ABSTRACT
The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants.
Subject(s)
Seedlings/metabolism , Tryptophan/metabolism , Amino Acids, Dicarboxylic/metabolism , Arabidopsis/metabolism , Cyclohexenes/metabolism , Phenylalanine/metabolism , Shikimic Acid/metabolism , Tyrosine/analogs & derivatives , Tyrosine/metabolismABSTRACT
Phenylacetic acid (PAA) is one type of natural auxin and widely exists in plants. Previous biochemical studies demonstrate that PAA in plants is synthesized from phenylalanine (Phe) via phenylpyruvate (PPA), but the PAA biosynthetic genes and its regulation remain unknown. In this article, we show that the AROGENATE DEHYDRATASE (ADT) family, which catalyzes the conversion of arogenate to Phe, can modulate the levels of PAA in Arabidopsis. We found that overexpression of ADT4 or ADT5 remarkably increased the amounts of PAA. Due to an increase in PAA levels, ADT4ox and ADT5ox plants can partially restore the auxin-deficient phenotypes caused by treatments with an inhibitor of the biosynthesis of indole-3-acetic acid (IAA), a main auxin in plants. In contrast, the levels of PAA were significantly reduced in adt multiple knockout mutants. Moreover, the levels of PPA are substantially increased in ADT4 or ADT5 overexpression plants but reduced in adt multiple knockout mutants, suggesting that PPA is a key intermediate of PAA biosynthesis. These results provide an evidence that members of the ADT family of Arabidopsis can modulate PAA level via the PPA-dependent pathway.
Subject(s)
Arabidopsis/genetics , Arabidopsis/metabolism , Hydro-Lyases/genetics , Hydro-Lyases/metabolism , Phenylacetates/metabolism , Amino Acids, Dicarboxylic/metabolism , Cyclohexenes/metabolism , Gene Expression Regulation, Plant , Gene Knockdown Techniques , Indoleacetic Acids/metabolism , Mutation , Phenylalanine/metabolism , Plants, Genetically Modified , Tyrosine/analogs & derivatives , Tyrosine/metabolismABSTRACT
BACKGROUND/AIMS: Hypoxia Inducible Factor-1α (HIF-1α) is involved in cancer progression and is stabilized by the chaperone HSP90 (Heat Shock Protein 90), preventing degradation. Previously identified HSP90 inhibitors bind to the N-terminal pocket of HSP90, which blocks binding to HIF-1α and induces HIF-1α degradation. N-terminal inhibitors have failed in the clinic as single therapy treatments partially because they induce a heat shock response. SM molecules are HSP90 inhibitors that bind to the C-terminus of HSP90 and do not induce a heat shock response. The effects of these C-terminal inhibitors on HIF-1α are unreported. METHODS: HCT116, MDA-MB-231, PC3, and HEK293T cells were treated with HSP90 inhibitors. qRT-PCR and western blotting was performed to assess mRNA and protein levels of HIF-1α, HSP- and RACK1-related genes. siRNA was used to knockdown RACK1, while MG262 was used to inhibit proteasome activity. Dimethyloxalylglycine (DMOG) was used to inhibit activity of the prolyl hydroxylases (PHDs). Anti-angiogenic activity of HSP90 inhibitors was assessed using a HUVEC tubule formation assay. RESULTS: We show that SM compounds decrease HIF-1α target expression at the mRNA and protein level under hypoxia in colorectal, breast and prostate cancer cells, leading to cell death, without inducing a heat shock response. Surprisingly, we found that when the C-terminal of HSP90 is inhibited, HIF-1α degradation occurs through the proteasome and prolyl hydroxylases in an oxygen-dependent manner even in very low levels of oxygen (tumor hypoxia levels). RACK1 was not required for proteasomal degradation of HIF-1α. CONCLUSION: Our results suggest that by targeting the C-terminus of HSP90 we can exploit the prolyl hydroxylase and proteasome pathway to induce HIF-1α degradation in hypoxic tumors.
