Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary ce...Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary cell wall formation. It has also been suggested that such three CesAs interact with each other to form plasma-membrane bound rosette complexes that are functional during cellulose production. However, in vivo demonstration of such assemblies of three CesAs into rosettes has not been possible. We used yeast two-hybrid assays to demonstrate the possible interactions among several CesAs from Arabidopsis and aspen via their N-terminal zinc-binding domains (ZnBDs). While strong positive interactions were detected among ZnBDs from secondary wall associated CesAs of both Arabidopsis and aspen, the intergeneric interactions between Arabidopsis and aspen CesAs were weak. Moreover, in aspen, three primary wall associated CesA ZnBDs positively interacted with each other as well as with secondary CesAs. These results suggest that ZnBDs from either primary or secondary CesAs, and even from different plant species could interact but are perhaps insufficient for specificities of such interactions among CesAs. These observations suggest that some other more specific interacting regions might exist within CesAs. It is also possible that some hitherto unknown mechanism exists in plants for assembling the rosette complexes with different compositions of CesAs. Understanding how cellulose is synthesized will have a direct impact on utilization of lignocellulosic biomass for bioenergy production.展开更多
Background:Endostatin(ES) is a well-established potent endogenous antiangiogenic factor.An ES variant,called zinc-binding protein-ES(ZBP-ES),is clinically available;however,its use is limited by rapid renal clearance ...Background:Endostatin(ES) is a well-established potent endogenous antiangiogenic factor.An ES variant,called zinc-binding protein-ES(ZBP-ES),is clinically available;however,its use is limited by rapid renal clearance and short residence time.PEGylation has been exploited to overcome these shortcomings,and mono-PEGylated ES(called M_2ES) as well as mono-PEGylated ZBP-ES(MZBP-ES) are developed in our study.This study aimed to compare the biophysical properties and biological effects of M_2ES and MZBP-ES to evaluate their druggability.Methods:Circular dichroism and tryptophan emission fluorescence were used to monitor the conformational changes of M_2ES and MZBP-ES.Their resistance to trypsin digestion and guanidinium chloride(GdmCl)-induced unfolding was examined by Coomassie staining and tryptophan emission fluorescence,respectively.The biological effects of M_2ES and MZBP-ES on endothelial cell migration were evaluated using Transwell migration and wound healing assays,and the uptake of M_2ES and MZBP-ES in endothelial cells was also compared by Western blotting and immunofluorescence.Results:Structural analyses revealed that M_2ES has a more compact tertiary structure than MZBP-ES.Moreover,M_2ES was more resistant to trypsin digestion and GdmCI-induced unfolding compared with MZBP-ES.In addition,although M_2ES and MZBP-ES showed comparable levels of inhibiting transwell migration and wound healing of endothelial cells,M_2ES displayed an increased ability to enter cells compared with MZBP-ES,possibly caused by the enhanced interaction with nucleolin.Conclusions:M_2ES has a more compact tertiary structure,is more stable for trypsin digestion and GdmCI-induced unfolding,exhibits increased cellular uptake and shows equivalent inhibitory effects on cell migration relative to MZBP-ES,indicating that M_2ES is a more promising candidate for anticancer drug development compared with MZBP-ES.展开更多
Importance:Transient neonatal zinc deficiency(TNZD)occurs in breastfed infants due to abnormally low breast milk zinc levels.Mutations in the solute carrier family 30 member 2(SLC30A2)gene,which encodes the zinc trans...Importance:Transient neonatal zinc deficiency(TNZD)occurs in breastfed infants due to abnormally low breast milk zinc levels.Mutations in the solute carrier family 30 member 2(SLC30A2)gene,which encodes the zinc transporter ZNT2,cause low zinc concentration in breast milk.Objective:This study aimed to provide further insights into TNZD pathophysiology.Methods:SLC30A2 sequencing was performed in three unrelated Japanese mothers,whose infants developed TNZD due to low-zinc milk consumption.The effects of the identified mutations were examined using cell-based assays and luciferase reporter analysis.Results:Novel SLC30A2 mutations were identified in each mother.One harbored a heterozygous missense mutation in the ZNT2 zinc-binding site,which resulted in defective zinc transport.The other two mothers exhibited multiple heterozygous mutations in the SLC30A2 promoter,the first mutations in the SLC30A2 regulatory region reported to date.Interpretation:This report provides new genetic insights into TNZD pathogenesis in breastfed infants.展开更多
文摘Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary cell wall formation. It has also been suggested that such three CesAs interact with each other to form plasma-membrane bound rosette complexes that are functional during cellulose production. However, in vivo demonstration of such assemblies of three CesAs into rosettes has not been possible. We used yeast two-hybrid assays to demonstrate the possible interactions among several CesAs from Arabidopsis and aspen via their N-terminal zinc-binding domains (ZnBDs). While strong positive interactions were detected among ZnBDs from secondary wall associated CesAs of both Arabidopsis and aspen, the intergeneric interactions between Arabidopsis and aspen CesAs were weak. Moreover, in aspen, three primary wall associated CesA ZnBDs positively interacted with each other as well as with secondary CesAs. These results suggest that ZnBDs from either primary or secondary CesAs, and even from different plant species could interact but are perhaps insufficient for specificities of such interactions among CesAs. These observations suggest that some other more specific interacting regions might exist within CesAs. It is also possible that some hitherto unknown mechanism exists in plants for assembling the rosette complexes with different compositions of CesAs. Understanding how cellulose is synthesized will have a direct impact on utilization of lignocellulosic biomass for bioenergy production.
