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1.
Cell Mol Life Sci ; 81(1): 216, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38740643

RESUMO

p50RhoGAP is a key protein that interacts with and downregulates the small GTPase RhoA. p50RhoGAP is a multifunctional protein containing the BNIP-2 and Cdc42GAP Homology (BCH) domain that facilitates protein-protein interactions and lipid binding and the GAP domain that regulates active RhoA population. We recently solved the structure of the BCH domain from yeast p50RhoGAP (YBCH) and showed that it maintains the adjacent GAP domain in an auto-inhibited state through the ß5 strand. Our previous WT YBCH structure shows that a unique kink at position 116 thought to be made by a proline residue between alpha helices α6 and α7 is essential for the formation of intertwined dimer from asymmetric monomers. Here we sought to establish the role and impact of this Pro116. However, the kink persists in the structure of P116A mutant YBCH domain, suggesting that the scaffold is not dictated by the proline residue at this position. We further identified Tyr124 (or Tyr188 in HBCH) as a conserved residue in the crucial ß5 strand. Extending to the human ortholog, when substituted to acidic residues, Tyr188D or Tyr188E, we observed an increase in RhoA binding and self-dimerization, indicative of a loss of inhibition of the GAP domain by the BCH domain. These results point to distinct roles and impact of the non-conserved and conserved amino acid positions in regulating the structural and functional complexity of the BCH domain.


Assuntos
Prolina , Prolina/metabolismo , Prolina/química , Prolina/genética , Tirosina/metabolismo , Tirosina/química , Tirosina/genética , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/química , Modelos Moleculares , Sequência Conservada , Humanos , Ligação Proteica
2.
Life Sci ; 348: 122700, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38724004

RESUMO

AIMS: To elucidate the impact of 10-(6-plastoquinonyl) decyltriphenylphosphonium (SkQ1) as an anti-colitogenic agent for maintenance of colon epithelial tract in ulcerated mice through recovery of mitochondrial dysfunction and mitochondrial stress by virtue of its free radical scavenging properties. MAIN METHODS: DSS induced ulcerated BALB/c mice were treated with SkQ1 for 14 days @ 30 nmol/kg/body wt./day/mice. Post-treatment, isolated colonic mitochondria were utilized for spectrophotometric and spectrofluorometric biochemical analysis of various mitochondrial functional variables including individual mitochondrial respiratory enzyme complexes. Confocal microscopy was utilized for measuring mitochondrial membrane potential in vivo. ELISA technique was adapted for measuring colonic nitrite and 3-nitrotyrosine (3-NT) content. Finally in vitro cell line study was carried out to substantiate in vivo findings and elucidate the involvement of free radicals in UC using antioxidant/free radical scavenging regimen. KEY FINDINGS: Treatment with SkQ1 in vivo reduced histopathological severity of colitis, induced recovery of mitochondrial respiratory complex activities and associated functional variables, improved oxidative stress indices and normalized mitochondrial cardiolipin content. Importantly, SkQ1 lowered nitrite concentration and 3-nitrotyrosine formation in vivo. In vitro SkQ1 restored mitochondrial functions wherein the efficacy of SkQ1 proved equal or better compared to SOD and DMSO indicating predominant involvement of O2- and OH in UC. However, NO and ONOO- also seemed to play a secondary role as MEG and L-NAME provided lesser protection as compared to SOD and DMSO. SIGNIFICANCE: SkQ1 can be considered as a potent anti-colitogenic agent by virtue of its free radical scavenging properties in treating UC.


Assuntos
Colite Ulcerativa , Colo , Camundongos Endogâmicos BALB C , Mitocôndrias , Estresse Oxidativo , Plastoquinona , Animais , Camundongos , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Plastoquinona/análogos & derivados , Plastoquinona/farmacologia , Colo/efeitos dos fármacos , Colo/patologia , Colo/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Tirosina/análogos & derivados , Tirosina/metabolismo , Tirosina/farmacologia , Antioxidantes/farmacologia , Sequestradores de Radicais Livres/farmacologia , Sulfato de Dextrana
3.
J Zhejiang Univ Sci B ; 25(5): 410-421, 2024 Mar 12.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-38725340

