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1.
Military Medical Sciences ; (12): 81-87, 2024.
Article in Chinese | WPRIM | ID: wpr-1018879

ABSTRACT

Objective To explore the characteristics and mechanism of phase separation between TAR DNA binding protein-43(TDP-43)and ubiquitin.Methods The TARDBP gene and its truncated genes were inserted into vectors to construct recombinant plasmids for expression and protein purification.The phase separation system of ubiquitin and TDP-43 was constructed in vitro.The characteristics of the droplets formed via liquid-liquid phase separation were observed by fluorescence microscopy.The plasmids of ubiquitin and TDP-43 were co-transfected into HEK293T cells to observe aggregates containing TDP-43 and ubiquitin and find out whether TDP-43 could be ubiquitinated.Results The GFP-8Ub,TDP-43 full-length(FL)and truncated proteins were purified.TDP-43 FL and C-terminal domain(CTD)proteins were able to form droplets via phase separation with ubiquitin.The droplets changed into solid-like aggregates after prolonged incubation.Insolvable aggregates containing TDP-43 and ubiquitin were formed.TDP-43 was ubiquitinated under stress conditions in HEK293T cells after being co-transfected with ubiquitin and TDP-43 recombinant plasmids.Conclusion TDP-43 undergoes co-phase separation with ubiquitin,mainly driven by the multivalent interaction between TDP-43′s CTD structural domain and ubiquitin.The droplets finally form aggregates with solid-like properties.Under stress conditions,especially when the protein homeostasis is disrupted,TDP-43 and ubiquitin form aggregates while TDP-43 is ubiquitinated.This study reveals the basic mechanism of TDP-43 co-phase separation with ubiquitin and liquid-solid transformation.

2.
Military Medical Sciences ; (12): 148-153, 2024.
Article in Chinese | WPRIM | ID: wpr-1018889

ABSTRACT

The important role of liquid-liquid phase separation in a series of biological processes,including regulation of gene transcription and translation,stress response,autophagy and the establishment of synaptic structure,has been widely accepted.Abnormal phase separation is associated with many human diseases,including neurodevelopmental disorders and neurodegenerative diseases.Studies have shown that some proteins associated with epigenetic modifications are also subject to liquid-liquid phase separation,suggesting that epigenetic modifications regulate the development and disease of the nervous system by regulating phase separation.This review summarized the important roles of epigenetic modification and phase separation in neurodevelopment and neurodiseases,and focused on the important roles of proteins related to epigenetic modification with phase separation characteristics.Understanding the correlation between epigenetic modification and phase separation will help fully understand the underlying mechanisms of neurodevelopment and neurodiseases,and will further provide new targets and strategies for the treatment of related diseases.

3.
Article in Chinese | WPRIM | ID: wpr-1039065

ABSTRACT

Cells not only contain membrane-bound organelles (MBOs), but also membraneless organelles (MLOs) formed by condensation of many biomacromolecules. Examples include RNA-protein granules such as nucleoli and PML nuclear bodies (PML-NBs) in the nucleus, as well as stress granules and P-bodies in the cytoplasm. Phase separation is the basic organizing principle of the form of the condensates or membraneless organelles (MLOs) of biomacromolecules including proteins and nucleic acids. In particular, liquid-liquid phase separation (LLPS) compartmentalises and concentrates biological macromolecules into liquid condensates. It has been found that phase separation of biomacromolecules requires some typical intrinsic characteristics, such as intrinsically disordered regions, modular domains and multivalent interactions. The phase separation of biomacromolecules plays a key role in many important cell activities. In recent years, the phase separation of biomacromolecules phase has become a focus of research in gene transcriptional regulation. Transcriptional regulatory elements such as RNA polymerases, transcription factors (TFs), and super enhancers (SEs) all play important roles through phase separation. Our group has previously reported for the first time that long-term inactivation or absence of assembly factors leads to the formation of condensates of RNA polymerase II (RNAPII) subunits in the cytoplasm, and this process is reversible, suggesting a novel regulatory model of eukaryotic transcription machinery. The phase separation of biomacromolecules provides a biophysical understanding for the rapid transmission of transcriptional signals by a large number of TFs. Moreover, phase separation during transcriptional regulation is closely related to the occurrence of cancer. For example, the activation of oncogenes is usually associated with the formation of phase separation condensates at the SEs. In this review, the intrinsic characteristics of the formation of biomacromolecules phase separation and the important role of phase separation in transcriptional regulation are reviewed, which will provide reference for understanding basic cell activities and gene regulation in cancer.