基金supported by the General Programs of the National Natural Science Foundation of China(No.81171998,81472667,and 81461148021)the National Science and Technology Major Project for "Major New Drugs Innovation and Development"(No.2013ZX09509103)supported by the General Programs of the National Natural Science Foundation of China(No. 81272529)
文摘Background:Endostatin(ES) is a well-established potent endogenous antiangiogenic factor.An ES variant,called zinc-binding protein-ES(ZBP-ES),is clinically available;however,its use is limited by rapid renal clearance and short residence time.PEGylation has been exploited to overcome these shortcomings,and mono-PEGylated ES(called M_2ES) as well as mono-PEGylated ZBP-ES(MZBP-ES) are developed in our study.This study aimed to compare the biophysical properties and biological effects of M_2ES and MZBP-ES to evaluate their druggability.Methods:Circular dichroism and tryptophan emission fluorescence were used to monitor the conformational changes of M_2ES and MZBP-ES.Their resistance to trypsin digestion and guanidinium chloride(GdmCl)-induced unfolding was examined by Coomassie staining and tryptophan emission fluorescence,respectively.The biological effects of M_2ES and MZBP-ES on endothelial cell migration were evaluated using Transwell migration and wound healing assays,and the uptake of M_2ES and MZBP-ES in endothelial cells was also compared by Western blotting and immunofluorescence.Results:Structural analyses revealed that M_2ES has a more compact tertiary structure than MZBP-ES.Moreover,M_2ES was more resistant to trypsin digestion and GdmCI-induced unfolding compared with MZBP-ES.In addition,although M_2ES and MZBP-ES showed comparable levels of inhibiting transwell migration and wound healing of endothelial cells,M_2ES displayed an increased ability to enter cells compared with MZBP-ES,possibly caused by the enhanced interaction with nucleolin.Conclusions:M_2ES has a more compact tertiary structure,is more stable for trypsin digestion and GdmCI-induced unfolding,exhibits increased cellular uptake and shows equivalent inhibitory effects on cell migration relative to MZBP-ES,indicating that M_2ES is a more promising candidate for anticancer drug development compared with MZBP-ES.
基金supported by a Grant-in-Aid for Scientific Research on Innovative Areas"Integrated Bio-metal Science"(MEXT KAKENHI Grant Number JP19H05768)from the Ministry of Education,Culture,Sports,Science,and Technologya Grant-in-Aid for Scientific Research(B)(JSPS KAKENHI Grant No.JP19H02883)from the Japan Society for the Promotion of Science(to Taiho Kambe).
文摘Importance:Transient neonatal zinc deficiency(TNZD)occurs in breastfed infants due to abnormally low breast milk zinc levels.Mutations in the solute carrier family 30 member 2(SLC30A2)gene,which encodes the zinc transporter ZNT2,cause low zinc concentration in breast milk.Objective:This study aimed to provide further insights into TNZD pathophysiology.Methods:SLC30A2 sequencing was performed in three unrelated Japanese mothers,whose infants developed TNZD due to low-zinc milk consumption.The effects of the identified mutations were examined using cell-based assays and luciferase reporter analysis.Results:Novel SLC30A2 mutations were identified in each mother.One harbored a heterozygous missense mutation in the ZNT2 zinc-binding site,which resulted in defective zinc transport.The other two mothers exhibited multiple heterozygous mutations in the SLC30A2 promoter,the first mutations in the SLC30A2 regulatory region reported to date.Interpretation:This report provides new genetic insights into TNZD pathogenesis in breastfed infants.
