ABSTRACT
Cu catalysts with different compositions and different Cu and promoter contents were prepared by precipitation-gel method and studied for the selective hydrogenation of syngas or biomass-based diethyl malonate (DEM) to valuable 1,3-propanediol (1,3-PDO). The Ga-promoted 70Cu6Ga/SiO2 catalyst was found to exhibit the highest catalytic performance, achieving 100 % DEM conversion and 76.6 % 1,3-PDO selectivity under reaction conditions of 160 °C and 8â MPa H2. The 70Cu6Ga/SiO2 bimetallic catalyst also presented obviously better stability than that of the monometallic 70Cu/SiO2 catalyst in a continuous flow reactor over 180â h time-on stream. Characterization results showed that the incorporation of Ga increased the interaction between Cu and Ga species, hindered the full reduction of Cu2+ species, and thus increased the proportion of Cu+ and the number of Lewis acidic sites on the catalyst surface. The synergistic effect between Cu0 and Cu+ enhanced the adsorption and activation of ester carbonyl groups and their subsequent hydrogenation, eventually contributed to the outstanding performances of the CuGa/SiO2 bimetallic catalysts.
ABSTRACT
Immunotherapy using monoclonal antibodies targeting the PD-1/PD-L1 interaction has shown enormous success for various cancers. Despite their encouraging results in clinics, antibody-based checkpoint inhibitors have several limitations, such as poor tumor penetration. To address these limitations of monoclonal antibodies, there is a growing interest in developing low-molecular-weight checkpoint inhibitors, such as antibody fragments. Several antibody fragments targeting PD-1/PD-L1 were recently discovered using phage libraries from camel or alpaca. However, animal-derived antibody fragments may elicit unwanted immune responses, which limit their therapeutic applications. For the first time, we used a human domain antibody phage library and discovered anti-human PD-L1 human single-domain antibodies (dAbs) that block the PD-1/PD-L1 interaction. Among them, the CLV3 dAb shows the highest affinity to PD-L1. The CLV3 dAb also exhibits the highest blocking efficacy of the PD-1/PD-L1 interaction. Moreover, the CLV3 dAb significantly inhibits tumor growth in mice implanted with CT26 colon carcinoma cells. These results suggest that CLV3 dAb can be potentially used as an anti-PD-L1 inhibitor for cancer immunotherapy.
Subject(s)
Antineoplastic Agents, Immunological , Colonic Neoplasms , Single-Domain Antibodies , Animals , Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents, Immunological/pharmacology , B7-H1 Antigen , Colonic Neoplasms/therapy , Humans , Immunotherapy/methods , Mice , Programmed Cell Death 1 ReceptorABSTRACT
The Tribbles (Trib) family of pseudokinase proteins regulate cell growth, proliferation, and differentiation during normal development and in response to environmental stress. Mutations in human Trib isoforms (Trib1, 2, and 3) have been associated with metabolic disease and linked to leukemia and the formation of solid tumors, including melanomas, hepatomas, and lung cancers. Drosophila Tribbles (Trbl) was the first identified member of this sub-family of pseudokinases and shares a conserved structure and similar functions to bind and direct the degradation of key mediators of cell growth and proliferation. Common Trib targets include Akt kinase (also known as protein kinase B), C/EBP (CAAT/enhancer binding protein) transcription factors, and Cdc25 phosphatases, leading to the notion that Trib family members stand athwart multiple pathways modulating their growth-promoting activities. Recent work using the Drosophila model has provided important insights into novel facets of conserved Tribbles functions in stem cell quiescence, tissue regeneration, metabolism connected to insulin signaling, and tumor formation linked to the Hippo signaling pathway. Here we highlight some of these recent studies and discuss their implications for understanding the complex roles Tribs play in cancers and disease pathologies.
