PTEN-induced putative kinase 1(PINK1),a mitochondrial kinase that phosphorylates Parkin and other proteins,plays a crucial role in mitophagy and protection against neurodegeneration.Mutations in PINK1 and Parkin can l...PTEN-induced putative kinase 1(PINK1),a mitochondrial kinase that phosphorylates Parkin and other proteins,plays a crucial role in mitophagy and protection against neurodegeneration.Mutations in PINK1 and Parkin can lead to loss of function and early onset Parkinson's disease.However,there is a lack of strong in vivo evidence in rodent models to support the theory that loss of PINK1 affects mitophagy and induces neurodegeneration.Additionally,PINK1 knockout pigs(Sus scrofa)do not appear to exhibit neurodegeneration.In our recent work involving non-human primates,we found that PINK1 is selectively expressed in primate brains,while absent in rodent brains.To extend this to other species,we used multiple antibodies to examine the expression of PINK1 in pig tissues.In contrast to tissues from cynomolgus monkeys(Macaca fascicularis),our data did not convincingly demonstrate detectable PINK1expression in pig tissues.Knockdown of PINK1 in cultured pig cells did not result in altered Parkin and BAD phosphorylation,as observed in cultured monkey cells.A comparison of monkey and pig striatum revealed more PINK1-phosphorylated substrates in the monkey brain.Consistently,PINK1 knockout in pigs did not lead to obvious changes in the phosphorylation of Parkin and BAD.These findings provide new evidence that PINK1expression is specific to primates,underscoring the importance of non-human primates in investigating PINK1function and pathology related to PINK1 deficiency.展开更多
Changes in protein abundance and reversible protein phosphorylation(RPP)play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes.To test the hypothesis ...Changes in protein abundance and reversible protein phosphorylation(RPP)play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes.To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation,we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog,Nanorana parkeri,living on the Qinghai-Xizang Plateau.In total,5170 proteins and 5695 phosphorylation sites in 1938 proteins were quantified.Based on proteomic analysis,674 differentially expressed proteins(438 up-regulated,236 down-regulated)were screened in hibernating N.parkeri versus summer individuals.Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways,whereas lower expressed proteins were mainly involved in metabolic processes.A total of 4251 modified sites(4147 up-regulated,104 down-regulated)belonging to 1638 phosphoproteins(1555 up-regulated,83 down-regulated)were significantly changed in the liver.During hibernation,RPP regulated a diverse array of proteins involved in multiple functions,including metabolic enzymatic activity,ion transport,protein turnover,signal transduction,and alternative splicing.These changes contribute to enhancing protection,suppressing energy-consuming processes,and inducing metabolic depression.Moreover,the activities of phosphofructokinase,glutamate dehydrogenase,and ATPase were all significantly lower in winter compared to summer.In conclusion,our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.展开更多
Inflammation, which is induced after infection of bacteria and tissue injury, is one of the important early stages of wound healing. Bradykinin is increased during acute and chronic inflammation. We previously reporte...Inflammation, which is induced after infection of bacteria and tissue injury, is one of the important early stages of wound healing. Bradykinin is increased during acute and chronic inflammation. We previously reported that bradykinin stimulation induces dephosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) after phosphorylation by ROCK leading neurite outgrowth in neuroblastoma SH-SY5Y cells. In this report we showed that knock-down of MARCKS by RNAi reduced cell migration. Wild-type MARCKS-overexpressed SH-SY5Y cells migrated faster than the control cells. Unphosphorylatable MARCKS-overexpressed cells notably migrated fast. Moreover, chronic MARCKS dephosphorylation by a ROCK inhibitor HA-1077 promoted the cell migration, on the other hand a PKC inhibitor Ro-31-8220 did not. After wounding, MARCKS was transiently phosphorylated and dephospho-rylated by 20 min. Immunocytochemistry showed that the dephosphorylated MARCKS was localized at neurite tips. These findings suggest that MARCKS dephosphorylation is important in wound-induced migration of SH-SY5Y cells. It indicates the possibility that MARCKS is associated with wound repair after inflammation.展开更多
Objective YAP1 plays a dual role as an oncogene and tumor suppressor gene in several tumors;differentiating between these roles may depend on the YAP1 phosphorylation pattern.The specific function of YAP1 in B cell ac...Objective YAP1 plays a dual role as an oncogene and tumor suppressor gene in several tumors;differentiating between these roles may depend on the YAP1 phosphorylation pattern.The specific function of YAP1 in B cell acute lymphoblastic leukemia(B-ALL),however,is currently unclear.Thus,in the present study,the role of YAP1 in B-ALL was investigated using relevant cell lines and patient datasets.Methods The effects of shRNA-mediated knockdown on YAP1 and LATS1 levels in the NALM6 and MOLT-4 cell lines were examined using Western blotting,quantitative real-time polymerase chain reaction,flow cytometry,immunostaining,and nude mouse subcutaneous tumorigenesis experiments.Gene expression levels of Hippo pathway-related molecules before and after verteporfin(VP)treatment were compared using RNA-Seq to identify significant Hippo pathway-related genes in NALM6 cells.Results Patients with ALL showing high YAP1 expression and low YAP1-Ser127 phosphorylation levels had worse prognoses than those with low YAP1 protein expression and high YAP1-Ser127 phosphorylation levels.YAP1-Ser127 phosphorylation levels were lower in NALM6 cells than in MOLT-4 and control cells;YAP1 was distributed in the nuclei in NALM6 cells.Knockdown of YAP1 inhibited MOLT-4 and NALM6 cell proliferation and arrested the NALM6 cell cycle in the G0/G1 phase.Before and after VP treatment,the expression of the upstream gene LATS1 was upregulated;its overexpression promoted YAP1-Ser127 phosphorylation.Further,YAP1 was distributed in the plasma.Conclusion LATS1 may downregulate YAP1-Ser127 phosphorylation and maintain B-ALL cell function;thus,VP,which targets this axis,may serve as a new therapeutic method for improving the outcomes for B-ALL patients.展开更多
Alzheimer’s disease is a neurodegenerative disorder characterized by the amyloid accumulation in the brains of patients with Alzheimer’s disease.The pathogenesis of Alzheimer’s disease is mainly mediated by the pho...Alzheimer’s disease is a neurodegenerative disorder characterized by the amyloid accumulation in the brains of patients with Alzheimer’s disease.The pathogenesis of Alzheimer’s disease is mainly mediated by the phosphorylation and aggregation of tau protein.Among the multiple causes of tau hyperphosphorylation,brain insulin resistance has generated much attention,and inositols as insulin sensitizers,are currently considered candidates for drug development.The present narrative review revises the interactions between these three elements:Alzheimer’s disease-tau-inositols,which can eventually identify targets for new disease modifiers capable of bringing hope to the millions of people affected by this devastating disease.展开更多
AIM: To study the effect of Hepatitis C virus non- structural 5A (HCV NS5A) on IFNα induced signal transducer and activator of transcription-1 (STAT1) phosphorylation and nuclear translocation. METHODS: Expression of...AIM: To study the effect of Hepatitis C virus non- structural 5A (HCV NS5A) on IFNα induced signal transducer and activator of transcription-1 (STAT1) phosphorylation and nuclear translocation. METHODS: Expression of STAT1 Tyr701 phosphorylation at different time points was confirmed by Western blot, and the time point when p-STAT1 expressed most, was taken as the IFN induction time for further studies. Immunocytochemistry was used to confirm the successful transient transfection of NS5A expression plasmid. Immunofluorescene was performed to observe if there was any difference in IFNα-induced STAT1 phosphorylation and nuclear translocation between HCV NS5A-expressed and non-HCV NS5A-expressed cells. Western blot was used to compare the phosphorylated STAT1 protein of the cells. RESULTS: Expression of HCV NS5A was found in the cytoplasm of PCNS5A-transfected Huh7 cells, but not in the PRC/ CMV transfected or non-transfected cells. STAT1 Tyr701 phosphorylation was found strongest in 30 min of IFN induction. STAT1 phosphorylation and nuclear import were much less in the presence of HCV NS5A protein in contrast to PRC/CMV-transfected and non-transfected cells under fluorescent microscopy, which was further confirmed by Western blot. CONCLUSION: HCV NS5A expression plasmid is successfully transfected into Huh7 cells and HCV NS5A protein is expressed in the cytoplasm of the cells. IFN-α is able to induce STAT1 phosphrylation and nuclear translocation, and this effect is inhibited by HCV NS5Aprotein, which might be another possible resistance mechanism to interferon alpha therapy.展开更多
