ABSTRACT
The inhibition of protein-protein interactions (PPIs) is an effective approach for therapy. Owing to their large binding surface areas to target proteins, macrocyclic peptides are suitable molecules for PPI inhibition. In this study, we developed single-chain tandem macrocyclic peptides (STaMPtides) that inhibits the vascular endothelial growth factor (VEGF) receptorâ 2 (VEGFR2). They were artificially designed to comprise two different VEGFR2-binding macrocyclic peptides linked in tandem by peptide linkers and secreted by Corynebacterium glutamicum. Most potent VEGFR2-inhibitory STaMPtides with length-optimized linkers exhibited >1000â times stronger inhibitory activity than their parental monomeric peptides, possibly due to the avidity effect of heterodimerization. Our approach of using STaMPtides for PPI inhibition may be used to inhibit other extracellular factors, such as growth factors and cytokines.
Subject(s)
Peptides , Vascular Endothelial Growth Factor A , Peptides/chemistry , Intercellular Signaling Peptides and ProteinsABSTRACT
Mimetics of growth factors and cytokines are promising tools for culturing large numbers of cells and manufacturing regenerative medicine products. In this study, we report single-chain tandem macrocyclic peptides (STaMPtides) as mimetics in a new multivalent peptide format. STaMPtides, which contain two or more macrocyclic peptides with a disulfide-closed backbone and peptide linkers, are successfully secreted into the supernatant by Corynebacterium glutamicum-based secretion technology. Without post-secretion modification steps, such as macrocyclization or enzymatic treatment, bacterially secreted STaMPtides form disulfide bonds, as designed; are biologically active; and show agonistic activities against respective target receptors. We also demonstrate, by cell-based assays, the potential of STaMPtides, which mimic growth factors and cytokines, in cell culture. The STaMPtide technology can be applied to the design, screening, and production of growth factor and cytokine mimetics.
Subject(s)
Biomimetics , Corynebacterium glutamicum , Cytokines/metabolism , Drug Design , Intercellular Signaling Peptides and Proteins/metabolism , Peptides/metabolismABSTRACT
Autism spectrum disorder (ASD) is characterized by persistent deficits in social communication and social interactions, as well as restricted, stereotyped patterns of behavior and interests. In addition, alterations in circadian sleep-wake rhythm are common in young children with ASD. Mutations in ATP binding cassette subfamily A member 13 (ABCA13) have been recently identified in a monkey that displays behavior associated with ASD. ABCA13, a member of the ABCA family of proteins, is predicted to transport lipid molecules and is expressed in the human trachea, testis, bone marrow, hippocampus, cortex, and other tissues. However, its physiological function remains unknown. Drosophila CG1718 shows high homology to human ABCA genes including ABCA13 and is thus designated as Drosophila ABCA (dABCA). To elucidate the physiological role of dABCA, we specifically knocked down dABCA in all neurons of flies and investigated their phenotypes. The pan-neuron-specific knockdown of dABCA resulted in increased social space with the closest neighbor in adult male flies but exerted no effect on their climbing ability, indicating that the increase in social space is not due to a defect in their climbing ability. An activity assay with adult male flies revealed that knockdown of dABCA in all neurons induces early onset of evening activity in adult flies followed by relatively high activity during morning peaks, evening peaks, and midday siesta. These phenotypes are similar to defects observed in human ASD patients, suggesting that the established dABCA knockdown flies are a promising model for ASD. In addition, an increase in satellite boutons in presynaptic terminals of motor neurons was observed in dABCA knockdown third instar larvae, suggesting that dABCA regulates the formation and/or maintenance of presynaptic terminals of motor neurons.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Autism Spectrum Disorder/genetics , Disease Models, Animal , Gene Targeting/methods , Locomotion/genetics , ATP-Binding Cassette Transporters/antagonists & inhibitors , Amino Acid Sequence , Animals , Animals, Genetically Modified , Circadian Rhythm/genetics , Drosophila , Gene Knockdown Techniques/methods , Humans , MaleABSTRACT
