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The binding of talin-F0 domain to ras-related protein 1b (Rap1b) plays an important role in the formation of thrombosis. However, since talin is a force-sensitive protein, it remains unclear whether and how force regulates the talin-F0/Rap1b interaction. To explore the effect of force on the binding affinity and the dynamics mechanisms of talin-F0/Rap1b, molecular dynamics simulation was used to observe and compare the changes in functional and conformational information of the complex under different forces. Our results showed that when the complex was subjected to tensile forces, there were at least two dissociation pathways with significantly different mechanical strengths. The key event determining the mechanical strength difference between the two pathways was whether the β4 sheet of the F0 domain was pulled away from the original β1-β4 parallel structure. As the force increased, the talin-F0/Rap1b interaction first strengthened and then weakened, exhibiting the signature of a transition from catch bonds to slip bonds. The mechanical load of 20 pN increased the interaction index of two residue pairs, ASP 54-ARG 41 and GLN 18-THR 65, which resulted in a significant increase in the affinity of the complex. This study predicts the regulatory mechanism of the talin-F0/Rap1b interaction by forces in the intracellular environment and provides novel ideas for the treatment of related diseases and drug development.
Subject(s)
Molecular Dynamics Simulation , TalinABSTRACT
ObjectiveTo explore the mechanism of Shenxiong glucose injection （SGI） in inhibiting hydrogen peroxide （H2O2）-induced oxidative damage in H9c2 cells by tandem mass tags （TMT）-labeled quantitative proteomics. MethodH9c2 cells cultured in vitro were exposed to H2O2 for inducing oxidative damage. The cell viability was measured by cell proliferation and cytotoxicity assay （MTS）， followed by peptide fractionation by high performance liquid chromatography （HPLC） and protein expression detection in H9c2 cells by ultrahigh performance liquid chromatography-mass spectrometry. MaxQuant （v1.5.2.8） was utilized for data retrieval， and the high-resolution mass spectrometry was conducted to screen out differentially expressed proteins， which were then subjected to gene ontology （GO） and Kyoto encyclopedia of genes and genomes （KEGG） enrichment analysis. The protein expression levels of perilipin 2 （Plin2） and tropomyosin 1 （Tpm1） in cells were measured by Western blot. ResultThe spectral analysis yielded 48 608 specific peptide fragments and 5 903 quantifiable proteins. Compared with the model group，the SGI group exhibited 82 differentially expressed proteins，of which 22 were up-regulated and 60 were down-regulated. GO analysis results showed that the differentially expressed proteins were mainly involved in biological processes such as programmed cell death regulation，regulation of cell proliferation，cardiovascular system development， and cell migration. As revealed by KEGG analysis， these proteins were mainly related to peroxisome proliferator-activated receptor （PPAR），focal adhesion，phosphatidylinositol 3-kinase/protein kinase B（PI3K/Akt），and Ras-related protein 1 （Rap1） pathways. Western blot results demonstrated that compared with the model group，SGI significantly increased the Plin2 protein expression and decreased the Tpm1 protein expression （P<0.01），consistent with the proteomics results. ConclusionSGI may inhibit cell apoptosis and antagonize H2O2-induced cell oxidative damage by regulating PPAR，focal adhesion，PI3K/Akt and Rap1 pathways，which should be further verified by subsequent experiments.
