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1.
J. appl. oral sci ; 27: e20180649, 2019. graf
Article in English | LILACS, BBO | ID: biblio-1040227

ABSTRACT

Abstract Objective: Cleft palate (CP) is a congenital birth defect caused by the failure of palatal fusion. Little is known about the potential role of DNA methylation in the pathogenesis of CP. This study aimed to explore the potential role of DNA methylation in the mechanism of CP. Methodology: We established an all-trans retinoic acid (ATRA)-induced CP model in C57BL/6J mice and used methylation-dependent restriction enzymes (MethylRAD, FspEI) combined with high-throughput sequencing (HiSeq X Ten) to compare genome-wide DNA methylation profiles of embryonic mouse palatal tissues, between embryos from ATRA-treated vs. untreated mice, at embryonic gestation day 14.5 (E14.5) (n=3 per group). To confirm differentially methylated levels of susceptible genes, real-time quantitative PCR (qPCR) was used to correlate expression of differentially methylated genes related to CP. Results: We identified 196 differentially methylated genes, including 17,298 differentially methylated CCGG sites between ATRA-treated vs. untreated embryonic mouse palatal tissues (P<0.05, log2FC>1). The CP-related genes Fgf16 (P=0.008, log2FC=1.13) and Tbx22 (P=0.011, log2FC=1.64,) were hypermethylated. Analysis of Fgf16 and Tbx22, using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), identified 3 GO terms and 1 KEGG pathway functionally related to palatal fusion. The qPCR showed that changes in expression level negatively correlated with methylation levels. Conclusions: Taken together, these results suggest that hypermethylation of Fgf16 and Tbx22 is associated with decreased gene expression, which might be responsible for developmental failure of palatal fusion, eventually resulting in the formation of CP.


Subject(s)
Animals , Male , Female , Cleft Palate/genetics , DNA Methylation , T-Box Domain Proteins/genetics , Fibroblast Growth Factors/genetics , Reference Values , Gene Expression , Cleft Palate/embryology , Cleft Palate/pathology , Sequence Analysis, DNA , T-Box Domain Proteins/analysis , Protein Interaction Domains and Motifs , Real-Time Polymerase Chain Reaction , Fibroblast Growth Factors/analysis , Mice, Inbred C57BL
2.
Frontiers of Medicine ; (4): 412-425, 2018.
Article in English | WPRIM | ID: wpr-771297

ABSTRACT

Transcription factor networks have evolved in order to control, coordinate, and separate, the functions of distinct network modules spatially and temporally. In this review we focus on the MYC network (also known as the MAX-MLX Network), a highly conserved super-family of related basic-helix-loop-helix-zipper (bHLHZ) proteins that functions to integrate extracellular and intracellular signals and modulate global gene expression. Importantly the MYC network has been shown to be deeply involved in a broad spectrum of human and other animal cancers. Here we summarize molecular and biological properties of the network modules with emphasis on functional interactions among network members. We suggest that these network interactions serve to modulate growth and metabolism at the transcriptional level in order to balance nutrient demand with supply, to maintain growth homeostasis, and to influence cell fate. Moreover, oncogenic activation of MYC and/or loss of a MYC antagonist, results in an imbalance in the activity of the network as a whole, leading to tumor initiation, progression and maintenance.


Subject(s)
Animals , Humans , Carcinogenesis , Metabolism , Disease Progression , Gene Expression Regulation , Gene Regulatory Networks , Physiology , Protein Interaction Domains and Motifs , Physiology , Proto-Oncogene Proteins c-myc , Metabolism
3.
Protein & Cell ; (12): 590-600, 2017.
Article in English | WPRIM | ID: wpr-756983

ABSTRACT

Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.