4.
Article in Chinese | WPRIM | ID: wpr-988189

ABSTRACT

ObjectiveTo investigate the effect of microemulsion on the distribution of index components in different phases of Zexietang extract based on high performance liquid chromatography(HPLC) and phase separation process. MethodParticle size meter and transmission electron microscope were used to characterize the colloidal particles in blank microemulsion, aqueous extract of Zexietang and microemulsion extract of Zexietang. The phase separation process was established by high-speed centrifugation and dialysis, and based on this process, the aqueous extract and microemulsion extract of Zexietang were separated into the true solution phase, the colloidal phase and the precipitation phase, respectively. The contents of six components, including atractylenolide Ⅲ, atractylenolide Ⅱ, 23-acetyl alisol C, alisol A, alisol B and alisol B 23-acetate, were determined by HPLC with the mobile phase of water(A)-acetonitrile(B) for gradient elution(0-5 min, 40%-43%B; 5-20 min, 43%-45%B; 20-45 min. 45%-60%B; 45-75 min, 60%-80%B). The solubility of the index components in water and microemulsion was determined by saturation solubility method. ResultThe colloidal particles in the aqueous extract, microemulsion extract and blank microemulsion were all spherical, and the particle size, polydispersity index(PDI) and Zeta potential of the colloidal particles were in the order of aqueous extract >microemulsion extract >blank microemulsion. The results of phase separation showed that the colloidal phase and the true solution phase could be completely separated by dialysis for 2.5 h, and the phase separation process was tested to be stable and feasible. Compared with the aqueous extract of Zexietang, the use of microemulsion as an extraction solvent could increase the contents of atractylenolide Ⅲ, 23-acetyl alisol C, atractylenolide Ⅱ , alisol A, alisol B and alisol B 23-acetate by 3.75, 6.82, 35.47, 10.66, 35.41, 27.75-fold, and could increase the extraction efficiencies of the latter five constituents by 2.03, 1.15, 1.70, 6.43, 5.53 times. The solubility test showed that the microemulsion could significantly improve the solubility of atractylenolide Ⅱ, alisol A, alisol B and alisol B 23-acetate, but it had less effect on the solubility of atractylenolide Ⅲ and 23-acetyl alisol C. ConclusionMicroemulsion can improve the extraction efficiency and increase the distribution of the index components in the colloidal phase state of Zexietang to different degrees, providing a reference for the feasibility of microemulsion as an extraction solvent for traditional Chinese medicine.

5.
Protein & Cell ; (12): 874-887, 2023.
Article in English | WPRIM | ID: wpr-1010762

ABSTRACT

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms. Current CRISPR-activation (CRISPRa) platforms often require multiple components because of inefficient transcriptional activation. Here, we fused different phase-separation proteins to dCas9-VPR (dCas9-VP64-P65-RTA) and observed robust increases in transcriptional activation efficiency. Notably, human NUP98 (nucleoporin 98) and FUS (fused in sarcoma) IDR domains were best at enhancing dCas9-VPR activity, with dCas9-VPR-FUS IDR (VPRF) outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity. dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR. These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.


