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1.
BMC Cancer ; 22(1): 864, 2022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35941558

RESUMO

BACKGROUND: IFN-γ has been traditionally recognized as an inflammatory cytokine that involves in inflammation and autoimmune diseases. Previously we have shown that sustained IFN-γ induced malignant transformation of bovine mammary epithelial cells (BMECs) via arginine depletion. However, the molecular mechanism underlying this is still unknown. METHODS: In this study, the amino acids contents in BMECs were quantified by a targeted metabolomics method. The acquisition of differentially expressed genes was mined from RNA-seq dataset and analyzed bioinformatically. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) assay were performed to detect gene mRNA and protein expression levels. CCK-8 and would healing assays were used to detect cell proliferation and migration abilities, respectively. Cell cycle phase alternations were analyzed by flow cytometry. RESULTS: The targeted metabolomics analysis specifically discovered IFN-γ induced arginine depletion through accelerating arginine catabolism and inhibiting arginine anabolism in BMECs. Transcriptome analysis identified leucine aminopeptidase 3 (LAP3), which was regulated by p38 and ERK MAPKs, to downregulate arginine level through interfering with argininosuccinate synthetase (ASS1) as IFN-γ stimulated. Moreover, LAP3 also contributed to IFN-γ-induced malignant transformation of BMECs by upregulation of HDAC2 (histone deacetylase 2) expression and promotion of cell cycle proteins cyclin A1 and D1 expressions. Arginine supplementation did not affect LAP3 and HDAC2 expressions, but slowed down cell cycle process of malignant BMECs. In clinical samples of patients with breast cancer, LAP3 was confirmed to be upregulated, while ASS1 was downregulated compared with healthy control. CONCLUSIONS: These results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.


Assuntos
Arginina , Neoplasias da Mama , Animais , Arginina/metabolismo , Argininossuccinato Sintase/metabolismo , Mama/metabolismo , Neoplasias da Mama/metabolismo , Bovinos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , Interferon gama/metabolismo
2.
J Vis Exp ; (185)2022 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-35938827

RESUMO

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often limited by the lack of a specific antibody recognizing the target arginine residue. Proximity ligation assay (PLA) was originally developed to study protein/protein interactions. Here, we describe in detail a PLA protocol dedicated to the detection of arginine methylation that we applied to the glucocorticoid receptor (GR). Having previously shown that PRMT5 dimethylates GRs in cells, we used PLA with a pan symmetrical dimethyl antibody and an anti-GR antibody to measure GR methylation in breast tumors. We demonstrate that PLA offers a unique approach to measure arginine methylation of a target protein, even when the site of methylation has not been identified. This technique could be extended to other post-translational modifications where effective pan antibodies are available. Hence, we detail the PLA technology used to detect arginine methylation in fixed tissue using GR as an example.


Assuntos
Arginina , Fenômenos Biológicos , Anticorpos/metabolismo , Arginina/metabolismo , Metilação , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas/metabolismo
3.
Gen Physiol Biophys ; 41(4): 329-338, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35938966

RESUMO

This study aims to explore the effect and mechanism of arginyl-fructosyl-glucose (AFG) on TGF-ß1-induced epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells. HK-2 cells were induced by TGF-ß1 and then co-cultured with AFG at different concentrations (0, 25, 50, and 100 µmol/l) for 48 h. The morphology of HK-2 cells was observed under an inverted microscope and the expressions of α-SMA, Vimentin, and E-cadherin were assessed by qRT-PCR, Western blot, and immunofluorescence. The mRNA expressions of ERK and STAT3 were also examined by qRT-PCR, and the protein levels of ERK, STAT3, p-ERK, and p-STAT3 were measured by Western blot. Finally, CCK-8 and transwell assays were used to detect cell proliferation and invasion. TGF-ß1 treatment significantly induced EMT in HK-2 cells. The expressions of p-ERK and p-STAT3 were signally increased after TGF-ß1 induction, while Mogrol treatment inhibited p-ERK, p-STAT3, α-SMA, and Vimentin expression levels, enhanced E-cadherin expression, and suppressed cell proliferation and invasion. AFG exposure could also inhibit p-ERK, p-STAT3, α-SMA, and Vimentin expressions, promote E-cadherin expression, and markedly inhibit HK-2 cell proliferation and invasion. AFG inhibited TGF-ß1-induced EMT of renal tubular epithelial cells by regulating phosphorylation of ERK and STAT3.


