OBJECTIVETo explore whether the membrane-associated protein Flotillin-1 has relationship with endocytosis of PrPc.

METHODSThe expression of Flotillin-1 in different cell lines was detected with the method of Western Blot; the interaction between Flotillin-1 and PrPc in Cells which were treated with copper ions was observed using immunoprecipitation method.

RESULTS(1) Flotillin-1 was widely expressed in many cell lines without significant difference in the amounts of expression level; (2) Only in the appearance of copper ions, the protein complexes of PrPc and Flotillin-1 can be detected with the method of IP, which were related to copper ions concentration and processing time.

CONCLUSIONThe membrane-associated protein Flotillin-1 has the relationship with the endocytosis of PrPc.

Cell Line ; Cell Membrane ; genetics ; metabolism ; Endocytosis ; Humans ; Membrane Proteins ; genetics ; metabolism ; PrPC Proteins ; genetics ; metabolism ; Protein Binding ; Protein Transport

The expression of a soluble protein on cell surface is often desirable for study of a functional protein, wide application of a protein or investigation of protein-protein interaction. The expression of a soluble protein on the surface of a cell is often achieved by genetically linking a protein to the extra-cellular fragment of a transmembrane partner. In this study, the myc epitope was linked with N terminal of transmembrane proteins either A2TM or deltaLNGFR amplified by overlapping PCR. The plasmids expressing fusion protein were transfected into 293FT cells and the expression of target proteins was evaluated by fluorescent microscope, flow cytometry and Western blotting. The results of flow cytometry revealed that both A2TM and deltaLNGFR were expressed on the cell surface, but A2TM could only be detected with high copy number. Western blotting showed that the expression level of deltaLNGFR was very high and protein was heavily glycosylated, by contrast the expression of A2TM was hardly detected. The results indicate that glycosylated deltaLNGFR is a good candidate partner for the expression of a soluble protein on the cell surface.
Apoptosis Regulatory Proteins ; biosynthesis ; genetics ; Cell Membrane ; metabolism ; Genes, MHC Class II ; genetics ; HLA-A2 Antigen ; biosynthesis ; genetics ; Humans ; Membrane Fusion Proteins ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism

LR11, also known as SorLA or SORL1, is a type-I membrane protein from which a large extracellular part, soluble LR11 (sLR11), is released by proteolytic shedding on cleavage with a disintegrin and metalloproteinase 17 (ADAM17). A shedding mechanism is presumed to have a key role in the functions of LR11, but the evidence for this has not yet been demonstrated. Tetraspanin CD9 has been recently shown to regulate the ADAM17-mediated shedding of tumor necrosis factor-alpha and intercellular adhesion molecule-1 on the cell surface. Here, we investigated the role of CD9 on the shedding of LR11 in leukocytes. LR11 was not expressed in THP-1 monocytes, but it was expressed and released in phorbol 12-myristate 13-acetate (PMA)-induced THP-1 macrophages (PMA/THP-1). Confocal microscopy showed colocalization of LR11 and CD9 proteins on the cell surface of PMA/THP-1. Ectopic neo-expression of CD9 in CCRF-SB cells, which are LR11-positive and CD9-negative, reduced the amount of sLR11 released from the cells. In contrast, incubation of LR11-transfected THP-1 cells with neutralizing anti-CD9 monoclonal antibodies increased the amount of sLR11 released from the cells. Likewise, the PMA-stimulated release of sLR11 increased in THP-1 cells transfected with CD9-targeted shRNAs, which was negated by treatment with the metalloproteinase inhibitor GM6001. These results suggest that the tetraspanin CD9 modulates the ADAM17-mediated shedding of LR11 in various leukemia cell lines and that the association between LR11 and CD9 on the cell surface has an important role in the ADAM17-mediated shedding mechanism.
ADAM Proteins/*metabolism ; Antigens, CD9/genetics/*metabolism ; Cell Line, Tumor ; Humans ; LDL-Receptor Related Proteins/genetics/*metabolism ; Leukocytes/*metabolism ; Macrophages/metabolism ; Membrane Transport Proteins/genetics/*metabolism ; Proteolysis

OBJECTIVETo explore the mechanism of miR-124 inhibiting the proliferative activity of prostate cancer PC3 cells.

