Nanoscale drug delivery systems(NDDSs)have emerged as promising carriers for combinational therapy by co-delivery of multiple drugs and modalities.However,most co-delivery systems require the use of complicated materi...Nanoscale drug delivery systems(NDDSs)have emerged as promising carriers for combinational therapy by co-delivery of multiple drugs and modalities.However,most co-delivery systems require the use of complicated materials and formulations.Herein,we report the single use of a polymeric material,namely mPEG-block-poly(phosphotyrosine)(mPEG-b-PpY),as a multi-functional carrier for the facile fabrication of NDDS Pt/Ce6@mPEG-b-PpY(PtCeNP)for the co-delivery of cisplatin and photosensitizer chlorin e6(Ce6)via phosphato-platinum coordination andπ-πstacking,respectively.PtCeNP improves the solubility,cellular uptake,and bioavailability of both parental drugs,and showed strong synergistic antitumor efficacy both in vitro and in vivo through combined chemo-photodynamic therapy.Our results indicate that PpY is a biocompatible,multifunctional,and promising carrier material suitable for a variety of drugs and may simplify the design for co-delivery systems.展开更多
BACKGROUND Adaptor protein,phosphotyrosine interacting with PH domain and leucine zipper 1(APPL1)plays a crucial role in regulating insulin signaling and glucose metabolism.Mutations in the APPL1 gene have been associ...BACKGROUND Adaptor protein,phosphotyrosine interacting with PH domain and leucine zipper 1(APPL1)plays a crucial role in regulating insulin signaling and glucose metabolism.Mutations in the APPL1 gene have been associated with the development of maturity-onset diabetes of the young type 14(MODY14).Currently,only two mutations[c.1655T>A(p.Leu552*)and c.281G>A p.(Asp94Asn)]have been identified in association with this disease.Given the limited understanding of MODY14,it is imperative to identify additional cases and carry out comprehensive research on MODY14 and APPL1 mutations.AIM To assess the pathogenicity of APPL1 gene mutations in diabetic patients and to characterize the functional role of the APPL1 domain.METHODS Patients exhibiting clinical signs and a medical history suggestive of MODY were screened for the study.Whole exome sequencing was performed on the patients as well as their family members.The pathogenicity of the identified APPL1 variants was predicted on the basis of bioinformatics analysis.In addition,the pathogenicity of the novel APPL1 variant was preliminarily evaluated through in vitro functional experiments.Finally,the impact of these variants on APPL1 protein expression and the insulin pathway were assessed,and the potential mechanism underlying the interaction between the APPL1 protein and the insulin receptor was further explored.RESULTS A total of five novel mutations were identified,including four missense mutations(Asp632Tyr,Arg633His,Arg532Gln,and Ile642Met)and one intronic mutation(1153-16A>T).Pathogenicity prediction analysis revealed that the Arg532Gln was pathogenic across all predictions.The Asp632Tyr and Arg633His variants also had pathogenicity based on MutationTaster.In addition,multiple alignment of amino acid sequences showed that the Arg532Gln,Asp632Tyr,and Arg633His variants were conserved across different species.Moreover,in in vitro functional experiments,both the c.1894G>T(at Asp632Tyr)and c.1595G>A(at Arg532Gln)mutations were found to downregulate the expression of APPL1 on both protein and mRNA levels,indicating their pathogenic nature.Therefore,based on the patient’s clinical and family history,combined with the results from bioinformatics analysis and functional experiment,the c.1894G>T(at Asp632Tyr)and c.1595G>A(at Arg532Gln)mutations were classified as pathogenic mutations.Importantly,all these mutations were located within the phosphotyrosinebinding domain of APPL1,which plays a critical role in the insulin sensitization effect.CONCLUSION This study provided new insights into the pathogenicity of APPL1 gene mutations in diabetes and revealed a potential target for the diagnosis and treatment of the disease.展开更多
