Iron is an essential element for plant growth and development. Iron homeostasis in plants is tightly regulated at both transcriptional and posttranscriptional level. Several bHLH transcription factors involved in iron...Iron is an essential element for plant growth and development. Iron homeostasis in plants is tightly regulated at both transcriptional and posttranscriptional level. Several bHLH transcription factors involved in iron homeostasis have been identified recently. However, their regulatory mechanisms remain unknown. In this work, we demonstrate that the transcription factor FIT interacted with AtbHLH38 and AtbHLH39 and directly conferred the expression regulation of iron uptake genes for iron homeostasis in Arabidopsis. Yeast two-hybrid analysis and transient expression in Arabidopsis protoplasts showed that AtbHLH38 or AtbHLH39 interacted with FIT, a central transcription factor involved in iron homeostasis in Arabidopsis. Expression of FIT/AtbHLH38 or FIT/AtbHLH39 in yeast cells activated GUS expression driven by ferric chelate reductase （FRO2） and ferrous transporter （IRT1） promoters. Overexpression of FITwith either AtbHLH38 or AtbHLH39 in plants converted the expression of the iron uptake genes FRO2 and IRT1 from induced to constitutive. Further analysis revealed that FRO2 and IRT1 were not regulated at the posttranscriptional level in these plants because IRT1 protein accumulation and high ferric chelate reductase activity were detected in the overexpression plants under both iron deficiency and iron sufficiency. The double overexpression plants accumulated more iron in their shoots than wild type or the plants overexpressing either AtbHLH38, AtbHLH39 or FIT. Our data support that ferric-chelate reductase FRO2 and ferrous-transporter IRT1 are the targets of the three transcription factors and the transcription of FRO2 and IRT1 is directly regulated by a complex of FIT/AtbHLH38 or FIT/AtbHLH39.展开更多
The eukaryotic transcription factor NF-Y consists of three subunits （A, B, and C）, which are encoded in Ara- bidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, al...The eukaryotic transcription factor NF-Y consists of three subunits （A, B, and C）, which are encoded in Ara- bidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, all potential combi- nations of the subunits are possible for the assembly of the heterotrimeric complex. We aimed at assessing the probability of each subunit to participate in the assembly of NF-Y. The evaluation of physical interactions among all members of the NF-Y subunit families indicate a strong requirement for NF-YB/NF-YC heterodimerization before the entire complex can be accomplished. By means of a modified yeast two-hybrid system assembly of all three subunits to a heterotrimeric complex was demonstrated. Using GFP fusion constructs, NF-YA and NF-YC localization in the nucleus was demonstrated, while NF- YB is solely imported into the nucleus as a NF-YC-associated heterodimer NF-YC. This piggyback transport of the two Arabidopsis subunits differs from the import of the NF-Y heterotrimer of heterotrophic organisms. Based on a peptide structure model of the histone-fold-motifs, disulfide bonding among intramolecular conserved cysteine residues of NF-YB, which is responsible for the redox-regulated assembly of NF-YB and NF-YC in human and Aspergillus nidulans, can be excluded for Arabidopsis NF-YB.展开更多
Cigarette smoking is a well-known risk factor for cardiovascular disease. Smoking can cause vascular endothelial dysfunction and consequently trigger haemostatic activation and thrombosis. However, the mechanism of ho...Cigarette smoking is a well-known risk factor for cardiovascular disease. Smoking can cause vascular endothelial dysfunction and consequently trigger haemostatic activation and thrombosis. However, the mechanism of how smoking promotes thrombosis is not fully understood. Thrombosis is associated with the imbalance of the coagulant system due to endothelial dysfunction. As a vital anticoagulation cofactor, thrombomodulin (TM) located on the endothelial cell surface is able to regulate intravascular coagulation by binding to thrombin, and the binding results in thrombosis inhibition. This work focused on the effects of cigarette smoke extract (CSE) on TM-thrombin binding by atomic force microscopy (AFM) based single-molecule force spectroscopy. The results from both in vitro and live-cell experiments indicated that CSE could notably reduce the binding probability of TM and thrombin. This study provided a new approach and new evidence for studying the mechanism of thrombosis triggered by cigarette smoking.展开更多
Functional protein microarray is an important tool for high-throughput and large-scale systems biology studies. Besides the progresses that have been made for protein microarray fabrication, significant advancements h...Functional protein microarray is an important tool for high-throughput and large-scale systems biology studies. Besides the progresses that have been made for protein microarray fabrication, significant advancements have also been achieved for applying protein microarrays on determining a variety of protein biochemical activities. Among these applications, detection of protein binding properties, such as protein-protein interactions （PPIs）, protein-DNA interactions （PDIs）, protein-RNA interactions, and antigen-antibody interactions, are straightforward and have substantial impacts on many research fields. In this review, we will focus on the recent progresses in protein-protein, protein-DNA, protein-RNA, protein-small molecule, protein-lipid, protein-glycan, and antigen-antibody interactions. We will also discuss the challenges and future directions of protein microarray technologies. We strongly believe that protein microarrays will soon become an indispensible tool for both basic research and clinical applications.展开更多
基金The authors thank ProfMary Lou Guerinot （Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire） for providing IRT1 peptide antibody and for the critical reading of the manuscript. We are also grateful to Drs Zhentao Lin and Yongfu Fu （Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing） for providing the BiFC assay system and technical supporting. This work was supported by the National Natural Science Foundation of China （Grant nos, 30530460 and 30521001） and the Ministry of Science and Technology of China （Grant nos, 2005cb20904 and 2006AA 10A 105） and Chinese Academy of Sciences （Grant no. KSCX2-YW-N- 001） as well as by the Harvest Plus-China Program.
