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Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent oxidation of L-proline to Δ(1)-pyrroline-5-carboxylate. PRODH plays a central role in the metabolic rewiring of cancer cells, which has motivated the discovery of inhibitors. Here, we studied the inhibition of PRODH by 18 proline-like compounds to understand the structural and chemical features responsible for the affinity of the best-known inhibitor, S-(–)-tetrahydro-2-furoic acid (1). The compounds were screened, and then six were selected for more thorough kinetic analysis: cyclobutane-1,1-dicarboxylic acid (2), cyclobutanecarboxylic acid (3), cyclopropanecarboxylic acid (4), cyclopentanecarboxylic acid (16), 2-oxobutyric acid (17), and (2S)-oxetane-2-carboxylic acid (18). These compounds are competitive inhibitors with inhibition constants in the range of 1.4 – 6 mM, compared to 0.3 mM for 1. Crystal structures of PRODH complexed with 2, 3, 4, and 18 were determined. All four inhibitors bind in the proline substrate site, but the orientations of their rings differ from that of 1. The binding of 3 and 18 is accompanied by compression of the active site to enable nonpolar contacts with Leu513. Compound 2 is unique in that the additional carboxylate displaces a structurally conserved water molecule from the active site. Compound 18 also destabilizes the conserved water, but by an unexpected non-steric mechanism. The results are interpreted using a chemical double mutant thermodynamic cycle. This analysis revealed unanticipated synergism between ring size and hydrogen bonding to the conserved water. These structure-affinity relationships provide new information relevant to the development of new inhibitor design strategies targeting PRODH.

Hyperprolinemia type II (HPII) is an inborn error of metabolism due to genetic variants in ALDH4A1, leading to a deficiency in Δ-1-pyrroline-5-carboxylate (P5C) dehydrogenase. This leads to an accumulation of toxic levels of P5C, an intermediate in proline catabolism. The accumulating P5C spontaneously reacts with, and inactivates, pyridoxal 5’-phosphate, a crucial cofactor for many enzymatic processes, which is thought to be the pathophysiological mechanism for HPII. Here, we describe the use of a combination of LC-QTOF untargeted metabolomics, NMR spectroscopy and infrared ion spectroscopy (IRIS) to identify and characterize biomarkers for HPII that result of the spontaneous reaction of P5C with malonic acid and acetoacetic acid. We show that these biomarkers can differentiate between HPI, caused by a deficiency of proline oxidase activity, and HPII. The elucidation of their molecular structures yields insights into the disease pathophysiology of HPII.

Proline biosynthesis and accumulation is a common response to unfavorable environment in many plants. This work aimed to elucidate the effects of boron (B)-deficiency and toxicity on proline metabolism and biosynthesis in Brassica napus in a hydroponic experiment. The results showed that B-deficiency and toxicity exert injurious impact on plant growth, accumulated high malondialdehyde (MDA) content, and caused the destruction of subcellular structure. Proline accumulated in both B deprivation and B toxicity plants, except B toxicity-treated root. In roots, B-deficiency increased ornithine content and pyrroline-5-carboxylate reductase (P5CR) activity, with the higher expression of BnaC03.P5CR, whilst decreased glutamate, glutamate-1-semialdehyde (GSA), pyrroline-5-carboxylate (P5C) contents and ornithine-δ-aminotransferase (δ-OAT), pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (ProDH) activities in terms of down-regulated the BnaC04.P5CS2, BnaA04.P5CS2, and BnaAnn.ProDH expression. The glutamate and GSA contents were decreased while P5C, arginine, and ornithine contents were enhanced in leaves under B-deficient and toxicity conditions. Lower glutamate pathway-related substance contents, P5CR, and δ-OAT activities while higher ProDH activity along with the same trend of related-gene expression were observed in B-toxicity-treated roots. Importantly, principal component analysis (PCA) in conjunction with correlation analysis indicated that ornithine pathway-related substances and enzymes contributed more to proline accumulation in B-deficient plant and B toxicity-treated leaves. Collectively, proline accumulation is caused by increased synthesis and decreased decomposition, and positively contributed, at least partly, by regulated ornithine pathway.

