Proline Oxidase Research Articles

Phenolic Compounds as Potential Antioxidant

An antioxidant may be roughly defined as a substance that when present at low concentrations, lower than the oxidizable compound to be protected, significantly delays or inhibits its oxidation. There are two basic categories of antioxidants, natural and synthetic, the second ones have been found to cause long-term toxicological effects, including carcinogenicity (1). Attentions have been paid to both natural and synthetic antioxidants. Antioxidants have been intensively studied over the past few decades. Antioxidants play a vital role against the deteriorating action of free radicals in the organisms. Deficiency of antioxidants in living organisms leads to oxidative stress. It has been found that antioxidant compounds present in food, and biological systems are fundamental to the protection of biomolecules from these free-radical redox reactions. There has been a great interest in phenolic compounds and their antioxidant activity among consumers and the scientific community in the past decade because of the epidemiological studies linking the consumption of diets rich in natural antioxidants with decreased risk of diseases associated with oxidative stress, such as cancer and cardiovascular disease (2). Phenolic compounds as bioactive substances are a large heterogeneous group of secondary plant metabolites that have been widely distributed in plants and are important constituents of human diet (3). The natural products rich in antioxidants are of great importance for scientists. For this reason antioxidant and antitumor activities of sulfated polysaccharide isolated from marine algae have been studied by many scientists. Marine resources have attracted a great attention in the search for bioactive substance to develop new drugs and healthy foods, because of their relatively low toxicity and high bioactivities (4). In particular, sulfated polysaccahrides from marine algae are known to exhibit various biological and physiological activities including antioxidant, anticoagulant, antiviral, antitumor, and anti-inflammatory activities. There is also an emerging interest in the use of naturally occurring antioxidants in foods. Honey a sweet natural product produced by honey bees form nectar, and other plant juices is also a good source of antioxidants. Antioxidant and antimicrobial properties of honey are due to presence of variety of compounds like phenolics, ascorbic acid, α-tocopherol, proline, vitamins, catalase and glucose oxidase. Extensive data is available for antioxidant properties of honeys from different origins of the world which evidenced that bioactivities of honeys vary from each other due to botanical and geographical variations (5). Due to importance of this class of compounds, assessment of antioxidant or radical-scavenging capacity has attracted increasing attention in a number of areas, which demands the availability of simple, convenient, rapid and reliable in vitro analytical methodologies. Many different methods are used to screen matrixes or to find specific antioxidants (6).

Salicylic acid alleviates adverse effects of heat stress on photosynthesis through changes in proline production and ethylene formation

We investigated the potential of salicylic acid (SA) in alleviating the adverse effects of heat stress on photosynthesis in wheat (Triticum aestivum L.) cv WH 711. Activity of ribulose 1,5-bisphosphate carboxylase (Rubisco), photosynthetic-nitrogen use efficiency (NUE), and net photosynthesis decreased in plants subjected to heat stress (40°C for 6 h), but proline metabolism increased. SA treatment (0.5 mM) alleviated heat stress by increasing proline production through the increase in γ-glutamyl kinase (GK) and decrease in proline oxidase (PROX) activity, resulting in promotion of osmotic potential and water potential necessary for maintaining photosynthetic activity. Together with this, SA treatment restricted the ethylene formation in heat-stressed plants to optimal range by inhibiting activity of 1-aminocyclopropane carboxylic acid (ACC) synthase (ACS). This resulted in improved proline metabolism, N assimilation and photosynthesis. The results suggest that SA interacts with proline metabolism and ethylene formation to alleviate the adverse effects of heat stress on photosynthesis in wheat.