Astrocytes and microglia play an orchestrated role following spinal cord injury;however,the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood.Herein...Astrocytes and microglia play an orchestrated role following spinal cord injury;however,the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood.Herein,microglia were pharmacologically depleted and the effects on the astrocytic response were examined.We further explored the potential mechanisms involving the signal transducers and activators of transcription 3(STAT3)pathway.For in vivo experiments,we constructed a contusion spinal cord injury model in C57BL/6 mice.To deplete microglia,all mice were treated with colony-stimulating factor 1 receptor inhibitor PLX3397,starting 2 weeks prior to surgery until they were sacrificed.Cell proliferation was examined by 5-ethynyl-2-deoxyuridine(EdU)and three pivotal inflammatory cytokines were detected by a specific Bio-Plex Pro^(TM) Reagent Kit.Locomotor function,neuroinflammation,astrocyte activation and phosphorylated STAT3(pSTAT3,a maker of activation of STAT3 signaling)levels were determined.For in vitro experiments,a microglia and astrocyte coculture system was established,and the small molecule STA21,which blocks STAT3 activation,was applied to investigate whether STAT3 signaling is involved in mediating astrocyte proliferation induced by microglia.PLX3397 administration disrupted glial scar formation,increased inflammatory spillover,induced diffuse tissue damage and impaired functional recovery after spinal cord injury.Microglial depletion markedly reduced EdU+proliferating cells,especially proliferating astrocytes at 7 days after spinal cord injury.RNA sequencing analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397.Double immunofluorescence staining confirmed that PLX3397 significantly decreased STAT3 expression in astrocytes.Importantly,in vitro coculture of astrocytes and microglia showed that microglia-induced astrocyte proliferation was abolished by STA21 administration.These findings suggest that microglial depletion impaired astrocyte proliferation and astrocytic scar formation,and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following spinal cord injury.展开更多
The degradation of titin could make the myofibrillar fragmentation to improve meat tenderization during postmortem.This study aimed to investigate effect of phosphorylation on titin degradation.Protein kinase A(PKA)an...The degradation of titin could make the myofibrillar fragmentation to improve meat tenderization during postmortem.This study aimed to investigate effect of phosphorylation on titin degradation.Protein kinase A(PKA)and alkaline phosphatase(AP)were added to crude titin extracted from ovine longissimus lumborum(LL)muscles.Phosphorylated/dephosphorylated titin were incubated withμ-calpain at 4℃ for 2 days.Results showed titin in AP group started degradation earlier than that in PKA and control groups.There were 20,16 and 12 phosphorylated sites identified by iTRAQ in the PKA,control and AP group,respectively.3D structure of dephosphorylated titin fragment was simulated and its molecular dynamics trajectory analysis was performed using Discovery StudioTM.The dihedral angle in AP group was less and the dephosphorylated fragment had a higher kinetic energy and total energy.We suggested that changes caused by AP treatment might make titin unstable,which easily degraded byμ-calpain.展开更多
The uptake of ammonium,nitrate,phosphorus,and potassium ions by roots is mediated by specific ion transporter or channel proteins,and protein phosphorylation regulation events occurring on these proteins and their reg...The uptake of ammonium,nitrate,phosphorus,and potassium ions by roots is mediated by specific ion transporter or channel proteins,and protein phosphorylation regulation events occurring on these proteins and their regulators determine their ultimate activity.Elucidating the mechanism by which protein phosphorylation modification regulates nutrient uptake will advance plant breeding for high nutrientuse efficiency.In this review,it is concluded that the root nutrient absorption system is composed of several,but not all,members of a specific ion transporter or channel family.Under nutrient-starvation conditions,protein phosphorylation-based regulation of these proteins and associated transcription factors increases ion transporter-or channel-mediated nutrient uptake capacity via direct function activity enhancement,allowing more protein trafficking to the plasma membrane,by strengthening the interaction of transporters and channels with partner proteins,by increasing their protein stability,and by transcriptional activation.Under excessive nutrient conditions,protein phosphorylation-based regulation suppresses nutrient uptake by reversing these processes.Strengthening phosphorylation regulation items that increase nutrient absorption and weakening phosphorylation modification items that are not conducive to nutrient absorption show potential as strategies for increasing nutrient use efficiency.展开更多
Benzo[a]pyrene(B[a]P)is a food contaminant toxic for cardiovascular diseases.The nuclear translocation of Arylhydrocarbon receptor(AhR)plays an important role in B[a]P-induced oxidative stress and vascular diseases.We...Benzo[a]pyrene(B[a]P)is a food contaminant toxic for cardiovascular diseases.The nuclear translocation of Arylhydrocarbon receptor(AhR)plays an important role in B[a]P-induced oxidative stress and vascular diseases.We confi rmed that B[a]P promoted ROS production in vascular smooth muscle cells(VSMCs)in vitro and in vivo,associated with the nuclear translocation of AhR.It is known that phosphorylation inhibits while dephosphorylation of AhR promotes nuclear translocation of AhR.However,from the posttranslational modifi cation level,the mechanism by which B[a]P activates and regulates the nuclear translocation of AhR is unclear.Co-immunoprecipitation results showed that cytoplasmic AhR was phosphorylated before B[a]P stimulation,and switched to O-GlcNAcylation upon B[a]P 1-h stimulation in VSMCs,suggesting there may be a competitively inhibitory relationship between O-GlcNAcylation and phosphorylation of AhR.Next,siRNAs of O-linked N-acetylglucosamine transferase(OGT),O-GlcNAcase(OGA)and OGA inhibitor PUGNAc were used.SiOGT blocks but siOGA and PUGNAc promote B[a]P-dependent AhR nuclear translocation and oxidative stress.Ser11 may be the competitive binding site for phosphorylation and O-GlcNAcylation of AhR.Phosphorylation-mimic variant inhibits but O-GlcNAcylation of AhR promotes AhR nuclear translocation and oxidative stress.Our fi ndings highlight a new perspective for AhR nuclear translocation regulated by the competitive modifi cation between phosphorylation and O-GlcNAcylation.展开更多
AIM: To examine phosphorylation of alpha B-crystallin(p-αBC), a vascular endothelial growth factor(VEGF)chaperone, and immunohistochemically investigate relationship between p-αBC, VEGF and phosphorylated p38-mitoge...AIM: To examine phosphorylation of alpha B-crystallin(p-αBC), a vascular endothelial growth factor(VEGF)chaperone, and immunohistochemically investigate relationship between p-αBC, VEGF and phosphorylated p38-mitogen-activated protein kinase(p-p38 MAPK) in the epiretinal membrane of human proliferative diabetic retinopathy(PDR).METHODS: Eleven epiretinal membranes of PDR surgically excised were included in this study. Two normal retinas were also collected from enucleation tissues due to choroidal melanoma. Paraformaldehyde-fixed, paraffin-embedded tissue sections were processed for immunohistochemistry with anti-p-αBC, VEGF, CD31,and p-p38 MAPK antibodies.RESULTS: Immunoreactivity for p-αBC was observed in all of the epiretinal membranes examined, where phosphorylation on serine(Ser) 59 showed strongest immunoreactivity in over 70% of the membranes. The immunolocalization of p-αBC was detected in the CD31-positive endothelial cells, and co-localized with VEGF and p-p38 MAPK in PDR membranes. Immunoreactivity for p-αBC, however, was undetectable in endothelial cells of the normal retinas, where p-p38 MAPK immunoreactivity was less marked than PDR membranes.CONCLUSION: Phosphorylation of αBC, in particular,phosphorylation on Ser59 by p-p38 MAPK may play a potential role as a molecular chaperon for VEGF in the pathogenesis of epiretinal membranes in PDR.展开更多
