RESUMO
Δ1-pyrroline-5-carboxylate reductase isoform 1 (PYCR1) is the last enzyme of proline biosynthesis and catalyzes the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate to L-proline. High PYCR1 gene expression is observed in many cancers and linked to poor patient outcomes and tumor aggressiveness. The knockdown of the PYCR1 gene or the inhibition of PYCR1 enzyme has been shown to inhibit tumorigenesis in cancer cells and animal models of cancer, motivating inhibitor discovery. We screened a library of 71 low molecular weight compounds (average MW of 131 Da) against PYCR1 using an enzyme activity assay. Hit compounds were validated with X-ray crystallography and kinetic assays to determine affinity parameters. The library was counter-screened against human Δ1-pyrroline-5-carboxylate reductase isoform 3 and proline dehydrogenase (PRODH) to assess specificity/promiscuity. Twelve PYCR1 and one PRODH inhibitor crystal structures were determined. Three compounds inhibit PYCR1 with competitive inhibition parameter of 100 µM or lower. Among these, (S)-tetrahydro-2H-pyran-2-carboxylic acid (70 µM) has higher affinity than the current best tool compound N-formyl-l-proline, is 30 times more specific for PYCR1 over human Δ1-pyrroline-5-carboxylate reductase isoform 3, and negligibly inhibits PRODH. Structure-affinity relationships suggest that hydrogen bonding of the heteroatom of this compound is important for binding to PYCR1. The structures of PYCR1 and PRODH complexed with 1-hydroxyethane-1-sulfonate demonstrate that the sulfonate group is a suitable replacement for the carboxylate anchor. This result suggests that the exploration of carboxylic acid isosteres may be a promising strategy for discovering new classes of PYCR1 and PRODH inhibitors. The structure of PYCR1 complexed with l-pipecolate and NADH supports the hypothesis that PYCR1 has an alternative function in lysine metabolism.
Assuntos
Inibidores Enzimáticos , Prolina , Pirrolina Carboxilato Redutases , delta-1-Pirrolina-5-Carboxilato Redutase , Pirrolina Carboxilato Redutases/metabolismo , Pirrolina Carboxilato Redutases/antagonistas & inibidores , Pirrolina Carboxilato Redutases/química , Pirrolina Carboxilato Redutases/genética , Humanos , Cristalografia por Raios X , Prolina/química , Prolina/análogos & derivados , Prolina/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Peso Molecular , Prolina Oxidase/metabolismo , Prolina Oxidase/química , Prolina Oxidase/antagonistas & inibidores , Prolina Oxidase/genética , Modelos MolecularesRESUMO
Hepatitis C virus (HCV) is an oncogenic virus that causes chronic liver disease in more than 80% of patients. During the last decade, efficient direct-acting antivirals were introduced into clinical practice. However, clearance of the virus does not reduce the risk of end-stage liver diseases to the level observed in patients who have never been infected. So, investigation of HCV pathogenesis is still warranted. Virus-induced changes in cell metabolism contribute to the development of HCV-associated liver pathologies. Here, we studied the impact of the virus on the metabolism of polyamines and proline as well as on the urea cycle, which plays a crucial role in liver function. It was found that HCV strongly suppresses the expression of arginase, a key enzyme of the urea cycle, leading to the accumulation of arginine, and up-regulates proline oxidase with a concomitant decrease in proline concentrations. The addition of exogenous proline moderately suppressed viral replication. HCV up-regulated transcription but suppressed protein levels of polyamine-metabolizing enzymes. This resulted in a decrease in polyamine content in infected cells. Finally, compounds targeting polyamine metabolism demonstrated pronounced antiviral activity, pointing to spermine and spermidine as compounds affecting HCV replication. These data expand our understanding of HCV's imprint on cell metabolism.