Proline dehydrogenase (PRODH) is a flavoenzyme that catalyzes the first step of proline catabolism, the oxidation of L-proline to Δ(1)-pyrroline-5-carboxylate. PRODH has emerged as a cancer therapy target because of its involvement in the metabolic reprogramming of cancer cells. Here we report the discovery of a new class of PRODH inactivator, which covalently and irreversibly modifies the FAD in a light-dependent manner. Two examples, 1,3-dithiolane-2-carboxylate and tetrahydrothiophene-2-carboxylate, have been characterized using X-ray crystallography (1.52 – 1.85 Å resolution), absorbance spectroscopy, and enzyme kinetics. The structures reveal that in the dark, these compounds function as classical reversible, proline analog inhibitors. However, exposure of enzyme-inhibitor co-crystals to bright white light induces decarboxylation of the inhibitor and covalent attachment of the residual S-heterocycle to the FAD N5 atom, locking the cofactor into a reduced, inactive state. Spectroscopic measurements of the inactivation process in solution confirm the requirement for light and show that blue light is preferred. Enzyme activity assays show that the rate of inactivation is enhanced by light and that the inactivation is irreversible. We also demonstrate the photosensitivity of cancer cells to one of these compounds. A possible mechanism is proposed involving photoexcitation of the FAD while the inhibitor is noncovalently bound in the active site, followed by electron transfer, decarboxylation, and radical combination steps. Our results could lead to the development of photopharmacological drugs targeting PRODH.

Cocaine is a commonly abused drug worldwide. Acute as well as repeated exposure to cocaine activates persistent cellular and molecular changes in the brain reward regions. The effects of cocaine are predominantly mediated via alterations in neuronal gene expression by chromatin remodeling. Poly(ADP-ribose) polymerase-1 (PARP-1) catalyzed PARylation of chromatin has been reported as an important regulator of cocaine-mediated gene expression. PARP-1 dependent ADP-ribosylation is an energy-dependent process. In this study, we investigated the cellular energy response to cocaine-induced upregulation of PARP-1 expression. Exposure of differentiated SH-SY5Y cells to varying concentrations of cocaine resulted in the induction of PARP-1 dependent PARylation of p53 tumor suppressor. Further analysis revealed that PARylation of p53 by cocaine treatment resulted in nuclear accumulation of p53. However, induction and nuclear accumulation of p53 did not correlate with neuronal apoptosis/cell death upon cocaine exposure. Interestingly, cocaine-induced p53 PARylation resulted in the induction of proline oxidase (POX)—a p53 responsive gene involved in cellular metabolism. Given that cocaine-induced p53 PARylation is an energy-dependent process, we observed that cocaine-induced PARP-1/p53/POX axes alters cellular energy metabolism. Accordingly, using pharmacological and genetic studies of PARP-1, p53, and POX, we demonstrated the contribution of POX in maintaining cellular energy during neuronal function. Collectively, these studies highlight activation of a novel metabolic pathway in response to cocaine treatment.

There are marked decreases in plasma concentrations of cortisol and arginine (an essential amino acid for neonates) as well as intestinal citrulline synthesis in piglets during the first 14 days of life. The objective of this study was to test the hypothesis that increasing plasma cortisol concentrations by cortisol administration may prevent the decline in intestinal citrulline and arginine synthesis from proline, thereby possibly increasing plasma arginine concentration in suckling piglets and their growth. Seven-day-old pigs reared by sows received daily intramuscular injections of hydrocortisone 21-acetate (25 mg/kg) or vehicle solution (saline) (n = 10/group). At 14 days of age, piglets were used to prepare jejunal enterocytes. Cells were incubated at 37 °C for 30 min in oxygenated Krebs buffer containing 5 mM glucose, 2 mM [U-14C]proline, and 2 mM glutamine. Cortisol treatment increased plasma cortisol concentration, mitochondrial proline oxidase and N-acetylglutamate synthase activities, cytosolic argininosuccinate lyase activity, and the intracellular concentrations of N-acetylglutamate and carbamoyl phosphate for citrulline and arginine synthesis. However, cortisol treatment induced the expression of intestinal arginase-II for arginine hydrolysis, resulting in no change in plasma arginine concentration. Administration of cortisol had no effect on milk consumption or the whole-body growth rate of piglets, but increased villus height in the jejunum and ileum. Collectively, these results suggest an important role for proline oxidase and N-acetylglutamate in regulating citrulline and arginine synthesis from proline in pig enterocytes. Because proline catabolism plays an important role in modulating protein synthesis, cell proliferation, and arginine production, our findings may have important implications for understanding the role of proline oxidase in the growth and health of the mammalian small intestine.

