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Exposure to xenobiotic such as benzo[a]pyrene (B[a]P) induces metabolic changes, which have a considerable impact on the cellular response. Nevertheless, we are just in the beginning to reach an understanding of these processes. In this study, a gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach was applied to distinguish the metabolic changes that bladder epithelia cells undergo upon B[a]P exposure. To closely reflect the epithelia cell conditions in vivo, freshly isolated primary porcine urinary bladder epithelial cells (PUBEC) were utilized for the current study. An untargeted metabolomics approach was used to characterize the time- (6 h, 24 h, 48 h) and dose-dependent (0.5 µM, 5 µM, 10 µM B[a]P) changes in the metabolome of PUBEC upon B[a]P exposure, which led to the profiling of more than 200 metabolites that differed significantly between control and exposed samples. Multivariate analysis of the data highlighted that in the experimental setup/model used other than the exposure concentration, it is the exposure time which seems to be most important for distinguishing between different groups and hence may have a bigger role in B[a]P-mediated toxicity but may be specific for cell model used and hence requires further investigations. Further, enrichment and pathway analysis using MetaboAnalyst highlighted that exposure to B[a]P mainly alters the cellular amino acid metabolism. Particularly, 1-pyrroline-5-carboxylic acid (P5C), an intermediate of the cycling of the amino acid proline, was identified as a differentially altered metabolite at all concentrations and exposure times used in the experiment. An increase in the activity of proline dehydrogenase/proline oxidase (PRODH/POX), which oxidizes proline to P5C, was also observed, further supporting our metabolomic data. Our findings contribute to an improved knowledge about the reprogramming of metabolism which is a fundamental element of the cellular response to B[a]P and draw attention to the role of proline in this context.

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

Polyamines (putrescine, spermidine, and spermine) are synthesized primarily from ornithine via ornithine decarboxylase (ODC) in mammals. Although avian tissues contain ODC activity, little is known about intracellular sources of ornithine for their polyamine synthesis. This study tested the hypothesis that arginase and proline oxidase contribute to polyamine synthesis in chickens. Kidney, jejunum, leg muscle, and liver from 0-, 7-, 14- and 21-day-old broiler chickens were assayed for the activities of arginase, proline oxidase (POX), ornithine aminotransferase (OAT), and ornithine decarboxylase (ODC). Kidney slices were also used to determine

Hyperprolinemia type I (HPI) is an autosomal recessive metabolic disorder caused by defects in proline oxidase. We herein describe a case of a patient with HPI and harboring the NM_016335.4 (PRODH_v001):c.1397 C > T (p.T466M) mutation and polymorphisms in the PRODH gene, as detected by plasma amino acid analysis and Sanger sequencing. The patient presented with short stature, carbohydrate-rich dietary preferences, and mild intellectual disability that was suggestive of a neurodevelopmental or learning disorder.

The pathogenic protist Trypanosoma cruzi uses kissing bugs as invertebrate hosts that vectorize the infection among mammals. This parasite oxidizes proline to glutamate through two enzymatic steps and one nonenzymatic step. In insect vectors, T. cruzi differentiates from a noninfective replicating form to nonproliferative infective forms. Proline sustains this differentiation, but to date, a link between proline metabolism and differentiation has not been established. In T. cruzi, the enzymatic steps of the proline-glutamate oxidation pathway are catalyzed exclusively by the mitochondrial enzymes proline dehydrogenase [TcPRODH, EC: 1.5.5.2] and Δ1-pyrroline-5-carboxylate dehydrogenase [TcP5CDH, EC: 1.2.1.88]. Both enzymatic steps produce reducing equivalents that are able to directly feed the mitochondrial electron transport chain (ETC) and thus produce ATP. In this study, we demonstrate the contribution of each enzyme of the proline-glutamate pathway to ATP production. In addition, we show that parasites overexpressing these enzymes produce increased levels of H2O2, but only those overexpressing TcP5CDH produce increased levels of superoxide anion. We show that parasites overexpressing TcPRODH, but not parasites overexpressing TcP5CDH, exhibit a higher rate of differentiation into metacyclic trypomastigotes in vitro. Finally, insect hosts infected with parasites overexpressing TcPRODH showed a diminished parasitic load but a higher percent of metacyclic trypomastigotes, when compared with controls. Our data show that parasites overexpressing both, PRODH and P5CDH had increased mitochondrial functions that orchestrated different oxygen signaling, resulting in different outcomes in relation to the efficiency of parasitic differentiation in the invertebrate host.