Assuntos
Hepacivirus , Poliaminas , Prolina , Ureia , Replicação Viral , Prolina/metabolismo , Humanos , Hepacivirus/fisiologia , Hepacivirus/efeitos dos fármacos , Poliaminas/metabolismo , Ureia/metabolismo , Ureia/farmacologia , Replicação Viral/efeitos dos fármacos , Arginase/metabolismo , Antivirais/farmacologia , Antivirais/metabolismo , Hepatite C/metabolismo , Hepatite C/virologia , Linhagem Celular Tumoral , Prolina Oxidase/metabolismoRESUMO
Metabolic reprogramming is critical in the onset of pressure overload-induced cardiac remodeling. Our study reveals that proline dehydrogenase (PRODH), the key enzyme in proline metabolism, reprograms cardiomyocyte metabolism to protect against cardiac remodeling. We induced cardiac remodeling using transverse aortic constriction (TAC) in both cardiac-specific PRODH knockout and overexpression mice. Our results indicate that PRODH expression is suppressed after TAC. Cardiac-specific PRODH knockout mice exhibited worsened cardiac dysfunction, while mice with PRODH overexpression demonstrated a protective effect. In addition, we simulated cardiomyocyte hypertrophy in vitro using neonatal rat ventricular myocytes treated with phenylephrine. Through RNA sequencing, metabolomics, and metabolic flux analysis, we elucidated that PRODH overexpression in cardiomyocytes redirects proline catabolism to replenish tricarboxylic acid cycle intermediates, enhance energy production, and restore glutathione redox balance. Our findings suggest PRODH as a modulator of cardiac bioenergetics and redox homeostasis during cardiac remodeling induced by pressure overload. This highlights the potential of PRODH as a therapeutic target for cardiac remodeling.
Assuntos
Camundongos Knockout , Miócitos Cardíacos , Prolina , Remodelação Ventricular , Animais , Prolina/metabolismo , Miócitos Cardíacos/metabolismo , Camundongos , Ratos , Prolina Oxidase/metabolismo , Prolina Oxidase/genética , Metabolismo Energético , Miocárdio/metabolismo , Miocárdio/patologia , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Cardiomegalia/etiologia , Modelos Animais de Doenças , Oxirredução , Masculino , Reprogramação MetabólicaRESUMO
Naive human embryonic stem cells (hESCs) that resemble the pre-implantation epiblasts are fueled by a combination of aerobic glycolysis and oxidative phosphorylation, but their mitochondrial regulators are poorly understood. Here we report that, proline dehydrogenase (PRODH), a mitochondria-localized proline metabolism enzyme, is dramatically upregulated in naive hESCs compared to their primed counterparts. The upregulation of PRODH is induced by a reduction in c-Myc expression that is dependent on PD0325901, a MEK inhibitor routinely present in naive hESC culture media. PRODH knockdown in naive hESCs significantly promoted mitochondrial oxidative phosphorylation (mtOXPHOS) and reactive oxygen species (ROS) production that triggered autophagy, DNA damage, and apoptosis. Remarkably, MitoQ, a mitochondria-targeted antioxidant, effectively restored the pluripotency and proliferation of PRODH-knockdown naive hESCs, indicating that PRODH maintains naive pluripotency by preventing excessive ROS production. Concomitantly, PRODH knockdown significantly slowed down the proteolytic degradation of multiple key mitochondrial electron transport chain complex proteins. Thus, we revealed a crucial role of PRODH in limiting mtOXPHOS and ROS production, and thereby safeguarding naive pluripotency of hESCs.