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OBJECTIVE@#To elucidate the pathogenic role of leukotriene B4 (LTB4) in pulmonary hyper-permeability and inflammation induced by lung-protective mechanical ventilation (LPMV) in rabbits.@*METHODS@#Thirty-two healthy Japanese white rabbits were randomized into 4 groups for treatment with vehicle or bestatin (a leukotriene A4 hydrolase inhibitor that inhibits LTB4 production) administered intragastrically at the daily dose of 8 mg/kg for 5 days, followed by sham operation (group S and group BS, respectively, in which the rabbits were anesthetized only) or LPMV (group PM and group BPM, respectively, in which the rabbits received ventilation with 50% oxygen at a tidal volume of 8 mL/kg for 5 h). The concentrations of LTB4 and cyclic adenosine monophosphate (cAMP) in the lung tissues were analyzed by ELISA. cAMP content, protein kinase A (PKA) protein expression and the Rap1-GTP protein to total Rap1 protein ratio were determined to assess the activities of cAMP/PKA and Rap1 signaling pathways. The lung injury was evaluated by assessing lung permeability index, lung wet/dry weight ratio, polymorphonuclear leukocyte (PMN) count in bronchoalveolar lavage fluid (BALF), pulmonary myeloperoxidase (MPO) activity and lung histological scores.@*RESULTS@#None of the examined parameters differed significantly between group S and group BS. All the parameters with the exception of lung histological score increased significantly in group PM and group BPM as compared to those in group S (@*CONCLUSIONS@#LPMV can induce LTB4 overproduction to down-regulate cAMP/PKA and Rap1 signaling pathways in the lungs of rabbits, which results in lung hyper-permeability and inflammation. Bestatin can inhibit LTB4 production in the lungs to protect against LPMV-induced lung hyper-permeability and inflammation.
Subject(s)
Animals , Rabbits , Bronchoalveolar Lavage Fluid , Leukotriene B4 , Lung , Lung Injury/prevention & control , Neutrophils , Respiration, Artificial/adverse effectsABSTRACT
RAP1 is a well-known telomere-binding protein, but its functions in human stem cells have remained unclear. Here we generated RAP1-deficient human embryonic stem cells (hESCs) by using CRISPR/Cas9 technique and obtained RAP1-deficient human mesenchymal stem cells (hMSCs) and neural stem cells (hNSCs) via directed differentiation. In both hMSCs and hNSCs, RAP1 not only negatively regulated telomere length but also acted as a transcriptional regulator of RELN by tuning the methylation status of its gene promoter. RAP1 deficiency enhanced self-renewal and delayed senescence in hMSCs, but not in hNSCs, suggesting complicated lineage-specific effects of RAP1 in adult stem cells. Altogether, these results demonstrate for the first time that RAP1 plays both telomeric and nontelomeric roles in regulating human stem cell homeostasis.
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Objective: To investigate the effects of Hedyotis diffusa Willd (HDW) on the proliferation, cell cycle, apoptosis, migration and invasion of human renal carcinoma cells, and to explore the possible mechanisms. Methods: After human renal adenocarcinoma ACHN cells and human renal proximal tubule HK-2 cells were treated with HDW for 24 h, the cell proliferation was detected by CCK-8 assay, and the cell cycle and apoptosis were detected by FCM assay. The phenotypes of ACHN and HK-2 cells treated with HDW were observed by light microscopy and rhodamine staining. The expressions of mesenchymal-epithelial transition (MET)-related proteins were detected by immunofluorescence staining and Western blotting, respectively. The effects of HDW on the migration and invasion of ACHN and HK-2 cells were detected by wound-healing and Transwell chamber assay. RNAsequencing was used to detect the effect of HDW on the transcriptome in ACHN cells. The expression levels of RAP1 pathway-related genes [RAP1 GTPase activating protein (RAP1GAP), RAS guanyl releasing protein 2 (RASGRP2), RAP guanine nucleotide exchange factor 3 (RAPGEF3), membrane-associated guanylate kinase, WW and PDZ domain containing 1 (MAGI1) and G protein subunit alpha i1 (GNAI1)] were detected by real-time fluorescent quantitative PCR. The expression levels of mitogen-activate protein kinase (MAPK) pathway-related proteins [c-Jun N -terminal kinase (JNK), phospho-JNK (p-JNK), protein kinase B (PKB, Akt), phospho-Akt (p-Akt), extracellular signalregulated kinase (ERK) and phospho-ERK (p-ERK)] were detected by Western blotting. Results: HDW selectively inhibited the proliferation of ACHN cells (P < 0.01), blocked cell cycle at S-phase (P < 0.01), and induced apoptosis (P < 0.01). After treatment with HDW, the morphology of ACHN cells significantly changed. HDW promoted the expressions of MET-related proteins (E-cadherin 1 and β-catenin) in ACHN cells, and inhibited the expressions of epithelial-mesenchymal transition (EMT)-related proteins (Vimentin and Snail1) (all P < 0.01). HDW inhibited the migration and invasion of ACHN and HK-2 cells (all P < 0.01), and there was no significant difference between ACHN and HK-2 cells. HDW affected the expressions of cell cycle-related genes and transcription factor E2F and Myc target genes, and activated the p53 signaling pathway. HDW significantly downregulated the expression levels of RAP1GAP, RASGRP2, RAPGEF3, MAGI1 and GNAI1 (all P < 0.000 1) and the phosphorylation level of JNK in ACHN cells. Conclusion: HDW may selectively inhibit cell proliferation, and promote MET, cell cycle arrest and apoptosis of ACHN cells by inhibiting RAP1-JNK signaling pathway.
