Endosomal sorting complex required for transport (ESCRT) system drives various cellular processes, including endosome sorting, organelle biogenesis, vesicle transport, maintenance of plasma membrane integrity, membrane fission during cytokinesis, nuclear membrane reformation after mitosis, closure of autophagic vacuoles, and enveloped virus budding. Increasing evidence suggests that the ESCRT system can be hijacked by different family viruses for their proliferation. At different stages of the virus life cycle, viruses can interfere with or exploit ESCRT-mediated physiological processes in various ways to maximize their chance of infecting the host. In addition, many retroviral and RNA viral proteins possess "late domain" motifs, which can recruit host ESCRT subunit proteins to assist in virus endocytosis, transport, replicate, budding and efflux. Therefore, the "late domain" motifs of viruses and ESCRT subunit proteins could serve as promising drug targets in antiviral therapy. This review focuses on the composition and functions of the ESCRT system, the effects of ESCRT subunits and virus "late domain" motifs on viral replication, and the antiviral effects mediated by the ESCRT system, aiming to provide a reference for the development and utilization of antiviral drugs.
Endosomal Sorting Complexes Required for Transport/metabolism* ; Viruses/metabolism* ; Protein Transport ; Virus Replication ; Endosomes/metabolism* ; Virus Release

Chemically defined medium is widely used for culturing mouse embryonic stem cells (mESCs), in which N2B27 works as a substitution for serum, and GSK3β and MEK inhibitors (2i) help to promote ground-state pluripotency. However, recent studies suggested that MEKi might cause irreversible defects that compromise the developmental potential of mESCs. Here, we demonstrated the deficient bone morphogenetic protein (BMP) signal in the chemically defined condition is one of the main causes for the impaired pluripotency. Mechanistically, activating the BMP signal pathway by BMP4 could safeguard the chromosomal integrity and proliferation capacity of mESCs through regulating downstream targets Ube2s and Chmp4b. More importantly, BMP4 promotes a distinct in vivo developmental potential and a long-term pluripotency preservation. Besides, the pluripotent improvements driven by BMP4 are superior to those by attenuating MEK suppression. Taken together, our study shows appropriate activation of BMP signal is essential for regulating functional pluripotency and reveals that BMP4 should be applied in the serum-free culture system.
Animals ; Bone Morphogenetic Protein 4/metabolism* ; Cell Differentiation ; Chromosomal Instability ; Endosomal Sorting Complexes Required for Transport ; Mice ; Mitogen-Activated Protein Kinase Kinases/metabolism* ; Mouse Embryonic Stem Cells/cytology* ; Pluripotent Stem Cells/cytology* ; Signal Transduction ; Ubiquitin-Conjugating Enzymes

OBJECTIVES: The effect of Vps4b gene mutation on the expressions of cytokeratin 14 (CK14) and proliferating cell nuclear antigen (PCNA) in the Hertwig's epithelial root sheath (HERS) is investigated. METHODS: The bilateral mandibular tissues of mouse on postnatal days 5, 9, 11, 15, and 19 were removed. The mandibular first molar tissue sections were obtained after paraffin embedding. The CK14 and PCNA expressions in the epithelial root sheath of the normal mouse and Vps4b knockout mouse were compared through immunohistochemistry. RESULTS: On postnatal day 5, the normal mouse began to form HERS and had a strong positive PCNA expression in the HERS cells; on postnatal day 9, the HERS structure was continuous, and PCNA was positive in the HERS cells; on postnatal day 11, a small portion of HERS began to break, and PCNA was weakly positive in the HERS cells; on postnatal day 15, HERS continued to fracture; PCNA was weakly and positively expressed in the HERS cells on the root surface; on postnatal day 19, the tooth root reached normal physiological length, and PCNA was positively expressed in the HERS cells of the terminal part. Similar to the normal mouse, the gene knockout mouse also formed a HERS structure on postnatal day 5. However, HERS began to break on postnatal day 9. On postnatal day 19, only a few fragments of HERS were found on the root surface, and the root development was immature. Moreover, the expression intensity of PCNA in the gene knockout mouse was decreased. CONCLUSIONS The Vps4b gene mutation may change the CK14 and PCNA expressions, leading to abnormal root development.
ATPases Associated with Diverse Cellular Activities ; Animals ; Endosomal Sorting Complexes Required for Transport ; Epithelial Cells ; Keratin-14 ; Mice ; Mice, Knockout ; Proliferating Cell Nuclear Antigen ; Tooth Root