LHCII is a crucial light-harvesting pigment/protein complex in photosystem II (PSII) supercomplex. It also participates in the light energy redistribution between photosystems and in the photoprotection via its revers...LHCII is a crucial light-harvesting pigment/protein complex in photosystem II (PSII) supercomplex. It also participates in the light energy redistribution between photosystems and in the photoprotection via its reversible dissociation with PSII and PSI (photosystem I). This reversible detachment of LHCII is regulated by phosphorylation of its own and PSII core protein. Under low light conditions, LHCII is phosphorylated and dissociated with PSII core protein complex and combined with PSI, which balances the excitation energy between PSII and PSI;Under high light environment, the phosphorylation of PSII core proteins makes LHCII detach from PSII. The dissociated LHCII presents in a free state, which involves in the thermal dissipation of excess excitation energy. During photodamage, dual phosphorylations of both PSII core proteins and LHCII complexes occur. The phosphorylation of D1 is conductive to the disintegration of photodamaged PSII and the cycle of repair. In this circumstance, the phosphorylation of LHCII is induced by reactive oxygen species (ROS) and then the phosphorylated LHCII migrates to PSI, into the repair cycle of damaged PSII. The ferredoxin (Fdr) and thioredoxin (Tdr) system may play a possible central role in the phosphorylation regulation on LHCII dissociation.展开更多
This study aimed to examine changes in phosphorylation of sarcoplasmic and myofibrillar proteins from longissimus lumborum,semitendinosus,and psoas major muscles during postmortem ageing for 5 d.These sarcoplasmic and...This study aimed to examine changes in phosphorylation of sarcoplasmic and myofibrillar proteins from longissimus lumborum,semitendinosus,and psoas major muscles during postmortem ageing for 5 d.These sarcoplasmic and myofibrillar proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with phosphorous and protein specific stains.Myofibril fragmentation index,pH,the content of lactic acid and the relative activity of μ-calpain in three ovine muscles were measured.These results showed that the relative phosphorylation level of sarcoplasmic and myofibrillar proteins of psoas major muscle were lower compared with longissimus lumborum and semitendinosus muscles(P<0.05).The pH of psoas major muscle was the lowest at 0.5 h postmortem,and the highest after 12 h postmortem(P<0.05).In addition,the relative activity of μ-calpain was higher within 5 d postmortem and myofibril fragmentation index was higher after 1 d postmortem in psoas major muscle than those of longissimus lumborum and semitendinosus muscles(P<0.05).The sarcoplasmic protein phosphorylation may regulate the rate of pH decline to influence the μ-calpain activity and then proteolysis of proteins consequently.This study gives a new perspective of the mechanism of postmortem meat tenderization.展开更多
Nowadays,the cumulative intake of glucocorticoids has become the most common pathogenic factor for non-traumatic osteonecrosis of the femoral head(ONFH).Apoptosis of osteoblasts is considered as the main reason of ONF...Nowadays,the cumulative intake of glucocorticoids has become the most common pathogenic factor for non-traumatic osteonecrosis of the femoral head(ONFH).Apoptosis of osteoblasts is considered as the main reason of ONFH at the molecular level.Glycogen synthase kinase 3β(GSK3β)is an important regulator of cellular differentiation and apoptosis pathway,which can modulate the balance between osteoblasts and osteoclasts.Several studies have reported about its function in osteoporosis,but little is known about it in osteonecrosis.In our study,lipopolysaccharide and methylprednisolone were utilized to establish a rat ONFH model.The phosphorylation of GSK3βSer-9 was decreased in the model.Western blotting examination ofβ-catenin,Bcl-2,Bax and caspase-3 revealed that the osteoblasts were apoptotic.In dexamethasone(Dex)-incubated primary osteoblasts,the expression profile of GSK3βphosphorylation and apoptotic factors were consistent with those in the rat ONFH model.To further investigate the regulation of osteonecrosis caused by GSK3β,the expression and function of GSK3βwere inhibited in Dex-incubated primary osteoblasts.The knockdown of GSK3βby siRNA decreased the expression of Bax and cleaved caspase-3,but increased Bcl-2 andβ-catenin.On the other hand,selective inhibition of GSK3βfunction by LiCl counteracted the activation of caspase-3 induced by Dex.Our work is the first study about the GSK3P phosphorylation in ONFH,and provides evidence for further therapeutic methods.展开更多
Dendritic cells (DCs) are the most potent antigen-presen ting cells that play crucial roles in the regulation of immune response. Triptol ide, an active component purified from the medicinal plant Tripterygium wilfor ...Dendritic cells (DCs) are the most potent antigen-presen ting cells that play crucial roles in the regulation of immune response. Triptol ide, an active component purified from the medicinal plant Tripterygium wilfor dii Hook F., has been demonstrated to act as a potent immunosuppressive drug c apab le of inhibiting T cell activation and proliferation. However, little is known a bout the effects of triptolide on DCs. The present study shows that triptolide d oes not affect phenotypic differentiation and LPS-induced maturation of murine DCs. But triptolide can dramatically reduce cell recovery by inducing apoptosis of DCs at concentration as low as 10 ng/ml, as demonstrated by phosphatidylserin e exposure, mitochondria potential decrease, and nuclear DNA condensation. Tript olide induces activation of p38 in DCs, which precedes the activation of caspase 3. SB203580, a specific kinase inhibitor for p38, can block the activation of caspase 3 and inhibit the resultant apoptosis of DCs. Our results suggest that t he anti-inflammatory and immunosuppressive activities of triptolide may be due, in part, to its apoptosis-inducing effects on DCs.展开更多
Parathyroid hormone(PTH) regulates bone remodeling by activating PTH type 1 receptor(PTH1R) in osteoblasts/osteocytes. Insulinlike growth factor type 1(IGF-1) stimulates mesenchymal stem cell differentiation to osteob...Parathyroid hormone(PTH) regulates bone remodeling by activating PTH type 1 receptor(PTH1R) in osteoblasts/osteocytes. Insulinlike growth factor type 1(IGF-1) stimulates mesenchymal stem cell differentiation to osteoblasts. However, little is known about the signaling mechanisms that regulates the osteoblast-to-osteocyte transition. Here we report that PTH and IGF-I synergistically enhance osteoblast-to-osteocyte differentiation. We identified that a specific tyrosine residue, Y494, on the cytoplasmic domain of PTH1R can be phosphorylated by insulin-like growth factor type I receptor(IGF1R) in vitro. Phosphorylated PTH1R localized to the barbed ends of actin filaments and increased actin polymerization during morphological change of osteoblasts into osteocytes.Disruption of the phosphorylation site reduced actin polymerization and dendrite length. Mouse models with conditional ablation of PTH1R in osteoblasts demonstrated a reduction in the number of osteoctyes and dendrites per osteocyte, with complete overlap of PTH1R with phosphorylated-PTH1R positioning in osteocyte dendrites in wild-type mice. Thus, our findings reveal a novel signaling mechanism that enhances osteoblast-to-osteocyte transition by direct phosphorylation of PTH1R by IGF1R.展开更多