Assuntos
Fosforilação Oxidativa , Prolina Oxidase , Humanos , Espécies Reativas de Oxigênio/metabolismo , Prolina Oxidase/genética , Prolina Oxidase/metabolismo , Mitocôndrias/metabolismo , ApoptoseRESUMO
Proline dehydrogenase (ProDH) and pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) catalyse the oxidation of proline into glutamate via the intermediates P5C and glutamate-semialdehyde (GSA), which spontaneously interconvert. P5C and GSA are also intermediates in the production of glutamate from ornithine and α-ketoglutarate catalysed by ornithine δ-aminotransferase (OAT). ProDH and P5CDH form a fused bifunctional PutA enzyme in Gram-negative bacteria and are associated in a bifunctional substrate-channelling complex in Thermus thermophilus; however, the physical proximity of ProDH and P5CDH in eukaryotes has not been described. Here, we report evidence of physical proximity and interactions between Arabidopsis ProDH, P5CDH, and OAT in the mitochondria of plants during dark-induced leaf senescence when all three enzymes are expressed. Pairwise interactions and localization of the three enzymes were investigated using bimolecular fluorescence complementation with confocal microscopy in tobacco and sub-mitochondrial fractionation in Arabidopsis. Evidence for a complex composed of ProDH, P5CDH, and OAT was revealed by co-migration of the proteins in native conditions upon gel electrophoresis. Co-immunoprecipitation coupled with mass spectrometry analysis confirmed the presence of the P5C metabolism complex in Arabidopsis. Pull-down assays further demonstrated a direct interaction between ProDH1 and P5CDH. P5C metabolism complexes might channel P5C among the constituent enzymes and directly provide electrons to the respiratory electron chain via ProDH.
Assuntos
Arabidopsis , Pirróis , Arabidopsis/metabolismo , Prolina Oxidase/química , Prolina Oxidase/metabolismo , Mitocôndrias/metabolismo , Glutamatos/metabolismo , Ornitina/metabolismo , Prolina/metabolismoRESUMO
Previous findings have shown that phospholipase D (PLD) contributes to the response to long-term chilling stress in barley by regulating the balance of proline (Pro) levels. Although Pro accumulation is one of the most prominent changes in barley roots exposed to this kind of stress, the regulation of its metabolism during recovery from stress remains unclear. Research has mostly focused on the responses to stress per se, and not much is known about the dynamics and mechanisms underlying the subsequent recovery. The present study aimed to evaluate how PLD, its product phosphatidic acid (PA), and diacylglycerol pyrophosphate (DGPP) modulate Pro accumulation in barley during recovery from long-term chilling stress. Pro metabolism involves different pathways and enzymes. The rate-limiting step is mediated by pyrroline-5-carboxylate synthetase (P5CS) in its biosynthesis, and by proline dehydrogenase (ProDH) in its catabolism. We observed that Pro levels decreased in recovering barley roots due to an increase in ProDH activity. The addition of 1-butanol, a PLD inhibitor, reverted this effect and altered the relative gene expression of ProDH. When barley tissues were treated with PA before recovery, the fresh weight of roots increased and ProDH activity was stimulated. These data contribute to our understanding of how acidic membrane phospholipids like PA help to control Pro degradation during recovery from stress.
Assuntos
Hordeum , Hordeum/metabolismo , Resposta ao Choque Frio , Transdução de Sinais , Prolina Oxidase/metabolismo , Ácidos Fosfatídicos/metabolismo , Prolina/metabolismoRESUMO
Regulation of the proline metabolic pathway is essential for the accumulation of proline under abiotic stress and for the amelioration of plant stress resistance. Δ1-pyrroline-5-carboxylate synthase (P5CS), pyrroline-5-carboxylate reductase (P5CR), ornithine transaminase (δ-OAT), proline dehydrogenase (PDH), pyrroline-5-carboxylate dehydrogenase (P5CDH), and proline transporter (ProT) are the key enzymes in the proline metabolic pathway. However, the gene families responsible for proline metabolism have not yet been identified or reported in alfalfa. In this study, a total of 12 MsP5CSs, 4 MsP5CRs, 3 MsOATs, 6 MsPDHs, 2 MsP5CDHs, and 5 MsProTs were identified in the genome of alfalfa, and the members of the same subfamily had similar gene structures and conserved motifs. Analysis of cis-regulatory elements revealed the presence of light-responsive, hormone-regulated, and stress-responsive elements in the promoter regions of alfalfa proline metabolism-related genes. Following treatment with saline-alkali, the expression of MsP5CSs, MsP5CRs, MsOATs, and MsProTs was significantly upregulated, whereas the expression of MsPDH1.1, MsPDH1.3, and MsP5CDH was significantly downregulated. The proline content and enzyme activity of P5CS gradually increased, whereas the enzyme activity of PDH gradually decreased as the duration of stress increased. Root growth rates decreased upon MsP5CS1a suppression (MsP5CS1a-RNAi) in the hairy roots of alfalfa compared to the empty vector line under saline-alkali stress. These results show that proline metabolism-related genes play an important role in the saline-alkali stress tolerance of alfalfa and provide a theoretical basis for further research on the functions of proline metabolism-related genes in alfalfa in response to saline-alkali stress.
