RESUMO
Δ1-pyrroline-5-carboxylate reductase isoform 1 (PYCR1) is the last enzyme of proline biosynthesis and catalyzes the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate to L-proline. High PYCR1 gene expression is observed in many cancers and linked to poor patient outcomes and tumor aggressiveness. The knockdown of the PYCR1 gene or the inhibition of PYCR1 enzyme has been shown to inhibit tumorigenesis in cancer cells and animal models of cancer, motivating inhibitor discovery. We screened a library of 71 low molecular weight compounds (average MW of 131 Da) against PYCR1 using an enzyme activity assay. Hit compounds were validated with X-ray crystallography and kinetic assays to determine affinity parameters. The library was counter-screened against human Δ1-pyrroline-5-carboxylate reductase isoform 3 and proline dehydrogenase (PRODH) to assess specificity/promiscuity. Twelve PYCR1 and one PRODH inhibitor crystal structures were determined. Three compounds inhibit PYCR1 with competitive inhibition parameter of 100 µM or lower. Among these, (S)-tetrahydro-2H-pyran-2-carboxylic acid (70 µM) has higher affinity than the current best tool compound N-formyl-l-proline, is 30 times more specific for PYCR1 over human Δ1-pyrroline-5-carboxylate reductase isoform 3, and negligibly inhibits PRODH. Structure-affinity relationships suggest that hydrogen bonding of the heteroatom of this compound is important for binding to PYCR1. The structures of PYCR1 and PRODH complexed with 1-hydroxyethane-1-sulfonate demonstrate that the sulfonate group is a suitable replacement for the carboxylate anchor. This result suggests that the exploration of carboxylic acid isosteres may be a promising strategy for discovering new classes of PYCR1 and PRODH inhibitors. The structure of PYCR1 complexed with l-pipecolate and NADH supports the hypothesis that PYCR1 has an alternative function in lysine metabolism.
Assuntos
Inibidores Enzimáticos , Prolina , Pirrolina Carboxilato Redutases , delta-1-Pirrolina-5-Carboxilato Redutase , Pirrolina Carboxilato Redutases/metabolismo , Pirrolina Carboxilato Redutases/antagonistas & inibidores , Pirrolina Carboxilato Redutases/química , Pirrolina Carboxilato Redutases/genética , Humanos , Cristalografia por Raios X , Prolina/química , Prolina/análogos & derivados , Prolina/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Peso Molecular , Prolina Oxidase/metabolismo , Prolina Oxidase/química , Prolina Oxidase/antagonistas & inibidores , Prolina Oxidase/genética , Modelos MolecularesRESUMO
Autosomal-recessive cutis laxa type 2 (ARCL2) is a rare genetic disorder caused by pyrroline-5-carboxylate reductase 1 (PYCR1) mutations and characterized by loose and sagging skin, typical facial features, intrauterine growth retardation, and developmental delay. To study the effect of PYCR1 mutations on protein function and clinical features, we identified a homozygous missense mutation c.559G > A (p.Ala187Thr) in PYCR1 in a Chinese child with typical clinical features, especially severe developmental delays. The three-dimensional (3D) model showed the modification of the hydrogen bonds produce a misfolding in the mutant PYCR1 protein. Mutagenesis and enzyme assay study revealed decreased activity of the mutant protein in vitro, indicating that this mutation impairs PYCR1 function. Our findings confirmed abnormal enzymatic activity and neurodevelopmental trajectory of this PYCR1 mutation.
