A L-proline/O2 biofuel cell using L-proline dehydrogenase (LPDH) from Aeropyrum pernix.

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

Prolidase-proline dehydrogenase/proline oxidase-collagen biosynthesis axis as a potential interface of apoptosis/autophagy.

Prolidase is a cytosolic imidodipeptidase that specifically splits imidodipeptides with C-terminal proline or hydroxyproline. The enzyme plays an important role in the recycling of proline from imidodipeptides for resynthesis of collagen and other proline-containing proteins. The mechanism of prolidase-dependent regulation of collagen biosynthesis was found at both transcriptional and post-transcriptional level. The increase in the enzyme activity is due to its phosphorylation on serine/threonine residues. Prolidase-dependent transcriptional regulation of collagen biosynthesis was found at the level of NF-κB, known inhibitor of type I collagen gene expression. Proline dehydrogenase/proline oxidase (PRODH/POX) is flavin-dependent enzyme associated with the inner mitochondrial membrane. The enzyme catalyzes conversion of proline into Δ(1) -pyrroline-5-carboxylate (P5C), during which reactive oxygen species (ROS) are produced, inducing intrinsic and extrinsic apoptotic pathways. Alternatively, under low glucose stress, PRODH/POX activation produces ATP for energy supply and survival. Of special interest is that PRODH/POX gene is induced by P53 and peroxisome proliferator-activated gamma receptor (PPARγ). Among down-regulators of PRODH/POX is an oncogenic transcription factor c-MYC and miR-23b*. On the other hand, PRODH/POX suppresses HIF-1α transcriptional activity, the MAPK pathway, cyclooxygenase-2, epidermal growth factor receptor and Wnt/b-catenin signaling. PRODH/POX expression is often down-regulated in various tumors, limiting mitochondrial proline utilization to P5C. It is accompanied by increased cytoplasmic level of proline. Proline availability for PRODH/POX-dependent ATP or ROS generation depends on activity of prolidase and utilization of proline in process of collagen biosynthesis. Therefore, Prolidase-PRODH/POX-Collagen Biosynthesis axis may represent potential interface that regulate apoptosis and survival. © 2016 BioFactors, 42(4):341-348, 2016.

Rapid reaction kinetics of proline dehydrogenase in the multifunctional proline utilization A protein.

The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli catalyzes the oxidation of proline to glutamate in two reaction steps using separate proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase domains. Here, the kinetic mechanism of PRODH in PutA is studied by stopped-flow kinetics to determine microscopic rate constants for the proline:ubiquinone oxidoreductase mechanism. Stopped-flow data for proline reduction of the flavin cofactor (reductive half-reaction) and oxidation of reduced flavin by CoQ(1) (oxidative half-reaction) were best-fit by a double exponential from which maximum observable rate constants and apparent equilibrium dissociation constants were determined. Flavin semiquinone was not observed in the reductive or oxidative reactions. Microscopic rate constants for steps in the reductive and oxidative half-reactions were obtained by globally fitting the stopped-flow data to a simulated mechanism that includes a chemical step followed by an isomerization event. A microscopic rate constant of 27.5 s(-1) was determined for proline reduction of the flavin cofactor followed by an isomerization step of 2.2 s(-1). The isomerization step is proposed to report on a previously identified flavin-dependent conformational change [Zhang, W. et al. (2007) Biochemistry 46, 483-491] that is important for PutA functional switching but is not kinetically relevant to the in vitro mechanism. Using CoQ(1), a soluble analogue of ubiquinone, a rate constant of 5.4 s(-1) was obtained for the oxidation of flavin, thus indicating that this oxidative step is rate-limiting for k(cat) during catalytic turnover. Steady-state kinetic constants calculated from the microscopic rate constants agree with the experimental k(cat) and k(cat)/K(m) parameters.

Oxidized low-density lipoproteins upregulate proline oxidase to initiate ROS-dependent autophagy.