Cancer cells often change their metabolism to support uncontrolled proliferation. Proline is the only proteogenic secondary amino acid that is abundant in the body. Recent studies have shown that proline metabolism plays an important role in metabolic reprogramming and affects the occurrence and development of cancer. Proline metabolism is related to ATP production, protein and nucleotide synthesis, and redox homeostasis in tumor cells. Proline can be synthesized by aldehyde dehydrogenase family 18 member A1 (ALDH18A1) and delta1-pyrroline-5-carboxylate reductase (PYCR), up-regulating ALDH18A1 and PYCR can promote the proliferation and invasion of cancer cells. As the main storage of proline, collagen can influence cancer cells proliferation, invasion, and metastasis. Its synthesis depends on the hydroxylation of proline catalyzed by prolyl 4-hydroxylases (P4Hs), which will affect the plasticity and metastasis of cancer cells. The degradation of proline occurs in the mitochondria and involves an oxidation step catalyzed by proline dehydrogenase/proline oxidase (PRODH/POX). Proline catabolism has a dual role in cancer, linking apoptosis with the survival and metastasis of cancer cells. In addition, it has been demonstrated that the regulation of proline metabolic enzymes at the genetic and post-translational levels is related to cancer. This article reviews the role of proline metabolic enzymes in cancer proliferation, apoptosis, metastasis, and development. Research on proline metabolism may provide a new strategy for cancer treatment.

L-proline catabolism is emerging as a key pathway that is critical to cellular metabolism, growth, survival, and death. Proline dehydrogenase (PRODH) enzyme, which catalyzes the first step of proline catabolism, has diverse functional roles in regulating many pathophysiological processes, including apoptosis, autophagy, cell senescence, and cancer metastasis. Notably, accumulated evidence demonstrated that PRODH plays complex role in many types of cancers. In this review, we briefly introduce the function of PRODH, then its expression in different types of cancer. We next discuss the regulation of PRODH in cancer, the downstream pathways of PRODH and the therapies that are under investigation. Finally, we propose novel insights for future perspectives on the modulation of PRODH.

Proline dehydrogenase (PRODH) is a mitochondrial inner membrane flavoprotein critical for cancer cell survival under stress conditions and newly recognized as a potential target for cancer drug development. Reversible (competitive) and irreversible (suicide) inhibitors of PRODH have been shown in vivo to inhibit cancer cell growth with excellent host tolerance. Surprisingly, the PRODH suicide inhibitor N-propargylglycine (N-PPG) also induces rapid decay of PRODH with concordant upregulation of mitochondrial chaperones (HSP-60, GRP-75) and the inner membrane protease YME1L1, signifying activation of the mitochondrial unfolded protein response (UPRmt) independent of anticancer activity. The present study was undertaken to address two aims: (i) use PRODH overexpressing human cancer cells (ZR-75-1) to confirm the UPRmt inducing properties of N-PPG relative to another equipotent irreversible PRODH inhibitor, thiazolidine-2-carboxylate (T2C); and (ii) employ biochemical and transcriptomic approaches to determine if orally administered N-PPG can penetrate the blood–brain barrier, essential for its future use as a brain cancer therapeutic, and also potentially protect normal brain tissue by inducing mitohormesis. Oral daily treatments of N-PPG produced a dose-dependent decline in brain mitochondrial PRODH protein without detectable impairment in mouse health; furthermore, mice repeatedly dosed with 50 mg/kg N-PPG showed increased brain expression of the mitohormesis associated protease, YME1L1. Whole brain transcriptome (RNAseq) analyses of these mice revealed significant gene set enrichment in N-PPG stimulated neural processes (FDR p N-PPG appears to be unique among anticancer agents and should be evaluated for repurposing as a pharmaceutical capable of mitigating the proteotoxic mechanisms driving neurodegenerative disorders.