Subject(s)
Animals , Humans , Mice , Amino Acid Sequence , Antibodies, Monoclonal , Chemistry , Genetics , Metabolism , Binding Sites , Cell Line , Crystallography, X-Ray , Enterovirus A, Human , Genetics , Allergy and Immunology , Fibroblasts , Virology , Gene Expression , HEK293 Cells , Immunoglobulin Fab Fragments , Chemistry , Genetics , Metabolism , Lysosomal Membrane Proteins , Chemistry , Genetics , Allergy and Immunology , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Receptors, Scavenger , Chemistry , Genetics , Allergy and Immunology , Receptors, Virus , Chemistry , Genetics , Allergy and Immunology , Recombinant Fusion Proteins , Chemistry , Genetics , Allergy and Immunology , Sequence Alignment , Sequence Homology, Amino Acid , Sf9 Cells , Spodoptera , Thermodynamics
4.
Article in Chinese | WPRIM | ID: wpr-300818

ABSTRACT

Autophagy is fundamental to maintain cellular homeostasis. As one kind of the most well-studied selective autophagy, autophagy of mitochondria (mitophagy)is crucial for the clearance of damaged mitochondria. Mitophagy dysfunction has been proved to be closely associated with many human diseases. Nix is a key protein for mitophagy during the maturation of reticulocytes. However, the detailed molecular mechanisms underlying Nix-mediated mitophagy are not fully understood. This article summarizes three possible working models of Nix in mitophagy induction. Firstly, Nix can interplay with Parkin, another important protein for mitophagy, to initiate mitophagy. Secondly, Nix can serve as a receptor for autophagy machinery by interacting with Atg8 family through its LIR motif. Finally, as a BH3-only protein, Nix can compete with Beclin-1 to bind other members of Bcl-2 family resulting in increased free Beclin-1 in cytosol, which further promotes autophagy flux.


Subject(s)
Autophagy , Genetics , Physiology , Autophagy-Related Protein 8 Family , Physiology , Beclin-1 , Physiology , Membrane Proteins , Physiology , Mitochondria , Genetics , Physiology , Mitophagy , Genetics , Physiology , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins , Physiology , Proto-Oncogene Proteins c-bcl-2 , Tumor Suppressor Proteins , Physiology , Ubiquitin-Protein Ligases , Physiology
5.
J. appl. oral sci ; 24(3): 250-257, tab, graf
Article in English | LILACS, BBO | ID: lil-787547

ABSTRACT

ABSTRACT A/J and 129P3/J mice strains have been widely studied over the last few years because they respond quite differently to fluoride (F) exposure. 129P3/J mice are remarkably resistant to the development of dental fluorosis, despite excreting less F in urine and having higher circulating F levels. These two strains also present different characteristics regardless of F exposure. Objective In this study, we investigated the differential pattern of protein expression in the liver of these mice to provide insights on why they have different responses to F. Material and Methods Weanling male A/J and 129P3/J mice (n=10 from each strain) were pared and housed in metabolic cages with ad libitum access to low-F food and deionized water for 42 days. Liver proteome profiles were examined using nLC-MS/MS. Protein function was classified by GO biological process (Cluego v2.0.7 + Clupedia v1.0.8) and protein-protein interaction network was constructed (PSICQUIC, Cytoscape). Results Most proteins with fold change were increased in A/J mice. The functional category with the highest percentage of altered genes was oxidation-reduction process (20%). Subnetwork analysis revealed that proteins with fold change interacted with Disks large homolog 4 and Calcium-activated potassium channel subunit alpha-1. A/J mice had an increase in proteins related to energy flux and oxidative stress. Conclusion This could be a possible explanation for the high susceptibility of these mice to the effects of F, since the exposure also induces oxidative stress.


Subject(s)
Animals , Male , Mice , Proteins/analysis , Genetic Predisposition to Disease , Proteome/drug effects , Fluorides/toxicity , Liver/drug effects , Liver/metabolism , Fluorosis, Dental/genetics , Reference Values , Mass Spectrometry/methods , Time Factors , Proteins/drug effects , Proteins/genetics , Gene Expression , Oxidative Stress/drug effects , Proteomics/methods , Protein Interaction Domains and Motifs , Mice, 129 Strain , Fluorides/analysis , Fluorides/metabolism , Mice, Inbred A
6.
Protein & Cell ; (12): 792-803, 2016.
Article in English | WPRIM | ID: wpr-757369

ABSTRACT

MRG proteins are conserved during evolution in fungi, flies, mammals and plants, and they can exhibit diversified functions. The animal MRGs were found to form various complexes to activate gene expression. Plant MRG1/2 and MRG702 were reported to be involved in the regulation of flowering time via binding to H3K36me3-marked flowering genes. Herein, we determined the crystal structure of MRG701 chromodomain (MRG701). MRG701 forms a novel dimerization fold both in crystal and in solution. Moreover, we found that the dimerization of MRG chromodomains is conserved in green plants. Our findings may provide new insights into the mechanism of MRGs in regulation of gene expression in green plants.