A splicing mutation in VPS4B can cause dentin dysplasia type I (DD-I), a hereditary autosomal-dominant disorder characterized by rootless teeth, the etiology of which is genetically heterogeneous. In our study, dental follicle cells (DFCs) were isolated and cultured from a patient with DD-I and compared with those from an age-matched, healthy control. In a previous study, this DD-I patient was confirmed to have a loss-of-function splicing mutation in VPS4B (IVS7 + 46C > G). The results from this study showed that the isolated DFCs were vimentin-positive and CK14-negative, indicating that the isolated cells were derived from the mesenchyme. DFCs harboring the VPS4B mutation had a significantly higher proliferation rate from day 3 to day 8 than control DFCs, indicating that VPS4B is involved in cell proliferation. The cells were then replenished with osteogenic medium to investigate how the VPS4B mutation affected osteogenic differentiation. Induction of osteogenesis, detected by alizarin red and alkaline phosphatase staining in vitro, was decreased in the DFCs from the DD-I patient compared to the control DFCs. Furthermore, we also found that the VPS4B mutation in the DD-I patient downregulated the expression of osteoblast-related genes, such as ALP, BSP, OCN, RUNX2, and their encoded proteins. These outcomes confirmed that the DD-I-associated VPS4B mutation could decrease the capacity of DFCs to differentiate during the mineralization process and may also impair physiological root formation and bone remodeling. This might provide valuable insights and implications for exploring the pathological mechanisms underlying DD-I root development.
ATPases Associated with Diverse Cellular Activities ; genetics ; Case-Control Studies ; Cell Differentiation ; genetics ; Cells, Cultured ; Dental Sac ; cytology ; Dentin Dysplasia ; genetics ; pathology ; physiopathology ; Endosomal Sorting Complexes Required for Transport ; genetics ; Humans ; Mutation ; genetics ; Osteogenesis ; genetics ; RNA Splicing ; genetics

OBJECTIVE: To verify the effect of the mutant gene vps4b on the expression of tooth development-related proteins, dentin sialophosphoprotein (DSPP) and collagenⅠ (COL-Ⅰ). METHODS: Paraffin tissue sections of the first molar tooth germ were obtained from the heads of fetal mice at the embryonic stages of 13.5, 14.5, and 16.5 days and from the mandibles of larvae aged 2.5 and 7 days after birth. The immunohistochemical method was used to detect the expression and location of DSPP and COL-Ⅰ in wild-type mouse and vps4b knockout mouse. RESULTS: DSPP and COL-Ⅰ were not found in the bud and cap stages of wild-type mouse molar germ. In the bell stage, DSPP was positively expressed in the inner enamel epithelium and dental papilla, whereas COL-Ⅰ was strongly expressed in the dental papilla and dental follicle. During the secretory and mineralized periods, DSPP and COL-Ⅰ were intensely observed in ameloblasts, odontoblasts, and dental follicles, but COL-Ⅰ was also expressed in the dental papilla. After vps4b gene knockout, DSPP was not expressed in the dental papilla of the bell stage and in the dental papilla and dental follicle of the secretory phase. The expression position of COL-Ⅰ in the bell and mineralization phase was consistent with that in the wild-type mice. Moreover, the expression of COL-Ⅰ in the dental papilla changed in the secretory stage. CONCLUSIONS Gene vps4b plays a significant role in the development of tooth germ. The expression of DSPP and COL-Ⅰ may be controlled by gene vps4b and regulates the development of tooth dentin and cementum together with vps4b.
ATPases Associated with Diverse Cellular Activities ; genetics ; Animals ; Collagen ; metabolism ; Endosomal Sorting Complexes Required for Transport ; genetics ; Extracellular Matrix Proteins ; metabolism ; Mice ; Mice, Knockout ; Molar ; Odontoblasts ; Phosphoproteins ; metabolism ; Sialoglycoproteins ; metabolism ; Tooth Germ

BACKGROUNDTwo recent whole-exome sequencing researches identifying somatic mutations in the ubiquitin-specific protease 8 (USP8) gene in pituitary corticotroph adenomas provide exciting advances in this field. These mutations drive increased epidermal growth factor receptor (EGFR) signaling and promote adrenocorticotropic hormone (ACTH) production. This study was to investigate whether the inhibition of USP8 activity could be a strategy for the treatment of Cushing's disease (CD).