Assuntos
Panax , Fator de Crescimento Transformador beta1 , Arginina/análogos & derivados , Caderinas/metabolismo , Linhagem Celular , Células Epiteliais , Transição Epitelial-Mesenquimal , Glucose , Panax/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Vimentina/metabolismo
4.
Int J Mol Sci ; 23(15)2022 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-35955653

RESUMO

We previously demonstrated the marked hepatosteatosis and endothelial dysfunction in hepatocyte-specific ERK2 knockout mice (LE2KO) with a high-fat/high-sucrose diet (HFHSD), but detailed metabolic changes and the characteristics in insulin-sensitive organs were not tested. This study aimed to characterize metabolic remodeling with changes in insulin-sensitive organs, which could induce endothelial dysfunction in HFHSD-LE2KO. The serum glucose and fatty acid (FA) were modestly higher in HFHSD-LE2KO than HFHSD-Control. FA synthesis genes were up-regulated, which was associated with the decreased phosphorylation of AMPK and ACC, and with the up-regulation of SREBP-1 in the liver from HFHSD-LE2KO. In FA and amino acids fraction analysis, arachidonic acid/eicosapentaenoic acid ratio, L-ornithine/arginine ratio, asymmetric dimethylarginine and homocysteine levels were elevated in HFHSD-LE2KO. Insulin-induced phosphorylation of AKT was blunted in skeletal muscle. Serum leptin and IL-1ß were elevated, and serum adiponectin was decreased with the enlargement of epididymal adipocytes. Finally, the enhanced superoxide levels in the aorta, which were blunted with CCCP, apocynin, and tempol, were observed in HFHSD-LE2KO. A pre-incubation of aortic rings with tempol improved endothelial dysfunction in HFHSD-LE2KO. HFHSD-LE2KO revealed an acceleration of FA synthesis in the liver leading to insulin resistance in skeletal muscle and the enlargement of visceral adipocytes. Global metabolic remodeling such as changes in arginine metabolism, ω3/ω6 ratio, and adipocytokines, could affect the vascular oxidative stress and endothelial dysfunction in HFHSD-LE2KO.


Assuntos
Dieta Hiperlipídica , Fígado , Animais , Arginina/metabolismo , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo , Fosforilação , Sacarose/metabolismo
5.
BMC Complement Med Ther ; 22(1): 208, 2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35927726

RESUMO

AIM: Acute pancreatitis is a common and potentially serious condition. However, a specific treatment for this condition is still lacking. Genistein, with its anti-oxidant and anti-inflammatory effects, could possibly be used to tackle the underlying pathophysiology of acute pancreatitis. Therefore, the aim of this study was to investigate the effects of genistein on oxidative stress, inflammation, and apoptosis in acute pancreatitis induced by L-arginine in mice. METHODS: Twenty-four male ICR mice were equally divided into 4 groups: Control (Con); Acute pancreatitis (AP) group: Two doses of i.p. 350 mg/100 g body weight (BW) of L-arginine were administered 1 h apart; AP and low-dose genistein (LG) group: mice were given i.p. injection of 10 mg/kg genistein 2 h prior to L-arginine injection followed by once-daily dosing for 3 days; and AP and high-dose genistein (HG) group: mice were given 100 mg/kg genistein with the similar protocol as the LG group. Pancreatic tissue was evaluated for histopathological changes and acinar cell apoptosis, malondialdehyde (MDA) levels, immunohistochemical staining for myeloperoxidase (MPO), nuclear factor-kappa beta (NF-kB), and 4-hydroxynonenal (4-HNE). Serum levels of amylase (AMY), c-reactive protein (CRP), and interleukin (IL)-6 were measured. RESULTS: Significant increases in the degree of acinar cell apoptosis, pancreatic MDA, serum IL-6 and amylase, MPO, NF-kB and 4-HNE positivity were observed in the AP group. All these parameters declined after low- and high-dose genistein treatment. Severe pancreatic inflammation, edema, and acinar cell necrosis were observed in the AP group. Significant improvement of histopathological changes was seen in both low- and high-dose genistein groups. There were no significant differences in any parameters between low and high doses of genistein. CONCLUSION: Genistein could attenuate the severity of histopathological changes in acute pancreatitis through its anti-oxidant, anti-inflammatory, and anti-apoptotic properties.


