A bifactorial endosperm box (EB), which contains an endosperm motif (EM) and a GCN4 motif, was found in rice Wx promoter. EB was found in 5′ upstream region of many seed storage protein genes accounting for these gen...A bifactorial endosperm box (EB), which contains an endosperm motif (EM) and a GCN4 motif, was found in rice Wx promoter. EB was found in 5′ upstream region of many seed storage protein genes accounting for these genes expression exclusive in endosperm among various cereals. Many reports demonstrated that the bZIP transcription activators isolated from wheat, barley and maize, etc. regulate the gene expression through binding to the GCN4 motif. In this research, we showed that GCN4 sequence could be recognized by nuclear proteins extracted from immature rice seeds. Furthermore, a rice bZIP protein, REB was isolated by using PCR method and REB fusion protein was expressed in E. coli. The results of gel shift analysis showed that REB could recognize and bind to the GCN4 motif in the Wx gene in addition to binding to the target sequence in the promoter of α-globulin.展开更多
The stability of GCN4 leucine zipper and its four mutants in guanidine hydrochloride was detected to verify the contributions of different a position amino acid residues in polypeptide sequences to the forming and sta...The stability of GCN4 leucine zipper and its four mutants in guanidine hydrochloride was detected to verify the contributions of different a position amino acid residues in polypeptide sequences to the forming and stability of parallel coiled coils. The changes of the circular dichroism spectra show that the displace- ment of the a position polar asparagine and the increase of asparagine in the GCN4 leucine zipper can reduce the α-helix content of the coiled coil structure. The mutants are less stable than the natural peptide in guanidine hydrochloride. The results show that the interaction between the polar asparagine contributes to the conformational stability of the coiled coil. Both the conformation and the number of polar residues in the coiled coil also affect the α-helix content and its resistance to the denaturant. The conclusions provide evidence describing the folding process of proteins including coiled coils in vivo.展开更多
GCN4是转录调节蛋白质二聚体,它可以在氨基酸饥饿的条件下控制酵母中histidine的生物合成。GCN4DNA结合区域是碱性区域/leucine拉链（bZIP）结构。GCN4的leucine拉链结构二聚成coiled-coil结构。GCN4的碱性区域结构光滑地插入AP-1DNA片...GCN4是转录调节蛋白质二聚体,它可以在氨基酸饥饿的条件下控制酵母中histidine的生物合成。GCN4DNA结合区域是碱性区域/leucine拉链（bZIP）结构。GCN4的leucine拉链结构二聚成coiled-coil结构。GCN4的碱性区域结构光滑地插入AP-1DNA片段的两边并与DNA主沟的对面结合。脚印跟踪（foot-printing）和one/two hybrid system protocols被用来研究GCN4和AP-1结合的定量构效关系。Max2-Jun是来自GCN4的变体之一。Max2-Jun的检测和表达的初步结果（Agro gel和HPLC）以及Max2-Jun和Ebox/XRE1的初步结合实验（foot-printing）表明Max2-Jun可能伴随一些不需要的组分。这些不需要的组分可能不能从需要的DNA片段/蛋白质中除去并且可能影响（正的/负的）需要的结合作用。进一步的GCN4蛋白质模型系统的设计正在进行中。展开更多
Gln, one of the most abundant amino acids(AA) in the body, performs a diverse range of fundamental physiological functions. However, information about the role of dietary Gln on AA levels, transporters,protein synthes...Gln, one of the most abundant amino acids(AA) in the body, performs a diverse range of fundamental physiological functions. However, information about the role of dietary Gln on AA levels, transporters,protein synthesis, and underlying mechanisms in vivo is scarce. The present study aimed to explore the effects of low-crude protein diet inclusion with differential doses of L-Gln on intestinal AA levels,transporters, protein synthesis, and potential mechanisms in weaned piglets. A total of 128 healthy weaned piglets(Landrace × Yorkshire) were randomly allocated into four treatments with four replicates. Pigs in the four groups were fed a low-crude protein diet containing 0%, 1%, 2%, or 3% L-Gln for28 d. L-Gln administration markedly(linear, P < 0.05) increased Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, Lys,Met, Orn, Phe, Ser, Thr, Tyr, and Val levels and promoted trypsin activity in the jejunal content of piglets.Moreover, L-Gln treatment significantly enhanced concentrations of colonic Gln and Trp, and serum Thr(linear, P < 0.01), and quadratically increased serum Lys and Phe levels(P < 0.05), and decreased plasma Glu, Ile, and Leu levels(linear, P < 0.05). Further investigation revealed that L-Gln administration significantly upregulated Atp1a1, Slc1a5, Slc3a2, Slc6a14, Slc7a5, Slc7a7, and Slc38a1 relative expressions in the jejunum(linear, P < 0.05). Additionally, dietary supplementation with L-Gln enhanced protein abundance of general control nonderepressible 2(GCN2, P = 0.010), phosphorylated eukaryotic initiation factor 2 subunit alpha(eIF2α, P < 0.001), and activating transcription factor 4(ATF4) in the jejunum of piglets(P = 0.008). These results demonstrated for the first time that a low crude protein diet with highlevel L-Gln inclusion exhibited side effects on piglets. Specifically, 2% and 3% L-Gln administration exceeded the intestinal utilization capacity and compromised the jejunal AA utilization efficiency, which is independent of digestive enzyme activities. A high level of L-Gln supplementation would inhibit protein synthesis by GCN2/eIF2α/ATF4 signaling in piglets fed low-protein diets, which, in turn, upregulates certain AA transporters to maintain AA homeostasis.展开更多
基金This work was supported by the National Natural Science Foundation of China (Grant No. 39893320).