Hyperprolinemia Type I and II are genetic metabolic disorders caused by disrupted proline degradation. It has been suggested that hyperprolinemia is associated with increased risk of developmental and mental disorders but detailed information on the psychiatric phenotype in hyperprolinemic patients is limited. Following PRISMA guidelines, we carried out a systematic review to clarify psychiatric phenotypes in patients with hyperprolinemia. We screened 1753 studies and included 35 for analysis, including 20 case reports and 15 case–control and cohort studies. From these studies, a common psychiatric phenotype is observed with a high prevalence of developmental delay, intellectual disability, autism spectrum disorders, and psychosis spectrum disorders. In most cases, a genetic cause of hyperprolinemia was known, these included mutations in the PRODH and ALDH4A1 genes and deletions of chromosome 22q11.2. No evidence for a biochemical phenotype-clinical phenotype correlation was found; that is, no association between higher proline levels and specific psychiatric phenotypes was observed. This suggests that genomic and environmental factors are likely to contribute to clinical outcomes. More studies are needed to clarify whether hyperprolinemia is a primary causal factor underlying the increased risk of developing psychiatric disorders seen in patients with hyperprolinemia, or whether hyperprolinemia and psychiatric disorders are both consequences of a shared underlying mechanism.

Background Lanthanum (La) is a rare earth element that can influence plant growth and development. However, the effect of La on growth, yield formation and 2-acetyl-1-pyrroline (2-AP, a key compound responsible for the aroma of rice) biosynthesis in aromatic rice (Oryza sativa L. subsp. japonica Kato) has not been reported. Therefore, the present study investigated the effects of La on growth, photosynthesis, yield formation and 2-AP biosynthesis in aromatic rice through three experiments. Results Two pot experiments and a two-year field trial were conducted with different rates of La application (20–120 LaCl3 mg kg−1 and 12 kg ha−1 LaCl3), and treatments without La application were used as controls. The results showed that the application of LaCl3 at 80 and 100 mg kg−1 and at 12 kg ha−1 greatly increased the 2-AP content (by 6.45–43.03%) in aromatic rice seedlings and mature grains compared with the control. The La treatments also increased the chlorophyll content, net photosynthetic rate and total aboveground biomass of rice seedlings. Higher antioxidant enzyme (superoxide, peroxidase, and catalase) activity was detected in the La treatments than in the control. The La treatments also increased the grain yield, grain number per panicle and seed-setting rate of aromatic rice relative to the control. Moreover, the grain proline and γ-aminobutyric acid contents and the activity of betaine aldehyde dehydrogenase significantly decreased under the La treatment. The application of La to soil enhanced the activity of proline dehydrogenase by 20.62–56.95%. Conclusions La improved the growth, yield formation and 2-AP content of aromatic rice and enhanced 2-AP biosynthesis by increasing the conversion of proline to 2-AP and decreasing the conversion of GABald to GABA.

Recent studies on the regulatory role of amino acids in cell metabolism have focused on the functional significance of proline degradation. The process is catalysed by proline dehydrogenase/proline oxidase (PRODH/POX), a mitochondrial flavin-dependent enzyme converting proline into ∆1-pyrroline-5-carboxylate (P5C). During this process, electrons are transferred to electron transport chain producing ATP for survival or they directly reduce oxygen, producing reactive oxygen species (ROS) inducing apoptosis/autophagy. However, the mechanism for switching survival/apoptosis mode is unknown. Although PRODH/POX activity and energetic metabolism were suggested as an underlying mechanism for the survival/apoptosis switch, proline availability for this enzyme is also important. Proline availability is regulated by prolidase (proline supporting enzyme), collagen biosynthesis (proline utilizing process) and proline synthesis from glutamine, glutamate, α-ketoglutarate (α-KG) and ornithine. Proline availability is dependent on the rate of glycolysis, TCA and urea cycles, proline metabolism, collagen biosynthesis and its degradation. It is well established that proline synthesis enzymes, P5C synthetase and P5C reductase as well as collagen prolyl hydroxylases are up-regulated in most of cancer types and control rates of collagen biosynthesis. Up-regulation of collagen prolyl hydroxylase and its exhaustion of ascorbate and α-KG may compete with DNA and histone demethylases (that require the same cofactors) to influence metabolic epigenetics. This knowledge led us to hypothesize that up-regulation of prolidase and PRODH/POX with inhibition of collagen biosynthesis may represent potential pharmacotherapeutic approach to induce apoptosis or autophagic death in cancer cells. These aspects of proline metabolism are discussed in the review as an approach to understand complex regulatory mechanisms driving PRODH/POX-dependent apoptosis/survival.

Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of l-proline to Δ1-pyrroline-5-carboxylate and is a target for inhibitor discovery because of its importance in cancer cell metabolism. Because human PRODH is challenging to purify, the PRODH domains of the bacterial bifunctional enzyme proline utilization A (PutA) have been used for inhibitor development. These systems have limitations due to large polypeptide chain length, conformational flexibility and the presence of domains unrelated to PRODH activity. Herein, we report the engineering of minimal PRODH domains for inhibitor discovery. The best designs contain one-third of the 1233-residue PutA from Sinorhizobium meliloti and include a linker that replaces the PutA α-domain. The minimal PRODHs exhibit near wild-type enzymatic activity and are susceptible to known inhibitors and inactivators. Crystal structures of minimal PRODHs inhibited by S-(−)-tetrahydro-2-furoic acid and 2-(furan-2-yl)acetic acid were determined at 1.23 and 1.72 Å resolution. Minimal PRODHs should be useful in chemical probe discovery.

Transcription factors (TFs) control an array of expressed genes. However, the specifics of how a gene is expressed in time and space as controlled by a TF remain largely unknown. Here, in TRPC6-regulated proline oxidase 1 (POX) transcription in human glioma, we report that OIP5-AS1, a long noncoding RNA, determines the specificity of p53-driven POX expression. The OIP5-AS1/p53 complex via its 24 nucleotides binds to the POX promoter and is necessary for POX expression but not for p21 transcription. An O-site in the POX promoter to which OIP5-AS1 binds was identified that is required for OIP5-AS1/p53 binding and POX transcription. Blocking OIP5-AS1 binding to the O-site inhibits POX transcription and promotes glioma development. Thus, the OIP5-AS1/O-site module decides p53-controlled POX expression as regulated by TRPC6 and affects glioma development.

22q11.2 deletion is a common microdeletion that causes an array of developmental defects including 22q11.2 deletion syndrome (22q11DS) or DiGeorge syndrome and velocardiofacial syndrome. About 30% of patients with 22q11.2 deletion develop schizophrenia. Mice with deletion of the ortholog region in mouse chromosome 16qA13 exhibit schizophrenia-like abnormal behaviors. It is suggested that the genes deleted in 22q11DS are involved in the pathogenesis of schizophrenia. Among these genes, COMT, ZDHHC8, DGCR8, and PRODH have been identified as schizophrenia susceptibility genes. And DGCR2 is also found to be associated with schizophrenia. In this review, we focused on these five genes and reviewed their functions in the brain and the potential pathophysiological mechanisms in schizophrenia, which will give us a deeper understanding of the pathology of schizophrenia.