文摘Iron is an essential element for plant growth and development. Iron homeostasis in plants is tightly regulated at both transcriptional and posttranscriptional level. Several bHLH transcription factors involved in iron homeostasis have been identified recently. However, their regulatory mechanisms remain unknown. In this work, we demonstrate that the transcription factor FIT interacted with AtbHLH38 and AtbHLH39 and directly conferred the expression regulation of iron uptake genes for iron homeostasis in Arabidopsis. Yeast two-hybrid analysis and transient expression in Arabidopsis protoplasts showed that AtbHLH38 or AtbHLH39 interacted with FIT, a central transcription factor involved in iron homeostasis in Arabidopsis. Expression of FIT/AtbHLH38 or FIT/AtbHLH39 in yeast cells activated GUS expression driven by ferric chelate reductase （FRO2） and ferrous transporter （IRT1） promoters. Overexpression of FITwith either AtbHLH38 or AtbHLH39 in plants converted the expression of the iron uptake genes FRO2 and IRT1 from induced to constitutive. Further analysis revealed that FRO2 and IRT1 were not regulated at the posttranscriptional level in these plants because IRT1 protein accumulation and high ferric chelate reductase activity were detected in the overexpression plants under both iron deficiency and iron sufficiency. The double overexpression plants accumulated more iron in their shoots than wild type or the plants overexpressing either AtbHLH38, AtbHLH39 or FIT. Our data support that ferric-chelate reductase FRO2 and ferrous-transporter IRT1 are the targets of the three transcription factors and the transcription of FRO2 and IRT1 is directly regulated by a complex of FIT/AtbHLH38 or FIT/AtbHLH39.
文摘The eukaryotic transcription factor NF-Y consists of three subunits （A, B, and C）, which are encoded in Ara- bidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, all potential combi- nations of the subunits are possible for the assembly of the heterotrimeric complex. We aimed at assessing the probability of each subunit to participate in the assembly of NF-Y. The evaluation of physical interactions among all members of the NF-Y subunit families indicate a strong requirement for NF-YB/NF-YC heterodimerization before the entire complex can be accomplished. By means of a modified yeast two-hybrid system assembly of all three subunits to a heterotrimeric complex was demonstrated. Using GFP fusion constructs, NF-YA and NF-YC localization in the nucleus was demonstrated, while NF- YB is solely imported into the nucleus as a NF-YC-associated heterodimer NF-YC. This piggyback transport of the two Arabidopsis subunits differs from the import of the NF-Y heterotrimer of heterotrophic organisms. Based on a peptide structure model of the histone-fold-motifs, disulfide bonding among intramolecular conserved cysteine residues of NF-YB, which is responsible for the redox-regulated assembly of NF-YB and NF-YC in human and Aspergillus nidulans, can be excluded for Arabidopsis NF-YB.
基金supported by the National Basic Research Program of China(Grant No.2011CB911001)National Natural Science Foundation of China(Grant Nos.21127901 and 21121063)Chinese Academy of Sciences
文摘Cigarette smoking is a well-known risk factor for cardiovascular disease. Smoking can cause vascular endothelial dysfunction and consequently trigger haemostatic activation and thrombosis. However, the mechanism of how smoking promotes thrombosis is not fully understood. Thrombosis is associated with the imbalance of the coagulant system due to endothelial dysfunction. As a vital anticoagulation cofactor, thrombomodulin (TM) located on the endothelial cell surface is able to regulate intravascular coagulation by binding to thrombin, and the binding results in thrombosis inhibition. This work focused on the effects of cigarette smoke extract (CSE) on TM-thrombin binding by atomic force microscopy (AFM) based single-molecule force spectroscopy. The results from both in vitro and live-cell experiments indicated that CSE could notably reduce the binding probability of TM and thrombin. This study provided a new approach and new evidence for studying the mechanism of thrombosis triggered by cigarette smoking.
文摘Functional protein microarray is an important tool for high-throughput and large-scale systems biology studies. Besides the progresses that have been made for protein microarray fabrication, significant advancements have also been achieved for applying protein microarrays on determining a variety of protein biochemical activities. Among these applications, detection of protein binding properties, such as protein-protein interactions （PPIs）, protein-DNA interactions （PDIs）, protein-RNA interactions, and antigen-antibody interactions, are straightforward and have substantial impacts on many research fields. In this review, we will focus on the recent progresses in protein-protein, protein-DNA, protein-RNA, protein-small molecule, protein-lipid, protein-glycan, and antigen-antibody interactions. We will also discuss the challenges and future directions of protein microarray technologies. We strongly believe that protein microarrays will soon become an indispensible tool for both basic research and clinical applications.