ABSTRACT
Acid-sensing ion channel (ASIC) subunits 1a and 3 are highly expressed in central and peripheral sensory neurons, respectively. Endogenous biomolecule zinc plays a critical role in physiological and pathophysiological conditions. Here, we found that currents recorded from heterologously expressed ASIC1a/3 channels using the whole-cell patch-clamp technique were regulated by zinc with dual effects. Co-application of zinc dose-dependently potentiated both peak amplitude and the sustained component of heteromeric ASIC1a/3 currents; pretreatment with zinc between 3 to 100 µM exerted the same potentiation as co-application. However, pretreatment with zinc induced a significant inhibition of heteromeric ASIC1a/3 channels when zinc concentrations were over 250 µM. The potentiation of heteromeric ASIC1a/3 channels by zinc was pH dependent, as zinc shifted the pH dependence of ASIC1a/3 currents from a pH50 of 6.54 to 6.77; whereas the inhibition of ASIC1a/3 currents by zinc was also pH dependent. Furthermore, we systematically mutated histidine residues in the extracellular domain of ASIC1a or ASIC3 and found that histidine residues 72 and 73 in both ASIC1a and ASIC3, and histidine residue 83 in the ASIC3 were responsible for bidirectional effects on heteromeric ASIC1a/3 channels by zinc. These findings suggest that histidine residues in the extracellular domain of heteromeric ASIC1a/3 channels are critical for zinc-mediated effects.
Subject(s)
Acid Sensing Ion Channels/chemistry , Acid Sensing Ion Channels/physiology , Acid Sensing Ion Channels/genetics , Animals , CHO Cells , Cations/metabolism , Cations/pharmacology , Cricetulus , Electric Conductivity , Histidine/chemistry , Histidine/genetics , Hydrogen-Ion Concentration , Mutagenesis, Site-Directed , Patch-Clamp Techniques , Protein Structure, Quaternary/drug effects , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/physiology , Sequence Alignment , Zinc/metabolism , Zinc/pharmacologyABSTRACT
BACKGROUND: Whitmania pigra Whitman (W pigra), a traditional Chinese medicine, has functions of breaking stagnant and eliminating blood stasis. The aim of this study was to investigate the underlying mechanism of W pigra against deep vein thrombosis (DVT). METHODS: A rat model of DVT induced by inferior vena cava stenosis was successfully established. Rats were administered vehicle (saline solution, p.o.), three doses of W pigra aqueous extract (34.7, 104.2, or 312.5 mg crude W pigra/kg, p.o.), heparin (200 U/kg, i.v.), or clopidogrel (25 mg/kg, p.o.) once daily for 2 d. Thrombus weight and histopathological changes were examined. Blood samples were collected to determine blood cell counts, blood viscosity, blood coagulation, blood fibrinolysis, serum levels of interleukin-1ß, and tumor necrosis factor-α. Protein expressions of Sirtuin1 (SIRT1), acetylated p65 (Ace-p65), and phosphorylated p65 (p-p65) were determined by Western blot. Furthermore, SIRT1-specific inhibitor EX527 was applied to confirm the role of SIRT1 in the antithrombotic effect of W pigra. RESULTS: W pigra significantly decreased thrombus weight. W pigra had no effects on blood cell counts, whole blood viscosity, blood coagulation, blood fibrinolysis. However, it reduced tissue factor protein expression in the vein wall and thrombus. Moreover, it sharply increased SIRT1 protein expression and decreased leukocytes recruitment in the thrombus and vein wall, serum levels of interleukin-1ß and tumor necrosis factor-α, and protein expressions of Ace-p65 and p-p65. Furthermore, the antithrombotic effect of W pigra was significantly abolished by EX527. CONCLUSIONS: Aqueous extract of W pigra effectively reduced DVT burden by inhibiting inflammation via SIRT1/nuclear factor-kappa B signaling pathway.