Assuntos
Cútis Laxa , Mutação de Sentido Incorreto , Pirrolina Carboxilato Redutases , delta-1-Pirrolina-5-Carboxilato Redutase , Humanos , Cútis Laxa/genética , Cútis Laxa/patologia , Pirrolina Carboxilato Redutases/genética , Pirrolina Carboxilato Redutases/metabolismo , Masculino , Feminino , Pré-Escolar , Modelos Moleculares , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/patologia , Homozigoto , Genes Recessivos , MutaçãoRESUMO
Pyrroline-5-carboxylate reductase (PYCR) is pivotal in converting pyrroline-5-carboxylate (P5C) to proline, the final step in proline synthesis. Three isoforms, PYCR1, PYCR2, and PYCR3, existed and played significant regulatory roles in tumor initiation and progression. In this study, we first assessed the molecular and immune characteristics of PYCRs by a pan-cancer analysis, especially focusing on their prognostic relevance. Then, a kidney renal clear cell carcinoma (KIRC)-specific prognostic model was established, incorporating pathomics features to enhance predictive capabilities. The biological functions and regulatory mechanisms of PYCR1 and PYCR2 were investigated by in vitro experiments in renal cancer cells. The PYCRs' expressions were elevated in diverse tumors, correlating with unfavorable clinical outcomes. PYCRs were enriched in cancer signaling pathways, significantly correlating with immune cell infiltration, tumor mutation burden (TMB), and microsatellite instability (MSI). In KIRC, a prognostic model based on PYCR1 and PYCR2 was independently validated statistically. Leveraging features from H&E-stained images, a pathomics feature model reliably predicted patient prognosis. In vitro experiments demonstrated that PYCR1 and PYCR2 enhanced the proliferation and migration of renal carcinoma cells by activating the mTOR pathway, at least in part. This study underscores PYCRs' pivotal role in various tumors, positioning them as potential prognostic biomarkers and therapeutic targets, particularly in malignancies like KIRC. The findings emphasize the need for a broader exploration of PYCRs' implications in pan-cancer contexts.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Pirrolina Carboxilato Redutases , Humanos , Pirrolina Carboxilato Redutases/metabolismo , Pirrolina Carboxilato Redutases/genética , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Prognóstico , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , delta-1-Pirrolina-5-Carboxilato Redutase , Proliferação de Células , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Transdução de SinaisRESUMO
Proline-5-carboxylate reductase 2, encoded by PYCR2 gene, is an enzyme that catalyzes the last step of proline synthesis from pyrroline-5-carboxylate synthetase to proline. PYCR2 gene defect causes hypomyelinating leukodystrophy 10. Up until now, to our knowledge around 38 patients with PYCR2 defect have been reported. Herein, we describe clinical, neuroradiological, biochemical findings, and metabolomic profiling of three new genetically related cases of PYCR2 defects from a large family. Cerebrospinal fluid (CSF) amino acid levels were measured and untargeted metabolomic profiling of plasma and CSF were conducted and evaluated together with the clinical findings in the patients. While plasma and CSF proline levels were found to be totally normal, untargeted metabolomic profiling revealed mild increases of glutamate, alpha-ketoglutarate, and l-glutamate semialdehyde and marked increases of inosine and xanthine. Our findings and all the previous reports suggest that proline auxotrophy is not the central disease mechanism. Untargeted metabolomics point to mild changes in proline pathway and also in purine/pyrimidine pathway.
Assuntos
Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central , Metabolômica , Prolina , Pirrolina Carboxilato Redutases , Criança , Feminino , Humanos , Masculino , delta-1-Pirrolina-5-Carboxilato Redutase , Ácido Glutâmico/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/diagnóstico , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/sangue , Imageamento por Ressonância Magnética , Redes e Vias Metabólicas/genética , Metaboloma/genética , Metabolômica/métodos , Mutação/genética , Linhagem , Prolina/líquido cefalorraquidiano , Purinas/metabolismo , Pirimidinas , Pirrolina Carboxilato Redutases/genética , Pirrolina Carboxilato Redutases/deficiência , Xantina/sangue , LactenteRESUMO
BACKGROUND: The basic helix-loop-helix family member e41 (BHLHE41) is frequently dysregulated in tumors and plays a crucial role in malignant progression of various cancers. Nevertheless, its specific function and underlying mechanism in bladder cancer (BCa) remain largely unexplored. METHODS: The expression levels of BHLHE41 in BCa tissues and cells were examined by qRT-PCR and western blot assays. BCa cells stably knocking down or overexpressing BHLHE41 were constructed through lentivirus infection. The changes of cell proliferation, cell cycle distribution, migration, and invasion were detected by CCK-8, flow cytometry, wound healing, transwell invasion assays, respectively. The expression levels of related proteins were detected by western blot assay. The interaction between BHLHE41 and PYCR1 was explored by co-immunoprecipitation analysis. RESULTS: In this study, we found that BHLHE41 was lowly expressed in bladder cancer tissues and cell lines, and lower expression of BHLHE41 was associated with poor overall survival in bladder cancer patients. Functionally, by manipulating the expression of BHLHE41, we demonstrated that overexpression of BHLHE41 significantly retarded cell proliferation, migration, invasion, and induced cell cycle arrest in bladder cancer through various in vitro and in vivo experiments, while silence of BHLHE41 caused the opposite effect. Mechanistically, we showed that BHLHE41 directly interacted with PYCR1, decreased its stability and resulted in the ubiquitination and degradation of PYCR1, thus inactivating PI3K/AKT signaling pathway. Rescue experiments showed that the effects induced by BHLHE41 overexpression could be attenuated by further upregulating PYCR1. CONCLUSION: BHLHE41 might be a useful prognostic biomarker and a tumor suppressor in bladder cancer. The BHLHE41/PYCR1/PI3K/AKT axis might be a potential therapeutic target for bladder cancer intervention.