Triple-negative breast cancers (TNBCs) lack specific targeted therapy options and have evolved into highly chemo-resistant tumors that metastasize to multiple organs. The present study demonstrated that the proline dehydrogenase (PRODH) mRNA level in paired (tumor vs. normal) human breast tissue samples (n=234) was 6.6-fold greater than normal cells (*p=0.021). We established stable PRODH-overexpressing TNBC (HS578T) cells, and the malignant phenotypes were evaluated using soft agar colony formation and Transwell migration assays. The results demonstrated that PRODH induced epithelial-mesenchymal transition in cancer cells and increased cell proliferation. The present study found that the tea polyphenol epigallocatechin-3-gallate (EGCG) significantly inhibited PRODH and its regulated proteins, such as alpha-smooth muscle actin (alpha-SMA) expression in TNBC cells. These findings support the targeting of the PRODH signaling pathway as a potential therapeutic strategy in preventing cancer cell metastasis. The patient-derived xenograft (PDX) mouse model is highly relevant to real human tumor growth. We established a TNBC-PDX (F4, n=4 in each group)mouse model. The PDX mice were treated with EGCG (50 mg/kg), and the results indicated that EGCG significantly inhibited PDX tumor growth (*p = 0.013). These experiments provide additional evidence to evaluate the antitumor effects of EGCG-induced PRODH inhibition for clinical therapeutic application, especially in TNBC patients.

Proline is one of the precursors of the biosynthesis of 2-acetyl-1-pyrroline (2-AP) which is the key and characteristic volatile component of fragrant rice aroma. In order to study the effects of exogenous proline on 2-AP biosynthesis and other grain quality attributes in fragrant rice, two indica fragrant rice cultivars, “Meixiangzhan-2” and “Xiangyaxiangzhan”, and one japonica fragrant rice, “Yunjingyou”, were used in present study. At initial heading stage, proline solutions at 0 (CK), 0.10 (Pro1), 0.20 (Pro2) and 0.50 (Pro3) g L-1 were applied as foliar spray solution to fragrant rice plants. Compared with CK, Pro1, Pro2 and Pro3 treatments significantly increased the grain 2-AP content. The significant up-regulation effects due to proline treatments were observed in the contents of proline, △1-pyrrolidine-5-carboxylic acid (P5C) and △1-pyrroline which involved in 2-AP formation. Exogenous proline application also significantly decreased the grain γ-aminobutyric acid (GABA) content. Furthermore, proline treatments enhanced the activity of proline dehydrogenase (ProDH) as well as transcript level of gene PRODH. On the other hand, the transcript level of gene BADH2 and activity of betaine aldehyde dehydrogenase (BADH) decreased under proline treatments. Proline treatments (Pro2 and Pro3) also increased the grain protein content by 3.57–6.51%. Moreover, 32.03–34.25% lower chalky rice rate and 30.80–48.88% lower chalkiness were recorded in proline treatments (Pro2 and Pro3) for both Meixiangzhan and Xiangyaxiangzhan whilst for Yunjingyou, foliar application of proline had no significant effect on chalky rice rate and chalkiness. There was no remarkable difference observed in grain milled quality (brown rice rate, milled rice rate and head rice rate) and amylose content between CK and proline treatments. In conclusion, exogenous proline enhanced the 2-AP biosynthesis and promoted some grain quality characters of fragrant rice.

Cyanobacteria have evolved mechanisms to adapt to environmental stress and nutrient availability, including accumulation of storage compounds in inclusions and granules. As arginine is a key building block of cyanophycin, a dynamic nitrogen reservoir in many cyanobacteria, arginine metabolism plays a key role in cyanobacterial nitrogen storage and remobilization. Recently, an arginine dihydrolase AgrE/ArgZ was identified as a major arginine-degrading enzyme in nondiazotrophic Synechocystis, which catalyzes the conversion of arginine into ornithine and ammonia. The N-terminal domain of AgrE/ArgZ is responsible for arginine dihydrolase activity. Burnat et al. (2019) identified the arginine catabolic pathway in diazotrophic Anabaena, which starts with the reaction catalyzed by AgrE/ArgZ. Moreover, this study identified the C-terminal domain of AgrE/ArgZ as an ornithine cyclodeaminase that catalyze the conversion of ornithine to proline. The results demonstrated that arginine is catabolized to generate glutamate by the concerted action of AgrE/ArgZ and bifunctional proline oxidase PutA in the vegetative cells of Anabaena. These findings expand our knowledge on nitrogen mobilization and redistribution in Anabaena under nitrogen-fixation conditions. AgrE/ArgZ is widely present in many diazotrophic cyanobacteria and may be important for their contribution to marine nitrogen fixation. AgrE/ArgZ may have potential applications in metabolic engineering and biotechnology.

