ABSTRACT
Here, we report a DNA-compatible reaction for the generation of cyclopropane derivatives using thiolides with α,ß-unsaturated ketones in the absence of transition metal and N2 protection, which is convenient for DNA encoded library (DEL) construction. This approach allows the rapid and efficient production of a series of DEL libraries of potentially biologically active cyclopropanes and spirocyclopropyl oxindole derivatives.
Subject(s)
Cyclopropanes , DNA , Cloning, Molecular , KetonesABSTRACT
Herein we report a DNA-compatible Biginelli reaction to construct isocytosine scaffolds. This reaction utilizes a one-pot reaction of DNA-conjugated guanidines with aldehydes and methyl cyanoacetates to give isocytosine derivatives, and the method is well compatible with different types of substrates. This is the first report on the synthesis of an isocytosine backbone in the field of DNA-compatible organic synthesis. The successful development of this reaction can widen the chemical space of DELs.
Subject(s)
Cytosine , DNA , Guanidine , AldehydesABSTRACT
Through a comprehensive analysis of the gene expression and dependency in HCC patients and cell lines, LAT1 was identified as the top amino acid transporter candidate supporting HCC tumorigenesis. To assess the suitability of LAT1 as a HCC therapeutic target, we used CRISPR/Cas9 to knockout (KO) LAT1 in the epithelial HCC cell line, Huh7. Knockout of LAT1 diminished its branched chain amino acid (BCAA) transport activity and significantly reduced cell proliferation in Huh7. Consistent with in vitro studies, LAT1 ablation led to suppression of tumor growth in a xenograft model. To elucidate the mechanism underlying the observed inhibition of cell proliferation upon LAT1 KO, we performed RNA-sequencing analysis and investigated the changes in the mTORC1 signaling pathway. LAT1 ablation resulted in a notable reduction in phosphorylation of p70S6K, a downstream target of mTORC1, as well as its substrate S6RP. This reduced cell proliferation and mTORC1 activity were rescued when LAT1 was overexpressed. These findings imply an essential role of LAT1 for maintenance of tumor cell growth and additional therapeutic angles against liver cancer.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Carcinoma, Hepatocellular/genetics , Liver Neoplasms/genetics , Signal Transduction , Cell Line , Mechanistic Target of Rapamycin Complex 1/genetics , Mechanistic Target of Rapamycin Complex 1/metabolismABSTRACT
Dysregulated cellular energetics has recently been recognized as a hallmark of cancer and garnered attention as a potential targeting strategy for cancer therapeutics. Cancer cells reprogram metabolic activities to meet bio-energetic, biosynthetic and redox requirements needed to sustain indefinite proliferation. In many cases, metabolic reprogramming is the result of complex interactions between genetic alterations in well-known oncogenes and tumor suppressors and epigenetic changes. While the metabolism of the two most abundant nutrients, glucose and glutamine, is reprogrammed in a wide range of cancers, accumulating evidence demonstrates that additional metabolic pathways are also critical for cell survival and growth. Proline metabolism is one such metabolic pathway that promotes tumorigenesis in multiple cancer types, including liver cancer, which is the fourth main cause of cancer mortality in the world. Despite the recent spate of approved treatments, including targeted therapy and combined immunotherapies, there has been no significant gain in clinical benefits in the majority of liver cancer patients. Thus, exploring novel therapeutic strategies and identifying new molecular targets remains a top priority for liver cancer. Two of the enzymes in the proline biosynthetic pathway, pyrroline-5-carboxylate reductase (PYCR1) and Aldehyde Dehydrogenase 18 Family Member A1 (ALDH18A1), are upregulated in liver cancer of both human and animal models, while proline catabolic enzymes, such as proline dehydrogenase (PRODH) are downregulated. Here we review the latest evidence linking proline metabolism to liver and other cancers and potential mechanisms of action for the proline pathway in cancer development.
