OBJECTIVETo study the expression and significance of the mammalian target of rapamycin (mTOR)/eukaryote initiating factor 4E binding protein 1(4EBP1)/hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathway in asthmatic mice.

METHODSForty SPF level 6-8 week-old female Balb/C mice were randomly divided into control, asthma, budesonide and mTOR inhibitor (rapamycin) intervention groups (n=10 each). The asthmatic mouse model was prepared via OVA induction and challenge test. The intervention groups were administered with rapamycin at the dosage of 3 mg/kg by an intraperitoneal injection or budesonide suspension at the dosage of l mg by aerosol inhalation respectively 30 minutes before the OVA challenge. The control and asthma groups were treated with normal saline instead. The concentrations of HIF-1α and VEGF in bronchoalveolar lavage fluid (BALF) were examined using ELISA 24 hours after the last challenge. The pathological changes of lung tissue were observed by hematoxylin-eosin (HE) staining. The p-mTOR and p-4EBP1 from the lung tissues were detected by immunohistochemistry and Western blot. Pearson analysis was used to study the correlation between p-mTOR, p-4EBP1, HIF-1α, and VEGF expression.

RESULTSCompared with the control group, inflammatory cell infiltration and secretions in the trachea increased in the asthma group. The levels of HIF-1α and VEGF in BALF and p-mTOR and p-4EBP1 expression in lung tissues also increased (P<0.01). Compared with the asthma group, inflammatory cell infiltration and secretions in the trachea were reduced in the two intervention groups, and the levels of HIF-1α and VEGF in BALF and p-mTOR and p-4EBP1 expression in lung tissues were also reduced (P<0.01). There were no significant differences in the above changes between the two intervention groups and control group (P>0.05). In the asthma group, there was a pairwise positive correlation between lung p-mTOR and p-4EBP1 expression and HIF-1α and VEGF levels in BALF (P<0.05). However, there were no correlations in the above indexes in the intervention groups and control group.

CONCLUSIONSp-mTOR, p-4EBP1, HIF-1α and VEGF together are involved in the pathogenesis of asthma. Rapamycin treatment can block this signaling pathway, suggesting that this pathway can be used as a novel target for asthma treatment.

Animals ; Asthma ; drug therapy ; metabolism ; Carrier Proteins ; analysis ; physiology ; Female ; Hypoxia-Inducible Factor 1, alpha Subunit ; analysis ; physiology ; Lung ; chemistry ; pathology ; Mice ; Mice, Inbred BALB C ; Phosphoproteins ; analysis ; physiology ; Signal Transduction ; physiology ; TOR Serine-Threonine Kinases ; analysis ; physiology ; Vascular Endothelial Growth Factor A ; analysis ; physiology

Angelica dahurica (A. dahurica) is a traditional Chinese medicinal plant being used in clinical practice. The present study demonstrated that A. dahurica could reduce white-fat weight in high-fat-diet hyperlipidemic mice, decrease total cholesterol and triglyceride concentrations in the livers of both high-fat-diet and Triton WR1339 induced hyperlipidemic mice, and enhance the total hepatic lipase activities of them. These findings were further supported by the results derived from the experiments with HepG2 cells in vitro. In addition, the proteins related to lipids metabolism were investigated using LC-MS/MS, indicating that genes of lipid metabolism and lipid transport were regulated by A. dhurica. The results from LC-MS/MS were further conformed by Western blot and real time PCR assays. A. dahurica could down-regulate the expression of catalase (CAT) and sterol carrier protein2 (SCP2) and up-regulate the expression of lipid metabolism related genes-lipase member C (LIPC) and peroxisome proliferator-activated receptor gamma (PPARγ). In the Triton WR1339 mouse liver and HepG2 cells in vitro, A. dahurica was able to increase the expression of LIPC and PPARγ, confirming the results from in vivo experiments. Imperatorin showed the same activity as A. dahurica, suggesting it was one of the major active ingredients of the herb. In conclusion, our work represented a first investigation demonstrating that A. dahurica was able to regulate lipid metabolism and could be developed as a novel approach to fighting against fatty liver and obesity.
Angelica ; chemistry ; Animals ; Carrier Proteins ; genetics ; metabolism ; Cholesterol ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Fatty Liver ; drug therapy ; genetics ; metabolism ; Humans ; Liver ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred ICR ; Obesity ; drug therapy ; genetics ; metabolism ; PPAR gamma ; genetics ; metabolism ; Triglycerides ; metabolism

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.
Animals ; Carrier Proteins/chemistry/genetics/*metabolism ; Disease Susceptibility ; GABAergic Neurons/*metabolism ; Gene Expression Regulation ; Humans ; Membrane Proteins/chemistry/genetics/*metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Processing, Post-Translational ; Protein Transport ; Synapses/*metabolism