To complete their life cycle and spread, viruses interfere with and gain control of diverse cellular processes, this most often occurring through interaction between viral proteins(VPs) and resident protein partners. ...To complete their life cycle and spread, viruses interfere with and gain control of diverse cellular processes, this most often occurring through interaction between viral proteins(VPs) and resident protein partners. Among the latter, Src family kinases(SFKs), a class of non-receptor tyrosine kinases that contributes to the conversion of extracellular signals into intracellular signaling cascades and is involved in virtually all cellular processes, have recently emerged as critical mediators between the cell's infrastructure and the viral demands. In this scenario, structural or ex novo synthesized VPs are able to bind to the different domains of these enzymes through specific short linear motifs present along their sequences. Proline-rich motifs displaying the conserved minimal consensus PxxP and recognizing the SFK Src homology(SH)3 domain constitute a cardinal signature for the formation of multiprotein complexes and this interaction may promote phosphorylation of VPs by SFKs, thus creating phosphotyrosine motifs that become a docking site for the SH2 domains of SFKs or other SH2 domain-bearing signaling molecules. Importantly, the formation of these assemblies also results in a change in the activity and/or location of SFKs, and these events are critical in perturbing key signalingpathways so that viruses can utilize the cell's machinery to their own benefit. In the light of these observations, although VPs as such, especially those with enzyme activity, are still regarded as valuable targets for therapeutic strategies, multiprotein complexes composed of viral and host cell proteins are increasingly becoming objects of investigation with a view to deeply characterize the structural aspects that favor their formation and to develop new compounds able to contrast viral diseases in an alternative manner.展开更多
The effect of okadaic acid (OA) on proto-oncogene protein expression of c-neu, c-myc, v-rasH, EGFR, and phosphotyrosine-containing phosphoproteins (P-Tyr) was investigated in rapidly growing (RG) normal human keratino...The effect of okadaic acid (OA) on proto-oncogene protein expression of c-neu, c-myc, v-rasH, EGFR, and phosphotyrosine-containing phosphoproteins (P-Tyr) was investigated in rapidly growing (RG) normal human keratinocytes (NHK) and in SV-40 virally-transformed keratinocytes (SVK) cultured in a growth factor supplemented serum-free medium as assessed by indirect immunofluorescence microscopy. P-Tyr positively stains cell surface antigens (cytoplasm) diffusely at monopolar sites in RG NHK cultures. OA-treatment intensifies cytoplasmic P-Tyr staining at localized monopolar intercellular focal adhesion (IFA) sites with reduced cytoplasmic staining. P-Tyr expression was predominate at IFA sites with little cytoplasmic staining in RG SVK cultures. OA-treatment increased monopolar P-Tyr staining and cytoplasmic staining. OA-treatment in RG NHK cultures intensified cytoplasmic staining of c-myc and EGFR (epidermal growth factor receptor) expression. OA-treatment in RG NHK and SVK cultures intensified c-neu staining at monopolar IFA sites and intensified c-neu staining at both cytoplasmic and bipolar IFA sites in RG SVK cells. OA was especially cytotoxic for SVK cells. RA treatment decreased c-neu expression in RG NHK cultures while TPA treatment has a lesser effect on both cytoplasmic and IFA sites. RA treatment also decreased P-Tyr staining in both NHK and SVK cells. Again, TPA had a lesser inhibitory effect on P-Tyr staining pattern. RA-treatment had a similar effect on P-Tyr staining of RG cultures of a mouse fibroblast cell line. These results confirm the generality of OA, RA and TPA on the regulation of oncogene expression in both normal and malignantly transformed keratinocytes.展开更多
基金supported by the National Key Research and Development Program of China(2016YFA0201400)the Na-tional Natural Science Foundation of China for Distinguished Young Investigators(22125101).
文摘Nanoscale drug delivery systems(NDDSs)have emerged as promising carriers for combinational therapy by co-delivery of multiple drugs and modalities.However,most co-delivery systems require the use of complicated materials and formulations.Herein,we report the single use of a polymeric material,namely mPEG-block-poly(phosphotyrosine)(mPEG-b-PpY),as a multi-functional carrier for the facile fabrication of NDDS Pt/Ce6@mPEG-b-PpY(PtCeNP)for the co-delivery of cisplatin and photosensitizer chlorin e6(Ce6)via phosphato-platinum coordination andπ-πstacking,respectively.PtCeNP improves the solubility,cellular uptake,and bioavailability of both parental drugs,and showed strong synergistic antitumor efficacy both in vitro and in vivo through combined chemo-photodynamic therapy.Our results indicate that PpY is a biocompatible,multifunctional,and promising carrier material suitable for a variety of drugs and may simplify the design for co-delivery systems.
基金Supported by the National Natural Science Foundation,No.81974124and Taishan Scholar Project,No.tsqn20161071.
