Prostate cancer is a major public health concern world-wide, being one of the most prevalent cancers in men. Great improvements have been made both in terms of early diagnosis and therapeutics. However, there is still...Prostate cancer is a major public health concern world-wide, being one of the most prevalent cancers in men. Great improvements have been made both in terms of early diagnosis and therapeutics. However, there is still an urgent need for reliable biomarkers that could overcome the lack of cancer-specifcity of prostate-specifc antigen, as well as alternative therapeutic targets for advanced metastatic cases. Reversible phosphorylation of proteins is a post-translational modifcation critical to the regulation of numerous cellular processes. Phosphoprotein phosphatase 1 （PPP1） is a major serine/threonine phos-phatase, whose specifcity is determined by its interacting proteins. These interactors can be PPP1 substrates, regulators, or even both. Deregulation of this protein-protein interaction network alters cell dynamics and underlies the development of several cancer hallmarks. Therefore, the identification of PPP1 interactome in specific cellular context is of crucial importance. The knowledge on PPP1 complexes in prostate cancer remains scarce, with only 4 holoenzymes characterized in human prostate cancer models. However, an increasing number of PPP1 interactors have been identifed as expressed in human prostate tissue, including the tumor suppressors TP53 and RB1. Efforts should be made in order to identify the role of such proteins in prostate carcinogenesis, since only 26 have yet well-recognized roles. Here, we revise literature and human protein databases to provide an in-depth knowledge on the biological significance of PPP1 complexes in human prostate carcinogenesis and their potential use as therapeutic targets for the development of new therapies for prostate cancer.展开更多
BACKGROUND Perianal fistulae are either primary(idiopathic)or secondary[commonly associated with Crohn’s disease,(CD)].It is assumed,although not proven,that the pathophysiology differs.AIM To systematically compare ...BACKGROUND Perianal fistulae are either primary(idiopathic)or secondary[commonly associated with Crohn’s disease,(CD)].It is assumed,although not proven,that the pathophysiology differs.AIM To systematically compare the clinical phenotypes,cytokine and phosphoprotein profiles of idiopathic and CD-related perianal fistulae.METHODS Sixty-one patients undergoing surgery for perianal fistula were prospectively recruited(48 idiopathic,13 CD)into a cohort study.Clinical data,including the Perineal Disease Activity Index(PDAI)and EQ-5D-5L were collected.Biopsies of the fistula tract,granulation tissue,internal opening mucosa and rectal mucosa were obtained at surgery.Concentrations of 30 cytokines and 39 phosphoproteins were measured in each biopsy using a magnetic bead multiplexing instrument and a chemiluminescent antibody array respectively.Over 12000 clinical and 23500 laboratory measurements were made.RESULTS The PDAI was significantly higher(indicating more active disease)in the CD group with a mean difference of 2.40(95%CI:0.52-4.28,P=0.01).Complex pathoanatomy was more prevalent in the CD group,namely more multiple fistulae,supralevator extensions,collections and rectal thickening.The IL-12p70 concentration at the internal opening specimen site was significantly higher(median difference 19.7 pg/mL,99%CI:0.2-40.4,P=0.008)and the IL-1RA/IL-1βratio was significantly lower in the CD group at the internal opening specimen site(median difference 15.0,99%CI=0.4-50.5,P=0.008).However in the remaining 27 cytokines and all 39 of the phosphoproteins across the four biopsy sites,no significant differences were found between the groups.CONCLUSION CD-related perianal fistulae are more clinically severe and anatomically complex than idiopathic perianal fistulae.However,overall there are no major differences in cytokine and phosphoprotein profiles.展开更多
AIM:To determine the effect and molecular mechanism of ezrin-radixin-moesin-binding phosphoprotein-50(EBP50) in hepatocellular carcinoma(HCC).METHODS:Three human HCC cell lines,i.e.,SMMC7721,HepG2 and Hep3B,were used....AIM:To determine the effect and molecular mechanism of ezrin-radixin-moesin-binding phosphoprotein-50(EBP50) in hepatocellular carcinoma(HCC).METHODS:Three human HCC cell lines,i.e.,SMMC7721,HepG2 and Hep3B,were used.We transfected the Pbk-CMV-HA-EBP50 plasmid into SMMC7721 cells with Lipofectamine 2000 to overexpress EBP50.Western blotting were performed to determine the effects of the plasmid on EBP50 expression and to detect the expression of β-catenin and E-cadherin before and after the transfection of the plasmid into SMMC7721 cells.In vitro cell proliferation was assessed with a Cell Counting Kit-8(CCK-8) assay.Cell cycle distribution was assessed with flow cytometry.Invasion and migration ability of before and after the transfection were determined with a transwell assay.Cell apoptosis was demonstrated with Annexin V-FITC.The effect of EBP50 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice.RESULTS:The transfection efficiency was confirmed with Western blotting(1.36 ± 0.07 vs 0.81 ± 0.09,P < 0.01).The CCK8 assay demonstrated that the growth of cells overexpressing EBP50 was significantly lower than control cells(P < 0.01).Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing EBP50(61.3% ± 3.1% vs 54.0% ± 2.4%,P < 0.05).The transwell assay showed that cell invasion and migration were significantly inhibited in cells overexpressing EBP50 compared with control cells(5.8 ± 0.8 vs 21.6 ± 1.3,P < 0.01).Annexin V-FITC revealed that apoptosis was significantly increased in cells overexpressing EBP50 compared with control cells(14.8% ± 2.7% vs 3.4% ± 1.3%,P < 0.05).The expression of β-catenin was downregulated and E-cadherin was upregulated in cells overexpressing EBP50 compared with control cells(0.28 ± 0.07 vs 0.56 ± 0.12,P < 0.05;0.55 ± 0.08 vs 0.39 ± 0.07,P < 0.05).In vivo tumor growth assay confirmed that up-regulation of EBP50 could obviously slow the growth of HCC derived from SMMC7721 cells(28.9 ± 7.2 vs 70.1 ± 7.2,P < 0.01).CONCLUSION:The overexpression of EBP50 could inhibit the growth of SMMC7721 cells and promote apoptosis by modulating β-catenin,E-cadherin.EBP50 may serve asa potential therapeutic target in HCC.展开更多