Protein phosphorylation,one of the major post-translational modifications,plays a crucial role in cell signaling,DNA replication,gene expression and differentiation;and alters enzyme activity and other biological acti...Protein phosphorylation,one of the major post-translational modifications,plays a crucial role in cell signaling,DNA replication,gene expression and differentiation;and alters enzyme activity and other biological activities;and regulates cell proliferation and enlargement,phytohormone biosynthesis and signaling,plant disease resistance,and grain filling and quality during rice seed development.Research work on protein phosphorylation started in the 1950 s with the discovery of phosphorylase a and phosphorylase b which are phospho and dephospho forms of the same enzyme.Over the last decade,rice proteomics has accomplished tremendous progress in setting up techniques to proteome nearly all tissues,organs and organelles.The progress made in this field is evident in number of research works.However,research on rice protein phosphorylation is still at its infancy and there are still many unanswered questions.In this review,the general description of protein phosphorylation,including history,structure,frequency of occurrence and function,are discussed.This work also elucidates the different methods for identification,qualification and finally,the progress in rice phosphoproteome research and perspectives.展开更多
Disturbances in constitutive nitric oxide synthase (cNOS) activation associated with H. pylori colonization of gastric mucosa are considered of major consequences in defining the extent of inflammatory involvement. As...Disturbances in constitutive nitric oxide synthase (cNOS) activation associated with H. pylori colonization of gastric mucosa are considered of major consequences in defining the extent of inflammatory involvement. As rapid changes in cNOS activation are linked to the enzyme phosphorylation at the specific Ser/Thr residues, we investigated the influence of H. pylori LPS and gastric hormone, ghrelin, on the processes of phosphorylation of these two critical sites in gastric mucosal cells. We show that the LPS-induced reduction in cNOS activity is reflected in the phosphorylation on Thr497, while the countering effect of ghrelin is associated with a rapid increase in cNOS phosphorylation on Ser1179. Further, we demonstrate that cNOS phosphorylation on Thr497 as well as Ser1179 displays dependence on PKCδ. However, while the LPS-induced suppression in cNOS activation shows reliance on the phosphorylation of PKCδ and PI3K on Ser, the effect of ghrelin is manifested by the increase in phosphorylation of PKCδ and PI3K on Tyr, as well as membrane translocation and phosphorylation of Akt on Ser493. Thus, our findings suggest that the LPS-induced suppression in cNOS activation is mediated by PKCδ-controlled phosphorylation of PI3K on Ser that interferes with the membrane recruitment of Akt and promotes cNOS phosphorylation on Thr497, and that ghrelin-elicited up-regulation in cNOS activation relies on the PKCδ-directed phosphorylation of PI3K on Tyr that stimulates the membrane localization of Akt and enhances cNOS phosphorylation on Ser1179.展开更多
Enamelin(ENAM) has three putative phosphoserines(p Sers) phosphorylated by a Golgi-associated secretory pathway kinase(FAM20 C) based on their distinctive Ser-x-Glu(S-x-E) motifs. Fam20 C-knockout mice show severe ena...Enamelin(ENAM) has three putative phosphoserines(p Sers) phosphorylated by a Golgi-associated secretory pathway kinase(FAM20 C) based on their distinctive Ser-x-Glu(S-x-E) motifs. Fam20 C-knockout mice show severe enamel defects similar to those in the Enam-knockout mice, implying an important role of the p Sers in ENAM. To determine the role of pSer^(55) in ENAM, we characterized ENAM^(Rgsc514) mice, in which Ser^(55) cannot be phosphorylated by FAM20 C due to an E^(57)>G^(57) mutation in the S-x-E motif. The enamel microstructure of 4-week-old mice was examined by scanning electron microscopy.The teeth of 6-day-old mice were characterized by histology and immunohistochemistry. The protein lysates of the first lower molars of 4-day-old mice were analyzed by Western immunoblotting using antibodies against ENAM, ameloblastin and amelogenin. ENAM^(Rgsc514) heterozygotes showed a disorganized enamel microstructure, while the homozygotes had no enamel on the dentin surface. The N-terminal fragments of ENAM in the heterozygotes were detained in the ameloblasts and localized in the mineralization front of enamel matrix, while those in the WT mice were secreted out of ameloblasts and distributed evenly in the outer 1/2 of enamel matrix. Surprisingly, the ~ 15 k Da C-terminal fragments of ameloblastin were not detected in the molar lysates of the homozygotes. These results suggest that the phosphorylation of Ser^(55) may be an essential posttranslational modification of ENAM and is required for the interaction with other enamel matrix molecules such as ameloblastin in mediating the structural organization of enamel matrix and protein-mineral interactions during enamel formation.展开更多
In order to confirm the hypothesis that during acute hypoxia, the antiarrhythmic peptide (AAP10) could improve conductance by changing the phosphorylation state of connexin43 (Cx43), isolated perfused rat hearts were ...In order to confirm the hypothesis that during acute hypoxia, the antiarrhythmic peptide (AAP10) could improve conductance by changing the phosphorylation state of connexin43 (Cx43), isolated perfused rat hearts were randomly divided into three groups: control, hypoxia and AAP10 (n=9 in each group). The change in Cx43 phosphorylation was tested by Western-blot; the distribu- tion of Cx43 was observed by confocal immunofluorescence microscopy. Western-blot analysis re- vealed that the expression of total Cx43 protein was significantly decreased during acute hypoxia, while nonphosphorylated Cx43 (NP-Cx43) was unchanged. AAP10 could increase the expression of total Cx43 protein, but had no effects on the NP-Cx43 protein. Immunofluorescence study showed that during acute hypoxia, both total Cx43 and NP-Cx43 proteins were greatly decreased, while AAP10 only increased the expression of total Cx43 protein, but had no effect of the NP-Cx43 protein expression. These findings suggested that the decrease of intercellular communication may be associ- ated with the reduction of phosphorylated Cx43 (p-Cx43) and translocation of NP-Cx43 from the surface of gap junction into intracellular pools during acute hypoxia. AAP10 can improve intercelluar communication by enhancing phosphorylation of Cx43.展开更多
基金supported by the National Natural Science Foundation of China (32070534,32370567,82371874,81830032,31872779,82071421,81873736)Key Field Research and Development Program of Guangdong Province (2018B030337001)+3 种基金Guangzhou Key Research Program on Brain Science (202007030008)Department of Science and Technology of Guangdong Province (2021ZT09Y007,2020B121201006)Guangdong Basic and Applied Basic Research Foundation (2023B1515020031,2022A1515012301)Fundamental Research Funds for the Central Universities (Jinan University,21620358)。
文摘PTEN-induced putative kinase 1(PINK1),a mitochondrial kinase that phosphorylates Parkin and other proteins,plays a crucial role in mitophagy and protection against neurodegeneration.Mutations in PINK1 and Parkin can lead to loss of function and early onset Parkinson's disease.However,there is a lack of strong in vivo evidence in rodent models to support the theory that loss of PINK1 affects mitophagy and induces neurodegeneration.Additionally,PINK1 knockout pigs(Sus scrofa)do not appear to exhibit neurodegeneration.In our recent work involving non-human primates,we found that PINK1 is selectively expressed in primate brains,while absent in rodent brains.To extend this to other species,we used multiple antibodies to examine the expression of PINK1 in pig tissues.In contrast to tissues from cynomolgus monkeys(Macaca fascicularis),our data did not convincingly demonstrate detectable PINK1expression in pig tissues.Knockdown of PINK1 in cultured pig cells did not result in altered Parkin and BAD phosphorylation,as observed in cultured monkey cells.A comparison of monkey and pig striatum revealed more PINK1-phosphorylated substrates in the monkey brain.Consistently,PINK1 knockout in pigs did not lead to obvious changes in the phosphorylation of Parkin and BAD.These findings provide new evidence that PINK1expression is specific to primates,underscoring the importance of non-human primates in investigating PINK1function and pathology related to PINK1 deficiency.
