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1.
Int J Mol Sci ; 25(2)2024 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-38255788

RESUMO

The identification of markers for early diagnosis, prognosis, and improvement of therapeutic options represents an unmet clinical need to increase survival in Non-Small Cell Lung Cancer (NSCLC), a neoplasm still characterized by very high incidence and mortality. Here, we investigated whether proline dehydrogenase (PRODH), a mitochondrial flavoenzyme catalyzing the key step in proline degradation, played a role in NSCLC tumorigenesis. PRODH expression was investigated by immunohistochemistry; digital PCR, quantitative PCR, immunoblotting, measurement of reactive oxygen species (ROS), and functional cellular assays were carried out. PRODH expression was found in the majority of lung adenocarcinomas (ADCs). Patients with PRODH-positive tumors had better cancer-free specific and overall survival compared to those with negative tumors. Ectopic modulation of PRODH expression in NCI-H1299 and the other tested lung ADC cell lines decreased cell survival. Moreover, cell proliferation curves showed delayed growth in NCI-H1299, Calu-6 and A549 cell lines when PRODH-expressing clones were compared to control clones. The 3D growth in soft agar was also impaired in the presence of PRODH. PRODH increased reactive oxygen species production and induced cellular senescence in the NCI-H1299 cell line. This study supports a role of PRODH in decreasing survival and growth of lung ADC cells by inducing cellular senescence.


Assuntos
Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Sobrevivência Celular/genética , Prolina Oxidase/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Espécies Reativas de Oxigênio , Neoplasias Pulmonares/genética , Adenocarcinoma de Pulmão/genética , Senescência Celular/genética
2.
J Exp Bot ; 75(3): 917-934, 2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-37843921

RESUMO

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/metabolismo
3.
Plant Physiol Biochem ; 206: 108208, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38039584

RESUMO

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/metabolismo
4.
G Ital Nefrol ; 40(6)2023 Dec 22.
Artigo em Italiano | MEDLINE | ID: mdl-38156540

RESUMO

Hyperprolinemia is a rare genetic condition due to mutations in proline metabolic pathway. Type I Hyperprolinemia (HPI) typically causes neuropsychiatric disorders, and diagnosis is usually confirmed in pediatric population with suggestive neuropsychiatric involvement by elevated serum proline levels and elevated urinary proline, hydroxyproline, and glycine levels. The possible coexistence of nephropathy in patients with HPI, often specified as malformative urinary disease, is often mentioned. However, reports of HPI diagnosis due to kidney impairment do not exist in scientific literature yet. Here we present the case of a patient presenting with chronic kidney disease secondary to obstructive nephropathy who received a HPI diagnosis in adulthood. Interestingly, the family study showed the same 22q11.21 deletion and elevated blood proline levels in the father, who had no clinical anomalies. We therefore suggest, in light of the high frequency of mutations involving 22q11 and PRODH in the general population, to consider these rare alterations in patients with congenital urinary malformations, even in the presence of nuanced neurological symptoms and negative family history.


Assuntos
Acidose , Erros Inatos do Metabolismo dos Aminoácidos , Humanos , Criança , Prolina Oxidase/genética , Mutação , Prolina/genética , Prolina/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/genética , Rim/metabolismo
5.
PLoS One ; 18(11): e0290901, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37967056

RESUMO

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/metabolismo
6.
Plant Physiol Biochem ; 205: 108182, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37977024

RESUMO

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/genética
8.
J Biol Chem ; 299(11): 105275, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37741457

RESUMO

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/metabolismo
9.
Front Immunol ; 14: 1201252, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37564635