Background Soil and water pollution due to nitrate are becoming increasingly serious worldwide. The government also put forward relevant governance policies, and a large number of scholars studied chemical physics and other methods to remove nitrate in water, but the cost was substantial. Studies have found that planting systems including grasses have the potential to remove nitrates. However, there are few studies on nitrate linked pathway and nitrate assimilation during its early growth. Results We have evaluated three different feed-plant species with three levels of overnight seed nitrate treatments along with a control. The activity of different enzymes from 2 weeks old shoots was measured to get a comprehension of proline-associated pentose phosphate pathway coupled with nitrate assimilation and phenolic-linked antioxidant response system in these species under nitrate treatments. All three feed-plant species showed high nitrate tolerance during germination and early growth stages. It is perceived that the accumulation of total soluble phenolics and total antioxidant activity was high in all three feed-plant species under high nitrate treatments. In terms of high G6PDH activity along with low SDH activity in alfalfa, there may be a shift of carbon flux in this species under high nitrate treatments. Higher activity of these enzymes along with higher SOD and GPX activity was observed in alfalfa. The efficient mechanism of nitrate stress tolerance of alfalfa also correlated with higher photochemical efficiency. Perennial ryegrass also showed excellent potential under high nitrate treatments by adopting an efficient mechanism to counter nitrate-induced oxidative stress. Conclusions Under the condition of nitrate treatment, the germination rates of the three feed-plant species are still ideal, and they have good enzyme activity and have the potential to remove nitrate.

Previous reports indicate that proline utilization A (PutA) is involved in the oxidation of proline to glutamate in many bacteria. We demonstrate here that in addition to its role in proline catabolism, PutA acts as a global regulator to control the important biological functions and virulence of Ralstonia solanacearum. PutA regulates target gene expression levels by directly binding to promoter DNA, and its regulatory activity is enhanced by L-proline. Intriguingly, we reveal that the cofactors NAD+ and FAD boost the enzymatic activity of PutA for converting L-proline to L-glutamic acid but inhibit the regulatory activity of PutA for controlling target gene expression. Our results present evidence that PutA is a proline metabolic enzyme that also functions as a global transcriptional regulator in response to its substrate and cofactors and provide insights into the complicated regulatory mechanism of PutA in bacterial physiology and pathogenicity.

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

The oxidation of proline to pyrroline-5-carboxylate (P5C) leads to the transfer of electrons to ubiquinone in mitochondria that express proline dehydrogenase (ProDH). This electron transfer supports Complexes CIII and CIV, thus generating the protonmotive force. Further catabolism of P5C forms glutamate, which fuels the citric acid cycle that yields the reducing equivalents that sustain oxidative phosphorylation. However, P5C and glutamate catabolism depend on CI activity due to NAD+ requirements. NextGen-O2k (Oroboros Instruments) was used to measure proline oxidation in isolated mitochondria of various mouse tissues. Simultaneous measurements of oxygen consumption, membrane potential, NADH, and the ubiquinone redox state were correlated to ProDH activity and F1FO-ATPase directionality. Proline catabolism generated a sufficiently high membrane potential that was able to maintain the F1FO-ATPase operation in the forward mode. This was observed in CI-inhibited mouse liver and kidney mitochondria that exhibited high levels of proline oxidation and ProDH activity. This action was not observed under anoxia or when either CIII or CIV were inhibited. The duroquinone fueling of CIII and CIV partially reproduced the effects of proline. Excess glutamate, however, could not reproduce the proline effect, suggesting that processes upstream of the glutamate conversion from proline were involved. The ProDH inhibitors tetrahydro-2-furoic acid and, to a lesser extent, S-5-oxo-2-tetrahydrofurancarboxylic acid abolished all proline effects. The data show that ProDH-directed proline catabolism could generate sufficient CIII and CIV proton pumping, thus supporting ATP production by the F1FO-ATPase even under CI inhibition.