Proline oxidase–adipose triglyceride lipase pathway restrains adipose cell death and tissue inflammation

The nutrient-sensing lipolytic enzyme adipose triglyceride lipase (ATGL) has a key role in adipose tissue function, and alterations in its activity have been implicated in many age-related metabolic disorders. In adipose tissue reduced blood vessel density is related to hypoxia state, cell death and inflammation. Here we demonstrate that adipocytes of poorly vascularized enlarged visceral adipose tissue (i.e. adipose tissue of old mice) suffer from limited nutrient delivery. In particular, nutrient starvation elicits increased activity of mitochondrial proline oxidase/dehydrogenase (POX/PRODH) that is causal in triggering a ROS-dependent induction of ATGL. We demonstrate that ATGL promotes the expression of genes related to mitochondrial oxidative metabolism (peroxisome proliferator-activated receptor-α, peroxisome proliferator-activated receptor-γ coactivator-1α), thus setting a metabolic switch towards fat utilization that supplies energy to starved adipocytes and prevents cell death, as well as adipose tissue inflammation. Taken together, these results identify ATGL as a stress resistance mediator in adipocytes, restraining visceral adipose tissue dysfunction typical of age-related metabolic disorders.

Δ1-Pyrroline-5-Carboxylate/Glutamate Biogenesis Is Required for Fungal Virulence and Sporulation

Proline dehydrogenase (Prodh) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5Cdh) are two key enzymes in the cellular biogenesis of glutamate. Recombinant Prodh and P5Cdh proteins of the chestnut blight fungus Cryphonectria parasitica were investigated and showed activity in in vitro assays. Additionally, the C. parasitica Prodh and P5Cdh genes were able to complement the Saccharomyces cerevisiae put1 and put2 null mutants, respectively, to allow these proline auxotrophic yeast mutants to grow on media with proline as the sole source of nitrogen. Deletion of the Prodh gene in C. parasitica resulted in hypovirulence and a lower level of sporulation, whereas deletion of P5Cdh resulted in hypovirulence though no effect on sporulation; both Δprodh and Δp5cdh mutants were unable to grow on minimal medium with proline as the sole nitrogen source. In a wild-type strain, the intracellular level of proline and the activity of Prodh and P5Cdh increased after supplementation of exogenous proline, though the intracellular Δ1-pyrroline-5-carboxylate (P5C) content remained unchanged. Prodh and P5Cdh were both transcriptionally down-regulated in cells infected with hypovirus. The disruption of other genes with products involved in the conversion of arginine to ornithine, ornithine and glutamate to P5C, and P5C to proline in the cytosol did not appear to affect virulence; however, asexual sporulation was reduced in the Δpro1 and Δpro2 mutants. Taken together, our results showed that Prodh, P5Cdh and related mitochondrial functions are essential for virulence and that proline/glutamate pathway components may represent down-stream targets of hypovirus regulation in C. parasitica.

Effect of exogenous application of salicylic acid on proline metabolism in salt stressed <i>Chamomile recutita</i>

The effect of exogenously applied salicylic acid (SA, 0.1 mM) on plant growth parameters, compatible solutes (proline, glycinebetaine) accumulation and on proline metabolism, in leaves and roots of Chamomile recutita plants grown in saline and non-saline control was investigated. Exogenously applied SA resulted increased plant growth significantly both in saline and non-saline conditions. The magnitude of increase in free proline accumulation was significantly increased in presence of NaCl but highly significant was found with the addition of SA to NaCl in leaves. Proline was found .5.3 folds increased in leaves and .1.3 folds only in root in presence of both treatments NaCl+SA with age of plant growth over that of non-saline control. Leaves tissue always maintained higher level of free proline contents than the roots. Anabolic enzymes such as pyrroline-5-carboxylate reductase and .-glutamyl kinase activities increased in all three treatments than the non-saline control and the magnitude of increase was found more pronounced in presence of both treatments than the others. In other way catabolic enzyme, proline oxidase activity was inhibited in all treatments. Nevertheless, the reduction in the activity was more in presence of both treatments than the others. Therefore, during the exogenously applied SA to salt stress, proline metabolism was significantly altered and the extent of alteration varied between the SA and salt stress, leading to the maintenance of the turgor by accumulating higher levels of free proline accumulation in C. recutita, supporting its protection from salt stress. Further, with addition of SA to salt of C. recutita was evident from the higher level of glycinebetaine (GB) compared with non-saline control or/others treatments. The enhanced activities of P-5-CR & .-glutamyl kinase, and proline content in response to NaCl and/or SA treatment, whereas their interaction had an additive/cumulative effect. Hence, SA could be used as a potential growth regulator to improve plant salt stress tolerance. It was, therefore, concluded that SA ameliorated the stress generated by salt through the alleviated proline metabolic pathway/system.Keywords- Chamomile recutita; Compatible osmolytes; glycinebetaine; osmotic pressure; proline; salicylic acid; salinity.