Subject(s)
Biological Products/therapeutic use , Leeches , NF-kappa B/metabolism , Sirtuin 1/metabolism , Venous Thrombosis/drug therapy , Animals , Biological Products/pharmacology , Carbazoles , Cytokines/blood , Drug Evaluation, Preclinical , Female , Inflammation/drug therapy , Male , Medicine, Chinese Traditional , Rats, Sprague-Dawley , Signal Transduction/drug effects , Sirtuin 1/antagonists & inhibitors , Thromboplastin/metabolism , Venous Thrombosis/metabolismABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Danhong Huayu Koufuye (DHK), a compound traditional Chinese medicine, is composed of Salvia miltiorrhiza radix (Salvia miltiorrhiza Bge.), Angelicae Sinensis radix (Angelicae Sinensis (Oliv.) Diels.), Chuanxiong rhizoma (Ligusticum chuanxiong Hort.), Persicae semen (Prunus persica (L.) Batsch), Carthami flos (Carthamus tinctorius L.), Bupleuri radix (Bupleurum chinense DC.) and Aurantii fructus (Citrus aurantium L.). DHK prevents deep vein thrombosis (DVT) through antiinflammation. However, the antiinflammatory mechanism of DHK is still unknown. OBJECTIVE: The aim of this study was to evaluate whether DHK prevented venous thrombosis through antiinflammation via Sirtuin 1 (SIRT1)/NF-κB signaling pathway. METHODS: Inferior vena cava (IVC) stenosis-induced DVT rat model was established. Rats were administered with DHK (1.6, 3.2 or 6.4â¯mL/kg/d, p.o.), heparin (200 U/kg/d, i.v.), clopidogrel (25â¯mg/kg/d, p.o.), resveratrol (50â¯mg/kg/d, p.o.) or vehicle (p.o.) once daily for two days. Blood coagulation, blood fibrinolysis, blood viscosity, blood cell counts and platelet activity were evaluated. Serum levels of inflammatory cytokines were analyzed by enzyme-linked immunosorbent assay. Pathological changes were observed by hematoxylin-eosin (HE) staining. Protein expressions in thrombosed IVCs were evaluated by Western blot and/or immunofluorescence analyses. SIRT1 mRNA expression was analyzed by real-time quantitative polymerase chain reaction. Besides, SIRT1-specific inhibitor EX527 was pretreated to confirm the role of SIRT1/NF-κB signaling pathway in the antithrombotic effect of DHK. RESULTS: DHK remarkably prevented DVT. DHK had no effects on blood coagulation, blood fibrinolysis, blood viscosity, blood cell counts or platelet activity. But DHK significantly up-regulated protein and mRNA expressions of SIRT1, and reduced leukocytes infiltration into thrombus and vein wall, serum levels of inflammatory cytokines, and protein expressions of acetylated p65 (Ace-p65), phosphorylated p65 (p-p65) and tissue factor (TF). Moreover, the antithrombotic effect of DHK was significantly abolished by EX527. CONCLUSION: DHK may prevent DVT by inhibiting inflammation via SIRT1/NF-κB signaling pathway.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Drugs, Chinese Herbal/therapeutic use , Fibrinolytic Agents/therapeutic use , Venous Thrombosis/drug therapy , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Blood Cell Count , Blood Coagulation/drug effects , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Female , Fibrinolytic Agents/chemistry , Fibrinolytic Agents/pharmacology , Interleukin-1beta/blood , Male , Phytochemicals/analysis , Phytochemicals/pharmacology , Phytochemicals/therapeutic use , Platelet Activation/drug effects , Rats, Sprague-Dawley , Signal Transduction/drug effects , Sirtuin 1/genetics , Sirtuin 1/metabolism , Tumor Necrosis Factor-alpha/blood , Venous Thrombosis/blood , Venous Thrombosis/genetics , Venous Thrombosis/metabolismABSTRACT
BACKGROUND: Inflammation plays an important role in thrombus formation, and Sirtuin 1 (SIRT1) negatively regulates inflammation via deacetylating nuclear factor-kappa B. However, the relationship between SIRT1-regulated inflammation and deep vein thrombosis (DVT) is still unknown. OBJECTIVE: The aim of this study was to investigate whether SIRT1 plays a critical role in inferior vena cava (IVC) stenosis-induced DVT. MATERIALS AND METHODS: Thrombus weight and histopathologic analysis of IVC were evaluated at different time points after IVC stenosis in rats. Serum levels of inflammatory cytokines and protein expressions of SIRT1, acetylated p65 (Ace-p65), phosphorylated p65 (p-p65) and tissue factor (TF) in thrombosed IVC were assessed. Besides, the effects of resveratrol (RES, a SIRT1 agonist) and EX527 (a selective SIRT1 inhibitor) on DVT were evaluated. RESULTS: Thrombus weight was increased from 1 to 3 days after IVC stenosis, and then was decreased afterwards. Leukocytes infiltration appeared and serum levels of cytokines were significantly increased in rats of IVC stenosis. SIRT1 protein expression was significantly down-regulated at 1 hour and 1 day after stenosis, while p-p65, Ace-p65 and TF protein expressions appeared a contrary trend. RES reduced thrombus weight, leukocytes infiltration, levels of tumour necrosis factor-α and interleukin-1ß and protein expressions of Ace-p65 and TF as well. Moreover, RES significantly increased the protein and messenger ribonucleic acid expressions of SIRT1, while EX527 abolished the protective effects of RES. CONCLUSION: SIRT1 activation attenuated IVC stenosis-induced DVT via anti-inflammation in rats. Therefore, SIRT1 may be a potential therapeutic target that could ameliorate DVT.