METHODSLuciferase reporter gene assay was used to examine the specific binding ability of miR-124 to PKM2 mRNA 3'-UTR. After miR-124 was transfected mimic to PC3 cells, the expression levels of PKM2 mRNA and protein were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, respectively. The effects of miR-124 mimic and PKM2 siRNA on the proliferative activity of the PC3 cells were determined by MTT assay.

RESULTSThe expressions of PKM2 mRNA and protein were upregulated (5.12 +/- 0.35) times and (4.05 +/- 0.20) times respectively in the PC3 cells as compared with those in the RWPE-1 cells (P < 0.05). Luciferase reporter gene assay demonstrated that miR-124 targeted PKM2 3'-UTR. At 24 hours after transfection with miR-124 mimic, the PKM2 protein expression in the PC3 cells was downregulated (0.16 +/- 0.04) times (P < 0.05), while the PKM2 mRNA level was not changed significantly (P > 0.05), as compared with the control group. MTT assay showed that both miRNA-124 mimic and PKM2 siRNA could inhibit the proliferation of the PC3 cells, but the former exhibited a greater inhibitory effect than the latter. After transfection with miR-124 mimic and PKM2 siRNA, the cell growth rates were (66.20 +/- 5.10)% vs (82.10 +/- 6.35)% at 24 hours (P < 0.05) and (49.34 +/- 2.37)% vs (70.10 +/- 5.80)% at 48 hours (P < 0.05).

CONCLUSIONmiR-124 can suppress the proliferation of PC3 cells by regulating the PKM2 gene.

Carrier Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; genetics ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; MicroRNAs ; genetics ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Thyroid Hormones ; genetics ; metabolism ; Transfection

Thanks to tremendous advances in sequencing technologies and in particular to whole exome sequencing (WES), many genes have now been linked to severe sperm defects. A precise genetic diagnosis is obtained for a minority of patients and only for the most severe defects like azoospermia or macrozoospermia which is very often due to defects in the aurora kinase C (AURKC gene. Here, we studied a subject with a severe oligozoospermia and a phenotypic diagnosis of macrozoospermia. AURKC analysis did not reveal any deleterious variant. WES was then initiated which permitted to identify a homozygous loss of function variant in the zinc finger MYND-type containing 15 (ZMYND15 gene. ZMYND15 has been described to serve as a switch for haploid gene expression, and mice devoid of ZMYND15 were shown to be sterile due to nonobstructive azoospermia (NOA). In man, ZMYND15 has been associated with NOA and severe oligozoospermia. We confirm here that the presence of a bi-allelic ZMYND15 variant induces a severe oligozoospermia. In addition, we show that severe oligozoospermia can be associated macrozoospermia, and that a phenotypic misdiagnosis is possible, potentially delaying the genetic diagnosis. In conclusion, genetic defects in ZMYND15 can induce complete NOA or severe oligozoospermia associated with a very severe teratozoospermia. In our experience, severe oligozoospermia is often associated with severe teratozoospermia and can sometimes be misinterpreted as macrozoospermia or globozoospermia. In these instances, specific AURKC or dpy-19 like 2 (DPY19L2) diagnosis is usually negative and we recommend the direct use of a pan-genomic techniques such as WES.
Animals ; Azoospermia/genetics* ; Humans ; Infertility, Male/genetics* ; Male ; Membrane Proteins/genetics* ; Mice ; Mutation ; Oligospermia/genetics* ; Repressor Proteins/metabolism* ; Teratozoospermia/genetics*

