Novel Fragment Inhibitors of PYCR1 from Docking-Guided X-ray Crystallography.

The proline biosynthetic enzyme Δ1-pyrroline-5-carboxylate (P5C) reductase 1 (PYCR1) is one of the most consistently upregulated enzymes across multiple cancer types and central to the metabolic rewiring of cancer cells. Herein, we describe a fragment-based, structure-first approach to the discovery of PYCR1 inhibitors. Thirty-seven fragment-like carboxylic acids in the molecular weight range of 143-289 Da were selected from docking and then screened using X-ray crystallography as the primary assay. Strong electron density was observed for eight compounds, corresponding to a crystallographic hit rate of 22%. The fragments are novel compared to existing proline analog inhibitors in that they block both the P5C substrate pocket and the NAD(P)H binding site. Four hits showed inhibition of PYCR1 in kinetic assays, and one has lower apparent IC50 than the current best proline analog inhibitor. These results show proof-of-concept for our inhibitor discovery approach and provide a basis for fragment-to-lead optimization.

PYCR2, induced by c-Myc, promotes the invasiveness and metastasis of breast cancer by activating AKT signalling pathway.

BACKGROUND: Pyrroline-5-carboxylate reductase 2 (PYCR2) expression is aberrantly upregulated in colon cancer. However, the functions and underlying mechanisms of PYCR2 in breast cancer remain elusive. The primary objective of the present study was to elucidate the function of PYCR2 in breast cancer and investigate whether PYCR2 may be transcriptionally regulated by c-Myc to activate the AKT signaling pathway. METHODS: Immunohistochemical analysis was performed to examine the expression of PYCR2 in breast cancer and adjacent non-cancerous tissues. Western blot and RT-qPCR were utilized to detect PYCR2 expression in breast cancer cells. Cellular functionalities were evaluated through Transwell assays in vitro and lung metastasis formation assays in vivo. Moreover, the impact of PYCR2 on the activation of AKT signaling was determined through western blot and immunohistochemistry analysis. The transcriptional regulation of PYCR2 expression by c-Myc was evaluated via both western blot analysis and luciferase gene reporter assay. RESULTS: PYCR2 overexpression was noted in breast cancer. Silencing PYCR2 expression attenuated the invasive and metastatic abilities of breast cancer cells. Furthermore, the activation of the AKT signaling pathway is indispensable for the promotion of invasion and metastasis mediated by PYCR2. Lastly, the binding of c-Myc to the promoter sequence of PYCR2 resulted in the upregulation of PYCR2 transcription. CONCLUSION: Taken together, these results indicate that PYCR2 is transcriptionally regulated by c-Myc and promotes invasion and metastasis in breast cancer through the activation of the AKT pathway.

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.

PYCR2 promotes growth and aerobic glycolysis in human liver cancer by regulating the AKT signaling pathway.

Hepatocellular carcinoma (HCC) is the world's third most fatal cancer. Because metabolic rewiring is a hallmark of HCC, studies into the causes of aberrant glycolysis could provide insight into novel HCC therapeutic strategies. Pyrroline-5-carboxylate reductase 2 (PYCR2), a key enzyme of proline synthesis, has previously been found to play vital roles in various malignancies regarding amino acid metabolism and oxidative stress response. Our study investigated the mechanistic function of PYCR2 in HCC. We used Gene Expression Profiling Interactive Analysis to perform bioinformatics analysis of PYCR2 expression and survival in human HCC patients based on the Cancer Genome Atlas database. The function of PYCR2 in cell viability and glycolysis was assessed using CCK-8 and ECAR assays. Transducing shRNA or overexpression vectors into the HCC cell line altered the expression status of PYCR2. PYCR2 expression was validated using quantitative real-time PCR and Western blot. In mouse xenograft models, the role of PYCR2 in HCC tumor formation was confirmed. PYCR2 was overexpressed in human HCC tumor tissue and was associated with a poor prognosis. The functional assay revealed that silencing PYCR2 inhibited cell viability, glycolysis, and AKT activation. Furthermore, the xenograft experiment demonstrated that silencing PYCR2 significantly inhibited tumor growth and Ki67 expression. On the other hand, PYCR2 overexpression significantly promoted cell viability and glycolysis, which could be inhibited by either a glycolysis inhibitor or an AKT inhibitor, indicating that PYCR2 may function via glycolysis and the AKT pathway. Moreover, despite the overexpression of PYCR2 in vivo, treatment with a glycolysis inhibitor may considerably suppress tumor growth. Our findings suggest that PYCR2 may play an oncogenic role in HCC growth by promoting glycolysis and activating AKT, emphasizing PYCR2's clinical relevance in HCC management as a novel potential therapeutic target.