Subject(s)
Inflammation Mediators/metabolism , Inflammation/enzymology , Signal Transduction , Sirtuin 1/metabolism , Transcription Factor RelA/metabolism , Vena Cava, Inferior/enzymology , Venous Thrombosis/enzymology , Acetylation , Animals , Anti-Inflammatory Agents/pharmacology , Carbazoles/pharmacology , Disease Models, Animal , Female , Fibrinolytic Agents/pharmacology , Histone Deacetylase Inhibitors/pharmacology , Inflammation/immunology , Inflammation/pathology , Inflammation/prevention & control , Inflammation Mediators/immunology , Male , Phosphorylation , Rats, Sprague-Dawley , Resveratrol/pharmacology , Signal Transduction/drug effects , Sirtuin 1/antagonists & inhibitors , Time Factors , Vena Cava, Inferior/drug effects , Vena Cava, Inferior/immunology , Vena Cava, Inferior/pathology , Venous Thrombosis/immunology , Venous Thrombosis/pathology , Venous Thrombosis/prevention & controlABSTRACT
It has been shown that oxidative damage and inflammation caused by hyperglycemia in endothelial cells are key factors triggering diabetic vascular complications. The aim of the present study was to investigate the antioxidant and antiinflammatory effects of Danhong Huayu Koufuye (DHK)medicated serum on high glucose (HG)induced injury in endothelial cells, and examine its underlying mechanisms. EA. hy926 cells were treated with normal glucose, HG, or HG with DHKmedicated serum. Cell viability was assessed using the MTT method. Apoptosis was detected using flow cytometry. Intracellular reactive oxygen species (ROS) levels were measured using the 2',7'dichlorodihydrofluorescein method. Cell culture supernatant was collected for detecting the activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD), and the levels of malondialdehyde (MDA). The protein expression levels of intercellular adhesion molecule1 (ICAM1), nuclear factorκB (NFκB), hypoxiainducible factor1α (HIF1α) and vascular endothelial growth factor (VEGF) were determined using western blot analysis. The results revealed that DHKmedicated serum accelerated the proliferation and inhibited the apoptosis of cells treated with HG (P<0.01) in a dosedependent manner. Compared with the HG group, the high levels of ROS and MDA were significantly reduced by DHKmedicated serum (P<0.01). A 10% concentration of DHKmedicated serum increased the activities of SOD and GPx by 59.4 and 95.5%, respectively. The high protein expression levels of ICAM1, NFκB, VEGF and HIF1α were significantly ameliorated by DHKmedicated serum (P<0.01, vs. HG group). These findings indicated that DHKmedicated serum protected EA. hy926 cells from HGinduced injury and apoptosis through antioxidation and antiinflammatory effects.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Antioxidants/pharmacology , Drugs, Chinese Herbal/pharmacology , Glucose/antagonists & inhibitors , Human Umbilical Vein Endothelial Cells/drug effects , Reactive Oxygen Species/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Survival/drug effects , Gene Expression Regulation , Glucose/toxicity , Glutathione Peroxidase/genetics , Glutathione Peroxidase/metabolism , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Male , Malondialdehyde/antagonists & inhibitors , Malondialdehyde/metabolism , Medicine, Chinese Traditional , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , NF-kappa B/metabolism , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Serum/chemistry , Signal Transduction , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
De novo synthesis of the sphingolipid sphingomyelin requires non-vesicular transport of ceramide from the endoplasmic reticulum to the Golgi by the multidomain protein ceramide transfer protein (CERT). CERT's N-terminal pleckstrin homology (PH) domain targets it to the Golgi by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane, whereas its C-terminal StAR-related lipid transfer domain (START) carries out ceramide transfer. Hyperphosphorylation of a serine-rich motif immediately after the PH domain decreases both PtdIns(4)P binding and ceramide transfer by CERT. This down-regulation requires both the PH and START domains, suggesting a possible inhibitory interaction between the two domains. In this study we show that