NRT1 family proteins play an important roles for absorbing and transporting of nitrate in different plants. In order to identify the NRT1 family genes of Rehmannia glutinosa, this study used 11 NRT1 homologous proteins of Arabidopsis as probe sequences and aligned with the transcriptome data of R. glutinosa by using NCBI BLASTN software. Resulting there were 18 NRT1 proteins were identified in R. glutinosa. On basis of this, a series of the molecular characteristics of R. glutinosa NRT1 proteins including the conserved domains, the transmembrane structure, the subcellular location and phylogenetic features were in detail analyzed. At same time, it were systematically analyzed that the temporal and spatial expression patterns and characteristics of R. glutinosa NRT1 family genes in response to different stress factors. The results indicated that 18 R. glutinosa NRT1 family genes with the length of coding region from 1 260 bp to 1 806 bp, encoded proteins ranging from 419 to 601 amino acids, and all of they owned the domains of typical peptide transporter with 7 to 12 transmembrane domains. These R. glutinosa NRT1 family proteins mostly were found to locate on cellular plasma membrane, and belonged to the hydrophobic proteins. Furthermore, the evolutionary analysis found that the 18 R. glutinosa NRT1 protein family could be divided into two subfamilies, of which 14 NRT1 family genes might occur the positive selection, and 4 genes occur the passivation selection during the evolution process of R. glutinosa. In addition the expression analysis showed that 18 R. glutinosa NRT1 family genes have the distinct expression patterns in different tissues of R. glutinosa, and their expression levels were also obvious difference in response to various stress. These findings infield that 18 R. glutinosa NRT1 family proteins might have obviously different functional roles in nitrate transport of R. glutinosa. In conclusion, this study lays a solid theoretical foundation for clarifying the absorption and transport molecular mechanism of N element during R. glutinosa growth and development, and at same time for deeply studying the molecular function of R. glutinosa NRT1 proteins in absorption and transport of nitrate.
Anion Transport Proteins ; Membrane Transport Proteins ; Nitrates ; Phylogeny ; Plant Proteins/metabolism* ; Rehmannia/genetics* ; Transcriptome

A specialized tissue type, the keratinizing epithelium, protects terrestrial mammals from water loss and noxious physical, chemical and mechanical insults. This barrier between the body and the environment is constantly maintained by reproduction of inner living epidermal keratinocytes which undergo a process of terminal differentiation and then migrate to the surface as interlocking layers of dead stratum corneum cells. These cells provide the bulwark of mechanical and chemical protection, and together with their intercellular lipid surroundings, confer water-impermeability. Much of this barrier function is provided by the cornified cell envelope (CE), an extremely tough protein/lipid polymer structure formed just below the cytoplasmic membrane and subsequently resides on the exterior of the dead cornified cells. It consists of two parts: a protein envelope and a lipid envelope. The protein envelope is thought to contribute to the biomechanical properties of the CE as a result of cross-linking of specialized CE structural proteins by both disulfide bonds and N(epsilon)-(gamma-glutamyl)lysine isopeptide bonds formed by transglutaminases. Some of the structural proteins involved include involucrin, loricrin, small proline rich proteins, keratin intermediate filaments, elafin, cystatin A, and desmosomal proteins. The lipid envelope is located on the exterior of and covalently attached by ester bonds to the protein envelope and consists of a monomolecular layer of omega-hydroxyceramides. These not only serve of provide a Teflon-like coating to the cell, but also interdigitate with the intercellular lipid lamellae perhaps in a Velcro-like fashion. In fact the CE is a common feature of all stratified squamous epithelia, although its precise composition, structure and barrier function requirements vary widely between epithelia. Recent work has shown that a number of diseases which display defective epidermal barrier function, generically known as ichthyoses, are the result of genetic defects of the synthesis of either CE proteins, the transglutaminase 1 cross-linking enzyme, or defective metabolism of skin lipids.
Animal ; Cell Membrane/metabolism ; Epidermis/metabolism* ; Epidermis/chemistry* ; Human ; Ichthyosis/metabolism ; Ichthyosis/genetics ; Keratinocytes/metabolism* ; Keratinocytes/chemistry ; Membrane Lipids/metabolism* ; Membrane Proteins/metabolism* ; Protein-Glutamine gamma-Glutamyltransferase/metabolism