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

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

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

Illumination conditions and acquisition of source status by winter oilseed rape leaves jointly impact on proline levels, expression levels of proline metabolism-related genes, and associated metabolic fluxes., Proline metabolism is an essential component of plant adaptation to multiple environmental stress conditions that is also known to participate in specific developmental phases, particularly in reproductive organs. Recent evidence suggested a possible role for proline catabolism in Brassica napus for nitrogen remobilization processes from source leaves at the vegetative stage. Here, we investigate transcript levels of Δ1-PYRROLINE-5-CARBOXYLATE SYNTHASE (P5CS) and PROLINE DEHYDROGENASE (ProDH) genes at the vegetative stage with respect to net proline biosynthesis and degradation fluxes in leaves having a different sink/source balance. We showed that the underexpression of three P5CS1 genes in source leaves was accompanied by a reduced commitment of de novo assimilated 15N towards proline biosynthesis and an overall depletion of free proline content. We found that the expression of ProDH genes was strongly induced by carbon starvation conditions (dark-induced senescence) compared with early senescing leaves. Our results suggested a role for proline catabolism in B. napus, but acting only at a late stage of senescence. In addition, we also identified some P5CS and ProDH genes that were differentially expressed during multiple processes (leaf status, dark to light transition, and stress response).

Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent 2-electron oxidation of l-proline to Δ(1)-pyrroline-5-carboxylate. PRODH has emerged as a possible cancer therapy target, and thus the inhibition of PRODH is of interest. Here we show that the proline analogue thiazolidine-2-carboxylate (T2C) is a mechanism-based inactivator of PRODH. Structures of the bifunctional proline catabolic enzyme proline utilization A (PutA) determined from crystals grown in the presence of T2C feature strong electron density for a 5-membered ring species resembling l-T2C covalently bound to the N5 of the FAD in the PRODH domain. The modified FAD exhibits a large butterfly bend angle, indicating that the FAD is locked into the 2-electron reduced state. Reduction of the FAD is consistent with the crystals lacking the distinctive yellow color of the oxidized enzyme and stopped-flow kinetic data showing that T2C is a substrate for the PRODH domain of PutA. A mechanism is proposed in which PRODH catalyzes the oxidation of T2C at the C atom adjacent to the S atom of the thiazolidine ring (C5). Then, the N5 atom of the reduced FAD attacks the C5 of the oxidized T2C species, resulting in the covalent adduct observed in the crystal structure. To our knowledge, this is the first report of T2C inactivating (or inhibiting) PRODH or any other flavoenzyme. These results may inform the design of new mechanism-based inactivators of PRODH for use as chemical probes to study the roles of proline metabolism in cancer.

Drought is one of the most serious environmental factors limiting production of sugarcane worldwide. In order to assess the influence of gibberellins (GA3 ) on drought and plant growth, along with associated physio-biochemical attributes, expression of eight drought-responsive genes were quantified and analysed. At grand growth stage (120 DAP) two sugarcane varieties (CoLk94184, CoPK05191) were exposed to drought by withholding irrigation. GA3 (35 ppm) was applied using battery-operated uniform controlled dispensing sprayer twice at 1-week intervals on 2-week drought-stressed plants. Physio-biochemical attributes including antioxidant enzyme activities were estimated following standard protocols. RT-PCR was performed to visualise the drought-associated gene expression patterns. Drought triggered a reduction in RWC and chlorophyll content but these recovered when droughted plants were exposed to GA3 . Proline content increased many fold in both varieties under stress, but decreased under the influence of GA3 . There was a mixed response of antioxidant enzyme activity, which distinctly declined after GA3 exposure, together with a lesser reduction in dry matter content over that of control plants. With increasing stress, expression of pyrroline-5-carboxylase synthetase (P5CS) and betaine-aldehyde dehydrogenase genes was observed, selectively up-regulated in CoPK05191. Expression of proline oxidase/transporter was high in CoPK05191 but diminished along with proline content after exposure to GA3. CoLk94184 showed no significant difference in P5CS gene expression under stress condition, whereas expression of betaine-aldehyde dehydrogenase gene was unchanged in response to stress. Results demonstrated that exposure of droughted plants to GA3 not only led to recovery of activity of drought-associated physio-biochemical attributes, but also minimised impact on cane dry weight and quality. Further, GA3 application caused differential gene expression that possibly triggers increased responsiveness towards drought tolerance in sugarcane.