文摘A bifactorial endosperm box (EB), which contains an endosperm motif (EM) and a GCN4 motif, was found in rice Wx promoter. EB was found in 5′ upstream region of many seed storage protein genes accounting for these genes expression exclusive in endosperm among various cereals. Many reports demonstrated that the bZIP transcription activators isolated from wheat, barley and maize, etc. regulate the gene expression through binding to the GCN4 motif. In this research, we showed that GCN4 sequence could be recognized by nuclear proteins extracted from immature rice seeds. Furthermore, a rice bZIP protein, REB was isolated by using PCR method and REB fusion protein was expressed in E. coli. The results of gel shift analysis showed that REB could recognize and bind to the GCN4 motif in the Wx gene in addition to binding to the target sequence in the promoter of α-globulin.
基金Supported by the National Natural Science Foundation of China (No. 30170199) and the Basic Research Foundation of Tsinghua University (No. JC2003050)
文摘The stability of GCN4 leucine zipper and its four mutants in guanidine hydrochloride was detected to verify the contributions of different a position amino acid residues in polypeptide sequences to the forming and stability of parallel coiled coils. The changes of the circular dichroism spectra show that the displace- ment of the a position polar asparagine and the increase of asparagine in the GCN4 leucine zipper can reduce the α-helix content of the coiled coil structure. The mutants are less stable than the natural peptide in guanidine hydrochloride. The results show that the interaction between the polar asparagine contributes to the conformational stability of the coiled coil. Both the conformation and the number of polar residues in the coiled coil also affect the α-helix content and its resistance to the denaturant. The conclusions provide evidence describing the folding process of proteins including coiled coils in vivo.
文摘GCN4是转录调节蛋白质二聚体,它可以在氨基酸饥饿的条件下控制酵母中histidine的生物合成。GCN4DNA结合区域是碱性区域/leucine拉链（bZIP）结构。GCN4的leucine拉链结构二聚成coiled-coil结构。GCN4的碱性区域结构光滑地插入AP-1DNA片段的两边并与DNA主沟的对面结合。脚印跟踪（foot-printing）和one/two hybrid system protocols被用来研究GCN4和AP-1结合的定量构效关系。Max2-Jun是来自GCN4的变体之一。Max2-Jun的检测和表达的初步结果（Agro gel和HPLC）以及Max2-Jun和Ebox/XRE1的初步结合实验（foot-printing）表明Max2-Jun可能伴随一些不需要的组分。这些不需要的组分可能不能从需要的DNA片段/蛋白质中除去并且可能影响（正的/负的）需要的结合作用。进一步的GCN4蛋白质模型系统的设计正在进行中。
基金supported by grants from the National Key R&D Program of China(2022YFF1100102,2022YFF1100104,2022YFC2105005)the National Natural Science Foundation of China(No.32172749,31625025).
文摘Gln, one of the most abundant amino acids(AA) in the body, performs a diverse range of fundamental physiological functions. However, information about the role of dietary Gln on AA levels, transporters,protein synthesis, and underlying mechanisms in vivo is scarce. The present study aimed to explore the effects of low-crude protein diet inclusion with differential doses of L-Gln on intestinal AA levels,transporters, protein synthesis, and potential mechanisms in weaned piglets. A total of 128 healthy weaned piglets(Landrace × Yorkshire) were randomly allocated into four treatments with four replicates. Pigs in the four groups were fed a low-crude protein diet containing 0%, 1%, 2%, or 3% L-Gln for28 d. L-Gln administration markedly(linear, P < 0.05) increased Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, Lys,Met, Orn, Phe, Ser, Thr, Tyr, and Val levels and promoted trypsin activity in the jejunal content of piglets.Moreover, L-Gln treatment significantly enhanced concentrations of colonic Gln and Trp, and serum Thr(linear, P < 0.01), and quadratically increased serum Lys and Phe levels(P < 0.05), and decreased plasma Glu, Ile, and Leu levels(linear, P < 0.05). Further investigation revealed that L-Gln administration significantly upregulated Atp1a1, Slc1a5, Slc3a2, Slc6a14, Slc7a5, Slc7a7, and Slc38a1 relative expressions in the jejunum(linear, P < 0.05). Additionally, dietary supplementation with L-Gln enhanced protein abundance of general control nonderepressible 2(GCN2, P = 0.010), phosphorylated eukaryotic initiation factor 2 subunit alpha(eIF2α, P < 0.001), and activating transcription factor 4(ATF4) in the jejunum of piglets(P = 0.008). These results demonstrated for the first time that a low crude protein diet with highlevel L-Gln inclusion exhibited side effects on piglets. Specifically, 2% and 3% L-Gln administration exceeded the intestinal utilization capacity and compromised the jejunal AA utilization efficiency, which is independent of digestive enzyme activities. A high level of L-Gln supplementation would inhibit protein synthesis by GCN2/eIF2α/ATF4 signaling in piglets fed low-protein diets, which, in turn, upregulates certain AA transporters to maintain AA homeostasis.
