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1.
Int J Mol Sci ; 25(15)2024 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-39125668

RESUMO

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 Sinais
2.
Mol Genet Genomics ; 299(1): 81, 2024 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-39172257

RESUMO

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 , Mutação
3.
Protein Sci ; 33(7): e5072, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-39133178

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 Moleculares
4.
Am J Med Genet A ; 194(9): e63645, 2024 09.
Artigo em Inglês | MEDLINE | ID: mdl-38709052

RESUMO

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 , Lactente
5.
Eur J Med Res ; 29(1): 302, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38811952

RESUMO

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 , Masculino
6.
Int J Biochem Cell Biol ; 171: 106581, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38642827

RESUMO

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 fármacos
7.
Int J Cancer ; 155(4): 742-755, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38647131

RESUMO

Alteration of cell metabolism is one of the essential characteristics of tumor growth. Cancer stem cells (CSCs) are the initiating cells of tumorigenesis, proliferation, recurrence, and other processes, and play an important role in therapeutic resistance and metastasis. Thus, identification of the metabolic profiles in prostate cancer stem cells (PCSCs) is critical to understanding prostate cancer progression. Using untargeted metabolomics and lipidomics methods, we show distinct metabolic differences between prostate cancer cells and PCSCs. Urea cycle is the most significantly altered metabolic pathway in PCSCs, the key metabolites arginine and proline are evidently elevated. Proline promotes cancer stem-like characteristics via the JAK2/STAT3 signaling pathway. Meanwhile, the enzyme pyrroline-5-carboxylate reductase 1 (PYCR1), which catalyzes the conversion of pyrroline-5-carboxylic acid to proline, is highly expressed in PCSCs, and the inhibition of PYCR1 suppresses the stem-like characteristics of prostate cancer cells and tumor growth. In addition, carnitine and free fatty acid levels are significantly increased, indicating reprogramming of fatty acid metabolism in PCSCs. Reduced sphingolipid levels and increased triglyceride levels are also observed. Collectively, our data illustrate the comprehensive landscape of the metabolic reprogramming of PCSCs and provide potential therapeutic strategies for prostate cancer.


Assuntos
Células-Tronco Neoplásicas , Neoplasias da Próstata , Pirrolina Carboxilato Redutases , Ureia , delta-1-Pirrolina-5-Carboxilato Redutase , Masculino , Humanos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Pirrolina Carboxilato Redutases/metabolismo , Ureia/metabolismo , Animais , Camundongos , Linhagem Celular Tumoral , Transdução de Sinais , Janus Quinase 2/metabolismo , Metabolômica/métodos , Prolina/metabolismo , Fator de Transcrição STAT3/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Proliferação de Células , Lipidômica/métodos
8.
Plant Physiol ; 195(2): 1038-1052, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38478428

RESUMO

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/metabolismo
9.
J Chem Inf Model ; 64(5): 1704-1718, 2024 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-38411104

RESUMO

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.


Assuntos
Pirrolina Carboxilato Redutases , delta-1-Pirrolina-5-Carboxilato Redutase , Pirrolina Carboxilato Redutases/química , Pirrolina Carboxilato Redutases/metabolismo , Cristalografia por Raios X , Sítios de Ligação , Prolina
10.
Int J Biochem Cell Biol ; 166: 106506, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38101533

RESUMO

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/metabolismo
11.
Plant Physiol Biochem ; 205: 108182, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37977024

RESUMO

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.


Assuntos
Medicago sativa , Prolina Oxidase , Medicago sativa/genética , Medicago sativa/metabolismo , Prolina Oxidase/genética , Prolina Oxidase/metabolismo , Pirrolina Carboxilato Redutases , Prolina/metabolismo , Biologia Computacional , Estresse Fisiológico/genética
12.
Biochem Biophys Res Commun ; 680: 15-24, 2023 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-37708598

RESUMO

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/metabolismo
13.
Cell Mol Biol (Noisy-le-grand) ; 69(4): 133-140, 2023 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-37329534

RESUMO

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/genética
14.
Eur J Paediatr Neurol ; 44: 51-56, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-37141741

RESUMO

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 , Adulto
15.
Arch Biochem Biophys ; 733: 109468, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36414121

RESUMO

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/metabolismo
16.
J Cell Mol Med ; 27(1): 89-100, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36495058

RESUMO

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 Redutase
17.
Hum Cell ; 35(6): 1900-1911, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36104652

RESUMO

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 Redutase
18.
J Mol Histol ; 53(4): 657-667, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35716330

RESUMO

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.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , Sirtuína 3 , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Pirrolina Carboxilato Redutases , Sirtuína 3/genética , Sirtuína 3/metabolismo , delta-1-Pirrolina-5-Carboxilato Redutase
19.
Nat Metab ; 4(6): 693-710, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35760868

RESUMO

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.


Assuntos
Neoplasias da Mama , Fibroblastos Associados a Câncer , Pirrolina Carboxilato Redutases/metabolismo , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Feminino , Glutamina/metabolismo , Humanos , Prolina , delta-1-Pirrolina-5-Carboxilato Redutase
20.
Biochim Biophys Acta Gene Regul Mech ; 1865(6): 194829, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35654390

RESUMO

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/genética
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