Moloney murine leukemia virus reverse transcriptase (MMLV RT) contains fingers, palm, thumb, and connection subdomains as well as an RNase H domain. The DNA polymerase active site resides in the palm subdomain, and the RNase H active site is located in the RNase H domain. The RNase H domain contains a positively charged α-helix called the C helix (H(594)GEIYRRR(601)), that is thought to be involved in substrate recognition. In this study, we expressed three versions of the RNase H domain in Escherichia coli, the wild-type domain (WT) (residues Ile498-Leu671) and two variants that lack the regions containing the C helix (Ile593-Leu603 and Gly595-Thr605, which we called ΔC1 and ΔC2, respectively) with a strep-tag at the N-terminus and a deca-histidine tag at the C-terminus. These peptides were purified from the cells by anion-exchange, Ni(2+) affinity, and Strep-Tactin affinity column chromatography, and then the tags were removed by proteolysis. In an RNase H assay using a 25-bp RNA-DNA heteroduplex, WT, ΔC1, and ΔC2 produced RNA fragments ranging from 7 to 16 nucleotides (nt) whereas the full-length MMLV RT (Thr24-Leu671) produced 14-20-nt RNA fragments, suggesting that elimination of the fingers, palm, thumb, and connection subdomains affects the binding of the RNase H domain to the RNA-DNA heteroduplex. The activity levels of WT, ΔC1, and ΔC2 were estimated to be 1%, 0.01%, and 0.01% of full-length MMLV RT activity, indicating that the C helix is important, but not critical, for the activity of the isolated RNase H domain.
Subject(s)
Moloney murine leukemia virus/genetics , RNA-Directed DNA Polymerase/metabolism , Recombinant Proteins/metabolism , Ribonuclease H/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Base Sequence , Escherichia coli/genetics , Molecular Sequence Data , Moloney murine leukemia virus/enzymology , RNA/metabolism , RNA-Directed DNA Polymerase/chemistry , RNA-Directed DNA Polymerase/genetics , RNA-Directed DNA Polymerase/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Ribonuclease H/chemistry , Ribonuclease H/genetics , Ribonuclease H/isolation & purification , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/isolation & purificationABSTRACT
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) possesses two distinct enzymatic activities: those of RNA- and DNA-dependent DNA polymerases and RNase H. In the current HIV-1 therapy, all HIV-1 RT inhibitors inhibit the activity of DNA polymerase, but not that of RNase H. We previously reported that ethanol and water extracts of Brasenia schreberi (Junsai) inhibited the DNA polymerase activity of HIV-1 RT [Hisayoshi et al. (2014) J Biol Macromol 14:59-65]. In this study, we screened 43 edible plants and found that ethanol and water extracts of Brasenia schreberi and water extract of Petasites japonicus strongly inhibit not only the activity of DNA polymerase to incorporate dTTP into poly(rA)-p(dT)15 but also the activity of RNase H to hydrolyze the RNA strand of an RNA/DNA hybrid. In addition, these three extracts inhibit HIV-1 replication in human cells, with EC50 values of 1-2 µg/ml. These results suggest that Brasenia schreberi and Petasites japonicus contain substances that block HIV-1 replication by inhibiting the DNA polymerase activity and/or RNase H activity of HIV-1 RT.
Subject(s)
Anti-HIV Agents/chemistry , HIV Reverse Transcriptase/antagonists & inhibitors , HIV-1/physiology , Petasites/chemistry , Plant Extracts/chemistry , Reverse Transcriptase Inhibitors/chemistry , Ribonuclease H/antagonists & inhibitors , Anti-HIV Agents/pharmacology , DNA-Directed DNA Polymerase/chemistry , Drug Evaluation, Preclinical , HIV Reverse Transcriptase/chemistry , HIV-1/drug effects , HIV-1/enzymology , HeLa Cells , Humans , Plant Extracts/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , Virus Replication/drug effectsABSTRACT
We have previously used site-directed mutagenesis to introduce basic residues (i.e., Arg; Lys) in the nucleic acid binding cleft of the Moloney murine leukemia virus reverse transcriptase (MMLV RT) in order to increase its template-primer (T/P) binding affinity. Three stabilizing mutations (i.e., E286R, E302K, and L435R) were identified (Yasukawa et al., 2010). Now, we studied the mechanism by which those mutations increase the thermal stability of the RT. The three single-mutants (E286R, E302K, and L435R), an RNase H-deficient MMLV RT (carrying the RNase H-inactivating mutation D524A), a quadruple mutant (E286R/E302K/L435R/D524A, designated as MM4) and the wild-type enzyme (WT) were produced in Escherichia coli. All RTs exhibited similar dissociation constants (Kd) for heteropolymeric DNA/DNA (2.9-6.5 nM) and RNA/DNA complexes (1.2-2.9 nM). Unlike the WT, mutant enzymes (E286R, E302K, L435R, D524A, and MM4) were devoid of RNase H activity, and were not able to degrade RNA in RNA/DNA complexes. These results suggest that the mutations, E286R, E302K, and L435R increase the thermostability of MMLV RT not by increasing its affinity for T/P but by abolishing its RNase H activity.