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Objective To observe the expression ofRapl,guanosine triphosphate-Rapl (GTP-Rapl),vascular endothelial growth factor (VEGF) and β-catenin in experimental choroidal neovascularization (CNV).Methods Forty-two brown Norwegian rats were randomly divided into a blank control group (7 rats) and a model group (35 rats).Both eyes were enrolled.The CNV model was established by holmium ion laser photocoagulation in the model group.At 3,7,14,21,and 28 days after photocoagulation,fluorescein fundus angiography (FFA) and choroidal vascular smear were performed to observe the degree of fluorescein leakage and CNV area in rats;Western blot and real-time quantitative polymerase chain reaction (RT-PCR) were used to detect the expression ofRap1,GTP-Rap1,VEGF,β-catenin and mRNA in CNV.Results The results of FFA examination showed that a large disc-shaped fluorescein leaked in the photo-condensation spot 14 days after photocoagulation.Laser confocal microscopy showed that compared with 7 days after photocoagulation,CNV area increased at 14,21,28 days after photocoagulation,and the difference were statistically significant (t=3.725,5.532,3.605;P＜0.05).Western blot showed that there was no significant difference in the relative expression of Rap1 protein in CNV at different time points after photocoagulation between the two groups (P=0.156).Compared with the blank control group,the relative expression of GTP-Rap1 protein was significantlydecreased,the relative expression of VEGF and β-catenin protein were significantly increased in the model group (P=0.000).The results of RT-PCR showed that there was no significant difference in the relative expression of Rap 1 mRNA at different time points after photocoagulation between the two groups (P=0.645),but there were significant difference in the relative expression of β-catenin mRNA (P=0.000).At 7,14,21 and 28 days after photocoagulation,there were significant difference in the relative expression of GTP-Rap 1 and VEGF mRNA between the two groups (P=0.000).Conclusions The expression of GTP-Rap1 in experimental CNV is significantly lower than that in normal rats.
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OBJECTIVE In this study we explored the role of Epac1-Rap1 pathway in the acute myocardial ischemia/reperfusion injury (MIRI) in vitro and in vivo. METHODS An acute myocardial ischemia/reperfusion injury model was established by the ligation of left anterior descending coronary. Myocardial architecture, fibers and apoptosis was evaluated by the Masson trichrome staining, Sirius red staining and TUNEL assay.H9c2 cells were subjected to hypoxia for 5 h followed by 1-h reoxygen-ation in vitro.Cell viability was measured by MTT assay and cellular injury was evaluated by measuring the release of lactate dehydrogenase (LDH). Western blot, real-time PCR and immunofluorescence were used to detect the expressions of Epac1 and relative downstream molecules.RESULTS Myocardial IR-induced cardiac apoptosis and accumulation of Epac1 and Rap1 in rat IR injury model.Direct Epac activation by 8-CPT(8-(4-chlorophenylthio)-2′-O-methyl-cAMP)exacerbated cardiomyocyte death and dysfunction following hypoxia-reoxygenation(H/R),selective activation of Epac in response to H/R was evident which enriched for cytosolic/membrane proteins and mRNA. Harmacological inhibitor of Epac (ESI-09)significantly ameliorated myocardial injury with the decline of Epac expression.Epac inhibitor and agonist studies also implicated the effect of Rap1, which is downstream of Epac in this pathway. The expression of Rap1 elevated when activated by Epac agonist and was blocked by Epac inhibitor. The same result was true for myocyte CaMK-II and intracellular calcium ions activation.Moreover,ESI-09 also increased ERK1/2 phosphorylation. CONCLUSION Our study reveal that Epac1/Rap1 signaling pathway is involved in the pathogenesis of myocardial I/R injury in rats,which provides evidence on the development of therapeutic strategies target this pathway for myocardial I/R injury.