RESUMO

Pheochromocytomas and paragangliomas (PPGLs) cause symptoms by altering the circulation levels of catecholamines and peptide hormones. Currently, the diagnosis of PPGLs relies on diagnostic imaging and the detection of catecholamines. In this study, we used ultra-performance liquid chromatography (UPLC)/quadrupole time-of-flight mass spectrometry (Q-TOF MS) analysis to identify and measure the perioperative differential metabolites in the plasma of adrenal pheochromocytoma patients. We identified differentially expressed genes by comparing the transcriptomic data of pheochromocytoma with the normal adrenal medulla. Through conducting two steps of metabolomics analysis, we identified 111 differential metabolites between the healthy group and the patient group, among which 53 metabolites were validated. By integrating the information of differential metabolites and differentially expressed genes, we inferred that the cysteine-methionine, pyrimidine, and tyrosine metabolism pathways were the three main metabolic pathways altered by the neoplasm. The analysis of transcription levels revealed that the tyrosine and cysteine-methionine metabolism pathways were downregulated in pheochromocytoma, whereas the pyrimidine pathway showed no significant difference. Finally, we developed an optimized diagnostic model of two metabolites, L-dihydroorotic acid and vanylglycol. Our results for these metabolites suggest that they may serve as potential clinical biomarkers and can be used to supplement and improve the diagnosis of pheochromocytoma.


Assuntos
Neoplasias das Glândulas Suprarrenais , Cisteína , Metionina , Feocromocitoma , Pirimidinas , Tirosina , Feocromocitoma/metabolismo , Feocromocitoma/sangue , Humanos , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/sangue , Pirimidinas/metabolismo , Metionina/metabolismo , Tirosina/metabolismo , Tirosina/sangue , Cisteína/metabolismo , Masculino , Metabolômica/métodos , Feminino , Pessoa de Meia-Idade , Adulto , Redes e Vias Metabólicas
4.
Nat Commun ; 15(1): 3975, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38729930

RESUMO

Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial LPMO, member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a tyrosine-histidine pair in the protein's surface. Real-time monitoring of radical formation reveals a clear correlation between the efficiency of hole transference and enzyme performance under oxidative stress. Residues involved in this pathway vary considerably between natural LPMOs, which could reflect adaptation to different ecological niches. Importantly, we show that enzyme activity is increased in a variant with slower radical transference, providing experimental evidence for a previously postulated trade-off between activity and redox robustness.


Assuntos
Proteínas de Bactérias , Oxigenases de Função Mista , Oxirredução , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Domínio Catalítico , Triptofano/metabolismo , Polissacarídeos/metabolismo , Mutação , Estresse Oxidativo , Tirosina/metabolismo , Modelos Moleculares , Histidina/metabolismo , Histidina/genética
5.
Biomolecules ; 14(5)2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38785983

RESUMO

BACKGROUND: Peroxynitrite (ONOO-) is an oxidant linked with several human pathologies. Apigenin, a natural flavonoid known for its health benefits, remains unexplored in relation to ONOO- effects. This study investigated the potential of apigenin to structurally protect fibrinogen, an essential blood clotting factor, from ONOO--induced damage. METHODS: Multi-approach analyses were carried out where fibrinogen was exposed to ONOO- generation while testing the efficacy of apigenin. The role of apigenin against ONOO--induced modifications in fibrinogen was investigated using UV spectroscopy, tryptophan or tyrosine fluorescence, protein hydrophobicity, carbonylation, and electrophoretic analyses. RESULTS: The findings demonstrate that apigenin significantly inhibits ONOO--induced oxidative damage in fibrinogen. ONOO- caused reduced UV absorption, which was reversed by apigenin treatment. Moreover, ONOO- diminished tryptophan and tyrosine fluorescence, which was effectively restored by apigenin treatment. Apigenin also reduced the hydrophobicity of ONOO--damaged fibrinogen. Moreover, apigenin exhibited protective effects against ONOO--induced protein carbonylation. SDS-PAGE analyses revealed that ONOO-treatment eliminated bands corresponding to fibrinogen polypeptide chains Aα and γ, while apigenin preserved these changes. CONCLUSIONS: This study highlights, for the first time, the role of apigenin in structural protection of human fibrinogen against peroxynitrite-induced nitrosative damage. Our data indicate that apigenin offers structural protection to all three polypeptide chains (Aα, Bß, and γ) of human fibrinogen. Specifically, apigenin prevents the dislocation or breakdown of the amino acids tryptophan, tyrosine, lysine, arginine, proline, and threonine and also prevents the exposure of hydrophobic sites in fibrinogen induced by ONOO-.


