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1.
Proc Natl Acad Sci U S A ; 117(14): 8083-8093, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32213586

RESUMO

Three-dimensional (3D) cell culture is well documented to regain intrinsic metabolic properties and to better mimic the in vivo situation than two-dimensional (2D) cell culture. Particularly, proline metabolism is critical for tumorigenesis since pyrroline-5-carboxylate (P5C) reductase (PYCR/P5CR) is highly expressed in various tumors and its enzymatic activity is essential for in vitro 3D tumor cell growth and in vivo tumorigenesis. PYCR converts the P5C intermediate to proline as a biosynthesis pathway, whereas proline dehydrogenase (PRODH) breaks down proline to P5C as a degradation pathway. Intriguingly, expressions of proline biosynthesis PYCR gene and proline degradation PRODH gene are up-regulated directly by c-Myc oncoprotein and p53 tumor suppressor, respectively, suggesting that the proline-P5C metabolic axis is a key checkpoint for tumor cell growth. Here, we report a metabolic reprogramming of 3D tumor cell growth by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Metabolomic analyses revealed that KSHV infection increased nonessential amino acid metabolites, specifically proline, in 3D culture, not in 2D culture. Strikingly, the KSHV K1 oncoprotein interacted with and activated PYCR enzyme, increasing intracellular proline concentration. Consequently, the K1-PYCR interaction promoted tumor cell growth in 3D spheroid culture and tumorigenesis in nude mice. In contrast, depletion of PYCR expression markedly abrogated K1-induced tumor cell growth in 3D culture, not in 2D culture. This study demonstrates that an increase of proline biosynthesis induced by K1-PYCR interaction is critical for KSHV-mediated transformation in in vitro 3D culture condition and in vivo tumorigenesis.


Assuntos
Transformação Celular Neoplásica/patologia , Herpesvirus Humano 8/metabolismo , Prolina/metabolismo , Pirrolina Carboxilato Redutases/metabolismo , Sarcoma de Kaposi/patologia , Proteínas Virais/metabolismo , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Metabolômica , Camundongos , Prolina Oxidase/metabolismo , Sarcoma de Kaposi/virologia , Esferoides Celulares , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Acta Biochim Biophys Sin (Shanghai) ; 51(10): 1064-1070, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31559416

RESUMO

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with poor clinical outcomes and without effective targeted therapies. Numerous studies have suggested that HDAC inhibitors (TSA/SAHA) may be effective in TNBCs. Proline oxidase, also known as proline dehydrogenase (POX/PRODH), is a key enzyme in the proline metabolism pathway and plays a vital role in tumorigenesis. In this study, we found that HDAC inhibitors (TSA/SAHA) significantly increased POX expression and autophagy through activating AMPK. Depletion of POX decreased autophagy and increased apoptosis induced by HDAC inhibitors in TNBC cells. These results suggest that POX contributes to cell survival under chemotherapeutic stresses and might serve as a potential target for treatment of TNBC.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Prolina Oxidase/genética , Ativação Transcricional/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias de Mama Triplo Negativas/genética
3.
BMC Plant Biol ; 19(1): 308, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299895

RESUMO

BACKGROUND: Land preparation is an important component of fragrant rice production. However, the effect of tillage on fragrant rice production is unclear, especially regarding the biosynthesis of 2-acetyl-1-pyrroline (2-AP), which is the main compound of the unique aroma of fragrant rice. This study aimed to explore 2-AP biosynthesis in fragrant rice under different tillage regimes. Three tillage methods were applied in the present study: conventional rotary tillage (CK) as the control, plough tillage (PT), and no-tillage (NT). RESULT: Compared with CK, the PT treatment increased 2-AP content in grain, upregulated the activity of ornithine aminotransferase (OAT) and increased contents of 1-pyrroline and pyrroline-5-carboxylic acid (P5C). Furthermore, the PT treatment increased the grain yield and nitrogen accumulation of fragrant rice. Meanwhile, the 2-AP content in the grain produced under the NT treatment was significantly higher than that in the grain produced under both the PT and CK treatments due to the enhancement of proline content and the activities of proline dehydrogenase (PDH) and △1-pyrroline-5-carboxylic acid synthetase (P5CS). However, the present study observed that the overall production of fragrant rice under NT conditions was inferior due to lower yield, nitrogen accumulation, and anti-oxidative enzymatic activities. Moreover, the organic matter content and soil microorganism quantity increased due to PT and NT treatments. CONCLUSIONS: Compared to CK, PT improved fragrant rice grain yield and nitrogen accumulation and induced an increase in OAT activity and led to an increase in 2-AP concentration. No-tillage also produced increased 2-AP content in grain by enhancing PDH and P5CS activities but limited yields and nitrogen accumulation in fragrant rice.