isolated PH and START domains interact with each other. The crystal structure of a PH-START complex revealed that the START domain binds to the PH domain at the same site for PtdIns(4)P-binding, suggesting that the START domain competes with PtdIns(4)P for association with the PH domain. We further report that mutations disrupting the PH-START interaction increase both PtdIns(4)P-binding affinity and ceramide transfer activity of a CERT-serine-rich phosphorylation mimic. We also found that these mutations increase the Golgi localization of CERT inside the cell, consistent with enhanced PtdIns(4)P binding of the mutant. Collectively, our structural, biochemical, and cellular investigations provide important structural insight into the regulation of CERT function and localization.
Subject(s)
Ceramides/metabolism , Golgi Apparatus/enzymology , Models, Molecular , Phosphatidylinositol Phosphates/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acid Substitution , Binding Sites , Binding, Competitive , Biological Transport , Crystallography, X-Ray , Fluorescence Resonance Energy Transfer , Golgi Apparatus/metabolism , HEK293 Cells , Humans , Microscopy, Fluorescence , Mutagenesis, Site-Directed , Mutation , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphatidylinositol Phosphates/chemistry , Protein Interaction Domains and Motifs , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Transport , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
Danhong Huayu Koufuye (DHK), a traditional Chinese prescription, is used to treat central retinal vein occlusion clinically. We previously reported that DHK prevented diabetic retinopathy (DR) in rats. Moreover, we found that it protected endothelial cells from hyperglycemia-induced apoptosis through antioxidation and anti-inflammation. Here, we investigated whether antioxidative and anti-inflammatory activities of DHK contributed to its therapeutic effect on DR in streptozotocin- (STZ-) induced diabetic rats. DHK significantly blocked the breakdown of the blood-retinal barrier (BRB) and increased the thickness of the inner nuclear layer (INL), as well as suppressed the swelling of the ganglion cell layer (GCL) in diabetic retinas. DHK remarkably increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in plasma, and decreased serum level of nitric oxide (NO). Moreover, DHK markedly reduced the serum levels of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1). Furthermore, DHK significantly downregulated protein expressions of VEGF and inducible NO synthase (iNOS) and mRNA expression of ICAM-1 in retinas. These results suggest that the antioxidative and anti-inflammatory activities of DHK may be important mechanisms involved in the protective effect of DHK on DR in STZ-induced diabetic rats.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/metabolism , Diabetic Retinopathy/drug therapy , Diabetic Retinopathy/metabolism , Drugs, Chinese Herbal/therapeutic use , Animals , Antioxidants/metabolism , Blood-Retinal Barrier/drug effects , Blood-Retinal Barrier/metabolism , Glutathione Peroxidase/metabolism , Intercellular Adhesion Molecule-1/metabolism , Male , Rats , Rats, Sprague-Dawley , Retina/drug effects , Retina/metabolism , Superoxide Dismutase/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
It was recently demonstrated that MCPIP1 is a critical factor that controls inflammation and immune homeostasis; however, the relationship between MCPIP1 and other members of this protein family is largely unknown. Here, we report that MCPIP1 interacts with MCPIP4 to form a protein complex, but acts independently in the regulation of IL-6 mRNA degradation. In an effort to identify MCPIP1-interacting proteins by co-immunoprecipitation (Co-IP) and mass-spec analysis, MCPIP4 was identified as a MCPIP1-interacting protein, which was further confirmed by Co-IP and mammalian two-hybrid assay. Immunofluorescence staining showed that MCPIP4 was co-localized with MCPIP1 in the GW-body, which features GW182 and Argonaute 2. Further studies showed that MCPIP1 and MCPIP4 act independently in regulation of IL-6 mRNA degradation. These results suggest that MCPIP1 and MCPIP4 may additively contribute to control IL-6 production in vivo.