Subject(s)
Cell Hypoxia/physiology , HSP90 Heat-Shock Proteins/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Amino Acids, Dicarboxylic/metabolism , Blotting, Western , Cell Hypoxia/genetics , Cell Survival/genetics , Cell Survival/physiology , HCT116 Cells , HEK293 Cells , HSP90 Heat-Shock Proteins/genetics , Human Umbilical Vein Endothelial Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , PC-3 Cells , Prolyl Hydroxylases/genetics , Prolyl Hydroxylases/metabolism , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismSubject(s)
Amino Acids, Dicarboxylic/chemistry , Angiogenesis Inducing Agents/chemistry , Coated Materials, Biocompatible/chemistry , Nanostructures/chemistry , Prostheses and Implants , Zeolites/chemistry , Amino Acids, Dicarboxylic/metabolism , Angiogenesis Inducing Agents/metabolism , Cell Adhesion/drug effects , Cell Line , Cell Proliferation/drug effects , Coated Materials, Biocompatible/metabolism , Endopeptidases/genetics , Endopeptidases/metabolism , Gene Expression Regulation/drug effects , Histones/genetics , Histones/metabolism , Human Umbilical Vein Endothelial Cells/cytology , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Molecular Structure , Osteogenesis , Surface Properties , Titanium/chemistry , Titanium/metabolism , Zeolites/metabolism , Zinc/metabolismABSTRACT
In addition to being a vital component of proteins, phenylalanine is also a precursor of numerous aromatic primary and secondary metabolites with broad physiological functions. In plants phenylalanine is synthesized predominantly via the arogenate pathway in plastids. Here, we describe the structure, molecular players and subcellular localization of a microbial-like phenylpyruvate pathway for phenylalanine biosynthesis in plants. Using a reverse genetic approach and metabolic flux analysis, we provide evidence that the cytosolic chorismate mutase is responsible for directing carbon flux towards cytosolic phenylalanine production via the phenylpyruvate pathway. We also show that an alternative transcription start site of a known plastidial enzyme produces a functional cytosolic prephenate dehydratase that catalyzes the conversion of prephenate to phenylpyruvate, the intermediate step between chorismate mutase and phenylpyruvate aminotransferase. Thus, our results complete elucidation of phenylalanine biosynthesis via phenylpyruvate in plants, showing that this pathway splits from the known plastidial arogenate pathway at chorismate, instead of prephenate as previously thought, and the complete pathway is localized in the cytosol.
Subject(s)
Biosynthetic Pathways , Chorismate Mutase/metabolism , Phenylalanine/metabolism , Phenylpyruvic Acids/metabolism , Plants/metabolism , Amino Acids, Dicarboxylic/metabolism , Cyclohexanecarboxylic Acids/metabolism , Cyclohexenes/metabolism , Cytosol/metabolism , Plants/genetics , Plastids/genetics , Plastids/metabolism , Prephenate Dehydratase/genetics , Prephenate Dehydratase/metabolism , Transaminases/metabolism , Transcription Initiation Site , Tyrosine/analogs & derivatives , Tyrosine/metabolismABSTRACT
Here we describe a simple method based on secreted luciferase driven by a hypoxia-inducible factor (HIF) response element (HRE) that allows the acquisition of dynamic and high-throughput data on HIF transcriptional activity during hypoxia and pharmacological activation of HIF. The sensitivity of the assay allows for the secreted luciferase to be consecutively sampled (as little as 1% of the total supernatant) over an extended time period, thus allowing the acquisition of time-resolved HIF transcriptional activity.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Luciferases/genetics , Response Elements , Amino Acids, Dicarboxylic/metabolism , Cell Hypoxia , HEK293 Cells , Humans , Promoter Regions, Genetic , Recombinant Proteins , Transcriptional ActivationABSTRACT
Differentiation of regulatory Treg (Treg) in the periphery is critical to control inflammatory processes. Although polarization of inducible Treg (iTreg) often occurs in an inflammatory environment, the effects exerted by inflammation on human iTreg differentiation have not been extensively studied. We observed that IL-1ß significantly reduced the frequency of FOXP3+ T cells under iTreg-polarizing conditions. Mechanistically, we show that IL-1ß activated mTORC1 and downstream upregulated hypoxia inducible factor-1 (HIF-1α) expression. Using specific inhibitors, we demonstrated that both steps were critical in the deleterious effect of IL-1ß on Treg differentiation. Chemical stabilization of HIF-1α by Dimethyloxalylglycine (DMOG) also significantly impaired iTreg differentiation. Interestingly, while IL-1ß-treated cells exhibited only minor changes in metabolism, DMOG treatment decreased iTreg mitochondrial respiration and increased their glycolytic capacity. In conclusion, exposure to inflammatory stimuli profoundly inhibits human Treg differentiation HIF-1α dependent, suggesting that targeting HIF-1α could be a strategy to foster iTreg differentiation in an inflammatory milieu. However, IL-1ß deleterious effect does not appear to be completely driven by metabolic changes. These data thus suggest that several mechanisms contribute to the regulation of iTreg differentiation, but the timing and respective requirement for each pathway vary depending on the milieu in which iTreg differentiate.