Subject(s)
Amino Acid Substitution , Moloney murine leukemia virus/enzymology , RNA-Directed DNA Polymerase/genetics , RNA-Directed DNA Polymerase/metabolism , Ribonuclease H/metabolism , Base Sequence , Catalytic Domain , DNA/analysis , DNA/metabolism , Escherichia coli/genetics , Moloney murine leukemia virus/chemistry , Moloney murine leukemia virus/genetics , Mutagenesis, Site-Directed , Protein Stability , RNA/analysis , RNA/metabolism , RNA-Directed DNA Polymerase/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TemperatureABSTRACT
After thermal incubation at 48 °C for 10 min, single variants of Moloney murine leukemia virus reverse transcriptase, V433R and V433K in which a surface hydrophobic residue, Val433, was mutated, retained 55% of initial reverse transcription activity, while the wild-type enzyme retained 17%. After thermal incubation at 50 °C for 10 min, multiple variants D108R/E286R/V433R and D108R/E286R/V433R/D524A, in which Val433âArg was combined with stabilizing mutations we identified previously, Asp108âArg and Glu286âArg, and RNase H activity-eliminating mutation Asp524âAla, retained 70% of initial activity, exhibiting higher stability than V433R or V433K.
Subject(s)
Moloney murine leukemia virus/enzymology , Mutagenesis, Site-Directed , RNA-Directed DNA Polymerase/chemistry , RNA-Directed DNA Polymerase/genetics , Valine , Enzyme Stability/genetics , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Mutation , Protein Conformation , TemperatureABSTRACT
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer containing 66 kDa p66 and 51 kDa p51 subunits. We previously showed that HIV-1 group M (HIV-1 M) RT and HIV-1 group O (HIV-1 O) RT have higher affinities for dTTP and template-primer (T/P) than Moloney murine leukemia virus RT, which is currently used for cDNA synthesis, suggesting that they might also be useful for cDNA synthesis (Konishi et al. Appl Biochem Biotechnol 2013, 169:77-87). Here, we have increased the thermostability of both HIV-1 M RT and HIV-1 O RT by site-directed mutagenesis. The Asp443 â Ala mutation, which abolishes RNase H activity, was introduced into the p66 subunits of HIV-1 M RT and HIV-1 O RT. The temperatures that reduced the initial activity by 50 % of the resulting mutants, HIV-1 M p66D443A/p51 and HIV-1 O p66D443A/p51, were 44 and 52 °C, respectively, which were higher than those of wild-type HIV-1 M p66/p51 (42 °C) and HIV-1 O p66/p51 (48 °C). The highest temperature at which both HIV-1 M p66D443A/p51 and HIV-1 O p66D443A/p51 exhibited cDNA synthesis activity was 68 °C, which was higher than for the wild-type enzymes (62 and 66 °C, respectively).