Bone sialoprotein-binding protein (Bbp), a MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) family protein expressed on the surface of Staphylococcus aureus (S. aureus), mediates adherence to fibrinogen α (Fg α), a component in the extracellular matrix of the host cell and is important for infection and pathogenesis. In this study, we solved the crystal structures of apo-Bbp(273-598) and Bbp(273-598)-Fg α(561-575) complex at a resolution of 2.03 Å and 1.45 Å, respectively. Apo-Bbp(273-598) contained the ligand binding region N2 and N3 domains, both of which followed a DE variant IgG fold characterized by an additional D1 strand in N2 domain and D1' and D2' strands in N3 domain. The peptide mapped to the Fg α(561-575) bond to Bbp(273-598) on the open groove between the N2 and N3 domains. Strikingly, the disordered C-terminus in the apo-form reorganized into a highly-ordered loop and a β-strand G'' covering the ligand upon ligand binding. Bbp(Ala298-Gly301) in the N2 domain of the Bbp(273-598)-Fg α(561-575) complex, which is a loop in the apo-form, formed a short α-helix to interact tightly with the peptide. In addition, Bbp(Ser547-Gln561) in the N3 domain moved toward the binding groove to make contact directly with the peptide, while Bbp(Asp338-Gly355) and Bbp(Thr365-Tyr387) in N2 domain shifted their configurations to stabilize the reorganized C-terminus mainly through strong hydrogen bonds. Altogether, our results revealed the molecular basis for Bbp-ligand interaction and advanced our understanding of S. aureus infection process.
Bacterial Proteins ; chemistry ; genetics ; metabolism ; Carrier Proteins ; chemistry ; genetics ; metabolism ; Crystallography, X-Ray ; Fibrinogen ; metabolism ; Ligands ; Models, Molecular ; Mutation ; Peptide Fragments ; chemistry ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; Staphylococcus aureus

Allosteric Regulation ; Carrier Proteins ; chemistry ; genetics ; metabolism ; Cell Proliferation ; Gene Expression ; Glycolysis ; genetics ; Humans ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Mutation ; Neoplasms ; enzymology ; genetics ; pathology ; Oxidative Phosphorylation ; Protein Conformation ; Protein Multimerization ; Protein Subunits ; chemistry ; genetics ; metabolism ; Thyroid Hormones ; chemistry ; genetics ; metabolism ; Tumor Cells, Cultured

Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17-Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.
Alanine ; chemistry ; metabolism ; Amino Acid Motifs ; Aspartic Acid ; chemistry ; metabolism ; Autophagy ; genetics ; Autophagy-Related Proteins ; Carrier Proteins ; chemistry ; metabolism ; Gene Expression Regulation, Fungal ; Membrane Proteins ; chemistry ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Nitrogen ; deficiency ; Phagosomes ; chemistry ; drug effects ; metabolism ; Phosphorylation ; Protein Transport ; Saccharomyces cerevisiae ; drug effects ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; chemistry ; genetics ; metabolism ; Serine ; chemistry ; metabolism ; Signal Transduction ; Sirolimus ; pharmacology

Pyruvate kinase isoform M2 (PKM2) converts phosphoenolpyruvate (PEP) to pyruvate and plays an important role in cancer metabolism. Here, we show that post-translational modifications and a patient-derived mutation regulate pyruvate kinase activity of PKM2 through modulating the conformation of the PKM2 tetramer. We determined crystal structures of human PKM2 mutants and proposed a "seesaw" model to illustrate conformational changes between an inactive T-state and an active R-state tetramers of PKM2. Biochemical and structural analyses demonstrate that PKM2(Y105E) (phosphorylation mimic of Y105) decreases pyruvate kinase activity by inhibiting FBP (fructose 1,6-bisphosphate)-induced R-state formation, and PKM2(K305Q) (acetylation mimic of K305) abolishes the activity by hindering tetramer formation. K422R, a patient-derived mutation of PKM2, favors a stable, inactive T-state tetramer because of strong intermolecular interactions. Our study reveals the mechanism for dynamic regulation of PKM2 by post-translational modifications and a patient-derived mutation and provides a structural basis for further investigation of other modifications and mutations of PKM2 yet to be discovered.
Acetylation ; Allosteric Regulation ; Carrier Proteins ; chemistry ; genetics ; metabolism ; Crystallography, X-Ray ; Fructosediphosphates ; chemistry ; metabolism ; Gene Expression ; Humans ; Kinetics ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Models, Molecular ; Mutation ; Neoplasms ; enzymology ; genetics ; pathology ; Phosphorylation ; Protein Conformation ; Protein Multimerization ; Protein Processing, Post-Translational ; Protein Subunits ; chemistry ; genetics ; metabolism ; Thyroid Hormones ; chemistry ; genetics ; metabolism ; Tumor Cells, Cultured