Subject(s)
Cell Differentiation , Forkhead Transcription Factors/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Interleukin-1beta/metabolism , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/metabolism , Amino Acids, Dicarboxylic/metabolism , Biomarkers , Cell Differentiation/drug effects , Cell Respiration , Gene Expression , Glycolysis , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Immunophenotyping , Mitochondria/genetics , Mitochondria/metabolism , Phenotype , T-Lymphocytes, Regulatory/immunology , TOR Serine-Threonine Kinases/metabolismABSTRACT
BACKGROUND: Endothelial colony-forming cells (ECFCs), also termed late outgrowth endothelial cells, are a well-defined circulating endothelial progenitor cell type with an established role in vascular repair. ECFCs have clear potential for cell therapy to treat ischaemic disease, although the precise mechanism(s) underlying their response to hypoxia remains ill-defined. METHODS: In this study, we isolated ECFCs from umbilical cord blood and cultured them on collagen. We defined the response of ECFCs to 1% O2 exposure at acute and chronic time points. RESULTS: In response to low oxygen, changes in ECFC cell shape, proliferation, size and cytoskeleton phenotype were detected. An increase in the number of senescent ECFCs also occurred as a result of long-term culture in 1% O2. Low oxygen exposure altered ECFC migration and tube formation in Matrigel®. Increases in angiogenic factors secreted from ECFCs exposed to hypoxia were also detected, in particular, after treatment with placental growth factor (PlGF). Exposure of cells to agents that stabilise hypoxia-inducible factors such as dimethyloxalylglycine (DMOG) also increased PlGF levels. Conditioned medium from both hypoxia-treated and DMOG-treated cells inhibited ECFC tube formation. This effect was reversed by the addition of PlGF neutralising antibody to the conditioned medium, confirming the direct role of PlGF in this effect. CONCLUSIONS: This study deepens our understanding of the response of ECFCs to hypoxia and also identifies a novel and important role for PlGF in regulating the vasculogenic potential of ECFCs.
Subject(s)
Endothelial Cells/metabolism , Endothelial Cells/physiology , Hypoxia/metabolism , Hypoxia/pathology , Placenta Growth Factor/metabolism , Placental Hormones/metabolism , Amino Acids, Dicarboxylic/metabolism , Cell Movement/physiology , Cell Proliferation/physiology , Cells, Cultured , Collagen/metabolism , Culture Media, Conditioned/metabolism , Drug Combinations , Endothelial Progenitor Cells/metabolism , Fetal Blood/metabolism , Fetal Blood/physiology , Humans , Laminin/metabolism , Neovascularization, Physiologic/physiology , Proteoglycans/metabolismABSTRACT
l-Phenylalanine serves as a building block for the biosynthesis of proteins, but also as a precursor for a wide range of plant-derived compounds essential for plants and animals. Plants can synthesize Phe within the plastids using arogenate as a precursor; however, an alternative pathway using phenylpyruvate as an intermediate, described for most microorganisms, has recently been proposed. The functionality of this pathway requires the existence of enzymes with prephenate dehydratase (PDT) activity (EC 4.2.1.51) in plants. Using phylogenetic studies, functional complementation assays in yeast and biochemical analysis, we have identified the enzymes displaying PDT activity in Pinus pinaster. Through sequence alignment comparisons and site-directed mutagenesis we have identified a 22-amino acid region conferring PDT activity (PAC domain) and a single Ala314 residue critical to trigger this activity. Our results demonstrate that all plant clades include PAC domain-containing ADTs, suggesting that the PDT activity, and thus the ability to synthesize Phe using phenylpyruvate as an intermediate, has been preserved throughout the evolution of plants. Moreover, this pathway together with the arogenate pathway gives plants a broad and versatile capacity to synthesize Phe and its derived compounds. PAC domain-containing enzymes are also present in green and red algae, and glaucophytes, the three emerging clades following the primary endosymbiont event resulting in the acquisition of plastids in eukaryotes. The evolutionary prokaryotic origin of this domain is discussed.