LncRNA MALAT1 regulates growth of carcinoma of the lung through modulating miR-338-3p/PYCR2 axis.

The progression of several cancers, including lung cancer, has been linked to long non-coding RNAs (lncRNAs) (LC). The current research concentrated on elucidating the effects of MALAT1 on the course of LC and investigating potential pathways. The qPCR and in situ hybridization (ISH) assays were used to measure MALAT1 expression in LC tissues. Additionally, the overall survival (OS), a percentage of LC patients with various MALAT1 levels was examined. Additionally, it was determined whether MALAT1 was expressed in LC cells through qPCR analysis. LC cells' proliferation, apoptosis, and metastasis were all examined concerning MALAT1 utilizing the following techniques: EdU, CCK-8, western blot and flow cytometry. This study predicted and verified the correlation between MALAT1, microRNA (miR)-338-3p as well as pyrroline-5-carboxylate reductase 2 using bioinformatics and dual-luciferase reporters (PYCR2). On the activity and function of MALAT1/miR-338-3p/PYCR2 in LC cell activities, more study was conducted. The amount of MALAT1 was raised in LC tissues and cells. Low OS was seen in patients with elevated MALAT1 expression. By inhibiting MALAT1, LC cells saw decreased migration, invasion, and proliferation as well as an increase in apoptosis. Additionally, PYCR2 appeared as an objective of miR-338-3p, while MALAT1 was a target of miR-338-3p. Additionally, the over-expression of miR-338-3p had effects that were comparable to those of MALAT1 down-regulation. The function of miR-338-3p inhibitor on the functional activities of LC cells co-transfected with sh-MALAT1 was partially recovered by PYCR2 inhibition. MALAT1/miR-338-3p/PYCR2 maybe the novel target for LC therapy.

Pyrroline-5-carboxylate reductase 2 (PYCR2) deficiency causes hereditary spastic paraplaegia in late childhood.

OBJECTIVES: PYCR2 gene variants are extremely rare condition which is associated with hypomyelinating leukodystrophy type 10 with microcephaly (HLD10). The aim of the present study is to report the clinical findings of patients having novel PYCR2 gene variant that manifest Hereditary Spastic Paraplegia (HSP) is the only symptom without hypomyelinating leukodystrophy. This is the first study that report the PYCR2 gene variants as a cause of HSP in late childhood. We believe it can contribute to expanding the spectrum of the phenotypes associated with PYCR2. METHODS: It is a retrospective study. Of the patients with similar clinical features from two related families, "patient 1" was designated as the index case, and was analyzed using Whole Exome Squence analysis (WES). The detected variation was investigated in the index case's parents, relatives, and sibling with a similar phenotype. Clinical, brain magnetic resonance (MR) images and MR spectroscopic findings of the patients were reported. RESULTS: A novel homozygous missense (NM_013328: c.383T > C, p.V128A) variant in the PYCR2 gene is detected in 5 patient from 2 related families. All the patients were male, their ages ranges from 6 to 26 years (15.58 ± 8,33 yrs). Developmantal milestones were normal without dysmorphic features. 4 (%80) patients exhibit mild intention tremor started at the age of approximately 6 years of age. 4 (%80) patients had gait difficulty and progressive lower limb spasticity started at the age of 8-12 years. White matter myelination was normal in all patients. Glycine peakes were detected on the MR spectroscopy in all patients. CONCLUSION: Some variants of PYCR2 gene are responsible for causing clinical features of HSP without hypomyelinating leukodystrophy in the pediatric patients.

Expression and kinetic characterization of PYCR3.

PYCRs are proline biosynthetic enzymes that catalyze the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline in humans. PYCRs - especially PYCR1 - are upregulated in many types of cancers and have been implicated in the altered metabolism of cancer cells. Of the three isoforms of PYCR, PYCR3 remains the least studied due in part to the lack of a robust recombinant expression. Herein, we describe a procedure for the expression of soluble SUMO-PYCR3 in Escherichia coli, purification of the fusion protein, and removal of the SUMO tag. PYCR3 is active with either NADPH or NADH as the coenzyme. Bi-substrate kinetic measurements obtained by varying the concentrations of both L-P5C and NADH, along with product inhibition data for l-proline, suggest a random ordered bi bi mechanism. A panel of 19 proline analogs was screened for inhibition, and the kinetics of competitive inhibition (with L-P5C) were measured for five of the compounds screened, including N-formyl-l-proline, a validated inhibitor of PYCR1. N-formyl-l-proline was found to be ten times more selective for PYCR1 over PYCR3. The SUMO-PYCR3 expression system should be useful for testing the isoform specificity of PYCR1 inhibitors.