The role of proline dehydrogenase/proline oxidase (PRODH/POX) in the mechanism of antineoplastic activity of metformin (MET) was studied in C32 melanoma cells. PRODH/POX is a mitochondrial enzyme-degrading proline that is implicated in the regulation of cancer cell survival/apoptosis. The enzyme is activated by AMP kinase (AMPK). It has been found that MET induced a significant decrease in cell viability and DNA biosynthesis accompanied by an increase in the expressions of AMPK and PRODH/POX in C32 cells. The mechanism for MET-dependent cytotoxicity on C32 cells was found at the level of PRODH/POX-induced ROS generation and activation of Caspase-3 and Caspase-9 expressions in these cells. The effects were not observed in MET-treated PRODH/POX knock-out C32 cells. Of interest is an MET-dependent increase in the concentration of proline, which is a substrate for PRODH/POX. This phenomenon is due to the MET-dependent inhibition of collagen biosynthesis, which is the main proline-utilizing process. It has been found that the underlying mechanism of anticancer activity of MET involves the activation of AMPK, PRODH/POX, increase in the cytoplasmic concentration of proline, inhibition of collagen biosynthesis, and stimulation of PRODH/POX-dependent ROS generation, which initiate the apoptosis of melanoma cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are considered to be therapeutics in cancer prevention because of their inhibitory effect on cyclooxygenases (COX), which are frequently overexpressed in many types of cancer. However, it was also demonstrated that NSAIDs provoked a proapoptotic effect in COX knocked-out cancer cells. Here, we suggest that this group of drugs may provoke antineoplastic activity through the activation of PPARγ, which induces proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis. PRODH/POX is a mitochondrial enzyme that catalyzes proline degradation, during which ATP or reactive oxygen species (ROS) are generated. We have found that NSAIDs induced PRODH/POX and PPARγ expressions (as demonstrated by Western Blot or immunofluorescence analysis) and cytotoxicity (as demonstrated by MTT, cytometric assay, and DNA biosynthesis assay) in breast cancer MCF7 cells. Simultaneously, the NSAIDs inhibited collagen biosynthesis, supporting proline for PRODH/POX-induced ROS-dependent apoptosis (as demonstrated by an increase in the expression of apoptosis markers). The data suggest that targeting proline metabolism and the PRODH/POX–PPARγ axis can be considered a novel approach for breast cancer treatment.

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

Simple Summary PRODH/POX (proline dehydrogenase/proline oxidase) is a mitochondrial enzyme that catalyzes proline degradation generating reactive oxygen species (ROS). Estrogens limit proline availability for PRODH/POX by stimulating collagen biosynthesis. It has been considered that estrogens determine efficiency of troglitazone (TGZ)-induced PRODH/POX-dependent apoptosis in breast cancer cells. The studies were performed in wild-type and PRODH/POX-silenced estrogen-dependent MCF-7 cells and estrogen-independent MDA-MB-231 cells. DNA and collagen biosynthesis were determined by radiometric method, ROS production was measured by fluorescence assay, protein expression was determined by Western blot and proline concentration by LC/MS analysis. We found that: i/TGZ-induced apoptosis in MDA-MB-231 occurs only in the absence of estradiol or ERβ, ii/the process is mediated by PRODH/POX, iii/and is facilitated by proline availability for PRODH/POX by TGZ-dependent inhibition of collagen biosynthesis (proline utilizing process). The data suggest that combined TGZ and anti-estrogen treatment could be considered in experimental therapy of ER negative breast cancers. Abstract The impact of estradiol on troglitazone (TGZ)-induced proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied in wild-type and PRODH/POX-silenced estrogen receptor (ER) dependent MCF-7 cells and ER-independent MDA-MB-231 cells. DNA and collagen biosynthesis were determined by radiometric method, prolidase activity evaluated by colorimetric method, ROS production was measured by fluorescence assay. Protein expression was determined by Western blot and proline concentration by LC/MS analysis. PRODH/POX degrades proline yielding reactive oxygen species (ROS). Estrogens stimulate collagen biosynthesis utilizing free proline and limiting its availability for PRODH/POX-dependent apoptosis. TGZ cytotoxicity was highly pronounced in wild-type MDA-MB-231 cells cultured in medium without estradiol or in the cells cultured in medium with estradiol but deprived of ERβ (by ICI-dependent degradation), while in PRODH/POX-silenced cells the process was not affected. The TGZ cytotoxicity was accompanied by increase in PRODH/POX expression, ROS production, expression of cleaved caspase-3, caspase-9 and PARP, inhibition of collagen biosynthesis, prolidase activity and decrease in intracellular proline concentration. The phenomena were not observed in PRODH/POX-silenced cells. The data suggest that TGZ-induced apoptosis in MDA-MB-231 cells cultured in medium without estradiol or deprived of ERβ is mediated by PRODH/POX and the process is facilitated by proline availability for PRODH/POX by TGZ-dependent inhibition of collagen biosynthesis. It suggests that combined TGZ and antiestrogen treatment could be considered in experimental therapy of estrogen receptor negative breast cancers.