基金supported by the National Natural Science Foundation of China(32001110)Training Program for Cultivating Highlevel Talents by the China Scholarship Council(2021lxjjw01)Open Project of State Key Laboratory of Plateau Ecology and Agriculture,Qinghai University(2021-KF-004)。
文摘Changes in protein abundance and reversible protein phosphorylation(RPP)play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes.To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation,we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog,Nanorana parkeri,living on the Qinghai-Xizang Plateau.In total,5170 proteins and 5695 phosphorylation sites in 1938 proteins were quantified.Based on proteomic analysis,674 differentially expressed proteins(438 up-regulated,236 down-regulated)were screened in hibernating N.parkeri versus summer individuals.Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways,whereas lower expressed proteins were mainly involved in metabolic processes.A total of 4251 modified sites(4147 up-regulated,104 down-regulated)belonging to 1638 phosphoproteins(1555 up-regulated,83 down-regulated)were significantly changed in the liver.During hibernation,RPP regulated a diverse array of proteins involved in multiple functions,including metabolic enzymatic activity,ion transport,protein turnover,signal transduction,and alternative splicing.These changes contribute to enhancing protection,suppressing energy-consuming processes,and inducing metabolic depression.Moreover,the activities of phosphofructokinase,glutamate dehydrogenase,and ATPase were all significantly lower in winter compared to summer.In conclusion,our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.
文摘Inflammation, which is induced after infection of bacteria and tissue injury, is one of the important early stages of wound healing. Bradykinin is increased during acute and chronic inflammation. We previously reported that bradykinin stimulation induces dephosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) after phosphorylation by ROCK leading neurite outgrowth in neuroblastoma SH-SY5Y cells. In this report we showed that knock-down of MARCKS by RNAi reduced cell migration. Wild-type MARCKS-overexpressed SH-SY5Y cells migrated faster than the control cells. Unphosphorylatable MARCKS-overexpressed cells notably migrated fast. Moreover, chronic MARCKS dephosphorylation by a ROCK inhibitor HA-1077 promoted the cell migration, on the other hand a PKC inhibitor Ro-31-8220 did not. After wounding, MARCKS was transiently phosphorylated and dephospho-rylated by 20 min. Immunocytochemistry showed that the dephosphorylated MARCKS was localized at neurite tips. These findings suggest that MARCKS dephosphorylation is important in wound-induced migration of SH-SY5Y cells. It indicates the possibility that MARCKS is associated with wound repair after inflammation.
文摘Objective YAP1 plays a dual role as an oncogene and tumor suppressor gene in several tumors;differentiating between these roles may depend on the YAP1 phosphorylation pattern.The specific function of YAP1 in B cell acute lymphoblastic leukemia(B-ALL),however,is currently unclear.Thus,in the present study,the role of YAP1 in B-ALL was investigated using relevant cell lines and patient datasets.Methods The effects of shRNA-mediated knockdown on YAP1 and LATS1 levels in the NALM6 and MOLT-4 cell lines were examined using Western blotting,quantitative real-time polymerase chain reaction,flow cytometry,immunostaining,and nude mouse subcutaneous tumorigenesis experiments.Gene expression levels of Hippo pathway-related molecules before and after verteporfin(VP)treatment were compared using RNA-Seq to identify significant Hippo pathway-related genes in NALM6 cells.Results Patients with ALL showing high YAP1 expression and low YAP1-Ser127 phosphorylation levels had worse prognoses than those with low YAP1 protein expression and high YAP1-Ser127 phosphorylation levels.YAP1-Ser127 phosphorylation levels were lower in NALM6 cells than in MOLT-4 and control cells;YAP1 was distributed in the nuclei in NALM6 cells.Knockdown of YAP1 inhibited MOLT-4 and NALM6 cell proliferation and arrested the NALM6 cell cycle in the G0/G1 phase.Before and after VP treatment,the expression of the upstream gene LATS1 was upregulated;its overexpression promoted YAP1-Ser127 phosphorylation.Further,YAP1 was distributed in the plasma.Conclusion LATS1 may downregulate YAP1-Ser127 phosphorylation and maintain B-ALL cell function;thus,VP,which targets this axis,may serve as a new therapeutic method for improving the outcomes for B-ALL patients.
基金supported by the European Regional Development Funds-European Union(ERDF-EU),FATZHEIMER project(EU-LAC HEALTH 2020,16/T010131 to FRdF),“Una manera de hacer Europa”Ministerio de Economía,Industria y Competitividad,Gobierno de Espa?a,Programa Estatal de Investigación,Desarrollo e Innovación Orientada a los Retos de la Sociedad(RTC2019-007329-1 to FRdF)+2 种基金Consejería de Economía,Conocimiento y Universidad,Junta de Andalucía,Plan Andaluz de Investigación,Desarrollo e Innovación(P18TP-5194 to FRdF)Instituto de Salud CarlosⅢ(DTS22/00021 to FRdF)DMV(FI20/00227)holds a“PFIS’’predoctoral contract from the National System of Health,EU-ERDF-Instituto de Salud CarlosⅢ。
文摘Alzheimer’s disease is a neurodegenerative disorder characterized by the amyloid accumulation in the brains of patients with Alzheimer’s disease.The pathogenesis of Alzheimer’s disease is mainly mediated by the phosphorylation and aggregation of tau protein.Among the multiple causes of tau hyperphosphorylation,brain insulin resistance has generated much attention,and inositols as insulin sensitizers,are currently considered candidates for drug development.The present narrative review revises the interactions between these three elements:Alzheimer’s disease-tau-inositols,which can eventually identify targets for new disease modifiers capable of bringing hope to the millions of people affected by this devastating disease.