Metabolomic and transcriptomic studies of improvements in myocardial infarction due to Pycr1 deletion.

Myocardial infarction (MI) remains a major challenge to cardiovascular health worldwide, with poor healing leaving a direct impact on patients' quality of life and survival. Metabolic abnormalities after MI are receiving increasing attention. Our previous studies showed that enhancing proline catabolism ameliorates hypoxic damage to myocardial cells; therefore, we sought to determine whether reducing the synthesis of endogenous proline also affects MI. We analysed GEO datasets associated with MI and western blot of mouse heart tissue in an MI model to demonstrate pyrroline-5-carboxylate reductase 1 (Pycr1) expression level after MI. We constructed Pycr1 KO mice by CRISPR/Cas9 technology to explore the effect of Pycr1 gene KO after MI using transcriptomic and metabolomic techniques. In this study, we found reduced mRNA and protein expression levels of Pycr1 in the hearts of mice after MI. We observed that Pycr1 gene KO has a protective effect against MI, reducing the area of MI and improving heart function. Using transcriptomics approaches, we found 215 upregulated genes and 247 downregulated genes after KO of the Pycr1 gene, indicating that unsaturated fatty acid metabolism was affected at the transcriptional level. Metabolomics results revealed elevated content for 141 metabolites and decreased content for 90 metabolites, among which the levels of fatty acids, glycerol phospholipids, bile acids, and other metabolites increased significantly. The changes in these metabolites may be related to the protective effect of Pycr1 KO on the heart after MI. Pycr1 gene KO has a protective effect against MI and our research will lay a solid foundation for the development of future Pycr1-related drug targets.

The role of PYCR1 in inhibiting 5-fluorouracil-induced ferroptosis and apoptosis through SLC25A10 in colorectal cancer.

Although PYCR1 is a well-recognized oncogenic gene for malignant tumors, the causal relationship of its expression with malignant growth and cytotoxic chemotherapeutics remains unclear. Therefore, this study aimed to clarify the role of PYCR1 and its interaction with SLC25A10 in a chemotherapeutic agent 5-fluorouracil (5-FU)'s toxicity to colorectal cancer cells. PYCR1 and SLC25A10 expressions were detected in The Cancer Genome Atlas database and colon adenocarcinoma (COAD) clinical samples. PYCR1 upregulation was associated with SLC25A10 expression and poor prognosis, and its high expression indicated decreased survival rates in patients with COAD. PYCR1 overexpression inhibited lipid reactive oxygen species production and promoted SLC25A10 expression in colorectal cancer cells. PYCR1 silencing enhanced the antitumor effects of 5-FU. Ferroptosis inhibitor deferoxamine suppressed the antitumor effects of PYCR1 silencing, whereas ferroptosis inducer erastin inhibited the protumor effects of PYCR1 overexpression. SLC25A10 overexpression reversed the antitumor effects of PYCR1 silencing in vitro and inhibited the antitumor effects of erastin in vivo. Therefore, PYCR1 is an oncogenic gene that promotes colorectal tumor growth and desensitizes colorectal cancer cells to 5-FU cytotoxicity by preventing apoptosis and ferroptosis.

The prognostic roles of PYCR2 and ZBTB18 expression in tissues of colorectal carcinoma and non-neoplastic tissues: an immunohistochemical study