METHODSThe anticancer effect of USP8 inhibitor was determined by testing cell viability, colony formation, apoptosis, and ACTH secretion. The immunoblotting and quantitative reverse transcription polymerase chain reaction were conducted to explore the signaling pathway by USP8 inhibition.

RESULTSInhibition of USP8-induced degradation of receptor tyrosine kinases including EGFR, EGFR-2 (ERBB2), and Met leading to a suppression of AtT20 cell growth and ACTH secretion. Moreover, treatment with USP8 inhibitor markedly induced AtT20 cells apoptosis.

CONCLUSIONSInhibition of USP8 activity could be an effective strategy for CD. It might provide a novel pharmacological approach for the treatment of CD.

Adrenocorticotropic Hormone ; metabolism ; Animals ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; physiology ; Cell Survival ; drug effects ; physiology ; Endopeptidases ; metabolism ; Endosomal Sorting Complexes Required for Transport ; antagonists & inhibitors ; metabolism ; Enzyme Inhibitors ; pharmacology ; Humans ; Indenes ; pharmacology ; Mice ; Pyrazines ; pharmacology ; Receptor, Epidermal Growth Factor ; metabolism ; Ubiquitin Thiolesterase ; antagonists & inhibitors ; metabolism

OBJECTIVETo assess the association of neural precursor cell expressed developmentally down-regulated 4 (NEDD4) with schizophrenia.

METHODSFive single nucleotide polymorphisms (SNPs) of the NEDD4 gene were genotyped by TaqMan SNP genotyping assay in an independent sample of 464 individuals with schizophrenia and 487 healthy controls from eastern Han Chinese population. Clinical data were collected with a general information questionnaire and Positive and Negative Syndrome Scale (PANSS).

RESULTSFrequencies of rs3088077 (allelic: χ2=18.024, P=0.000; genotypic: χ2=16.634, P=0.000), rs7162435 (allelic: χ2=6.771, P=0.009; genotypic: χ2=7.352, P=0.025) and rs2303579 (allelic: χ2=11.253, P=0.001; genotypic: χ2=12.248, P=0.002) were found to be significant different between the two groups. Moreover, TT of rs7162435 was significantly correlated with scores of factors of excitement and hostility (14.53±3.925, F=3.551, P=0.029).

CONCLUSIONrs3088077, rs7162435 and rs2303579 of the NEDD4 gene may be associated with schizophrenia. Moreover, the TT genotype of rs7162435 may increase the severity of excitement and hostility. Our results may provide a clue for delineating the connection between the glutamate hypothesis of schizophrenia and ubiquitination.

Adolescent ; Adult ; Aged ; Alleles ; Asian Continental Ancestry Group ; ethnology ; genetics ; China ; ethnology ; Endosomal Sorting Complexes Required for Transport ; genetics ; Female ; Genetic Predisposition to Disease ; Genotype ; Humans ; Male ; Middle Aged ; Nedd4 Ubiquitin Protein Ligases ; Polymorphism, Single Nucleotide ; Schizophrenia ; enzymology ; ethnology ; genetics ; Ubiquitin-Protein Ligases ; genetics ; Young Adult

OBJECTIVETo assess the association of single nucleotide polymorphisms (SNPs) of ubiquitin-specific protease 8 gene (USP8) with male infertility among ethnic Han Chinese from Sichuan.

METHODSA total of 316 infertile males were recruited (case group), which included 72 severe oligozoospermic (SO) cases and 244 non-obstructive azoospermic (NOA) cases. The control group consisted of 149 fertile males. The genotypes of 4 SNPs (rs2241769, rs11857513, rs7174015 and rs3743044) were determined with a Sequenom MassArray technique. The frequencies of genotype, allele and haploptye were analyzed.

RESULTSNo significant difference was detected in the allelic or genotypic frequencies of the 4 SNPs between the two groups (P>0.05). Based on linkage disequilibrium analysis and haplotype construction, the frequency distribution of haplotype CAAG showed a significant difference between non-obstructive azoospermic patients and the controls (P=0.021).

CONCLUSIONThe 4 SNPs (rs2241769, rs11857513, rs7174015 and rs3743044) of USP8 gene may not be associated with male infertility in ethnic Hans from Sichuan. While the haplotype CAAG may be a down-regulating factor for the risk of NOA.