RESUMO

Background: The shortened life expectancy in schizophrenia (SCZ) patients may be correlated with most cancers, yet there is heterogeneity in the studies examining these correlations. This study explored the expression of SCZ-related genes (HTR2A, COMT, and PRODH) in pan-cancer analysis. It helped to enhance the mechanistic understanding of the SCZ-cancer relationship and their immune mechanisms at the genetic level. Additionally, this study established a survival prediction model for glioblastoma and low-grade glioma (GBMLGG). Methods and results: SCZ-associated genes (HTR2A, COMT, and PRODH) were subjected to pan-cancer analysis. COX regression analysis and survival analysis were carried out for differentially expressed genes in multiple cancers, and finally, GBMLGG was derived as the focus for further detailed analysis. The immune scores and immune cell infiltration analyses were performed. All three genes were considerably linked with immune infiltration in GBMLGG, consistent with survival analysis. Based on the immunocyte analysis, it was observed that CD8+ T cells might be critically involved in the survival of GBMLGG. Genomic heterogeneity studies identified correlations of three genes with GBMLGG in tumor mutational burden (TMB) and mutant-allele tumor heterogeneity (MATH). HTR2A and COMT were significantly negatively correlated in TMB. Furthermore, it was found that HTR2A had a significant positive correlation with MATH, whereas PRODH had a significant negative correlation with MATH. Accordingly, a survival prediction model was constructed for GBMLGG using these three genes and clinical data, with better results obtained when evaluated in two separate datasets. Finally, gene expression validation and further immunocyte analysis were carried out in the single-cell RNA sequencing (scRNA-seq) data of glioma. Conclusion: SCZ-associated genes (HTR2A, COMT, and PRODH) were significantly differentially expressed in the carcinogenesis and survival of multiple cancers. The up or downregulation of gene expression varied across cancer types. In the GBMLGG analysis, upregulation of HTR2A and COMT was significantly positively correlated with carcinogenesis, while the opposite was noted for PRODH. Furthermore, a negative correlation was found between the upregulation of HTR2A and COMT and the survival of GBMLGG, and the opposite was also noted for PRODH. As reflected in the immunocyte analysis, abnormal expression of the three genes might be linked with CD8+ T cell infiltration, which might be critically involved in the survival of GBMLGG patients. The expression of HTR2A and COMT may inversely affect the efficacy of immunotherapy through the TMB pathway and further affect the prognosis of patient survival. The expression of HTR2A might positively indicate the degree of tumor heterogeneity through MATH and further affect the survival and prognosis of patients. The negative correlation of PRODH led to the opposite effect. Finally, the constructed survival prediction model demonstrated good predictive value, which was well validated in scRNA-seq analysis.


Assuntos
Catecol O-Metiltransferase , Glioma , Prolina Oxidase , Receptor 5-HT2A de Serotonina , Esquizofrenia , Humanos , Carcinogênese , Catecol O-Metiltransferase/genética , Linfócitos T CD8-Positivos , Glioma/genética , Prognóstico , Prolina Oxidase/genética , Esquizofrenia/genética , Receptor 5-HT2A de Serotonina/genética
10.
BMC Plant Biol ; 23(1): 194, 2023 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-37041465

RESUMO

BACKGROUND: Improving the yield and aroma content of fragrant rice is the focus of fragrant rice research. Light and Zinc (Zn) management generally cause regulations in the 2-acetyl-1-pyrroline (2AP) accumulation in fragrant rice. In addition, Zn promotes rice growth and improves rice yield, which has the potential to compensate for the negative impact of low light on fragrant rice yield. However, the potential of Zn to improve fragrant rice yield and 2AP content under shading conditions has not been verified. METHODS: Field experiments were conducted in the rice season (May-September) in 2019 to 2021. Two light i.e., normal light (NL) and low light (LL) and four Zn levels i.e., 0 kg Zn ha- 1 (N0), 1 kg Zn ha- 1 (Zn1), 2 kg Zn ha- 1(Zn2), and 3 kg Zn ha- 1 (Zn3), which applied at booting stage was set up. The grain yield, 2AP contents, Zn content in polished rice, photosynthesis related indicators, MDA content, antioxidant enzyme activity and the biochemical parameters related to 2AP formation were investigated. RESULTS: Shading reduced yield by 8.74% and increased 2AP content by 24.37%. In addition, shading reduced net photosynthetic rate (Pn), superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and increased proline, γ-aminobutyric acid (GABA), and pyrroline-5-carboxylic acid (P5C), proline dehydrogenase (PDH), △1-pyrroline-5-carboxylic acid synthetase (P5CS), malondialdehyde (MDA). With increasing Zn application levels, yield, 2AP, Zn content in polished rice, Pn, proline, P5C, GABA, PDH, P5CS, SOD, CAT and POD increased, and MDA decreased. Significant Light and Zn interaction effect on 2AP content was detected, and both shading and increasing Zn application increased the 2AP content. CONCLUSION: Shading can increase the 2AP content but reduce the yield of fragrant rice. Increasing Zn application under shading conditions can further promote the biosynthesis of 2AP, but the effect of improving yield is limited.