Background: It is important to detect novel biomarkers responsible for the progression and spread of colorectal cancer (CRC) to better evaluate the prognosis of the patients, provide better management, and foster the development of therapeutic targets. In humans, pyrroline-5-carboxylate reductase 2 (PYCR2) is encoded on chromosome 1q42.12, and its metabolic activity has been linked to oncogenesis in many cancers. Zinc finger and broad-complex, tramtrack, and bric-à-brac (BTB) domain-containing protein 18 (ZBTB18), a zinc finger transcriptional repressor, has been found to have a tumor-suppressor role and to be methylated in CRCs. To date, the prognostic roles of PYCR2 and ZBTB18 in CRC patients have not been thoroughly studied. Objective: To evaluate the tissue protein expression of PYCR2 and ZBTB18 in CRC and adjacent non-neoplastic intestinal tissues, to detect their roles in CRC carcinogenesis, progression and metastases. Patients and methods: After applying the inclusion criteria, 60 CRC patients were included in the study. Tissue samples from the tumor and the adjacent non-neoplastic tissues were stained with PYCR2 and ZBTB18. The patients were followed up for about 30 months (range: 10 to 36 months). We performed a correlation regarding the expression of the markers, and clinicopathological and prognostic parameters. Results Upregulation of PYCR2 and downregulation of ZBTB18 were found to be higher in CRC tissue than in the adjacent non-neoplastic colonic mucosa (p = 0.026 and p < 0.001 respectively). High expression of PYCR2 and low expression of ZBTB18 were positively correlated with large tumor size, higher tumor grade, advanced tumor stage, presence of spread to lymph nodes, and presence of distant metastases (p < 0.001). High PYCR2 and low ZBTB18 expressions were significantly associated with poor response to therapy (p = 0.008 and 0.0.17 respectively), as well as high incidence of progression and recurrence (p = 0.005), and unfavorable overall survival (OS) rates (p = 0.001). Conclusion: High expression of PYCR2 and low expression of ZBTB18 were independent predictors of CRC, progression, poor prognosis and unfavorable patient OS and progression-free survival (PFS) rates. (AU)

Cancer-associated fibroblasts require proline synthesis by PYCR1 for the deposition of pro-tumorigenic extracellular matrix.

Elevated production of collagen-rich extracellular matrix is a hallmark of cancer-associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. Here we find that proline, a highly abundant amino acid in collagen proteins, is newly synthesized from glutamine in CAFs to make tumour collagen in breast cancer xenografts. PYCR1 is a key enzyme for proline synthesis and highly expressed in the stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. Both collagen and glutamine-derived proline synthesis in CAFs are epigenetically upregulated by increased pyruvate dehydrogenase-derived acetyl-CoA levels. PYCR1 is a cancer cell vulnerability and potential target for therapy; therefore, our work provides evidence that targeting PYCR1 may have the additional benefit of halting the production of a pro-tumorigenic extracellular matrix. Our work unveils new roles for CAF metabolism to support pro-tumorigenic collagen production.

CRIF1 promotes the progression of non-small-cell lung cancer by SIRT3- mediated deacetylation of PYCR1.

Lung cancer is the cancer with the highest mortality in the world. So further exploration of the pathogenesis of lung cancer is of great significance. In this study, the specific role and related mechanism of CRIF1 in non-small cell lung cancer (NSCLC) were explored in this research. TheRT-PCR, western blot and IHC assays were used to examine the expression level of CRIF1 in NSCLC tissue, tissue adjacent to carcinoma, NSCLC cell lines and human normal lung epithelial cells. Next, colony formation assay, Alamar blue Kit and EdU assays were employed to examine the proliferation of transfected A549 and NCI-H2009 cells. Measurement of mitochondrial permeability transition pore opening, ATP production and cellular oxygen consumption were used to evaluate the mitochondrial apoptosis of transfected NSCLC cells. Enzymatic activity assays for PYCR1, western blot and flow cytometry assays were used to explore the relationship between PYCR1 and CRIF1. The subcutaneous xenograft tumor mice model was established to explore the role of CRIF1 in vivo. Collectively, results revealed that CRIF1 was upregulated in NSCLC cells and tissues (p < 0.001). CRIF1 promoted proliferation of NSCLC cells (p < 0.001). CRIF1 inhibited mitochondrial apoptosis in NSCLC cells (p < 0.05). Moreover, CRIF1 promoted PYCR1 deacetylation and increased its activity through SIRT3 (p < 0.05). Deacetylation of PYCR1 reversed the antitumor effect of CRIF1 knockdown (p < 0.05). Finally, knockdown of CRIF1 inhibited the tumor growth of NSCLC in vivo (p < 0.05).This research found that CRIF1 promoted the progression of non-small-cell lung cancer by SIRT3- mediated deacetylation of PYCR1.

Epigenetic modification facilitates proline synthase PYCR1 aberrant expression in gastric cancer.