Subject(s)
Pinus/genetics , Prephenate Dehydratase/genetics , Amino Acids, Dicarboxylic/metabolism , Cyclohexenes/metabolism , Genes, Plant/physiology , Metabolic Networks and Pathways/physiology , Phenylalanine/metabolism , Phenylpyruvic Acids/metabolism , Phylogeny , Pinus/enzymology , Pinus/metabolism , Plants , Prephenate Dehydratase/metabolism , Tyrosine/analogs & derivatives , Tyrosine/metabolismABSTRACT
OBJECTIVES: This study examines the relationship between synovial hypoxia and cellular bioenergetics with synovial inflammation. METHODS: Primary rheumatoid arthritis synovial fibroblasts (RASF) were cultured with hypoxia, dimethyloxalylglycine (DMOG) or metabolic intermediates. Mitochondrial respiration, mitochondrial DNA mutations, cell invasion, cytokines, glucose and lactate were quantified using specific functional assays. RASF metabolism was assessed by the XF24-Flux Analyzer. Mitochondrial structural morphology was assessed by transmission electron microscopy (TEM). In vivo synovial tissue oxygen (tpO2 mmHg) was measured in patients with inflammatory arthritis (n=42) at arthroscopy, and markers of glycolysis/oxidative phosphorylation (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), PKM2, GLUT1, ATP) were quantified by immunohistology. A subgroup of patients underwent contiguous MRI and positron emission tomography (PET)/CT imaging. RASF and human dermal microvascular endothelial cells (HMVEC) migration/angiogenesis, transcriptional activation (HIF1α, pSTAT3, Notch1-IC) and cytokines were examined in the presence of glycolytic inhibitor 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). RESULTS: DMOG significantly increased mtDNA mutations, mitochondrial membrane potential, mitochondrial mass, reactive oxygen species and glycolytic RASF activity with concomitant attenuation of mitochondrial respiration and ATP activity (all p<0.01). This was coupled with altered mitochondrial morphology. Hypoxia-induced lactate levels (p<0.01), which in turn induced basic fibroblast growth factor (bFGF) secretion and RASF invasiveness (all p<0.05). In vivo glycolytic markers were inversely associated with synovial tpO2 levels <20â mmâ Hg, in contrast ATP was significantly reduced (all p<0.05). Decrease in GAPDH and GLUT1 was paralleled by an increase in in vivo tpO2 in tumour necrosis factor alpha inhibitor (TNFi) responders. Novel PET/MRI hybrid imaging demonstrated close association between metabolic activity and inflammation. 3PO significantly inhibited RASF invasion/migration, angiogenic tube formation, secretion of proinflammatory mediators (all p<0.05), and activation of HIF1α, pSTAT3 and Notch-1IC under normoxic and hypoxic conditions. CONCLUSIONS: Hypoxia alters cellular bioenergetics by inducing mitochondrial dysfunction and promoting a switch to glycolysis, supporting abnormal angiogenesis, cellular invasion and pannus formation.
Subject(s)
Arthritis, Rheumatoid/physiopathology , Energy Metabolism/physiology , Fibroblasts/metabolism , Amino Acids, Dicarboxylic/metabolism , Cell Movement/physiology , Cells, Cultured , Cytokines/analysis , DNA, Mitochondrial/metabolism , Glucose/analysis , Humans , Hypoxia/metabolism , Joints/metabolism , Lactic Acid/analysis , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Synovial Membrane/cytologyABSTRACT
Hypoxic preconditioning is a promising strategy to prevent hypoxia-induced damages to several tissues. This effect is related to prior stabilization of the hypoxia-inducible factor-1α via inhibition of the prolyl-hydroxylases (PHDs), which are responsible for its degradation under normoxia. Although PHD inhibition has been shown to increase endurance performance in rodents, potential side effects of such a therapy have not been explored. Here, we investigated the effects of 1 wk of dimethyloxalylglycine (DMOG) treatment (150 mg/kg) on exercise capacity, as well as on cardiac and skeletal muscle function in sedentary and endurance-trained rats. DMOG improved maximal aerobic velocity and endurance in both sedentary and trained rats. This effect was associated with an increase in red blood cells without significant alteration of skeletal muscle contractile properties. In sedentary rats, DMOG treatment resulted in enhanced left ventricle (LV) weight together with impairment in diastolic function, LV relaxation, and pulse pressure. Moreover, DMOG decreased maximal oxygen uptake (state 3) of isolated mitochondria from skeletal muscle. Importantly, endurance training reversed the negative effects of DMOG treatment on cardiac function and restored maximal mitochondrial oxygen uptake to the level of sedentary placebo-treated rats. In conclusion, we provide here evidence that the PHD inhibitor DMOG has detrimental influence on myocardial and mitochondrial function in healthy rats. However, one may suppose that the deleterious influence of PHD inhibition would be potentiated in patients with already poor physical condition. Therefore, the present results prompt us to take into consideration the potential side effects of PHD inhibitors when administrated to patients.