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Objective To explore the methylation status of Rap1 GTPase activating protein (Rap1GAP) promoter in colon cancer, and to provide the oretical basis and research direction for the early diagnosis, targeted therapy, anti-multidrug resistance of colon cancer and so on. Methods The paraffin embedded specimens of 33 patients with colonic adenocarcinoma diagnosed by pathology were analyzed from Department of Pathology of Xinzhou City People′s Hospital from January 2010 to September 2014, including 19 males and 14 females, and aged 41-72 years old. The paraffin embedded specimens of 16 patients with colonic adenoma were enrolled, including 9 males and 7 females, and aged 34-58 years old. 13 normal tissues from the tumor distal margin (from the tumor > 15 cm) were selected. Quantitative methylation specific PCR (q-MSP) was applied to detect methylation level of Rap1GAP gene promoter. The methylation level differences of Rap1GAP gene promoter region among 3 groups or between different clinicopathologic factor subgroups were compared. Results The methylation rates [median (interquartile range)] of Rap1GAP promoter were 65.43 % (50.35 %), 21.37 % (8.39 %) and 17.43 % (15.71 %) in colonic adenocarcinoma group, colonic adenoma group and adjacent normal tissue group, respectively. The methylation rate of colonic adenocarcinoma group was significantly higher than that of colon adenoma group or that of adjacent normal tissue group (P60yearsold:36.26%(62.62%)and26.23%(76.42 %);well-differentiated vs. moderately/poorly-differentiated: 21.98 % (40.32 %) vs. 42.74 % (74.20 %); TNM Ⅰ-Ⅱ vsⅢ-Ⅳ: 25.31 % (48.27 %) vs. 36.26 % (75.55 %); all P> 0.05]. Conclusion The methylation status of RAP1GAP promoter maybe associate with genesis and development of colon cancer, which might be used as a target for early diagnose of colon cancer.
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Objective To choose zebrafish as the experimental animal model for studying the spatiotemporal expression rule of rap1 gen in zebrafish embryo early development process .Methods The Rap1 gene fragment was cloned from the zebrafish emby‐oscDNA ,then the Rap1 gene fragment and pCS2+ plasmid were performed the in vitro connetion and recombination was extracted , the combinant plasmid was correct after the double enzyme digestion ,colony PCR and sequencing identification .T3 RNA polymer‐ase in vitro transcription system was used to obtain the digoxin (DIG)‐labeled anti‐sense mRNA probe of Rap1 gene .The whole mount in situ hybridization method was adopted to detect the Rap1 expression in zebrafish embryo early development process . Results The positive hybridization signal of Rap1 gene was detected at the cell division junction region of 0 .75 hpf ,animal pole of 3 .70 hpf and 6 .00 hpf ,and notochord of 12 .00 -72 .00 hpf .Conclusion Rap1 gene might be involved in the early development process of notochord nervous system in zebrafish .