Assuntos
Oryza/fisiologia , Prolina Oxidase/metabolismo , Pirróis/metabolismo , Odorantes , Oryza/enzimologia , Oryza/genética , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prolina/análise , Prolina Oxidase/genética , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/fisiologia
4.
Eur J Med Genet ; 62(8): 103705, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31229682

RESUMO

22q11.2 deletion syndrome (22q11DS) is a genetic disorder caused by a hemizygous microdeletion on the long arm of chromosome 22 and is associated with a high risk for psychosis and cognitive impairment. One of the genes located in the deleted region of 22q11DS is Proline Dehydrogenase (PRODH) which is important for conversion of proline to glutamate. Glutamate is the primary excitatory neurotransmitter and is involved in the pathophysiology of psychosis as well as in cognition. Excessive concentrations are toxic. Possibly, neuroprotective drugs modulating glutamatergic neurotransmission could be effective in treating psychotic symptoms and cognitive enhancement in patients with 22q11DS. Riluzole is a potent anti-glutamatergic drug that reduces glutamatergic neurotransmission. Here we report acute (single dose) and long-term effects of riluzole on glutamate and GABA levels in the anterior cingulate cortex (ACC) and striatum (measured with magnetic resonance spectroscopy, 1H-MRS) as well as on psychotic symptoms and cognitive functioning in a medication-free 23-year old woman with 22q11DS. Patient presented with frequent auditory and visual hallucinations and mild paranoid ideas. The 1H-MRS measurements showed that after a single dose riluzole (50 mg), glutamate in the ACC and striatum was reduced whereas striatal GABA increased compared to baseline. Strikingly, hallucinations and paranoia disappeared. Therefore, riluzole treatment was initiated and patient was followed up after 18 months of treatment. At follow-up, patient reported no hallucinations or paranoia and several cognitive functions were improved. Furthermore, glutamate concentrations in the ACC and striatum decreased whereas GABA concentrations increased in the striatum but decreased in the ACC. These results suggests that riluzole may be an effective treatment option for psychotic symptoms and cognitive enhancement in 22q11DS. Results warrant replication in a bigger sample.


Assuntos
Síndrome de DiGeorge/tratamento farmacológico , Transtornos Psicóticos/tratamento farmacológico , Riluzol/administração & dosagem , Adulto , Cromossomos Humanos Par 22/genética , Síndrome de DiGeorge/genética , Síndrome de DiGeorge/patologia , Feminino , Ácido Glutâmico/metabolismo , Humanos , Prolina/metabolismo , Prolina Oxidase/genética , Transtornos Psicóticos/genética , Transtornos Psicóticos/patologia , Adulto Jovem
5.
Appl Biochem Biotechnol ; 189(2): 498-510, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31053997

RESUMO

Empirical modeling the partition behavior and recovery of a recombinant Pseudomonas putida POS-F84 proline dehydrogenase (ProDH) in aqueous two-phase systems (ATPS) was carried out by response surface methodology (RSM). Polyethylene glycol 1000 (PEG-1000) concentration, sodium carbonate concentration, and pH, which were the most important factors, were chosen for modeling the partition feature of enzyme. The adequacy of the models was investigated by means of variance analysis. Also, to confirm the efficiency of the ATPS in partition and purification of recombinant ProDH, purity and enzymatic activity was studied. After numerical optimization, an optimal ATPS composed of 14.33% PEG-1000 and 11.79% sodium carbonate at pH 7.48 was achieved. Yield, purification factor, and recovery were 81.41%, 60.82, and 270.82%, respectively. Purified recombinant ProDH was found as a single protein band into the upper PEG-rich phase and the specific activity was calculated to be 46.23 ± 2.1 U/mg. Collectively, our data showed that the RSM could be an appropriate and powerful tool to define the best ATPS system for recovery and purification of P. putida ProDH.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Microrganismos Geneticamente Modificados/enzimologia , Prolina Oxidase/isolamento & purificação , Pseudomonas putida/enzimologia , Proteínas de Bactérias/genética , Carbonatos/química , Concentração de Íons de Hidrogênio , Microrganismos Geneticamente Modificados/genética , Polietilenoglicóis/química , Prolina Oxidase/química , Pseudomonas putida/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
6.
Toxins (Basel) ; 11(4)2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995739