It has been considered that proline dehydrogenase/proline oxidase (PRODH/POX) is involved in antineoplastic activity of metformin (MET). The aim of this study is identification of key metabolites of glycolysis, pentose phosphate pathway (PPP), tricarboxylic acids (TCA), urea cycles (UC) and some amino acids in MET-treated MCF-7 cells and PRODH/POX-knocked out MCF-7 (MCF-7crPOX) cells. MCF-7crPOX cells were generated by using CRISPR-Cas9. Targeted metabolomics was performed by LC-MS/MS/QqQ. Expression of pro-apoptotic proteins was evaluated by Western blot. In the absence of glutamine, MET treatment or PRODH/POX-knock out of MCF-7 cells contributed to similar inhibition of glycolysis (drastic increase in intracellular glucose and pyruvate) and increase in the utilization of phospho-enol-pyruvic acid, glucose-6-phosphate and some metabolites of TCA and UC, contributing to apoptosis. However, in the presence of glutamine, MET treatment or PRODH/POX-knock out of MCF-7 cells contributed to utilization of some studied metabolites (except glucose), facilitating pro-survival phenotype of MCF-7 cells in these conditions. It suggests that MET treatment or PRODH/POX-knock out induce similar metabolic effects (glucose starvation) and glycolysis is tightly linked to glutamine metabolism in MCF-7 breast cancer cells. The data provide insight into mechanism of anticancer activity of MET as an approach to further studies on experimental breast cancer therapy.

Proline degradation by proline dehydrogenase/proline oxidase (PRODH/POX) contributes to apoptosis or autophagy. The identification of specific pathway of apoptosis/survival regulation is the aim of this study. We generated knocked-down PRODH/POX MCF-7 breast cancer cells (MCF-7shPRODH/POX). PRODH/POX silencing did not affect cell viability. However, it contributed to decrease in DNA and collagen biosynthesis, increase in prolidase activity and intracellular proline concentration as well as increase in the expression of iNOS, NF-κB, mTOR, HIF-1α, COX-2, AMPK, Atg7 and Beclin-1 in MCF-7shPRODH/POX cells. In these cells, glycyl-proline (GlyPro, substrate for prolidase) further inhibited DNA and collagen biosynthesis, maintained high prolidase activity, intracellular concentration of proline and up-regulated HIF-1α, AMPK, Atg7 and Beclin-1, compared to GlyPro-treated MCF-7 cells. In MCF-7 cells, GlyPro increased collagen biosynthesis, concentration of proline and expression of caspase-3, cleaved caspases -3 and -9, iNOS, NF-κB, COX-2 and AMPKβ. PRODH/POX knock-down contributed to pro-survival autophagy pathways in MCF-7 cells and GlyPro-derived proline augmented this process. However, GlyPro induced apoptosis in PRODH/POX-expressing MCF-7 cells as detected by up-regulation of active caspases -3 and -9. The data suggest that PRODH/POX silencing induces autophagy in MCF-7 cells and GlyPro-derived proline supports this process.

The hypO gene from Sinorhizobium meliloti, located within the trans-4-hydroxy-L-proline metabolic gene cluster, was first successfully expressed in the host Pseudomonas putida. Purified HypO protein functioned as a FAD-containing cis-4-hydroxy-D-proline dehydrogenase with a homomeric structure. In contrast to other known enzymes, significant activity for D-proline was found, confirming a previously proposed potential involvement in D-proline metabolism.

Escherichia coli proline utilization A (EcPutA) is the archetype of trifunctional PutA flavoproteins, which function both as regulators of the proline utilization operon and bifunctional enzymes that catalyze the four-electron oxidation of proline to glutamate. EcPutA shifts from a self-regulating transcriptional repressor to a bifunctional enzyme in a process known as functional switching. The flavin redox state dictates the function of EcPutA. Upon proline oxidation, the flavin becomes reduced, triggering a conformational change that causes EcPutA to dissociate from the put regulon and bind to the cellular membrane. Major structure/function domains of EcPutA have been characterized, including the DNA-binding domain, proline dehydrogenase (PRODH) and L-glutamate-γ-semialdehyde dehydrogenase catalytic domains, and an aldehyde dehydrogenase superfamily fold domain. Still lacking is an understanding of the membrane-binding domain, which is essential for EcPutA catalytic turnover and functional switching. Here, we provide evidence for a conserved C-terminal motif (CCM) in EcPutA having a critical role in membrane binding. Deletion of the CCM or replacement of hydrophobic residues with negatively charged residues within the CCM impairs EcPutA functional and physical membrane association. Furthermore, cell-based transcription assays and limited proteolysis indicate that the CCM is essential for functional switching. Using fluorescence resonance energy transfer involving dansyl-labeled liposomes, residues in the α-domain are also implicated in membrane binding. Taken together, these experiments suggest that the CCM and α-domain converge to form a membrane-binding interface near the PRODH domain. The discovery of the membrane-binding region will assist efforts to define flavin redox signaling pathways responsible for EcPutA functional switching.

