CURRENT BIOLOGY
·
2024
·
IF 8.1
Xueyi Xue,Lu Wang,Aobo Huang,Zehao Liu,Xiaoyu Guo,Yuying Sang,Jian-Kang Zhu,Huiling Xue,Juan Dong
In Arabidopsis , stomatal development and patterning require tightly regulated cell division and cell-fate differentiation that are controlled by key transcription factors and signaling molecules. To identify new regulators of stomatal development, we assay the transcriptomes of plants bearing enriched stomatal lineage cells that undergo active division. A member of the novel regulators at the plasma membrane (NRPM) family annotated as hydroxyproline-rich glycoproteins was identified to highly express in stomatal lineage cells. Overexpressing each of the four NRPM genes suppressed stomata formation, while the loss-of-function nrpm triple mutants generated severely overproduced stomata and abnormal patterning, mirroring those of the erecta receptor family and MAPKKK yoda null mutants. Manipulation of the subcellular localization of NRPM1 surprisingly revealed its regulatory roles as a peripheral membrane protein instead of a predicted cell wall protein. Further functional characterization suggests that NRPMs function downstream of the EPF1/2 peptide ligands and upstream of the YODA MAPK pathway. Genetic and cell biological analyses reveal that NRPM may promote the localization and function of the ERECTA receptor proteins at the cell surface. Therefore, we identify NRPM as a new class of signaling molecules at the plasma membrane to regulate many aspects of plant growth and development.
研究领域:
植物学
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遗传学
·
分子生物学
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产品关联
DOI
PubMed
高影响因子
PROTEIN EXPRESSION AND PURIFICATION
·
2025
·
IF 1.2
Hao Wang,Xiaohui Zhang,Cunlong Yin,Lu Liu,Liang Chen,Hengfang Tang,Bo Wu,Junfeng Wang
Tumor necrosis factor receptor superfamily member 4 (OX40) is essential for the activation and maintenance of T cell immune function because of its recognition and regulation in signal transduction, therefore understanding the structure and dynamics of the membrane protein OX40 in a near-native membrane environment is critical for elucidating its functions. However, efforts to prepare OX40 in a stable, functional form and reconstitute it into a membrane-mimicking lipid system face persistent challenges, limiting progress in its structural and functional characterization. Here, we developed an efficient method for the expression in E. coli and purification of the N-terminal truncated transmembrane and cytoplasmic domains of OX40 (OX40-TC) using a TrpLE fusion system. High-purity OX40-TC was achieved and successfully reconstituted into membrane scaffold protein (MSP)-nanodiscs for the first time. Characterization through size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy confirmed their stability and homogeneity. This study provides a reliable platform for investigating the structure and function of OX40-TC, advancing our understanding of OX40-mediated signaling and its therapeutic potential.
研究领域:
免疫学
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结构生物学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
ACS Nano
·
2025
·
IF 16
Jinyang Liu,Xuebo Zhang,Bingjie Zhao,Huan Ling,Yanzhong Li,Kuangshi Sun,Song Chen,Yanxin Zhang,Tianli Zhai,Yunxiang Zhang,Fuyou Li,Qian Liu
Single-molecule tracking offers nanometer resolution for studying individual molecule dynamics but is often limited by sparse labeling to avoid signal overlap. We present Red-Light-Activated Single-molecule Tracking (RE-LAST) strategy to address this challenge utilizing a photoactivatable probe, SiR670. SiR670 combines traditional silicon rhodamine with a photocage called SO, quenching fluorescence via photoinduced electron transfer (PET). Red light triggers SiR670 excitation, generating singlet oxygen that oxidizes the SO cage, halting PET and restoring fluorescence. RE-LAST used red light for both activation and imaging, eliminating harmful UV exposure. This method enables high-throughput single-molecule tracking, achieving approximately 9 times more tracks than conventional methods and allowing detailed classification of CD56 membrane protein motion. Furthermore, in situ imaging of single live cells revealed the effects of triplet quencher and oxygen scavenging system (OSS) on membrane protein dynamics. While triplet quenchers like Trolox had minimal impact on protein movement patterns, OSS significantly accelerated protein movement and increased the proportion of mobile proteins. This approach provides a comprehensive method for investigating membrane protein dynamics in living cells, contributing to further developments in cellular and molecular biology.