Assuntos
Apigenina , Fibrinogênio , Estresse Nitrosativo , Ácido Peroxinitroso , Fibrinogênio/metabolismo , Fibrinogênio/química , Apigenina/farmacologia , Apigenina/química , Humanos , Ácido Peroxinitroso/química , Estresse Nitrosativo/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Carbonilação Proteica/efeitos dos fármacos , Tirosina/química , Tirosina/metabolismo , Estresse Oxidativo/efeitos dos fármacos
6.
Elife ; 132024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38780416

RESUMO

Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above-which were used to identify endogenous PANX1 phosphorylation at these two sites-are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.


Assuntos
Conexinas , Proteínas do Tecido Nervoso , Quinases da Família src , Fosforilação , Conexinas/metabolismo , Conexinas/genética , Humanos , Quinases da Família src/metabolismo , Quinases da Família src/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Tirosina/metabolismo , Animais , Células HEK293 , Camundongos
7.
Mol Med Rep ; 30(1)2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38757335

RESUMO

Thrombin, which plays a crucial role in hemostasis, is also implicated in cancer progression. In the present study, the effects of the thrombin­targeting recombinant tyrosine­sulfated madanin­1 on cancer cell behavior and signaling pathways compared with madanin­1 wild­type (WT) were investigated. Recombinant madanin­1 2 sulfation (madanin­1 2S) and madanin­1 WT proteins were generated using Escherichia coli. SKOV3 and MDA­MB­231 cells were treated with purified recombinant proteins with or without thrombin stimulation. Migration and invasion of cells were analyzed by wound healing assay and Transwell assay, respectively. Thrombin markedly increased cell migration and invasion in both SKOV3 and MDA­MB­231 cells, which were significantly suppressed by madanin­1 2S (P<0.05). Madanin­1 2S also significantly suppressed thrombin­induced expression of phosphorylated (p)­Akt and p­extracellular signal­regulated kinase in both cell lines (P<0.05), whereas madanin­1 WT had no effect on the expression levels of these proteins in MDA­MB­231 cells. Furthermore, madanin­1 2S significantly reversed the effects of thrombin on E­cadherin, N­cadherin and vimentin expression in MDA­MB­231 cells (P<0.05), whereas madanin­1 WT did not show any effect. In conclusion, madanin­1 2S suppressed the migration and invasion of cancer cells more effectively than madanin­1 WT. It is hypothesized that inhibiting thrombin via the sulfated form of madanin­1 may be a potential candidate for enhanced cancer therapy; however, further in vivo validation is required.


Assuntos
Movimento Celular , Proteínas Recombinantes , Trombina , Humanos , Movimento Celular/efeitos dos fármacos , Trombina/farmacologia , Linhagem Celular Tumoral , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tirosina/metabolismo , Tirosina/farmacologia , Caderinas/metabolismo , Caderinas/genética
8.
Proc Natl Acad Sci U S A ; 121(21): e2322501121, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38748578

RESUMO

Biological regulation often depends on reversible reactions such as phosphorylation, acylation, methylation, and glycosylation, but rarely halogenation. A notable exception is the iodination and deiodination of thyroid hormones. Here, we report detection of bromotyrosine and its subsequent debromination during Drosophila spermatogenesis. Bromotyrosine is not evident when Drosophila express a native flavin-dependent dehalogenase that is homologous to the enzyme responsible for iodide salvage from iodotyrosine in mammals. Deletion or suppression of the dehalogenase-encoding condet (cdt) gene in Drosophila allows bromotyrosine to accumulate with no detectable chloro- or iodotyrosine. The presence of bromotyrosine in the cdt mutant males disrupts sperm individualization and results in decreased fertility. Transgenic expression of the cdt gene in late-staged germ cells rescues this defect and enhances tolerance of male flies to bromotyrosine. These results are consistent with reversible halogenation affecting Drosophila spermatogenesis in a process that had previously eluded metabolomic, proteomic, and genomic analyses.


