Pyrroline-5-carboxylate Reductase Research Articles

Tanshinone IIA alleviates pulmonary fibrosis by modulating glutamine metabolic reprogramming based on [U-13C5]-glutamine metabolic flux analysis

IntroductionGlutamine metabolic reprogramming, mediated by glutaminase (GLS), is an important signal during pulmonary fibrosis (PF) progression. Tanshinone IIA (Tan IIA) is a naturally lipophilic diterpene with antioxidant and antifibrotic properties. However, the potential mechanisms of Tan IIA for regulating glutamine metabolic reprogramming are not yet clear. ObjectivesThis study aimed was to evaluate the role of Tan IIA in intervening in glutamine metabolic reprogramming to exert anti-PF and to explore the potential new mechanisms of metabolic regulation. MethodsFibrotic characteristics was detected via immunofluorescence and western blotting analysis. Cell proliferation was examined with EdU Assay. Cell metabolites were labeled by using stable isotope [U-13C5]-glutamine. By utilizing 100% 13C glutamine tracers and employing network analysis to investigate the activation of metabolic pathways in fibroblasts, as well as evaluating the impact of Tan IIA on these pathways, we accurately quantified the absolute flux of glutaminolysis, proline synthesis, and the TCA cycle pathway using isotopomer network compartmental analysis (INCA), a user-friendly software tool for 13C metabolic flux analysis (13C-MFA). Molecular docking was used for identifying the binding of Tan IIA with target protein. ResultsTan IIA ameliorate TGF-β1-induced myofibroblast proliferation, reduce collagen I and III and α-SMA protein expression in MRC-5 and NIH-3T3 cells. Furthermore, Tan IIA regulate mitochondrial energy metabolism by modulating TGF-β1-stimulated glutamine metabolic reprogramming in NIH-3T3 cells and inhibiting GLS1 expression, which reduced the metabolic flux of glutamine into mitochondria in myofibroblasts, and also targeted inhibited the expression of Δ1-pyrroline-5-carboxylate synthase (P5CS), P5C reductase 1 (PYCR1), and phosphoserine aminotransferase 1 (PSAT1), and reduced proline hydroxylation and blocked the collagen synthesis pathway. ConclusionTan IIA reverses glutamine metabolic reprogramming, reduces mitochondrial energy expenditure, and inhibits collagen matrix synthesis by modulating potential targets in glutamine metabolism. This novel perspective sheds light on the essential role of glutamine metabolic reprogramming in PF.

Effect of Drought Stress on Proline Gene Expression, Enzyme Activity, and Physiological Responses in Thai Mulberry (Morus spp.)

Mulberries are vital for the silk industry as the sole natural food for silkworms, but their quality and quantity can be greatly impacted by environmental factors, notably water shortages or droughts. In this study, the proline content and expression levels of the Pyrroline-5-carboxylate reductase (P5CR) gene in four recommended Thai mulberry varieties (Sakhonnakorn, Sakhonnakorn 85, Burirum 60, and Srisaket 84) and one standard drought tolerant variety (SRCM9809-34) were measured under drought stress. Additionally, physiological data and antioxidative enzymatic activities were also examined. The findings revealed that SRCM9809-34, a drought-tolerant variety, had the lowest proline content, followed by Sakhonnakorn 85, Burirum 60, while the highest proline content was observed in Srisaket 84. Although there was no correlation between the expression level of the P5CR gene and proline content, the overall trend in all varieties was the same: proline content increased after drought conditions. Regarding physiological responses, the wilting score showed similar results to proline content, with SRCM9809-34 having the lowest proline content and wilting score. Moreover, SRCM9809-34 exhibited the highest RWC, Pn and WUE values, as well as the lowest level of MDA and H2O2. Our results validated and indicated that SRCM9809-34 is a drought-tolerant variety. From this finding, among the four Thai mulberry varieties, Sakhonnakorn 85 exhibited the highest potential for drought tolerance, and this potential can be enhanced through crossbreeding with SRCM9809-34.