Subject(s)
Carcinogenesis/metabolism , Cellular Reprogramming/physiology , Liver Neoplasms/metabolism , Liver/metabolism , Liver/pathology , Mitochondria/metabolism , Proline/metabolism , Animals , Humans , Liver Neoplasms/pathology , Mitochondria/pathologyABSTRACT
Tumor-specific metabolic rewiring, acquired to confer a proliferative and survival advantage over nontransformed cells, represents a renewed focus in cancer therapy development. Hepatocellular carcinoma (HCC), a malignancy that has hitherto been resistant to compounds targeting oncogenic signaling pathways, represents a candidate cancer to investigate the efficacy of selectively antagonizing such adaptive metabolic reprogramming. To this end, we sought to characterize metabolic changes in HCC necessary for tumorigenesis. We analyzed gene expression profiles in three independent large-scale patient cohorts who had HCC. We identified a commonly deregulated purine metabolic signature in tumors with the extent of purine biosynthetic enzyme up-regulation correlated with tumor grade and a predictor of clinical outcome. The functional significance of enhanced purine metabolism as a hallmark in human HCC was then validated using a combination of HCC cell lines, patient-derived xenograft (PDX) organoids, and mouse models. Targeted ablation of purine biosynthesis by knockdown of the rate-limiting enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) or using the drug mycophenolate mofetil (MMF) reduced HCC proliferation in vitro and decreased the tumor burden in vivo. In comparing the sensitivities of PDX tumor organoids to MMF therapy, we found that HCC tumors defined by high levels of IMPDH and guanosine nucleosides were most susceptible to treatment. Mechanistically, a phosphoinositide 3-kinase (PI3K)-E2F transcription factor 1 (E2F1) axis coordinated purine biosynthetic enzyme expression, deregulation of which altered the activity of mitogen-activated protein kinase/RAS signaling. Simultaneously abolishing PI3K signaling and IMPDH activity with clinically approved inhibitors resulted in greatest efficacy in reducing tumor growth in a PDX mouse model. Conclusion: Enhanced purine metabolic activity regulated by PI3K pathway-dependent activation of E2F1 promotes HCC carcinogenesis, suggesting the potential for targeting purine metabolic reprogramming as a precision therapeutic strategy for patients with HCC.
ABSTRACT
BACKGROUND & AIM: Under the regulation of various oncogenic pathways, cancer cells undergo adaptive metabolic programming to maintain specific metabolic states that support their uncontrolled proliferation. As it has been difficult to directly and effectively inhibit oncogenic signaling cascades with pharmaceutical compounds, focusing on the downstream metabolic pathways that enable indefinite growth may provide therapeutic opportunities. Thus, we sought to characterize metabolic changes in hepatocellular carcinoma (HCC) development and identify metabolic targets required for tumorigenesis. METHODS: We compared gene expression profiles of Morris Hepatoma (MH3924a) and DEN (diethylnitrosamine)-induced HCC models to those of liver tissues from normal and rapidly regenerating liver models, and performed gain- and loss-of-function studies of the identified gene targets for their roles in cancer cell proliferation in vitro and in vivo. RESULTS: The proline biosynthetic enzyme PYCR1 (pyrroline-5-carboxylate reductase 1) was identified as one of the most upregulated genes in the HCC models. Knockdown of PYCR1 potently reduced cell proliferation of multiple HCC cell lines in vitro and tumor growth in vivo. Conversely, overexpression of PYCR1 enhanced the proliferation of the HCC cell lines. Importantly, PYCR1 expression was not elevated in the regenerating liver, and KD or overexpression of PYCR1 had no effect on proliferation of non-cancerous cells. Besides PYCR1, we found that additional proline biosynthetic enzymes, such as ALDH18A1, were upregulated in HCC models and also regulated HCC cell proliferation. Clinical data demonstrated that PYCR1 expression was increased in HCC, correlated with tumor grade, and was an independent predictor of clinical outcome. CONCLUSION: Enhanced expression of proline biosynthetic enzymes promotes HCC cell proliferation. Inhibition of PYCR1 or ALDH18A1 may be a novel therapeutic strategy to target HCC. LAY SUMMARY: Even with the recently approved immunotherapies against liver cancer, currently available medications show limited clinical benefits or efficacy in the majority of patients. As such, it remains a top priority to discover new targets for effective liver cancer treatment. Here, we identify a critical role for the proline biosynthetic pathway in liver cancer development, and demonstrate that targeting key proteins in the pathway, namely PYCR1 and ALDH18A1, may be a novel therapeutic strategy for liver cancer.