Assuntos
Proteínas de Drosophila , Fertilidade , Espermatogênese , Tirosina , Animais , Masculino , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Tirosina/metabolismo , Tirosina/análogos & derivados , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Drosophila/genética , Drosophila/metabolismo , Animais Geneticamente Modificados , Hidrolases/metabolismo , Hidrolases/genética
9.
J Agric Food Chem ; 72(19): 11278-11291, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38708781

RESUMO

Moringa seeds are an excellent dietary source of phytochemicals (i.e., glucosinolates, GSLs; isothiocyanates, ITCs) with health-beneficial effects. Although numerous studies have been conducted on moringa seeds, the effect of germination on the regulation of GSLs remains scarcely explored. The present study investigated the dynamic changes of GSLs in moringa seeds during germination (at 25, 30, and 35 °C for 6 days in the dark) through an untargeted metabolomics approach and compared the antioxidant capacity of ungerminated and germinated moringa seeds. Our results showed that germination significantly increased the total GSL content from 150 (day 0) to 323 µmol/g (35 °C, day 6) on a dry weight (DW) basis, especially glucomoringin (GMG), the unique glucosinolate in moringa seeds, which was significantly upregulated from 61 (day 0) to 149 µmol/g DW (35 °C, day 4). The upregulation of GMG corresponded to the metabolism of tyrosine, which might be the initial precursor for the formation of GMG. In addition, germination enhanced the total ITC content from 85 (day 0) to 239 µmol SE/g DW (35 °C, day 6), indicating that germination may have also increased the activity of myrosinase. Furthermore, germination remarkably increased the total phenolic content (109-507 mg GAE/100 g DW) and antioxidant capacity of moringa seeds. Our findings suggest that moringa sprouts could be promoted as a novel food and/or ingredient rich in GMG.


Assuntos
Germinação , Glucosinolatos , Moringa , Sementes , Tirosina , Sementes/química , Sementes/metabolismo , Sementes/crescimento & desenvolvimento , Tirosina/metabolismo , Tirosina/análise , Moringa/química , Moringa/metabolismo , Moringa/crescimento & desenvolvimento , Glucosinolatos/metabolismo , Glucosinolatos/análise , Glucosinolatos/química , Antioxidantes/metabolismo , Antioxidantes/química , Antioxidantes/análise
10.
Nature ; 629(8014): 1174-1181, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38720073

RESUMO

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.


Assuntos
Fosfotirosina , Proteínas Tirosina Quinases , Especificidade por Substrato , Tirosina , Animais , Humanos , Motivos de Aminoácidos , Evolução Molecular , Espectrometria de Massas , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteoma/química , Proteoma/metabolismo , Proteômica , Transdução de Sinais , Domínios de Homologia de src , Tirosina/metabolismo , Tirosina/química
11.
Nature ; 630(8015): 206-213, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38778111

RESUMO

Targeted radionuclide therapy, in which radiopharmaceuticals deliver potent radionuclides to tumours for localized irradiation, has addressed unmet clinical needs and improved outcomes for patients with cancer1-4. A therapeutic radiopharmaceutical must achieve both sustainable tumour targeting and fast clearance from healthy tissue, which remains a major challenge5,6. A targeted ligation strategy that selectively fixes the radiopharmaceutical to the target protein in the tumour would be an ideal solution. Here we installed a sulfur (VI) fluoride exchange (SuFEx) chemistry-based linker on radiopharmaceuticals to prevent excessively fast tumour clearance. When the engineered radiopharmaceutical binds to the tumour-specific protein, the system undergoes a binding-to-ligation transition and readily conjugates to the tyrosine residues through the 'click' SuFEx reaction. The application of this strategy to a fibroblast activation protein (FAP) inhibitor (FAPI) triggered more than 80% covalent binding to the protein and almost no dissociation for six days. In mice, SuFEx-engineered FAPI showed 257% greater tumour uptake than did the original FAPI, and increased tumour retention by 13-fold. The uptake in healthy tissues was rapidly cleared. In a pilot imaging study, this strategy identified more tumour lesions in patients with cancer than did other methods. SuFEx-engineered FAPI also successfully achieved targeted ß- and α-radionuclide therapy, causing nearly complete tumour regression in mice. Another SuFEx-engineered radioligand that targets prostate-specific membrane antigen (PSMA) also showed enhanced therapeutic efficacy. Considering the broad scope of proteins that can potentially be ligated to SuFEx warheads, it might be possible to adapt this strategy to other cancer targets.