Assuntos
Medicago sativa , Prolina Oxidase , Medicago sativa/genética , Medicago sativa/metabolismo , Prolina Oxidase/genética , Prolina Oxidase/metabolismo , Pirrolina Carboxilato Redutases , Prolina/metabolismo , Biologia Computacional , Estresse Fisiológico/genéticaRESUMO
L-Proline dehydrogenase (ProDH) is a flavin-dependent oxidoreductase, which catalyzes the oxidation of L-proline to (S)-1-pyrroline-5-carboxylate. Based on the experimental studies, a stepwise proton and hydride transfer mechanism is supported. According to this mechanism, the amino group of L-proline is deprotonated by a nearby Lys residue, which is followed by the hydride transfer process from C5 position of L-proline to N5 position of isoalloxazine ring of FAD. It was concluded that the hydride transfer step is rate limiting in the reductive half-reaction, however, in the overall reaction, the oxidation of FAD is the rate limiting step. In this study, we performed a computational mechanistic investigation based on ONIOM method to elucidate the mechanism of the reductive half-reaction corresponding to the oxidation of L-proline into iminoproline. Our calculations support the stepwise mechanism in which the deprotonation occurs initially as a fast step as result of a proton transfer from L-proline to the Lys residue. Subsequently, a hydride ion transfers from L-proline to FAD with a higher activation barrier. The enzyme-product complex showed a strong interaction between reduced FAD and iminoproline, which might help to explain why a step in the oxidative half-reaction is rate-limiting.
Assuntos
Prolina Oxidase , Prótons , Prolina Oxidase/genética , Prolina Oxidase/metabolismo , Oxirredutases , Oxirredução , Prolina , Cinética , Flavina-Adenina Dinucleotídeo/metabolismoRESUMO
It is known that metabolic defects in the retinal pigment epithelium (RPE) can cause degeneration of its neighboring photoreceptors in the retina, leading to retinal degenerative diseases such as age-related macular degeneration. However, how RPE metabolism supports the health of the neural retina remains unclear. The retina requires exogenous nitrogen sources for protein synthesis, neurotransmission, and energy metabolism. Using 15N tracing coupled with mass spectrometry, we found human RPE can utilize the nitrogen in proline to produce and export 13 amino acids, including glutamate, aspartate, glutamine, alanine, and serine. Similarly, we found this proline nitrogen utilization in the mouse RPE/choroid but not in the neural retina of explant cultures. Coculture of human RPE with the retina showed that the retina can take up the amino acids, especially glutamate, aspartate, and glutamine, generated from proline nitrogen in the RPE. Intravenous delivery of 15N proline in vivo demonstrated 15N-derived amino acids appear earlier in the RPE before the retina. We also found proline dehydrogenase, the key enzyme in proline catabolism is highly enriched in the RPE but not the retina. The deletion of proline dehydrogenase blocks proline nitrogen utilization in RPE and the import of proline nitrogen-derived amino acids in the retina. Our findings highlight the importance of RPE metabolism in supporting nitrogen sources for the retina, providing insight into understanding the mechanisms of the retinal metabolic ecosystem and RPE-initiated retinal degenerative diseases.