RESUMO

The toxicity and related mechanisms of aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) in the mouse kidney were studied, and the role of l-proline in alleviating kidney damage was investigated. In a 28-day toxicity mouse model, thirty mice were divided into six groups: control (without treatment), l-proline group (10 g/kg body weight (b.w.)), AFB1 group (0.5 mg/kg b.w.), AFM1 (3.5 mg/kg b.w.), AFB1 + l-proline group and AFM1 + l-proline group. Kidney index and biochemical indicators were detected, and pathological staining was observed. Using a human embryonic kidney 293 (HEK 293) cell model, cell apoptosis rate and apoptotic proteins expressions were detected. The results showed that AFB1 and AFM1 activated pathways related with oxidative stress and caused kidney injury; l-proline significantly alleviated abnormal expressions of biochemical parameters and pathological kidney damage, as well as excessive cell apoptosis in the AF-treated models. Moreover, proline dehydrogenase (PRODH) was verified to regulate the levels of l-proline and downstream apoptotic factors (Bax, Bcl-2, and cleaved Caspase-3) compared with the control (p < 0.05). In conclusion, l-proline could protect mouse kidneys from AFB1 and AFM1 through alleviating oxidative damage and decreasing downstream apoptosis, which deserves further research and development.


Assuntos
Lesão Renal Aguda/tratamento farmacológico , Aflatoxina B1/toxicidade , Aflatoxina M1/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Prolina/uso terapêutico , Substâncias Protetoras/uso terapêutico , Lesão Renal Aguda/induzido quimicamente , Lesão Renal Aguda/metabolismo , Lesão Renal Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Células HEK293 , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Prolina/farmacologia , Prolina Oxidase/metabolismo , Substâncias Protetoras/farmacologia , Testes de Toxicidade Subaguda
7.
Prog Cardiovasc Dis ; 62(2): 193-202, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30867130

RESUMO

The benefits of physical activity in cardiovascular diseases have long been appreciated. However, the molecular mechanisms that trigger and sustain the cardiac benefits of exercise are poorly understood, and it is anticipated that unveiling these mechanisms will identify novel therapeutic targets. In search of these mechanisms we took advantage of unbiased RNA-sequencing (RNA-seq) technology to discover cardiac gene targets whose expression is disrupted in heart failure (HF) and rescued by exercise in a rat model. Upon exhaustive validation in a separate rat cohort (qPCR) and human datasets, we shortlisted 16 targets for a cell-based screening, aiming to evaluate whether targeted disruption of these genes with silencing RNA would affect the abundance of a CVD biomarker (BNP, B-type natriuretic peptide) in human cardiomyocytes. Overall, these experiments showed that Proline Dehydrogenase (PRODH) expression is reduced in human failing hearts, rescued by exercise in a rat model of HF, and its targeted knockdown increases BNP expression in human cardiomyocytes. On the other hand, overexpression of PRODH increases the abundance of metabolism-related gene transcripts, and PRODH appears to be crucial to sustain normal mitochondrial function and maintenance of ATP levels in human cardiomyocytes in a hypoxic environment, as well as for redox homeostasis in both normoxic and hypoxic conditions. Altogether our findings show that PRODH is a novel molecular target of exercise in failing hearts and highlight its role in cardiomyocyte physiology, thereby proposing PRODH as a potential experimental target for gene therapy in HF.


Assuntos
Exercício Físico/fisiologia , Insuficiência Cardíaca , Prolina Oxidase/metabolismo , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/prevenção & controle , Humanos , Mitocôndrias Cardíacas/metabolismo , Ratos , Transdução de Sinais
8.
Biotechnol J ; 14(5): e1800540, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30791229

RESUMO

Thermus thermophilus proline dehydrogenase ( TtProDH) catalyzes the first step in proline catabolism. The thermostable flavoenzyme consists of a distorted triosephosphate isomerase (TIM) barrel and three N-terminal helices: αA, αB, and αC. Using maltose-binding protein (MBP) fused constructs, it has been recently demonstrated that helix αC is crucial for TtProDH catalysis and for tetramerization through positioning of helix α8. Here, the structural features that determine the thermostability of TtProDH are reported. Selective disruption of two ion pairs in the dimerization interface of several MBP-TtProDH variants result in the formation of monomers. The newly created monomers have improved catalytic properties but their melting temperatures are decreased by more than 20 °C. Sequence comparison suggests that one of the ion-pairs involved in dimerization is unique for ProDHs from Thermus species. In summary, intermolecular ion-pairs improve the thermostability of TtProDH and a trade-off is made between thermostability and catalytic activity.