BACKGROUND & AIMS: Pyrroline-5-carboxylate reductase 1 (PYCR1) upregulation contributes to the progression of gastric cancer (GC) and indicates poor survival. However, PYCR1 expression profile in GC subtypes and the mechanism behind its upregulation are not well-studied. METHODS: PYCR1 expression profiles in GC subtypes and different stages of gastric carcinogenesis were assessed in different GC cohorts. Genetic alterations and epigenetic modulation in PYCR1 regulation were further investigated using bioinformatics analysis and in vitro experiments. RESULTS: PYCR1 expression was significantly higher in intestinal-type GC and associated molecular subtypes in TCGA and ACRG GC cohorts. During the cascade of intestinal-type GC, PYCR1 was continuously increased from normal gastric tissues through to atrophic gastritis, to intraepithelial neoplasia, and to GC. Copy number alterations in PYCR1 were associated with PYCR1 transcript expression. One CpG island was observed in PYCR1 promoter region, and the hypomethylation occurred at this region could contribute to PYCR1 transcriptional activation in GC. Besides, H3K27ac combination was found in PYCR1 promoter, and acetyltransferase p300 induced H3K27ac could promote PYCR1 expression in GC. CONCLUSIONS: PYCR1 expression varies across GC subtypes, with intestinal-type GC and associated molecular subtypes having the highest expression. Hypomethylation at CpG sites and p300-induced H3K27ac modification within PYCR1 promoter could contribute to maintaining PYCR1 overexpression in GC. These results provide us with a new insight into epigenetic modulation in mitochondrial proline metabolism.

Shikonin induces apoptosis and autophagy via downregulation of pyrroline-5-carboxylate reductase1 in hepatocellular carcinoma cells.

Shikonin(SK) is a natural small molecule naphthoquinone compound, which has anti-cancer activity in various human malignant tumors. Pyrroline-5-carboxylate reductase 1(PYCR1) is involved in tumorigenesis and regulates various cellular processes, including growth, invasion, migration, and apoptosis. However, the effect of SK and PYCR1 on apoptosis and autophagy in hepatocellular carcinoma are unclear. Our goal is to determine the internal molecular mechanism of the interaction between SK and PYCR1 and its role in the occurrence and development of liver cancer. The CCK8 assay, wound healing assay, and transwell assays show that SK and siPYCR1(gene silence PYCR1) inhibited the malignant phenotype of HCC cells, including cell viability, colony formation, migration, and invasion, respectively. The flow cytometry assays and immunofluorescence show that SK and siPYCR1 activated apoptosis and autophagy, respectively. SK induces apoptosis and autophagy in a dose-dependent manner. In addition, HCC cells were transfected with small interference fragment PYCR1 siRNA to construct siPYCR1 and SK single treatment group and co-treatment group to verify the interaction between SK and PYCR1. The Western blot identified that PI3K/Akt/mTOR signal pathway protein expression was significantly downregulated in HCC cells treated with SK and siPYCR1 together. Collectively, SK may induce apoptosis and autophagy by reducing the expression of PYCR1 and suppressing PI3K/Akt/mTOR. Thus, SK may be a promising antineoplastic drug in Hepatocellular carcinoma (HCC). SK downregulating PYCR1 might supply a theoretical foundation for the potential therapeutic application in hepatocellular carcinoma.

Pyrroline-5-Carboxylate Reductase 1: a novel target for sensitizing multiple myeloma cells to bortezomib by inhibition of PRAS40-mediated protein synthesis.

BACKGROUND: Multiple myeloma (MM) remains an incurable cancer despite advances in therapy. Therefore, the search for new targets is still essential to uncover potential treatment strategies. Metabolic changes, induced by the hypoxic bone marrow, contribute to both MM cell survival and drug resistance. Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) are two mitochondrial enzymes that facilitate the last step in the glutamine-to-proline conversion. Overexpression of PYCR1 is involved in progression of several cancers, however, its' role in hematological cancers is unknown. In this study, we investigated whether PYCR affects MM viability, proliferation and response to bortezomib. METHODS: Correlation of PYCR1/2 with overall survival was investigated in the MMRF CoMMpass trial (653 patients). OPM-2 and RPMI-8226 MM cell lines were used to perform in vitro experiments. RPMI-8226 cells were supplemented with 13C-glutamine for 48 h in both normoxia and hypoxia (< 1% O2, by chamber) to perform a tracer study. PYCR1 was inhibited by siRNA or the small molecule inhibitor pargyline. Apoptosis was measured using Annexin V and 7-AAD staining, viability by CellTiterGlo assay and proliferation by BrdU incorporation. Differential protein expression was evaluated using Western Blot. The SUnSET method was used to measure protein synthesis. All in vitro experiments were performed in hypoxic conditions. RESULTS: We found that PYCR1 and PYCR2 mRNA expression correlated with an inferior overall survival. MM cells from relapsed/refractory patients express significantly higher levels of PYCR1 mRNA. In line with the strong expression of PYCR1, we performed a tracer study in RPMI-8226 cells, which revealed an increased conversion of 13C-glutamine to proline in hypoxia. PYCR1 inhibition reduced MM viability and proliferation and increased apoptosis. Mechanistically, we found that PYCR1 silencing reduced protein levels of p-PRAS40, p-mTOR, p-p70, p-S6, p-4EBP1 and p-eIF4E levels, suggesting a decrease in protein synthesis, which we also confirmed in vitro. Pargyline and siPYCR1 increased bortezomib-mediated apoptosis. Finally, combination therapy of pargyline with bortezomib reduced viability in CD138+ MM cells and reduced tumor burden in the murine 5TGM1 model compared to single agents. CONCLUSIONS: This study identifies PYCR1 as a novel target in bortezomib-based combination therapies for MM.