Assuntos
Proliferação de Células , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Pirrolina Carboxilato Redutases , Transdução de Sinais , Neoplasias da Bexiga Urinária , delta-1-Pirrolina-5-Carboxilato Redutase , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/genética , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirrolina Carboxilato Redutases/metabolismo , Pirrolina Carboxilato Redutases/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Movimento Celular/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Camundongos , Animais , MasculinoRESUMO
Triple-negative breast cancer (TNBC) poses significant challenges in treatment due to its aggressive nature and limited therapeutic targets. Understanding the underlying molecular mechanisms driving TNBC progression and chemotherapy resistance is imperative for developing effective therapeutic strategies. Thus, in this study, we aimed to elucidate the role of pyrroline-5-carboxylate reductase 3 (PYCR3) in TNBC pathogenesis and therapeutic response. We observed that PYCR3 is significantly upregulated in TNBC specimens compared to normal breast tissues, correlating with a poorer prognosis in TNBC patients. Knockdown of PYCR3 not only suppresses TNBC cell proliferation but also reverses acquired resistance of TNBC cells to doxorubicin, a commonly used chemotherapeutic agent. Mechanistically, we identified the mitochondrial localization of PYCR3 in TNBC cells and demonstrated its impact on TNBC cell proliferation and sensitivity to doxorubicin through the regulation of mtDNA copy number and mitochondrial respiration. Importantly, Selective reduction of mtDNA copy number using the mtDNA replication inhibitor 2', 3'-dideoxycytidine effectively recapitulates the phenotypic effects observed in PYCR3 knockout, resulting in decreased TNBC cell proliferation and the reversal of doxorubicin resistance through apoptosis induction. Thus, our study underscores the clinical relevance of PYCR3 and highlight its potential as a therapeutic target in TNBC management. By elucidating the functional significance of PYCR3 in TNBC, our findings contribute to a deeper understanding of TNBC biology and provide a foundation for developing novel therapeutic strategies aimed at improving patient outcomes.
Assuntos
Proliferação de Células , DNA Mitocondrial , Doxorrubicina , Resistencia a Medicamentos Antineoplásicos , Pirrolina Carboxilato Redutases , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Doxorrubicina/farmacologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Pirrolina Carboxilato Redutases/metabolismo , Pirrolina Carboxilato Redutases/genética , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Drought and soil salinization substantially impact agriculture. While proline's role in enhancing stress tolerance is known, the exact molecular mechanism by which plants process stress signals and control proline synthesis under stress is still not fully understood. In tomato (Solanum lycopersicum L.), drought and salt stress stimulate nitric oxide (NO) production, which boosts proline synthesis by activating Δ1-pyrroline-5-carboxylate synthetase (SlP5CS) and Δ1-pyrroline-5-carboxylate reductase (SlP5CR) genes and the P5CR enzyme. The crucial factor is stress-triggered NO production, which regulates the S-nitrosylation of SlP5CR at Cys-5, thereby increasing its NAD(P)H affinity and enzymatic activity. S-nitrosylation of SlP5CR enables tomato plants to better adapt to changing NAD(P)H levels, boosting both SlP5CR activity and proline synthesis during stress. By comparing tomato lines genetically modified to express different forms of SlP5CR, including a variant mimicking S-nitrosylation (SlP5CRC5W), we found that SlP5CRC5W plants show superior growth and stress tolerance. This is attributed to better P5CR activity, proline production, water use efficiency, reactive oxygen species scavenging, and sodium excretion. Overall, this study demonstrates that tomato engineered to mimic S-nitrosylated SlP5CR exhibits enhanced growth and yield under drought and salt stress conditions, highlighting a promising approach for stress-tolerant tomato cultivation.