AIM： To elucidate the localization of vasodilator stimulated phosphoprotein （VASP）, a cytoskeletal organizing protein and a substrate of protein kinases A and G in mitotic gastric cancer cells. METHODS： Immunofluo...AIM： To elucidate the localization of vasodilator stimulated phosphoprotein （VASP）, a cytoskeletal organizing protein and a substrate of protein kinases A and G in mitotic gastric cancer cells. METHODS： Immunofluorescence microscopy was used to observe the localization of α-tubulin, VASP and Ser157 phosphorylated VASP （p-VASP） in interphase of mitotic gastric cancer of the cell line SGC-7901. RESULTS： Immunofluorescence staining showed that p-VASP but not VASP was co-localized with α-tubulin on spindle poles and fibers in prophase, metaphase and anaphase of the mitotic process of the gastric cancer cell line SGC-7901. H89, an inhibitor of protein kinases A and G, had no effect on the localization of p-VASP on the spindles. CONCLUSION： VASP may play a role in assembling and stabilizing the mitotic spindle of cells, and phosphorylation of the protein is the precondition for it to exert this function.展开更多
A sensitive method based on the fluorescence quenching effect of the Tb^3＋-Tiron complex is proposed for the determination of alkali-labile phosphoprotein phosphorus （ALP） released from fish plasma. The detection l...A sensitive method based on the fluorescence quenching effect of the Tb^3＋-Tiron complex is proposed for the determination of alkali-labile phosphoprotein phosphorus （ALP） released from fish plasma. The detection limit was 5.4 ng/ml （S/N=2）, and the relative standard deviation of the quenching effect （6 replicates） was 4.6%. The results obtained by the proposed method were in good agreement with those obtained by the colorimetric assay. The advantages of the present method are its relatively simple detection procedure, the lack of toxic organic solvents, and high sensitivity.展开更多
Human parainfluenza virus type 3(HPIV3),a member of the Paramyxoviridae family,can cause lower respiratory disease in infants and young children.The phosphoprotein(P)of HPIV3 is an essential cofactor of the viral RNA-...Human parainfluenza virus type 3(HPIV3),a member of the Paramyxoviridae family,can cause lower respiratory disease in infants and young children.The phosphoprotein(P)of HPIV3 is an essential cofactor of the viral RNA-dependent RNA polymerase large protein(L).P connects nucleocapsid protein(N)with L to initiate genome transcription and replication.Sumoylation influences many important pathways of the target proteins,and many viral proteins are also themselves sumoylated.In this study,we found that the P of HPIV3 could be sumoylated,and mutation of K492 and K532 to arginine(PK492 R/K532 R)failed to be sumoylated within P,which enhances HPIV3 minigenome activity.Biochemical studies showed that PK492 R/K532 Rhad no effect on its interactions with N,formation of homo-tetramers and formation of inclusion bodies.Finally,we found that incorporation of K492 R/K532 R into a recombinant HPIV3(rHPIV3-PK492 R/K532 R)increased viral production in culture cells,suggesting that sumoylation attenuates functions of P and down-regulates viral replication.展开更多
Objective:To investigate the potential role of human cytomegalovirus lower matrix phosphoprotein 65(HCMV-pp65) in murine systemic lupus erythematosus(SLE).Methods:The prokaryotic plasmid pET-28b-pp65 was constructed t...Objective:To investigate the potential role of human cytomegalovirus lower matrix phosphoprotein 65(HCMV-pp65) in murine systemic lupus erythematosus(SLE).Methods:The prokaryotic plasmid pET-28b-pp65 was constructed to express the HCMVpp65 protein.BXSB mice and C57BL/6 mice were inoculated with pp65 eukarvotic plasmid pcDNA3.0-pp65 intramuscularly 5 times at 2-week intervals,and then the blood of the mice was subsequently collected via the retro-orbital vein.Indirect ELISAs were used to evaluate the concentration of anti-pp65 immunoglobulin G,anti-double-stranded DNA and antinuclear antibodies.lnterleukin-1β and tumor necrosis factor-α were also determined by competitive ELISA.At the same time,3 major SLE-related circulating microRNAs were examined by quantitative RT-PCR.Results:The early production of autoantibodies was observed in pp65-immunized male BXSB as well as C57BL/6 mice.Overexpression of interleukin-1β and tumor necrosis factor-a were detected in pp65-immunized male BXSB mice.Quantitative RT-PCR analyses showed that three SLE related microRNAs(microRNA-126,microRNA-125 a,and microRKA-146a) were dovvnrcgulatcd in peripheral blood mononuclear cells of pp65-immunizcd mice.Conclusions:Our findings indicate that HCMV-pp65 immunization strongly triggers the development and progression of" SLE-like disease in both BXSB and C57BL/6 mice,which indicates that the immune responses induced by HCMV-pp65 may be involved in the development of SLE.展开更多
A novel phosphoprotein separation material was developed, which is constructed by a magnetic mesoporous Fe3 O4@TiO2（Fe3 O4@mTiO2） microsphere and a 5-aminoisophthalic acid（AIPA） monolayer that provides additional ...A novel phosphoprotein separation material was developed, which is constructed by a magnetic mesoporous Fe3 O4@TiO2（Fe3 O4@mTiO2） microsphere and a 5-aminoisophthalic acid（AIPA） monolayer that provides additional binding sites toward phosphate groups. The results of characteristic experiments demonstrated that Fe3 O4@mTiO2-AIPA had good dispersability, high magnetic susceptibility, and satisfactory grafting ratio of AIPA, ascribed to the large specific surface area of the inorganic substrate. Taking advantages of these features, Fe3 O4@mTiO2-AIPA was successfully utilized to separate α-casein（a typical phosphoprotein） and bovine serum albumin（BSA, a typical non-phosphoprotein） from their mixtures（molar ratio = 1:2）. Through adjusting pH and polarity of solutions, the BSA and α-casein were respectively enriched in washing fraction and elution fraction. This result displays the good potential of Fe3 O4@mTiO2-AIPA for application in phosphoprotein enrichment.展开更多