Bioinformatics and expression analysis of proline metabolism-related gene families in alfalfa under saline-alkali stress

Regulation of the proline metabolic pathway is essential for the accumulation of proline under abiotic stress and for the amelioration of plant stress resistance. Δ1-pyrroline-5-carboxylate synthase (P5CS), pyrroline-5-carboxylate reductase (P5CR), ornithine transaminase (δ-OAT), proline dehydrogenase (PDH), pyrroline-5-carboxylate dehydrogenase (P5CDH), and proline transporter (ProT) are the key enzymes in the proline metabolic pathway. However, the gene families responsible for proline metabolism have not yet been identified or reported in alfalfa. In this study, a total of 12 MsP5CSs, 4 MsP5CRs, 3 MsOATs, 6 MsPDHs, 2 MsP5CDHs, and 5 MsProTs were identified in the genome of alfalfa, and the members of the same subfamily had similar gene structures and conserved motifs. Analysis of cis-regulatory elements revealed the presence of light-responsive, hormone-regulated, and stress-responsive elements in the promoter regions of alfalfa proline metabolism-related genes. Following treatment with saline-alkali, the expression of MsP5CSs, MsP5CRs, MsOATs, and MsProTs was significantly upregulated, whereas the expression of MsPDH1.1, MsPDH1.3, and MsP5CDH was significantly downregulated. The proline content and enzyme activity of P5CS gradually increased, whereas the enzyme activity of PDH gradually decreased as the duration of stress increased. Root growth rates decreased upon MsP5CS1a suppression (MsP5CS1a-RNAi) in the hairy roots of alfalfa compared to the empty vector line under saline-alkali stress. These results show that proline metabolism-related genes play an important role in the saline-alkali stress tolerance of alfalfa and provide a theoretical basis for further research on the functions of proline metabolism-related genes in alfalfa in response to saline-alkali stress.

Arbuscular mycorrhizal fungi as an effective approach to enhance the growth and metabolism of soybean plants under thallium (TI) toxicity

Thallium (TI) is a toxic metal that can trigger harmful impacts on growth and metabolism of plants. Utilizing arbuscular mycorrhizal fungi (AMF) proves to be an effective strategy for alleviating heavy metal toxicity in plants. To this end, AMF were applied to mitigate TI toxic effects on the growth, primary and secondary metabolism of soybean plants. Here, TI stress inhibited the growth and photosynthetic parameters of soybean plants. It also increased the oxidative damage as demonstrated by increased levels of oxidative markers, (MDA and lipoxygenase (LOX) activity). However, AMF could mitigate the reduction in growth and photosynthesis induced by TI, as well as the induction of oxidative damage. To overcome TI toxicity, AMF increased the levels and metabolism of osmolytes such as proline in soybean plants. This was in line with the increased activities of key enzymes that involved in proline biosynthesis (e.g., P5CS (pyrroline-5-carboxylate synthetase), P5CR (pyrroline-5-carboxylate reductase) and OAT (ornithine aminotransferase) under the AMF and/or TI treatments. Furthermore, soybean plants could benefit from the synergism between AMF and TI to enhance the contents of individual (e.g., spermine and spermidine) and total polyamines as well as their metabolic enzymes (e.g., arginine decarboxylase and ornithine decarboxylase). Overall, the combined application of AMF emerges as a viable approach for alleviating TI toxicity in soybean plants.

Proline Metabolism Process and Antioxidant Potential of Lycium ruthenicum Murr. in Response to NaCl Treatments.

Salinity influences the level of antioxidants and proline content, which are both involved in the regulation of stress responses in plants. To examine the interplay between the antioxidant system and proline metabolism in plant stress acclimation, explants of Lycium ruthenicum were subjected to NaCl treatments, and the growth characteristics, antioxidant enzyme activities, proline accumulation, and metabolic enzyme content were analyzed. The results revealed that NaCl concentrations between 50 to 150 mM have a positive effect on the growth of L. ruthenicum explants. Increasing NaCl concentrations elevated the activities of superoxide dismutase (SOD) and catalase (CAT), while hydrogen peroxide (H2O2) content was inhibited, suggesting that the elevated antioxidants play a central protective role in superoxide anion (O2•-) and H2O2 scavenging processes in response to NaCl treatments. Also, high proline levels also protect antioxidant enzyme machinery, thus protecting the plants from oxidative damage and enhancing osmotic adjustment. Increasing levels of pyrroline-5-carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR), and ornithine-δ-aminotransferase (δ-OAT) were observed, resulting in elevated level of proline. In addition, the expression levels of LrP5CS1, -2, -3, LrOAT-1, and -2 were promoted in NaCl treatments. According to the combined analysis of metabolic enzyme activities and their relative expression, it is confirmed that the glutamate (Glu) pathway is activated in L. ruthenicum faced with different levels of NaCl concentrations. However, Glu supplied by δ-OAT is fed back into the main pathway for proline metabolism.