Assuntos
Pancreatite , Doença Aguda , Amilases/metabolismo , Amilases/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/metabolismo , Apoptose , Arginina/metabolismo , Arginina/farmacologia , Arginina/uso terapêutico , Genisteína/farmacologia , Inflamação/tratamento farmacológico , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Estresse Oxidativo , Pancreatite/induzido quimicamente , Pancreatite/tratamento farmacológico , Pancreatite/patologia
6.
Bioconjug Chem ; 33(7): 1363-1376, 2022 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-35793523

RESUMO

Many low-molecular weight targeted radiotherapeutics (TRTs) are capable of rapidly achieving exceptional tumor to non-target ratios shortly after administration. However, the low tumor residence time of many TRTs limits therapeutic dose delivery and has become the Achilles heel to their clinical translation. To combat the tumor efflux of these otherwise promising agents, we have previously presented a strategy of equipping low-molecular weight TRTs with irreversible cysteine cathepsin inhibitors (e.g., E-64 analogues). These inhibitors are capable of forming irreversible adducts with cysteine proteases within the endolysosomal compartments of cells. Using these endolysosomal trapping agents (ETs), the receptor-targeted constructs are able to increase tumor retention and, thus, deliverable therapeutic doses. In this study, we examine this approach in the development of agents targeting the neurotensin receptor subtype 1 (NTSR1), a receptor overexpressed in numerous cancers. Using an antagonistic NTSR1-targeting vector, we explore the impact of charge modification of the ETs on the in vitro and in vivo biological performance of the constructs using HT-29 colon cancer models. Four ETs (based on the epoxysuccinyl peptide E-64) with various charge states were synthesized and incorporated into the structures of the NTSR1-targeted antagonist. These four 177Lu-labeled, ET-enhanced, NTSR1-targeted agents (177Lu-NA-ET1-4), along with the structurally analogous 177Lu-3BP-227, currently in clinical trials, underwent a battery of in vitro assays using HT-29 xenograft colon cancer cells to examine their NTSR1 binding, internalization and efflux, inhibition, and adduct formation properties. The biodistribution profile of these constructs was studied in an HT-29 mouse model. Charge modification of the terminal carboxylic acid and arginine of the ETs had deleterious effects on inhibition kinetics and in vitro adduct formation. Contrastingly, deletion of the arginine resulted in a modest increase in inhibition kinetics. Incorporation of ETs into the NTSR1-targeted agents was well-tolerated with minimal impact on the in vivo NTSR1 targeting but resulted in increased renal uptake. This study demonstrates that the ETs can be successfully incorporated into antagonistic NTSR1-targeted constructs without compromising their adduct formation capabilities. Based on these results, further exploration of the endolysosomal trapping approach is warranted in NTSR1- and other receptor-targeted antagonistic constructs.


Assuntos
Antineoplásicos , Neoplasias do Colo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Arginina/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Células HT29 , Humanos , Camundongos , Receptores de Neurotensina/metabolismo , Distribuição Tecidual
7.
Food Chem ; 395: 133582, 2022 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-35779509

RESUMO

This study aimed to investigate the individual effects of rosemary extract and green tea polyphenols on the stability of the soybean oil-myosin emulsions with l-arginine or l-lysine. The results showed that l-arginine or l-lysine increased the physical stability of emulsion in all cases. In the presence of metallic cations, rosemary extract increased the physical stability, while green tea polyphenols decreased the physical stability. l-Arginine or l-lysine retarded the lipid and protein oxidation of emulsion in the absence of metallic cations during storage, but accelerated it in the presence of metallic cations. The two antioxidants delayed l-arginine- or l-lysine-induced lipid and protein oxidation in the presence of metallic cations. The results provide a new method for improving the physical and chemical stability of emulsion sausages in which l-arginine or l-lysine is applied to improve the quality attributes of emulsion sausage.