Assuntos
Compostos Radiofarmacêuticos , Animais , Camundongos , Humanos , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/uso terapêutico , Compostos Radiofarmacêuticos/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Feminino , Masculino , Ligantes , Linhagem Celular Tumoral , Proteínas de Membrana/metabolismo , Proteínas de Membrana/química , Neoplasias/radioterapia , Neoplasias/metabolismo , Radioisótopos/uso terapêutico , Fluoretos/química , Fluoretos/metabolismo , Tirosina/metabolismo , Tirosina/química , Antígenos de Superfície , Glutamato Carboxipeptidase II
12.
Mol Pharm ; 21(6): 2795-2812, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38747353

RESUMO

O-([18F]Fluoroethyl)-l-tyrosine ([18F]FET) is actively transported into the brain and cancer cells by LAT1 and possibly other amino acid transporters, which enables brain tumor imaging by positron emission tomography (PET). However, tumor delivery of this probe in the presence of competing amino acids may be limited by a relatively low affinity for LAT1. The aim of the present work was to evaluate the meta-substituted [18F]FET analog m-[18F]FET and the methyl ester [18F]FET-OMe, which were designed to improve tumor delivery by altering the physicochemical, pharmacokinetic, and/or transport properties. Both tracers could be prepared with good radiochemical yields of 41-56% within 66-90 min. Preclinical evaluation with [18F]FET as a reference tracer demonstrated reduced in vitro uptake of [18F]FET-OMe by U87 glioblastoma cells and no advantage for in vivo tumor imaging. In contrast, m-[18F]FET showed significantly improved in vitro uptake and accelerated in vivo tumor accumulation in an orthotopic glioblastoma model. As such, our work identifies m-[18F]FET as a promising alternative to [18F]FET for brain tumor imaging that deserves further evaluation with regard to its transport properties and in vivo biodistribution.


Assuntos
Neoplasias Encefálicas , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Tirosina , Animais , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/metabolismo , Humanos , Camundongos , Tirosina/análogos & derivados , Tirosina/química , Linhagem Celular Tumoral , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/síntese química , Distribuição Tecidual , Radioisótopos de Flúor/química , Glioblastoma/diagnóstico por imagem , Glioblastoma/metabolismo , Camundongos Nus , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo
13.
Int J Mol Sci ; 25(7)2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38612864

RESUMO

Flavonoids exhibit various bioactivities including anti-oxidant, anti-tumor, anti-inflammatory, and anti-viral properties. Methylated flavonoids are particularly significant due to their enhanced oral bioavailability, improved intestinal absorption, and greater stability. The heterologous production of plant flavonoids in bacterial factories involves the need for enough biosynthetic precursors to allow for high production levels. These biosynthetic precursors are malonyl-CoA and l-tyrosine. In this work, to enhance flavonoid biosynthesis in Streptomyces albidoflavus, we conducted a transcriptomics study for the identification of candidate genes involved in l-tyrosine catabolism. The hypothesis was that the bacterial metabolic machinery would detect an excess of this amino acid if supplemented with the conventional culture medium and would activate the genes involved in its catabolism towards energy production. Then, by inactivating those overexpressed genes (under an excess of l-tyrosine), it would be possible to increase the intracellular pools of this precursor amino acid and eventually the final flavonoid titers in this bacterial factory. The RNAseq data analysis in the S. albidoflavus wild-type strain highlighted the hppD gene encoding 4-hydroxyphenylpyruvate dioxygenase as a promising target for knock-out, exhibiting a 23.2-fold change (FC) in expression upon l-tyrosine supplementation in comparison to control cultivation conditions. The subsequent knock-out of the hppD gene in S. albidoflavus resulted in a 1.66-fold increase in the naringenin titer, indicating enhanced flavonoid biosynthesis. Leveraging the improved strain of S. albidoflavus, we successfully synthesized the methylated flavanones hesperetin, homoeriodictyol, and homohesperetin, achieving titers of 2.52 mg/L, 1.34 mg/L, and 0.43 mg/L, respectively. In addition, the dimethoxy flavanone homohesperetin was produced as a byproduct of the endogenous metabolism of S. albidoflavus. To our knowledge, this is the first time that hppD deletion was utilized as a strategy to augment the biosynthesis of flavonoids. Furthermore, this is the first report where hesperetin and homoeriodictyol have been synthesized from l-tyrosine as a precursor. Therefore, transcriptomics is, in this case, a successful approach for the identification of catabolism reactions affecting key precursors during flavonoid biosynthesis, allowing the generation of enhanced production strains.