Subject(s)
Carcinogenesis/metabolism , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms/metabolism , Proline/biosynthesis , Signal Transduction/genetics , Aldehyde Dehydrogenase/deficiency , Aldehyde Dehydrogenase/genetics , Animals , Carcinogenesis/genetics , Carcinoma, Hepatocellular/chemically induced , Carcinoma, Hepatocellular/pathology , Cell Proliferation/genetics , Diethylnitrosamine/adverse effects , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , HEK293 Cells , HaCaT Cells , Hep G2 Cells , Humans , Liver Neoplasms/chemically induced , Liver Neoplasms/pathology , Liver Neoplasms, Experimental/genetics , Liver Neoplasms, Experimental/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , Mice, SCID , Pyrroline Carboxylate Reductases/deficiency , Pyrroline Carboxylate Reductases/genetics , Rats , Transcriptome , Transfection , Tumor Burden/genetics , Xenograft Model Antitumor Assays , delta-1-Pyrroline-5-Carboxylate ReductaseABSTRACT
Cancer cells are known to adopt aerobic glycolysis in order to fuel tumor growth, but the molecular basis of this metabolic shift remains largely undefined. O-GlcNAcase (OGA) is an enzyme harboring O-linked ß-N-acetylglucosamine (O-GlcNAc) hydrolase and cryptic lysine acetyltransferase activities. Here, we report that OGA is upregulated in a wide range of human cancers and drives aerobic glycolysis and tumor growth by inhibiting pyruvate kinase M2 (PKM2). PKM2 is dynamically O-GlcNAcylated in response to changes in glucose availability. Under high glucose conditions, PKM2 is a target of OGA-associated acetyltransferase activity, which facilitates O-GlcNAcylation of PKM2 by O-GlcNAc transferase (OGT). O-GlcNAcylation inhibits PKM2 catalytic activity and thereby promotes aerobic glycolysis and tumor growth. These studies define a causative role for OGA in tumor progression and reveal PKM2 O-GlcNAcylation as a metabolic rheostat that mediates exquisite control of aerobic glycolysis.
Subject(s)
Antigens, Neoplasm/metabolism , Carrier Proteins/metabolism , Histone Acetyltransferases/metabolism , Hyaluronoglucosaminidase/metabolism , Membrane Proteins/metabolism , N-Acetylglucosaminyltransferases/metabolism , Neoplasms/pathology , Thyroid Hormones/metabolism , Acetylation , Acetylglucosamine/metabolism , Animals , Cell Line, Tumor , Datasets as Topic , Disease Progression , Female , Gene Expression Profiling , Glycolysis , HEK293 Cells , Humans , Male , Mice , Neoplasm Grading , Neoplasm Staging , Neoplasms/metabolism , Protein Processing, Post-Translational , Tissue Array Analysis , Up-Regulation , Xenograft Model Antitumor Assays , Thyroid Hormone-Binding ProteinsABSTRACT
Tumors display profound changes in cellular metabolism, yet how these changes aid the development and growth of tumors is not fully understood. Here we use a multi-omic approach to examine liver carcinogenesis and regeneration, and find that progressive loss of branched-chain amino acid (BCAA) catabolism promotes tumor development and growth. In human hepatocellular carcinomas and animal models of liver cancer, suppression of BCAA catabolic enzyme expression led to BCAA accumulation in tumors, though this was not observed in regenerating liver tissues. The degree of enzyme suppression strongly correlated with tumor aggressiveness, and was an independent predictor of clinical outcome. Moreover, modulating BCAA accumulation regulated cancer cell proliferation in vitro, and tumor burden and overall survival in vivo. Dietary BCAA intake in humans also correlated with cancer mortality risk. In summary, loss of BCAA catabolism in tumors confers functional advantages, which could be exploited by therapeutic interventions in certain cancers.