Trastuzumab resistance is one of the causes of poor prognosis in patients with human epidermal growth factor receptor 2(HER2)-positive(HER2+)breast cancer(BC).The truncated isoform of dopamine-and cAMPregulated phosph...Trastuzumab resistance is one of the causes of poor prognosis in patients with human epidermal growth factor receptor 2(HER2)-positive(HER2+)breast cancer(BC).The truncated isoform of dopamine-and cAMPregulated phosphoprotein(t-DARPP)has been reported to be involved in trastuzumab therapy resistance and promoting tumor progression.To evaluate the t-DARPP expression in BC,paired tumors and surrounding normal tissues were analyzed by real-time polymerase chain reaction and confirmed higher DARPP-32 kDa family mRNA expression in HER2+BC tumor tissues.We established 2 patient-derived xenografts(PDX)mice models to test the efficacy of trastuzumab,named model 1(non-responder)and model 2(responder).t-DARPP and p95-HER2 protein-protein interactions were detected in PDX tumor tissue from non-responders using Förster resonance energy transfer assays.Instead,there is no response from the responder.Furthermore,mechanistic studies using transwell and western blot assays demonstrated that t-DARPP could upregulate epithelial-mesenchymal transition signaling proteins,enhance p95-HER2 expression and promote cell migration.We found that quercetin effectively reduced t-DARPP expression in HER2+BC cells.In t-DARPP ShRNA-suppressed cells,quercetin synergistically enhanced trastuzumab-induced apoptotic cell death and G2/M phase arrest.In conclusion,the combination of quercetin and trastuzumab treatment by targeting t-DARPP in HER2+BC patients has the potential as a biomarker for mitigating drug resistance.展开更多
BACKGROUND Dopamine and cyclic adenosine monophosphate(cAMP)-regulated phosphop-rotein with an apparent Mr of 32000(DARPP-32)is a protein that is involved in regulating dopamine and cAMP signaling pathways in the brai...BACKGROUND Dopamine and cyclic adenosine monophosphate(cAMP)-regulated phosphop-rotein with an apparent Mr of 32000(DARPP-32)is a protein that is involved in regulating dopamine and cAMP signaling pathways in the brain.However,recent studies have shown that DARPP-32 is also expressed in other tissues,including colorectal cancer(CRC),where its function is not well understood.AIM To explore the effect of DARPP-32 on CRC progression.METHODS The expression levels of DARPP-32 were assessed in CRC tissues using both quantitative polymerase chain reaction and immunohistochemistry assays.The proliferative capacity of CRC cell lines was evaluated with Cell Counting Kit-8 and 5-ethynyl-2’-deoxyuridine assays,while apoptosis was measured by flow cytometry.The migratory and invasive potential of CRC cell lines were deter-mined using wound healing and transwell chamber assays.In vivo studies involved monitoring the growth rate of xenograft tumors.Finally,the underlying molecular mechanism of DARPP-32 was investigated through RNA-sequencing and western blot analyses.RESULTS DARPP-32 was frequently upregulated in CRC and associated with abnormal clinicopathological features in CRC.Overexpression of DARPP-32 was shown to promote cancer cell proliferation,migration,and invasion and reduce apoptosis.DARPP-32 knockdown resulted in the opposite functional effects.Mechanistically,DARPP-32 may regulate the phosphoinositide 3-kinase(PI3K)/AKT signaling pathway in order to carry out its biological function.CONCLUSION DARPP-32 promotes CRC progression via the PI3K/AKT signaling pathway.展开更多
Reversible protein phosphorylation is a central regulatory mechanism of cell function. Deregulation of the balanced actions of protein kinases and phosphatases has been frequently associated with several pathological ...Reversible protein phosphorylation is a central regulatory mechanism of cell function. Deregulation of the balanced actions of protein kinases and phosphatases has been frequently associated with several pathological conditions, including cancer. Many studies have already addressed the role of protein kinases misregulation in cancer. However, much less is known about protein phosphatases influence. Phosphoprotein Phosphatase 1 (PPP1) is one of the major serine/threonine protein phosphatases who has three catalytic isoforms: PPP1CA, PPP1CB, and PPP1CC. Its function is achieved by binding to regulatory subunits, known as PPP1-interacting proteins (PIPs), which may prefer a catalytic isoform. Also, some inhibitors/enhancers may exhibit isoform specificity. Here we show that, prodigiosin (PG), a molecule with anticancer properties, promotes the formation of PPP1CA-AKT complex and not of PPP1CC-MAPK complex. Both, AKT and MAPK, are well-known PIPs from two pathways that crosstalk and regulate melanoma cells survival. In addition, the analysis performed using surface plasmon resonance (SPR) technology indicates that PPP1 interacts with obatoclax (OBX), a drug that belongs to the same family of PG. Overall, these results suggest that PG might, at least in part, act through PPP1C/PIPs. Also, this study is pioneer in demonstrating PPP1 isoform-specific modulation by small molecules.展开更多
Background:Cancer-associated fibroblasts(CAFs)play an important role in the induction of chemo-resistance.This study aimed to clarify the mechanism underlying CAF-mediated resistance to two tyrosine kinase inhibitors(...Background:Cancer-associated fibroblasts(CAFs)play an important role in the induction of chemo-resistance.This study aimed to clarify the mechanism underlying CAF-mediated resistance to two tyrosine kinase inhibitors(TKIs),sorafenib and lenvatinib,and to identify a novel therapeutic target for overcoming TKI resistance in hepatocellular carcinoma(HCC).Methods:We performed a systematic integrative analysis of publicly available gene expression datasets and whole-transcriptome sequencing data from 9 pairs of CAFs and para-cancer fibroblasts isolated from human HCC and para-tumor tissues,respectively,to identify key molecules that might induce resistance to TKIs.We then performed in vitro and in vivo experiments to validate selected targets and related mechanisms.The associations of plasma secreted phosphoprotein 1(SPP1)expression levels before sorafenib/lenvatinib treatment with progression-free survival(PFS)and overall survival(OS)of 54 patients with advanced HCC were evaluated using Kaplan-Meier and Cox regression analysis.Results:Bioinformatic analysis identified CAF-derived SPP1 as a candidate molecule driving TKI resistance.SPP1 inhibitors reversed CAF-induced TKI resistance in vitro and in vivo.CAF-derived SPP1 activated rapidly accelerated fibrosarcoma(RAF)/mitogen-activated protein kinase(MAPK)and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)through the integrin-protein kinase C-alpha(PKCα)signaling pathway and promoted epithelial-to-mesenchymal transition(EMT).A high plasma SPP1 level before TKI treatment was identified as an independent predictor of poor PFS(P=0.026)and OS(P=0.047)in patients with advanced HCC after TKI treatment.Conclusions:CAF-derived SPP1 enhances TKI resistance in HCC via bypass activation of oncogenic signals and EMT promotion.Its inhibition represents a promising therapeutic strategy against TKI resistance inHCC.Moreover,plasma SPP1 level before TKI treatment represents a potential biomarker for treatment response prediction.展开更多