Proline has important roles in multiple biological processes such as cellular bioenergetics, cell growth, oxidative and osmotic stress response, protein folding and stability, and redox signaling. The proline catabolic pathway, which forms glutamate, enables organisms to utilize proline as a carbon, nitrogen, and energy source. FAD-dependent proline dehydrogenase (PRODH) and NAD+-dependent glutamate semialdehyde dehydrogenase (GSALDH) convert proline to glutamate in two sequential oxidative steps. Depletion of PRODH and GSALDH in humans leads to hyperprolinemia, which is associated with mental disorders such as schizophrenia. Also, some pathogens require proline catabolism for virulence. A unique aspect of proline catabolism is the multifunctional proline utilization A (PutA) enzyme found in Gram-negative bacteria. PutA is a large (> 1000 residues) bifunctional enzyme that combines PRODH and GSALDH activities into one polypeptide chain. In addition, some PutAs function as a DNA-binding transcriptional repressor of proline utilization genes. This review describes several attributes of PutA that make it a remarkable flavoenzyme: (1) diversity of oligomeric state and quaternary structure; (2) substrate channeling and enzyme hysteresis; (3) DNA-binding activity and transcriptional repressor function; and (4) flavin redox dependent changes in subcellular location and function in response to proline (functional switching).

Metastases are the leading cause of mortality in patients with cancer. Metastasis formation requires cancer cells to adapt their cellular phenotype. However, how metabolism supports this adaptation of cancer cells is poorly defined. We use 2D versus 3D cultivation to induce a shift in the cellular phenotype of breast cancer cells. We discover that proline catabolism via proline dehydrogenase (Prodh) supports growth of breast cancer cells in 3D culture. Subsequently, we link proline catabolism to in vivo metastasis formation. In particular, we find that PRODH expression and proline catabolism is increased in metastases compared to primary breast cancers of patients and mice. Moreover, inhibiting Prodh is sufficient to impair formation of lung metastases in the orthotopic 4T1 and EMT6.5 mouse models, without adverse effects on healthy tissue and organ function. In conclusion, we discover that Prodh is a potential drug target for inhibiting metastasis formation., Metastasizing cancer cells rewire their metabolism to support their malignant phenotypes. Here, the authors show that the acquisition of a metastatic phenotype in breast cancer cell lines results in increased proline catabolism and that inhibition of this pathway decreases lung metastasis formation in two mouse models.

Proline dehydrogenase (ProDH) is a flavoenzyme that catalyzes the oxidation of proline (Pro) into Δ1-pyrroline-5-carboxylate (P5C). In eukaryotes, ProDH coordinates with different Pro metabolism enzymes to control energy supply or stress responses signaling. Heterologous expression and crystallization of prokaryotic enzymes provided key data on their active center, folding capacity and oligomerization status. In contrast, eukaryotic ProDHs have not been crystallized so far, and their study as recombinant proteins remains limited. Plants contain two isoforms of ProDH with non-redundant functions. To contribute to the study of these enzymes, we describe the modeling, expression in E. coli, purification, and characterization of the Arabidopsis isoenzymes, AtProDH1 and AtProDH2. The 3D model suggested that both proteins adopt a distorted barrel structure (βα) with a cap formed by N-terminal α helices. The expression of two types of N-terminal deletion proteins indicated that this domain affected enzyme activity. Full-length enzymes had Km values similar to those of native proteins, whereas truncated proteins were inactive. Moreover, the first α helix proved to be necessary for AtProDH1 and AtProDH2 activities. Interestingly, both isoenzymes were able to oligomerize and this also required the first N-terminal α helix. Thus, we report the first insights into structure-function relationship of plant ProDHs demonstrating that the N-terminus, although not directly involved in catalysis, controls enzyme arrangement and activity. The resources generated here could be useful to analyze other plant ProDH features, such as its coordination with other enzymes, and differences between ProDH1 and ProDH2, providing new information on its effects on stress tolerance.