Assuntos
Antioxidantes , Rosmarinus , Antioxidantes/química , Arginina , Emulsões/química , Lisina , Miosinas , Extratos Vegetais/química , Polifenóis/química , Rosmarinus/química , Óleo de Soja/química , Chá/química
8.
Int J Biol Sci ; 18(11): 4341-4356, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35864952

RESUMO

Background and aims: Vasoactive intestinal polypeptide type-I receptor (VIPR1) overexpression has been reported in numerous types of malignancies and utilized to develop novel target therapeutics and radiolabeled VIP analogue-based tumor imaging technology, but its role in liver carcinogenesis has not been explored. In the current study, we investigated the role of the VIP/VIPR1 signaling in controlling hepatocellular carcinoma (HCC) progression. Approach and results: By analyzing clinical samples, we found the expression level of VIPR1 was downregulated in human HCC tissues, which was correlated with advanced clinical stages, tumor growth, recurrence, and poor outcomes of HCC clinically. In vitro and in vivo studies revealed that activation of VIPR1 by VIP markedly inhibited HCC growth and metastasis. Intriguingly, transcriptome sequencing analyses revealed that activation of VIPR1 by VIP regulated arginine biosynthesis. Mechanistical studies in cultured HCC cells demonstrated that VIP treatment partially restored the expression of arginine anabolic key enzyme argininosuccinate synthase (ASS1), and to some extent, inhibited de novo pyrimidine synthetic pathway by downregulating the activation of CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase). VIP treatment upregulated ASS1 and subsequently suppressed CAD phosphorylation in an mTOR/p70S6K signaling dependent manner. Clinically, we found human HCC samples were associated with downregulation of ASS1 but upregulation of CAD phosphorylation, and that VIPR1 levels positively correlated with ASS1 levels and serum levels of urea, the end product of the urea cycle and arginine metabolism in HCC. Conclusions: Loss of VIPR1 expression in HCC facilitates CAD phosphorylation and tumor progression, and restoration of VIPR1 and treatment with the VIPR1 agonist may be a promising approach for HCC treatment.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Arginina/uso terapêutico , Argininossuccinato Sintase/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/metabolismo , Pirimidinas/uso terapêutico , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo , Ureia/uso terapêutico
9.
Bioorg Med Chem ; 70: 116921, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35863237

RESUMO

>90% of genes in the human body undergo alternative splicing (AS) after transcription, which enriches protein species and regulates protein levels. However, there is growing evidence that various genetic isoforms resulting from dysregulated alternative splicing are prevalent in various types of cancers. Dysregulated alternative splicing leads to cancer generation and maintenance of cancer properties such as proliferation differentiation, apoptosis inhibition, invasion metastasis, and angiogenesis. Serine/arginine-rich proteins and SR protein-associated kinases mediate splice site recognition and splice complex assembly during variable splicing. Based on the impact of dysregulated alternative splicing on disease onset and progression, the search for small molecule inhibitors targeting alternative splicing is imminent. In this review, we discuss the structure and specific biological functions of SR proteins and describe the regulation of SR protein function by SR protein related kinases meticulously, which are closely related to the occurrence and development of various types of cancers. On this basis, we summarize the reported small molecule inhibitors targeting SR proteins and SR protein related kinases from the perspective of medicinal chemistry. We mainly categorize small molecule inhibitors from four aspects, including targeting SR proteins, targeting Serine/arginine-rich protein-specific kinases (SRPKs), targeting Cdc2-like kinases (CLKs) and targeting dual-specificity tyrosine-regulated kinases (DYRKs), in terms of structure, inhibition target, specific mechanism of action, biological activity, and applicable diseases. With this review, we are expected to provide a timely summary of recent advances in alternative splicing regulated by kinases and a preliminary introduction to relevant small molecule inhibitors.


Assuntos
Processamento Alternativo , Serina , Arginina/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Serina/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo
10.
Oxid Med Cell Longev ; 2022: 5339361, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35847590

RESUMO

Background: Asymmetric dimethylarginine (ADMA) is a nonselective nitric oxide synthase inhibitor. ADMA is thought to inhibit the production of nitric oxide (NO) by neurons after oxygen-glucose deprivation (OGD). The gap junction protein Connexin-36 (cx-36) is involved in the pathophysiology of stroke. We investigated whether ADMA could protect neurons from OGD insults by regulating the expression of cx-36. Methods: Cultured rat cortical neuronal cells were used. Neurons were treated with OGD with or without ADMA pretreatment. The lactate dehydrogenase (LDH) release rate was used to assess neuronal injury. Intracellular NO levels were determined using 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. Western blotting was performed to detect cx-36 expression. Results: The LDH release rate increased in the supernatant of neurons after the OGD insult, whereas ADMA treatment reduced the LDH release rate. Intracellular NO levels increased following OGD treatment, and this increase was not inhibited by ADMA treatment. Expression of cx-36 was upregulated in neurons under OGD conditions, and treatment with ADMA downregulated the expression of cx-36. Conclusions: ADMA protects neurons from OGD insult, and cx-36 downregulation may be a possible pathway involved in ADMA-mediated neuronal protection.