Subject(s)
Amino Acid Sequence , Arabidopsis , Genetics , Metabolism , Arabidopsis Proteins , Chemistry , Genetics , Metabolism , Binding Sites , Chromosomal Proteins, Non-Histone , Chemistry , Genetics , Metabolism , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli , Genetics , Metabolism , Gene Expression , Histones , Chemistry , Genetics , Metabolism , Models, Molecular , Oryza , Genetics , Metabolism , Peptides , Chemistry , Genetics , Metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Isoforms , Chemistry , Genetics , Metabolism , Protein Multimerization , Protein Structure, Secondary , Recombinant Proteins , Chemistry , Genetics , Metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Viridiplantae , Genetics , Metabolism
7.
Braz. j. med. biol. res ; 48(8): 683-690, 08/2015. tab, graf
Article in English | LILACS | ID: lil-753056

ABSTRACT

NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.


Subject(s)
Bacterial Proteins/genetics , Escherichia coli/genetics , Herbaspirillum/genetics , Transcription Factors/genetics , Bacterial Proteins/chemistry , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Herbaspirillum/metabolism , Nitrogen Fixation/genetics , Point Mutation , Protein Interaction Domains and Motifs , Transcription Factors/chemistry
8.
Article in Korean | WPRIM | ID: wpr-147140

ABSTRACT

Gephyrin is a central element that anchors, clusters and stabilizes glycine and gamma-aminobutyric acid type A receptors at inhibitory synapses of the mammalian brain. It self-assembles into a hexagonal lattice and interacts with various inhibitory synaptic proteins. Intriguingly, the clustering of gephyrin, which is regulated by multiple posttranslational modifications, is critical for inhibitory synapse formation and function. In this review, we summarize the basic properties of gephyrin and describe recent findings regarding its roles in inhibitory synapse formation, function and plasticity. We will also discuss the implications for the pathophysiology of brain disorders and raise the remaining open questions in this field.


Subject(s)
Animals , Humans , Carrier Proteins/chemistry , Disease Susceptibility , GABAergic Neurons/metabolism , Gene Expression Regulation , Membrane Proteins/chemistry , Protein Binding , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Protein Transport , Synapses/metabolism
9.
Article in Korean | WPRIM | ID: wpr-147141

ABSTRACT

Viral infection induces numerous tripartite motif (TRIM) proteins to control antiviral immune signaling and viral replication. Particularly, SPRY-containing TRIM proteins are found only in vertebrates and they control target protein degradation by their RING-finger and SPRY domains, and proper cytoplasmic localization. To understand TRIM30 function, we analyzed its localization pattern and putative roles of its RING-finger and SPRY domains. We found that TRIM30 is located in actin-mediated cytoplasmic bodies and produces colocalized ubiquitin chains in SPRY domain- and RING-finger domain-dependent ways that are degraded by autophagy and the proteasome. These results suggest a TRIM protein-dependent degradation mechanism by cytoplasmic body formation with actin networks.


Subject(s)
Animals , Mice , Amino Acid Sequence , Autophagy , Cell Line , Inclusion Bodies/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Molecular Sequence Data , Polyubiquitin/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Interaction Domains and Motifs , Protein Transport , Proteolysis , RING Finger Domains
10.
Acta Physiologica Sinica ; (6): 48-58, 2015.
Article in Chinese | WPRIM | ID: wpr-255973

ABSTRACT

The p26, a multifunctional ubiquitin-binding protein, has been proposed to be involved in protein degradation as a component within the ubiquitin-proteasome and autophagy-lysosome systems. As a scaffolding protein with several different kinds of protein-protein interaction domains, p62 mediates various cellular functions. Importantly, p62 plays a critical role in cell's selective autophagy and oxidative stress response, which are associated with the pathogenesis of several human diseases. In this review, we describe the structure of p62 and the mechanism of connection between p62 and ubiquitin-proteasome system/autophagy, so as to provide some perspectives on p62 research.