基金Supported by National Natural Science Foundation of ChinaNo. 39670671, No. 30471531
文摘AIM: To study the effect of Hepatitis C virus non- structural 5A (HCV NS5A) on IFNα induced signal transducer and activator of transcription-1 (STAT1) phosphorylation and nuclear translocation. METHODS: Expression of STAT1 Tyr701 phosphorylation at different time points was confirmed by Western blot, and the time point when p-STAT1 expressed most, was taken as the IFN induction time for further studies. Immunocytochemistry was used to confirm the successful transient transfection of NS5A expression plasmid. Immunofluorescene was performed to observe if there was any difference in IFNα-induced STAT1 phosphorylation and nuclear translocation between HCV NS5A-expressed and non-HCV NS5A-expressed cells. Western blot was used to compare the phosphorylated STAT1 protein of the cells. RESULTS: Expression of HCV NS5A was found in the cytoplasm of PCNS5A-transfected Huh7 cells, but not in the PRC/ CMV transfected or non-transfected cells. STAT1 Tyr701 phosphorylation was found strongest in 30 min of IFN induction. STAT1 phosphorylation and nuclear import were much less in the presence of HCV NS5A protein in contrast to PRC/CMV-transfected and non-transfected cells under fluorescent microscopy, which was further confirmed by Western blot. CONCLUSION: HCV NS5A expression plasmid is successfully transfected into Huh7 cells and HCV NS5A protein is expressed in the cytoplasm of the cells. IFN-α is able to induce STAT1 phosphrylation and nuclear translocation, and this effect is inhibited by HCV NS5Aprotein, which might be another possible resistance mechanism to interferon alpha therapy.
基金supported by the Natural Science Foundation of Guangdong Province,No.2020A1515010090(to ZLZ)the Science and Technology Project Foundation of Guangzhou City,No.202002030004(to HZ).
文摘Astrocytes and microglia play an orchestrated role following spinal cord injury;however,the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood.Herein,microglia were pharmacologically depleted and the effects on the astrocytic response were examined.We further explored the potential mechanisms involving the signal transducers and activators of transcription 3(STAT3)pathway.For in vivo experiments,we constructed a contusion spinal cord injury model in C57BL/6 mice.To deplete microglia,all mice were treated with colony-stimulating factor 1 receptor inhibitor PLX3397,starting 2 weeks prior to surgery until they were sacrificed.Cell proliferation was examined by 5-ethynyl-2-deoxyuridine(EdU)and three pivotal inflammatory cytokines were detected by a specific Bio-Plex Pro^(TM) Reagent Kit.Locomotor function,neuroinflammation,astrocyte activation and phosphorylated STAT3(pSTAT3,a maker of activation of STAT3 signaling)levels were determined.For in vitro experiments,a microglia and astrocyte coculture system was established,and the small molecule STA21,which blocks STAT3 activation,was applied to investigate whether STAT3 signaling is involved in mediating astrocyte proliferation induced by microglia.PLX3397 administration disrupted glial scar formation,increased inflammatory spillover,induced diffuse tissue damage and impaired functional recovery after spinal cord injury.Microglial depletion markedly reduced EdU+proliferating cells,especially proliferating astrocytes at 7 days after spinal cord injury.RNA sequencing analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397.Double immunofluorescence staining confirmed that PLX3397 significantly decreased STAT3 expression in astrocytes.Importantly,in vitro coculture of astrocytes and microglia showed that microglia-induced astrocyte proliferation was abolished by STA21 administration.These findings suggest that microglial depletion impaired astrocyte proliferation and astrocytic scar formation,and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following spinal cord injury.
基金financially supported by National Natural Science Foundation(32102035)the Agricultural Science and Technology Innovation Program(CAAS-ASTIP-2020-IFST-03)Central Public-interest Scientific Institution Basal Research(S2019RCJC01,S2020JBKY-16).
文摘The degradation of titin could make the myofibrillar fragmentation to improve meat tenderization during postmortem.This study aimed to investigate effect of phosphorylation on titin degradation.Protein kinase A(PKA)and alkaline phosphatase(AP)were added to crude titin extracted from ovine longissimus lumborum(LL)muscles.Phosphorylated/dephosphorylated titin were incubated withμ-calpain at 4℃ for 2 days.Results showed titin in AP group started degradation earlier than that in PKA and control groups.There were 20,16 and 12 phosphorylated sites identified by iTRAQ in the PKA,control and AP group,respectively.3D structure of dephosphorylated titin fragment was simulated and its molecular dynamics trajectory analysis was performed using Discovery StudioTM.The dihedral angle in AP group was less and the dephosphorylated fragment had a higher kinetic energy and total energy.We suggested that changes caused by AP treatment might make titin unstable,which easily degraded byμ-calpain.
基金supported by the Jiangsu Provincial DoubleInnovation Doctor Program(JSSCBS20221643)the Jiangsu Institute of Botany Talent Fund(JIBTF202210)+2 种基金the Program for the Young Innovative Talents of Jiangsu Vocational College of Agriculture and Forest(2021kj26)the National Natural Science Foundation of China(32101429)Natural Science Foundation of Jiangsu Province,China(BK20200288)。
文摘The uptake of ammonium,nitrate,phosphorus,and potassium ions by roots is mediated by specific ion transporter or channel proteins,and protein phosphorylation regulation events occurring on these proteins and their regulators determine their ultimate activity.Elucidating the mechanism by which protein phosphorylation modification regulates nutrient uptake will advance plant breeding for high nutrientuse efficiency.In this review,it is concluded that the root nutrient absorption system is composed of several,but not all,members of a specific ion transporter or channel family.Under nutrient-starvation conditions,protein phosphorylation-based regulation of these proteins and associated transcription factors increases ion transporter-or channel-mediated nutrient uptake capacity via direct function activity enhancement,allowing more protein trafficking to the plasma membrane,by strengthening the interaction of transporters and channels with partner proteins,by increasing their protein stability,and by transcriptional activation.Under excessive nutrient conditions,protein phosphorylation-based regulation suppresses nutrient uptake by reversing these processes.Strengthening phosphorylation regulation items that increase nutrient absorption and weakening phosphorylation modification items that are not conducive to nutrient absorption show potential as strategies for increasing nutrient use efficiency.
基金supported by the National Key Research Project of China(2022YFF1100300)National Natural Science Foundation of China(32272328)+5 种基金Natural Science Foundation of Hebei Province(B2022321001)Major Public Welfare Projects in Henan Province(201300110200)National Key Research Project of Hebei Province(20375502D)National Key Research Project of Hebei Province(H2021206427)University Science and Technology Research Project of Hebei Province(QN2017107)Postdoctoral Research Funds of Hebei Medical University(307050100163759).