Subject(s)
Amino Acids, Dicarboxylic/metabolism , Heart/physiology , Hypoxia/physiopathology , Muscle, Skeletal/physiology , Physical Conditioning, Animal/physiology , Physical Endurance/physiology , Animals , Hypoxia/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Male , Mice, Inbred C57BL , Mitochondria/metabolism , Mitochondria/physiology , Muscle, Skeletal/metabolism , Muscular Diseases/metabolism , Muscular Diseases/physiopathology , Musculoskeletal Physiological Phenomena , Myocardium/metabolism , Rats , Rats, WistarABSTRACT
Oxidative stress has been implicated in a variety of conditions, including cancer, heart failure, diabetes, neurodegeneration and other diseases. A potential biomarker for oxidative stress is the cystine/glutamate transporter, system x(C)(-). L-Aminosuberic acid (L-ASu) has been identified as a system x(C)(-) substrate. Here we report a facile method for [(11)C]N-Me labeling of L-ASu, automation of the radiochemical process, and preliminary PET imaging with EL4 tumor bearing mice. The results demonstrate uptake in the tumor above background, warranting further studies on the use of radiolabeled analogs of L-ASu as a PET imaging agent for system x(C)(-).
Subject(s)
Amino Acids, Dicarboxylic/metabolism , Diagnostic Imaging/methods , Neoplasms/metabolism , Amino Acid Transport System y+/metabolism , Animals , Cells, Cultured , Membrane Transport Proteins , Mice , Molecular Structure , Oxidative Stress , Up-RegulationABSTRACT
The aromatic amino acid Phe is required for protein synthesis and serves as the precursor of abundant phenylpropanoid plant natural products. While Phe is synthesized from prephenate exclusively via a phenylpyruvate intermediate in model microbes, the alternative pathway via arogenate is predominant in plant Phe biosynthesis. However, the molecular and biochemical evolution of the plant arogenate pathway is currently unknown. Here, we conducted phylogenetically informed biochemical characterization of prephenate aminotransferases (PPA-ATs) that belong to class-Ib aspartate aminotransferases (AspAT Ibs) and catalyze the first committed step of the arogenate pathway in plants. Plant PPA-ATs and succeeding arogenate dehydratases (ADTs) were found to be most closely related to homologs from Chlorobi/Bacteroidetes bacteria. The Chlorobium tepidum PPA-AT and ADT homologs indeed efficiently converted prephenate and arogenate into arogenate and Phe, respectively. A subset of AspAT Ib enzymes exhibiting PPA-AT activity was further identified from both Plantae and prokaryotes and, together with site-directed mutagenesis, showed that Thr-84 and Lys-169 play key roles in specific recognition of dicarboxylic keto (prephenate) and amino (aspartate) acid substrates. The results suggest that, along with ADT, a gene encoding prephenate-specific PPA-AT was transferred from a Chlorobi/Bacteroidetes ancestor to a eukaryotic ancestor of Plantae, allowing efficient Phe and phenylpropanoid production via arogenate in plants today.