In many bacteria, the reactions of proline catabolism are catalyzed by the bifunctional enzyme known as proline utilization A (PutA). PutA catalyzes the two-step oxidation of l -proline to l -glutamate using distinct proline dehydrogenase (PRODH) and l -glutamate-γ-semialdehyde dehydrogenase (GSALDH) active sites, which are separated by over 40 A and connected by a complex tunnel system. The tunnel system consists of a main tunnel that connects the two active sites and functions in substrate channeling, plus six ancillary tunnels whose functions are unknown. Here we used tunnel-blocking mutagenesis to probe the role of a dynamic ancillary tunnel (tunnel 2a) whose shape is modulated by ligand binding to the PRODH active site. The 1.90 A resolution crystal structure of Geobacter sulfurreducens PutA variant A206W verified that the side chain of Trp206 cleanly blocks tunnel 2a without perturbing the surrounding structure. Steady-state kinetic measurements indicate the mutation impaired PRODH activity without affecting the GSALDH activity. Single-turnover experiments corroborated a severe impairment of PRODH activity with flavin reduction decreased by nearly 600-fold in A206W relative to wild-type. Substrate channeling is also significantly impacted as A206W exhibited a 3000-fold lower catalytic efficiency in coupled PRODH-GSALDH activity assays, which measure NADH formation as a function of proline. The structure suggests that Trp206 inhibits binding of the substrate l -proline by preventing the formation of a conserved glutamate-arginine ion pair and closure of the PRODH active site. Our data are consistent with tunnel 2a serving as an open space through which the glutamate of the ion pair travels during the opening and closing of the active site in response to binding l -proline. These results confirm the essentiality of the conserved ion pair in binding l -proline and support the hypothesis that the ion pair functions as a gate that controls access to the PRODH active site.

Loss of pod dehiscence was a key step in soybean [Glycine max (L.) Merr.] domestication. Genome-wide association analysis for soybean shattering identified loci harboring Pdh1, NST1A and SHAT1-5. Pairwise epistatic interactions were observed, and the dehiscent Pdh1 overcomes resistance conferred by NST1A or SHAT1-5 locus. Further candidate gene association analysis identified a nonsense mutation in NST1A associated with pod dehiscence. Geographic analysis showed that in Northeast China (NEC), indehiscence at both Pdh1 and NST1A were required in cultivated soybean, while indehiscent Pdh1 alone is capable of preventing shattering in Huang-Huai-Hai (HHH) valleys. Indehiscent Pdh1 allele was only identified in wild soybean (Glycine soja L.) accession from HHH valleys suggesting that it may have originated in this region. No specific indehiscence was required in Southern China. Geo-climatic investigation revealed strong correlation between relative humidity and frequency of indehiscent Pdh1 across China. This study demonstrates that epistatic interaction between Pdh1 and NST1A fulfills a pivotal role in determining the level of resistance against pod dehiscence, and humidity shapes the distribution of indehiscent alleles. Our results give further evidence to the hypothesis that HHH valleys was at least one of the origin centers of cultivated soybean.

Zinc (Zn) is an important microelement for rice and plays a key role in many physiological processes. This study assessed the physio-biochemical responses involved in biosynthesis of 2-acety-1-pyrroline (2-AP), which is a key compound in the aroma of fragrant rice, in four different fragrant rice varieties, i.e., Meixiangzhan-2, Xiangyaxiangzhan, Ruanhuayou-134, and Yunjingyou. Four concentrations (0, 0.50, 1.00 and 2.00 g L−1) of zinc chloride were applied to fragrant rice foliage at the heading stage and named CK, Zn1, Zn2 and Zn3, respectively. Our results showed that compared with CK, the Zn1, Zn2 and Zn3 treatments all significantly increased the 2-AP concentration in mature grains of the four fragrant rice genotypes. Furthermore, exogenous application of Zn not only enhanced the activities of enzymes, including proline dehydrogenase (PDH), △1-pyrroline-5-carboxylic acid synthetase (P5CS), and diamine oxidase (DAO), which are involved in 2-AP biosynthesis, but also improved the contents of the related precursors, such as Δ1-pyrroline, proline and pyrroline-5-carboxylic acid (P5C). In addition, compared to the CK treatment, the Zn2 treatment markedly increased the net photosynthetic rate of fragrant rice during the grain filling stage and increased the seed-setting rate, 1000-grain weight and grain yield in all fragrant rice genotypes. Foliar application of Zn also markedly increased the grain Zn content. In general, 1.00 g L−1 seemed to be the most suitable application concentration because the highest 2-AP content and grain weight were recorded with this treatment.