Aim:Extracellular matrix(ECM)-adhesions and their interaction with actin cytoskeleton are fundamental for hepatocellular carcinoma(HCC).Fascin-1,an actin-bundling protein,is correlated with poor HCC prognosis,and is k...Aim:Extracellular matrix(ECM)-adhesions and their interaction with actin cytoskeleton are fundamental for hepatocellular carcinoma(HCC).Fascin-1,an actin-bundling protein,is correlated with poor HCC prognosis,and is known regarding the molecular mechanism of its action.In this study,the authors investigated Fascin-1 basic molecular mechanism and cellular properties in HCC cells.Methods:Fascin-1 was silenced by small interfering RNA and the expression of actin.The ECM-adhesion-related proteins were assessed along with the cells’adhesion capacity in two cell lines that differ in terms of aggressiveness;the hepatoma cell line PLC/PRF/5(Alexander)and the highly invasive HCC cell line HepG2.Results:This study shows that Fascin-1 is upregulated in HepG2 cells compared to Alexander cells and when silenced leads to increased cell adhesion only in HepG2,while at the same time is associated with reduced migfilin and vasodilator-stimulated phosphoprotein(VASP)expression.Conclusion:This is the first study to show that Fascin-1 contributes to a more aggressive phenotype in HCC cells and acts through migfilin and VASP.展开更多
Studies on the mechanism of protein phosphorylation and therapeutic interventions of its related molecular processes are limited by the difficulty in the production of purpose-built phosphoproteins harboring site-spec...Studies on the mechanism of protein phosphorylation and therapeutic interventions of its related molecular processes are limited by the difficulty in the production of purpose-built phosphoproteins harboring site-specific phosphorylated amino acids or their nonhydrolyzable analogs.Here we address this limitation by customizing the cell-free protein synthesis(CFPS)machinery via chassis strain selection and orthogonal translation system(OTS)reconfiguration screening.The suited chassis strains and reconfigured OTS combinations with high orthogonality were consequently picked out for individualized phosphoprotein synthesis.Specifically,we synthesized the sfGFP protein and MEK1 protein with site-specific phosphoserine(O-pSer)or its nonhydrolyzable analog,2-amino-4-phosphonobutyric acid(C-pSer).This study successfully realized building cell-free systems for site-specific incorporation of phosphonate mimics into the target protein.Our work lays the foundation for developing a highly expansible CFPS platform and the streamlined production of user-defined phosphoproteins,which can facilitate research on the physiological mechanism and potential interference tools toward protein phosphorylation.展开更多
Rabies virus(RABV)phosphoprotein(P)plays an important role in disrupting host interferon(IFN)-mediated antiviral immune response.ABCE1 is known as an RNase L inhibitor that negatively regulates the 2′,5′-oligoadenyl...Rabies virus(RABV)phosphoprotein(P)plays an important role in disrupting host interferon(IFN)-mediated antiviral immune response.ABCE1 is known as an RNase L inhibitor that negatively regulates the 2′,5′-oligoadenylate(2-5A)/RNase L antiviral system related to the IFN signaling pathway.In this study,we screened the host factors associated with RABV P protein,while focusing on ABCE1 because of its role in the life cycle of several viruses.Our results showed that knockout of ABCE1 in HEK293T cells inhibited RABV replication.In contrast,the overexpression of ABCE1 in HEK293T cells enhanced RABV replication.Notably,the co-immunoprecipitation assay showed that ABCE1 interacted with RABV P protein.Since ABCE1 and RABV P proteins are related to the IFN signaling pathway,we propose that the interaction of viral P protein with ABCE1 leads to the disruption of host antiviral immune response.In summary,our research showed that ABCE1 is an important host protein that interacts with viral P protein and regulates RABV replication.Moreover,the interaction between ABCE1 and RABV P protein may facilitate the viral P protein-mediated disruption of host antiviral immune response.展开更多
Background and aims:Secreted phosphoprotein 1(SPP1)functions in several physiological processes.The role of SPP1 expression in the prognosis and tumor immunity of hepatocellular carcinoma(HCC)is unknown.The aim of thi...Background and aims:Secreted phosphoprotein 1(SPP1)functions in several physiological processes.The role of SPP1 expression in the prognosis and tumor immunity of hepatocellular carcinoma(HCC)is unknown.The aim of this study was to investigate the expression pattern of SPP1 in HCC and its correlation with prognosis and tumor immunity.Methods:Clinical and gene expression data of The Cancer Genome Atlas-liver hepatocellular carcinoma(LIHC)cohort and 11 other HCC datasets were collected.The Kaplan–Meier method and Cox regression analysis were used to analyze the prognostic value of SPP1.The DESeq2 package in R was used to analyze SPP1-related genes.Gene Ontology analysis and gene set enrichment analysis were used to determine the biological function of SPP1 in HCC.The single sample Gene Set Enrichment Analysis(ssGSEA)method was used to analyze the immune infiltrates of HCC.Illumina human methylation 450 data and level 3 HTSeq-FPKM data from The Cancer Genome Atlas-LIHC were used to analyze the effects of DNA methylation level on SPP1 expression.Results:SPP1 was overexpressed in HCC and correlated with T stage,histological grade,adjacent hepatic tissue inflammation,and vascular invasion in HCC.The analysis of survival rates indicated that high SPP1 levels were associated with poor overall survival in HCC.Functional analysis showed that SPP1 is related to tumor immunity,especially macrophage infiltration.Aberrant demethylation of the promoter region is one of the mechanisms underlying the increase of SPP1 in HCC.Conclusion:Our results indicate that SPP1 is an independent prognostic factor for HCC and is correlated with the clinical features and macrophage infiltration in HCC.展开更多