Assuntos
Glucose , Oxigênio , Animais , Arginina/análogos & derivados , Células Cultivadas , Conexinas/metabolismo , Glucose/metabolismo , L-Lactato Desidrogenase/metabolismo , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Ratos
11.
Sensors (Basel) ; 22(14)2022 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-35890916

RESUMO

One of the hallmarks of diabetes is an increased modification of cellular proteins. The most prominent type of modification stems from the reaction of methylglyoxal with arginine and lysine residues, leading to structural and functional impairments of target proteins. For lysine glycation, several algorithms allow a prediction of occurrence; thus, making it possible to pinpoint likely targets. However, according to our knowledge, no approaches have been published for predicting the likelihood of arginine glycation. There are indications that arginine and not lysine is the most prominent target for the toxic dialdehyde. One of the reasons why there is no arginine glycation predictor is the limited availability of quantitative data. Here, we used a recently published high-quality dataset of arginine modification probabilities to employ an artificial neural network strategy. Despite the limited data availability, our results achieve an accuracy of about 75% of correctly predicting the exact value of the glycation probability of an arginine-containing peptide without setting thresholds upon whether it is decided if a given arginine is modified or not. This contribution suggests a solution for predicting arginine glycation of short peptides.


Assuntos
Arginina , Produtos Finais de Glicação Avançada , Produtos Finais de Glicação Avançada/química , Lisina/química , Redes Neurais de Computação , Peptídeos/química , Proteínas , Aldeído Pirúvico/química , Aldeído Pirúvico/metabolismo
12.
Anal Chem ; 94(28): 10271-10277, 2022 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-35804490

RESUMO

Arginine is considered as a biomarker of cystinuria and other diseases, and thus, it is of urgency to develop a simple and rapid method with high sensitivity and selectivity for arginine detection to meet the demand of on-site analysis and bedside diagnosis. In this work, a lanthanide metal-organic framework, La(TATB), was prepared using a triazine-based planar ligand, 4,4',4″-s-triazine-2,4,6-triyltribenzoate (H3TATB), and lanthanide ion (La3+). La(TATB) can be used as a highly photosensitive agent to activate molecular oxygen to 1O2 to achieve efficient photosensitive oxidation of arginine accompanied by strong blue fluorescence emission under 302 nm UV irradiation. Due to the porous structure and high specific surface area of La(TATB), short-life 1O2 can effectively approach and react with amino acid substrate molecules, thus leading to higher sensitivity than other systems. Therefore, the "turn-on" fluorescence sensing of trace arginine can be realized, with a measured linear response range of 10-20,000 nM and a limit of detection as low as 7 nM. This method can be used for the detection of trace arginine in urine, which is conducive to the bedside diagnosis and rapid screening of cystinuria and other diseases. The proposed method not only expands the application scope of Ln-MOFs but also provides a new construction strategy for "turn-on" luminescence sensors.


Assuntos
Cistinúria , Elementos da Série dos Lantanídeos , Estruturas Metalorgânicas , Arginina , Humanos , Elementos da Série dos Lantanídeos/química , Luminescência , Estruturas Metalorgânicas/química , Fármacos Fotossensibilizantes , Triazinas
13.
Int J Mol Sci ; 23(14)2022 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-35887012