Reactive Oxygen Species Homeostasis and Virulence of the Fungal PathogenCryptococcus neoformansRequires an Intact Proline Catabolism Pathway

Degradation of the multifunctional amino acid proline is associated with mitochondrial oxidative respiration. The two-step oxidation of proline is catalyzed by proline oxidase and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase, which produce P5C and glutamate, respectively. In animal and plant cells, impairment of P5C dehydrogenase activity results in P5C-proline cycling when exogenous proline is supplied via the actions of proline oxidase and P5C reductase (the enzyme that converts P5C to proline). This proline is oxidized by the proline oxidase-FAD complex that delivers electrons to the electron transport chain and to O2, leading to mitochondrial reactive oxygen species (ROS) overproduction. Coupled activity of proline oxidase and P5C dehydrogenase is therefore important for maintaining ROS homeostasis. In the genome of the fungal pathogen Cryptococcus neoformans, there are two paralogs (PUT1 and PUT5) that encode proline oxidases and a single ortholog (PUT2) that encodes P5C dehydrogenase. Transcription of all three catabolic genes is inducible by the presence of proline. However, through the creation of deletion mutants, only Put5 and Put2 were found to be required for proline utilization. The put2Δ mutant also generates excessive mitochondrial superoxide when exposed to proline. Intracellular accumulation of ROS is a critical feature of cell death; consistent with this fact, the put2Δ mutant exhibits a slight, general growth defect. Furthermore, Put2 is required for optimal production of the major cryptococcal virulence factors. During murine infection, the put2Δ mutant was discovered to be avirulent; this is the first report highlighting the importance of P5C dehydrogenase in enabling pathogenesis of a microorganism.

Abstract 4613: Effects of co-regulation of proline oxidase and succinate dehydrogenase on oxidative respiration.

Abstract Proline oxidase (POX) is an inner mitochondrial membrane protein that oxidizes proline to generate superoxide as well as ATP. Work in our laboratory has documented that POX activity is highly regulated during genotoxic and metabolic stress. Inducers of POX include p53, and proline-dependent ROS play a role in p53- and mitochondrial-mediated apoptosis. Succinate dehydrogenase, also known as Complex II of the electron transport chain (ETC), oxidizes the TCA cycle metabolite succinate to fumarate as it reduces ubiquinone to ubiquinol, generating ATP through oxidative respiration. Complex II and the p53-DNA damage repair pathway are intimately linked. Mutations in Complex II have been shown to increase risk of breast and thyroid cancer through destabilization of p53, and tocopherol-succinate has been demonstrated to reduce radiation-induced apoptosis. Thus, the ETC and activities of p53 appear to be linked through Complex II. Because POX can generate ATP from proline, we explored the relationship between POX and the ETC. We found that POX binds directly to ubiquinone, and that ubiquinone is a very effective acceptor of proline-derived electrons. Increased POX catalytic activity due to ubiquinone required Complex III and Complex IV of the ETC to be functional, indicating that ATP generated by POX is through oxidative respiration. In addition, experiments using the Seahorse XF24 analyzer confirmed that while POX can utilize proline as a short term source of electrons during nutrient stress conditions, long term expression of POX results in reactive oxygen species (ROS)-dependent suppression of respiration, consistent with its role in mitochondrial-mediated apoptosis. We found that the POX and Complex II were linked functionally, and that the presence of succinate dramatically affects POX activity. Lineweaver-Burk analysis showed that succinate inhibits POX catalytic activity using anti-competitive inhibition, a rare mechanism of enzymatic inhibition. Two highly selective inhibitors of the Complex II holoenzyme, TTFA and carboxin, inhibit the ubiquinone-mediated activities of POX; in addition, POX co-immunoprecipitates with Complex II, indicating a physical interaction between these two enzymes. Interestingly, proline appears to stimulate oxidation of succinate by Complex II in vitro, and acute co-administration of proline and succinate to POX-expressing cells results in a burst of oxidative respiration well above that seen with either substrate alone. Finally, addition of succinate to cell culture inhibits ROS generation by POX and inhibits the long-term effects of POX on respiration. Together, these data support a model in which POX is an integral contributor in oxidative respiration during nutrient stress, and in which respiration is regulated by level and duration of POX expression, as well as levels of proline relative to the TCA cycle metabolite, and Complex II substrate, succinate. Citation Format: Chad N. Hancock, James M. Phang. Effects of co-regulation of proline oxidase and succinate dehydrogenase on oxidative respiration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4613. doi:10.1158/1538-7445.AM2013-4613