Melatonin treatment alleviates chilling injury in mango fruit ‘Keitt’ by modulating proline metabolism under chilling stress

Mangoes often suffer from low temperature-induced chilling injury (CI) during postharvest cold storage. Therefore, advanced techniques are crucial and in high demand to solve the chilling stress of mango fruit for a higher value. This study addresses chilling stress modulation by investigating the effects of melatonin treatment on CI, proline metabolism, and related gene expressions of ‘Keitt’ mango during cold storage after dipped in 0 (control), 0.1 (MT1), and 0.2 mmol L-1 (MT2) melatonin solution for 30 min. The results revealed that melatonin treatment in MT1 significantly reduced CI development and increased proline content in mango fruit during cold storage compared to the control. These changes were along with increases in the activity of critical enzymes as well as the expression of encoding genes involved in proline biosynthesis, such as pyrroline-5-carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR), ornithine D-aminotransferase (OAT), P5CS2, P5CR2, and OAT3. Additionally, proline dehydrogenase (PDH) activity and the expression of the PDH3 gene associated with proline dehydrogenation were lower in MT1-treated mangoes than the controlled group. Thus, melatonin treatment has regulated proline metabolism resulting in the accumulation of proline, subsequently contributing to enhancing the chilling tolerance of ‘Keitt’ mango fruit.

The regulatory mechanisms of proline and hydroxyproline metabolism: Recent advances in perspective.

For diverse human tumors, growth and metastasis are dependent on proline synthesis, but the mechanisms underlying this association are not clear. Proline incorporated into collagen is primarily synthesized from glutamine. Thus, rates of collagen synthesis are modulated by the enzymes of proline synthesis. On the other hand, the hydroxylation of collagen proline requires αKG, ascorbate and ferrous iron, substrates necessary for the epigenetic demethylation of DNA and histones. The metabolic relationship of proline and hydroxyproline degradation are initiated by distinct dehydrogenases but the respective oxidized products, P5C and OH-P5C are substrates for P5C Reductase and P5C Dehydrogenase allowing for mutual competition. This provides a model by which proline synthesis in cancer plays a role in reprogramming gene expression. The metabolism of proline and hydroxyproline are also linked to the HIF response to hypoxia. Hypoxia increased the expression of ALDH18A1, which is the limiting step in proline and collagen synthesis. Hydroxyproline increases levels of HIF-1α presumably by inhibiting its degradation. These new findings allow the suggestion that there is a regulatory axis from glutamine to proline and collagen synthesis, and the release of free hydroxyproline can feed back on the HIF pathway.

Functional Impact of a Cancer-Related Variant in Human Δ1-Pyrroline-5-Carboxylate Reductase 1.

Pyrroline-5-carboxylate reductase (PYCR) is a proline biosynthetic enzyme that catalyzes the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline. Humans have three PYCR isoforms, with PYCR1 often upregulated in different types of cancers. Here, we studied the biochemical and structural properties of the Thr171Met variant of PYCR1, which is found in patients with malignant melanoma and lung adenocarcinoma. Although PYCR1 is strongly associated with cancer progression, characterization of a PYCR1 variant in cancer patients has not yet been reported. Thr171 is conserved in all three PYCR isozymes and is located near the P5C substrate binding site. We found that the amino acid replacement does not affect thermostability but has a profound effect on PYCR1 catalytic activity. The k cat of the PYCR1 variant T171M is 100- to 200-fold lower than wild-type PYCR1 when P5C is the variable substrate, and 10- to 25-fold lower when NAD(P)H is varied. A 1.84 Å resolution X-ray crystal structure of T171M reveals that the Met side chain invades the P5C substrate binding site, suggesting that the catalytic defect is due to steric clash preventing P5C from achieving the optimal pose for hydride transfer from NAD(P)H. These results suggest that any impact on PYCR1 function associated with T171M in cancer does not derive from increased catalytic activity.

PYCR in Kidney Renal Papillary Cell Carcinoma: Expression, Prognosis, Gene Regulation Network, and Regulation Targets.