Subject(s)
Amino Acids, Branched-Chain/metabolism , Carcinogenesis/metabolism , Carcinoma, Hepatocellular/metabolism , Disease Progression , Down-Regulation , Liver Neoplasms/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Aged , Aged, 80 and over , Amino Acids, Branched-Chain/administration & dosage , Amino Acids, Branched-Chain/pharmacology , Animals , Carcinogenesis/drug effects , Carcinoma, Hepatocellular/pathology , Cell Proliferation/drug effects , Cohort Studies , Disease Models, Animal , Female , Hep G2 Cells , Humans , Liver Neoplasms/pathology , Male , Mice , Mice, Inbred C57BL , Middle Aged , Rats , Rats, Inbred ACIABSTRACT
We investigated the changes in amino acid (AA) metabolism induced in MCF10A, a human mammary epithelial cell line, by the sequential knock-in of K-Ras and PI3K mutant oncogenes. Differentially regulated genes associated to AA pathways were identified on comparing gene expression patterns in the isogenic cell lines. Additionally, we monitored the changes in the levels of AAs and transcripts in the cell lines treated with kinase inhibitors (REGO: a multi-kinase inhibitor, PI3K-i: a PI3K inhibitor, and MEK-i: a MEK inhibitor). In total, 19 AAs and 58 AA-associated transcripts were found to be differentially regulated by oncogene knock-in and by drug treatment. In particular, the multi-kinase and MEK inhibitor affected pathways in K-Ras mutant cells, whereas the PI3K inhibitor showed a major impact in the K-Ras/PI3K double mutant cells. These findings may indicate the dependency of AA metabolism on the oncogene mutation pattern in human cancer. Thus, future therapy might include combinations of kinase inhibitors and drug targeting enzymes of AA pathways.
Subject(s)
Amino Acids/metabolism , Antineoplastic Agents/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Mutation , Oncogenes/genetics , Protein Kinase Inhibitors/pharmacology , Cell Line, Tumor , Female , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Intracellular Space/metabolism , Metabolic Networks and Pathways/drug effects , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolismSubject(s)
Genetic Therapy/methods , Phenylketonurias/pathology , Phenylketonurias/therapy , Pigmentation , Animals , MiceABSTRACT
BACKGROUND AND AIM: Hepatic fibrosis is a worldwide healthy burden associated with significant morbidity and mortality. It is caused by a variety of chronic liver injuries. There is currently no effective treatment for liver fibrosis. In this report, we tested an imidazolium salt, 1,3-diisopropylimidazolium tetrafluoroborate (DPIM), for its anti-fibrotic properties in the thioacetamide-induced mouse model. METHODS: DPIM was orally delivered to the thioacetamide-treated mice via drinking water for 12 weeks at the onset of thioacetamide treatment at a concentration of 0.1% (prevention group), and for 4 weeks starting at the 8(th) week at a concentration of 0.1% or 0.2% (attenuation group), respectively. Messenger RNA and protein were determined by real-time polymerase chain reaction and Western blotting, matrix metalloproteinase (MMP) activities were measured by fluorogenic peptide substrate and zymography. Mitogen-activated protein kinase (MAPK) and PI3K inhibitors were applied in HSC-T6 cells in combination of DPIM to probe possible signal pathways underlying the compound's action. RESULTS: We observed a significant reduction in collagen deposition in both prevention and attenuation groups. The α-smooth muscle actin (SMA) and transforming growth factor (TGF)-ß gene expressions were also reduced in both groups. The reduction of collagen deposition could be in part attributed to the suppression of CCR-2 expression and the enhanced matrix protein remodeling by metalloproteinases, especially MMP-3. MAPK and PI3K signaling pathways may be partially participated in DPIM's molecular action. CONCLUSION: DPIM reduced fibrosis in the thioacetamide-induced mouse liver fibrosis model, and warranted further studies for possible clinical application in the future.