Subject(s)
Humans , Adaptor Proteins, Signal Transducing , Physiology , Autophagy , Oxidative Stress , Proteasome Endopeptidase Complex , Protein Interaction Domains and Motifs , Proteolysis , Ubiquitin
11.
Article in Chinese | WPRIM | ID: wpr-266715

ABSTRACT

Human lymphocyte function-associated antigen 3 (hLFA3) has been identified as an important T cell accessory molecule. Rhesus monkeys (Macaca mulatta) have been widely used as animal models for human immune disorders. Due to the species-specificity of immune system, it is necessary to study M. mulatta LFA3 (mmLFA3). In this study, the gene encoding mmLFA3 CD2-binding domain (mmLFA3Sh) was amplified by polymerase chain reaction (PCR) and genetically fused to human IgG1 Fc fragment in pPIC9K to construct the expression plasmid pPIC9K-mmLFA3Sh-Ig. Approximately 3-4 mg mmLFA3Sh-Ig protein was recovered from 1 L of inductive media, and mmLFA3Sh-Ig produced by the P. pastoris can bind to the CD2 positive cells, and suppress the monkey and human lymphocytes proliferation induced by Con A and alloantigen in a dose-dependent manner. These results suggested that mmLFA3Sh-Ig might be used as a novel tool for pathogenesis and experimental immunotherapy of Rhesus monkey immune disorders.


Subject(s)
Animals , Humans , CD58 Antigens , Immunoglobulin G , Lymphocyte Activation , Macaca mulatta , Pichia , Plasmids , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins , T-Lymphocytes
12.
Indian J Biochem Biophys ; 2014 Dec ; 51 (6): 559-566
Article in English | IMSEAR | ID: sea-156537

ABSTRACT

In addition to well-known process of proteasome-mediated degradation of polyubiquitinated proteins, monoubiquitination of proteins is also an important post-translational modification that regulates various non-degradative cellular processes like protein trafficking, cellular signalling, DNA replication and DNA repair. We have previously characterized a multi-domain cycling sequence binding protein LdCSBP from Leishmania donovani, which binds specifically to a conserved CAUAGAAG octamer containing RNAs via its uniquely arranged CCCH type Zn-fingers and degrades them using its Smr endonuclease domain, indicative of its potential role in the turnover of the S-phase mRNAs. Remarkably, its riboendonuclease activity is inhibited due to the incorporation of a monoubiquitin residue in the ZnF domain, though the target Lys residue remains unknown. Here, we report through systematic mutation of Lys residue to Ala that Lys-413 in LdCSBP is the site of monoubiquitination. However, the amino acid motif around the target Lys in LdCSBP is not consensus with any previously known monoubiquitination site, though partial homology is observed with a subset of recently identified mammalian ubiquitination target sites. Interestingly, Lys-413 of LdCSBP is conserved in the homologous annotated proteins from the related kinetoplastida parasites, suggesting similar monoubiquitination-mediated regulation of RNA endonuclease activity in the organisms.


Subject(s)
Amino Acid Sequence , Binding Sites , Endonucleases/chemistry , Endonucleases/genetics , Endonucleases/metabolism , Leishmania donovani/cytology , Leishmania donovani/physiology , Lysine/chemistry , Lysine/genetics , Lysine/metabolism , Molecular Sequence Data , Protein Binding , Protein Interaction Domains and Motifs , Protozoan Proteins/metabolism , RNA-Binding Proteins , S Phase/physiology , Structure-Activity Relationship , Ubiquitination , Zinc Fingers
13.
Article in English | WPRIM | ID: wpr-819708