Pyrroline-5-carboxylate reductase (PYCR) includes three human genes encoding three isozymes, PYCR1, PYCR2, and PYCR3 (or PYCRL), which facilitate the final step in the conversion of glutamine to proline. These genes play important roles in regulating the cell cycle and redox homeostasis as well as promoting growth signaling pathways. Proline is abnormally upregulated in a variety of cancers, and as the last key enzyme in proline production, PYCR plays an integral role in promoting tumorigenesis and cancer progression. However, its role in patients with kidney renal papillary cell carcinoma (KIRP) has not been fully elucidated. In this study, we aimed to systematically analyze the expression, gene regulatory network, prognostic value, and target prediction of PYCR in patients with KIRP, elucidate the association between PYCR expression and KIRP, and identify potential new targets for the clinical treatment of KIRP. We systematically analyzed the expression, prognosis, gene regulatory network, and regulatory targets of PYCR1, PYCR2, and PYCRL in KIRP using multiple online databases including cBioPortal, STRING, MethSurv, GeneMANIA, Gene Expression Profiling Interactive Analysis (GEPIA), Metascape, UALCAN, LinkedOmics, and TIMER. The expression levels of PYCR1, PYCR2, and PYCRL were considerably upregulated in patients with KIRP based on sample type, sex, age, and individual cancer stage. PYCR1 and PYCR2 transcript levels were markedly upregulated in females than in males, and patients aged 21-40 years had higher PYCR1 and PYCR2 transcript levels than those in other age groups. Interestingly, PYCR2 transcript levels gradually decreased with age. In addition, the expressions of PYCR1 and PYCR2 were notably correlated with the pathological stage of KIRP. Patients with KIRP with low PYCR1 and PYCR2 expression had longer survival than those with high PYCR1 and PYCR2 expression. PYCR1, PYCR2, and PYCRL were altered by 4%, 7%, and 6%, respectively, in 280 patients with KIRP. The methylation levels of cytosine-phosphate-guanine (CpG) sites in PYCR were markedly correlated with the prognosis of patients with KIRP. PYCR1, PYCR2, PYCRL, and their neighboring genes form a complex network of interactions. The molecular functions of the genes, as demonstrated by their corresponding Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, included calcium channel activity, phospholipid binding, RNA polymerase II-specificity, and kinase and GTPase-regulatory activities. PYCR1, PYCR2, and PYCRL targeted miR-21, miR-221, and miR-222, resulting in a better prognosis of KIRP. We analyzed mRNA sequencing data from 290 patients with KIRP and found that ADA, NPM3, and TKT were positively associated with PYCR1 expression; PFDN2, JTB, and HAX1 were positively correlated with PYCR2 expression; SHARPIN, YDJC, and NUBP2 were positively correlated with PYCRL expression; PYCR1 was positively correlated with B cell and CD8+ T-cell infiltration levels; macrophage infiltration was negatively correlated with PYCR2 expression; and PYCRL expression was negatively correlated with B-cell, CD8+ T cell, and dendritic cell infiltration levels. PYCR1, PYCR2, and PYCRL may be potential therapeutic and prognostic biomarkers for patients with KIRP. The regulation of microRNAs (miRNAs), including miR-21, miR-221, and miR-222, may prove an important strategy for KIRP treatment.

Combined Metabolomic and Quantitative RT-PCR Analyses Revealed the Synthetic Differences of 2-Acetyl-1-pyrroline in Aromatic and Non-Aromatic Vegetable Soybeans

Aroma is an important economic trait of vegetable soybeans, which greatly influences their market value. The 2-acetyl-1-pyrroline (2AP) is considered as an important substance affecting the aroma of plants. Although the 2AP synthesis pathway has been resolved, the differences of the 2AP synthesis in the aromatic and non-aromatic vegetable soybeans are unknown. In this study, a broad targeted metabolome analysis including measurement of metabolites levels and gene expression levels was performed to reveal pathways of aroma formation in the two developmental stages of vegetable soybean grains [35 (S5) and 40 (S6) days after anthesis] of the 'Zhexian No. 8' (ZX8, non-aromatic) and ZK1754 (aromatic). The results showed that the differentially accumulated metabolites (DAMs) of the two varieties can be classified into nine main categories including flavonoids, lipids, amino acids and derivatives, saccharides and alcohols, organic acids, nucleotides and derivatives, phenolic acids, alkaloids and vitamin, which mainly contributed to their phenotypic differences. Furthermore, in combination with the 2AP synthesis pathway, the differences of amino acids and derivatives were mainly involved in the 2AP synthesis. Furthermore, 2AP precursors' analysis revealed that the accumulation of 2AP mainly occurred from 1-pyrroline-5-carboxylate (P5C), not 4-aminobutyraldehyde (GABald). The quantitative RT-PCR showed that the associated synthetic genes were 1-pyrroline-5-carboxylate dehydrogenase (P5CDH), ∆1-pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (PRODH) and pyrroline-5-carboxylate reductase (P5CR), which further verified the synthetic pathway of 2AP. Furthermore, the betaine aldehyde dehydrogenase 2 (GmBADH2) mutant was not only vital for the occurrence of 2AP, but also for the synthesis of 4-aminobutyric acid (GABA) in vegetable soybean. Therefore, the differences of 2AP accumulation in aromatic and non-aromatic vegetable soybeans have been revealed, and it also provides an important theoretical basis for aromatic vegetable soybean breeding.