Epidemiological studies showed that high levels of oxidized low-density lipoproteins (oxLDLs) are associated with increased cancer risk. We examined the direct effect of physiologic concentrations oxLDL on cancer cells. OxLDLs were cytotoxic and activate both apoptosis and autophagy. OxLDLs have ligands for peroxisome proliferator-activated receptor gamma and upregulated proline oxidase (POX) through this nuclear receptor. We identified 7-ketocholesterol (7KC) as a main component responsible for the latter. To elucidate the role of POX in oxLDL-mediated cytotoxicity, we knocked down POX via small interfering RNA and found that this (i) further reduced viability of cancer cells treated with oxLDL; (ii) decreased oxLDL-associated reactive oxygen species generation; (iii) decreased autophagy measured via beclin-1 protein level and light-chain 3 protein (LC3)-I into LC3-II conversion. Using POX-expressing cell model, we established that single POX overexpression was sufficient to activate autophagy. Thus, it led to autophagosomes accumulation and increased conversion of LC3-I into LC3-II. Moreover, beclin-1 gene expression was directly dependent on POX catalytic activity, namely the generation of POX-dependent superoxide. We conclude that POX is critical in the cellular response to the noxious effects of oxLDL by activating protective autophagy.

Proline oxidase functions as a mitochondrial tumor suppressor in human cancers.

Tumor metabolism and bioenergetics have become important topics for cancer research and are promising targets for anticancer therapy. Although glucose serves as the main source of energy, proline, an alternative substrate, is important, especially during nutrient stress. Proline oxidase (POX), catalyzing the first step in proline catabolism, is induced by p53 and can regulate cell survival as well as mediate programmed cell death. In a mouse xenograft tumor model, we found that POX greatly reduced tumor formation by causing G2 cell cycle arrest. Furthermore, immunohistochemical staining showed decreased POX expression in tumor tissues. Importantly, HIF-1alpha signaling was impaired with POX expression due to the increased production of alpha-ketoglutarate, a critical substrate for prolyl hydroxylation and degradation of HIF-1alpha. Combined with previous in vitro findings and reported clinical genetic associations, these new findings lead us to propose POX as a mitochondrial tumor suppressor and a potential target for cancer therapy.

Regulation and function of proline oxidase under nutrient stress.

Under conditions of nutrient stress, cells switch to a survival mode catabolizing cellular and tissue constituents for energy. Proline metabolism is especially important in nutrient stress because proline is readily available from the breakdown of extracellular matrix (ECM), and the degradation of proline through the proline cycle initiated by proline oxidase (POX), a mitochondrial inner membrane enzyme, can generate ATP. This degradative pathway generates glutamate and alpha-ketoglutarate, products that can play an anaplerotic role for the TCA cycle. In addition the proline cycle is in a metabolic interlock with the pentose phosphate pathway providing another bioenergetic mechanism. Herein we have investigated the role of proline metabolism in conditions of nutrient stress in the RKO colorectal cancer cell line. The induction of stress either by glucose withdrawal or by treatment with rapamycin, stimulated degradation of proline and increased POX catalytic activity. Under these conditions POX was responsible, at least in part, for maintenance of ATP levels. Activation of AMP-activated protein kinase (AMPK), the cellular energy sensor, by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), also markedly upregulated POX and increased POX-dependent ATP levels, further supporting its role during stress. Glucose deprivation increased intracellular proline levels, and expression of POX activated the pentose phosphate pathway. Together, these results suggest that the induction of proline cycle under conditions of nutrient stress may be a mechanism by which cells switch to a catabolic mode for maintaining cellular energy levels.

Proline affects brain function in 22q11DS children with the low activity COMT 158 allele.

