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A practical guide for fast implementation of SNARE-mediated liposome fusion

biophysics Reports 2024年第1期10卷 31-40页

作者： Shen Wang Cong Ma Key laboratory of Molecular biophysics of the Ministry of Education College of Life Science and TechnologyHuazhong University of Science and Technology

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor(SNAER) family proteins are the engines of most intra-cellular and exocytotic membrane fusion pathways(Jahn and Scheller 2006).Over the past two decades, in-vitro liposome fusion has been proven to be a powerful tool to reconstruct physiological SNARE-mediated membrane fusion processes(Liu et al. 2017). The reconstitution of the membrane fusion process not only provides direct evidence of the capability of the cognate SNARE complex in driving membrane fusion but also allows researchers to study the functional mechanisms of regulatory proteins in related pathways(Wickner and Rizo 2017). Heretofore, a variety of delicate methods for in-vitro SNARE-mediated liposome fusion have been established(Bao et al. 2018;Diao et al. 2012; Duzgunes 2003; Gong et al. 2015; Heo et al. 2021; Kiessling et al. 2015; Kreye et al.2008; Kyoung et al. 2013; Liu et al. 2017; Scott et al. 2003). Although technological advances have made reconstitution more physiologically relevant, increasingly elaborate experimental procedures, instruments, and data processing algorithms nevertheless hinder the non-experts from setting up basic SNARE-mediated liposome fusion assays. Here, we describe a low-cost, timesaving, and easy-to-handle protocol to set up a foundational in-vitro SNARE-mediated liposome fusion assay based on our previous publications(Liu et al. 2023; Wang and Ma 2022). The protocol can be readily adapted to assess various types of SNARE-mediated membrane fusion and the actions of fusion regulators by using appropriate alternative additives(e.g., proteins, macromolecules, chemicals, etc.). The total time required for one round of the assay is typically two days and could be extremely compressed into one day.

关键词： Liposome fusion SNARE In-vitro reconstitution Protocol

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Silence of Synaptotagmin VII inhibits release of dense core vesicles in PC12 cells

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Science China(Life Sciences) 2009年第12期52卷 1156-1163页

作者： LI JiangLi1, XIAO Yang1, ZHOU Wei1, WU ZhengXing1, ZHANG RongYing1· & XU Tao1,2· 1 Key laboratory of Molecular biophysics of the Ministry of Education, Institute of biophysics and Biochemistry, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China 2 National Key laboratory of Biomacromolecules, Institute of biophysics, Chinese Academy of Sciences, Beijing 100101, China. Key laboratory of Molecular biophysics of the Ministry of Education Institute of Biophysics and Biochemistry College of Life Science and Technology Huazhong University of Science and Technology Wuhan China National Key laboratory of Biomacromolecules Institute of Biophysics Chinese Academy of Sciences Beijing China

Synaptotagmin VII (Syt VII), which has a higher Ca2+ affinity and slower disassembly kinetics with lipid than Syt I and Syt IX, was regarded as being uninvolved in synaptic vesicle (SV) exocytosis but instead possibly as a calcium sensor for the slower kinetic phase of dense core vesicles (DCVs) release. By using high temporal resolution capacitance and amperometry measurements, it was demonstrated that the knockdown of endogenous Syt VII attenuated the fusion of DCV with the plasma membrane, reduced the amplitude of the exocytotic burst of the Ca2+-triggered DCV release without affecting the slope of the sustained component, and blocked the fusion pore expansion. This suggests that Syt VII is the Ca2+ sensor of DCV fusion machinery and is an essential factor for the establishment and maintenance of the pool size of releasable DCVs in PC12 cells.

关键词： Synaptotagmin VII dense core vesicles exocytosis RNA-mediated interference (RNAi)

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Targeting calcium signaling in Alzheimer’s disease: challenges and promising therapeutic avenues

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Neural Regeneration Research 2024年第3期19卷 501-502页

作者： LinLin Song YongPei Tang Betty Yuen Kwan Law Neher’s biophysics laboratory for Innovative Drug Discovery State Key Laboratory of Quality Research in Chinese MedicineMacao University of Science and TechnologyMacao Special Administration RegionChina

The critical role of calcium dyshomeostasis in the pathogenesis of Alzheimer’s disease(AD):AD is a progressive neurodegenerative disease characterized by cognitive decline,memory impairment,and behavioral changes.With an estimated 50 million people being affected worldwide,the incidence of AD is constantly increasing globally.The hallmark of AD is the accumulation of amyloid-beta protein(Aβ)in the form of amyloid plaques and hyperphosphorylated tau protein in the form of neurofibrillary tangles.However,increasing evidence suggests that calcium ion(Ca2+)dysregulation also plays a crucial role in the pathogenesis of AD(Calvo-Rodriguez and Bacskai,2021).As a key second messenger,Ca2+regulates a wide range of cellular processes,including the release of neurotransmitters,gene expression,and cell death.Ca^(2+)also regulates the activity of Calcium/calmodulin-dependent protein kinase II,which is critical for synaptic plasticity,learning,and memory(Kaushik et al.,2022).Alternation in the Ca^(2+)signal is an early event in the pathogenesis of AD,which can lead to synaptic dysfunction,neuronal loss,and cognitive impairment.