Valeriana wallichii, a perennial herb belonging to family Valerianaceae, is an important medicinal herb of the Himalayan region. The incessant exploitation of nature for meeting the demands of the pharmaceutical industry has put unbearable pressure on its natural habitats. A study on its physiological, biochemical, growth and reproductive attributes was planned. Physiological study revealed that ex-situ (outside their natural habitat) populations faced severe stress as compared to in-situ (natural habitat) plants. The difference in the performance of these habitat plants was related to superoxide and H2O2 in the leaves. Photosynthetic attributes were increased in in-situ populations. Proline content and its biosynthetic enzymes ornithine aminotransferase, and pyrroline-5-carboxylate reductase showed an increase in ex-situ plants, proline oxidase decreased. Glucose-6-phosphate dehydrogenase, shikimic acid dehydrogenese, phenylalanine lyase, and flavonoids content showed an increment in ex-situ plants. Antioxidants enzyme superoxide dismutase, catalase, ascorbate peroxidase and reduced glutathione showed an increment in ex-situ conditions. Growth and reproductive attributes were more in ex-situ plants. The observations made are suggestive that a comprehensive conservation programme involving in-situ as well as ex-situ strategies will be effective for the conservation and long term survival of the species.

Background/aims Proline availability for proline dehydrogenase/proline oxidase (PRODH/POX) may represent switching mechanism between PRODH/POX-dependent apoptosis and autophagy. The aim of the study was to evaluate the impact of overexpression of prolidase (proline releasing enzyme) on apoptosis/autophagy in breast cancer MCF-7 cells. Methods The model of MCF-7 cells with prolidase overexpression (MCF-7PL) was obtained. In order to targeting proline for PRODH/POX-dependent pathways substrate for prolidase, glycyl-proline (GP) was provided and proline utilization for collagen biosynthesis was blocked using 2-methoxyestradiol (MOE). Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. DNA, collagen and total protein biosynthesis were determined by radiometric method. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. Results Prolidase overexpression in MCF-7PL cells contributed to 10-fold increase in the enzyme activity, 3-fold increase in cytoplasmic proline level and decrease in cell viability and DNA biosynthesis compared to wild type MCF-7 cells. In MCF-7PL cells MOE and GP significantly decreased the number of living cells. MOE inhibited DNA biosynthesis in both cell lines while GP evoked inhibitory effect on the process only in MCF-7PL cells. In both cell lines, MOE or MOE+GP inhibited DNA and collagen biosynthesis. Although GP in MCF-7 cells stimulated collagen biosynthesis, it inhibited the process in MCF-7PL cells. The effects of studied compounds in MCF-7PL cells were accompanied by increase in the expression of Atg7, LC3A/B, Beclin-1, HIF-1α and decrease in the expression of PRODH/POX, active caspases-3 and -9. Conclusion The data suggest that overexpression of prolidase in MCF-7 cells contributes to increase in intracellular proline concentration and PRODH/POX-dependent autophagic cell death.

DNA methylation changes during aging, but it remains unclear whether the effect of DNA methylation on lung cancer survival varies with age. Such an effect could decrease prediction accuracy and treatment efficacy. We performed a methylation-age interaction analysis using 1,230 early-stage lung adenocarcinoma patients from five cohorts. A Cox proportional hazards model was used to investigate lung adenocarcinoma and squamous cell carcinoma patients for methylation-age interactions, which were further confirmed in a validation phase. We identified one adenocarcinoma-specific CpG probe, cg14326354PRODH, with effects significantly modified by age (HRinteraction = 0.989; 95% CI: 0.986-0.994; P = 9.18×10-7). The effect of low methylation was reversed for young and elderly patients categorized by the boundary of 95% CI standard (HRyoung = 2.44; 95% CI: 1.26-4.72; P = 8.34×10-3; HRelderly = 0.58; 95% CI: 0.42-0.82; P = 1.67×10-3). Moreover, there was an antagonistic interaction between low cg14326354PRODH methylation and elderly age (HRinteraction = 0.21; 95% CI: 0.11-0.40; P = 2.20×10-6). In summary, low methylation of cg14326354PRODH might benefit survival of elderly lung adenocarcinoma patients, providing new insight to age-specific prediction and potential drug targeting.