Subject(s)
Enzyme Stability/genetics , HIV Reverse Transcriptase/chemistry , HIV Reverse Transcriptase/genetics , Mutagenesis, Site-Directed/methods , Amino Acid Sequence , Base Sequence , Electrophoresis, Polyacrylamide Gel , HIV Reverse Transcriptase/metabolism , Hot Temperature , Kinetics , Molecular Sequence Data , Mutation/genetics , RNA, Viral/analysis , RNA, Viral/genetics , Sequence Analysis, DNAABSTRACT
The aim of this study is to explore the advantages of using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in cDNA synthesis. Recombinant HIV-1 group M (HIV-1 M) RT and HIV-1 group O (HIV-1 O) RT were produced in an Escherichia coli expression system. In the incorporation of dTTP into poly(rA)-p(dT)(15) (T/P), the K (m) values for dTTP of HIV-1 M RT and HIV-1 O RT were 8 and 12 % of that of Moloney murine leukemia virus (MMLV) RT, respectively, and the K (m) values for T/P were 25 and 23 % of that of MMLV RT, respectively. Compared with MMLV RT, HIV-1 M RT and HIV-1 O RT were less susceptible to formamide, which is frequently used for cDNA synthesis with a G + C-rich RNA to improve specificity. The high substrate affinity and low susceptibility to formamide of HIV-1 RT might be advantageous for its use in cDNA synthesis.
Subject(s)
DNA, Complementary/metabolism , HIV Reverse Transcriptase/chemistry , HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , DNA, Complementary/genetics , HIV Reverse Transcriptase/genetics , Humans , Kinetics , Nucleotides/metabolism , Substrate SpecificityABSTRACT
Some bacterial thermostable, wild-type or genetically engineered family A DNA polymerases have reverse transcriptase activity. However, difference in reverse transcriptase activities of family A DNA polymerases and retroviral reverse transcriptases (RTs) is unclear. In this study, comparative kinetic analysis was performed for the reverse transcriptase activities of the wild-type enzyme of family A DNA polymerase (M1pol(WT)) from Thermus thermophilus M1 and the variant enzyme of family A DNA polymerase (K4pol(L329A)), in which the mutation of Leu329âAla is undertaken, from Thermotoga petrophila K4. In the incorporation of dTTP into poly(rA)-p(dT)(45), the reaction rates of K4pol(L329A) and M1pol(WT) exhibited a saturated profile of the Michaelis-Menten kinetics for dTTP concentrations but a substrate inhibition profile for poly(rA)-p(dT)(45) concentrations. In contrast, the reaction rates of Moloney murine leukemia virus (MMLV) RT exhibited saturated profiles for both dTTP and poly(rA)-p(dT)(45) concentrations. This suggests that high concentrations of DNA-primed RNA template decrease the efficiency of cDNA synthesis with bacterial family A DNA polymerases.
Subject(s)
DNA-Directed DNA Polymerase/chemistry , RNA-Directed DNA Polymerase/chemistry , Thermus thermophilus/enzymology , DNA-Directed DNA Polymerase/genetics , Kinetics , Moloney murine leukemia virus/enzymology , Mutation , RNA-Directed DNA Polymerase/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Thymine Nucleotides/chemistryABSTRACT
Avian myeloblastosis virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63 kDa α-subunit and a 95 kDa ß subunit. Moloney murine leukaemia virus reverse transcriptase (MMLV RT) is a 75 kDa monomer. These two RTs are the most extensively used for conversion of RNA to DNA. We previously developed several mutations that increase the thermostability of MMLV RT and generated a highly stable MMLV RT variant E286R/E302K/L435R/D524A by combining three of them (Glu286âArg, Glu302âLys, and Leu435âArg) and the mutation to abolish RNase H activity (Asp524âAla) [Yasukawa et al. (2010) J Biotechnol 150:299-306]. To generate a highly stable AMV RT variant, we have introduced the triple mutation of Val238âArg, Leu388âArg, and Asp450âAla into AMV RT α-subunit and the resulted variant V238R/L388R/D450A, was expressed in insect cells and purified. The temperature decreasing the initial activity by 50 %, measured over 10 min, of the variant with or without template primer (T/P), poly(rA)-p(dT)(15), was 50 °C; for the wild-type AMV RT α-subunit (WT) this was 44 °C. The highest temperature at which the variant exhibited cDNA synthesis activity was 64 °C; the WT was 60 °C. A highly stable AMV RT α-subunit is therefore generated by the same mutation strategy as applied to MMLV RT and that positive charges are introduced into RT at positions that have been implicated to interact with T/P by site-directed mutagenesis.