Assuntos
Anormalidades Craniofaciais , Flavonas , Flavonoides , Perfilação da Expressão Gênica , Hesperidina , Streptomyces , Aminoácidos , Tirosina
14.
Front Endocrinol (Lausanne) ; 15: 1379231, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38638139

RESUMO

Receptor tyrosine kinases (RTKs) mediate the actions of growth factors in metazoans. In decapod crustaceans, RTKs are implicated in various physiological processes, such molting and growth, limb regeneration, reproduction and sexual differentiation, and innate immunity. RTKs are organized into two main types: insulin receptors (InsRs) and growth factor receptors, which include epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR). The identities of crustacean RTK genes are incomplete. A phylogenetic analysis of the CrusTome transcriptome database, which included all major crustacean taxa, showed that RTK sequences segregated into receptor clades representing InsR (72 sequences), EGFR (228 sequences), FGFR (129 sequences), and PDGFR/VEGFR (PVR; 235 sequences). These four receptor families were distinguished by the domain organization of the extracellular N-terminal region and motif sequences in the protein kinase catalytic domain in the C-terminus or the ligand-binding domain in the N-terminus. EGFR1 formed a single monophyletic group, while the other RTK sequences were divided into subclades, designated InsR1-3, FGFR1-3, and PVR1-2. In decapods, isoforms within the RTK subclades were common. InsRs were characterized by leucine-rich repeat, furin-like cysteine-rich, and fibronectin type 3 domains in the N-terminus. EGFRs had leucine-rich repeat, furin-like cysteine-rich, and growth factor IV domains. N-terminal regions of FGFR1 had one to three immunoglobulin-like domains, whereas FGFR2 had a cadherin tandem repeat domain. PVRs had between two and five immunoglobulin-like domains. A classification nomenclature of the four RTK classes, based on phylogenetic analysis and multiple sequence alignments, is proposed.


Assuntos
Furina , Insulina , Furina/genética , Filogenia , Insulina/genética , Transcriptoma , Cisteína , Leucina/genética , Fator A de Crescimento do Endotélio Vascular/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores ErbB/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Tirosina
15.
Am J Bot ; 111(4): e16308, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38581167

RESUMO

PREMISE: Better understanding of the relationship between plant specialized metabolism and traditional medicine has the potential to aid in bioprospecting and untangling of cross-cultural use patterns. However, given the limited information available for metabolites in most plant species, understanding medicinal use-metabolite relationships can be difficult. The order Caryophyllales has a unique pattern of lineages of tyrosine- or phenylalanine-dominated specialized metabolism, represented by mutually exclusive anthocyanin and betalain pigments, making Caryophyllales a compelling system to explore the relationship between medicine and metabolites by using pigment as a proxy for dominant metabolism. METHODS: We compiled a list of medicinal species in select tyrosine- or phenylalanine-dominant families of Caryophyllales (Nepenthaceae, Polygonaceae, Simmondsiaceae, Microteaceae, Caryophyllaceae, Amaranthaceae, Limeaceae, Molluginaceae, Portulacaceae, Cactaceae, and Nyctaginaceae) by searching scientific literature until no new uses were recovered. We then tested for phylogenetic clustering of uses using a "hot nodes" approach. To test potential non-metabolite drivers of medicinal use, like how often humans encounter a species (apparency), we repeated the analysis using only North American species across the entire order and performed phylogenetic generalized least squares regression (PGLS) with occurrence data from the Global Biodiversity Information Facility (GBIF). RESULTS: We hypothesized families with tyrosine-enriched metabolism would show clustering of different types of medicinal use compared to phenylalanine-enriched metabolism. Instead, wide-ranging, apparent clades in Polygonaceae and Amaranthaceae are overrepresented across nearly all types of medicinal use. CONCLUSIONS: Our results suggest that apparency is a better predictor of medicinal use than metabolism, although metabolism type may still be a contributing factor.