研究领域:
细胞生物学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF MEMBRANE BIOLOGY
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2016
·Vol. 249
·期 6
·743-756
·
IF 1.991
Yan Qiongxian,Tang Shaoxun,Han Xuefeng,Bamikole Musibau Adungbe,Zhou Chuanshe,Kang Jinhe,Wang Min,Tan Zhiliang
Free fatty acids (FFAs) in plasma are essential substrates for de novo synthesis of milk fat, or directly import into mammary cells. The physico-chemical properties of mammary cells membrane composition affected by FFAs with different chain lengths and saturability are unclear yet. Employing GC, FTIR and fluorescence spectroscopy, the adsorption capacity, phospholipids content, membrane proteins conformation, lipid peroxidation product, and free sulfhydryl of plasma membranes (PMs) interacted with different FFAs were determined. The mammary cells PMs at 38 and 39.5 °C showed different adsorption capacities: acetic acid (Ac) > stearic acid (SA) > β-hydroxybutyric acid (BHBA) > trans 10, cis 12 CLA. In the FTIR spectrum, the major adsorption peaks appeared at 2920 and 2850 cm −1 for phospholipids, and at 1628 and 1560 cm −1 for membrane proteins. The intensities of PMs-FFAs complexes were varied with the FFAs species and their initial concentrations. The β-sheet and turn structures of membrane proteins were transferred into random coil and α-helix after BHBA, SA and trans 10, cis 12 CLA treatments compared with Ac treatment. The quenching effects on the fluorescence of endogenous membrane protein, 1, 8-ANS, NBD-PE, and DHPE entrapped in PMs by LCFA were different from those of short chain FFAs. These results indicate that the adsorption of FFAs could change membrane protein conformation and polarity of head group in phospholipids. This variation of the mammary cells PMs was regulated by carbon chain length and saturability of FFAs.
研究领域:
生物化学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
Bioresources and Bioprocessing
·
2017
·Vol. 4
·期 1
·1-10
·
IF 0
Lyu Fei,Gao Fei,Wei Qianqian,Liu Lin
Background In order to detect the antimicrobial mechanism of combined treatment of cinnamon oil and gamma irradiation (GI), the membrane fatty acids and proteins characteristics of Shewanella putrefaciens ( S. putrefaciens ) treated with cinnamon oil and GI, and the distribution of cinnamon oil in S. putrefaciens were observed in this study. Results The membrane lipid profile of S. putrefaciens was notably damaged by treatments of cinnamon oil and the combination of cinnamon oil and GI, with significantly fatty acids decrease in C14:0, C16:0, C16:1, C17:1, C18:1 ( p < 0.05). The SDS-PAGE result showed that GI did not have obvious effect on membrane proteins (MP), but GI combined with cinnamon oil changed the MP subunits. Cinnamaldehyde, the main component of cinnamon oil, can not transport into S. putrefaciens obviously. It was transformed into cinnamyl alcohol in the nutrient broth with the action of S. putrefaciens . This indicated that the antimicrobial action of cinnamon oil mainly happened on the membrane of S. putrefaciens.
Conclusion Cinnamon oil could act on the membrane of S. putrefaciens with the damage of fatty acids and proteins, and GI would increase the destructive capability of cinnamon oil on the membrane fatty acids and proteins of S. putrefaciens .
研究领域:
微生物学
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食品科学
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化学
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产品关联
DOI
PubMed
ACS Omega
·
2021
·
IF 3.512
Lu Liu,Zhihao Zhu,Fang Zhou,Dongxiang Xue,Tao Hu,Weiling Luo,Yanli Qiu,Dong Wu,Fei Zhao,Zhiping Le,Houchao Tao
Throughout the in vitro studies of membrane proteins (MPs), proper detergents are essential for the preparation of stable aqueous samples. To date, universally applicable detergents have not yet been reported to accommodate the distinct requirements for the highly diversified MPs and at the different stages of MP manipulation. Detergent exchange often has to be performed. We report herein the catalytically cleavable detergents (CatCDs) that can be efficiently removed to facilitate a complete exchange. To this end, functional groups, like propargyl and allyl, are introduced as branched chains or built in the hydrophobic chain close to the hydrophilic head. The representative CatCDs can be used as usual detergents in the extraction and purification of MPs and later be removed upon the addition of catalytic palladium. Mediated by CatCD-1, reconstitution of a transporter protein MsbA into a series of detergents was achieved. The extension of these designs could facilitate the future optimization of other biophysics studies.