The association between the 22q11.2 deletion syndrome (22q11DS) and psychiatric disorders, particularly psychosis, suggests a causal relationship between 22q11DS genes and abnormal brain function. The genes catechol-O-methyl-transferase (COMT) and proline dehydrogenase both reside within the commonly deleted region of 22q11.2. COMT activity and proline levels may therefore be altered in 22q11DS individuals. Associations of both COMT(158) genotype and elevated serum proline levels with abnormal brain function have been reported. Fifty-six 22q11DS children and 75 healthy controls were assessed on physiological measures of brain function, including prepulse inhibition (PPI) of startle, P50 auditory sensory gating and smooth pursuit eye movements (SPEM). COMT(158) genotype and plasma proline levels were determined in the 22q11DS children. We hypothesized an interaction between the COMT(158) genotype and proline, predicting the strongest negative effect of high proline on brain function to occur in 22q11DS children who are carriers of the COMT(met) allele. Of the three physiological measures, only SPEM and PPI were abnormal in the patient sample. With regard to the SPEM performance, there was a significant interaction between the COMT(158) genotype and proline level with significantly decreased SPEM performance in children with high plasma proline levels and the low activity COMT(met) allele. A similar interaction effect was not observed with regard to PPI. These findings are consistent with a model in which elevated proline negatively affects brain function by an increase in dopamine in the prefrontal cortex. 22q11DS patients with low dopamine catabolic capacity are therefore especially vulnerable to this functional disruption.

Combinatorial control of Arabidopsis proline dehydrogenase transcription by specific heterodimerisation of bZIP transcription factors.

Proline metabolism has been implicated in plant responses to abiotic stresses. The Arabidopsis thaliana proline dehydrogenase (ProDH) is catalysing the first step in proline degradation. Transcriptional activation of ProDH by hypo-osmolarity is mediated by an ACTCAT cis element, a typical binding site of basic leucine zipper (bZIP) transcription factors. In this study, we demonstrate by gain-of-function and loss-of-function approaches, as well as chromatin immunoprecipitation (ChIP), that ProDH is a direct target gene of the group-S bZIP factor AtbZIP53. Dimerisation studies making use of yeast and Arabidopsis protoplast-based two-hybrid systems, as well as bimolecular fluorescence complementation (BiFC) reveal that AtbZIP53 does not preferentially form dimers with group-S bZIPs but strongly interacts with members of group-C. In particular, a synergistic interplay of AtbZIP53 and group-C AtbZIP10 was demonstrated by colocalisation studies, strong enhancement of ACTCAT-mediated transcription as well as complementation studies in atbzip53 atbzip10 T-DNA insertion lines. Heterodimer mediated activation of transcription has been found to operate independent of the DNA-binding properties and is described as a crucial mechanism to modulate transcription factor activity and function.

Proline oxidase, a proapoptotic gene, is induced by troglitazone: evidence for both peroxisome proliferator-activated receptor gamma-dependent and -independent mechanisms.

Proline oxidase (POX) is a redox enzyme localized in the mitochondrial inner membrane. We and others have shown that POX is a p53-induced gene that can mediate apoptosis through generation of reactive oxygen species (ROS). The peroxisome proliferator-activated receptor gamma (PPARgamma) ligand troglitazone was found to activate the POX promoter in colon cancer cells. PPARgamma ligands have been reported to induce apoptosis in a variety of cancer cells. In HCT116 cells expressing a wild-type PPARgamma, troglitazone enhanced the binding of PPARgamma to PPAR-responsive element in the POX promoter and increased endogenous POX expression. Blocking of PPARgamma activation either by antagonist GW9662 or deletion of PPAR-responsive element in the POX promoter only partially decreased the POX promoter activation in response to troglitazone, indicating also the involvement of PPARgamma-independent mechanisms. Further, troglitazone also induced p53 protein expression in HCT116 cells, which may be the possible mechanism for PPARgamma-independent POX activation, since POX has been shown to be a downstream mediator in p53-induced apoptosis. In HCT15 cells, with both mutant p53 and mutant PPARgamma, there was no effect of troglitazone on POX activation, whereas in HT29 cells, with a mutant p53 and wild type PPARgamma, increased activation was observed by ligand stimulation, indicating that both PPARgamma-dependent and -independent mechanisms are involved in the troglitazone-induced POX expression. A time- and dose-dependent increase in POX catalytic activity was obtained in HCT116 cells treated with troglitazone with a concomitant increase in the production of intracellular ROS. Our results suggest that the induction of apoptosis by troglitazone may, at least in part, be mediated by targeting POX gene expression for generation of ROS by POX both by PPARgamma-dependent and -independent mechanisms.

The p53-induced gene-6 (proline oxidase) mediates apoptosis through a calcineurin-dependent pathway.