Subject(s)
Humans , Transcriptional Activation , RNA, Guide, CRISPR-Cas Systems , Gene Expression Regulation , CRISPR-Cas Systems/genetics
6.
Frontiers of Medicine ; (4): 855-866, 2023.
Article in English | WPRIM | ID: wpr-1010821

ABSTRACT

Biomolecular condensates formed by phase separation are widespread and play critical roles in many physiological and pathological processes. cGAS-STING signaling functions to detect aberrant DNA signals to initiate anti-infection defense and antitumor immunity. At the same time, cGAS-STING signaling must be carefully regulated to maintain immune homeostasis. Interestingly, exciting recent studies have reported that biomolecular phase separation exists and plays important roles in different steps of cGAS-STING signaling, including cGAS condensates, STING condensates, and IRF3 condensates. In addition, several intracellular and extracellular factors have been proposed to modulate the condensates in cGAS-STING signaling. These studies reveal novel activation and regulation mechanisms of cGAS-STING signaling and provide new opportunities for drug discovery. Here, we summarize recent advances in the phase separation of cGAS-STING signaling and the development of potential drugs targeting these innate immune condensates.


Subject(s)
Humans , Nucleotidyltransferases/chemistry , Signal Transduction/physiology , Membrane Proteins/chemistry , Phase Separation
7.
Article in Chinese | WPRIM | ID: wpr-1038325

ABSTRACT

Objective @# To investigate the effect of SHP2 protein phase separation induced by activation mutation on cell proliferation and its mechanism through construct a mouse model of SHP2E76K mutation . @*Methods @#Hybrid PTPN11 E76K⁃NEO/ + C57BL/6J mouse were hybridizedwith Mx1 ⁃cre tool mice to obtain the required Mx1 ⁃cre;Ptpn11 + / + and Mx1 ⁃cre;Ptpn11 E76K/ + . The later genotype mice were injected with pI⁃pC to induce the expression of Cre enzyme and mutate Ptpn11E76K in bone marrow mesenchymal stem cells(MSC) . The Mx1 ⁃cre ; Ptpn11 + / + and Mx1 ⁃cre;Ptpn11 E76K/ + genotype mice ′s cells were isolated and cultured in vitro and identified as MSCs by immunofluorescence staining . With Ptpn11 + / + MSC as the control group and Ptpn11 E76K/ + MSC as the experimental group , the two kinds of cells were divided into 6 groups by adding drugs : Ptpn11 + / + group ; Ptpn11 + / + + SHP099 group ; Ptpn11 + / + + ET070 group ; Ptpn11 E76K/ + group ; Ptpn11 E76K/ + + SHP099 group ; Ptpn11 E76K/ + + ET070 group . The differences of SHP2 protein phase separation in the six groups were observed by immunofluorescencestaining, and the differences of SHP2 protein expression were detectedby Western blot . CCK⁃8 was used to observe the changes of cell proliferation after phase separation was affected . Western blot was used to detect the expression levels of ERK/ cell proliferation after phase separation was affected . Western blot was used to detect the expression levels of ERK/ p ⁃ERK , AMPK/p⁃AMPK , mTOR/p⁃mTOR and other molecules between the six groups . @*Results @# Genotypes Mx1 ⁃cre ; Ptpn11 E76K/ + and Mx1 ⁃cre ; Ptpn11 + / + mice were obtained by genotyping , and the primary MSCs were isolated . Compared with Ptpn11 + / + group , SHP2 proteins in the Ptpn11 E76K/ + group produced more phase separation condensates , and compared with Ptpn11 E76K/ + group , the SHP2 proteins in the Ptpn11 E76K/ + + SHP099 and Ptpn11 E76K/ ++ ET070 groups produced less phase separation condensates . No difference in SHP2 protein expression levels between groups was detected by Western blot . Compared with Ptpn11 + / + group , the proliferation ability of MSC in Ptpn11 + / + + SHP099 and Ptpn11 + / + + ET070 groups decreased , the expression of p ⁃ERK and p ⁃mTOR decreased, and the expression of p ⁃AMPK protein increased . Compared with Ptpn11 E76K/ + group , the proliferation ability of MSC in Ptpn11 E76K/ + + SHP099 and Ptpn11 E76K/ + + ET070 groups decreased , the expression of p ⁃ERK and p ⁃ mTOR decreased , and the expression of p⁃AMPK protein increased . @*Conclusion @# SHP2 phase isolation is involved in the alteration of proliferative capacity of SHP2E76K ⁃activated MSCs by stimulating the expression of AMPK⁃mTOR signaling pathway .