Assuntos
Aminoácidos , Epitélio Pigmentado da Retina , Animais , Humanos , Camundongos , Aminoácidos/metabolismo , Ácido Aspártico/metabolismo , Glutamatos/metabolismo , Glutamina/metabolismo , Nitrogênio/metabolismo , Prolina/metabolismo , Prolina Oxidase/metabolismo , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismoRESUMO
Amino acids in the bone marrow microenvironment (BMME) are a critical factor for multiple myeloma (MM) progression. Here, we have determined that proline is elevated in BMME of MM patients and links to poor prognosis in MM. Moreover, exogenous proline regulates MM cell proliferation and drug resistance. Elevated proline in BMME is due to bone collagen degradation and abnormal expression of the key enzyme of proline catabolism, proline dehydrogenase (PRODH). PRODH is downregulated in MM patients, mainly as a result of promoter hypermethylation with high expression of DNMT3b. Thus, overexpression of PRODH suppresses cell proliferation and drug resistance of MM and exhibits therapeutic potential for treatment of MM. Altogether, we identify proline as a key metabolic regulator of MM, unveil PRODH governing MM progression and provide a promising therapeutic strategy for MM treatment.
Assuntos
Mieloma Múltiplo , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Prolina Oxidase/genética , Prolina Oxidase/metabolismo , Prolina/metabolismo , Regulação para Baixo , Resistência a Medicamentos , Proliferação de Células , Microambiente TumoralRESUMO
Proline is an amino acid that is degraded in the mitochondria by the sequential action of proline dehydrogenase (ProDH) and pyrroline-5-carboxylate dehydrogenase (P5CDH) to form glutamate. We investigated the phenotypes of Arabidopsis wild-type plants, the knockout prodh1 prodh2 double-mutant, and knockout p5cdh allelic mutants grown at low and high nitrate supplies. Surprisingly, only p5cdh presented lower seed yield and produced lighter seeds. Analyses of elements in above-ground organs revealed lower C concentrations in the p5cdh seeds. Determination of C, N, and dry matter partitioning among the above-ground organs revealed a major defect in stem-to-seed resource allocations in this mutant. Again surprisingly, defects in C, N, and biomass allocation to seeds dramatically increased in high-N conditions. 15N-labelling consistently confirmed the defect in N remobilization from the rosette and stem to seeds in p5cdh. Consequently, the p5cdh mutants produced morphologically abnormal, C-depleted seeds that displayed very low germination rates. The most striking result was the strong amplification of the N-remobilization defects in p5cdh under high nitrate supply, and interestingly this phenotype was not observed in the prodh1 prodh2 double-mutant irrespective of nitrate supply. This study reveals an essential role of P5CDH in carbon and nitrogen remobilization for reserve accumulation during seed development in Arabidopsis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Carbono/metabolismo , Nitratos/metabolismo , Nitrogênio/metabolismo , Plantas/metabolismo , Prolina Oxidase/genética , Prolina Oxidase/metabolismo , SementesRESUMO
During leaf senescence, nitrogen is remobilized and carbon backbones are replenished by amino acid catabolism, with many of the key reactions occurring in mitochondria. The intermediate Δ1 -pyrroline-5-carboxylate (P5C) is common to some catabolic pathways, thus linking the metabolism of several amino acids, including proline and arginine. Specifically, mitochondrial proline catabolism involves sequential action of proline dehydrogenase (ProDH) and P5C dehydrogenase (P5CDH) to produce P5C and then glutamate. Arginine catabolism produces urea and ornithine, the latter in the presence of α-ketoglutarate being converted by ornithine δ-aminotransferase (OAT) into P5C and glutamate. Metabolic changes during dark-induced leaf senescence (DIS) were studied in Arabidopsis thaliana leaves of Col-0 and in prodh1prodh2, p5cdh and oat mutants. Progression of DIS was followed by measuring chlorophyll and proline contents for 5 days. Metabolomic profiling of 116 compounds revealed similar profiles of Col-0 and oat metabolism, distinct from prodh1prodh2 and p5cdh metabolism. Metabolic dynamics were accelerated in p5cdh by 1 day. Notably, more P5C and proline accumulated in p5cdh than in prodh1prodh2. ProDH1 enzymatic activity and protein amount were significantly down-regulated in p5cdh mutant at Day 4 of DIS. Mitochondrial P5C levels appeared critical in determining the flow through interconnected amino acid remobilization pathways to sustain senescence.