Subject(s)
Interleukin-6/metabolism , Macrophages/metabolism , Proteins/metabolism , RNA Stability/genetics , RNA, Messenger/metabolism , Ribonucleases/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , COS Cells , Cell Cycle Proteins , Cell Line , Chlorocebus aethiops , Endonucleases , Endoribonucleases , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Humans , Immunoprecipitation , Interleukin-6/genetics , Macrophages/cytology , Mice , Molecular Sequence Data , Plasmids/chemistry , Plasmids/metabolism , Protein Binding , Proteins/genetics , RNA, Messenger/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ribonucleases/genetics , Signal Transduction , Transcription Factors/genetics , Two-Hybrid System TechniquesABSTRACT
The complement system plays a central role in a number of human inflammatory diseases, and there is a significant need for development of complement-directed therapies. The discovery of an arsenal of anti-complement proteins secreted by the pathogen Staphylococcus aureus brought with it the potential for harnessing the powerful inhibitory properties of these molecules. One such family of inhibitors, the SCINs, interact with a functional "hot-spot" on the surface of C3b. SCINs not only stabilize an inactive form of the alternative pathway (AP) C3 convertase (C3bBb), but also overlap the C3b binding site of complement factors B and H. Here we determined that a conserved Arg residue in SCINs is critical for function of full-length SCIN proteins. Despite this, we also found SCIN-specific differences in the contributions of other residues found at the C3b contact site, which suggested that a more diverse repertoire of residues might be able to recognize this region of C3b. To investigate this possibility, we conducted a phage display screen aimed at identifying SCIN-competitive 12-mer peptides. In total, seven unique sequences were identified and all exhibited direct C3b binding. A subset of these specifically inhibited the AP in assays of complement function. The mechanism of AP inhibition by these peptides was probed through surface plasmon resonance approaches, which revealed that six of the seven peptides disrupted C3bBb formation by interfering with factor B/C3b binding. To our knowledge this study has identified the first small molecules that retain inhibitory properties of larger staphylococcal immune evasion proteins.
Subject(s)
Bacterial Proteins/metabolism , Complement Pathway, Alternative/drug effects , Peptides/pharmacology , Staphylococcus aureus/metabolism , Animals , Arginine/metabolism , Binding, Competitive/drug effects , Complement C3 Convertase, Alternative Pathway , Complement C3b/metabolism , Complement Factor B/metabolism , Conserved Sequence , Humans , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Peptide Library , Peptides/chemistry , Protein Binding/drug effects , Protein Structure, Secondary , Rabbits , Solutions , Surface Plasmon ResonanceABSTRACT
Alternative splicing and proteolytic processing of VEGFs generate proteins with distinct physiological roles. In this issue of Structure, Parker et al. show that proteolysis of an isoform of the VEGF-C coreceptor Nrp2 produces a soluble receptor that inhibits VEGF-C/Nrp2 interactions.
Subject(s)
Neuropilin-2/chemistry , Vascular Endothelial Growth Factor C/chemistry , HumansABSTRACT
Ceramide transfer protein (CERT) is responsible for the nonvesicular trafficking of ceramide from the endoplasmic reticulum (ER) to the trans Golgi network where it is converted to sphingomyelin (SM). The N-terminal pleckstrin homology (PH) domain is required for Golgi targeting of CERT by recognizing the phosphatidylinositol 4-phosphate (PtdIns(4)P) enriched in the Golgi membrane. We report a crystal structure of the CERT PH domain. This structure contains a sulfate that is hydrogen bonded with residues in the canonical ligand-binding pocket of PH domains. Our nuclear magnetic resonance (NMR) chemical shift perturbation (CSP) analyses show sulfate association with CERT PH protein resembles that of PtdIns(4)P, suggesting that the sulfate bound structure likely mimics the holo form of CERT PH protein. Comparison of the sulfate bound structure with the apo form solution structure shows structural rearrangements likely occur upon ligand binding, suggesting conformational flexibility in the ligand-binding pocket. This structural flexibility likely explains CERT PH domain's low affinity for PtdIns(4)P, a property that is distinct from many other PH domains that bind to their phosphoinositide ligands tightly. This unique structural feature of CERT PH domain is probably tailored towards the transfer activity of CERT protein where it needs to shuttle between ER and Golgi and therefore requires short resident time on ER and Golgi membranes.