关键词： Alzheimer homeostasis globally

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Dynamic tracking and mobility analysis of single GLUT4 storage vesicle in live 3T3-L1 cells

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Cell Research 2004年第6期14卷 480-486页

作者： Chen Hong LI, Li BAI, Dong Dong LI, Sheng XIA, Tao XU Institute of biophysics and Biochemistry, Huazhong University of Science and Technology, Wuhan 430074, China National Key laboratory of Biomacromoleculars, Institute of biophysics, Chinese Academy of Sciences, Beijing 100101, China Institute of biophysics and Biochemistry Huazhong University of Science and Technology Wuhan China National Key laboratory of Biomacromoleculars Institute of Biophysics Chinese Academy of Sciences Beijing China

Glucose transporter 4 (GLUT4) is responsible for insulin-stimulated glucose transporting into the insulin-sensitive fat and muscle cells. The dynamics of GLUT4 storage vesicles (GSVs) remains to be explored and it is unclear how GSVs are arranged based on their mobility. We examined this issue in 3T3-L1 cells via investigating the three-dimensional mobility of single GSV labeled with EGFP-fused GLUT4. A thin layer of cytosol right adjacent to the plasma membrane was illuminated and successively imaged at 5 Hz under a total internal reflection fluorescence microscope with a penetration depth of 136 nm. Employing single particle tracking, the three-dimensional subpixel displacement of single GSV was tracked at a spatial precision of 22 nm. Both the mean square displacement and the diffusion coefficient were calculated for each vesicle. Tracking results revealed that vesicles moved as if restricted within a cage that has a mean radius of 160 nm, suggesting the presence of some intracellular tethering matrix

关键词： Insulin GLUT4 GLUT4 storage vesicle (GSV) 3T3-L1 total internal reflection fluorescence microscopy single particle tracking.

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Efficient adsorption separation of methane from C_(2)-C_(3) hydrocarbons in a Co(Ⅱ)-nodes metal-organic framework

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Chinese Journal of Chemical Engineering 2024年第5期69卷 192-198页

作者： Jie Zhang Xingzhe Guo Bing Lin Guangzu Xiong Hanshuang Wang Min Zhang Liwen Fan Bingwen Li Shuisheng Chen Department of Chemistry and Chemical Engineering Taiyuan Institute of TechnologyTaiyuan 030008China Engineering Research Center of Oligosaccharides Fuyang Normal UniversityFuyang 236041China Shandong Key laboratory of biophysics Institute of BiophysicsDezhou UniversityDezhou 253023China

Methane(CH_(4))as a substitute for other mineral fuels plays a crucial role in reducing energy consumption and preventing environmental pollution.The present study employs a solvothermal method to fabricate a porous framework Co-metal-organic framework(Co-MOF)containing two distinct secondary building units(SBUs):an anionic[Co_(2)(μ_(2)-OH)(COO)_(4)(H_(2)O)]and a neutral[CoN_(2)(COO)_(2)].Notably,within the anionic SBUs,the coordinated water molecules induce the generation of divergent unsaturated Co(Ⅱ)centers in the unidirectional porous channels,thereby creating open metal sites.The adsorption performance of Co-MOF towards pure component gases was systematically investigated.The results demonstrated that Co-MOF exhibits superior adsorption capacity for C_(2)-C_(3) hydrocarbons compared to CH_(4),which offers the potential for efficient adsorption and separation of CH_(4) from C_(2)-C_(3) hydrocarbons.The gas selectivity separation ratios of Co-MOF for C_(2)H_(6)/CH_(4) and C_(3)H_8/CH_(4) were calculated using the ideal adsorbed solution theory method at 273/298 K and 0.1 MPa.The results revealed that Co-MOF achieved remarkable equilibrium separation selectivity for CH_(4) and C_(2)-C_(3) hydrocarbon gases among non-modified MOFs,signifying the potential of the synthesized Co-MOF for efficient recovery and purification of CH_(4) from C_(2)-C_(3) hydrocarbons.Breakthrough experiments further demonstrate the ability of Co-MOF to purify methane from C_(2)-C_(3) hydrocarbons in practical gas separation scenarios.Additionally,molecular simulation calculations further substantiate the propensity of anionic SBUs to interact with C_(2)-C_(3) hydrocarbon compounds.This study provides a novel paradigm for the development of porous MOF materials in the application of gas mixture separation.

关键词： Co-MOF Separation hydrocarbons Dynamic breakthrough curves Molecular simulations adsorbent Monte Carlo simulation

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Tripartite motif-containing protein 11(TRIM11):a novel weapon against Alzheimer’s disease

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Signal Transduction and Targeted Therapy 2024年第2期9卷 339-340页

作者： Yichu Fu Bi Zhang Jianfeng Liu College of Life Science and Technology Key Laboratory of Molecular Biophysics of MOEHuazhong University of Science and TechnologyWuhanHubei 430074China

In a recent study published in Science,Zhang et al.discovered that tripartite motif-containing(TRIM)protein 11(TRIM11)exhibited strong inhibition on tau aggregation,which is a pathological characteristic of tauopathies including Alzheimer’s disease(AD).1 This makes TRIM11 a promising novel target for the treatment of AD and other tauopathies.