研究领域:
生物化学
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分子生物学
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DOI
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2024
·
IF 7.7
Hua Yang,Long Liu,Ling Chen,Chunlin Yang,Qiong Huang,Nanxi Wang,Hongling Hu
Sensory neuron membrane proteins (SNMPs) play critical roles in insect olfactory system. However, functional studies outside of Drosophila remain limited, especially in Coleoptera species. In our previous study, a SNMP1 (CbuqSNMP1b) was identified from Cyrtotrachelus buqueti (Coleoptera: Curculionidae), an insect pest that seriously influence the development of the bamboo industry. Here in a membrane protein yeast two-hybrid system, protein interactions between CbuqSNMP1b as a bait protein and a cDNA library of antenna of male C. buqueti adults as prey protein were assessed. Of 29 proteins identified as putative interactors, the Minus-C odorant-binding protein (CbuqOBP1) was selected for further analysis. The interaction between CbuqSNMP1b and CbuqOBP1 was further confirmed by both the in vivo yeast spotting analysis and the in vitro glutathione-S-transferase pull-down assay. Fluorescence binding assays indicated that the interaction between CbuqSNMP1b and CbuqOBP1 could enhance the binding abilities of CbuqOBP1 to four adult C. buqueti biologically active volatiles. The knockdown of CbuqSNMP1b + CbuqOBP1 expression by RNA interference significantly reduced the behavior responses of male adults to ethyl hexanoate and trans , trans -2,4-Nonadienal. These results increase our understanding of insect SNMP1 and will aid in exploring the underlying mechanisms of CbuqSNMP1b functions in the future.
研究领域:
昆虫学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2024
·
IF 13.3
Jing Huang,Tucan Chen,Yunong Zhao,Dandan Li,Qing Huang,Lina Cao,Jingqiu Chen,Dongjuan Chen,Liang Hu,Huan Liu
Small extracellular vesicles (sEVs), primarily referring to exosomes, have emerged as potent tools for investigating pathological mechanisms and facilitating precise diagnostics owing to their ability to transfer informative molecules between cells and their accessibility from diverse biological fluids. Despite the multitude of biomedical applications, achieving enhanced specificity and sensitivity in detecting trace sEV samples continues to pose a significant challenge. Considering the potential of electrochemical sensing in efficient molecular detection, this study proposes a viable strategy for specifically recognizing sEVs by targeting their membrane proteins. Additionally, it demonstrates sensitive and quantitative detection of sEVs based on the unique performance of PbS colloidal quantum dots (CQDs) nanomaterials. The good immobilization of target proteins on the chemically modified electrode is achieved through the synergistic effect of the charge–discharge mechanism and strong suspension bonds of PbS CQDs. The results obtained from the analysis of differential pulse voltammetry demonstrate rapid and highly sensitive electrochemical responses. The CD63 antibody-modified sensor can achieve highly sensitive detection of sEVs, providing a wide linear detection range (10 2 ∼ 10 8 particles mL −1 ) and a low detection limit (19 particles mL −1 ). The established method is ultimately employed to differentiate the sEVs originating from distinct tumor cells, thereby demonstrating its potential in membrane-biomarker-based diagnostics. Consequently, the presented electrochemical sensor demonstrates remarkable efficiency in quantifying EVs with exceptional specificity and sensitivity, thereby offering extensive prospects for applications in EV analysis and point-of-care tests.