Subject(s)
Aspartate Aminotransferases/genetics , Phenylalanine/metabolism , Plants/enzymology , Transaminases/genetics , Amino Acid Sequence , Amino Acids, Dicarboxylic/metabolism , Aspartate Aminotransferases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biosynthetic Pathways , Chlorobium/enzymology , Chlorobium/genetics , Conserved Sequence , Cyclohexenes/metabolism , Evolution, Molecular , Hydro-Lyases/genetics , Hydro-Lyases/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Phylogeny , Plant Proteins/genetics , Plant Proteins/metabolism , Plants/genetics , Sequence Alignment , Transaminases/metabolism , Tyrosine/analogs & derivatives , Tyrosine/metabolismABSTRACT
OBJECTIVE: Prolyl hydroxylases (PHD) are oxygen sensors and therefore pharmacological targets to stimulate periodontal regeneration. Here we evaluate the release profile of the PHD inhibitors dimethyloxaloylglycine and l-mimosine from bone substitutes. MATERIALS: Dimethyloxaloylglycine and l-mimosine were lyophilised onto bone substitutes including bovine bone mineral, beta-tricalcium phosphate, and hydroxyapatite. Release kinetic was evaluated by bioassays with gingival and periodontal ligament fibroblasts. We determined the capacity of PHD inhibitors to provoke VEGF expression and to repress metabolic activity and proliferation as assessed by immunoassay, MTT conversion and (3)[H]thymidine incorporation, respectively. RESULTS: We found that the PHD inhibitors are released from bovine bone mineral as indicated by the increase of VEGF production in gingival and periodontal ligament fibroblasts. Supernatants obtained after 1h also decreased metabolic activity and proliferation of the fibroblasts. A fibrin matrix prolonged the release of PHD inhibitors up to 192h. A similar cellular response was found when supernatants from PHD inhibitors loaded beta-tricalcium phosphate and hydroxyapatite embedded in fibrin were assessed. CONCLUSIONS: In conclusion bone substitutes can serve as carriers for PHD inhibitors that maintain their capacity to provoke a pro-angiogenic response in vitro. These findings provide the basis for preclinical studies to evaluate if this release kinetic can stimulate periodontal regeneration.
Subject(s)
Amino Acids, Dicarboxylic/metabolism , Bone Substitutes/pharmacology , Fibroblasts/metabolism , Mimosine/metabolism , Minerals/pharmacology , Animals , Cattle , Gingiva/cytology , In Vitro Techniques , Microscopy, Electron, Scanning , Periodontal Ligament/cytology , Vascular Endothelial Growth Factor A/metabolismABSTRACT
The aromatic amino acids phenylalanine and tyrosine represent essential sources of high value natural aromatic compounds for human health and industry. Depending on the organism, alternative routes exist for their synthesis. Phenylalanine and tyrosine are synthesized either via phenylpyruvate/4-hydroxyphenylpyruvate or via arogenate. In arogenate-competent microorganisms, an aminotransferase is required for the transamination of prephenate into arogenate, but the identity of the genes is still unknown. We present here the first identification of prephenate aminotransferases (PATs) in seven arogenate-competent microorganisms and the discovery that PAT activity is provided by three different classes of aminotransferase, which belong to two different fold types of pyridoxal phosphate enzymes: an aspartate aminotransferase subgroup 1ß in tested α- and ß-proteobacteria, a branched-chain aminotransferase in tested cyanobacteria, and an N-succinyldiaminopimelate aminotransferase in tested actinobacteria and in the ß-proteobacterium Nitrosomonas europaea. Recombinant PAT enzymes exhibit high activity toward prephenate, indicating that the corresponding genes encode bona fide PAT. PAT functionality was acquired without other modification of substrate specificity and is not a general catalytic property of the three classes of aminotransferases.
Subject(s)
Amino Acids, Dicarboxylic , Bacteria , Bacterial Proteins , Cyclohexenes , Evolution, Molecular , Transaminases , Tyrosine/analogs & derivatives , Amino Acids, Dicarboxylic/chemistry , Amino Acids, Dicarboxylic/genetics , Amino Acids, Dicarboxylic/metabolism , Bacteria/enzymology , Bacteria/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cyclohexenes/chemistry , Cyclohexenes/metabolism , Humans , Pyridoxal Phosphate/chemistry , Pyridoxal Phosphate/genetics , Pyridoxal Phosphate/metabolism , Transaminases/chemistry , Transaminases/genetics , Transaminases/metabolism , Tyrosine/chemistry , Tyrosine/genetics , Tyrosine/metabolismABSTRACT
JMJD5 is a Jumonji C (JmjC) protein that has been implicated in breast cancer tumorigenesis, circadian rhythm regulation, embryological development, and osteoclastogenesis. Recently, JMJD5 (also called KDM8) has been reported to demethylate dimethylated Lys-36 in histone H3 (H3K36me2), regulating genes that control cell cycle progression. Here, we report high-resolution crystal structures of the human JMJD5 catalytic domain in complex with the substrate 2-oxoglutarate (2-OG) and the inhibitor N-oxalylglycine (NOG). The structures reveal a ß-barrel fold that is conserved in the JmjC family and a long shallow cleft that opens into the enzyme's active site. A comparison with other JmjC enzymes illustrates that JMJD5 shares sequence and structural homology with the asparaginyl and histidinyl hydroxylase FIH-1 (factor inhibiting hypoxia-inducible factor 1 [HIF-1]), the lysyl hydroxylase JMJD6, and the RNA hydroxylase TYW5 but displays limited homology to JmjC lysine demethylases (KDMs). Contrary to previous findings, biochemical assays indicate that JMJD5 does not display demethylase activity toward methylated H3K36 nor toward the other methyllysines in the N-terminal tails of histones H3 and H4. Together, these results imply that JMJD5 participates in roles independent of histone demethylation and may function as a protein hydroxylase given its structural homology with FIH-1 and JMJD6.