Subject(s)
Hepatic Stellate Cells/drug effects , Imidazoles/therapeutic use , Liver Cirrhosis, Experimental/drug therapy , Liver Cirrhosis, Experimental/prevention & control , Actins/genetics , Actins/metabolism , Alanine Transaminase/blood , Analysis of Variance , Animals , Aspartate Aminotransferases/blood , Cells, Cultured , Collagen/metabolism , Gene Expression , Hepatic Stellate Cells/enzymology , Imidazoles/pharmacology , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/metabolism , MAP Kinase Signaling System , Male , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 3/metabolism , Mice , Mice, Inbred BALB C , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase Inhibitors/pharmacology , RNA, Messenger/metabolism , Receptors, CCR2/genetics , Receptors, CCR2/metabolism , Thioacetamide , Transforming Growth Factor beta/geneticsABSTRACT
Hepatic fibrosis is the result of chronic liver injuries underlined by diverse etiologies. The massive accumulation of extracellular matrix (ECM) proteins during fibrogenesis leads to structural distortion and functional disruption of the liver. There is currently no effective standard treatment for liver fibrosis. We previously identified a class of imidazolium salts (IMSs) with anti-fibrotic properties in a cell-based screen. In this report, we investigated the anti-fibrotic efficacy and mechanisms of a small IMS, 1,3-diisopropylimidazolium tetrafluoroborate (DPIM), in a hepatic fibrosis model induced by bile duct ligation (BDL) in mice. The orally available DPIM was administered to BDL mice via drinking water at three concentrations (0.5, 0.75, and 1 g/l) for 4 weeks. We observed a significant reduction in inflammation and collagen deposition in the liver, which could be mediated by a reduction in the expression of monocyte chemoattractant protein-1 (MCP-1) and by an enhancement in the matrix metalloproteinase-mediated ECM remodeling. The current findings highlight the importance for simultaneously targeting multiple pathways to more effectively attenuate and resolve liver fibrosis and warrant further studies on this compound in additional models of hepatic fibrosis.
Subject(s)
Cholestasis/drug therapy , Fibrosis/drug therapy , Imidazoles/therapeutic use , Inflammation/drug therapy , Administration, Oral , Animals , Cell Line , Collagen/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Imidazoles/administration & dosage , Immunohistochemistry , In Situ Nick-End Labeling , Male , Matrix Metalloproteinases/metabolism , Mice , Polymerase Chain Reaction , Tissue Inhibitor of Metalloproteinase-1/metabolismABSTRACT
Phenylketonuria (PKU) is caused by hepatic phenylalanine hydroxylase (PAH) deficiency and is associated with systemic accumulation of phenylalanine (Phe). Previously we demonstrated correction of murine PKU after intravenous injection of a recombinant type 2 adeno-associated viral vector pseudotyped with type 8 capsid (rAAV2/8), which successfully directed hepatic transduction and Pah gene expression. Here, we report that liver PAH activity and phenylalanine clearance were also restored in PAH-deficient mice after simple intramuscular injection of either AAV2 pseudotype 1 (rAAV2/1) or rAAV2/8 vectors. Serotype 2 AAV vector (rAAV2/2) was also investigated, but long-term phenylalanine clearance has been observed only for pseudotypes 1 and 8. Therapeutic correction was shown in both male and female mice, albeit more effectively in males, in which correction lasted for the entire period of the experiment (>1 year). Although phenylalanine levels began to rise in female mice at about 8-10 months after rAAV2/8 injection they remained only mildly hyperphenylalaninemic thereafter and subsequent supplementation with synthetic tetrahydrobiopterin resulted in a transient decrease in blood phenylalanine. Alternatively, subsequent administration of a second vector with a different AAV pseudotype to avoid immunity against the previously administrated vector was also successful for long-term treatment of female PKU mice. Overall, this relatively less invasive gene transfer approach completes our previous studies and allows comparison of complementary strategies in the development of efficient PKU gene therapy protocols.