Assuntos
Caryophyllales , Plantas Medicinais , Caryophyllales/metabolismo , Caryophyllales/genética , Plantas Medicinais/metabolismo , Medicina Tradicional , Filogenia , Tirosina/metabolismo , Betalaínas/metabolismo , Fenilalanina/metabolismo
16.
Drug Des Devel Ther ; 18: 1165-1174, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38623566

RESUMO

Purpose: Citric acid (CA) is a tricarboxylic acid with antioxidant and antimicrobial properties. Based on previous studies, the small compound with its three carboxylic groups can be considered a protein tyrosine phosphatase inhibitor. YopH, a protein tyrosine phosphatase, is an essential virulence factor in Yersinia bacteria. Materials and Methods: We performed enzymatic activity assays of YopH phosphatase after treatment with citric acid in comparison with the inhibitory compound trimesic acid, which has a similar structure. We also measured the cytotoxicity of these compounds in Jurkat T E6.1 and macrophage J774.2 cell lines. We performed molecular docking analysis of the binding of citric acid molecules to YopH phosphatase. Results: Citric acid and trimesic acid reversibly reduced the activity of YopH enzyme and decreased the viability of Jurkat and macrophage cell lines. Importantly, these two compounds showed greater inhibitory properties against bacterial YopH activity than against human CD45 phosphatase activity. Molecular docking simulations confirmed that citric acid could bind to YopH phosphatase. Conclusion: Citric acid, a known antioxidant, can be considered an inhibitor of bacterial phosphatases.


Assuntos
Antioxidantes , Proteínas Tirosina Fosfatases , Ácidos Tricarboxílicos , Humanos , Simulação de Acoplamento Molecular , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Tirosina
17.
Sci Adv ; 10(15): eadk8157, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38598628

RESUMO

Redesigning protein-protein interfaces is an important tool for developing therapeutic strategies. Interfaces can be redesigned by in silico screening, which allows for efficient sampling of a large protein space before experimental validation. However, computational costs limit the number of combinations that can be reasonably sampled. Here, we present combinatorial tyrosine (Y)/serine (S) selection (combYSelect), a computational approach combining in silico determination of the change in binding free energy (ΔΔG) of an interface with a highly restricted library composed of just two amino acids, tyrosine and serine. We used combYSelect to design two immunoglobulin G (IgG) heterodimers-combYSelect1 (L368S/D399Y-K409S/T411Y) and combYSelect2 (D399Y/K447S-K409S/T411Y)-that exhibit near-optimal heterodimerization, without affecting IgG stability or function. We solved the crystal structures of these heterodimers and found that dynamic π-stacking interactions and polar contacts drive preferential heterodimeric interactions. Finally, we demonstrated the utility of our combYSelect heterodimers by engineering both a bispecific antibody and a cytokine trap for two unique therapeutic applications.


Assuntos
Anticorpos Biespecíficos , Imunoglobulina G , Dimerização , Tirosina/metabolismo , Serina/metabolismo , Biologia Computacional
18.
Adv Exp Med Biol ; 1446: 135-154, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38625527

RESUMO

The hair and skin of domestic cats or dogs account for 2% and 12-24% of their body weight, respectively, depending on breed and age. These connective tissues contain protein as the major constituent and provide the first line of defense against external pathogens and toxins. Maintenance of the skin and hair in smooth and elastic states requires special nutritional support, particularly an adequate provision of amino acids (AAs). Keratin (rich in cysteine, serine and glycine) is the major protein both in the epidermis of the skin and in the hair. Filaggrin [rich in some AAs (e.g., serine, glutamate, glutamine, glycine, arginine, and histidine)] is another physiologically important protein in the epidermis of the skin. Collagen and elastin (rich in glycine and proline plus 4-hydroxyproline) are the predominant proteins in the dermis and hypodermis of the skin. Taurine and 4-hydroxyproline are abundant free AAs in the skin of dogs and cats, and 4-hydroxyproline is also an abundant free AA in their hair. The epidermis of the skin synthesizes melanin (the pigment in the skin and hair) from tyrosine and produces trans-urocanate from histidine. Qualitative requirements for proteinogenic AAs are similar between cats and dogs but not identical. Both animal species require the same AAs to nourish the hair and skin but the amounts differ. Other factors (e.g., breeds, coat color, and age) may affect the requirements of cats or dogs for nutrients. The development of a healthy coat, especially a black coat, as well as healthy skin critically depends on AAs [particularly arginine, glycine, histidine, proline, 4-hydroxyproline, and serine, sulfur AAs (methionine, cysteine, and taurine), phenylalanine, and tyrosine] and creatine. Although there are a myriad of studies on AA nutrition in cats and dogs, there is still much to learn about how each AA affects the growth, development and maintenance of the hair and skin. Animal-sourced foodstuffs (e.g., feather meal and poultry by-product meal) are excellent sources of the AAs that are crucial to maintain the normal structure and health of the skin and hair in dogs and cats.