Proline oxidase is a p53-induced redox gene that can generate reactive oxygen species (ROS) and mediate apoptosis in tumor cells. We report that proline oxidase is a downstream effector in p53-mediated activation of the calcium/calmodulin-dependent phosphatase calcineurin in lung, renal, colon, and ovarian carcinoma cells. The activation of calcineurin by p53 and proline oxidase was detected by activation of the nuclear factor of activated T cells (NFAT), an established indicator of activated calcineurin. Both proline oxidase- and p53-induced activation of NFAT were sensitive to the calcineurin inhibitors cyclosporin A and FK-506, to scavengers of ROS, and to inhibitors of calcium mobilization. A proline oxidase antisense vector suppressed the ability of p53 to up-regulate proline oxidase, activate calcineurin, and induce apoptosis. Moreover, two renal carcinoma-derived mutant p53 proteins were deficient in inducing proline oxidase expression and in activating calcineurin. Inhibitors of calcineurin and calcium mobilization abolished proline oxidase-mediated apoptosis and reduced p53-induced apoptosis. Treatment of colon and ovarian carcinoma cells with the anticancer genotoxic agent etoposide up-regulated both p53 and proline oxidase, activated calcineurin, and induced apoptosis. The etoposide-mediated activation of calcineurin and induction of apoptosis was markedly suppressed by FK-506 calcineurin inhibitor. We propose that proline oxidase mediates apoptosis through the generation of proline-dependent ROS, which then mobilize calcium and activate calcineurin. The activation of calcineurin-regulated transcription factor pathways by proline oxidase might affect gene expression events important to p53 regulation of cell growth and apoptosis.

Functional consequences of PRODH missense mutations.

PRODH maps to 22q11 in the region deleted in the velocardiofacial syndrome/DiGeorge syndrome (VCFS/DGS) and encodes proline oxidase (POX), a mitochondrial inner-membrane enzyme that catalyzes the first step in the proline degradation pathway. At least 16 PRODH missense mutations have been identified in studies of type I hyperprolinemia (HPI) and schizophrenia, 10 of which are present at polymorphic frequencies. The functional consequences of these missense mutations have been inferred by evolutionary conservation, but none have been tested directly. Here, we report the effects of these mutations on POX activity. We find that four alleles (R185Q, L289M, A455S, and A472T) result in mild (<30%), six (Q19P, A167V, R185W, D426N, V427M, and R431H) in moderate (30%-70%), and five (P406L, L441P, R453C, T466M, and Q521E) in severe (>70%) reduction in POX activity, whereas one (Q521R) increases POX activity. The POX encoded by one severe allele (T466M) shows in vitro responsiveness to high cofactor (flavin adenine dinucleotide) concentrations. Although there is limited information on plasma proline levels in individuals of known PRODH genotype, extant data suggest that severe hyperprolinemia (>800 microM) occurs in individuals with large deletions and/or PRODH missense mutations with the most-severe effect on function (L441P and R453C), whereas modest hyperprolinemia (300-500 microM) is associated with PRODH alleles with a moderate reduction in activity. Interestingly, three of the four alleles associated with or found in schizophrenia (V427M, L441P, and R453C) resulted in severe reduction of POX activity and hyperprolinemia. These observations plus the high degree of polymorphism at the PRODH locus are consistent with the hypothesis that reduction in POX function is a risk factor for schizophrenia.

Proline oxidase induces apoptosis in tumor cells, and its expression is frequently absent or reduced in renal carcinomas.

Proline oxidase is a p53-induced gene that can mediate apoptosis in lung carcinoma cells. Here, we provide evidence implicating a role for proline oxidase in renal carcinoma. We observed absent or reduced expression of proline oxidase in 8 of 12 primary renal cell carcinomas, with respect to their normal tissue counterparts. Two renal cell carcinomas, which displayed little or no expression of proline oxidase, expressed p53s that were less capable of inducing proline oxidase than p53 isolated from normal renal tissue. One of those tumor-derived p53s contained a double transition mutation at amino acid residues 125 (Ala to Thr) and 193 (Arg to His), and the other exhibited a single transition mutation at amino acid 149 (Ser to Phe). Forced up-regulation of proline oxidase induced the formation of reactive oxygen species and mediated apoptosis in the 786-0 renal cell carcinoma cell line. A proline oxidase antisense vector repressed p53-induced up-regulation of proline oxidase, release of cytochrome c from mitochondria, and apoptosis in 786-0 renal carcinoma cells. Taken together, these findings support a role for proline oxidase as a downstream effector in p53-mediated apoptosis. We hypothesize that its altered expression can contribute to the development of renal carcinomas. The presence of proline oxidase in mitochondria, a primary organelle that regulates apoptosis, places this molecule in a subcellular localization that can directly influence the apoptotic pathway and thus tumorigenesis.