In recent years, human cancer genome projects provide unprecedented opportunities for the discovery of cancer genes and signaling pathways that contribute to tumor development. While numerous gene mutations can be identified from each cancer genome, what these mutations mean for cancer is a challenging question to address, especially for those from less understood putative new cancer genes. As a powerful approach, in silico bioinformatics analysis could efficiently sort out mutations that are predicted to damage gene function. Such an analysis of human large tumor suppressor genes, LATS1 and LATS2, has been carried out and the results support a role of hLATS1//2 as negative growth regulators and tumor suppressors.
Adaptor Proteins, Signal Transducing ; chemistry ; metabolism ; Animals ; Carrier Proteins ; chemistry ; metabolism ; Computational Biology ; Genes, Neoplasm ; Humans ; LIM Domain Proteins ; chemistry ; metabolism ; Mice ; Mutation ; Neoplasms ; genetics ; pathology ; Phosphoproteins ; chemistry ; metabolism ; Phosphorylation ; Protein Binding ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases ; chemistry ; genetics ; metabolism ; Transferases (Other Substituted Phosphate Groups) ; chemistry ; metabolism ; Tumor Suppressor Proteins ; chemistry ; genetics ; metabolism

OBJECTIVE: To investigate the potential effect of NALP3 inflammasome on the occurrence and development of nonalcoholic steatohepatitis (NASH). METHODS: THP-1 macrophages were cultured for 24 h by palmitic acid at various concentrations. The THP-1 macrophages were pretreated with N-acetyl-cysteine at different doses for 24 h before the palmitic acid cultivation. ROS production was determined by flow cytometry. The expression of IL- 1β was detected by ELISA; the expressions of NALP3 protein and caspase-1 protein were detected by immunofluorescence; NALP3, ASC, and caspase-1 mRNA were measured by real-time PCR. RESULTS: Compared with the THP-1 macrophages without palmitic acid, the level of ROS, NALP3 protein and caspase-1 protein, and the expression of IL-1β were increased after palmitic acid treatment in a dose dependent manner (P<0.05). Compared with the THP-1 macrophages with palmitic acid (400 μmol/L), the level of NALP3 mRNA (P<0.05), the level of NALP3 protein and caspase-1 protein (P<0.05), the expression of IL-1β (P<0.05) were decreased after preadministration of N-acetyl-cysteine in a dose dependent manner. CONCLUSION ROS induced by free fatty acid can regulate the activation of NALP3 inflammasome signaling pathway leading to the release of inflammatory cytokines. This pathway may be the possible mechanism of NASH.
Carrier Proteins ; metabolism ; Caspase 1 ; metabolism ; Cell Line ; Fatty Acids, Nonesterified ; chemistry ; Humans ; Inflammasomes ; metabolism ; Interleukin-1beta ; metabolism ; Macrophages ; cytology ; NLR Family, Pyrin Domain-Containing 3 Protein ; Reactive Oxygen Species ; metabolism ; Real-Time Polymerase Chain Reaction ; Signal Transduction

Escherichia coli (E. coli) FadR regulator plays dual roles in fatty acid metabolism, which not only represses the fatty acid degradation (fad) system, but also activates the unsaturated fatty acid synthesis pathway. Earlier structural and biochemical studies of FadR protein have provided insights into interplay between FadR protein with its DNA target and/or ligand, while the missing knowledge gap (esp. residues with indirect roles in DNA binding) remains unclear. Here we report this case through deep mapping of old E. coli fadR mutants accumulated. Molecular dissection of E. coli K113 strain, a fadR mutant that can grow on decanoic acid (C10) as sole carbon sources unexpectedly revealed a single point mutation of T178G in fadR locus (W60G in FadRk113). We also observed that a single genetically-recessive mutation of W60G in FadR regulatory protein can lead to loss of its DNA-binding activity, and thereby impair all the regulatory roles in fatty acid metabolisms. Structural analyses of FadR protein indicated that the hydrophobic interaction amongst the three amino acids (W60, F74 and W75) is critical for its DNA-binding ability by maintaining the configuration of its neighboring two β-sheets. Further site-directed mutagenesis analyses demonstrated that the FadR mutants (F74G and/or W75G) do not exhibit the detected DNA-binding activity, validating above structural reasoning.
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase ; genetics ; metabolism ; Amino Acid Sequence ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; DNA, Bacterial ; chemistry ; metabolism ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; Fatty Acid Synthase, Type II ; genetics ; metabolism ; Fatty Acids ; metabolism ; Gene Expression Regulation, Bacterial ; Hydro-Lyases ; genetics ; metabolism ; Hydrophobic and Hydrophilic Interactions ; Lipid Metabolism ; Models, Molecular ; Molecular Sequence Data ; Mutation ; Protein Binding ; Protein Structure, Secondary ; Repressor Proteins ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Sequence Homology, Amino Acid ; Signal Transduction