Assuntos
Secas , Engenharia Genética , Plantas Geneticamente Modificadas , Pirrolina Carboxilato Redutases , Solanum lycopersicum , Solanum lycopersicum/genética , Pirrolina Carboxilato Redutases/genética , Pirrolina Carboxilato Redutases/metabolismo , delta-1-Pirrolina-5-Carboxilato Redutase , Tolerância ao Sal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Estresse Fisiológico/genética , Regulação da Expressão Gênica de Plantas , Plantas Tolerantes a Sal/genética , Plantas Tolerantes a Sal/metabolismoRESUMO
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.
Assuntos
Neoplasias da Mama , Proteínas Proto-Oncogênicas c-akt , Humanos , Feminino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias da Mama/patologia , Transdução de Sinais , Regulação para Cima , Linhagem Celular Tumoral , Invasividade Neoplásica/genética , Movimento Celular , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , delta-1-Pirrolina-5-Carboxilato Redutase , Pirrolina Carboxilato Redutases/genética , Pirrolina Carboxilato Redutases/metabolismoRESUMO
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.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Camundongos , Humanos , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Modelos Animais de Doenças , Proliferação de Células , Glicólise , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Pirrolina Carboxilato Redutases/genética , Pirrolina Carboxilato Redutases/metabolismoRESUMO
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.
Assuntos
Carcinoma , Neoplasias Pulmonares , MicroRNAs , Pirrolina Carboxilato Redutases , RNA Longo não Codificante , Humanos , Carcinoma/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Pirrolina Carboxilato Redutases/genética , RNA Longo não Codificante/genética , Neoplasias Pulmonares/genéticaRESUMO
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.
Assuntos
Espasticidade Muscular , Paraplegia Espástica Hereditária , Criança , Humanos , Masculino , Mutação/genética , Oxirredutases/genética , Linhagem , Fenótipo , Pirrolina Carboxilato Redutases/genética , Estudos Retrospectivos , Paraplegia Espástica Hereditária/genética , Adolescente , Adulto Jovem , AdultoRESUMO
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.
Assuntos
NAD , Pirrolina Carboxilato Redutases , Humanos , Pirrolina Carboxilato Redutases/genética , Pirrolina Carboxilato Redutases/química , Cinética , NAD/metabolismo , Prolina/química , NADP/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
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.
Assuntos
Infarto do Miocárdio , Pirrolina Carboxilato Redutases , Animais , Camundongos , Metabolômica , Infarto do Miocárdio/genética , Prolina , Pirrolina Carboxilato Redutases/genética , Transcriptoma/genética , delta-1-Pirrolina-5-Carboxilato RedutaseRESUMO
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.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Transportadores de Ácidos Dicarboxílicos , Ferroptose , Pirrolina Carboxilato Redutases , Adenocarcinoma , Apoptose/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Desferroxamina/farmacologia , Desferroxamina/uso terapêutico , Transportadores de Ácidos Dicarboxílicos/genética , Ferroptose/genética , Fluoruracila/farmacologia , Humanos , Lipídeos/farmacologia , Lipídeos/uso terapêutico , Pirrolina Carboxilato Redutases/genética , Espécies Reativas de Oxigênio/metabolismo , delta-1-Pirrolina-5-Carboxilato RedutaseRESUMO
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.
Assuntos
Neoplasias Gástricas , Ilhas de CpG/genética , Epigênese Genética , Humanos , Prolina/genética , Prolina/metabolismo , Pirrolina Carboxilato Redutases/genética , Pirrolina Carboxilato Redutases/metabolismo , Neoplasias Gástricas/genéticaRESUMO
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.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Naftoquinonas , Apoptose , Autofagia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo , Humanos , Neoplasias Hepáticas/metabolismo , Naftoquinonas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirróis , Pirrolina Carboxilato Redutases/genética , Pirrolina Carboxilato Redutases/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
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.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , MicroRNAs , Masculino , Feminino , Humanos , Redes Reguladoras de Genes , Carcinoma de Células Renais/genética , MicroRNAs/genética , Neoplasias Renais/genética , Rim/metabolismo , Prolina/química , Prolina/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Pirrolina Carboxilato Redutases/genética , Pirrolina Carboxilato Redutases/metabolismoRESUMO
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.