Association of 757 C/T polymorphismin PRODH gene with Schizophrenia in Iranian population

Evidence is emerging for the association of polymorphisms in PRODH gene and increased risk of schizophrenia. In this project, peripheral blood sampling was obtained from 175 schizophrenia patients that their diseases were confirmed by psychiatrists. 185 healthy individuals were considered as control group. The related tests were administered on the basis of PCR-RFLP. In the continuation, statistical analysis was made on the basis of obtained genotypes from two groups of healthy and patient groups using SPSS16.0 software. The administration of this project aims at investigating the hypothesis that proline dehydrogenase gene, as one of the most important genes involved in schizophrenia incidence which is proved in various populations [outside Iran], may also be involved in incidence of schizophrenia in Iran. This study has analyzed one single nucleotide polymorphism in the PRODH gene, including 757C/T in the incidence of this disease in the given statistical population. According to our results, SNP 757 C/T may be effective in incidence of the disease since P value was < 0.01. Ultimately, our results suggest that outbreak of mutation in PRODH gene in our population can be one of causes of increasing risk of Schizophrenia in this population.

Potassium starvation-induced oxidative stress and antioxidant defense responses in Brassica juncea

In this study, changes in growth, chlorophyll pigments, proline, hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents, and antioxidative enzyme activities were investigated in the seedlings of four different cultivars (cvs) of mustard [Brassica juncea (L.) Czern. & Coss.], i.e. Varuna, RH-30, Rohini, and Vaibhave under potassium (K) nutrition-deficient conditions. K deficiency induced a significant decrease in concentrations of photosynthetic pigments in all four cvs, however, this decrease was higher in cvs. Varuna and RH-30. During K deficiency, proline concentration increased in all mustard cvs, but a maximum increase in this parameter was shown by cvs. Varuna and RH-30. The activity of the key proline metabolizing enzyme γ-glutamyl kinase increased more in cvs. Varuna and RH-30 compared to cvs. Rohini and Vaibhave. The proline oxidase activity showed greater increase in cvs. Vaibhave and Rohini compared to cvs. Varuna and RH-30. K deficiency increased the concentrations of H2O2 and the activities of anti-oxidative defense system enzymes like superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) in the seedlings of all mustard cvs, but higher activities of these enzymes were observed in cvs. Varuna and RH-30 compared to cvs. Rohini and Vaibhave. A significant lipid peroxidation in terms of MDA contents was also observed in the K-deficient seedlings of all four mustard cvs. This study suggests that K-starvation-induced oxidative stress through the high generation of reactive oxygen species (ROS). All mustard cvs counteracted to some extent the effects of ROS by activation of antioxidant machinery. Overall, the results indicate that of all four mustard cvs, Varuna and RH-30 were tolerant to K deficiency.