基金Supported by Fundao para a Ciência e Tecnologia(FCT)(PTDC/QUI-BIQ/118492/2010)Fundo Europeu de Desenvolvimento Regional(FEDER)(FCOMP-01-0124-FEDER-020895),Portugal
文摘Prostate cancer is a major public health concern world-wide, being one of the most prevalent cancers in men. Great improvements have been made both in terms of early diagnosis and therapeutics. However, there is still an urgent need for reliable biomarkers that could overcome the lack of cancer-specifcity of prostate-specifc antigen, as well as alternative therapeutic targets for advanced metastatic cases. Reversible phosphorylation of proteins is a post-translational modifcation critical to the regulation of numerous cellular processes. Phosphoprotein phosphatase 1 （PPP1） is a major serine/threonine phos-phatase, whose specifcity is determined by its interacting proteins. These interactors can be PPP1 substrates, regulators, or even both. Deregulation of this protein-protein interaction network alters cell dynamics and underlies the development of several cancer hallmarks. Therefore, the identification of PPP1 interactome in specific cellular context is of crucial importance. The knowledge on PPP1 complexes in prostate cancer remains scarce, with only 4 holoenzymes characterized in human prostate cancer models. However, an increasing number of PPP1 interactors have been identifed as expressed in human prostate tissue, including the tumor suppressors TP53 and RB1. Efforts should be made in order to identify the role of such proteins in prostate carcinogenesis, since only 26 have yet well-recognized roles. Here, we revise literature and human protein databases to provide an in-depth knowledge on the biological significance of PPP1 complexes in human prostate carcinogenesis and their potential use as therapeutic targets for the development of new therapies for prostate cancer.
基金Supported by Bowel and Cancer Research charity,No.MMBG1J3R
文摘BACKGROUND Perianal fistulae are either primary(idiopathic)or secondary[commonly associated with Crohn’s disease,(CD)].It is assumed,although not proven,that the pathophysiology differs.AIM To systematically compare the clinical phenotypes,cytokine and phosphoprotein profiles of idiopathic and CD-related perianal fistulae.METHODS Sixty-one patients undergoing surgery for perianal fistula were prospectively recruited(48 idiopathic,13 CD)into a cohort study.Clinical data,including the Perineal Disease Activity Index(PDAI)and EQ-5D-5L were collected.Biopsies of the fistula tract,granulation tissue,internal opening mucosa and rectal mucosa were obtained at surgery.Concentrations of 30 cytokines and 39 phosphoproteins were measured in each biopsy using a magnetic bead multiplexing instrument and a chemiluminescent antibody array respectively.Over 12000 clinical and 23500 laboratory measurements were made.RESULTS The PDAI was significantly higher(indicating more active disease)in the CD group with a mean difference of 2.40(95%CI:0.52-4.28,P=0.01).Complex pathoanatomy was more prevalent in the CD group,namely more multiple fistulae,supralevator extensions,collections and rectal thickening.The IL-12p70 concentration at the internal opening specimen site was significantly higher(median difference 19.7 pg/mL,99%CI:0.2-40.4,P=0.008)and the IL-1RA/IL-1βratio was significantly lower in the CD group at the internal opening specimen site(median difference 15.0,99%CI=0.4-50.5,P=0.008).However in the remaining 27 cytokines and all 39 of the phosphoproteins across the four biopsy sites,no significant differences were found between the groups.CONCLUSION CD-related perianal fistulae are more clinically severe and anatomically complex than idiopathic perianal fistulae.However,overall there are no major differences in cytokine and phosphoprotein profiles.
基金Supported by Fundamental Research Funds for the Central Universities of China,No.201130202020005
文摘AIM:To determine the effect and molecular mechanism of ezrin-radixin-moesin-binding phosphoprotein-50(EBP50) in hepatocellular carcinoma(HCC).METHODS:Three human HCC cell lines,i.e.,SMMC7721,HepG2 and Hep3B,were used.We transfected the Pbk-CMV-HA-EBP50 plasmid into SMMC7721 cells with Lipofectamine 2000 to overexpress EBP50.Western blotting were performed to determine the effects of the plasmid on EBP50 expression and to detect the expression of β-catenin and E-cadherin before and after the transfection of the plasmid into SMMC7721 cells.In vitro cell proliferation was assessed with a Cell Counting Kit-8(CCK-8) assay.Cell cycle distribution was assessed with flow cytometry.Invasion and migration ability of before and after the transfection were determined with a transwell assay.Cell apoptosis was demonstrated with Annexin V-FITC.The effect of EBP50 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice.RESULTS:The transfection efficiency was confirmed with Western blotting(1.36 ± 0.07 vs 0.81 ± 0.09,P < 0.01).The CCK8 assay demonstrated that the growth of cells overexpressing EBP50 was significantly lower than control cells(P < 0.01).Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing EBP50(61.3% ± 3.1% vs 54.0% ± 2.4%,P < 0.05).The transwell assay showed that cell invasion and migration were significantly inhibited in cells overexpressing EBP50 compared with control cells(5.8 ± 0.8 vs 21.6 ± 1.3,P < 0.01).Annexin V-FITC revealed that apoptosis was significantly increased in cells overexpressing EBP50 compared with control cells(14.8% ± 2.7% vs 3.4% ± 1.3%,P < 0.05).The expression of β-catenin was downregulated and E-cadherin was upregulated in cells overexpressing EBP50 compared with control cells(0.28 ± 0.07 vs 0.56 ± 0.12,P < 0.05;0.55 ± 0.08 vs 0.39 ± 0.07,P < 0.05).In vivo tumor growth assay confirmed that up-regulation of EBP50 could obviously slow the growth of HCC derived from SMMC7721 cells(28.9 ± 7.2 vs 70.1 ± 7.2,P < 0.01).CONCLUSION:The overexpression of EBP50 could inhibit the growth of SMMC7721 cells and promote apoptosis by modulating β-catenin,E-cadherin.EBP50 may serve asa potential therapeutic target in HCC.