8.
Chinese Journal of Neuromedicine ; (12): 1281-1285, 2022.
Article in Chinese | WPRIM | ID: wpr-1035772

ABSTRACT

Numerous studies have suggested that liquid-liquid phase separation (LLPS) may be involved in occurrence and progression of neurodegenerative diseases through mediating immune inflammation, transcriptional regulation, protein homeostasis, genomic stability, and oxidative stress, and regulation of LLPS-mediated protein homeostasis has attracted particular attention. Therefore, this paper reviews the research progress of mechanism of protein homeostasis regulation in neurodegenerative diseases in recent years, and discusses the prospect of LLPS related research.

9.
Protein & Cell ; (12): 680-694, 2021.
Article in English | WPRIM | ID: wpr-888723

ABSTRACT

Signaling pathways in innate and adaptive immunity play vital roles in pathogen recognition and the functions of immune cells. Higher-order assemblies have recently emerged as a central principle that governs immune signaling and, by extension, cellular communication in general. There are mainly two types of higher-order assemblies: 1) ordered, solid-like large supramolecular complexes formed by stable and rigid protein-protein interactions, and 2) liquid-like phase-separated condensates formed by weaker and more dynamic intermolecular interactions. This review covers key examples of both types of higher-order assemblies in major immune pathways. By placing emphasis on the molecular structures of the examples provided, we discuss how their structural organization enables elegant mechanisms of signaling regulation.

10.
Yao Xue Xue Bao ; (12): 585-592, 2021.
Article in Chinese | WPRIM | ID: wpr-873768

ABSTRACT

Puerarin (PUE), as an isoflavone component, has a wide range of pharmacological activities, while its poorly aqueous solubility limits the development of solid oral dosage forms. In this study, PUE along with nicotinamide (NIC) were prepared into the coamorphous system by solvent-evaporation method and characterized by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR). In addition, its dissolution behavior and solubilization mechanism were also investigated. PUE-NIC coamorphous was a single homogeneous binary system, with a single glass transition temperature at 35.1 ℃. In comparison to crystalline PUE, during the dissolution process, coamorphous PUE-NIC not only exhibited the "liquid-liquid phase separation" (LLPS) phenomenon, but the formation of Ap type complexation (1∶1 and 1∶2) between PUE and NIC molecules was also verified, which significantly improved the solubility of PUE and prolonged the supersaturation time, and would benefit its absorption.

11.
Article in Chinese | WPRIM | ID: wpr-1015839

ABSTRACT

Aberrant expression or mutation of many genes that are essential for embryonic development, are closely associated with human diseases, one of which is SPOP (speckle type BTB/POZ protein). SPOP is an E3 ubiquitin ligase adaptor protein and mainly composed of MATH, BTB and BACK domains, which plays distinct roles to fulfill the proper function of SPOP. SPOP usually targets its substrates for degradation via the ubiquitin-proteasome pathway. More than thirty substrates of SPOP have been identified by far, most of which are associated with tumorigenesis of prostate, endometrial and kidney cancers. SPOP also plays an important role during development. Genomic loss or mutation of SPOP locus leads to postnatal lethality in mice, while de novo variants in SPOP cause neurodevelopmental disorders in children. Similarly, SPOP regulates a variety of developmental processes via targeting its substrates for degradation, including Gli2/3, PDX1, NANOG and SENP7 which are involved in neural, skeletal and pancreatic development as well as senescence. In addition, recent studies have revealed that SPOP co-localizes with its substrates into membraneless organelles such as nuclear speckles, and promotes ubiquitination and degradation of its substrates. Oligomerization of SPOP and liquid-liquid phase separation (LLPS) triggered by multivalent interactions between SPOP and substrates play a pivotal role in this process. BTB or BACK mutants, which are defective in SPOP oligomerization, are also defective in driving LLPS of SPOP and recruiting SPOP into membraneless organelles. In this review, we summarized and discussed the recent progress on the essential role of SPOP during development.