Assuntos
Dimerização , Estabilidade Enzimática , Prolina Oxidase/metabolismo , Thermus thermophilus/enzimologia , Thermus thermophilus/metabolismo , Sequência de Aminoácidos , Catálise , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Temperatura Alta , Cinética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Mutagênese , Prolina Oxidase/genética , Conformação Proteica , Dobramento de Proteína , Análise de Sequência , Temperatura , Thermus thermophilus/genética , Triose-Fosfato Isomerase
9.
PLoS Genet ; 15(2): e1007976, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30742618

RESUMO

Amino acids are among the earliest identified inducers of yeast-to-hyphal transitions in Candida albicans, an opportunistic fungal pathogen of humans. Here, we show that the morphogenic amino acids arginine, ornithine and proline are internalized and metabolized in mitochondria via a PUT1- and PUT2-dependent pathway that results in enhanced ATP production. Elevated ATP levels correlate with Ras1/cAMP/PKA pathway activation and Efg1-induced gene expression. The magnitude of amino acid-induced filamentation is linked to glucose availability; high levels of glucose repress mitochondrial function thereby dampening filamentation. Furthermore, arginine-induced morphogenesis occurs more rapidly and independently of Dur1,2-catalyzed urea degradation, indicating that mitochondrial-generated ATP, not CO2, is the primary morphogenic signal derived from arginine metabolism. The important role of the SPS-sensor of extracellular amino acids in morphogenesis is the consequence of induced amino acid permease gene expression, i.e., SPS-sensor activation enhances the capacity of cells to take up morphogenic amino acids, a requisite for their catabolism. C. albicans cells engulfed by murine macrophages filament, resulting in macrophage lysis. Phagocytosed put1-/- and put2-/- cells do not filament and exhibit reduced viability, consistent with a critical role of mitochondrial proline metabolism in virulence.


Assuntos
Candida albicans/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Prolina/metabolismo , Proteínas ras/metabolismo , Trifosfato de Adenosina/metabolismo , Aminoácidos/metabolismo , Animais , Candida albicans/genética , Candida albicans/patogenicidade , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Fúngicas/genética , Humanos , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Macrófagos/microbiologia , Camundongos , Mitocôndrias/metabolismo , Morfogênese , Prolina Oxidase/genética , Prolina Oxidase/metabolismo , Células RAW 264.7 , Transdução de Sinais , Virulência/fisiologia , Proteínas ras/genética
10.
Mol Microbiol ; 111(4): 883-897, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30636068

RESUMO

Arginine participates widely in metabolic processes. The heterocyst-forming cyanobacterium Anabaena catabolizes arginine to produce proline and glutamate, with concomitant release of ammonium, as major products. Analysis of mutant Anabaena strains showed that this catabolic pathway is the product of two genes, agrE (alr4995) and putA (alr0540). The predicted PutA protein is a conventional, bifunctional proline oxidase that produces glutamate from proline. In contrast, AgrE is a hitherto unrecognized enzyme that contains both an N-terminal α/ß propeller domain and a unique C-terminal domain of previously unidentified function. In vitro analysis of the proteins expressed in Escherichia coli or Anabaena showed arginine dihydrolase activity of the N-terminal domain and ornithine cyclodeaminase activity of the C-terminal domain, overall producing proline from arginine. In the diazotrophic filaments of Anabaena, ß-aspartyl-arginine dipeptide is transferred from the heterocysts to the vegetative cells, where it is cleaved producing aspartate and arginine. Both agrE and putA were found to be expressed at higher levels in vegetative cells than in heterocysts, implying that arginine is catabolized by the AgrE-PutA pathway mainly in the vegetative cells. Expression in Anabaena of a homolog of the C-terminal domain of AgrE obtained from Methanococcus maripaludis enabled us to identify an archaeal ornithine cyclodeaminase.