Subject(s)
Dependovirus/classification , Dependovirus/physiology , Genetic Vectors/administration & dosage , Phenylalanine Hydroxylase/metabolism , Phenylketonurias/therapy , Animals , Dependovirus/genetics , Female , Genetic Therapy , Genetic Vectors/classification , Genetic Vectors/genetics , Humans , Injections, Intramuscular , Liver/enzymology , Liver/metabolism , Liver/virology , Male , Mice , Phenylalanine Hydroxylase/genetics , Phenylketonurias/virology , Serotyping , Treatment OutcomeABSTRACT
A class of imidazolium salts (IMSs) is routinely used in organic synthetic chemistry as precursors to generate N-heterocyclic carbenes (NHCs) with catalytic activity. However, their biological properties are largely unknown. The current study investigates the biological activity of a typical NHC precursor DBZIM and its trimer TDBZIM in hepatic stellate cells (HSCs), which is an in vitro model for studying liver fibrosis. The results show that HSCs treated with IMSs have an enhanced GSH/GSSG ratio and a reduced level of reactive oxygen species (ROS), which may consequently contribute to the attenuation in gene expression of fibrogenic molecules such as smooth muscle actin-alpha (SMAA), transforming growth factor-beta 1 (TGF-beta1), procollagen alphaI(I) and fibronectin. Further, the in vivo experiments demonstrate that DBZIM is an anti-fibrotic agent in a mouse model of liver fibrosis. These findings suggest that the versatile IMSs could be a potential source for developing novel therapeutics to treat liver fibrosis and other fibrogenic disorders caused by oxidative stress and TGF-beta1 mal-signalling.
Subject(s)
Antioxidants/pharmacology , Hepatic Stellate Cells/drug effects , Imidazolines/pharmacology , Animals , Antioxidants/chemistry , Cell Culture Techniques , Cell Line , Cell Proliferation/drug effects , Gene Expression/drug effects , Glutathione/genetics , Glutathione/metabolism , Glutathione Disulfide/metabolism , Hepatic Stellate Cells/enzymology , Hepatic Stellate Cells/metabolism , Hydrogen Peroxide/antagonists & inhibitors , Hydrogen Peroxide/pharmacology , Imidazolines/chemistry , Male , Mice , Oxidative Stress/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/genetics , Reactive Oxygen Species/metabolism , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Receptors, Transforming Growth Factor beta/biosynthesis , Receptors, Transforming Growth Factor beta/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta1/antagonists & inhibitors , Transforming Growth Factor beta1/biosynthesis , Transforming Growth Factor beta1/geneticsABSTRACT
Phenylketonuria (PKU) caused by phenylalanine hydroxylase (PAH) deficiency leads to toxic accumulation of phenylalanine (Phe). PAH is predominantly expressed in liver and its activity requires a supply of tetrahydrobiopterin (BH(4)) cofactor, but we propose that expression of a complete Phe hydroxylating system (PAH plus BH(4) synthetic enzymes) in skeletal muscle will lead to therapeutic reduction of blood Phe levels in Pah(enu2) mice, a model of human PKU. In order to test this hypothesis, we first developed transgenic Pah(enu2) mice that lack liver PAH activity but coexpress, in their skeletal muscle, PAH and guanosine triphosphate cyclohydrolase I (GTPCH). The latter is responsible for the committing enzymatic step in BH(4) biosynthesis. Despite sufficient muscle enzyme expression, these mice remained hyperphenylalaninemic, thereby suggesting that expression of additional BH(4) synthetic enzymes would be necessary. A recombinant triple-cistronic adeno-associated virus-2 (AAV2) pseudotype 1 vector expressing PAH along with GTPCH and 6-pyruvoyltetrahydrobiopterin synthase (PTPS), the next step in BH(4) synthesis, was generated. Injection of this vector into the gastrocnemius muscles of Pah(enu2) mice led to stable and long-term reduction of blood Phe and reversal of PKU-associated coat hypopigmentation. We propose that muscle-directed gene therapy will be a viable alternative treatment approach to PKU and other inborn errors of metabolism.