Subject(s)
Amino Acids, Dicarboxylic/metabolism , Histone Demethylases/chemistry , Histone Demethylases/metabolism , Ketoglutaric Acids/metabolism , Catalytic Domain , Crystallography, X-Ray , Histone Demethylases/antagonists & inhibitors , Histone Demethylases/genetics , Humans , Jumonji Domain-Containing Histone Demethylases/chemistry , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , Models, Molecular , Phylogeny , Protein Conformation , Structural Homology, Protein , Substrate SpecificityABSTRACT
2-Oxoglutarate or α-ketoglutarate (αKG) is a substrate of HIF prolyl hydroxylases 1-3 that decrease cellular levels of the hypoxia-inducible factor 1α (HIF-1α) in the presence of oxygen. αKG analogs are applied to stabilize HIF-1α even in the presence of oxygen and thus provide a novel therapeutic option in treating kidney diseases. In the kidneys, the organic anion transporters 1 and 3 (OAT1 and OAT3, respectively) in cooperation with the sodium-dependent dicarboxylate transporter 3 (NaDC3) and the OAT4 might be responsible for the uptake of αKG analogs into and the efflux out of the tubular cells. Using the radiolabelled substrates p-aminohippurate (PAH, OAT1), estrone-3-sulfate (ES; OAT3, OAT4), and succinate (NaDC3), N-oxalylglycine (NOG), dimethyloxalyl glycine (DMOG), 2,4-diethylpyridine dicarboxylate (2,4-DPD), and pyridine-2,4-dicarboxylic acid (PDCA) were tested in cis-inhibition and trans-stimulation experiments. None of these αKG analogs interacted with NaDC3. 2,4-DPD and PDCA inhibited ES uptake by OAT3 moderately. NOG, 2,4-DPD and PDCA, but not DMOG, inhibited PAH uptake by OAT1 significantly. trans-Stimulation experiments and experiments demonstrating stabilization of HIF-1α revealed that NOG and PDCA, but not 2,4-DPD, are translocated by OAT1. All compounds trans-stimulated ES uptake by OAT4, but only PDCA stabilized HIF-1α. The data suggest that OAT1 is involved in the uptake of NOG and PDCA across the basolateral membrane of proximal tubule cells, whereas OAT4 may release these compounds into the primary urine.
Subject(s)
Dioxygenases/antagonists & inhibitors , Ketoglutaric Acids/metabolism , Organic Anion Transport Protein 1/metabolism , Organic Anion Transporters/metabolism , Procollagen-Proline Dioxygenase/antagonists & inhibitors , Amino Acids, Dicarboxylic/metabolism , Biological Transport, Active , Estrone/analogs & derivatives , Estrone/metabolism , HEK293 Cells , Humans , Hypoxia-Inducible Factor 1/metabolism , Organic Anion Transporters/drug effects , Organic Anion Transporters, Sodium-Dependent/drug effects , Organic Anion Transporters, Sodium-Dependent/metabolism , Pyridines/metabolism , Succinic Acid/metabolism , Symporters/drug effects , Symporters/metabolism , p-Aminohippuric Acid/metabolismABSTRACT
The joint substitution of three active-site residues in Escherichia coli (L)-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10(5)-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k(cat)' for desulfination of l-cysteine sulfinate increased to 0.5s(-1) (from 0.05s(-1) in wild-type enzyme), whereas k(cat)' for transamination of the same substrate was reduced from 510s(-1) to 0.05s(-1). Similarly, k(cat)' for ß-decarboxylation of l-aspartate increased from<0.0001s(-1) to 0.07s(-1), whereas k(cat)' for transamination was reduced from 530s(-1) to 0.13s(-1). l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate ß-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.