Assuntos
Amônia-Liases/metabolismo , Anabaena/enzimologia , Arginina/metabolismo , Prolina/metabolismo , Amônia-Liases/genética , Anabaena/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Redes e Vias Metabólicas , Fixação de Nitrogênio , Prolina Oxidase/genética , Prolina Oxidase/metabolismo
11.
Protoplasma ; 256(2): 449-457, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30206687

RESUMO

Oxidative stress can occur in different parts of plant cells. We employed two oxidants that induce reactive oxygen species (ROS) in different intracellular compartments: methyl viologen (MV, in chloroplasts) and menadione (MD, in mitochondria). The responses of pea (Pisum sativum) leaf discs to MV or MD after 4-h incubation in dark or moderate (300 µE m-2 s-1) or high light (1200 µE m-2 s-1) were examined. Marked increase in ROS levels was observed, irrespective of compartment targeted. The levels of proline, a compatible solute, increased markedly much more than that of ascorbate or glutathione during oxidative/photo-oxidative stress, emphasizing the importance of proline. Further, the activities and transcripts of enzymes involved in biosynthesis or oxidation of proline were studied. An upregulation of biosynthesis and downregulation of oxidation was the basis of proline accumulation. Pyrroline-5-carboxylate synthetase (P5CS, involved in biosynthesis) and proline dehydrogenase (PDH, involved in oxidation) were the key enzymes regulated under oxidative stress. Since these two enzymes-P5CS and PDH-are located in chloroplasts and mitochondria, respectively, we suggest that proline metabolism can help to mediate inter-organelle interactions and achieve redox homeostasis under photo-oxidative stress.


Assuntos
Cloroplastos/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Ervilhas/metabolismo , Folhas de Planta/metabolismo , Prolina/metabolismo , Ácido Ascórbico/metabolismo , Regulação da Expressão Gênica de Plantas , Glutationa/metabolismo , Oxirredução , Ervilhas/genética , Ervilhas/crescimento & desenvolvimento , Prolina Oxidase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo
12.
Antioxid Redox Signal ; 30(4): 650-673, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28990412

RESUMO

SIGNIFICANCE: Proline catabolism refers to the 4-electron oxidation of proline to glutamate catalyzed by the enzymes proline dehydrogenase (PRODH) and l-glutamate γ-semialdehyde dehydrogenase (GSALDH, or ALDH4A1). These enzymes and the intermediate metabolites of the pathway have been implicated in tumor growth and suppression, metastasis, hyperprolinemia metabolic disorders, schizophrenia susceptibility, life span extension, and pathogen virulence and survival. In some bacteria, PRODH and GSALDH are combined into a bifunctional enzyme known as proline utilization A (PutA). PutAs are not only virulence factors in some pathogenic bacteria but also fascinating systems for studying the coordination of metabolic enzymes via substrate channeling. Recent Advances: The past decade has seen an explosion of structural data for proline catabolic enzymes. This review surveys these structures, emphasizing protein folds, substrate recognition, oligomerization, kinetic mechanisms, and substrate channeling in PutA. CRITICAL ISSUES: Major unsolved structural targets include eukaryotic PRODH, the complex between monofunctional PRODH and monofunctional GSALDH, and the largest of all PutAs, trifunctional PutA. The structural basis of PutA-membrane association is poorly understood. Fundamental aspects of substrate channeling in PutA remain unknown, such as the identity of the channeled intermediate, how the tunnel system is activated, and the roles of ancillary tunnels. FUTURE DIRECTIONS: New approaches are needed to study the molecular and in vivo mechanisms of substrate channeling. With the discovery of the proline cycle driving tumor growth and metastasis, the development of inhibitors of proline metabolic enzymes has emerged as an exciting new direction. Structural biology will be important in these endeavors.


Assuntos
Prolina Oxidase/metabolismo , Prolina/biossíntese , Animais , Biocatálise , Humanos , Modelos Moleculares , Estrutura Molecular , Prolina/química , Prolina Oxidase/química
13.
Antioxid Redox Signal ; 30(4): 635-649, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28990419