Assuntos
Carcinogênese/metabolismo , MicroRNAs/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Proteínas de Neoplasias/metabolismo , Pirrolina Carboxilato Redutases/metabolismo , RNA Neoplásico/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Humanos , MicroRNAs/genética , Neoplasias Nasofaríngeas/genética , Proteínas de Neoplasias/genética , Pirrolina Carboxilato Redutases/genética , RNA Neoplásico/genética , delta-1-Pirrolina-5-Carboxilato RedutaseRESUMO
AIM: The aim of this study was to investigate the effect and underlying pathway of pyrroline-5-carboxylate reductase-2 (PYCR2) on colorectal cancer (CRC). METHODS: The Cancer Genome Atlas (TCGA) database was used to analyze PYCR2 expression levels and clinical information. Cell proliferation was evaluated using colony forming and EdU assay. Cell apoptosis rate was determined using flow cytometry. Cell migration and invasion were measured by performing a Transwell assay, and PYCR2, MMP-2, MMP-9, Bax, cleaved caspase-3, Bcl-2, cleaved PARP, p-PI3K, PI3K, p-AKT, AKT, p-mTOR, and mTOR protein levels were detected by Western blot. RESULTS: A review of the TCGA database revealed that PYCR2 was highly expressed in CRC patients and that high PYCR2 expression was associated with advanced stage, adenocarcinoma, nodal metastasis, and poor survival rate. Moreover, PYCR2 knockdown reduced cell viability, proliferation, migration, and invasion and increased apoptosis. Additionally, PYCR2 knockdown increased Bax, cleaved caspase-3, and cleaved PARP levels and decreased Bcl-2, MMP-2, MMP-9, p-PI3K, p-AKT, and p-mTOR levels in CRC cells. Effects of silencing PYCR2 on proliferation, migration, invasion, apoptosis, and the PI3K/AKT/mTOR pathway in CRC cells were all reversed using a PI3K activator (740Y-P). CONCLUSION: PYCR2 was highly expressed in CRC, and its knockdown suppressed CRC tumorigenesis via inhibiting the activation of PI3K/AKT/mTOR pathway. This finding provides a new theoretical foundation for the treatment of CRC.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirrolina Carboxilato Redutases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Inativação Gênica , Humanos , Fosfatidilinositol 3-Quinases/genética , Prognóstico , Proteínas Proto-Oncogênicas c-akt/genética , Pirrolina Carboxilato Redutases/antagonistas & inibidores , Pirrolina Carboxilato Redutases/genética , RNA Longo não Codificante/genética , Transdução de Sinais , Taxa de Sobrevida , Serina-Treonina Quinases TOR/genética , Células Tumorais CultivadasRESUMO
Prostate cancer, the most common non-cutaneous male cancer, is a public health problem with a third prevalence worldwide. PYCR1 and miR-1207-5p dysregulations were found in cancer progression. Our study aims to reveal the biological role of miR-1207-5p-PYCR1 axis in prostate cancer progression. First, we investigated the expression of miR-1207-5p in prostate cancer tissues and cell lines by RT-qPCR. Next, we confirmed miR-1207-5p targeting PYCR1 by luciferase assay. CCK-8 assay, BrdU assay, flow cytometry, and tanswell assay were applied for examining cell proliferation, apoptosis, and invasion in prostate cancer cells, respectively. In the present study, decreased miR-1207-5p expression was obviously observed in prostate cancer tissues and cells. Upregulation of miR-1207-5p hampered cellular proliferation and invasion, while enhanced cellular apoptosis. In addition, upregulation of PYCR1 elevated cell proliferation and invasion, but repressed apoptosis of prostate cancer cells. Moreover, miR-1207-5p inhibited the expression of PYCR1 to repress prostate cancer tumorigenesis. MiR-1207-5p inhibited the expression of PYCR1 to repress the progression of prostate cancer by inhibiting cell growth and elevating cell apoptosis. Overall, our study clarifies the biological role of miR-1207-5p-PYCR1 axis in prostate cancer progression, which might be effective biomarkers for clinical treatment of prostate cancer.