基金Supported by National Natural Science Foundation of China, No. 30340036 and 30470891 Startup Grant from Jiangsu University, and Grant of Zhenjiang Key Institute of Clinical Laboratory Medicine (SH2006066)
文摘AIM： To elucidate the localization of vasodilator stimulated phosphoprotein （VASP）, a cytoskeletal organizing protein and a substrate of protein kinases A and G in mitotic gastric cancer cells. METHODS： Immunofluorescence microscopy was used to observe the localization of α-tubulin, VASP and Ser157 phosphorylated VASP （p-VASP） in interphase of mitotic gastric cancer of the cell line SGC-7901. RESULTS： Immunofluorescence staining showed that p-VASP but not VASP was co-localized with α-tubulin on spindle poles and fibers in prophase, metaphase and anaphase of the mitotic process of the gastric cancer cell line SGC-7901. H89, an inhibitor of protein kinases A and G, had no effect on the localization of p-VASP on the spindles. CONCLUSION： VASP may play a role in assembling and stabilizing the mitotic spindle of cells, and phosphorylation of the protein is the precondition for it to exert this function.
基金Project supported by the Hi-Tech Research and Development Program (863) of China (No. 2006AA06Z424) the National Basic Research Program (973) of China (No. 2003CB415001)the Pilot Project of Knowledge Innovation Program of Chinese Academy of Sciences (No. KJCX2-SW-H06).
文摘A sensitive method based on the fluorescence quenching effect of the Tb^3＋-Tiron complex is proposed for the determination of alkali-labile phosphoprotein phosphorus （ALP） released from fish plasma. The detection limit was 5.4 ng/ml （S/N=2）, and the relative standard deviation of the quenching effect （6 replicates） was 4.6%. The results obtained by the proposed method were in good agreement with those obtained by the colorimetric assay. The advantages of the present method are its relatively simple detection procedure, the lack of toxic organic solvents, and high sensitivity.
基金supported by grants from National Key R&D Program of China(2017YFA0505801)the National Natural Science Foundation of China(81825015,81871650 and 31630086)+2 种基金National Science and Technology Major Project(2018ZX10101004)the Natural Science Foundation of Hubei Province Innovation Group(2017CFA022)Advanced Customer Cultivation Project of Wuhan National Biosafety Laboratory(2019ACCP-MS06)。
文摘Human parainfluenza virus type 3(HPIV3),a member of the Paramyxoviridae family,can cause lower respiratory disease in infants and young children.The phosphoprotein(P)of HPIV3 is an essential cofactor of the viral RNA-dependent RNA polymerase large protein(L).P connects nucleocapsid protein(N)with L to initiate genome transcription and replication.Sumoylation influences many important pathways of the target proteins,and many viral proteins are also themselves sumoylated.In this study,we found that the P of HPIV3 could be sumoylated,and mutation of K492 and K532 to arginine(PK492 R/K532 R)failed to be sumoylated within P,which enhances HPIV3 minigenome activity.Biochemical studies showed that PK492 R/K532 Rhad no effect on its interactions with N,formation of homo-tetramers and formation of inclusion bodies.Finally,we found that incorporation of K492 R/K532 R into a recombinant HPIV3(rHPIV3-PK492 R/K532 R)increased viral production in culture cells,suggesting that sumoylation attenuates functions of P and down-regulates viral replication.
基金Supported by National High Technology Research and Development Program of China(863 program No.2011AA02A116)
文摘Objective:To investigate the potential role of human cytomegalovirus lower matrix phosphoprotein 65(HCMV-pp65) in murine systemic lupus erythematosus(SLE).Methods:The prokaryotic plasmid pET-28b-pp65 was constructed to express the HCMVpp65 protein.BXSB mice and C57BL/6 mice were inoculated with pp65 eukarvotic plasmid pcDNA3.0-pp65 intramuscularly 5 times at 2-week intervals,and then the blood of the mice was subsequently collected via the retro-orbital vein.Indirect ELISAs were used to evaluate the concentration of anti-pp65 immunoglobulin G,anti-double-stranded DNA and antinuclear antibodies.lnterleukin-1β and tumor necrosis factor-α were also determined by competitive ELISA.At the same time,3 major SLE-related circulating microRNAs were examined by quantitative RT-PCR.Results:The early production of autoantibodies was observed in pp65-immunized male BXSB as well as C57BL/6 mice.Overexpression of interleukin-1β and tumor necrosis factor-a were detected in pp65-immunized male BXSB mice.Quantitative RT-PCR analyses showed that three SLE related microRNAs(microRNA-126,microRNA-125 a,and microRKA-146a) were dovvnrcgulatcd in peripheral blood mononuclear cells of pp65-immunizcd mice.Conclusions:Our findings indicate that HCMV-pp65 immunization strongly triggers the development and progression of" SLE-like disease in both BXSB and C57BL/6 mice,which indicates that the immune responses induced by HCMV-pp65 may be involved in the development of SLE.
基金Funded by the National Natural Science Foundation of China(Nos.51473131,21275114,51533007 and 51521001)the Major State Basic Research Development Program of China(973 Program)(No.2013CB933002)+1 种基金Hubei Provincial Department of Education for Financial Assistance Through the “Chutian Scholar” ProgramHubei Provincial Natural Science Foundation of China(No.2014CFA039)
文摘A novel phosphoprotein separation material was developed, which is constructed by a magnetic mesoporous Fe3 O4@TiO2（Fe3 O4@mTiO2） microsphere and a 5-aminoisophthalic acid（AIPA） monolayer that provides additional binding sites toward phosphate groups. The results of characteristic experiments demonstrated that Fe3 O4@mTiO2-AIPA had good dispersability, high magnetic susceptibility, and satisfactory grafting ratio of AIPA, ascribed to the large specific surface area of the inorganic substrate. Taking advantages of these features, Fe3 O4@mTiO2-AIPA was successfully utilized to separate α-casein（a typical phosphoprotein） and bovine serum albumin（BSA, a typical non-phosphoprotein） from their mixtures（molar ratio = 1:2）. Through adjusting pH and polarity of solutions, the BSA and α-casein were respectively enriched in washing fraction and elution fraction. This result displays the good potential of Fe3 O4@mTiO2-AIPA for application in phosphoprotein enrichment.