12.
J Biosci ; 2020 Jan; : 1-5
Article | IMSEAR | ID: sea-214363

ABSTRACT

The organization of chromatin into different types of compact versus open states provides a means to fine tune generegulation. Recent studies have suggested a role for phase-separation in chromatin compaction, raising new possibilities for regulating chromatin compartments. This perspective discusses some specific molecular mechanismsthat could leverage such phase-separation processes to control the functions and organization of chromatin

13.
J Biosci ; 2020 Jan; : 1-8
Article | IMSEAR | ID: sea-214351

ABSTRACT

For mammals to develop properly, master regulatory genes must be repressed appropriately in a heritable manner. This review concerns the Polycomb Repressive Complex 1 (PRC1) family and the relationshipbetween the establishment of repression and memory of the repressed state. The primary focus is on the CBXfamily of proteins in PRC1 complexes and their role in both chromatin compaction and phase separation.These two activities are linked and might contribute to both repression and memory.

14.
J Biosci ; 2020 Jan; : 1-17
Article | IMSEAR | ID: sea-214336

ABSTRACT

In order to improve crop plants in terms of their yield, drought resistance, pest resistance, nutritional value, etc.,modern agriculture has relied upon plant genetic engineering. Since the advent of recombinant DNA technology, several tools have been used for genetic transformations in plants such as Agrobacterium tumefaciens,virus-mediated gene transfer, direct gene transfer systems such as electroporation, particle gun, microinjectionand chemical methods. All these traditional methods lack specificity and the transgenes are integrated atrandom sites in the plant DNA. Recently novel techniques for gene targeting have evolved such as engineerednucleases such as Zinc Finger Nucleases, Transcription Activator like effector nucleases, Clustered regularinterspaced short palindromic repeats. Other advances include improvement in tools for delivery of geneediting components which include carrier proteins, and carbon nanotubes. The present review focuses on thelatest techniques for target specific gene delivery in plants, their expression and future directions in plantbiotechnology

15.
Article in Chinese | WPRIM | ID: wpr-847291

ABSTRACT

BACKGROUND: Bionic porous scaffolds used in bone tissue engineering requires extracellular matrix-like nanofibrous and connected macroporous structure to effectively support cell implantation, adhesion, proliferation and other behaviors, and promote tissue regeneration. OBJECTIVE: To summarize the research progress in nanorfibrous macroporous scaffold preparation technology for tissue engineering based on the latest relevant research trends. METHODS: The first author searched Web of Science, CNKI and Baidu academic databases to retrieve papers published from 2000 to 2019 with the search terms “bone tissue engineering, nanofibrous, macroporous, scaffolds” in English and Chinese, respectively. Finally, 58 articles were included in result analysis. RESULTS AND CONCLUSION: The scaffolds with nanofibrous structures are fabricated using three strategies, including electrospinning, thermally induced phase separation, and self-assembly process. However, bone scaffold fabricated by a single strategy failed to provide interconnected macropores to simulate the microenvironment in the body, which was necessary for cell migration, growth, differentiation, proliferation, and tissue and organ regeneration. Therefore, it is now of great practical and scientific significance to develop macroporous nanofibrous scaffold using a combination of several strategies. Three-dimensional printing technique can provide precise structure and enables the customization of the internal structure and external shape of the scaffold, which promotes the development of bone tissue engineering technique.

16.
Chinese Journal of Biotechnology ; (12): 1261-1268, 2020.
Article in Chinese | WPRIM | ID: wpr-826851

ABSTRACT

The phenomenon of phase separation of intracellular biological macromolecules is an emerging research field that has received great attention in recent years. As an aggregation and compartment mechanism of cell biochemical reactions, it widely exists in nature and participates in important physiological processes such as gene transcription and regulation, as well as influences organism's response to external stimuli. Disequilibrium of phase separation may lead to the occurrence of some major diseases. Researchers in cross-cutting fields are trying to examine dementia and other related diseases from a new perspective of phase separation, exploring its molecular mechanism and the potential possibility of intervention and treatment. This review intends to introduce the latest research progress in this field, summarize the major research directions, biochemical basis, its relationship with disease occurrence, and giving a future perspective of key problems to focus on.