Targeting PYCR2 inhibits intraperitoneal metastatic tumors of mouse colorectal cancer in a proline-independent approach.

Whether proline deficiency is a metabolic vulnerability in colorectal tumors is unknown. The aim of this study was to investigate the effects of proline metabolism-related genes and exogenous proline on the progression of colorectal cancer (CRC). We aimed to further clarify the role of pyrroline-5-carboxylate reductase (PYCR) 2, a key enzyme of proline synthesis, in the regulation of colorectal intraperitoneal metastatic tumors. This study was carried out based on The Cancer Genome Atlas (TCGA) data, database analysis, single-cell functional analysis, tissue microarray, cell experiments, and animal models. We found that, PYCR2 mRNA and protein levels were upregulated in CRC. The mRNA level of PYCR2 was closely related to the prognosis and tumor metastasis of CRC patients. The upregulated PYCR2 expression was at least partly due to low promoter methylation levels. The nomogram constructed based on PYCR2 expression and clinical characteristics of CRC showed good accuracy in predicting lymph node metastasis. Pycr2 knockdown inhibited epithelial-mesenchymal transition (EMT) of mouse CRC cells. Proline supplementation did not rescue the inhibition of mouse CRC cell proliferation and migration by Pycr2 knockdown. Proline supplementation also did not rescue the suppression of subcutaneous tumors and intraperitoneal metastatic tumors in mice by Pycr2 knockdown. PYCR2 co-expressed genes in TCGA-CRC were enriched in epigenetic modification-related biological processes and molecular functions. Four small molecules with the lowest binding energy to the PYCR2 protein were identified. Collectively, Pycr2 knockdown inhibited mouse CRC progression in a proline-independent approach. PYCR2 may be a promising tumor metastasis predictor and therapeutic target in CRC.

Functional Characterization of Saccharomyces cerevisiae P5C Reductase, the Enzyme at the Converging Point of Proline and Arginine Metabolism.

The enzyme that, in Saccharomyces cerevisiae, catalyzes the last step in both proline synthesis and arginine catabolism, δ1-pyrroline-5-carboxylate (P5C) reductase, was purified to near homogeneity and characterized thoroughly. Retention patterns upon gel permeation chromatography were consistent with a homodecameric composition of the holomer. High lability of the purified preparations and stabilization by reducing compounds suggested susceptibility to reactive-oxygen-species-mediated damage. Both NADH and NADPH were used as the electron donor, the latter resulting in a 3-fold higher Vmax. However, a higher affinity toward NADH was evident, and the NADPH-dependent activity was inhibited by NAD+, proline, arginine, and a variety of anions. With proline and arginine, the inhibition was of the competitive type with respect to the specific substrate, and of the uncompetitive- or mixed-type with respect to NADPH, respectively. The results suggest that, contrary to the enzyme from higher plants, yeast P5C reductase may preferentially use NADH in vivo. An in silico analysis was also performed to investigate the structural basis of such enzyme features. Superposition of the protein model with the experimental structure of P5C reductase from Medicago truncatula allowed us to hypothesize on the possible allosteric sites for arginine and anion binding, and the cysteine pairs that may be involved in disulfide formation.

Transcriptomic analysis reveals the mechanism of low/high temperature resistance in an outstanding diet alga Nannochloropsis oceanica