RESUMO

SIGNIFICANCE: It is increasingly clear that proline metabolism plays an important role in metabolic reprogramming, not only in cancer but also in related fields such as aging, senescence, and development. Although first focused on proline catabolism, recent studies from a number of laboratories have emphasized the regulatory effects of proline synthesis and proline cycling. Recent Advances: Although proline dehydrogenase/proline oxidase (PRODH/POX) has been known as a tumor protein 53 (P53)-activated source of redox signaling for initiating apoptosis and autophagy, senescence has been added to the responses. On the biosynthetic side, two well-recognized oncogenes, c-MYC and phosphoinositide 3-kinase (PI3K), markedly upregulate enzymes of proline synthesis; mechanisms affected include augmented redox cycling and maintenance of pyridine nucleotides. The reprogramming has been shown to shift in clonogenesis and/or metastasis. CRITICAL ISSUES: Although PRODH/POX generates reactive oxygen species (ROS) for signaling, the cellular endpoint is variable and dependent on metabolic context; the switches for these responses remain unknown. On the synthetic side, the enzymes require more complete characterization in various cancers, and demonstration of coupling of proline metabolites to other pathways may require studies of protein-protein interactions, membrane transporters, and shuttles. FUTURE DIRECTIONS: The proline metabolic axis can serve as a scaffold on which a variety of regulatory mechanisms are integrated. Once understood as a central mechanism in cancer metabolism, proline metabolism may be a good target for adjunctive cancer therapy.


Assuntos
Neoplasias/metabolismo , Prolina/metabolismo , Humanos , Neoplasias/patologia , Oxirredução , Prolina/química , Prolina Oxidase/metabolismo , Proteína Supressora de Tumor p53/metabolismo
14.
Antonie Van Leeuwenhoek ; 112(2): 237-251, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30159626

RESUMO

Paenarthrobacter aurescens (formerly called Arthrobacter aurescens) strain TC1 is a high G + C Gram-positive aerobic bacterium that can degrade the herbicide atrazine. Analysis of its genome indicated strain TC1 has the potential to form a bifunctional PutA protein containing L-proline dehydrogenase and L-glutamate-γ-semialdehyde dehydrogenase (L-Δ1-pyrroline-5-carboxylate dehydrogenase) activities. P. aurescens strain TC1 grew well in minimal media with L-Proline as a supplemental nutrient, the nitrogen source, or the sole carbon and nitrogen source. Multicellular myceloids induced by NaCl or citrate also grew on L-proline. The specific activity of L-proline dehydrogenase in whole cells was higher whenever L-proline was added to the medium. Both L-proline dehydrogenase and L-glutamate-γ-semialdehyde dehydrogenase (L-Δ1-pyrroline-5-carboxylate dehydrogenase) activities were found primarily in a membrane fraction from exponential-phase cells. The two activities co-eluted from a Bio-Gel P-60 column after precipitation of proteins with ammonium sulfate and solubilization with 0.1% Tween 20. The PutA protein in the active fraction also oxidized 3,4-dehydro-DL-proline, but there was no activity with other L-proline analogues. When P. aurescens strain TC1 was grown in minimal media containing increasing concentrations of NaCl, there was a progressive decrease in the specific activity of L-proline dehydrogenase and a concomitant increase in the intracellular concentration of L-proline. These results indicate that P. aurescens strain TC1 can use L-proline as a nutrient in a regulated fashion. Because this bacterium also showed the ability to degrade most of the other common amino acids, it can serve as a useful model for the control of amino acid catabolism in the high G + C Actinobacteria.


Assuntos
Arthrobacter/genética , Arthrobacter/metabolismo , Prolina/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Composição de Bases , Prolina Oxidase/genética , Prolina Oxidase/metabolismo , Cloreto de Sódio/metabolismo
15.
Mol Biol Rep ; 45(6): 1821-1825, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30143972

RESUMO

To utilize amino acids from food waste as an energy source, L-proline/O2 biofuel cell was constructed using a stable enzyme from hyperthermophilic archaeon for long-term operation. On the anode, the electrocatalytic oxidation of L-proline by L-proline dehydrogenase from Aeropyrum pernix was observed in the presence of ferrocenecarboxylic acid as mediator. On the cathode, electrocatalytic oxygen reduction was detected. Ketjenblack modification of carbon cloth substrate increased the current density due to increased laccase loading and enhanced electron transfer reaction. The biofuel cell using these electrodes achieved a current density of 6.00 µA/cm2. We successfully constructed the first biofuel cell that generates power from L-proline.