Subject(s)
Avian Myeloblastosis Virus/enzymology , RNA-Directed DNA Polymerase/metabolism , Amino Acid Substitution , Animals , Avian Myeloblastosis Virus/genetics , Cell Line , Enzyme Stability/radiation effects , Gene Expression , Hot Temperature , Insecta , Mutagenesis, Site-Directed , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Stability/radiation effects , Protein Subunits/genetics , Protein Subunits/metabolism , RNA-Directed DNA Polymerase/geneticsABSTRACT
Avian myeloblastosis virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63-kDa α subunit and a 95-kDa ß subunit. In this study, we explored the role of the interaction between the α and ß subunits on AMV RT stability. The recombinant AMV RT α subunit was expressed in insect cells and purified. It exhibited lower thermal stability than the native AMV RT αß heterodimer. Unlike the αß heterodimer, the α subunit was not stabilized by template-primer. These results suggest that interaction between the α and ß subunits is important for AMV RT stability.
Subject(s)
Avian Myeloblastosis Virus/chemistry , Protein Binding/genetics , Protein Subunits/chemistry , RNA-Directed DNA Polymerase/chemistry , Recombinant Proteins/chemistry , Animals , Avian Leukosis/virology , Avian Myeloblastosis Virus/genetics , Avian Myeloblastosis Virus/metabolism , Baculoviridae , Base Sequence , Birds , Cell Line , Cloning, Molecular , Dimerization , Enzyme Stability , Molecular Sequence Data , Plasmids , Protein Subunits/genetics , Protein Subunits/metabolism , RNA, Viral/chemistry , RNA, Viral/genetics , RNA-Directed DNA Polymerase/genetics , RNA-Directed DNA Polymerase/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spodoptera , Transfection , Transformation, BacterialABSTRACT
We hypothesized that the thermal stability of Moloney murine leukaemia virus reverse transcriptase (MMLV RT) will increase with increases in its ability to bind with a template-primer (T/P). To test this hypothesis, we introduced positive charges into MMLV RT by site-directed mutagenesis at positions that have been implicated in the interaction with T/P. Thirty-six variants were constructed in which one of the twelve residues (Glu69, Gln84, Asp108, Asp114, Glu117, Glu123, Asp124, Glu286, Glu302, Trp313, Leu435, and Asn454) was replaced with Lys, Arg, or Ala, and these were expressed in Escherichia coli. In about half of these 36 variants, thermal inactivation at 50°C was reduced in the presence of the T/P, which suggested that this strategy was effective at stabilizing MMLV RT. We next combined three of the 36 mutations, Glu286âAla, Glu302âLys, and Leu435âArg, and the mutation, Asp524âAla, which is known to abolish the RNase H activity and increase the stability. Temperatures of 54 and 56°C reduced the initial reverse transcription activity by 50% over a 10-min incubation in the triple variant E286R/E302K/L435R and quadruple variant E286R/E302K/L435R/D524A, respectively. These temperatures were higher than that observed for WT (45°C). The highest temperature at which the triple and quadruple variants exhibited cDNA synthesis activity was 60°C, which was again higher than for WT (54°C). Thus, highly stable MMLV RT variants were generated by this mutation strategy.
Subject(s)
Moloney murine leukemia virus/enzymology , Mutagenesis, Site-Directed/methods , RNA-Directed DNA Polymerase/chemistry , DNA, Complementary/metabolism , Electrophoresis, Polyacrylamide Gel , Hot Temperature , Kinetics , Models, Molecular , Moloney murine leukemia virus/genetics , Mutation/genetics , Protein Stability , Protein Structure, Tertiary , RNA-Directed DNA Polymerase/genetics , RNA-Directed DNA Polymerase/metabolismABSTRACT
The use of certain organic chemicals has been found to improve yields and specificity in PCR. In this study, we examined the effects of dimethyl sulfoxide (DMSO), formamide, and glycerol on the reverse transcription reaction catalyzed by reverse transcriptases (RTs) from avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MMLV). At 42 °C, DMSO at 24% v/v and formamide at 12-14% inhibited the cDNA synthesis reaction, but DMSO at 12% and formamide at 6-8% improved the efficiency of the cDNA synthesis reaction at low temperatures (25-34 °C). Glycerol at 10% improved the efficiency of the cDNA synthesis reaction at high temperatures (49-61 °C). The effects of DMSO and formamide appeared to be accompanied by decreases in the melting temperatures of the primers, and the effect of glycerol was due to increases in the thermal stabilities of AMV RT and MMLV RT.