Assuntos
Arabidopsis , Aminoácidos/metabolismo , Arabidopsis/metabolismo , Arginina/metabolismo , Glutamatos/metabolismo , Ornitina/metabolismo , Prolina/metabolismo , Prolina Oxidase/genética , Prolina Oxidase/metabolismoRESUMO
Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of l-proline to Δ1-pyrroline-5-carboxylate and is a target for inhibitor discovery because of its importance in cancer cell metabolism. Because human PRODH is challenging to purify, the PRODH domains of the bacterial bifunctional enzyme proline utilization A (PutA) have been used for inhibitor development. These systems have limitations due to large polypeptide chain length, conformational flexibility and the presence of domains unrelated to PRODH activity. Herein, we report the engineering of minimal PRODH domains for inhibitor discovery. The best designs contain one-third of the 1233-residue PutA from Sinorhizobium meliloti and include a linker that replaces the PutA α-domain. The minimal PRODHs exhibit near wild-type enzymatic activity and are susceptible to known inhibitors and inactivators. Crystal structures of minimal PRODHs inhibited by S-(-)-tetrahydro-2-furoic acid and 2-(furan-2-yl)acetic acid were determined at 1.23 and 1.72 Å resolution. Minimal PRODHs should be useful in chemical probe discovery.
Assuntos
Prolina Oxidase , Prolina , Humanos , Prolina Oxidase/genética , Prolina Oxidase/química , Prolina Oxidase/metabolismo , Prolina/química , Prolina/metabolismo , Proteínas de Bactérias/químicaRESUMO
Hyperprolinemia type II (HPII) is an inborn error of metabolism due to genetic variants in ALDH4A1, leading to a deficiency in Δ-1-pyrroline-5-carboxylate (P5C) dehydrogenase. This leads to an accumulation of toxic levels of P5C, an intermediate in proline catabolism. The accumulating P5C spontaneously reacts with, and inactivates, pyridoxal 5'-phosphate, a crucial cofactor for many enzymatic processes, which is thought to be the pathophysiological mechanism for HPII. Here, we describe the use of a combination of LC-QTOF untargeted metabolomics, NMR spectroscopy and infrared ion spectroscopy (IRIS) to identify and characterize biomarkers for HPII that result of the spontaneous reaction of P5C with malonic acid and acetoacetic acid. We show that these biomarkers can differentiate between HPI, caused by a deficiency of proline oxidase activity, and HPII. The elucidation of their molecular structures yields insights into the disease pathophysiology of HPII.
Assuntos
Prolina Oxidase , Prolina , 1-Pirrolina-5-Carboxilato Desidrogenase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos , Biomarcadores , Fosfatos , Prolina/metabolismo , Prolina Oxidase/genética , Prolina Oxidase/metabolismo , Piridoxal , PirróisRESUMO
Previous reports indicate that proline utilization A (PutA) is involved in the oxidation of proline to glutamate in many bacteria. We demonstrate here that in addition to its role in proline catabolism, PutA acts as a global regulator to control the important biological functions and virulence of Ralstonia solanacearum. PutA regulates target gene expression levels by directly binding to promoter DNA, and its regulatory activity is enhanced by L-proline. Intriguingly, we reveal that the cofactors NAD+ and FAD boost the enzymatic activity of PutA for converting L-proline to L-glutamic acid but inhibit the regulatory activity of PutA for controlling target gene expression. Our results present evidence that PutA is a proline metabolic enzyme that also functions as a global transcriptional regulator in response to its substrate and cofactors and provide insights into the complicated regulatory mechanism of PutA in bacterial physiology and pathogenicity.