Assuntos
Aeropyrum/metabolismo , Biocombustíveis/microbiologia , Prolina Oxidase/metabolismo , Fontes de Energia Bioelétrica , Eletrodos , Compostos Ferrosos/metabolismo , Lacase/química , Metalocenos , Oxirredução , Oxigênio/metabolismo , Prolina/metabolismo , Prolina Oxidase/fisiologia
16.
ACS Chem Neurosci ; 9(9): 2101-2113, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30044078

RESUMO

The potential role in neuropsychiatric disease risk arising from alterations and derangements of endogenous small-molecule metabolites remains understudied. Alterations of endogenous metabolite concentrations can arise in multiple ways. Marked derangements of single endogenous small-molecule metabolites are found in a large group of rare genetic human diseases termed "inborn errors of metabolism", many of which are associated with prominent neuropsychiatric symptomology. Whether such metabolites act neuroactively to directly lead to distinct neural dysfunction has been frequently hypothesized but rarely demonstrated unequivocally. Here we discuss this disease concept in the context of our recent findings demonstrating that neural dysfunction arising from accumulation of the schizophrenia-associated metabolite l-proline is due to its structural mimicry of the neurotransmitter GABA that leads to alterations in GABA-ergic short-term synaptic plasticity. For cases in which a similar direct action upon neurotransmitter binding sites is suspected, we lay out a systematic approach that can be extended to assessing the potential disruptive action of such candidate disease metabolites. To address the potentially important and broader role in neuropsychiatric disease, we also consider whether the more subtle yet more ubiquitous variations in endogenous metabolites arising from natural allelic variation may likewise contribute to disease risk but in a more complex and nuanced manner.


Assuntos
Erros Inatos do Metabolismo/metabolismo , Prolina/metabolismo , Ácido gama-Aminobutírico/metabolismo , Síndrome de DiGeorge/metabolismo , Síndrome de DiGeorge/psicologia , Humanos , Erros Inatos do Metabolismo/psicologia , Metabolômica , Mimetismo Molecular , Plasticidade Neuronal , Neurotransmissores , Prolina Oxidase/metabolismo , Esquizofrenia/metabolismo , Psicologia do Esquizofrênico
17.
Cell Rep ; 23(13): 3960-3974, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29949777

RESUMO

Proline accumulation is one of the most important adaptation mechanisms for plants to cope with environmental stresses, such as drought and freezing. However, the molecular mechanism of proline homeostasis under these stresses is largely unknown. Here, we identified a mitochondrial protein, DFR1, involved in the inhibition of proline degradation in Arabidopsis. DFR1 was strongly induced by drought and cold stresses. The dfr1 knockdown mutants showed hypersensitivity to drought and freezing stresses, whereas the DFR1 overexpression plants exhibited enhanced tolerance, which was positively correlated with proline levels. DFR1 interacts with proline degradation enzymes PDH1/2 and P5CDH and compromises their activities. Genetic analysis showed that DFR1 acts upstream of PDH1/2 and P5CDH to positively regulate proline accumulation. Our results demonstrate a regulatory mechanism by which, under drought and freezing stresses, DFR1 interacts with PDH1/2 and P5CDH to abrogate their activities to maintain proline homeostasis, thereby conferring drought and freezing tolerance.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Secas , Proteínas Mitocondriais/metabolismo , Prolina/metabolismo , 1-Pirrolina-5-Carboxilato Desidrogenase/química , 1-Pirrolina-5-Carboxilato Desidrogenase/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Congelamento , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Prolina Oxidase/química , Prolina Oxidase/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , Estresse Fisiológico
18.
PLoS One ; 13(4): e0196283, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29694413

RESUMO

L-Proline is a multifunctional amino acid that plays an essential role in primary metabolism and physiological functions. Proline is oxidized to glutamate in the mitochondria and the FAD-containing enzyme proline oxidase (PO) catalyzes the first step in L-proline degradation pathway. Alterations in proline metabolism have been described in various human diseases, such as hyperprolinemia type I, velo-cardio-facial syndrome/Di George syndrome, schizophrenia and cancer. In particular, the mutation giving rise to the substitution Leu441Pro was identified in patients suffering of schizophrenia and hyperprolinemia type I. Here, we report on the expression of wild-type and L441P variants of human PO in a U87 glioblastoma human cell line in an attempt to assess their effect on glutamate metabolism. The subcellular localization of the flavoenzyme is not altered in the L441P variant, for which specific activity is halved compared to the wild-type PO. While this decrease in activity is significantly less than that previously proposed, an effect of the substitution on the enzyme stability is also apparent in our studies. At 24 hours of growth from transient transfection, the intracellular level of proline, glutamate, and glutamine is decreased in cells expressing the PO variants as compared to control U87 cells, reaching a similar figure at 72 h. On the other hand, the extracellular levels of the three selected amino acids show a similar time course for all clones. Furthermore, PO overexpression does not modify to a significant extent the expression of GLAST and GLT-1 glutamate transporters. Altogether, these results demonstrate that the proline pathway links cellular proline levels with those of glutamate and glutamine. On this side, PO might play a regulatory role in glutamatergic neurotransmission by affecting the cellular concentration of glutamate.