Subject(s)
Animals , Humans , Chemistry Techniques, Analytical , Cytoplasm , Chemistry , Metabolism , Macromolecular Substances , Research
17.
Article in English | WPRIM | ID: wpr-644865

ABSTRACT

In this study, an attempt was made to develop bi-functional constructs serving both as scaffolds and potential delivery systems for application in neural tissue engineering. The constructs were prepared in two steps. In the first step, the bulks of poly (L-lactic acid) (PLLA) in 1, 4-dioxane/water (87:13) were fabricated using liquid-liquid thermally induced phase separation technique. In the next step, the prepared bulks were coated with chitosan nanoparticles produced by two different techniques of ultrasonication and ionic gelation by grafting-coating technique. In ultrasonication technique, the chitosan solution (2 mg/mL) in acetic acid/sodium acetate buffer (90:10) was irradiated by an ultrasound generator at 20 kHz and power output of 750 W for 100 s. In ionic gelation technique, the tripolyphosphate in water solution (1 mg/mL) was added to the same chitosan solution. The physicochemical properties of the products were characterized by Scanning Electron Microscopy, Attenuated Total Reflection Fourier Transform-Infrared, liquid displacement technique, contact angle measurement, compressive and tensile tests, as well as zeta potential and particle size analysis using dynamic light scattering. Moreover, the cell proliferation and attachment on the scaffolds were evaluated through human glioblastoma cell line (U-87 MG) and human neuroblastoma cell line [BE (2)-C] culture respectively. The results showed that the samples coated with chitosan nanoparticles prepared by ultrasonication possessed enhanced hydrophilicity, biodegradation and cytocompatibility compared with pure PLLA and PLLA coated with chitosan nanoparticles prepared by ionic gelation. This study suggests successful nanoparticles-scaffold systems which can act simultaneously as potential delivery systems and tissue engineering scaffolds.


Subject(s)
Humans , Cell Line , Cell Proliferation , Chitosan , Dynamic Light Scattering , Glioblastoma , Hydrophobic and Hydrophilic Interactions , Microscopy, Electron, Scanning , Nanoparticles , Neuroblastoma , Particle Size , Tissue Engineering , Ultrasonography , Water
18.
Acta Pharmaceutica Sinica B ; (6): 18-25, 2014.
Article in English | WPRIM | ID: wpr-329759

ABSTRACT

The solid dispersion has become an established solubilization technology for poorly water soluble drugs. Since a solid dispersion is basically a drug-polymer two-component system, the drug-polymer interaction is the determining factor in its design and performance. In this review, we summarize our current understanding of solid dispersions both in the solid state and in dissolution, emphasizing the fundamental aspects of this important technology.

19.
Article in English | IMSEAR | ID: sea-157637

ABSTRACT

In the present study, gelatin microspheres containing ofloxacin were prepared by coacervation phase separation method and characterized by optical microscopy and scanning electron microscopy. The microspheres were analyzed for drug entrapment, bulk density, angle of repose, particle size and In-vitro release pattern. The effect of process variables on microsphere size was studied and based on these preliminary studies, different batches of microspheres were prepared by altering the drug: polymer ratio and cross-linking with glutaraldehyde. The size of microspheres was in range of 42- 45μm. They were spherical in shape as evidenced by photomicrographs and scanning electron microscopy. The percent drug entrapment was in the range of 78-90 % and they could sustain drug release over a period of 8 hrs.

20.
Article in Chinese | WPRIM | ID: wpr-404808

ABSTRACT

Phase separation immunoassay (PSI) combines some advantages of both homogeneous and heterogeneous immunoassays by carrying out a fast homogeneous immune reaction and a simple heterogeneous separation process. The method is readily amenable to automation. The principle of PSI is introduced. The carriers, the immobilization modes, the labels, the applications and the development of PSI are comprehensively reviewed with 60 References.

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