Low/high temperature stress is one of the key abiotic factors restricting the implementation of microalgal large-scale production. Metabolism, involved in abiotic stress resistances, have been reported in plants, but the related research in microalgae is far behind. This study analyzed the transcriptional changes in Nannochloropsis oceanica under low and high temperature stresses by RNA-seq technology. Results indicated that the total differentially expressed gene (DEG) amount under high temperature stress was much more than low temperature stress. GO enrichment analysis suggested that DEGs under low temperature were mainly concentrated in protein modification and phosphorus metabolic process, while transmembrane transport, lipid localization and transport, and cellular homeostasis were the most enriched terms under high temperature. Moreover, “membrane” was the most enriched term in both low and high temperature groups. The gene expressions of 37 stress resistance-related enzymes/proteins (172 genes) were analyzed concretely in N. oceanica for the first time. Results revealed that ornithine decarboxylase (ODC) was significantly down-regulated, while choline dehydrogenase (CHDH), late embryogenesis abundant (LEA), dehydrin (DHN), chitinase (CHI), calmodulin (CaM), and lipid transfer protein (LTP) were extremely up-regulated under low temperature. Under high temperature, more genes with drastic changes were identified. Superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione synthetase (GSS), and glyoxalase II (Gly II) exhibited down-regulated significantly, while glutathione S-transferase (GST), CHDH, L-asparaginase (L-ASP), LEA, DHN, early response to dehydration protein (ERD), heat shock protein (HSP), and CaM were dramatically up-regulated. No significant changes were detected in glutathione peroxidase (GPX), tocopherol cyclase (TC), gamma-glutamylcysteine synthetase (γ-GCS), pyrroline‐5–carboxylate reductase (P5CR), asparagine synthetase (ASN ), succinate-semialdehyde dehydrogenase (SSADH), pyruvate decarboxylase (PDC), and S-adenosylmethionine synthetase (SAMS) under low or high temperature. Furthermore, 166 transcription factor (TF) genes related to plant stress resistance were characterized and categorized into 20 TF families. MYB was the most abundant and active one, revealing that the MYB family played a critical role of temperature resistance in N. oceanica. These new findings could benefit the understanding of the temperature resistance mechanism in Nannochloropsis. The significantly changed genes might be ideal candidates for breeding elite Nannochloropsis strains with stronger resistance to temperature stress using genetic engineering methods.

Selection of Newly Identified Growth-Promoting Archaea Haloferax Species With a Potential Action on Cobalt Resistance in Maize Plants.

Soil contamination with cobalt (Co) negatively impacts plant growth and production. To combat Co toxicity, plant growth-promoting microorganisms for improving plant growth are effectively applied. To this end, unclassified haloarchaeal species strain NRS_31 (OL912833), belonging to Haloferax genus, was isolated, identified for the first time, and applied to mitigate the Co phytotoxic effects on maize plants. This study found that high Co levels in soil lead to Co accumulation in maize leaves. Co accumulation in the leaves inhibited maize growth and photosynthetic efficiency, inducing oxidative damage in the tissue. Interestingly, pre-inoculation with haloarchaeal species significantly reduced Co uptake and mitigated the Co toxicity. Induced photosynthesis improved sugar metabolism, allocating more carbon to defend against Co stress. Concomitantly, the biosynthetic key enzymes involved in sucrose (sucrose-P-synthase and invertases) and proline (pyrroline-5- carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR)) biosynthesis significantly increased to maintain plant osmotic potential. In addition to their osmoregulation potential, soluble sugars and proline can contribute to maintaining ROS hemostasis. Maize leaves managed their oxidative homeostasis by increasing the production of antioxidant metabolites (such as phenolics and tocopherols) and increasing the activity of ROS-scavenging enzymes (such as POX, CAT, SOD, and enzymes involved in the AsA/GSH cycle). Inside the plant tissue, to overcome heavy Co toxicity, maize plants increased the synthesis of heavy metal-binding ligands (metallothionein, phytochelatins) and the metal detoxifying enzymes (glutathione S transferase). Overall, the improved ROS homeostasis, osmoregulation, and Co detoxification systems were the basis underlying Co oxidative stress, mitigating haloarchaeal treatment's impact.

Staphylococcus aureus Does Not Synthesize Arginine from Proline under Physiological Conditions.

The Gram-positive pathogen Staphylococcus aureus is the only bacterium known to synthesize arginine from proline via the arginine-proline interconversion pathway despite having genes for the well-conserved glutamate pathway. Since the proline-arginine interconversion pathway is repressed by CcpA-mediated carbon catabolite repression (CCR), CCR has been attributed to the arginine auxotrophy of S. aureus. Using ribose as a secondary carbon source, here, we demonstrate that S. aureus arginine auxotrophy is not due to CCR but due to the inadequate concentration of proline degradation product. Proline is degraded by proline dehydrogenase (PutA) into pyrroline-5-carboxylate (P5C). Although the PutA expression was fully induced by ribose, the P5C concentration remained insufficient to support arginine synthesis because P5C was constantly consumed by the P5C reductase ProC. When the P5C concentration was artificially increased by either PutA overexpression or proC deletion, S. aureus could synthesize arginine from proline regardless of carbon source. In contrast, when the P5C concentration was reduced by overexpression of proC, it inhibited the growth of the ccpA deletion mutant without arginine. Intriguingly, the ectopic expression of the glutamate pathway enzymes converted S. aureus into arginine prototroph. In an animal experiment, the arginine-proline interconversion pathway was not required for the survival of S. aureus. Based on these results, we concluded that S. aureus does not synthesize arginine from proline under physiological conditions. We also propose that arginine auxotrophy of S. aureus is not due to the CcpA-mediated CCR but due to the inactivity of the conserved glutamate pathway. IMPORTANCE Staphylococcus aureus is a versatile Gram-positive human pathogen infecting various human organs. The bacterium's versatility is partly due to efficient metabolic regulation via the carbon catabolite repression system (CCR). S. aureus is known to interconvert proline and arginine, and CCR represses the synthesis of both amino acids. However, when CCR is released by a nonpreferred carbon source, S. aureus can synthesize proline but not arginine. In this study, we show that, in S. aureus, the intracellular concentration of pyrroline-5-carboxylate (P5C), the degradation product of proline and the substrate of proline synthesis, is too low to synthesize arginine from proline. These results call into question the notion that S. aureus synthesizes arginine from proline.