Subject(s)
Avian Myeloblastosis Virus/enzymology , Moloney murine leukemia virus/enzymology , Organic Chemicals/pharmacology , Polymerase Chain Reaction/methods , RNA-Directed DNA Polymerase/metabolism , Reverse Transcription/drug effects , Solvents/pharmacology , Catalysis , Dimethyl Sulfoxide/pharmacology , Formamides/pharmacology , Glycerol/pharmacology , RNA-Directed DNA Polymerase/drug effectsABSTRACT
In vascular smooth muscle cells, Axl is a key receptor tyrosine kinase, because it is upregulated in injury, increases migration and neointima formation, and is activated by reactive oxygen species. Reaction of glutathione with cysteine residues (termed "glutathiolation") is an important posttranslational redox modification that may alter protein activity and protein-protein interactions. To investigate the mechanisms by which reactive oxygen species increase Axl-dependent vascular smooth muscle cell function we assayed for glutathiolated proteins that associated with Axl in a redox-dependent manner. We identified glutathiolated nonmuscle myosin heavy chain (MHC)-IIB as a novel Axl interacting protein. This interaction was specific in that other myosins did not interact with Axl. The endogenous ligand for Axl, Gas6, increased production of reactive oxygen species in vascular smooth muscle cells and also increased the association of Axl with MHC-IIB. Antioxidants ebselen and N-acetylcysteine decreased the association of Axl with MHC-IIB in response to both Gas6 and reactive oxygen species. Blocking the Axl-MHC-IIB interaction with the specific myosin II inhibitor blebbistatin decreased phosphorylation of Axl and activation of extracellular signal-regulated kinase 1/2 and Akt. Association of MHC-IIB with Axl was increased in balloon-injured rat carotid vessels. Finally, expression of MHC-IIB was upregulated in the neointima of the carotid artery after balloon injury similar to upregulation of Axl protein expression, as shown in our previous studies. These results demonstrate a novel interaction between Axl and MHC-IIB in response to reactive oxygen species. This interaction provides a direct link between Axl and molecular motors crucial for directed cell migration, which may mediate increased migration in vascular dysfunction.
Subject(s)
Carotid Artery Diseases/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Muscle, Smooth, Vascular/metabolism , Nonmuscle Myosin Type IIB/metabolism , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Animals , Blotting, Western , Carotid Artery Diseases/pathology , Cells, Cultured , Disease Models, Animal , Immunohistochemistry , Immunoprecipitation , Male , Muscle, Smooth, Vascular/pathology , Oxidation-Reduction , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Axl Receptor Tyrosine KinaseABSTRACT
The administration of L-isoleucine (isoleucine) has been shown to induce hypoglycemia in normal rats. However, it remains to be elucidated whether isoleucine can improve the blood glucose level in glucose-intolerant or diabetic animals. In the present study, oral isoleucine significantly reduced the blood glucose level after an oral glucose challenge in normal mice, as well as in glucose-intolerant mice fed a high-fat diet (HFD) and db/db mice, a model of severe type 2 diabetes. Isoleucine treatment significantly augmented the blood insulin level after an oral glucose load in HFD mice, but not in normal or db/db mice, suggesting that its hypoglycemic activity was attributable to both insulinotropic and non-insulinotropic mechanisms. Chronic supplementation of isoleucine in mice on a high-fat/high-sucrose diet significantly reduced insulin release after an oral glucose challenge without any change in glucose tolerance curve, suggesting that isoleucine might have an insulin-sensitizing effect along with its acute hypoglycemic effect. These results indicate that both acute and chronic treatment with isoleucine is beneficial for glucose metabolism in glucose-intolerant and diabetic animals.
Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Type 2/drug therapy , Glucose/metabolism , Hypoglycemic Agents/therapeutic use , Isoleucine/therapeutic use , Animals , Blood Glucose/analysis , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/metabolism , Dietary Fats/administration & dosage , Drug Administration Schedule , Female , Glucose Tolerance Test , Hypoglycemic Agents/administration & dosage , Insulin/blood , Isoleucine/administration & dosage , Male , Mice , Mice, Inbred C57BL , Sucrose/administration & dosageABSTRACT
OBJECTIVE: The receptor tyrosine kinase Axl and its ligand Gas6 are involved in the development of renal diabetic disease. In vascular smooth muscle cells (VSMCs) Axl is activated by reactive oxygen species and stimulates migration and cell survival, suggesting a role for Axl in the vascular complications of diabetes. METHODS AND RESULTS: We investigated the effect of varying glucose concentration on Axl signaling in VSMCs. Glucose exerted powerful effects on Gas6-Axl signaling with greater activation of Akt and mTOR in low glucose, and greater activation of ERK1/2 in high glucose. Plasma membrane distribution and tyrosine phosphorylation of Axl were not affected by glucose. However, coimmunoprecipitation studies demonstrated that glucose changed the interaction of Axl with its binding partners. Specifically, binding of Axl to the p85 subunit of PI3-kinase was increased in low glucose, whereas binding to SHP-2 was increased in high glucose. Furthermore, Gas6-Axl induced migration was increased in high glucose, whereas Gas6-Axl mediated inhibition of apoptosis was greater in low glucose. CONCLUSIONS: This study demonstrates a role for glucose in altering Axl signaling through coupling to binding partners and suggests a mechanism by which Axl contributes to VSMC dysfunction in diabetes.
Subject(s)
Glucose/physiology , Intercellular Signaling Peptides and Proteins/metabolism , Muscle, Smooth, Vascular/metabolism , Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Muscle, Smooth, Vascular/pathology , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinases/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Proto-Oncogene Proteins , Proto-Oncogene Proteins c-akt/metabolism , Rats , Signal Transduction/physiology , TOR Serine-Threonine Kinases , Axl Receptor Tyrosine KinaseABSTRACT
Axl, a receptor tyrosine kinase, is involved in cell survival, proliferation, and migration. We have shown that Axl expression increases in the neointima of balloon-injured rat carotids. Because oxidative stress is known to play a major role in remodeling of injured vessels, we hypothesized that H(2)O(2) might activate Axl by promoting autophosphorylation. H(2)O(2) rapidly stimulated Axl tyrosine phosphorylation in rat vascular smooth muscle cells within 1 min that was maximal at 5 min (6-fold). The response to H(2)O(2) was concentration-dependent with EC(50) of approximately 500 microm. Axl phosphorylation was partly dependent on production of its endogenous ligand, growth arrest gene 6 (Gas6), because Axl-Fc, a fragment of Axl extracellular domain that neutralizes Gas6, inhibited H(2)O(2)-induced Axl phosphorylation by 50%. Axl phosphorylation by H(2)O(2) was also attenuated by warfarin, which inhibits Gas6 activity by preventing post-translational modification. In intact vessels Axl was phosphorylated by H(2)O(2), and Axl phosphorylation was inhibited by warfarin treatment in balloon-injured carotids. Akt, a downstream target of Axl, was phosphorylated by H(2)O(2)in Axl(+/+) mouse aorta but significantly inhibited in Axl(-/-) aorta. Intimal proliferation was decreased significantly in a cuff injury model in Axl(-/-) mice compared with Axl(+/+) mice. In summary, Axl is an important signaling mediator for oxidative stress in cultured vascular smooth muscle cells and intact vessels and may represent an important therapeutic target for vascular remodeling and response to injury.