Assuntos
Prolina Oxidase , Prolina , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Prolina/genética , Prolina Oxidase/genética , Prolina Oxidase/metabolismo , VirulênciaRESUMO
The oxidation of proline to pyrroline-5-carboxylate (P5C) leads to the transfer of electrons to ubiquinone in mitochondria that express proline dehydrogenase (ProDH). This electron transfer supports Complexes CIII and CIV, thus generating the protonmotive force. Further catabolism of P5C forms glutamate, which fuels the citric acid cycle that yields the reducing equivalents that sustain oxidative phosphorylation. However, P5C and glutamate catabolism depend on CI activity due to NAD+ requirements. NextGen-O2k (Oroboros Instruments) was used to measure proline oxidation in isolated mitochondria of various mouse tissues. Simultaneous measurements of oxygen consumption, membrane potential, NADH, and the ubiquinone redox state were correlated to ProDH activity and F1FO-ATPase directionality. Proline catabolism generated a sufficiently high membrane potential that was able to maintain the F1FO-ATPase operation in the forward mode. This was observed in CI-inhibited mouse liver and kidney mitochondria that exhibited high levels of proline oxidation and ProDH activity. This action was not observed under anoxia or when either CIII or CIV were inhibited. The duroquinone fueling of CIII and CIV partially reproduced the effects of proline. Excess glutamate, however, could not reproduce the proline effect, suggesting that processes upstream of the glutamate conversion from proline were involved. The ProDH inhibitors tetrahydro-2-furoic acid and, to a lesser extent, S-5-oxo-2-tetrahydrofurancarboxylic acid abolished all proline effects. The data show that ProDH-directed proline catabolism could generate sufficient CIII and CIV proton pumping, thus supporting ATP production by the F1FO-ATPase even under CI inhibition.
Assuntos
Prolina Oxidase , Ubiquinona , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Complexo I de Transporte de Elétrons/metabolismo , Ácido Glutâmico/metabolismo , Camundongos , Mitocôndrias/metabolismo , Prolina/metabolismo , Prolina Oxidase/metabolismo , Ubiquinona/metabolismoRESUMO
Non-steroidal anti-inflammatory drugs (NSAIDs) are considered in cancer therapy for their inhibitory effect on cyclooxygenase-2 (COX-2), which is overexpressed in most cancers. However, we found that NSAIDs as ligands of peroxisome proliferator-activated receptor-γ (PPARγ)-induced apoptosis independent of the COX-2 inhibition, and the process was mediated through activation of proline dehydrogenase/proline oxidase (PRODH/POX)-dependent generation of reactive oxygen species (ROS). This mitochondrial enzyme converts proline to ∆1-pyrroline-5-carboxylate (P5C) during which ATP or ROS is generated. To confirm the role of PRODH/POX in the mechanism of NSAID-induced apoptosis we obtained an MCF7 CRISPR/Cas9 PRODH/POX knockout breast cancer cell model (MCF7POK-KO). Interestingly, the studied NSAIDs (indomethacin and diclofenac) in MCF7POK-KO cells contributed to a more pronounced pro-apoptotic phenotype of the cells than in PRODH/POX-expressing MCF7 cells. The observed effect was independent of ROS generation, but it was related to the energetic disturbances in the cells as shown by an increase in the expression of AMPKα (sensor of cell energy status), GLUD1/2 (proline producing enzyme from glutamate), prolidase (proline releasing enzyme), PPARδ (growth supporting transcription factor) and a decrease in the expression of proline cycle enzymes (PYCR1, PYCRL), mammalian target of rapamycin (mTOR), and collagen biosynthesis (the main proline utilizing process). The data provide evidence that the studied NSAIDs induce PRODH/POX-dependent and independent apoptosis in MCF7 breast cancer cells.