基金The National Science and Technology Council of Taiwan funded this study.
文摘Trastuzumab resistance is one of the causes of poor prognosis in patients with human epidermal growth factor receptor 2(HER2)-positive(HER2+)breast cancer(BC).The truncated isoform of dopamine-and cAMPregulated phosphoprotein(t-DARPP)has been reported to be involved in trastuzumab therapy resistance and promoting tumor progression.To evaluate the t-DARPP expression in BC,paired tumors and surrounding normal tissues were analyzed by real-time polymerase chain reaction and confirmed higher DARPP-32 kDa family mRNA expression in HER2+BC tumor tissues.We established 2 patient-derived xenografts(PDX)mice models to test the efficacy of trastuzumab,named model 1(non-responder)and model 2(responder).t-DARPP and p95-HER2 protein-protein interactions were detected in PDX tumor tissue from non-responders using Förster resonance energy transfer assays.Instead,there is no response from the responder.Furthermore,mechanistic studies using transwell and western blot assays demonstrated that t-DARPP could upregulate epithelial-mesenchymal transition signaling proteins,enhance p95-HER2 expression and promote cell migration.We found that quercetin effectively reduced t-DARPP expression in HER2+BC cells.In t-DARPP ShRNA-suppressed cells,quercetin synergistically enhanced trastuzumab-induced apoptotic cell death and G2/M phase arrest.In conclusion,the combination of quercetin and trastuzumab treatment by targeting t-DARPP in HER2+BC patients has the potential as a biomarker for mitigating drug resistance.
基金Supported by Chongqing Key Diseases Research and Application Demonstration Program,No.2019ZX003General Project of Chongqing Nature Science Foundation,No.cstc2021jcyj-msxmX0283.
文摘BACKGROUND Dopamine and cyclic adenosine monophosphate(cAMP)-regulated phosphop-rotein with an apparent Mr of 32000(DARPP-32)is a protein that is involved in regulating dopamine and cAMP signaling pathways in the brain.However,recent studies have shown that DARPP-32 is also expressed in other tissues,including colorectal cancer(CRC),where its function is not well understood.AIM To explore the effect of DARPP-32 on CRC progression.METHODS The expression levels of DARPP-32 were assessed in CRC tissues using both quantitative polymerase chain reaction and immunohistochemistry assays.The proliferative capacity of CRC cell lines was evaluated with Cell Counting Kit-8 and 5-ethynyl-2’-deoxyuridine assays,while apoptosis was measured by flow cytometry.The migratory and invasive potential of CRC cell lines were deter-mined using wound healing and transwell chamber assays.In vivo studies involved monitoring the growth rate of xenograft tumors.Finally,the underlying molecular mechanism of DARPP-32 was investigated through RNA-sequencing and western blot analyses.RESULTS DARPP-32 was frequently upregulated in CRC and associated with abnormal clinicopathological features in CRC.Overexpression of DARPP-32 was shown to promote cancer cell proliferation,migration,and invasion and reduce apoptosis.DARPP-32 knockdown resulted in the opposite functional effects.Mechanistically,DARPP-32 may regulate the phosphoinositide 3-kinase(PI3K)/AKT signaling pathway in order to carry out its biological function.CONCLUSION DARPP-32 promotes CRC progression via the PI3K/AKT signaling pathway.
基金supported by grants from Fundacao para a Ciencia e Tecnologia(FCT)of the Portuguese Ministry of Science and Higher Education(PTDC/DTP-PIC/0460/2012)by FEDER through Eixo I do Programa Operacional Fatores de Competitividade(POFC)(FCOMP-01-0124-FEDER-028692)co-funded by QREN
文摘Reversible protein phosphorylation is a central regulatory mechanism of cell function. Deregulation of the balanced actions of protein kinases and phosphatases has been frequently associated with several pathological conditions, including cancer. Many studies have already addressed the role of protein kinases misregulation in cancer. However, much less is known about protein phosphatases influence. Phosphoprotein Phosphatase 1 (PPP1) is one of the major serine/threonine protein phosphatases who has three catalytic isoforms: PPP1CA, PPP1CB, and PPP1CC. Its function is achieved by binding to regulatory subunits, known as PPP1-interacting proteins (PIPs), which may prefer a catalytic isoform. Also, some inhibitors/enhancers may exhibit isoform specificity. Here we show that, prodigiosin (PG), a molecule with anticancer properties, promotes the formation of PPP1CA-AKT complex and not of PPP1CC-MAPK complex. Both, AKT and MAPK, are well-known PIPs from two pathways that crosstalk and regulate melanoma cells survival. In addition, the analysis performed using surface plasmon resonance (SPR) technology indicates that PPP1 interacts with obatoclax (OBX), a drug that belongs to the same family of PG. Overall, these results suggest that PG might, at least in part, act through PPP1C/PIPs. Also, this study is pioneer in demonstrating PPP1 isoform-specific modulation by small molecules.
文摘Background:Cancer-associated fibroblasts(CAFs)play an important role in the induction of chemo-resistance.This study aimed to clarify the mechanism underlying CAF-mediated resistance to two tyrosine kinase inhibitors(TKIs),sorafenib and lenvatinib,and to identify a novel therapeutic target for overcoming TKI resistance in hepatocellular carcinoma(HCC).Methods:We performed a systematic integrative analysis of publicly available gene expression datasets and whole-transcriptome sequencing data from 9 pairs of CAFs and para-cancer fibroblasts isolated from human HCC and para-tumor tissues,respectively,to identify key molecules that might induce resistance to TKIs.We then performed in vitro and in vivo experiments to validate selected targets and related mechanisms.The associations of plasma secreted phosphoprotein 1(SPP1)expression levels before sorafenib/lenvatinib treatment with progression-free survival(PFS)and overall survival(OS)of 54 patients with advanced HCC were evaluated using Kaplan-Meier and Cox regression analysis.Results:Bioinformatic analysis identified CAF-derived SPP1 as a candidate molecule driving TKI resistance.SPP1 inhibitors reversed CAF-induced TKI resistance in vitro and in vivo.CAF-derived SPP1 activated rapidly accelerated fibrosarcoma(RAF)/mitogen-activated protein kinase(MAPK)and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)through the integrin-protein kinase C-alpha(PKCα)signaling pathway and promoted epithelial-to-mesenchymal transition(EMT).A high plasma SPP1 level before TKI treatment was identified as an independent predictor of poor PFS(P=0.026)and OS(P=0.047)in patients with advanced HCC after TKI treatment.Conclusions:CAF-derived SPP1 enhances TKI resistance in HCC via bypass activation of oncogenic signals and EMT promotion.Its inhibition represents a promising therapeutic strategy against TKI resistance inHCC.Moreover,plasma SPP1 level before TKI treatment represents a potential biomarker for treatment response prediction.