RESUMO

Membrane-less organelles (MLOs) are formed by biomolecular liquid-liquid phase separation (LLPS). Proteins with charged low-complexity domains (LCDs) are prone to phase separation and localize to MLOs, but the mechanism underlying the distributions of such proteins to specific MLOs remains poorly understood. Recently, proteins with Arg-enriched mixed-charge domains (R-MCDs), primarily composed of R and Asp (D), were found to accumulate in nuclear speckles via LLPS. However, the process by which R-MCDs selectively incorporate into nuclear speckles is unknown. Here, we demonstrate that the patterning of charged amino acids and net charge determines the targeting of specific MLOs, including nuclear speckles and the nucleolus, by proteins. The redistribution of R and D residues from an alternately sequenced pattern to uneven blocky sequences caused a shift in R-MCD distribution from nuclear speckles to the nucleolus. In addition, the incorporation of basic residues in the R-MCDs promoted their localization to the MLOs and their apparent accumulation in the nucleolus. The R-MCD peptide with alternating amino acids did not undergo LLPS, whereas the blocky R-MCD peptide underwent LLPS with affinity to RNA, acidic poly-Glu, and the acidic nucleolar protein nucleophosmin, suggesting that the clustering of R residues helps avoid their neutralization by D residues and eventually induces R-MCD migration to the nucleolus. Therefore, the distribution of proteins to nuclear speckles requires the proximal positioning of D and R for the mutual neutralization of their charges.


Assuntos
Arginina , Nucléolo Celular , Arginina/metabolismo , Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Organelas/metabolismo , RNA/metabolismo
14.
Nat Struct Mol Biol ; 29(7): 628-638, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35835870

RESUMO

Glycogen synthase (GYS1) is the central enzyme in muscle glycogen biosynthesis. GYS1 activity is inhibited by phosphorylation of its amino (N) and carboxyl (C) termini, which is relieved by allosteric activation of glucose-6-phosphate (Glc6P). We present cryo-EM structures at 3.0-4.0 Å resolution of phosphorylated human GYS1, in complex with a minimal interacting region of glycogenin, in the inhibited, activated and catalytically competent states. Phosphorylations of specific terminal residues are sensed by different arginine clusters, locking the GYS1 tetramer in an inhibited state via intersubunit interactions. The Glc6P activator promotes conformational change by disrupting these interactions and increases the flexibility of GYS1, such that it is poised to adopt a catalytically competent state when the sugar donor UDP-glucose (UDP-glc) binds. We also identify an inhibited-like conformation that has not transitioned into the activated state, in which the locking interaction of phosphorylation with the arginine cluster impedes subsequent conformational changes due to Glc6P binding. Our results address longstanding questions regarding the mechanism of human GYS1 regulation.


Assuntos
Glucose-6-Fosfato , Glicogênio Sintase , Arginina/metabolismo , Glucose-6-Fosfato/metabolismo , Glicogênio Sintase/química , Glicogênio Sintase/metabolismo , Humanos , Fosforilação , Difosfato de Uridina/metabolismo
15.
J Clin Invest ; 132(14)2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35838048

RESUMO

The metabolic dependencies of cancer cells have substantial potential to be exploited to improve the diagnosis and treatment of cancer. Creatine riboside (CR) is identified as a urinary metabolite associated with risk and prognosis in lung and liver cancer. However, the source of high CR levels in patients with cancer as well as their implications for the treatment of these aggressive cancers remain unclear. By integrating multiomics data on lung and liver cancer, we have shown that CR is a cancer cell-derived metabolite. Global metabolomics and gene expression analysis of human tumors and matched liquid biopsies, together with functional studies, revealed that dysregulation of the mitochondrial urea cycle and a nucleotide imbalance were associated with high CR levels and indicators of a poor prognosis. This metabolic phenotype was associated with reduced immune infiltration and supported rapid cancer cell proliferation that drove aggressive tumor growth. CRhi cancer cells were auxotrophic for arginine, revealing a metabolic vulnerability that may be exploited therapeutically. This highlights the potential of CR not only as a poor-prognosis biomarker but also as a companion biomarker to inform the administration of arginine-targeted therapies in precision medicine strategies to improve survival for patients with cancer.