Adult ; Asian Continental Ancestry Group ; ethnology ; genetics ; Azoospermia ; genetics ; Base Sequence ; Case-Control Studies ; China ; ethnology ; Endopeptidases ; genetics ; Endosomal Sorting Complexes Required for Transport ; genetics ; Genetic Predisposition to Disease ; ethnology ; Genotype ; Humans ; Infertility, Male ; ethnology ; genetics ; Male ; Molecular Sequence Data ; Polymorphism, Single Nucleotide ; Ubiquitin Thiolesterase ; genetics

OBJECTIVETo study the feasibility of using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) for the detection of DNA methylation in placenta tissue.

METHODSFor blood cells from 13 non-pregnant women and 9 euploid placenta, the ratios of DNA methylation were evaluated for 4 genes including CGI149, CGI113, HLCS and ACTB with MS-MLPA and bisulfite sequencing, respectively.

RESULTSThe methylation ratio of the ACTB gene was 0-0.1 for the blood cells when the digestion control was completely digested. The cutoff value for the methylation ratio of MS-MLPA has been determined as 0.1. For the 9 placenta samples, results of MS-MLPA and bisulfite sequencing were concordant for all of the four genes.

CONCLUSIONMS-MLPA is an effective alternative to bisulfite sequencing for the assessment of methylation ratios in placental tissues.

Actins ; genetics ; Adult ; Carbon-Nitrogen Ligases ; genetics ; CpG Islands ; genetics ; DNA Methylation ; Endosomal Sorting Complexes Required for Transport ; genetics ; Feasibility Studies ; Female ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Placenta ; metabolism ; Pregnancy ; Reproducibility of Results ; Ribosomal Proteins ; genetics ; Young Adult

OBJECTIVETo explore whether docetaxel-resistant cells (MCF-7/Doc) and doxorubicin-resistant cells (MCF-7/ADM) can secrete Exosomes and their potential role in cell-cell drug-resistance transfer.

METHODSExosomes were extracted from the cell culture supernatants of MCF-7/Doc and MCF-7/ADM cells by fractionation ultracentrifugation, and were identified by transmission electron microscopy and Western blot analysis. GFP-MCF-7/S, a breast cancer parental sensitive cell line stably expressing green fluorescent protein (GFP), was constructed by recombinant lentiviral vector with GFP. Then the resistance experiment of cells and the experiment of resistance transfer by exosomes were designed to observe the phenomenon of cell-to-cell drug-resistance transfer.

RESULTSSimilar to the breast cancer parental sensitive cells (MCF-7/S), the breast cancer resistant sublines could secrete exosomes, which exhibited round or elliptic shape ranging from 30 to 100 nm in diameter with intact membrane, and only expressed the protein marker of exosomes, Tsg101, did not express the endoplasmic reticulum marker calnexin. After MCF-7/S, MCF-7/DOC and MCF-7/ADM cells we cocultured with GFP-MCF-7/S cells for 72 h, there were no significant differences in the expression of fluorescence-labeled cells among the four groups. When treated by the drug ADM or DOC for 24 hours, the MCF-7/DOC+GFP-MCF-7/S group was in favor of a significant higher survival rate of fluorescence-labeled cells compared with the MCF-7/S+GFP-MCF-7/S group (65.5% vs. 25.5%, P < 0.001), and so did the MCF-7/ADM+GFP-MCF-7/S group (53.6% vs. 25.4%, P < 0.001). The exosomes extracted from MCF-7/S, MCF-7/DOC and MCF-7/ADM cells were cultured with the GFP-MCF-7/S cells for 48 h. Among these groups, no significant differences in the expression of fluorescence-labeled cells were found. After treated by the drug ADM or DOC for 24 hours, the exosomes extracted from MCF-7/DOC+GFP-MCF-7/S group was associated with a significant higher survival rate of fluorescence-labeled cells compared with the exosomes extracted from MCF-7/S+GFP-MCF-7/S group (59.9% vs. 32.4%, P < 0.001), and so did the exosomes extracted from the MCF-7/ADM)+GFP-MCF-7/S group (58.3% vs. 27.2%, P < 0.001).

CONCLUSIONOur results suggest that drug-resistance can be transferred between breast cancer cells, and exosomes are probably the transporter of the drug resistance.

Antibiotics, Antineoplastic ; pharmacology ; Antineoplastic Agents ; pharmacology ; Cell Survival ; Coculture Techniques ; DNA-Binding Proteins ; metabolism ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Endosomal Sorting Complexes Required for Transport ; metabolism ; Exosomes ; metabolism ; pathology ; Green Fluorescent Proteins ; metabolism ; Humans ; MCF-7 Cells ; pathology ; Taxoids ; pharmacology ; Transcription Factors ; metabolism