CO2 Enrichment Differentially Upregulated Sugar, Proline, and Polyamine Metabolism in Young and Old Leaves of Wheat and Sorghum to Mitigate Indium Oxide Nanoparticles Toxicity.

Soil contamination with indium oxide nanoparticles (In2O3-NPs) is a challenge for plant growth and productivity. Despite In2O3-NPs toxicity, their effects on plant growth and metabolism are largely unknown, particularly under future climate CO2 (eCO2). Therefore, the In2O3-NPs toxicity and stress mitigating impact of eCO2 in the young and old leaves of C3 (wheat) and C4 (sorghum) plants were investigated. Overall, In2O3-NPs significantly retard the biomass and photosynthetic machinery of all tested crops, particularly the young leaves of C3 plants. Consequently, In2O3-NPs altered C and N metabolism in C3 and C4 plants. On the other hand, eCO2 contrarily alleviated the hazardous effects of In2O3-NPs on growth and photosynthesis, especially in the young leaves of C4 plants. Increased photosynthesis consequently enhanced the soluble sugars’ accumulation and metabolism (e.g., sucrose P synthase, cytosolic, and vacuolar invertase) in all stressed plants, but to a greater extent in C4 young leaves. High sugar availability also induced TCA organic and fatty acids’ accumulation. This also provided a route for amino acids and polyamines biosynthesis, where a clear increase in proline biosynthetic enzymes [e.g., pyrroline-5-carboxylate synthetase (P5CS), ornithine aminotransferase (OAT), Pyrroline-5-carboxylate reductase (P5CR), pyrroline-5-carboxylate dehydrogenase (P5CDH), and proline dehydrogenase (PRODH)] and polyamine metabolic enzymes (e.g., spermine and spermidine synthases, ornithine decarboxylase, and adenosyl methionine decarboxylase) were mainly recorded in C4 young leaves. The observed increases in these metabolites involved in osmo- and redox-regulation to reduce In2O3-NPs induced oxidative damage. Overall, our study, for the first time, shed light on how eCO2 differentially mitigated In2O3-NPs stress in old and young leaves of different species groups under the threat of In2O3-NPs contamination.

Increased mitochondrial proline metabolism sustains proliferation and survival of colorectal cancer cells.

Research into the metabolism of the non-essential amino acid (NEAA) proline in cancer has gained traction in recent years. The last step in the proline biosynthesis pathway is catalyzed by pyrroline-5-carboxylate reductase (PYCR) enzymes. There are three PYCR enzymes: mitochondrial PYCR1 and 2 and cytosolic PYCR3 encoded by separate genes. The expression of the PYCR1 gene is increased in numerous malignancies and correlates with poor prognosis. PYCR1 expression sustains cancer cells’ proliferation and survival and several mechanisms have been implicated to explain its oncogenic role. It has been suggested that the biosynthesis of proline is key to sustain protein synthesis, support mitochondrial function and nucleotide biosynthesis. However, the links between proline metabolism and cancer remain ill-defined and are likely to be tissue specific. Here we use a combination of human dataset, human tissue and mouse models to show that the expression levels of the proline biosynthesis enzymes are significantly increased during colorectal tumorigenesis. Functionally, the expression of mitochondrial PYCRs is necessary for cancer cells’ survival and proliferation. However, the phenotypic consequences of PYCRs depletion could not be rescued by external supplementation with either proline or nucleotides. Overall, our data suggest that, despite the mechanisms underlying the role of proline metabolism in colorectal tumorigenesis remain elusive, targeting the proline biosynthesis pathway is a suitable approach for the development of novel anti-cancer therapies.