Proline Oxidase Promotes Tumor Cell Survival in Hypoxic Tumor Microenvironments

Proline is a readily released stress substrate that can be metabolized by proline oxidase (POX) to generate either reactive oxygen species (ROS) to induce apoptosis or autophagy or ATP during times of nutrient stress. However, the contribution of proline metabolism to tumorigenesis in hypoxic microenvironments has not been explored. In this study, we investigated the different functions of POX under hypoxia and glucose depletion. We found that hypoxia induced POX expression in cancer cells in vitro and that POX upregulation colocalized with hypoxic tissues in vivo. In addition, the combination of hypoxia and low glucose showed additive effects on POX expression. Similar to conditions of low glucose, hypoxia-mediated POX induction was dependent on AMP-activated protein kinase activation but was independent of HIF-1α and HIF-2α. Under low-glucose and combined low-glucose and hypoxic conditions, proline catabolized by POX was used preferentially for ATP production, whereas under hypoxia, POX mediated autophagic signaling for survival by generating ROS. Although the specific mechanism was different for hypoxia and glucose deprivation, POX consistently contributed to tumor cell survival under these conditions. Together, our findings offer new insights into the metabolic reprogramming of tumor cells present within a hostile microenvironment and suggest that proline metabolism is a potential target for cancer therapeutics.

Reprogramming of proline and glutamine metabolism contributes to the proliferative and metabolic responses regulated by oncogenic transcription factor c-MYC

In addition to glycolysis, the oncogenic transcription factor c-MYC (MYC) stimulates glutamine catabolism to fuel growth and proliferation of cancer cells through up-regulating glutaminase (GLS). Glutamine is converted to glutamate by GLS, entering the tricarboxylic acid cycle as an important energy source. Less well-recognized, glutamate can also be converted to proline through Δ(1)-pyrroline-5-carboxylate (P5C) and vice versa. This study suggests that some MYC-induced cellular effects are due to MYC regulation of proline metabolism. Proline oxidase, also known as proline dehydrogenase (POX/PRODH), the first enzyme in proline catabolism, is a mitochondrial tumor suppressor that inhibits proliferation and induces apoptosis. MiR-23b* mediates POX/PRODH down-regulation in human kidney tumors. MiR-23b* is processed from the same transcript as miR-23b; the latter inhibits the translation of GLS. Using MYC-inducible human Burkitt lymphoma model P493 and PC3 human prostate cancer cells, we showed that MYC suppressed POX/PRODH expression primarily through up-regulating miR-23b*. The growth inhibition in the absence of MYC was partially reversed by POX/PRODH knockdown, indicating the importance of suppression of POX/PRODH in MYC-mediated cellular effects. Interestingly, MYC not only inhibited POX/PRODH, but also markedly increased the enzymes of proline biosynthesis from glutamine, including P5C synthase and P5C reductase 1. MYC-induced proline biosynthesis from glutamine was directly confirmed using (13)C,(15)N-glutamine as a tracer. The metabolic link between glutamine and proline afforded by MYC emphasizes the complexity of tumor metabolism. Further studies of the relationship between glutamine and proline metabolism should provide a deeper understanding of tumor metabolism while enabling the development of novel therapeutic strategies.

Abstract 1119: The oxidation of proline by proline oxidase provides a regulated source of ROS for mitochondria derived cellular signaling

Abstract A role for reactive oxygen species (ROS) in intracellular signaling has been generally accepted, but the organellar localization, the enzymatic source and the regulation of ROS generation are not well understood. Recent work by others has focused on mitochondria as a source of ROS and specifically on the reduction of oxygen by electrons from complex III to form superoxide. The accessibility of complex III to the mitochondrial intermembrane space and cytosol provides an attractive hypothesis for mitochondrial ROS as regulators of cellular redox signaling. We now report that the regulated metabolism of proline makes it a special substrate for this process. Proline oxidase (POX) is an inner mitochondrial membrane protein that oxidizes proline to generate superoxide as well as ATP. Work in our laboratory has documented that POX activity is highly regulated during genotoxic and metabolic stress. Inducers of POX include p53, and proline-dependent ROS play a role in mitochondrial apoptosis. PPAR-γ and its ligands also induce POX but depending on the metabolic context, POX initiates autophagy as well as apoptosis. Mechanistic models for ROS generation by POX have come from microorganisms. PutA, the prokaryotic homolog of POX can generate ROS by electrons reducing oxygen directly from its catalytic center. On the other hand, in S. cerevisiae, the POX homolog, Put1, has been shown to directly interact with ubiquinone, thereby contributing electrons derived from proline oxidation into the electron transport chain. In this work, we now report that human POX interacts directly with ubiquinone and that POX activity is markedly increased by the presence of ubiquinone. In addition, by using selective inhibitors of electron transport, we demonstrate that electrons derived from proline can be converted to superoxide by complex III of the electron transport chain. These radicals are directed into both the mitochondrial matrix as well as into the intermembrane space and the cytosol. Thus, POX is not only specifically regulated but also specially located and coupled to the mitochondrial electron transport chain to mediate ROS signaling. These POX- mediated mechanisms provide important insights into its novel apoptotic and metabolic tumor suppressive functions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1119. doi:1538-7445.AM2012-1119