NADPH oxidase-mediated H2O2 maintains proline concentration under NaCl stress through regulating its biosynthesis and degradation, conferring salt tolerance to wheat plants. Considerable attention has been paid to the specific role of hydrogen peroxide (H2O2) in plant stress responses. Here, using microscopic, pharmacological and biochemical approaches, we explored H2O2 production and its roles in redox control under salt stress in wheat roots. Exogenous H2O2 pretreatment decreased salt-induced lipid peroxidation, while increased proline content in wheat roots. Salt stress led to a transient increase in NADPH oxidase activity accompanied by accumulation of H2O2 and proline in roots. The elevated proline accumulation in the presence of NaCl was significantly suppressed by diphenyleneiodonium, an inhibitor of NADPH oxidase, and dimethylthiourea, a scavenger of H2O2. The rate-limiting enzyme involved in proline biosynthesis, Δ1-pyrroline-5-carboxylate synthetase (P5CS), was induced by NaCl, whereas the house-keeping enzyme in proline degradation, proline dehydrogenase (ProDH), was inhibited. After 6 h, the activity of P5CS increased by 1.5-fold, whereas ProDH decreased by 13.9%. The levels of these enzymes, however, were restored by NADPH oxidase inhibitor or H2O2 scavenger. After treatment with H2O2, the effects of diphenyleneiodonium and or dimethylthiourea on proline content and activities of P5CS and ProDH were reversed. These results suggested that NADPH oxidase-mediated H2O2 alleviates oxidative damage induced by salt stress through regulating proline biosynthesis and degradation.

In stress conditions, as neoplastic transformation, amino acids serve not only as nutrients to maintain the cell survival but also as mediators of several regulatory pathways which are involved in apoptosis and autophagy. Especially, under glucose deprivation, in order to maintain the cell survival, proline and glutamine together with other glutamine-derived products such as glutamate, alpha-ketoglutarate, and ornithine serve as alternative sources of energy. They are substrates for production of pyrroline-5-carboxylate which is the product of conversion of proline by proline dehydrogenase/ proline oxidase (PRODH/POX) to produce ATP for protective autophagy or reactive oxygen species for apoptosis. Interconversion of proline, ornithine, and glutamate may therefore regulate PRODH/POX-dependent apoptosis/autophagy. The key amino acid is proline, circulating between mitochondria and cytoplasm in the proline cycle. This shuttle is known as proline cycle. It is coupled to pentose phosphate pathway producing nucleotides for DNA biosynthesis. PRODH/POX is also linked to p53 and AMP-activated protein kinase (AMPK)-dependent pathways. Proline availability for PRODH/POX-dependent apoptosis/autophagy is regulated at the level of collagen biosynthesis (proline utilizing process) and prolidase activity (proline supporting process). In this review, we suggest that amino acid metabolism linking TCA and Urea cycles affect PRODH/POX-dependent apoptosis/autophagy and the knowledge might be useful to targeted cancer therapy.

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

Two mungbean (Vigna radiata L.) cultivars, namely, Punt Mung (salt-tolerant) and Samrat (salt-sensitive), were tested for their response to salinity (50 mM NaCl) and sulfur (1.0 mM SO42− and 2.0 mM SO42−) supplied alone or in combination. Compared to Punt Mung, the extent of 50 mM NaCl-accrued decreases in leaf gas exchange parameters (net photosynthetic rate, intercellular CO2, stomatal conductance, intrinsic water-use efficiency and Rubisco activity), stomatal behavior (frequency and diameter), and plant dry mass was higher in Samrat. However, contents of proline and reduced glutathione (GSH), and the activity of glutathione reductase (GR) and nitrate reductase (NR) were higher in Punt Mung. Notably, the salt-sensitive Samrat exhibited the higher content of glucose and activity of proline oxidase under 50 mM NaCl exposure. The effects of 2.0 mM SO42− in alleviating 50 mM NaCl impact on leaf gas exchange parameters and plant dry mass were more conspicuous in Punt Mung. Overall, SO42− helped Punt Mung to maintain higher cellular levels of GSH and proline; a higher activity of GR and NR; decreased activity of proline oxidase, lower content of glucose, and controlled stomatal behavior. Eventually, SO42−-mediated aforesaid changes culminated into a higher tolerance of Punt Mung to 50 mM NaCl stress when compared to Samrat. Thus, the strategy of sulfur-mediated control of salinity in V. radiata may be adopted for salinity prone areas.