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Objective To investigate the expression of Rap1 GTPase-activating protein 1 (Rap1GAP),matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9),and their relation with clinical patterns in colorectal carcinoma.Methods Immunohistochemistry was used to detect the expression of Rap1GAP,MMP-2 and MMP-9 in colorectal carcinoma,villous adenoma,tubular adenoma and normal colorectal tissue,and their relationship with clinicopathological parameters was analyzed.Results The positive rate of Rap1GAP expression was 30.4 ％ (14/46),77.8 ％ (14/18),69.6 ％ (16/23) and 95.2 ％ (20/21) in colorectal carcinoma,villous adenoma,tubular adenoma,and normal colorectal tissue,respectively (x2 =30.659,P=0.000).The figures were 71.7 ％ (33/46),55.6 ％ (10/18),52.2 ％ (12/16) and 9.5 ％ (2/21) for the positive rate of MMP-2 expression (x2 =22.459,P =0.000),as well as for 76.1％ (35/46),61.1％ (11/18),56.5 ％ (13/23) and 14.3 ％ (3/21) for the positive rate of MMP-9 expression,respectively (x2 =22.643,P =0.000).In patients with colorectal carcinoma,the expression of Rap1GAP was correlated with tumor differentiation (x2 =5.275,P =0.022),but not sex,age,or lymphatic metastasis (all P ＞ 0.05).The expression of MMP-2 and MMP-9 were correlated with lymphatic metastasis (x2 =6.661,P =0.010;x2 =8.475,P =0.040),but not sex,age or tumor differentiation(all P ＞ 0.05).There was a negative correlation between expression of Rap1GAP and MMP-2,MMP-9 in colorectal carcinoma,respectively (r =-0.424,P =0.003;r =-0.294,P =0.048),but no correlation between the expression of MMP-2 and MMP-9 (r =0.101,P =0.505).Conclusions Rap1GAP,MMP-2 and MMP-9 play important roles in the malignant biological behavior of colorectal carcinoma,and the expression of Rap1GAP is negatively correlated with MMP-2 and MMP-9.The interactions among the three affect the occurrence and development of colorectal carcinoma.
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Objective To explore the over-expression of the CCAAT enhancer binding proteinα(C/EBPα)in hepatic stellate cells (HSC-T6) which activated by protease-activated receptor 1(PAR1) ,to explore its possible relationship with cell proliferation ,cell activation and collagen secretion of HSC-T6 cell .Methods HSC-T6 cells were divided into 4 groups ,control group ,SFLLR group;vehicle plasmid+SFLLR group ,C/EBPαplasmid+SFLLR group .The eukaryotic vector harboring the full length of C/EBPαcD-NA was transfected into PAR1 activated HSC-T6 cell ,then Western blot was applied to detect the expression of α-smooth muscle actin(α-SMA) and collagen I and evaluate the effect of transfection on proliferation of HSC-T6 by MTT .Results The expression of α-SMA and collagen I in C/EBPαplasmid+SFLLR group were dramatically decreased compared with other 3 groups ,as well as proliferation after 48 h(P<0 .05) .Conclusion Over-expression of C/EBPαgene by transfection had inhibitory influence on prolif-eration ,activation and collagen secretion of HSC-T6 which were activated by PAR1 .
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The expression of Rap1 GAP protein was detected in 69 cases of papillary thyroid carcinoma and adjacent normal thyroid tissues with immunohistochemistry method.Methylation of Rapl GAP gene was analyzed by methylation-specific-PCR (MSP) in these tissues.The immunohistochemistry results indicated that the Rap1GAP protein was down-regulated in tumor tissues of 54 ( 78％ ) cases compared with normal thyroid tissues.Statistical analysis demonstrated that the decreased level of Rap1 GAP protein expression was significantly correlated with the tumor stage T according to American Joint Committee on Cancer ( AJCC,P =0.043 ).The MSP results demonstrated that 46 cases of Rap1GAP gene methylation were detected in 54 cases with down-regulated expression of Rap1GAP (66.7％),but only 1 case of methylation was found in 15 cases without obvious change of Rap1GAP expression (6.67％),showing significant difference ( P＜0.01 ).There was no methylation in normal thyroid tissues.These results suggest that raised methylation of promoter region may contribute to the low expression of Rap1GAP protein in papillary thyroid carcinoma.