Assuntos
Neoplasias da Mama , Prolina Oxidase , Anti-Inflamatórios não Esteroides/farmacologia , Apoptose , Neoplasias da Mama/tratamento farmacológico , Ciclo-Oxigenase 2/farmacologia , Feminino , Humanos , Células MCF-7 , Oxirredutases , Prolina/metabolismo , Prolina Oxidase/genética , Prolina Oxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Nonsteroidal anti-inflammatory drugs (NSAIDs) are considered to be therapeutics in cancer prevention because of their inhibitory effect on cyclooxygenases (COX), which are frequently overexpressed in many types of cancer. However, it was also demonstrated that NSAIDs provoked a proapoptotic effect in COX knocked-out cancer cells. Here, we suggest that this group of drugs may provoke antineoplastic activity through the activation of PPARγ, which induces proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis. PRODH/POX is a mitochondrial enzyme that catalyzes proline degradation, during which ATP or reactive oxygen species (ROS) are generated. We have found that NSAIDs induced PRODH/POX and PPARγ expressions (as demonstrated by Western Blot or immunofluorescence analysis) and cytotoxicity (as demonstrated by MTT, cytometric assay, and DNA biosynthesis assay) in breast cancer MCF7 cells. Simultaneously, the NSAIDs inhibited collagen biosynthesis, supporting proline for PRODH/POX-induced ROS-dependent apoptosis (as demonstrated by an increase in the expression of apoptosis markers). The data suggest that targeting proline metabolism and the PRODH/POX-PPARγ axis can be considered a novel approach for breast cancer treatment.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , PPAR gama/metabolismo , Prolina Oxidase/metabolismo , Apoptose , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno/biossíntese , Colágeno/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Fosforilação Oxidativa/efeitos dos fármacos , PPAR gama/agonistas , Prolina/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The role of proline dehydrogenase/proline oxidase (PRODH/POX) in the mechanism of antineoplastic activity of metformin (MET) was studied in C32 melanoma cells. PRODH/POX is a mitochondrial enzyme-degrading proline that is implicated in the regulation of cancer cell survival/apoptosis. The enzyme is activated by AMP kinase (AMPK). It has been found that MET induced a significant decrease in cell viability and DNA biosynthesis accompanied by an increase in the expressions of AMPK and PRODH/POX in C32 cells. The mechanism for MET-dependent cytotoxicity on C32 cells was found at the level of PRODH/POX-induced ROS generation and activation of Caspase-3 and Caspase-9 expressions in these cells. The effects were not observed in MET-treated PRODH/POX knock-out C32 cells. Of interest is an MET-dependent increase in the concentration of proline, which is a substrate for PRODH/POX. This phenomenon is due to the MET-dependent inhibition of collagen biosynthesis, which is the main proline-utilizing process. It has been found that the underlying mechanism of anticancer activity of MET involves the activation of AMPK, PRODH/POX, increase in the cytoplasmic concentration of proline, inhibition of collagen biosynthesis, and stimulation of PRODH/POX-dependent ROS generation, which initiate the apoptosis of melanoma cells.
Assuntos
Apoptose , Melanoma/tratamento farmacológico , Metformina/farmacologia , Prolina Oxidase/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Humanos , Melanoma/enzimologia , Melanoma/fisiopatologia , Metformina/uso terapêutico , Mitocôndrias/enzimologiaRESUMO
Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent oxidation of L-proline to Δ1-pyrroline-5-carboxylate. PRODH plays a central role in the metabolic rewiring of cancer cells, which has motivated the discovery of inhibitors. Here, we studied the inhibition of PRODH by 18 proline-like compounds to understand the structural and chemical features responsible for the affinity of the best-known inhibitor, S-(-)-tetrahydro-2-furoic acid (1). The compounds were screened, and then six were selected for more thorough kinetic analysis: cyclobutane-1,1-dicarboxylic acid (2), cyclobutanecarboxylic acid (3), cyclopropanecarboxylic acid (4), cyclopentanecarboxylic acid (16), 2-oxobutyric acid (17), and (2S)-oxetane-2-carboxylic acid (18). These compounds are competitive inhibitors with inhibition constants in the range of 1.4-6 mM, compared to 0.3 mM for 1. Crystal structures of PRODH complexed with 2, 3, 4, and 18 were determined. All four inhibitors bind in the proline substrate site, but the orientations of their rings differ from that of 1. The binding of 3 and 18 is accompanied by compression of the active site to enable nonpolar contacts with Leu513. Compound 2 is unique in that the additional carboxylate displaces a structurally conserved water molecule from the active site. Compound 18 also destabilizes the conserved water, but by an unexpected non-steric mechanism. The results are interpreted using a chemical double mutant thermodynamic cycle. This analysis revealed unanticipated synergism between ring size and hydrogen bonding to the conserved water. These structure-affinity relationships provide new information relevant to the development of new inhibitor design strategies targeting PRODH.