Subject(s)
Dependovirus/genetics , GTP Cyclohydrolase/genetics , Gene Transfer Techniques , Phenylalanine Hydroxylase/genetics , Phenylalanine/metabolism , Phenylketonurias/therapy , Phosphorus-Oxygen Lyases/genetics , Animals , GTP Cyclohydrolase/metabolism , Genetic Vectors , Hydroxylation , Injections, Intramuscular , Liver/enzymology , Mice , Mice, Transgenic , Muscle, Skeletal/enzymology , Phenylalanine/blood , Phenylalanine Hydroxylase/metabolism , Phosphorus-Oxygen Lyases/metabolismABSTRACT
Tetrahydrobiopterin (BH4) supplementation in patients with BH4-responsive phenylalanine hydroxylase (PAH) deficiency is an alternative to low-phenylalanine diet. To further investigate hepatic BH4-responsiveness, oral administration of 50 mg BH4/kg/day for 5 weeks was performed in wild-type mice. We observed a 2-fold increase in PAH protein by quantitative Western blot analysis and a 1.7-fold increase in enzyme activity, but no change in Pah-mRNA expression by quantitative real-time PCR analysis in treated mice compared to controls. Our findings support the proposed chemical-chaperone effect of BH4 to protect PAH.
Subject(s)
Biopterins/analogs & derivatives , Gene Expression Regulation, Enzymologic , Phenylalanine Hydroxylase/metabolism , Administration, Oral , Animals , Biopterins/administration & dosage , Biopterins/pharmacology , Liver/drug effects , Liver/enzymology , Mice , Phenylalanine Hydroxylase/biosynthesis , Phenylalanine Hydroxylase/genetics , RNA, Messenger/biosynthesisABSTRACT
The natural cofactor of phenylalanine hydroxylase (PAH), tetrahydrobiopterin (BH4), regulates the enzyme activity as well as being essential in catalysis. BH4-responsive PAH deficiency is a variant of hyperphenylalaninemia or phenylketonuria (PKU) caused by mutations in the human PAH gene that respond to oral BH4 loading by stimulating enzyme activity and therefore lowering serum phenylalanine. Here, we showed in a coupled transcription-translation in vitro assay that upon expression in the presence of BH4, wild-type PAH enzyme activity was enhanced. We then investigated the effect of BH4 on PAH activity in transgenic mice that had a complete or partial deficiency in the endogenous cofactor biosynthesis. The rate of hepatic PAH enzyme activity increased significantly with BH4 content without affecting gene expression or Pah-mRNA stability. These results indicate that BH4 has a chaperon-like effect on PAH synthesis and/or is a protecting cofactor against enzyme auto-inactivation and degradation also in vivo. Our findings thus contribute to the understanding of the regulation of PAH by its cofactor BH4 on an additional level and provide a molecular explanation for cofactor-responsive PKU.