关键词： Alzheimer tau TRIM

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Enrichment of ER tubule-derived microsomes by differential centrifugation and immunoprecipitation

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biophysics Reports 2024年第2期10卷 61-66页

作者： Yiduo Liu Junjie Hu Bing Yan National laboratory of Biomacromolecules CAS Center for Excellence in Biomacromolecules Institute of Biophysics Chinese Academy of Sciences University of Chinese Academy of Sciences

The endoplasmic reticulum(ER) is an essential component of the endomembrane system in eukaryotes and plays a crucial role in protein and lipid synthesis, as well as the maintenance of calcium homeostasis. Morphologically, the ER is composed primarily of sheets and tubules. The tubular ER is composed of a network of tubular membrane structures, each with diameters ranging from 30 to 50nanometers. In recent years, there has been in-depth research on the molecular mechanisms of membrane shaping and membrane fusion of the tubular ER. However, there is still limited understanding of the specific physiological functions of the tubular ER. Here, we report a protocol that combines differential centrifugation and immunoprecipitation to specifically enrich microsomes originating from the tubular ER in yeast. The ER tubule-derived microsomes can be further used for proteomic and lipidomic studies or other biochemical analyses.

关键词： Endoplasmic reticulum Microsome Differential centrifugation Immunoprecipitation

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Generation and characterization of nanobodies targeting GPCR

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biophysics Reports 2024年第1期10卷 22-30页

作者： Shenglan Zhang Zhiran Fan Jianfeng Liu Bioland laboratory (Guangzhou Regenerative Medicine and Health Guangdong laboratory) Cellular Signaling laboratory International Research Center for Sensory Biology and Technology of MOST Key Laboratory of Molecular Biophysics of MOE College of Life Science and Technology Huazhong University of Science and Technology

G protein-coupled receptors(GPCRs) are a large family of cell membrane proteins that are important targets for drug discovery. Nanobodies, also known as VHH(variable domains of heavy chain-only antibodies, HcAbs) antibodies, are small antibody fragments derived from camelids that have gained significant attention as potential therapeutics for targeting GPCRs due to their advantages over conventional antibodies. However, there are challenges in developing nanobodies targeting GPCRs, among which epitope accessibility is the most significant because the cell membrane partially shields the GPCR surface. We developed a universal protocol for making nanobodies targeting GPCRs using the cell membrane extract of GPCR-overexpressing HEK293 cells as the llama/alpaca immunization antigen. We constructed an immune VHH library and identified nanobodies by phage display bio-panning. The monoclonal nanobodies were recombinantly expressed in Escherichia coli(E. coli) and purified to characterize their binding potency.

关键词： Nanobody Single domain antibody Heavy chain-only antibody GPCR G protein-coupled receptor

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Complicated target recognition by archaeal box C/D guide RNAs

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Science China(Life Sciences) 2024年第4期67卷 631-644页

作者： Jiayin Wang Songlin Wu Keqiong Ye Key laboratory of RNA Science and Engineering CAS Center for Excellence in BiomacromoleculesInstitute of BiophysicsChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China

Box C/D RNAs guide the site-specific formation of 2′-O-methylated nucleotides(Nm)of RNAs in eukaryotes and archaea.Although C/D RNAs have been profiled in several archaea,their targets have not been experimentally determined.Here,we mapped Nm in r RNAs,t RNAs,and abundant small RNAs(s RNAs)and profiled C/D RNAs in the crenarchaeon Sulfolobus islandicus.The targets of C/D RNAs were assigned by analysis of base-pairing interactions,in vitro modification assays,and gene deletion experiments,revealing a complicated landscape of C/D RNA-target interactions.C/D RNAs widely use dual antisense elements to target adjacent sites in r RNAs,enhancing modification at weakly bound sites.Two consecutive sites can be guided with the same antisense element upstream of box D or D′,a phenomenon known as doublespecificity that is exclusive to internal box D′in eukaryotic C/D RNAs.Several C/D RNAs guide modification at a single non-canonical site.This study reveals the global landscape of RNA-guided 2′-O-methylation in an archaeon and unexpected targeting rules employed by C/D RNA.

关键词： C/D RNA 2′-O-methylation archaea guide RNA RNA modification

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FLEXIBILITY OF ENZYME ACTIVE SITES

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Chinese Science Bulletin 1989年第10期34卷 793-799页

作者：邹承鲁 laboratory of Biomacromolecules Institute of Biophysics Academia Sinica Beijing

The effects of structural changes on the activity of enzymes have been extensively studied. However, previous authors concentrated largely on the modification of the primary structures of the enzymes. On the other han... 详细信息

关键词： conformational enzymes concentrated reversible residues folding integrity largely monomer buried

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