文摘Benzo[a]pyrene(B[a]P)is a food contaminant toxic for cardiovascular diseases.The nuclear translocation of Arylhydrocarbon receptor(AhR)plays an important role in B[a]P-induced oxidative stress and vascular diseases.We confi rmed that B[a]P promoted ROS production in vascular smooth muscle cells(VSMCs)in vitro and in vivo,associated with the nuclear translocation of AhR.It is known that phosphorylation inhibits while dephosphorylation of AhR promotes nuclear translocation of AhR.However,from the posttranslational modifi cation level,the mechanism by which B[a]P activates and regulates the nuclear translocation of AhR is unclear.Co-immunoprecipitation results showed that cytoplasmic AhR was phosphorylated before B[a]P stimulation,and switched to O-GlcNAcylation upon B[a]P 1-h stimulation in VSMCs,suggesting there may be a competitively inhibitory relationship between O-GlcNAcylation and phosphorylation of AhR.Next,siRNAs of O-linked N-acetylglucosamine transferase(OGT),O-GlcNAcase(OGA)and OGA inhibitor PUGNAc were used.SiOGT blocks but siOGA and PUGNAc promote B[a]P-dependent AhR nuclear translocation and oxidative stress.Ser11 may be the competitive binding site for phosphorylation and O-GlcNAcylation of AhR.Phosphorylation-mimic variant inhibits but O-GlcNAcylation of AhR promotes AhR nuclear translocation and oxidative stress.Our fi ndings highlight a new perspective for AhR nuclear translocation regulated by the competitive modifi cation between phosphorylation and O-GlcNAcylation.
基金Supported by the Research foundation of the Japan Society for the Promotion of Science(JSPS)(No.15K10856)Scientific Research from The Ministry of Education,Culture,Sports,Science,and Technology(MEXT)
文摘AIM: To examine phosphorylation of alpha B-crystallin(p-αBC), a vascular endothelial growth factor(VEGF)chaperone, and immunohistochemically investigate relationship between p-αBC, VEGF and phosphorylated p38-mitogen-activated protein kinase(p-p38 MAPK) in the epiretinal membrane of human proliferative diabetic retinopathy(PDR).METHODS: Eleven epiretinal membranes of PDR surgically excised were included in this study. Two normal retinas were also collected from enucleation tissues due to choroidal melanoma. Paraformaldehyde-fixed, paraffin-embedded tissue sections were processed for immunohistochemistry with anti-p-αBC, VEGF, CD31,and p-p38 MAPK antibodies.RESULTS: Immunoreactivity for p-αBC was observed in all of the epiretinal membranes examined, where phosphorylation on serine(Ser) 59 showed strongest immunoreactivity in over 70% of the membranes. The immunolocalization of p-αBC was detected in the CD31-positive endothelial cells, and co-localized with VEGF and p-p38 MAPK in PDR membranes. Immunoreactivity for p-αBC, however, was undetectable in endothelial cells of the normal retinas, where p-p38 MAPK immunoreactivity was less marked than PDR membranes.CONCLUSION: Phosphorylation of αBC, in particular,phosphorylation on Ser59 by p-p38 MAPK may play a potential role as a molecular chaperon for VEGF in the pathogenesis of epiretinal membranes in PDR.
文摘LHCII is a crucial light-harvesting pigment/protein complex in photosystem II (PSII) supercomplex. It also participates in the light energy redistribution between photosystems and in the photoprotection via its reversible dissociation with PSII and PSI (photosystem I). This reversible detachment of LHCII is regulated by phosphorylation of its own and PSII core protein. Under low light conditions, LHCII is phosphorylated and dissociated with PSII core protein complex and combined with PSI, which balances the excitation energy between PSII and PSI;Under high light environment, the phosphorylation of PSII core proteins makes LHCII detach from PSII. The dissociated LHCII presents in a free state, which involves in the thermal dissipation of excess excitation energy. During photodamage, dual phosphorylations of both PSII core proteins and LHCII complexes occur. The phosphorylation of D1 is conductive to the disintegration of photodamaged PSII and the cycle of repair. In this circumstance, the phosphorylation of LHCII is induced by reactive oxygen species (ROS) and then the phosphorylated LHCII migrates to PSI, into the repair cycle of damaged PSII. The ferredoxin (Fdr) and thioredoxin (Tdr) system may play a possible central role in the phosphorylation regulation on LHCII dissociation.
基金funded by the National Agricultural Science and Technology Innovation Program in China
文摘This study aimed to examine changes in phosphorylation of sarcoplasmic and myofibrillar proteins from longissimus lumborum,semitendinosus,and psoas major muscles during postmortem ageing for 5 d.These sarcoplasmic and myofibrillar proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with phosphorous and protein specific stains.Myofibril fragmentation index,pH,the content of lactic acid and the relative activity of μ-calpain in three ovine muscles were measured.These results showed that the relative phosphorylation level of sarcoplasmic and myofibrillar proteins of psoas major muscle were lower compared with longissimus lumborum and semitendinosus muscles(P<0.05).The pH of psoas major muscle was the lowest at 0.5 h postmortem,and the highest after 12 h postmortem(P<0.05).In addition,the relative activity of μ-calpain was higher within 5 d postmortem and myofibril fragmentation index was higher after 1 d postmortem in psoas major muscle than those of longissimus lumborum and semitendinosus muscles(P<0.05).The sarcoplasmic protein phosphorylation may regulate the rate of pH decline to influence the μ-calpain activity and then proteolysis of proteins consequently.This study gives a new perspective of the mechanism of postmortem meat tenderization.
文摘Nowadays,the cumulative intake of glucocorticoids has become the most common pathogenic factor for non-traumatic osteonecrosis of the femoral head(ONFH).Apoptosis of osteoblasts is considered as the main reason of ONFH at the molecular level.Glycogen synthase kinase 3β(GSK3β)is an important regulator of cellular differentiation and apoptosis pathway,which can modulate the balance between osteoblasts and osteoclasts.Several studies have reported about its function in osteoporosis,but little is known about it in osteonecrosis.In our study,lipopolysaccharide and methylprednisolone were utilized to establish a rat ONFH model.The phosphorylation of GSK3βSer-9 was decreased in the model.Western blotting examination ofβ-catenin,Bcl-2,Bax and caspase-3 revealed that the osteoblasts were apoptotic.In dexamethasone(Dex)-incubated primary osteoblasts,the expression profile of GSK3βphosphorylation and apoptotic factors were consistent with those in the rat ONFH model.To further investigate the regulation of osteonecrosis caused by GSK3β,the expression and function of GSK3βwere inhibited in Dex-incubated primary osteoblasts.The knockdown of GSK3βby siRNA decreased the expression of Bax and cleaved caspase-3,but increased Bcl-2 andβ-catenin.On the other hand,selective inhibition of GSK3βfunction by LiCl counteracted the activation of caspase-3 induced by Dex.Our work is the first study about the GSK3P phosphorylation in ONFH,and provides evidence for further therapeutic methods.
文摘Dendritic cells (DCs) are the most potent antigen-presen ting cells that play crucial roles in the regulation of immune response. Triptol ide, an active component purified from the medicinal plant Tripterygium wilfor dii Hook F., has been demonstrated to act as a potent immunosuppressive drug c apab le of inhibiting T cell activation and proliferation. However, little is known a bout the effects of triptolide on DCs. The present study shows that triptolide d oes not affect phenotypic differentiation and LPS-induced maturation of murine DCs. But triptolide can dramatically reduce cell recovery by inducing apoptosis of DCs at concentration as low as 10 ng/ml, as demonstrated by phosphatidylserin e exposure, mitochondria potential decrease, and nuclear DNA condensation. Tript olide induces activation of p38 in DCs, which precedes the activation of caspase 3. SB203580, a specific kinase inhibitor for p38, can block the activation of caspase 3 and inhibit the resultant apoptosis of DCs. Our results suggest that t he anti-inflammatory and immunosuppressive activities of triptolide may be due, in part, to its apoptosis-inducing effects on DCs.