Subject(s)
Aspartate Aminotransferases/chemistry , Carbon-Sulfur Lyases/chemistry , Carbon-Sulfur Lyases/genetics , Catalytic Domain/genetics , Amino Acid Sequence , Amino Acid Substitution , Amino Acids, Dicarboxylic/metabolism , Aspartate Aminotransferases/genetics , Aspartate Aminotransferases/metabolism , Biocatalysis , Carbon-Sulfur Lyases/metabolism , Crystallography, X-Ray , Escherichia coli , Hydrogen Bonding , Ligands , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Engineering , Pyridoxal Phosphate/chemistry , Pyridoxal Phosphate/metabolism , Pyridoxamine/analogs & derivatives , Pyridoxamine/chemistry , Pyridoxamine/metabolism , Substrate SpecificityABSTRACT
Altering expression of transcription factors can be an effective means to coordinately modulate entire metabolic pathways in plants. It can also provide useful information concerning the identities of genes that constitute metabolic networks. Here, we used ectopic expression of a MYB transcription factor, Petunia hybrida ODORANT1, to alter Phe and phenylpropanoid metabolism in tomato (Solanum lycopersicum) fruits. Despite the importance of Phe and phenylpropanoids to plant and human health, the pathway for Phe synthesis has not been unambiguously determined. Microarray analysis of ripening fruits from transgenic and control plants permitted identification of a suite of coregulated genes involved in synthesis and further metabolism of Phe. The pattern of coregulated gene expression facilitated discovery of the tomato gene encoding prephenate aminotransferase, which converts prephenate to arogenate. The expression and biochemical data establish an arogenate pathway for Phe synthesis in tomato fruits. Metabolic profiling and ¹³C flux analysis of ripe fruits further revealed large increases in the levels of a specific subset of phenylpropanoid compounds. However, while increased levels of these human nutrition-related phenylpropanoids may be desirable, there were no increases in levels of Phe-derived flavor volatiles.
Subject(s)
Fruit/metabolism , Gene Expression Regulation, Plant , Phenylalanine/metabolism , Plant Proteins/metabolism , Proto-Oncogene Proteins c-myb/metabolism , Solanum lycopersicum/genetics , Solanum lycopersicum/metabolism , Amino Acids, Dicarboxylic/chemistry , Amino Acids, Dicarboxylic/metabolism , Cyclohexanecarboxylic Acids/chemistry , Cyclohexanecarboxylic Acids/metabolism , Cyclohexenes/chemistry , Cyclohexenes/metabolism , Fruit/chemistry , Fruit/genetics , Humans , Solanum lycopersicum/chemistry , Metabolic Networks and Pathways/genetics , Microarray Analysis , Molecular Sequence Data , Molecular Structure , Petunia/genetics , Phenylpyruvic Acids/chemistry , Phenylpyruvic Acids/metabolism , Phylogeny , Plant Extracts/chemistry , Plant Extracts/metabolism , Plant Proteins/classification , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Proto-Oncogene Proteins c-myb/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transaminases/classification , Transaminases/genetics , Transaminases/metabolism , Tyrosine/analogs & derivatives , Tyrosine/chemistry , Tyrosine/metabolism , Volatile Organic Compounds/chemistry , Volatile Organic Compounds/metabolismABSTRACT
The crystal structures of both isoforms of the aminoaldehyde dehydrogenase from pea (PsAMADH) have been solved recently [Tylichováet al. (2010) J Mol Biol396, 870-882]. The characterization of the PsAMADH2 proteins, altered here by site-directed mutagenesis, suggests that the D110 and D113 residues at the entrance to the substrate channel are required for high-affinity binding of ω-aminoaldehydes to PsAMADH2 and for enzyme activity, whereas N162, near catalytic C294, contributes mainly to the enzyme's catalytic rate. Inside the substrate cavity, W170 and Y163, and, to a certain extent, L166 and M167 probably preserve the optimal overall geometry of the substrate channel that allows for the appropriate orientation of the substrate. Unconserved W288 appears to affect the affinity of the enzyme for the substrate amino group through control of the substrate channel diameter without affecting the reaction rate. Therefore, W288 may be a key determinant of the differences in substrate specificity found among plant AMADH isoforms when they interact with naturally occurring substrates such as 3-aminopropionaldehyde and 4-aminobutyraldehyde.