Subject(s)
Amino Acid Metabolism, Inborn Errors , Biopterins/analogs & derivatives , Biopterins/metabolism , Gene Expression Regulation, Enzymologic , Phenylalanine Hydroxylase/metabolism , Phenylketonurias/genetics , Animals , Animals, Newborn , Biopterins/deficiency , Blotting, Western , Gene Deletion , Genes, Recessive , Heterozygote , Humans , Kinetics , Liver/enzymology , Mice , Mice, Knockout , Mice, Transgenic , Phenylalanine/genetics , Phenylalanine/metabolism , Phenylalanine Hydroxylase/genetics , Protein Biosynthesis/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, GeneticABSTRACT
Phenylketonuria (or PKU) is a well-known and widespread genetic disease for which many countries perform newborn screening, and life-long dietary restriction is still the ultimate and effective therapy. However, the diet is complicated, unpalatable, and expensive. The long-term effects of diet discontinuation in adults, except for the serious adverse effects of maternal hyperphenylalaninemia upon the developing fetus, have not been systematically studied, but cognitive decline and neurologic abnormalities have been anecdotally reported. Thus, alternative approaches for PKU therapy, including gene therapy, must be further explored. Here we summarize past present nonviral and viral gene transfer approaches, both in vitro studies and preclinical animal trials, to delivering the PAH gene into liver or other organs as potential alternatives to life-long phenylalanine-restricted dietary therapy.
Subject(s)
Gene Transfer Techniques , Genetic Therapy , Phenylketonurias/therapy , Adenoviridae/genetics , Animals , Genetic Vectors , Humans , Liver/metabolism , Mice , Phenylalanine Hydroxylase/geneticsABSTRACT
Sex-Peptide (SP) and the peptide DUP99B elicit two postmating responses in Drosophila melanogaster females: receptivity is reduced and oviposition is increased. Both are synthesized in the male genital tract and transferred into the female during copulation. To elucidate their function, we characterized the binding properties of SP and DUP99B in females. Cryostat sections of adult females were incubated with alkaline phosphatase (AP)-tagged peptides. In virgin females, both peptides have specific target sites in the nervous system and in the genital tract. The binding pattern is almost identical for both peptides. Incubation of sections of mated females confirm that some of these target sites correspond to the in vivo targets of the two peptides. Neuronal binding is dependent on an intact C-terminal sequence of SP, binding in the genital tract is less demanding in terms of amino acid sequence requirement. On affinity blots the AP-SP probe binds to membrane proteins extracted from abdomen and head plus thorax, respectively. The binding proteins in the nervous system and the genital tract differ in their molecular properties. Calculation of dissociation constants (K(d)), and also determination of the minimal peptide concentrations necessary for binding, indicate that SP is the more important peptide inducing the postmating responses. Our results suggest that binding of SP in the nervous system is responsible for eliciting the postmating responses, whereas binding in the genital tract reflects the presence of a peptide transporter.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/physiology , Peptides/metabolism , Sex Attractants/metabolism , Sexual Behavior, Animal/physiology , Animals , Female , Genitalia, Female/metabolism , Immunoblotting , Intercellular Signaling Peptides and Proteins , Nervous System/metabolism , Protein BindingABSTRACT
We have characterized a glycosylated, 31 amino-acid peptide of 4932 Da isolated from Drosophila melanogaster males. The mature peptide contains a sugar moiety of 1184 Da at a NDT consensus glycosylation site and a disulfide bond. It is synthesized in the male ejaculatory duct via a 54 amino-acid precursor containing an N-terminal signal peptide and Arg-Lys at the C-terminus which is cleaved off during maturation. The gene contains an intron of 53 bp and is localized in the cytological region 99B of the D. melanogaster genome. The peptide is therefore named DUP99B (for ductus ejaculatorius peptide, cytological localization 99B). The C-terminal parts of mature DUP99B and D. melanogaster sex-peptide (ACP70A) are highly homologous. Injected into virgin females, DUP99B elicits the same postmating responses as sex-peptide (increased oviposition, reduced receptivity). These effects are also induced by de-glycosylated native peptide or synthetic DUP99B lacking the sugar moiety. Presence of the glycosyl group, however, decreases the amount needed to elicit the postmating responses. Homologies in the coding regions of the two exons of DUP99B and sex-peptide, respectively, suggest that the two genes have evolved by gene duplication. Thus, we consider these two genes to be members of the new sex-peptide gene family.