ABSTRACT

OBJECTIVE@#To investigate the multienzyme complex formation of human malaria parasite Plasmodium falciparum (P. falciparum) orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC), the fifth and sixth enzyme of the de novo pyrimidine biosynthetic pathway. Previously, we have clearly established that the two enzymes in the malaria parasite exist physically as a heterotetrameric (OPRT)2(OMPDC)2 complex containing two subunits each of OPRT and OMPDC, and that the complex have catalytic kinetic advantages over the monofunctional enzyme.@*METHODS@#Both enzymes were cloned and expressed as recombinant proteins. The protein-protein interaction in the enzyme complex was identified using bifunctional chemical cross-linker, liquid chromatography-mass spectrometric analysis and homology modeling.@*RESULTS@#The unique insertions of low complexity region at the α 2 and α 5 helices of the parasite OMPDC, characterized by single amino acid repeat sequence which was not found in homologous proteins from other organisms, was located on the OPRT-OMPDC interface. The structural models for the protein-protein interaction of the heterotetrameric (OPRT)2(OMPDC)2 multienzyme complex were proposed.@*CONCLUSIONS@#Based on the proteomic data and structural modeling, it is surmised that the human malaria parasite low complexity region is responsible for the OPRT-OMPDC interaction. The structural complex of the parasite enzymes, thus, represents an efficient functional kinetic advantage, which in line with co-localization principles of evolutional origin, and allosteric control in protein-protein-interactions.


Subject(s)
Humans , Amino Acid Sequence , Malaria, Falciparum , Parasitology , Models, Molecular , Molecular Sequence Data , Orotate Phosphoribosyltransferase , Chemistry , Genetics , Metabolism , Orotidine-5'-Phosphate Decarboxylase , Chemistry , Genetics , Metabolism , Plasmodium falciparum , Genetics , Protein Binding , Protein Interaction Domains and Motifs , Protozoan Proteins , Chemistry , Genetics , Metabolism , Recombinant Proteins , Chemistry , Genetics , Metabolism , Sequence Alignment
14.
Chinese Journal of Biotechnology ; (12): 920-930, 2014.
Article in Chinese | WPRIM | ID: wpr-279460

ABSTRACT

Chloroplast-based expression system is promising for the hyper-expression of plant-derived recombinant therapeutic proteins and vaccines. To verify the feasibility of obtaining high-level expression of the SARS subunit vaccine and to provide a suitable plant-derived vaccine production platform against the severe acute respiratory syndrome coronavirus (SARS-CoV), a 193-amino acid fragment of SARS CoV spike protein receptor-binding domain (RBD), fused with the peptide vector cholera toxin B subunit (CTB), was expressed in tobacco chloroplasts. Codon-optimized CTB-RBD sequence was integrated into the chloroplast genome and homoplasmy was obtained, as confirmed by PCR and Southern blot analysis. Western blot showed expression of the recombinant fusion protein mostly in soluble monomeric form. Quantification of the recombinant fusion protein CTB-RBD was conducted by ELISA analysis from the transplastomic leaves at different developmental stages, attachment positions and time points in a day and the different expression levels of the CTB-RBD were observed with the highest expression of 10.2% total soluble protein obtained from mature transplastomic leaves. Taken together, our results demonstrate the feasibility of highly expressing SARS subunit vaccine RBD, indicating its potential in subsequent development of a plant-derived recombinant subunit vaccine and reagents production for antibody detection in SARS serological tests.


Subject(s)
Chloroplasts , Metabolism , Cholera Toxin , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins , Severe acute respiratory syndrome-related coronavirus , Spike Glycoprotein, Coronavirus , Nicotiana , Metabolism , Vaccines, Subunit
15.
Chinese Journal of Biotechnology ; (12): 504-513, 2014.
Article in Chinese | WPRIM | ID: wpr-279499