研究领域:
生物医学
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纳米技术
·
诊断学
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产品关联
DOI
高影响因子
Insects
·
2024
·Vol. 15
·期 2
·111
·
IF 2.7
Hua Yang,Long Liu,Fan Wang,Wei Yang,Qiong Huang,Nanxi Wang,Hongling Hu
Simple SummaryThe bamboo weevil beetle,Cyrtotrachelus buqueti(Coleoptera: Curculionidae) is the primary bamboo pest and seriously influences the development of the bamboo industry. Sensory neuron membrane proteins (SNMPs) play important roles in insect pheromone communication. However, SNMPs ofC. buquetiare still uncharacterized. With the aim of developing control techniques to control this pest, one novelSNMPfromC. buqueti,CbuqSNMP1b, was functionally characterized in this study. The results indicated thatCbuqSNMP1bwas predominantly expressed in the antennae of both sexes and showed significantly higher transcription levels in the adult stage, suggesting thatCbuqSNMP1bis involved in the process of olfaction. Fluorescence binding assays revealed that CbuqSNMP1b could bind to three out of fourteen volatile compounds emitted fromC. buquetiand showed the strongest binding affinity to dibutyl phthalate, demonstrating the role of SNMP1 in detecting odorants. Molecular docking was performed to further understand the binding mode between CbuqSNMP1b and these three ligands, and the results showed that hydrophobic interactions were the prevailing forces within the binding cavities of CbuqSNMP1b. The results of this study will be helpful to understand the function of SNMP1 inC. buquetiand lay a foundation for developing new methods to control this pest.AbstractSensory neuron membrane proteins (SNMPs) play important roles in insect chemoreception and SNMP1s have been reported to be essential in detecting sex pheromones inDrosophilaand some lepidopteran species. However, SNMPs forCyrtotrachelus buqueti(Coleoptera: Curculionidae), a major insect pest of bamboo plantations, remain uncharacterized. In this study, a novelSNMPgene,CbuqSNMP1b, fromC. buquetiwas functionally characterized. The expression ofCbuqSNMP1bwas significantly higher in antennae than in other tissues of both sexes and the expression level was significantly male-biased. Additionally,CbuqSNMP1bshowed significantly higher transcription levels in the adult stage and very low transcription levels in other stages, suggesting thatCbuqSNMP1bis involved in the process of olfaction. Fluorescence binding assays indicated that CbuqSNMP1b displayed the strongest binding affinity to dibutyl phthalate (Ki= 9.03 μM) followed by benzothiazole (Ki= 11.59 μM) and phenol (Ki= 20.95 μM) among fourteenC. buquetivolatiles. Furthermore, molecular docking revealed key residues in CbuqSNMP1b that interact with dibutyl phthalate, benzothiazole, and phenol. In conclusion, these findings will lay a foundation to further understand the olfactory mechanisms ofC. buquetiand promote the development of novel methods for controlling this pest.Keywords:Cyrtotrachelus buqueti;sensory neuron membrane protein;expression pattern;binding assay;molecular docking
研究领域:
昆虫学
·
分子生物学
·
害虫管理
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产品关联
DOI
PubMed
高影响因子
STAR Protocols
·
2025
·
IF 1.3
Peng Xu,Jianbo Yue
Summary Here, we present a protocol to study epidermal growth factor (EGF) receptor (EGFR) or transferrin trafficking in mammalian cells. We describe steps for using fluorescent ligands or antibodies, confocal imaging, and quantitative analysis to track their movement. We detail procedures for cell culture preparation, labeling membrane proteins, optimizing imaging, and isolating cell lysates for the biochemical analysis of EGFR degradation after EGF treatment. This protocol is adaptable to various cell types and for assessing genetic or pharmacological impacts on endosomal trafficking. For complete details on the use and execution of this protocol, please refer to Ye et al., 1 Ye et al., 2 and Wang et al. 3
研究领域:
细胞生物学
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分子生物学
·
生物化学
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产品关联
DOI
PubMed
高影响因子
Nature Protocols
·
2017
·Vol. 12
·期 12
·2554-2569
·
IF 10.032
Tang Shan,Zuo Chao,Huang Dong-Liang,Cai Xiao-Ying,Zhang Long-Hua,Tian Chang-Lin,Zheng Ji-Shen,Liu Lei
Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.
研究领域:
生物化学
·
结构生物学
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DOI
PubMed
高影响因子
RSC Advances
·
2016
·Vol. 6
·期 10
·7955-7960
·
IF 3.289
Weiwei He,Jiaqi Ma,Xiaowen Shu,Jun Qian,Daoben Hua
Membrane proteins (MPs) play essential roles in determining the interactions of biological systems with their surroundings, and their reconstitution determines the application in a variety of areas. Artificial polymers are pursued in MP reconstitution for fabricating bio-hybrid materials due to their adjustable properties. In this work, a cationic amphiphilic copolymer was synthesized by RAFT polymerization of dimethylaminoethyl acrylate (DMAEA) and methylacrylate, followed by quaternization of the PDMAEA segments. The copolymer could self-assemble in water displaying a sphere morphology. As an example, MP bacteriorhodopsin could insert in the assembled micelles of the copolymer spontaneously when incubated together, which showed an affinity towards the surface of poly(ethylene terephthalate) film. This work may provide a facile strategy to prepare bio-hybrid materials for promising applications of membrane proteins.
研究领域:
生物化学
·
材料科学
·
高分子化学
✓ 已验证
DOI
高影响因子
危险品化学品经营许可证（带存储）