding potentially two transmembrane domains are present. The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu). Immunoprecipitation assay confirmed the interaction of ASY and ASYIP in mammalian cells. Compared with asy gene, overexpression of asyip gene can inhibit growth of tumor cell Saos2 and induce cell apoptosis with a low efficiency.
基金supported by the National Natural Science Foundation of China (Grant nos.81370089,81529003,81621064 and 81361138020)the Foundation for Innovative Research Groups and the Funds for International Cooperation and Exchange between China–Sweden and CAMS Initiative for Innovative Medicine (2016-12M-3-014)
文摘Gram-negative bacteria have become the main pathogens and cause serious clinical problems with increased morbidity and mortality. However, the slow discovery of new antimicrobial agents is unable to meet the need for the treatment of bacterial infections caused by drug-resistant strains. The interaction of L12 and L10 is essential for ribosomal function and protein synthesis. In this study, a yeast two-hybrid system was established to successfully detect the interaction between L12 and L10 proteins from gram-negative bacteria Escherichia coli, which allows us to screen compounds that specifically disrupt this interaction. With this system, we identified two compounds IMB-84 and IMB-87 that block L12-L10 interaction and show bactericidal activity against E. coli. We used glutathione-S-transferase(GST) pull-down and surface plasmon resonance(SPR) assays to demonstrate that these compounds disrupt L12-L10 interaction in vitro and the target of compounds was further confirmed by the overexpression of target proteins. Moreover, protein synthesis and elongation factor G-dependent GTPase activities are inhibited by two compounds. Therefore, we have identified two antibacterial agents that disrupt L12-L10 interaction by using yeast two-hybrid system.
基金This work was supported by grants from the National Natural Science Foundation of China (No. 30371288) and Beijing Natural Science Foundation (No. 5042024).
文摘Background The hepatitis B virus （HBV） genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 （human gene 5 transactivated by pre-S1 protein of HBV） is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory （GenBank accession： AY427953）. In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS1TP5 protein. Methods The reverse transcription polymerase chain reaction （RT-PCR） was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then ＂bait＂ plasmid pGBKT7-PS1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blotting hybridization. After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH109 with Y187 containing a leukocyte cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis. Results Forty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes with known functions were
基金CAMS Innovation Fund for Medical Sciences(CIFMS)(2021-I2M-1-026,2022-I2M-2-002,2021I2M-JB-011,China)National Natural Science Foundation of China(No.81773784)Beijing Key Laboratory of NonClinical Drug Metabolism and PK/PD study(Z141102004414062,China)。
文摘Tuberculosis(TB)is one of the deadly diseases caused by Mycobacterium tuberculosis(Mtb),which presents a significant public health challenge.Treatment of TB relies on the combination of several anti-TB drugs to create shorter and safer regimens.Therefore,new anti-TB agents working by different mechanisms are urgently needed.FtsZ,a tubulin-like protein with GTPase activity,forms a dynamic Z-ring in cell division.Most of FtsZ inhibitors are designed to inhibit GTPase activity.In Mtb,the function of Z-ring is modulated by SepF,a FtsZ binding protein.The FtsZ/SepF interaction is essential for FtsZ bundling and localization at the site of division.Here,we established a yeast twohybrid based screening system to identify inhibitors of FtsZ/SepF interaction in M.tuberculosis.Using this system,we found compound T0349 showing strong anti-Mtb activity but with low toxicity to other bacteria strains and mice.Moreover,we have demonstrated that T0349 binds specifically to SepF to block FtsZ/SepF interaction by GST pull-down,fluorescence polarization(FP),surface plasmon resonance(SPR)and CRISPRi knockdown assays.Furthermore,T0349 can inhibit bacterial cell division by inducing filamentation and abnormal septum.Our data demonstrated that FtsZ/SepF interaction is a promising anti-TB drug target for identifying agents with novel mechanisms.
基金funded by the National Natural Science Foundation of China(Grant Nos.31870694,31870279,32002083)the Natural Science Fund of Jiangsu Province(Grant No.BK20190076)+2 种基金the National Key R&D Program of China(Grant No.2018YFD1000403)the Foundation of Central Laboratory of Xinyang Agriculture and Forestry University(Grant No.FCL202002)a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institution。
文摘Mitogen-activated protein kinases(MAPKs,also known as MPKs)regulate diverse cellular and physiological functions,and dual-specificity MAPK phosphatases(MKPs)modulate MAPK signalling through MAPK dephosphorylation and inactivation.Due to lacking of overall understanding for the regulatory networks between Chrysanthemum morifolium MKPs(CmMKPs)and C.morifolium MAPKs(CmMPKs),we systematically studied the interactions between four groups of CmMPKs and eight identified CmMKPs in chrysanthemum and found that the interaction between the specific CmMKP and the specific CmMPK differed from those in other plants.Furthermore,the expression of CmMKP1 and CmMKP1-LIKE1showed opposite trends during the development of chrysanthemum flower buds under salt treatment and Alternaria alternata inoculation,but these genes could interact with the same CmMPKs,providing insight into the subfunctionalization of paralogues.Amino acid variations(M87V,T277P and V6L)in dual-specificity protein phosphatases(DsPTP1)-LIKE1/2/3 changed the interactions of these proteins with the four CmMPK groups in chrysanthemum,providing evidence for the de/neofunctionalization of paralogues in polyploids,suggesting that we can identify the key functional sites of proteins by studying polyploid paralogues.