In many plants and microorganisms, intracellular proline has a protective role against various stresses, including heat-shock, oxidation and osmolarity. Environmental stresses induce cellular senescence in a variety of eukaryotes. Here we showed that intracellular proline regulates the replicative lifespan in the budding yeast Saccharomyces cerevisiae. Deletion of the proline oxidase gene PUT1 and expression of the γ-glutamate kinase mutant gene PRO1-I150T that is less sensitive to feedback inhibition accumulated proline and extended the replicative lifespan of yeast cells. Inversely, disruption of the proline biosynthetic genes PRO1, PRO2, and CAR2 decreased stationary proline level and shortened the lifespan of yeast cells. Quadruple disruption of the proline transporter genes unexpectedly did not change intracellular proline levels and replicative lifespan. Overexpression of the stress-responsive transcription activator gene MSN2 reduced intracellular proline levels by inducing the expression of PUT1, resulting in a short lifespan. Thus, the intracellular proline levels at stationary phase was positively correlated with the replicative lifespan. Furthermore, multivariate analysis of amino acids in yeast mutants deficient in proline metabolism showed characteristic metabolic profiles coincident with longevity: acidic and basic amino acids and branched-chain amino acids positively contributed to the replicative lifespan. These results allude to proline metabolism having a physiological role in maintaining the lifespan of yeast cells.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with poor clinical outcomes and without effective targeted therapies. Numerous studies have suggested that HDAC inhibitors (TSA/SAHA) may be effective in TNBCs. Proline oxidase, also known as proline dehydrogenase (POX/PRODH), is a key enzyme in the proline metabolism pathway and plays a vital role in tumorigenesis. In this study, we found that HDAC inhibitors (TSA/SAHA) significantly increased POX expression and autophagy through activating AMPK. Depletion of POX decreased autophagy and increased apoptosis induced by HDAC inhibitors in TNBC cells. These results suggest that POX contributes to cell survival under chemotherapeutic stresses and might serve as a potential target for treatment of TNBC.

Proline dehydrogenase (PRODH) is a p53-inducible inner mitochondrial membrane flavoprotein linked to electron transport for anaplerotic glutamate and ATP production, most critical for cancer cell survival under microenvironmental stress conditions. Proposing that PRODH is a unique mitochondrial cancer target, we structurally model and compare its cancer cell activity and consequences upon exposure to either a reversible (S-5-oxo: S-5-oxo-2-tetrahydrofurancarboxylic acid) or irreversible (N-PPG: N-propargylglycine) PRODH inhibitor. Unlike 5-oxo, the suicide inhibitor N-PPG induces early and selective decay of PRODH protein without triggering mitochondrial destruction, consistent with N-PPG activation of the mitochondrial unfolded protein response. Fly and breast tumor (MCF7)-xenografted mouse studies indicate that N-PPG doses sufficient to phenocopy PRODH knockout and induce its decay can be safely and effectively administered in vivo. Among breast cancer cell lines and tumor samples, PRODH mRNA expression is subtype dependent and inversely correlated with glutaminase (GLS1) expression; combining inhibitors of PRODH (S-5-oxo and N-PPG) and GLS1 (CB-839) produces additive if not synergistic loss of cancer cell (ZR-75-1, MCF7, DU4475, and BT474) growth and viability. Although PRODH knockdown alone can induce cancer cell apoptosis, the anticancer potential of either reversible or irreversible PRODH inhibitors is strongly enhanced when p53 is simultaneously upregulated by an MDM2 antagonist (MI-63 and nutlin-3). However, maximum anticancer synergy is observed in vitro when the PRODH suicide inhibitor, N-PPG, is combined with both GLS1-inhibiting and a p53-upregulating MDM2 antagonist. These findings provide preclinical rationale for the development of N-PPG–like PRODH inhibitors as cancer therapeutics to exploit synthetic lethal interactions with p53 upregulation and GLS1 inhibition.