Enterovirus 71 (EV71) is a neurotropic pathogen that can induce hand, foot and mouth disease in children. There is an appreciable mortality rate after EV71 infections. The mechanism of action of EV71 replication is not known. Recent work has identified some of cell factors of the host that participate in the synthesis of the RNA and proteins of EV71 (e.g., hnRNP K, reticulon 3 (RTN 3)). In that work, researchers used a competitive assay to show that hnRNP K can interact with EV71 5' UTR, which is required for efficient synthesis of viral RNA. Using a yeast two-hybrid system, other researchers demonstrated that RTN 3 interacts with the N-terminal domain of EV71 2C, which is crucial for replication of viral RNA. Here, we discuss recent work focusing on the molecular mechanisms of hnRNP K and RTN 3 in the synthesis of the RNA and proteins of EV71.
Animals ; Carrier Proteins ; genetics ; metabolism ; Enterovirus A, Human ; genetics ; physiology ; Enterovirus Infections ; genetics ; metabolism ; virology ; Heterogeneous-Nuclear Ribonucleoprotein K ; Host-Pathogen Interactions ; Humans ; Membrane Proteins ; genetics ; metabolism ; Nerve Tissue Proteins ; genetics ; metabolism ; Ribonucleoproteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Replication

OBJECTIVETo investigate the role of mitofusin-2 gene (mfn2) in apoptosis in human breast carcinoma cell line MCF-7 cells after in vitro transfection.

METHODSpEGFP mfn2 was transfected by sofast in vitro. Expression of GFP was observed by Western blot, and the MCF-7 cell proliferation was measured by MTT and cell counting. Apoptosis in MCF-7 cells was observed in annexin-V/PI and chondrosome transmembrane potential of MCF-7 marked in JC-1 by FCM. The Ultrastructure of cells was observed by transmission electron microscopy.

RESULTSThe stable expression of GFP in MCF-7 cells was confirmed by Western blot. Mfn2 significantly inhibited cell proliferation, revealed by MTT, and decrease chondrosome transmembrane potential. Exogenous mfn2 gene significantly induced apoptosis. The apoptotic rate was increased from 3.6% to 16.0% (P < 0.05). Mfn2 gene induced break down and loss of mitochondrial cristae, and rarefaction of mitochondrial ground substance. Swollen mitochondria intensely aggregated around the cell nuclei.

CONCLUSIONMfn2 can strongly induce apoptosis in MCF-7 cells, which may be associated with decrease of mitochondrial transmembrane potential.

Apoptosis ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Female ; GTP Phosphohydrolases ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Membrane Potential, Mitochondrial ; Membrane Proteins ; genetics ; metabolism ; Mitochondria ; ultrastructure ; Mitochondrial Proteins ; genetics ; metabolism ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo study the changes of the intramembrane protein particles of erythrocyte from Duchenne muscular dystrophy (DMD) patients and the gene carriers and to explore the pathogenesis of DMD and the diagnostic value of erythrocyte freeze-fracture technology.

METHODSThe fixed erythrocyte mass was treated to form replica membrane by means of the freeze-fracture technology. Then the replica membrane was observed and a picture was taken under electron microscope. The protein particles of extracellular face(EF) and protoplasmic face(PF) per square were counted. The statistical comparative analysis was performed.

RESULTSThe protein particle counts of EF face and PF face of erythrocyte membrane from DMD patients and DMD carriers decreased obviously in comparison with the normal control group (P<0.001).

CONCLUSIONThe erythrocyte freeze-fracture electron microscopic technology may serve as a method for accessory examination of diagnosing DMD patients and a method for detecting DMD carriers. This investigation material supplies reliable evidence for the theory of the systemic membrane defect of DMD.

Erythrocyte Membrane ; metabolism ; ultrastructure ; Female ; Heterozygote ; Humans ; Male ; Membrane Proteins ; metabolism ; Microscopy, Electron ; Muscular Dystrophy, Duchenne ; diagnosis ; genetics ; metabolism