文摘BACKGROUND Adaptor protein,phosphotyrosine interacting with PH domain and leucine zipper 1(APPL1)plays a crucial role in regulating insulin signaling and glucose metabolism.Mutations in the APPL1 gene have been associated with the development of maturity-onset diabetes of the young type 14(MODY14).Currently,only two mutations[c.1655T>A(p.Leu552*)and c.281G>A p.(Asp94Asn)]have been identified in association with this disease.Given the limited understanding of MODY14,it is imperative to identify additional cases and carry out comprehensive research on MODY14 and APPL1 mutations.AIM To assess the pathogenicity of APPL1 gene mutations in diabetic patients and to characterize the functional role of the APPL1 domain.METHODS Patients exhibiting clinical signs and a medical history suggestive of MODY were screened for the study.Whole exome sequencing was performed on the patients as well as their family members.The pathogenicity of the identified APPL1 variants was predicted on the basis of bioinformatics analysis.In addition,the pathogenicity of the novel APPL1 variant was preliminarily evaluated through in vitro functional experiments.Finally,the impact of these variants on APPL1 protein expression and the insulin pathway were assessed,and the potential mechanism underlying the interaction between the APPL1 protein and the insulin receptor was further explored.RESULTS A total of five novel mutations were identified,including four missense mutations(Asp632Tyr,Arg633His,Arg532Gln,and Ile642Met)and one intronic mutation(1153-16A>T).Pathogenicity prediction analysis revealed that the Arg532Gln was pathogenic across all predictions.The Asp632Tyr and Arg633His variants also had pathogenicity based on MutationTaster.In addition,multiple alignment of amino acid sequences showed that the Arg532Gln,Asp632Tyr,and Arg633His variants were conserved across different species.Moreover,in in vitro functional experiments,both the c.1894G>T(at Asp632Tyr)and c.1595G>A(at Arg532Gln)mutations were found to downregulate the expression of APPL1 on both protein and mRNA levels,indicating their pathogenic nature.Therefore,based on the patient’s clinical and family history,combined with the results from bioinformatics analysis and functional experiment,the c.1894G>T(at Asp632Tyr)and c.1595G>A(at Arg532Gln)mutations were classified as pathogenic mutations.Importantly,all these mutations were located within the phosphotyrosinebinding domain of APPL1,which plays a critical role in the insulin sensitization effect.CONCLUSION This study provided new insights into the pathogenicity of APPL1 gene mutations in diabetes and revealed a potential target for the diagnosis and treatment of the disease.
文摘To complete their life cycle and spread, viruses interfere with and gain control of diverse cellular processes, this most often occurring through interaction between viral proteins(VPs) and resident protein partners. Among the latter, Src family kinases(SFKs), a class of non-receptor tyrosine kinases that contributes to the conversion of extracellular signals into intracellular signaling cascades and is involved in virtually all cellular processes, have recently emerged as critical mediators between the cell's infrastructure and the viral demands. In this scenario, structural or ex novo synthesized VPs are able to bind to the different domains of these enzymes through specific short linear motifs present along their sequences. Proline-rich motifs displaying the conserved minimal consensus PxxP and recognizing the SFK Src homology(SH)3 domain constitute a cardinal signature for the formation of multiprotein complexes and this interaction may promote phosphorylation of VPs by SFKs, thus creating phosphotyrosine motifs that become a docking site for the SH2 domains of SFKs or other SH2 domain-bearing signaling molecules. Importantly, the formation of these assemblies also results in a change in the activity and/or location of SFKs, and these events are critical in perturbing key signalingpathways so that viruses can utilize the cell's machinery to their own benefit. In the light of these observations, although VPs as such, especially those with enzyme activity, are still regarded as valuable targets for therapeutic strategies, multiprotein complexes composed of viral and host cell proteins are increasingly becoming objects of investigation with a view to deeply characterize the structural aspects that favor their formation and to develop new compounds able to contrast viral diseases in an alternative manner.
文摘The effect of okadaic acid (OA) on proto-oncogene protein expression of c-neu, c-myc, v-rasH, EGFR, and phosphotyrosine-containing phosphoproteins (P-Tyr) was investigated in rapidly growing (RG) normal human keratinocytes (NHK) and in SV-40 virally-transformed keratinocytes (SVK) cultured in a growth factor supplemented serum-free medium as assessed by indirect immunofluorescence microscopy. P-Tyr positively stains cell surface antigens (cytoplasm) diffusely at monopolar sites in RG NHK cultures. OA-treatment intensifies cytoplasmic P-Tyr staining at localized monopolar intercellular focal adhesion (IFA) sites with reduced cytoplasmic staining. P-Tyr expression was predominate at IFA sites with little cytoplasmic staining in RG SVK cultures. OA-treatment increased monopolar P-Tyr staining and cytoplasmic staining. OA-treatment in RG NHK cultures intensified cytoplasmic staining of c-myc and EGFR (epidermal growth factor receptor) expression. OA-treatment in RG NHK and SVK cultures intensified c-neu staining at monopolar IFA sites and intensified c-neu staining at both cytoplasmic and bipolar IFA sites in RG SVK cells. OA was especially cytotoxic for SVK cells. RA treatment decreased c-neu expression in RG NHK cultures while TPA treatment has a lesser effect on both cytoplasmic and IFA sites. RA treatment also decreased P-Tyr staining in both NHK and SVK cells. Again, TPA had a lesser inhibitory effect on P-Tyr staining pattern. RA-treatment had a similar effect on P-Tyr staining of RG cultures of a mouse fibroblast cell line. These results confirm the generality of OA, RA and TPA on the regulation of oncogene expression in both normal and malignantly transformed keratinocytes.