基金supported by the European Association for the Study of the Liver Sheila Sherlock fellowship 2012.
文摘Aim:Extracellular matrix(ECM)-adhesions and their interaction with actin cytoskeleton are fundamental for hepatocellular carcinoma(HCC).Fascin-1,an actin-bundling protein,is correlated with poor HCC prognosis,and is known regarding the molecular mechanism of its action.In this study,the authors investigated Fascin-1 basic molecular mechanism and cellular properties in HCC cells.Methods:Fascin-1 was silenced by small interfering RNA and the expression of actin.The ECM-adhesion-related proteins were assessed along with the cells’adhesion capacity in two cell lines that differ in terms of aggressiveness;the hepatoma cell line PLC/PRF/5(Alexander)and the highly invasive HCC cell line HepG2.Results:This study shows that Fascin-1 is upregulated in HepG2 cells compared to Alexander cells and when silenced leads to increased cell adhesion only in HepG2,while at the same time is associated with reduced migfilin and vasodilator-stimulated phosphoprotein(VASP)expression.Conclusion:This is the first study to show that Fascin-1 contributes to a more aggressive phenotype in HCC cells and acts through migfilin and VASP.
基金supported by the National Natural Science Foundation of China (21878173,22177059)National Key R&D Program of China (2018YFA0901700,2021YFC2103900)a grant from the Institute Guo Qiang,Tsinghua University (2021GQG1016).
文摘Studies on the mechanism of protein phosphorylation and therapeutic interventions of its related molecular processes are limited by the difficulty in the production of purpose-built phosphoproteins harboring site-specific phosphorylated amino acids or their nonhydrolyzable analogs.Here we address this limitation by customizing the cell-free protein synthesis(CFPS)machinery via chassis strain selection and orthogonal translation system(OTS)reconfiguration screening.The suited chassis strains and reconfigured OTS combinations with high orthogonality were consequently picked out for individualized phosphoprotein synthesis.Specifically,we synthesized the sfGFP protein and MEK1 protein with site-specific phosphoserine(O-pSer)or its nonhydrolyzable analog,2-amino-4-phosphonobutyric acid(C-pSer).This study successfully realized building cell-free systems for site-specific incorporation of phosphonate mimics into the target protein.Our work lays the foundation for developing a highly expansible CFPS platform and the streamlined production of user-defined phosphoproteins,which can facilitate research on the physiological mechanism and potential interference tools toward protein phosphorylation.
基金supported by the National Key R&D Program of China(2018YFC1200601)Fundamental Research Funds for Central Non-profit Scientific Institutiona grant from the State Key Laboratory of Veterinary Biotechnology Program(SKLVBP201801).
文摘Rabies virus(RABV)phosphoprotein(P)plays an important role in disrupting host interferon(IFN)-mediated antiviral immune response.ABCE1 is known as an RNase L inhibitor that negatively regulates the 2′,5′-oligoadenylate(2-5A)/RNase L antiviral system related to the IFN signaling pathway.In this study,we screened the host factors associated with RABV P protein,while focusing on ABCE1 because of its role in the life cycle of several viruses.Our results showed that knockout of ABCE1 in HEK293T cells inhibited RABV replication.In contrast,the overexpression of ABCE1 in HEK293T cells enhanced RABV replication.Notably,the co-immunoprecipitation assay showed that ABCE1 interacted with RABV P protein.Since ABCE1 and RABV P proteins are related to the IFN signaling pathway,we propose that the interaction of viral P protein with ABCE1 leads to the disruption of host antiviral immune response.In summary,our research showed that ABCE1 is an important host protein that interacts with viral P protein and regulates RABV replication.Moreover,the interaction between ABCE1 and RABV P protein may facilitate the viral P protein-mediated disruption of host antiviral immune response.
基金supported by Shandong Natural Science Foundation[grant number ZR2021MH339].
文摘Background and aims:Secreted phosphoprotein 1(SPP1)functions in several physiological processes.The role of SPP1 expression in the prognosis and tumor immunity of hepatocellular carcinoma(HCC)is unknown.The aim of this study was to investigate the expression pattern of SPP1 in HCC and its correlation with prognosis and tumor immunity.Methods:Clinical and gene expression data of The Cancer Genome Atlas-liver hepatocellular carcinoma(LIHC)cohort and 11 other HCC datasets were collected.The Kaplan–Meier method and Cox regression analysis were used to analyze the prognostic value of SPP1.The DESeq2 package in R was used to analyze SPP1-related genes.Gene Ontology analysis and gene set enrichment analysis were used to determine the biological function of SPP1 in HCC.The single sample Gene Set Enrichment Analysis(ssGSEA)method was used to analyze the immune infiltrates of HCC.Illumina human methylation 450 data and level 3 HTSeq-FPKM data from The Cancer Genome Atlas-LIHC were used to analyze the effects of DNA methylation level on SPP1 expression.Results:SPP1 was overexpressed in HCC and correlated with T stage,histological grade,adjacent hepatic tissue inflammation,and vascular invasion in HCC.The analysis of survival rates indicated that high SPP1 levels were associated with poor overall survival in HCC.Functional analysis showed that SPP1 is related to tumor immunity,especially macrophage infiltration.Aberrant demethylation of the promoter region is one of the mechanisms underlying the increase of SPP1 in HCC.Conclusion:Our results indicate that SPP1 is an independent prognostic factor for HCC and is correlated with the clinical features and macrophage infiltration in HCC.