ABSTRACT

Anthocyanins are a ubiquitous group of water-soluble plant pigments of the flavonoid family, with anticancer property through HER-2 signaling pathway. Nowadays, molecular docking plays an important role in exposing the active sites and obtaining the bioactive conformation involving protein-ligand interactions. According to the crystal structure of HER-2 kinase domain and 12 main antitumor compounds of anthocyanins as well as ATP, a molecular docking study was performed by MVD program. All 12 compounds could bind to the same cavity of HER-2 kinase domain by high affinity (MolDock Score < -105 kJ/mol for anthocyanidins, < -130 kJ/mol for anthocyanidins-glc), where hydrophobic force and hydrogen bond played key roles. Additionally, this cavity overlapped with ATP binding (MolDock Score = -161 kJ/mol) domain; the binding of anthocyanins presumably interfered the H bond formation between ATP and HER-2. These results indicate that anthocyanins may competitively bind to ATP binding site in HER-2 kinase domain by suppressing HER-2 activation and downstream signaling cascade. This may provide useful theoretical instruction for the molecular mechanism of HER-2 kinase activity inhibition by anthocyanins in cancer prevention and treatment.


Subject(s)
Anthocyanins , Chemistry , Catalytic Domain , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Molecular Docking Simulation , Phosphorylation , Protein Interaction Domains and Motifs , Receptor, ErbB-2 , Chemistry
16.
Article in Chinese | WPRIM | ID: wpr-352134

ABSTRACT

Proteins are the final executive actor of cell viability and function. Protein-protein interactions determine the complexity of the organism. Research on the protein interactions can help us understand the function of the protein at the molecular level, learn the cell growth, development, differentiation, apoptosis and understand biological regulation mechanisms and other activities. They are essential for understanding the pathologies of diseases and helpful in the prevention and treatment of diseases, as well as in the development of new drugs. In this paper, we employ the single decision-tree classification model to predict protein-protein interactions in the yeast. The original data came from the existing literature. Using software Clementine, this paper analyzes how these attributes affect the accuracy of the model by adjusting the predicted attributes. The result shows that a single decision tree is a good classification model and it has higher accuracy compared to those in the previous researches.


Subject(s)
Algorithms , Decision Trees , Fungal Proteins , Chemistry , Models, Theoretical , Protein Interaction Domains and Motifs , Protein Interaction Maps
17.
Article in Chinese | WPRIM | ID: wpr-352152

ABSTRACT

Nowadays various protein medicines are increasingly playing significant roles in the treatment of many diseases, but the bioactive structures of such kinds of protein medicines are unstable because they are heat sensitive. Therefore, it is very important to explore a protective method and to explain the protective mechanism of protein medicines. In the present research, insulin was chosen as a heat-sensitive protein medicine, and a Group 3 late embryogenesis abundant (LEA) protein was chosen as its bioactive protectant during desiccation. The results of replica exchange molecular dynamics simulation suggest that comparing with insulin without any protection, the bioactive 3D structure and secondary structure of the insulin protected by LEA protein were preserved very well. All analyzing results proved that the LEA protein was a good bioactive protectant for heat sensitive protein medicines.


Subject(s)
Animals , Cold Temperature , Drug Stability , Freeze Drying , Helminth Proteins , Chemistry , Insulin , Chemistry , Nematoda , Plant Proteins , Pharmacology , Protein Interaction Domains and Motifs , Protein Structure, Secondary
18.
Protein & Cell ; (12): 711-721, 2013.
Article in English | WPRIM | ID: wpr-757563

ABSTRACT

Mutations in LR RK2 (Leucine rich repeat kinase 2) are a major cause of Parkinson's disease (PD). We and others reported recently that expression of the pathogenic gainof-function mutant form of LRRK2, LRRK2 G2019S, induces mitochondrial fission in neurons through DLP1. Here we provide evidence that expression of LRRK2 G2019S stimulates mitochondria loss or mitophagy. We have characterized several LRRK2 interacting proteins and found that LRRK2 interacts with ULK1 which plays an essential role in autophagy. Knockdown of either ULK1 or DLP1 expression with shRNAs suppresses LRRK2 G2019S expression-induced mitochondrial clearance, suggesting that LRRK2 G2019S expression induces mitochondrial fission through DLP1 followed by mitophagy via an ULK1 dependent pathway. In addition to ULK1, we found that LRRK2 interacts with the endogenous MKK4/7, JIP3 and coordinates with them in the activation of JNK signaling. Interestingly, LRRK2 G2019S-induced loss of mitochondria can also be suppressed by 3 different JNK inhibitors, implying the involvement of the JNK pathway in the pathogenic mechanism of mutated LRRK2. Thus our findings may provide an insight into the complicated pathogenesis of PD as well as some clues to the development of novel therapeutic strategies.