Assuntos
Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Prolina Oxidase/metabolismo , Prolina/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Regulação para Baixo , Transportador 1 de Aminoácido Excitatório/genética , Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Glioblastoma , Ácido Glutâmico/análise , Glutamina/análise , Humanos , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Mutagênese Sítio-Dirigida , Prolina/análise , Prolina Oxidase/genética
19.
Biochemistry ; 57(25): 3433-3444, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29648801

RESUMO

Interest in how proline contributes to cancer biology is expanding because of the emerging role of a novel proline metabolic cycle in cancer cell survival, proliferation, and metastasis. Proline biosynthesis and degradation involve the shared intermediate Δ1-pyrroline-5-carboxylate (P5C), which forms l-glutamate-γ-semialdehyde (GSAL) in a reversible non-enzymatic reaction. Proline is synthesized from glutamate or ornithine through GSAL/P5C, which is reduced to proline by P5C reductase (PYCR) in a NAD(P)H-dependent reaction. The degradation of proline occurs in the mitochondrion and involves two oxidative steps catalyzed by proline dehydrogenase (PRODH) and GSAL dehydrogenase (GSALDH). PRODH is a flavin-dependent enzyme that couples proline oxidation with reduction of membrane-bound quinone, while GSALDH catalyzes the NAD+-dependent oxidation of GSAL to glutamate. PRODH and PYCR form a metabolic relationship known as the proline-P5C cycle, a novel pathway that impacts cellular growth and death pathways. The proline-P5C cycle has been implicated in supporting ATP production, protein and nucleotide synthesis, anaplerosis, and redox homeostasis in cancer cells. This Perspective details the structures and reaction mechanisms of PRODH and PYCR and the role of the proline-P5C cycle in cancer metabolism. A major challenge in the field is to discover inhibitors that specifically target PRODH and PYCR isoforms for use as tools for studying proline metabolism and the functions of the proline-P5C cycle in cancer. These molecular probes could also serve as lead compounds in cancer drug discovery targeting the proline-P5C cycle.


Assuntos
Neoplasias/metabolismo , Prolina/metabolismo , Animais , Vias Biossintéticas , Proliferação de Células , Humanos , Simulação de Acoplamento Molecular , Oxirredução , Prolina Oxidase/metabolismo , Pirrolina Carboxilato Redutases/metabolismo
20.
Molecules ; 23(1)2018 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-29337919

RESUMO

Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme that catalyzes the oxidation of proline to Δ¹-pyrroline-5-carboxylate. Thermus thermophilus ProDH (TtProDH) contains in addition to its flavin-binding domain an N-terminal arm, consisting of helices αA, αB, and αC. Here, we report the biochemical properties of the helical arm truncated TtProDH variants ΔA, ΔAB, and ΔABC, produced with maltose-binding protein as solubility tag. All three truncated variants show similar spectral properties as TtProDH, indicative of a conserved flavin-binding pocket. ΔA and ΔAB are highly active tetramers that rapidly react with the suicide inhibitor N-propargylglycine. Removal of the entire N-terminal arm (ΔABC) results in barely active dimers that are incapable of forming a flavin adduct with N-propargylglycine. Characterization of V32D, Y35F, and V36D variants of ΔAB established that a hydrophobic patch between helix αC and helix α8 is critical for TtProDH catalysis and tetramer stabilization.


Assuntos
Prolina Oxidase/química , Prolina Oxidase/metabolismo , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , Catálise , Ativação Enzimática , Expressão Gênica , Hidrodinâmica , Modelos Anatômicos , Estrutura Molecular , Prolina Oxidase/genética , Prolina Oxidase/isolamento & purificação , Conformação Proteica , Engenharia de Proteínas , Multimerização Proteica , Análise Espectral , Thermus thermophilus/genética
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