Assuntos
Doenças do Gato , Doenças do Cão , Gatos , Cães , Animais , Aminoácidos , Histidina , Cisteína , Hidroxiprolina , Cabelo , Glicina , Tirosina , Taurina , Serina , Prolina , Arginina
19.
Biophys Chem ; 309: 107234, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38603989

RESUMO

Activation of heterotrimeric G-proteins (Gαßγ) downstream to receptor tyrosine kinases (RTKs) is a well-established crosstalk between the signaling pathways mediated by G-protein coupled receptors (GPCRs) and RTKs. While GPCR serves as a guanine exchange factor (GEF) in the canonical activation of Gα that facilitates the exchange of GDP for GTP, the mechanism through which RTK phosphorylations induce Gα activation remains unclear. Recent experimental studies revealed that the epidermal growth factor receptor (EGFR), a well-known RTK, phosphorylates the helical domain tyrosine residues Y154 and Y155 and accelerates the GDP release from the Gαi3, a subtype of Gα-protein. Using well-tempered metadynamics and extensive unbiased molecular dynamics simulations, we captured the GDP release event and identified the intermediates between bound and unbound states through Markov state models. In addition to weakened salt bridges at the domain interface, phosphorylations induced the unfolding of helix αF, which contributed to increased flexibility near the hinge region, facilitating a greater distance between domains in the phosphorylated Gαi3. Although the larger domain separation in the phosphorylated system provided an unobstructed path for the nucleotide, the accelerated release of GDP was attributed to increased fluctuations in several conserved regions like P-loop, switch 1, and switch 2. Overall, this study provides atomistic insights into the activation of G-proteins induced by RTK phosphorylations and identifies the specific structural motifs involved in the process. The knowledge gained from the study could establish a foundation for targeting non-canonical signaling pathways and developing therapeutic strategies against the ailments associated with dysregulated G-protein signaling.


Assuntos
Fatores de Troca do Nucleotídeo Guanina , Transdução de Sinais , Fosforilação , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Tirosina/metabolismo
20.
Front Cell Infect Microbiol ; 14: 1264525, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38585651

RESUMO

Introduction: Dengue is an arboviral disease causing severe illness in over 500,000 people each year. Currently, there is no way to constrain dengue in the clinic. Host kinase regulators of dengue virus (DENV) infection have the potential to be disrupted by existing therapeutics to prevent infection and/or disease progression. Methods: To evaluate kinase regulation of DENV infection, we performed kinase regression (KiR), a machine learning approach that predicts kinase regulators of infection using existing drug-target information and a small drug screen. We infected hepatocytes with DENV in vitro in the presence of a panel of 38 kinase inhibitors then quantified the effect of each inhibitor on infection rate. We employed elastic net regularization on these data to obtain predictions of which of 291 kinases are regulating DENV infection. Results: Thirty-six kinases were predicted to have a functional role. Intriguingly, seven of the predicted kinases - EPH receptor A4 (EPHA4), EPH receptor B3 (EPHB3), EPH receptor B4 (EPHB4), erb-b2 receptor tyrosine kinase 2 (ERBB2), fibroblast growth factor receptor 2 (FGFR2), Insulin like growth factor 1 receptor (IGF1R), and ret proto-oncogene (RET) - belong to the receptor tyrosine kinase (RTK) family, which are already therapeutic targets in the clinic. We demonstrate that predicted RTKs are expressed at higher levels in DENV infected cells. Knockdown of EPHB4, ERBB2, FGFR2, or IGF1R reduces DENV infection in hepatocytes. Finally, we observe differential temporal induction of ERBB2 and IGF1R following DENV infection, highlighting their unique roles in regulating DENV. Discussion: Collectively, our findings underscore the significance of multiple RTKs in DENV infection and advocate further exploration of RTK-oriented interventions against dengue.


Assuntos
Vírus da Dengue , Dengue , Humanos , Vírus da Dengue/fisiologia , Receptor EphA1 , Hepatócitos/metabolismo , Tirosina , Replicação Viral
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