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Rap 1 is expressed in human umbilical vein endothelial cells (HUVECs).Rap1-GTPase activating protein (Rap 1 GAP),with its specific target,Rap 1,has been shown to be important in the regulation of many physiological and certain pathological processes.In this study,we investigated the effect of RaplGAP expression on endothelial cell function,or,more specifically,proliferation and migration of endothelial cells.HUVECs were transfected with pcDNA3.1 (empty vector),pcDNA3.1 containing Flag-tagged-Rap1GAP or Myc-tagged-Rap1N17.The proliferation,migration and tube formation were examined and compared among the 3 groups.Expression of Rap1,Rap1GAP,extracellular signal-regulated kinase (ERK),phospho-ERK,Akt,phosphor-Akt was detected by Western blotting.The results showed that the proliferation,migration and tube formation were significantly reduced in Rap1GAP- and Rap1N17-transfected HUVECs as compared with empty vector-transfected control.These changes were coincident with increased expression of Rap 1 GAP and decreased expression of activated Rap 1,phospho-ERK and -Akt.After treatment of Rap 1 GAP-transfected HUVECs with a stimulator of Rapl guanine-nucleotide-exchange factor (Rap1GEF) 8CPT-2'OMe-cAMP,it was found that Rap1 activity was decreased as compared with empty vector-transfected control.Pretreatment of HUVECs with an ERK inhibitor PD98059 or a PI3K inhibitor LY294002 prior to stimulation not only blocked 8CPT-2'OMe-cAMP-induced phosphorylation of ERK and Akt,but also significantly reduced cell proliferation and migration.Finally,we examined the effect of vascular endothelial growth factor (VEGF) on HUVECs overexpressing Rap1GAP.VEGF-stimulated Rap1 activity,phosphorylation of ERK and Akt,cyclin D1 expression and cell proliferation were repressed in HUVECs overexpressing Rap 1 GAP as compared to empty vector-transfected control.Taken together,our findings demonstrate that Rap1GAP/Rap1 and their downstream effectors regulate proliferation and migration of HUVECs via ERK and Akt pathways.
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Rat pheochromocytoma (PC12) cells have been used to investigate neurite outgrowth. Nerve growth factor (NGF) has been well known to induce neurite outgrowth from PC12 cells. RhoA belongs to Ras-related small GTP-binding proteins, which regulate a variety of cellular processes, including cell morphology alteration, actin dynamics, and cell migration. NGF suppressed GTP-RhoA levels after 12 h in PC12 cells and was consistently required for a long time to induce neurite outgrowth. Constitutively active (CA)-RhoA suppressed neurite outgrowth from PC12 cells in response to NGF, whereas dominant-negative (DN)-RhoA stimulated it, suggesting that RhoA inactivation is essential for neurite outgrowth. Here, we investigated the mechanism of RhoA inactivation. DN-p190RhoGAP abrogated neurite outgrowth, whereas wild-type (WT)-p190RhoGAP and WT-Src synergistically stimulated it along with accelerating RhoA inactivation, suggesting that p190RhoGAP, which can be activated by Src, is a major component in inhibiting RhoA in response to NGF in PC12 cells. Contrary to RhoA, Rap1 was activated by NGF, and DN-Rap1 suppressed neurite outgrowth, suggesting that Rap1 is also essential for neurite outgrowth. RhoA was co-immunoprecipitated with Rap1, suggesting that Rap1 interacts with RhoA. Furthermore, a DN-Rap-dependent RhoGAP (ARAP3) prevented RhoA inactivation, abolishing neurite formation from PC12 cells in response to NGF. These results suggest that NGF activates Rap1, which, in turn, up-regulates ARAP3 leading to RhoA inactivation and neurite outgrowth from PC12 cells. Taken together, p190RhoGAP and ARAP3 seem to be two main factors inhibiting RhoA activity during neurite outgrowth in PC12 cells in response to NGF.
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To find the underlying causes of primary myelodysplastic syndrome (MDS), the gene expression profiling of both CD34+ cells and bone marrow mononuclear cells from MDS patients was performed using oligonucleotide microarray and cDNA microarrays, respectively. Several candidate genes which were differentially expressed in MDS patients versus normal controls were selected and confirmed in expanding samples by quantitative real-time reverse transcription-polymerase chain reaction after clustering and bioinformatics analysis. one of these genes, RAP1GAP, was found to be expressed at a significantly higher level in patients with MDS in comparison with those suffering from other hematopoietic diseases including leukemia (P < 0.01). We propose that over-expression of RAP1GAP gene may play a role in the pathogenesis of MDS.