Assuntos
Oryza , Zinco , Zinco/farmacologia , Odorantes , Antioxidantes/farmacologia , Superóxido Dismutase , Peroxidases , Prolina Oxidase , Prolina , Ácidos Carboxílicos , Ácido gama-Aminobutírico/farmacologia , Suplementos Nutricionais
11.
Br J Haematol ; 201(4): 704-717, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36755409

RESUMO

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 Tumoral
12.
J Exp Bot ; 74(5): 1489-1500, 2023 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-36528796

RESUMO

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 , Sementes
13.
Plant Cell Environ ; 46(3): 901-917, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36583533

RESUMO

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/metabolismo
14.
Protein Eng Des Sel ; 352022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-36448708

RESUMO

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/química
15.
Commun Biol ; 5(1): 997, 2022 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-36131087

RESUMO

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 , Pirróis
16.
Int J Mol Sci ; 23(9)2022 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-35563503

RESUMO

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/metabolismo
17.
Appl Biochem Biotechnol ; 194(9): 4135-4146, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35635604

RESUMO

D-proline and N-boc-5-hydroxy-L-proline are key chiral intermediates in the production of eletriptan and saxagliptin, respectively. An efficient proline racemase-proline dehydrogenase cascade was developed for the enantioselective production of D-proline. It included the racemization of L-proline to DL-proline and the enantioselective dehydrogenation of L-proline in DL-proline. The racemization of L-proline to DL-proline used an engineered proline racemase (ProR). L-proline up to 1000 g/L could be racemized to DL-proline with 1 g/L of wet Escherichia coli cells expressing ProR within 48 h. The efficient dehydrogenation of L-proline in DL-proline was achieved using whole cells of proline dehydrogenase-producing Pseudomonas pseudoalcaligenes XW-40. Moreover, using a cell-recycling strategy, D-proline was obtained in 45.7% yield with an enantiomeric excess of 99.6%. N-boc-5-hydroxy-L-proline was also synthesized from L-glutamate semialdehyde, a dehydrogenated product of L-proline, in a 16.7% yield. The developed proline racemase-proline dehydrogenase cascade exhibits great potential and economic competitiveness for manufacturing D-proline and N-boc-5-hydroxy-L-proline from L-proline.


Assuntos
Isomerases de Aminoácido , Prolina , Escherichia coli/genética , Prolina Oxidase , Racemases e Epimerases
18.
Commun Biol ; 5(1): 496, 2022 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-35614320

RESUMO

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 , Virulência
19.
Int J Mol Sci ; 23(7)2022 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-35409177

RESUMO

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/metabolismo
20.
Arch Med Res ; 53(4): 388-398, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35367090

RESUMO

BACKGROUND: Cognitive impairment in schizophrenia (SCZ) is a core feature, relevant for the disease prognosis and functional capacity of the patients. It has also been identified as an endophenotype and proposed as a genetic mechanism of risk for schizophrenia. AIM OF THE STUDY: We aimed to evaluate the association of genetic variants in COMT, PRODH, and DISC1 with the cognitive performance of Mexican-Mestizo adult patients with SCZ in order to identify endophenotypes. SUBJECTS AND METHODS: The association of seven variants in COMT, 15 in PRODH, and three in DISC1 was evaluated in 150 patients and 150 control volunteers. The MATRICS Consensus Cognitive Battery was administered to a subset of 44 patients and 42 controls. RESULTS: COMT rs4633 was related to MATRICS global assessment, while in the multi-phenotype analysis, PRODH rs2870984 was associated with processing speed, working memory, verbal learning, and social cognition. In addition, the association of variants in COMT and PRODH with the risk for SCZ was also found in Mexican-Mestizo patients. CONCLUSION: COMT might be a potential biomarker of cognitive impairment in Mexican-Mestizo patients with SCZ, supporting the relevance of this gene for drug design.


Assuntos
Esquizofrenia , Catecol O-Metiltransferase/genética , Cognição , Genótipo , Humanos , Proteínas do Tecido Nervoso/genética , Prolina Oxidase/genética , Esquizofrenia/complicações , Esquizofrenia/genética
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