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

The 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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Kinetics of human pyrroline-5-carboxylate reductase in L-thioproline metabolism.

L-Thioproline (L-thiazolidine-4-carboxylate, L-T4C) is a cyclic sulfur-containing analog of L-proline found in multiple kingdoms of life. The oxidation of L-T4C leads to L-cysteine formation in bacteria, plants, mammals, and protozoa. The conversion of L-T4C to L-Cys in bacterial cell lysates has been attributed to proline dehydrogenase and L-Δ1-pyrroline-5-carboxylate (P5C) reductase (PYCR) enzymes but detailed kinetic studies have not been conducted. Here, we characterize the dehydrogenase activity of human PYCR isozymes 1 and 2 with L-T4C using NAD(P)+ as the hydride acceptor. Both PYCRs exhibit significant L-T4C dehydrogenase activity; however, PYCR2 displays nearly tenfold higher catalytic efficiency (136 M-1 s-1) than PYCR1 (13.7 M-1 s-1). Interestingly, no activity was observed with either L-Pro or the analog DL-thiazolidine-2-carboxylate, indicating that the sulfur at the 4-position is critical for PYCRs to utilize L-T4C as a substrate. Inhibition kinetics show that L-Pro is a competitive inhibitor of PYCR1 [Formula: see text] with respect to L-T4C, consistent with these ligands occupying the same binding site. We also confirm by mass spectrometry that L-T4C oxidation by PYCRs leads to cysteine product formation. Our results suggest a new enzyme function for human PYCRs in the metabolism of L-T4C.

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.

MicroRNA hsa-miR-150-5p inhibits nasopharyngeal carcinogenesis by suppressing PYCR1 (pyrroline-5-carboxylate reductase 1).

Nasopharyngeal cancer is a rare cancer type, but with a low five-year survival rate. Dysregulation of pyrroline-5-carboxylate reductase 1 (PYCR1) and microRNA hsa-miR-150-5p is involved in the development of various cancers. However, the molecular mechanism of the hsa-miR-150-5p-PYCR1 axis in nasopharyngeal cancer remains unclear. To identify the mechanism of the hsa-miR-150-5p-PYCR1 axis, the expression of hsa-miR-150-5p and PYCR1 in nasopharyngeal cancer tissues and cells was first measured by reverse transcription quantitative polymerase chain reaction. The luciferase and RNA pull-down assays were used to confirm the interaction between hsa-miR-150-5p and PYCR1. The overexpression of hsa-miR-150-5p and PYCR1 was detected by cell viability, proliferation, western blotting, migration, and invasion in nasopharyngeal cancer cells. The expression levels of hsa-miR-150-5p was reduced in the nasopharyngeal cancer tissues and cells and were negatively correlated with the PYCR1 levels. The upregulation of hsa-miR-150-5p significantly repressed cell growth and promoted apoptosis. However, the upregulation of PYCR1 expression significantly promoted nasopharyngeal carcinogenesis, which could abolish the inhibitory effect of hsa-miR-150-5p. In conclusion, we clarified that hsa-miR-150-5p attenuated nasopharyngeal carcinogenesis by reducing the PYCR1 expression levels. This provides a new perspective of nasopharyngeal cancer involving both hsa-miR-150-5p and PYCR1 for the treatment of nasopharyngeal cancer.