Subject(s)
Blood Proteins/chemistry , Phosphoproteins/chemistry , Protein Serine-Threonine Kinases/metabolism , Blood Proteins/metabolism , Crystallography, X-Ray , Humans , Magnetic Resonance Spectroscopy , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Structure, SecondaryABSTRACT
Norovirus protease is an essential enzyme for proteolytic maturation of norovirus nonstructural proteins and has been implicated as a potential target for antiviral drug development. Although X-ray structural studies of the protease give us wealth of structural information including interactions of the protease with its substrate and dimeric overall structure, the role of protein dynamics in the substrate recognition and the biological relevance of the protease dimer remain unclear. Here we determined the solution NMR structure of the 3C-like protease from Norwalk virus (NV 3CLpro), a prototype strain of norovirus, and analyzed its backbone dynamics and hydrodynamic behavior in solution. ¹5N spin relaxation and analytical ultracentrifugation analyses demonstrate that NV 3CLpro is predominantly a monomer in solution. Solution structure of NV 3CLpro shows significant structural variation in C-terminal domain compared with crystal structures and among lower energy structure ensembles. Also, ¹5N spin relaxation and Carr-Purcell-Meiboom-Gill (CPMG)-based relaxation dispersion analyses reveal the dynamic properties of residues in the C-terminal domain over a wide range of timescales. In particular, the long loop spanning residues T123-G133 show fast motion (ps-ns), and the residues in the bII-cII region forming the large hydrophobic pocket (S2 site) undergo conformational exchanges on slower timescales (µs-ms), suggesting their important role in substrate recognition.
Subject(s)
Cysteine Endopeptidases/chemistry , Norovirus/enzymology , Viral Proteins/chemistry , Binding Sites , Biocatalysis , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Dimerization , Hydrodynamics , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Solubility , Substrate Specificity , Ultracentrifugation , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Genetically encoded protein photosensors are promising tools for engineering optical control of cellular behavior; we are only beginning to understand how to couple these light detectors to effectors of choice. Here we report a method that increases the dynamic range of an artificial photoswitch based on the LOV2 domain of Avena sativa phototropin 1 (AsLOV2). This approach can potentially be used to improve many AsLOV2-based photoswitches.
Subject(s)
Light , Photochemistry/methods , Phototropins/chemistry , Protein Engineering/methods , Avena , Phototropins/radiation effects , Protein Structure, Tertiary , Proteins/radiation effectsABSTRACT
Protein photosensors are versatile tools for studying ligand-regulated allostery and signaling. Fundamental to these processes is the amount of energy that can be provided by a photosensor to control downstream signaling events. Such regulation is exemplified by the phototropins--plant serine/threonine kinases that are activated by blue light via conserved LOV (light, oxygen and voltage) domains. The core photosensor of oat phototropin 1 is a LOV domain that interacts in a light-dependent fashion with an adjacent alpha-helix (J alpha) to control kinase activity. We used solution NMR measurements to quantify the free energy of the LOV domain-J alpha-helix binding equilibrium in the dark and lit states. These data indicate that light shifts this equilibrium by approximately 3.8 kcal mol(-1), thus quantifying the energy available through LOV-J alpha for light-driven allosteric regulation. This study provides insight into the energetics of light sensing by phototropins and benchmark values for engineering photoswitchable systems based on the LOV-J alpha interaction.