Subject(s)
Humans , Amino Acid Substitution , Autophagosomes , Metabolism , Pathology , Autophagy-Related Protein-1 Homolog , Chemistry , Genetics , Metabolism , GTP Phosphohydrolases , Genetics , Metabolism , Gene Knockdown Techniques , HeLa Cells , Intracellular Signaling Peptides and Proteins , Chemistry , Genetics , Metabolism , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Chemistry , Genetics , Metabolism , MAP Kinase Signaling System , Microtubule-Associated Proteins , Genetics , Metabolism , Mitophagy , Genetics , Physiology , Mitochondrial Proteins , Genetics , Metabolism , Mutant Proteins , Chemistry , Genetics , Metabolism , Mutation , Parkinson Disease , Genetics , Metabolism , Pathology , Protein Interaction Domains and Motifs , Recombinant Proteins , Chemistry , Genetics , Metabolism
19.
Protein & Cell ; (12): 695-701, 2013.
Article in English | WPRIM | ID: wpr-757771

ABSTRACT

The F-BAR domain containing proteins PACSINs are cytoplasmic phosphoproteins involved in various membrane deformations, such as actin reorganization, vesicle transport and microtubule movement. Our previous study shows that all PACSINs are composed of crescent shaped dimers with two wedge loops, and the wedge loop-mediated lateral interaction between neighboring dimers is important for protein packing and tubulation activity. Here, from the crystal packing of PACSIN 2, we observed a tight tip-to-tip interaction, in addition to the wedge loop-mediated lateral interaction. With this tip-to-tip interaction, the whole packing of PACSIN 2 shows a spiral-like assembly with a central hole from the top view. Elimination of this tip-to-tip connection inhibited the tubulation function of PACSIN 2, indicating that tip-to-tip interaction plays an important role in membrane deformation activity. Together with our previous study, we proposed a packing model for the assembly of PACSIN 2 on membrane, where the proteins are connected by tip-to-tip and wedge loop-mediated lateral interactions on the surface of membrane to generate various diameter tubules.


Subject(s)
Humans , Adaptor Proteins, Signal Transducing , Chemistry , Genetics , Cell Membrane , Chemistry , Crystallography, X-Ray , Liposomes , Chemistry , Models, Molecular , Mutagenesis, Site-Directed , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Quaternary , Recombinant Proteins , Chemistry , Genetics , Static Electricity
20.
Article in Chinese | WPRIM | ID: wpr-322017

ABSTRACT

<p><b>OBJECTIVE</b>To screen the hepatocyte proteins that interact with hepatitis B virus X protein (HBx).</p><p><b>METHODS</b>The recombinant plasmid pSos-HBx was constructed by inserting Sos-HBx fragment into the bait vector, and after sequence verification the plasmid was transformed into competent yeast cells. The expression and self-activation of Sos-HBx protein was detected in the yeast cells. The hepatocyte proteins interacting with the bait protein was screened with CytoTrap yeast two-hybrid technique.</p><p><b>RESULTS</b>The reconstructed plasmid harboring HBx gene expressed Sos-HBx protein in the yeast cells without self-activation of the protein. CytoTrap yeast two-hybrid system identified 6 hepatocyte proteins that interacted with HBx, including fibronectin 1, translationally controlled tumor protein, IQ motif and WD repeats 1, follistatin, orosomucoid 1, and disulfide isomerase family A member 3.</p><p><b>CONCLUSION</b>Six HBx-binding hepatocyte proteins have been identified using the CytoTrap yeast two-hybrid system, which provides clues for further investigation of the role of HBx protein in hepatitis and liver cancer.</p>


Subject(s)
Humans , Genetic Vectors , Hepatocytes , Metabolism , Plasmids , Protein Interaction Domains and Motifs , Proteins , Metabolism , Trans-Activators , Metabolism , Two-Hybrid System Techniques
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