Assuntos
Neoplasias Hepáticas , Ribonucleosídeos , Arginina/metabolismo , Creatina/análogos & derivados , Creatina/urina , Humanos , Ribonucleosídeos/urina
16.
Proc Natl Acad Sci U S A ; 119(31): e2209597119, 2022 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-35878037

RESUMO

N-degron pathways are proteolytic systems that target proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Nt-Arg of a protein is among Nt-residues that can be recognized as destabilizing ones by the Arg/N-degron pathway. A proteolytic cleavage of a protein can generate Arg at the N terminus of a resulting C-terminal (Ct) fragment either directly or after Nt-arginylation of that Ct-fragment by the Ate1 arginyl-tRNA-protein transferase (R-transferase), which uses Arg-tRNAArg as a cosubstrate. Ate1 can Nt-arginylate Nt-Asp, Nt-Glu, and oxidized Nt-Cys* (Cys-sulfinate or Cys-sulfonate) of proteins or short peptides. Ate1 genes of fungi, animals, and plants have been cloned decades ago, but a three-dimensional structure of Ate1 remained unknown. A detailed mechanism of arginylation is unknown as well. We describe here the crystal structure of the Ate1 R-transferase from the budding yeast Kluyveromyces lactis. The 58-kDa R-transferase comprises two domains that recognize, together, an acidic Nt-residue of an acceptor substrate, the Arg residue of Arg-tRNAArg, and a 3'-proximal segment of the tRNAArg moiety. The enzyme's active site is located, at least in part, between the two domains. In vitro and in vivo arginylation assays with site-directed Ate1 mutants that were suggested by structural results yielded inferences about specific binding sites of Ate1. We also analyzed the inhibition of Nt-arginylation activity of Ate1 by hemin (Fe3+-heme), and found that hemin induced the previously undescribed disulfide-mediated oligomerization of Ate1. Together, these results advance the understanding of R-transferase and the Arg/N-degron pathway.


Assuntos
Aminoaciltransferases , Hemina , Aminoaciltransferases/metabolismo , Animais , Arginina/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Proteólise , RNA de Transferência de Arginina/metabolismo
17.
Arch Razi Inst ; 77(1): 241-247, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35891771

RESUMO

Cancer is one of the most serious diseases facing humanity; accordingly, it is urgent to find a cure that is rarely harmful to the patient as much as possible. It has been approved that arginine deiminase (ADI) can hydrolyze the plasma arginine to citrulline. This hydrolysis activity and reduction in the amount of intercellular arginine suppress lipopolysaccharide-induced nitric oxide synthesis. On the other hand, arginine depletion arrests the cell cycle at the G1 phase; therefore, ADI has been considered a powerful anticancer agent. The current study aimed to investigate the lethal effects of ADI purified from the Lactobacillus plantarum p5 strain on murine mammary adenocarcinoma and Vero cell lines. Anti-proliferative activity of ADI against murine mammary adenocarcinoma) AMN3) cell line was evaluated after different incubation times (3, 6, 24, 48, and 72 h) of exposure to 1 µg/mL of ADI, compared to Vero (non-cancer cell line) transformed cell line with same conditions. The autophagy process in cancer cells was recognized after three hours of incubation with ADI which was clearly observed in the AMN3 cell line under an inverted microscope. The first stages of the programmed cell death (apoptosis) pathway were only observed in AMN3 cells after 24 h of incubation with ADI, and this process continued with the time until they reached the last stages of apoptosis after 72 h of incubation. The results of the current study showed that the AMN3 cell line was auxotrophic for arginine because it could not produce it in the presence of enzyme which had a robust activity to kill these cancer cells; however, Vero non-cancer cell line survived in the presence of ADI because it had the ability to produce arginine.


Assuntos
Adenocarcinoma , Neoplasias da Mama , Hidrolases , Lactobacillus plantarum , Adenocarcinoma/tratamento farmacológico , Animais , Arginina , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Chlorocebus aethiops , Hidrolases/farmacologia , Lactobacillus plantarum/enzimologia , Camundongos , Células Vero
18.
Oncogene ; 41(32): 3912-3924, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35798877

RESUMO

Metastasis accounts for the major cause of cancer-related mortality. How disseminated tumor cells survive under suspension conditions and avoid anoikis is largely unknown. Here, using a metabolic enzyme-centered CRISPR-Cas9 genetic screen, we identified methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1 (MTHFD1) as a novel suppressor of anoikis. MTHFD1 depletion obviously restrained the capacity of cellular antioxidant defense and inhibited tumor distant metastasis. Mechanistically, MTHFD1 was found to bind the protein arginine methyltransferase 5 (PRMT5) and then undergo symmetric dimethylation on R173 by PRMT5. Under suspension conditions, the interaction between MTHFD1 and PRMT5 was strengthened, which increased the symmetric dimethylation of MTHFD1. The elevated methylation of MTHFD1 largely augmented its metabolic activity to generate NADPH, therefore leading to anoikis resistance and distant organ metastasis. Therapeutically, genetic depletion or pharmacological inhibition of PRMT5 declined tumor distant metastasis. And R173 symmetric dimethylation status was associated with metastasis and prognosis of ESCC patients. In conclusion, our study uncovered a novel regulatory role and therapeutic implications of PRMT5/MTHFD1 axis in facilitating anoikis resistance and cancer metastasis.