Proline synthesis through PYCR1 is required to support cancer cell proliferation and survival in oxygen-limiting conditions.

SummaryThe demands of cancer cell proliferation alongside an inadequate angiogenic response lead to insufficient oxygen availability in the tumor microenvironment. Within the mitochondria, oxygen is the major electron acceptor for NADH, with the result that the reducing potential produced through tricarboxylic acid (TCA) cycle activity and mitochondrial respiration are functionally linked. As the oxidizing activity of the TCA cycle is required for efficient synthesis of anabolic precursors, tumoral hypoxia could lead to a cessation of proliferation without another means of correcting the redox imbalance. We show that in hypoxic conditions, mitochondrial pyrroline 5-carboxylate reductase 1 (PYCR1) activity is increased, oxidizing NADH with the synthesis of proline as a by-product. We further show that PYCR1 activity is required for the successful maintenance of hypoxic regions by permitting continued TCA cycle activity, and that its loss leads to significantly increased hypoxia in vivo and in 3D culture, resulting in widespread cell death.

P5C as an Interface of Proline Interconvertible Amino Acids and Its Role in Regulation of Cell Survival and Apoptosis.

Studies of cancer metabolism have focused on the production of energy and the interconversion of carbons between cell cycles. More recently, amino acid metabolism, especially non-essential amino acids (NEAAs), has been investigated, underlining their regulatory role. One of the important mediators in energy production and interconversion of carbons in the cell is Δ1-pyrroline-5-carboxylate (P5C)—the physiological intracellular intermediate of the interconversion of proline, ornithine, and glutamate. As a central component of these conversions, it links the tricarboxylic acid cycle (TCA), urea cycle (UC), and proline cycle (PC). P5C has a cyclic structure containing a tertiary nitrogen atom (N) and is in tautomeric equilibrium with the open-chain form of L-glutamate-γ-semialdehyde (GSAL). P5C is produced by P5C synthase (P5CS) from glutamate, and ornithine via ornithine δ-amino acid transferase (δOAT). It can also be converted to glutamate by P5C dehydrogenase (P5CDH). P5C is both a direct precursor of proline and a product of its degradation. The conversion of P5C to proline is catalyzed by P5C reductase (PYCR), while proline to P5C by proline dehydrogenase/oxidase (PRODH/POX). P5C-proline-P5C interconversion forms a functional redox couple. Their transformations are accompanied by the transfer of a reducing-oxidizing potential, that affect the NADP+/NADPH ratio and a wide variety of processes, e.g., the synthesis of phosphoribosyl pyrophosphate (PRPP), and purine ribonucleotides, which are crucial for DNA synthesis. This review focuses on the metabolism of P5C in the cell as an interconversion mediator of proline, glutamate, and ornithine and its role in the regulation of survival and death with particular emphasis on the metabolic context.

Expression of the pyrroline-5-carboxylate reductase (P5CR) gene from the wild grapevine Vitis yeshanensis promotes drought resistance in transgenic Arabidopsis

Proline accumulation is one of the most common reactions in plants under drought stress. Pyrroline-5-carboxylate reductase (P5CR) is the final enzyme and plays an important role in proline biosynthesis. The Chinese wild grapevine Vitis yeshanensis J.X. Chen accession 'Yanshan-1' is highly resistant to drought, but the genetic and molecular mechanisms associated with this resistance have not been elucidated. Here, we cloned a VyP5CR gene (Genbank ID: MZ226960) from 'Yanshan-1', and evaluated its transcriptional response to drought, NaCl, cold, as well as exogenous ABA, MeJA and SA. Tissue specific analysis showed that VyP5CR could be expressed in various organs and was highly expressed in roots. To gain insight into the roles of VyP5CR, we overexpressed VyP5CR in Arabidopsis thaliana (Arabidopsis). Transgenic Arabidopsis plants expressing VyP5CR showed enhanced survival rate, smaller stomata in response to severe drought, as well as stronger root growth on a medium containing mannitol. Under drought stress, VyP5CR-OE plants showed reduced levels of MDA, H2O2 and O2−, and higher proline content, SOD and POD activity. In addition, VyP5CR-OE plants showed increased induction of the drought-related genes COR15A, COR47, DREB2A, KIN1, NCED3 and RD29A. Taken together, these experiments reveal that VyP5CR can promote the drought tolerance of transgenic Arabidopsis. Besides, an interacting protein with VyP5CR, VyCSN5B (COP9 signalosome complex subunit 5b), was screened out by yeast two-hybrid and verified by bimolecular fluorescence complementation assay.