Subject(s)
Hydrogen Peroxide/pharmacology , Intercellular Signaling Peptides and Proteins/metabolism , Muscle, Smooth, Vascular/cytology , Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Animals , Blotting, Western , Cell Division , Cell Movement , Cells, Cultured , Dose-Response Relationship, Drug , Femoral Artery/pathology , Hydrogen Peroxide/metabolism , Immunoblotting , Ligands , Male , Mice , Mice, Transgenic , Oxidative Stress , Phosphorylation , Precipitin Tests , Proto-Oncogene Proteins , Rats , Rats, Sprague-Dawley , Time Factors , Tyrosine/chemistry , Tyrosine/metabolism , Vascular Endothelial Growth Factor A/metabolism , Warfarin/pharmacology , Axl Receptor Tyrosine KinaseABSTRACT
We hypothesized that glucose-mediated alterations in vascular smooth muscle cell signal transduction contribute to diabetic complications. We found enhanced AngII activation of Akt and extracellular ERK1/2 in vascular smooth muscle cells incubated with high glucose (27.5 mM) compared with low glucose (5.5 mM). Because AngII-mediated transactivation of the epidermal growth factor receptor (EGFR) is important in Akt and ERK1/2 activation, we studied the effects of glucose on EGFR function. The EGFR in cells cultured for 48 h in low glucose was smaller (145 kDa) than the EGFR in cells cultured with high glucose (170 kDa). The shift from the 170-kDa isoform to the 145-kDa isoform was reversible and dependent upon glucose concentration with EC50 approximately 1 mM. N-Glycosylation was responsible because peptide N-glycosidase F treatment of isolated 170-kDa EGFR yielded a single band at 145 kDa. Cell surface biotinylation showed that the 145-kDa EGFR was present on plasma membrane. AngII and other G-protein-coupled receptor ligands known to transactivate EGFR phosphorylated the 170-kDa EGFR but not the 145-kDa EGFR, whereas EGF, heparin-binding EGF-like growth factor, and transforming growth factor-alpha phosphorylated both receptors. Subcellular fractionation showed that the 145-kDa receptor localized to a different plasma membrane domain than the 170-kDa receptor. These results establish a novel mechanism by which glucose-dependent EGFR N-glycosylation modulates AngII signal transduction and suggest a potential mechanism for pathogenic effects of AngII in diabetic vasculopathy.
Subject(s)
ErbB Receptors/genetics , Glucose/pharmacology , Muscle, Smooth, Vascular/physiology , Protein Serine-Threonine Kinases , Transcriptional Activation/drug effects , Angiotensin II/pharmacology , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/physiology , Glycosylation , Kinetics , Male , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Models, Biological , Molecular Weight , Muscle, Smooth, Vascular/drug effects , Phosphorylation , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Rats , Rats, Sprague-Dawley , Transcriptional Activation/geneticsABSTRACT
BACKGROUND: APC0576, 5-(((S)-2,2-dimethylcyclopropanecarbonyl)amino)-2-(4-(((S)-2,2-dimethylcyclopropanecarbonyl)amino)phenoxy)pyridine is a novel synthetic compound with an inhibitory activity on NF-kappaB-dependent gene activation and chemokine synthesis in human endothelial cells. This article describes the effect of APC0576 on T-cell-dependent immune functions in vitro and in vivo in primate models, because NF-kappaB is known to be one of the potent mediators in T-cell activation. METHODS: The effects of APC0576 on interleukin-2 production and proliferative responses in human peripheral blood mononuclear cells were studied under various stimuli with in vitro culture assay. Next, female rhesus monkeys were immunized with tetanus toxoid (TTx), and APC0576 was orally administered for 4 weeks. Serum-specific antibody for TTx was monitored weekly using an enzyme-linked immunosorbent assay, and delayed-type hypersensitivity reaction was examined after 4 weeks of APC0576 treatment. To evaluate the immunosuppressive activity, APC0576 was orally administered for 32 days to rhesus monkeys that received transplants of allogeneic kidney. RESULTS: APC0576 effectively suppressed interleukin-2 production and proliferation in activated human peripheral blood mononuclear cells. Both delayed-type hypersensitivity reaction and specific antibody formation evoked by TTx was significantly and dose-dependently attenuated by 4 weeks treatment of APC0576 without any serious toxicologic signs. Allogeneic kidneys grafted in rhesus monkeys were not rejected and fully functioned during the 32 days of APC0576 treatment, although they were rapidly rejected after the withdrawal of the drug. CONCLUSIONS: A novel, orally available immunosuppressive agent, APC0576, effectively inhibited T-cell-based immune responses both in vitro and in vivo. APC0576 may have potential for a therapeutic agent in clinical organ transplantation and various cytokine-mediated diseases.