Neurotransmitter levels and synaptic strength at the Drosophila larval neuromuscular junction are not altered by mutation in the sluggish-A gene, which encodes proline oxidase and affects adult locomotion.

The sluggish-A (slgA) gene of Drosophila melanogaster has been shown to encode for the enzyme proline oxidase, a mitochondrial enzyme which catalyzes the first step in the conversion of L-proline to L-glutamate. The slgA transcript is expressed in both larval and adult Drosophila melanogaster. Mutations in this gene lead to reduced proline oxidase activity and an elevation of free proline levels. Adult mutant flies show a striking reduction of motor activity. Since proline oxidase may contribute to the supply of the neurotransmitter glutamate in the nervous system, a reduction in proline oxidase activity could reduce neural glutamate pools and affect synaptic transmission in neurons utilizing glutamate as a transmitter, including peripheral motor neurons. We tested the hypothesis that glutamate, and synaptic transmission mediated by glutamate, are reduced at synapses of glutamatergic motor neurons in slgA mutants. Levels of glutamate and proline in different cell compartments, and functional properties of synaptic transmission were compared in slgA and control specimens. Proline is elevated in muscle cells of slgA mutants, indicating that the slgA gene regulates tissue proline levels. In nerve terminal varicosities, proline levels were low in both mutants and controls. Glutamate levels in nerve terminal varicosities of slgA mutants and controls were similar. In addition, we found that glutamatergic synaptic transmission at individual nerve endings and at the whole-cell level was similar in slgA mutants and controls. Thus, proline oxidase does not play a major role in generating neuronal glutamate pools at the Drosophila larval neuromuscular junction, and larval neuromuscular performance is not altered significantly in slgA mutants. Metabolic pathways other than that involving proline oxidase are able to sustain glutamatergic synaptic function in Drosophila larvae.

Sinorhizobium meliloti putA gene regulation: a new model within the family Rhizobiaceae.

Proline dehydrogenase (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. In Sinorhizobium meliloti, as in other microorganisms, the putA gene is transcriptionally activated in response to proline. In Rhodobacter capsulatus, Agrobacterium, and most probably in Bradyrhizobium, this activation is dependent on an Lrp-like protein encoded by the putR gene, located immediately upstream of putA. Interestingly, sequence and genetic analysis of the region upstream of the S. meliloti putA gene did not reveal such a putR locus or any other encoded transcriptional activator of putA. Furthermore, results obtained with an S. meliloti putA null mutation indicate the absence of any proline-responsive transcriptional activator and that PutA serves as an autogenous repressor. Therefore, the model of S. meliloti putA regulation completely diverges from that of its Rhizobiaceae relatives and resembles more that of enteric bacteria. However, some differences have been found with the latter model: (i) S. meliloti putA gene is not catabolite repressed, and (ii) the gene encoding for the major proline permease (putP) does not form part of an operon with the putA gene.

[Metabolism of L-thiazolidine-4-carboxylic acid]. / Metabolismus der L-Thiazolidincarbonsäure-(4).

L-thiazolidine-(4)-carboxylic acid (TAC) has proven to be a good substrate for long-term parenteral nutrition. In this study the metabolism of TAC in the rat is examined at subcellular level. TAC is oxidized by mitochondrial proline oxidase of liver and kidney to L-thiazoline-(4)-carboxylic acid, which then is hydrolyzed to N-formyl-cysteine (FCYS). FCYS is hydrolyzed to cysteine and formic acid by a cytosolic enzyme.