基金provided by K01-AR060433 (T.Q.)K08-AR064833 (J.C)R01-AR063943 (X.C)
文摘Parathyroid hormone(PTH) regulates bone remodeling by activating PTH type 1 receptor(PTH1R) in osteoblasts/osteocytes. Insulinlike growth factor type 1(IGF-1) stimulates mesenchymal stem cell differentiation to osteoblasts. However, little is known about the signaling mechanisms that regulates the osteoblast-to-osteocyte transition. Here we report that PTH and IGF-I synergistically enhance osteoblast-to-osteocyte differentiation. We identified that a specific tyrosine residue, Y494, on the cytoplasmic domain of PTH1R can be phosphorylated by insulin-like growth factor type I receptor(IGF1R) in vitro. Phosphorylated PTH1R localized to the barbed ends of actin filaments and increased actin polymerization during morphological change of osteoblasts into osteocytes.Disruption of the phosphorylation site reduced actin polymerization and dendrite length. Mouse models with conditional ablation of PTH1R in osteoblasts demonstrated a reduction in the number of osteoctyes and dendrites per osteocyte, with complete overlap of PTH1R with phosphorylated-PTH1R positioning in osteocyte dendrites in wild-type mice. Thus, our findings reveal a novel signaling mechanism that enhances osteoblast-to-osteocyte transition by direct phosphorylation of PTH1R by IGF1R.
文摘Protein phosphorylation,one of the major post-translational modifications,plays a crucial role in cell signaling,DNA replication,gene expression and differentiation;and alters enzyme activity and other biological activities;and regulates cell proliferation and enlargement,phytohormone biosynthesis and signaling,plant disease resistance,and grain filling and quality during rice seed development.Research work on protein phosphorylation started in the 1950 s with the discovery of phosphorylase a and phosphorylase b which are phospho and dephospho forms of the same enzyme.Over the last decade,rice proteomics has accomplished tremendous progress in setting up techniques to proteome nearly all tissues,organs and organelles.The progress made in this field is evident in number of research works.However,research on rice protein phosphorylation is still at its infancy and there are still many unanswered questions.In this review,the general description of protein phosphorylation,including history,structure,frequency of occurrence and function,are discussed.This work also elucidates the different methods for identification,qualification and finally,the progress in rice phosphoproteome research and perspectives.
文摘Disturbances in constitutive nitric oxide synthase (cNOS) activation associated with H. pylori colonization of gastric mucosa are considered of major consequences in defining the extent of inflammatory involvement. As rapid changes in cNOS activation are linked to the enzyme phosphorylation at the specific Ser/Thr residues, we investigated the influence of H. pylori LPS and gastric hormone, ghrelin, on the processes of phosphorylation of these two critical sites in gastric mucosal cells. We show that the LPS-induced reduction in cNOS activity is reflected in the phosphorylation on Thr497, while the countering effect of ghrelin is associated with a rapid increase in cNOS phosphorylation on Ser1179. Further, we demonstrate that cNOS phosphorylation on Thr497 as well as Ser1179 displays dependence on PKCδ. However, while the LPS-induced suppression in cNOS activation shows reliance on the phosphorylation of PKCδ and PI3K on Ser, the effect of ghrelin is manifested by the increase in phosphorylation of PKCδ and PI3K on Tyr, as well as membrane translocation and phosphorylation of Akt on Ser493. Thus, our findings suggest that the LPS-induced suppression in cNOS activation is mediated by PKCδ-controlled phosphorylation of PI3K on Ser that interferes with the membrane recruitment of Akt and promotes cNOS phosphorylation on Thr497, and that ghrelin-elicited up-regulation in cNOS activation relies on the PKCδ-directed phosphorylation of PI3K on Tyr that stimulates the membrane localization of Akt and enhances cNOS phosphorylation on Ser1179.
基金supported by NIH grant DE026461start funding of Texas A&M University College of Dentistry
文摘Enamelin(ENAM) has three putative phosphoserines(p Sers) phosphorylated by a Golgi-associated secretory pathway kinase(FAM20 C) based on their distinctive Ser-x-Glu(S-x-E) motifs. Fam20 C-knockout mice show severe enamel defects similar to those in the Enam-knockout mice, implying an important role of the p Sers in ENAM. To determine the role of pSer^(55) in ENAM, we characterized ENAM^(Rgsc514) mice, in which Ser^(55) cannot be phosphorylated by FAM20 C due to an E^(57)>G^(57) mutation in the S-x-E motif. The enamel microstructure of 4-week-old mice was examined by scanning electron microscopy.The teeth of 6-day-old mice were characterized by histology and immunohistochemistry. The protein lysates of the first lower molars of 4-day-old mice were analyzed by Western immunoblotting using antibodies against ENAM, ameloblastin and amelogenin. ENAM^(Rgsc514) heterozygotes showed a disorganized enamel microstructure, while the homozygotes had no enamel on the dentin surface. The N-terminal fragments of ENAM in the heterozygotes were detained in the ameloblasts and localized in the mineralization front of enamel matrix, while those in the WT mice were secreted out of ameloblasts and distributed evenly in the outer 1/2 of enamel matrix. Surprisingly, the ~ 15 k Da C-terminal fragments of ameloblastin were not detected in the molar lysates of the homozygotes. These results suggest that the phosphorylation of Ser^(55) may be an essential posttranslational modification of ENAM and is required for the interaction with other enamel matrix molecules such as ameloblastin in mediating the structural organization of enamel matrix and protein-mineral interactions during enamel formation.
基金a grant from National Natu-ral Sciences Foundation of China (No. 30370573)
文摘In order to confirm the hypothesis that during acute hypoxia, the antiarrhythmic peptide (AAP10) could improve conductance by changing the phosphorylation state of connexin43 (Cx43), isolated perfused rat hearts were randomly divided into three groups: control, hypoxia and AAP10 (n=9 in each group). The change in Cx43 phosphorylation was tested by Western-blot; the distribu- tion of Cx43 was observed by confocal immunofluorescence microscopy. Western-blot analysis re- vealed that the expression of total Cx43 protein was significantly decreased during acute hypoxia, while nonphosphorylated Cx43 (NP-Cx43) was unchanged. AAP10 could increase the expression of total Cx43 protein, but had no effects on the NP-Cx43 protein. Immunofluorescence study showed that during acute hypoxia, both total Cx43 and NP-Cx43 proteins were greatly decreased, while AAP10 only increased the expression of total Cx43 protein, but had no effect of the NP-Cx43 protein expression. These findings suggested that the decrease of intercellular communication may be associ- ated with the reduction of phosphorylated Cx43 (p-Cx43) and translocation of NP-Cx43 from the surface of gap junction into intracellular pools during acute hypoxia. AAP10 can improve intercelluar communication by enhancing phosphorylation of Cx43.