Enhancement of the proline and nitric oxide synthetic pathway improves fermentation ability under multiple baking-associated stress conditions in industrial baker's yeast

BackgroundDuring the bread-making process, industrial baker's yeast, mostly Saccharomyces cerevisiae, is exposed to baking-associated stresses, such as air-drying and freeze-thaw stress. These baking-associated stresses exert severe injury to yeast cells, mainly due to the generation of reactive oxygen species (ROS), leading to cell death and reduced fermentation ability. Thus, there is a great need for a baker's yeast strain with higher tolerance to baking-associated stresses. Recently, we revealed a novel antioxidative mechanism in a laboratory yeast strain that is involved in stress-induced nitric oxide (NO) synthesis from proline via proline oxidase Put1 and N-acetyltransferase Mpr1. We also found that expression of the proline-feedback inhibition-less sensitive mutant γ-glutamyl kinase (Pro1-I150T) and the thermostable mutant Mpr1-F65L resulted in an enhanced fermentation ability of baker's yeast in bread dough after freeze-thaw stress and air-drying stress, respectively. However, baker's yeast strains with high fermentation ability under multiple baking-associated stresses have not yet been developed.ResultsWe constructed a self-cloned diploid baker's yeast strain with enhanced proline and NO synthesis by expressing Pro1-I150T and Mpr1-F65L in the presence of functional Put1. The engineered strain increased the intracellular NO level in response to air-drying stress, and the strain was tolerant not only to oxidative stress but also to both air-drying and freeze-thaw stresses probably due to the reduced intracellular ROS level. We also showed that the resultant strain retained higher leavening activity in bread dough after air-drying and freeze-thaw stress than that of the wild-type strain. On the other hand, enhanced stress tolerance and fermentation ability did not occur in the put1-deficient strain. This result suggests that NO is synthesized in baker's yeast from proline in response to oxidative stresses that induce ROS generation and that increased NO plays an important role in baking-associated stress tolerance.ConclusionsIn this work, we clarified the importance of Put1- and Mpr1-mediated NO generation from proline to the baking-associated stress tolerance in industrial baker's yeast. We also demonstrated that baker's yeast that enhances the proline and NO synthetic pathway by expressing the Pro1-I150T and Mpr1-F65L variants showed improved fermentation ability under multiple baking-associated stress conditions. From a biotechnological perspective, the enhancement of proline and NO synthesis could be promising for breeding novel baker's yeast strains.

Investigation of genetic association between PRODH gene and schizophrenia in Iranian population

Schizophrenia is a complicated, debilitative mental disorder. Evidence is emerging for the association of polymorphisms in PRODH gene and increased risk of schizophrenia. In the present research, we investigated relationship between of this gene and schizophrenia disease by means of a gene polymorphism using PCRRFLP technique.150 persons suffering from acute Schizophrenia and 160 healthy persons volunteering for this project were bled. . Based on intended SNP, pair of primers was designed by Oligo7 program and polymerase chain reaction (PCR) was performed by thermo cycler. Then the resulted reactive mixture was exposed to a special enzyme, which we had intended for our study. Finally, the fragments of enzyme cut were transferred on the gel (4%) and migration pattern of resulted components were compared in healthy and patient subjects , whereby obtaining genotypes of different persons in polymorphic position. We utilized SPSS 16.0 program for statistical investigation of the work and studied SNP 1945T>C and its relation with the disease in statistical population. Our findings showed a meaningful relation between the occurrence of this nucleotide mutation and its frequency in patients (given P value=0.00). Results of this work indicate that PRODH gene can be considered to be a significant candidate in our population as a factor influencing the occurrence of Schizophrenia. Keyword: Schizophrenia, PRODH, Proline, Hyperprolinemia