Subject(s)
Allosteric Regulation/radiation effects , Flavoproteins/chemistry , Flavoproteins/radiation effects , Light , Thermodynamics , Cryptochromes , Enzyme Activation/radiation effects , Oxygen , Plant Proteins , Protein Serine-Threonine Kinases , Protein Structure, Tertiary/radiation effectsABSTRACT
One of the main mechanisms of membrane protein folding is by spontaneous insertion into the lipid bilayer from the aqueous environment. The bacterial toxin, colicin Ia, is one such protein. To shed light on the conformational changes involved in this dramatic transfer from the polar to the hydrophobic milieu, we carried out 2D magic-angle spinning (13)C NMR experiments on the water-soluble and membrane-bound states of the channel-forming domain of colicin Ia. Proton-driven (13)C spin diffusion spectra of selectively (13)C-labeled protein show unequivocal attenuation of cross-peaks after membrane binding. This attenuation can be assigned to distance increases but not reduction of the diffusion coefficient. Analysis of the statistics of the interhelical and intrahelical (13)C-(13)C distances in the soluble protein structure indicates that the observed cross-peak reduction is well correlated with a high percentage of short interhelical contacts in the soluble protein. This suggests that colicin Ia channel domain becomes open and extended upon membrane binding, thus lengthening interhelical distances. In comparison, cross-peaks with similar intensities between the two states are dominated by intrahelical contacts in the soluble state. This suggests that the membrane-bound structure of colicin Ia channel domain may be described as a "molten globule", in which the helical secondary structure is retained while the tertiary structure is unfolded. This study demonstrates that (13)C spin diffusion NMR is a valuable tool for obtaining qualitative long-range distance constraints on membrane protein folding.
Subject(s)
Colicins/chemistry , Ion Channels/chemistry , Escherichia coli/chemistry , Membrane Proteins/chemistry , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Conformation , Protein Folding , Protein Structure, TertiaryABSTRACT
We describe a new experiment for measuring homonuclear-decoupled anisotropic chemical shift patterns in doubly 13C-labeled compounds under magic-angle spinning. The experiment combines a pair of selective and non-selective 180 degrees pulses to suppress the 13C-13C scalar and dipolar interactions. This is combined with the recently developed SUPER technique to recouple the chemical shift anisotropy. Demonstrations on 13Calpha and 13CO-labeled amino acids and peptides show that accurate chemical shift powder patterns can be obtained. This permits the use of chemical shift anisotropy for conformational studies of suitably extensively 13C-labeled peptides and proteins.
Subject(s)
Carbon Isotopes/chemistry , Magnetic Resonance Spectroscopy , Peptides/chemistry , Anisotropy , Magnetic Resonance Spectroscopy/methods , Models, MolecularABSTRACT
The C(alpha) chemical shift tensors of proteins contain information on the backbone conformation. We have determined the magnitude and orientation of the C(alpha) chemical shift tensors of two peptides with a-helical torsion angles: the Ala residue in G*AL (phi = -65.7 degrees, psi = -40 degrees), and the Val residue in GG*V (phi = -81.5 degrees, psi = -50.7 degrees). The magnitude of the tensors was determined from quasi-static powder patterns recoupled under magic-angle spinning, while the orientation of the tensors was extracted from C(alpha)-H(alpha) and C(alpha)-N dipolar modulated powder patterns. The helical Ala C(alpha) chemical shift tensor has a span of 36 ppm and an asymmetry parameter of 0.89. Its sigma11 axis is 116 degrees +/- 5 degrees from the C(alpha)-H(alpha) bond while the sigma22 axis is 40 degrees +/- 5 degrees from the C(alpha)-N bond. The Val tensor has an anisotropic span of 25 ppm and an asymmetry parameter of 0.33, both much smaller than the values for beta-sheet Val found recently (Yao and Hong, 2002). The Val sigma33 axis is tilted by 115 degrees +/- 50 from the Ca-Halpha bond and 98 degrees +/- 5 degrees from the C(alpha)-N bond. These represent the first completely experimentally determined C(alpha) chemical shift tensors of helical peptides. Using an icosahedral representation, we compared the experimental chemical shift tensors with quantum chemical calculations and found overall good agreement. These solid-state chemical shift tensors confirm the observation from cross-correlated relaxation experiments that the projection of the C(alpha) chemical shift tensor onto the C(alpha)-H(alpha) bond is much smaller in alpha-helices than in beta-sheets.