Assuntos
Formiato-Tetra-Hidrofolato Ligase , Neoplasias , Anoikis/genética , Arginina/genética , Arginina/metabolismo , Formiato-Tetra-Hidrofolato Ligase/metabolismo , Humanos , Metilação , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Neoplasias/genética , Proteína-Arginina N-Metiltransferases/metabolismo
19.
Cell Metab ; 34(8): 1151-1167.e7, 2022 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-35839757

RESUMO

Hepatocellular carcinoma (HCC) is a typically fatal malignancy exhibiting genetic heterogeneity and limited therapy responses. We demonstrate here that HCCs consistently repress urea cycle gene expression and thereby become auxotrophic for exogenous arginine. Surprisingly, arginine import is uniquely dependent on the cationic amino acid transporter SLC7A1, whose inhibition slows HCC cell growth in vitro and in vivo. Moreover, arginine deprivation engages an integrated stress response that promotes HCC cell-cycle arrest and quiescence, dependent on the general control nonderepressible 2 (GCN2) kinase. Inhibiting GCN2 in arginine-deprived HCC cells promotes a senescent phenotype instead, rendering these cells vulnerable to senolytic compounds. Preclinical models confirm that combined dietary arginine deprivation, GCN2 inhibition, and senotherapy promote HCC cell apoptosis and tumor regression. These data suggest novel strategies to treat human liver cancers through targeting SLC7A1 and/or a combination of arginine restriction, inhibition of GCN2, and senolytic agents.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Arginina/metabolismo , Arginina/farmacologia , Carcinoma Hepatocelular/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Proteínas Serina-Treonina Quinases , Senoterapia
20.
Nutrients ; 14(13)2022 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-35807771

RESUMO

Acute pancreatitis (AP) is one of the most common causes of hospitalization for gastrointestinal diseases, with high morbidity and mortality. Endoplasmic reticulum stress (ERS) and Gasdermin D (GSDMD) mediate AP, but little is known about their mutual influence on AP. Diosgenin has excellent anti-inflammatory and antioxidant effects. This study investigated whether Diosgenin derivative D (Drug D) inhibits L-arginine-induced acute pancreatitis through meditating GSDMD in the endoplasmic reticulum (ER). Our studies were conducted in a mouse model of L-arginine-induced AP as well as in an in vitro model on mouse pancreatic acinar cells. The GSDMD accumulation in ER was found in this study, which caused ERS of acinar cells. GSDMD inhibitor Disulfiram (DSF) notably decreased the expression of GSDMD in ER and TXNIP/HIF-1α signaling. The molecular docking study indicated that there was a potential interaction between Drug D and GSDMD. Our results showed that Drug D significantly inhibited necrosis of acinar cells dose-dependently, and we also found that Drug D alleviated pancreatic necrosis and systemic inflammation by inhibiting the GSDMD accumulation in the ER of acinar cells via the TXNIP/HIF-1α pathway. Furthermore, the level of p-IRE1α (a marker of ERS) was also down-regulated by Drug D in a dose-dependent manner in AP. We also found that Drug D alleviated TXNIP up-regulation and oxidative stress in AP. Moreover, our results revealed that GSDMD-/- mitigated AP by inhibiting TXNIP/HIF-1α. Therefore, Drug D, which is extracted from Dioscorea zingiberensis, may inhibit L-arginine-induced AP by meditating GSDMD in the ER by the TXNIP /HIF-1α pathway.


Assuntos
Diosgenina , Pancreatite , Doença Aguda , Animais , Apoptose , Arginina/farmacologia , Proteínas de Transporte , Diosgenina/efeitos adversos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Pancreatite/induzido quimicamente , Pancreatite/tratamento farmacológico , Pancreatite/metabolismo , Proteínas Serina-Treonina Quinases , Tiorredoxinas/metabolismo
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