Expression in Escherichia coli of the catalytic domain of human proline oxidase

The human PRODH gene has been shown to have unique roles in regulating cell survival and apoptotic pathways and it has been related to velocardiofacial syndrome/DiGeorge syndrome and increased susceptibility to schizophrenia. It encodes for the flavoprotein proline oxidase (PO), which catalyzes the conversion of l-proline to Δ1-pyrroline-5-carboxylate. Despite the important physiological and medical interest in human PO, up to now only microbial homologues of PO have been expressed as recombinant protein and fully characterized. By using a bioinformatics analysis aimed at identifying the catalytic domain and the regions with a high intrinsic propensity to structural disorder, we designed deletion variants of human PO that were successfully expressed in Escherichia coli as soluble proteins in fairly high amounts (up to 10mg/L of fermentation broth). The His-tagged PO-barrelN protein was isolated as an active (the specific activity is 0.032U/mg protein), dimeric holoenzyme showing the typical spectral properties of FAD-containing flavoprotein oxidases. These results pave the way for elucidating structure–function relationships of this human flavoenzyme and clarifying the effect of the reported polymorphisms associated with disease states.

Proline metabolism and cancer

Proline plays a special role in cancer metabolism. Proline oxidase (POX), a.k.a. proline dehydrogenase (PRODH), is among a few genes induced rapidly and robustly by P53, the tumor suppressor. Ectopic expression of POX under control of tet-off promoter initiated mitochondrial apoptosis. The mechanism activated by POX is mediated by its production of ROS. In immunodeficient mice, POX overexpression markedly retarded growth of xenograft tumors. In human tumors of the digestive tract and kidney, POX was markedly decreased, suggesting that the suppressive effect of POX was downregulated. This was not due to POX gene mutations or hypermethylation. Instead, a microRNA, miR-23b*, expressed at high levels in tumors, was a potent inhibitor of POX expression. Furthermore, antagomirs of miR-23b* reversed the downregulated expression of POX and its tumor-suppressive effect, thereby providing a therapeutic strategy. POX not only responds to genotoxic stress, but also to inflammatory and metabolic stress. Depending on microenvironmental and temporal factors, POX can mediate oppositely-directed responses-programmed cell death, on the one hand, and survival, on the other.

Proline dehydrogenase: a key enzyme in controlling cellular homeostasis

Proline dehydrogenase (ProDH), also called proline oxidase (POX), is a universal enzyme in living organisms. It catalyzes the oxidation of L-proline to delta1-pyrroline-5-carboxylate leading to the release of electrons, which can be transferred to either electron transfer systems or to molecular oxygen. ProDH is not only essential for proline catabolism but also plays key roles in providing energy, shuttling redox potential between cellular compartments and reactive oxygen species production. Structural analysis of prokaryotic ProDHs already gives some insights into the biochemical activity and biological functions of this enzyme, which can be extended to eukaryotic ProDHs based on sequence similarities. Here we report the most recent investigations on the biochemical and regulation of ProDH at transcriptional, post-transcriptional and translational levels. The biological roles of ProDH in cell homeostasis and adaptation through energetic, developmental, adaptive, physiological and pathological processes in eukaryotes are presented and discussed to create a framework for future research direction.

Enzymes of the ornithine–glutamate–proline pathway in the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta

A fully functional ornithine–glutamate–proline pathway was detected in L3 and adult Haemonchus contortus and Teladorsagia circumcincta, making the parasites capable of interconversion of these amino acids. Ornithine aminotransferase (OAT) (E.C. 2.6.1.13) was a reversible pyridoxal-5-phosphate (PLP)-dependent enzyme with an optimum pH 8.5. Hydroxylamine completely inhibited OAT activity in both parasites. For all five enzymes, substrate affinity was similar for each species and life cycle stage, the notable exceptions being the nearly 10-fold lower affinity for Δ1-pyrroline-5-carboxylate (P5C) of P5C reductase (E.C. 1.5.1.2) in adult T. circumcincta and about half for P5C for L3 H. contortus P5C dehydrogenase (E.C. 1.5.1.12). P5C synthase (E.C. 1.2.1.41) activity was similar with either NADPH or NADH as co-factor. Proline oxidase (E.C. 1.5.99.8) was a co-factor independent enzyme with an optimal pH 8.5. Despite similarities to those in the host, enzymes of this pathway may still be useful as control targets if they differ antigenically, as a supply of proline is necessary for cuticle formation.

Abstract 2067: Tumor cell survival under microenvironmental stress: Hypoxia and/or low glucose upregulate poline oxidase

Abstract Proline is a stress substrate which can be readily released from collagen in extracelluar matrix (ECM) initiated by matrix metalloproteinases accompanying tumor progression and invasion. Proline oxidase (POX) can metabolize proline to generate reactive oxygen species (ROS) for inducing apoptosis or autophagy; it also is a source of energy (ATP) under metabolic stress. However, the contribution of proline metabolism to tumorigenesis under concurrent hypoxia and low-glucose in the tumor microenvironment, has not been explored. In the present study, we showed that hypoxia and low glucose induced POX expression in HT29 colorectal cancer cells. The combination of low-glucose and hypoxia had additive effects on POX expression. Knockdown of HIF-1α, an important mediator of the hypoxic response, did not alter POX expression. Instead, inhibition of the activation of AMPK, the celluar energy sensor, by its antagonist (Compound C) blocked the upregulation of POX under conditions of either hypoxia or low glucose. Functional studies showed POX was consistently responsible for the maintenance of cell growth under hypoxia, low-glucose or with the combination of both. However, ATP and ROS assays showed that ATP production from POX was favored under low-glucose condition with or without hypoxia, whereas POX preferred ROS production with hypoxia. POX knockdown partially decreased hypoxia-induced prosurvival autophagy, which was also decreased by the antioxidant, N-acetylcysteine. Taken together, these results suggest that the induction of proline metabolism with hypoxia and/or low-glucose contributes an important mechanism by which tumor cells switch to a survival mode. These findings offer new insights into our understanding of energy metabolism in tumor cells beyond the well-recognized Warburg effect of aerobic glycolysis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2067. doi:10.1158/1538-7445.AM2011-2067

An antioxidative mechanism mediated by the yeast N-acetyltransferase Mpr1: oxidative stress-induced arginine synthesis and its physiological role

Saccharomyces cerevisiaeSigma1278b has the MPR1 gene encoding the N-acetyltransferase Mpr1 that acetylates the proline metabolism intermediate Delta(1)-pyrroline-5-carboxylate (P5C)/glutamate-gamma-semialdehyde (GSA) in vitro. In addition, Mpr1 protects cells from various oxidative stresses by regulating the levels of intracellular reactive oxygen species (ROS). However, the relationship between P5C/GSA acetylation and antioxidative mechanism involving Mpr1 remains unclear. Here, we report the synthesis of oxidative stress-induced arginine via P5C/GSA acetylation catalyzed by Mpr1. Gene disruption analysis revealed that Mpr1 converts P5C/GSA into N-acetyl-GSA for arginine synthesis in the mitochondria, indicating that Mpr1 mediates the proline and arginine metabolic pathways. More importantly, Mpr1 regulate ROS generation by acetylating toxic P5C/GSA. Under oxidative stress conditions, the transcription of PUT1 encoding the proline oxidase Put1 and MPR1 was strongly induced, and consequently, the arginine content was significantly increased. We also found that two deletion mutants (Deltampr1/2 and Deltaput1) were more sensitive to high-temperature stress than the wild-type strain, but that direct treatment with arginine restored the cell viability of these mutants. These results suggest that Mpr1-dependent arginine synthesis confers stress tolerance. We propose an antioxidative mechanism that is involved in stress-induced arginine synthesis requiring Mpr1 and Put1.