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1.
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
2.
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
3.
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
4.
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
5.
Int J Mol Sci ; 25(8)2024 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-38673925

RESUMO

The protective effects of hydrogen sulfide (H2S) against ischemic brain injury and its role in promoting angiogenesis have been established. However, the specific mechanism underlying these effects remains unclear. This study is designed to investigate the regulatory impact and mechanism of H2S on VEGFR2 phosphorylation. Following expression and purification, the recombinant His-VEGFR2 protein was subjected to LC-PRM/MS analysis to identify the phosphorylation sites of VEGFR2 upon NaHS treatment. Adenovirus infection was used to transfect primary rat brain artery endothelial cells (BAECs) with the Ad-VEGFR2WT, Ad-VEGFR2Y797F, and Ad-VEGFR2S799A plasmids. The expression of VEGFR2 and recombinant Flag-VEGFR2, along with Akt phosphorylation, cell proliferation, and LDH levels, was assessed. The migratory capacity and tube-forming potential of BAECs were assessed using wound healing, transwell, and tube formation assays. NaHS notably enhanced the phosphorylation of VEGFR2 at Tyr797 and Ser799 sites. These phosphorylation sites were identified as crucial for mediating the protective effects of NaHS against hypoxia-reoxygenation (H/R) injury. NaHS significantly enhanced the Akt phosphorylation, migratory capacity, and tube formation of BAECs and upregulated the expression of VEGFR2 and recombinant proteins. These findings suggest that Tyr797 and Ser799 sites of VEGFR2 serve as crucial mediators of H2S-induced pro-angiogenic effects and protection against H/R injury.


Assuntos
Células Endoteliais , Sulfeto de Hidrogênio , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Fosforilação/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Sulfeto de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/metabolismo , Animais , Ratos , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Ratos Sprague-Dawley , Hipóxia Celular , Proliferação de Células/efeitos dos fármacos , Tirosina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/genética , Indutores da Angiogênese/farmacologia , Indutores da Angiogênese/metabolismo , Serina/metabolismo , Hipóxia/metabolismo
6.
Proc Natl Acad Sci U S A ; 121(19): e2300606121, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38683979

RESUMO

ß-catenin has influential roles affecting embryonic development, tissue homeostasis, and human diseases including cancer. Cellular ß-catenin levels are exquisitely controlled by a variety of regulatory mechanisms. In the course of exploring the functions of the Nek10 tyrosine kinase, we observed that deletion of Nek10 in lung adenocarcinoma cells resulted in dramatic stabilization of ß-catenin, suggestive of a Nek10 role in the control of ß-catenin turnover. Nek10-deficient cells exhibited diminished ability to form tumorspheres in suspension, grow in soft agar, and colonize mouse lung tissue following tail vein injection. Mechanistically, Nek10 associates with the Axin complex, responsible for ß-catenin degradation, where it phosphorylates ß-catenin at Tyr30, located within the regulatory region governing ß-catenin turnover. In the absence of Nek10 phosphorylation, GSK3-mediated phosphorylation of ß-catenin, a prerequisite for its turnover, is impaired. This represents a divergent function within the Nek family, whose other members are serine-threonine kinases involved in different elements of the centrosomal cycle, primary cilia function, and DNA damage responses.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Quinases Relacionadas a NIMA , beta Catenina , Animais , Humanos , Camundongos , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , beta Catenina/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Quinases Relacionadas a NIMA/metabolismo , Quinases Relacionadas a NIMA/genética , Fosforilação , Tirosina/metabolismo
7.
BMC Plant Biol ; 24(1): 276, 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38605285

RESUMO

BACKGROUND: Stephania kwangsiensis Lo (Menispermaceae) is a well-known Chinese herbal medicine, and its bulbous stems are used medicinally. The storage stem of S. kwangsiensis originated from the hypocotyls. To date, there are no reports on the growth and development of S. kwangsiensis storage stems. RESULTS: The bulbous stem of S. kwangsiensis, the starch diameter was larger at the stable expanding stage (S3T) than at the unexpanded stage (S1T) or the rapidly expanding stage (S2T) at the three different time points. We used ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and Illumina sequencing to identify key genes involved in bulbous stem development. A large number of differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs) were identified. Based on the differential expression profiles of the metabolites, alkaloids, lipids, and phenolic acids were the top three differentially expressed classes. Compared with S2T, significant changes in plant signal transduction and isoquinoline alkaloid biosynthesis pathways occurred at both the transcriptional and metabolic levels in S1T. In S2T compared with S3T, several metabolites involved in tyrosine metabolism were decreased. Temporal analysis of S1T to S3T indicated the downregulation of phenylpropanoid biosynthesis, including lignin biosynthesis. The annotation of key pathways showed an up-down trend for genes and metabolites involved in isoquinoline alkaloid biosynthesis, whereas phenylpropanoid biosynthesis was not completely consistent. CONCLUSIONS: Downregulation of the phenylpropanoid biosynthesis pathway may be the result of carbon flow into alkaloid synthesis and storage of lipids and starch during the development of S. kwangsiensis bulbous stems. A decrease in the number of metabolites involved in tyrosine metabolism may also lead to a decrease in the upstream substrates of phenylpropane biosynthesis. Downregulation of lignin synthesis during phenylpropanoid biosynthesis may loosen restrictions on bulbous stem expansion. This study provides the first comprehensive analysis of the metabolome and transcriptome profiles of S. kwangsiensis bulbous stems. These data provide guidance for the cultivation, breeding, and harvesting of S. kwangsiensis.


Assuntos
Alcaloides , Plantas Medicinais , Stephania , Stephania/química , Stephania/metabolismo , Plantas Medicinais/metabolismo , Cromatografia Líquida/métodos , Lignina/metabolismo , Espectrometria de Massas em Tandem , Melhoramento Vegetal , Perfilação da Expressão Gênica , Transcriptoma , Alcaloides/metabolismo , Amido/metabolismo , Isoquinolinas/metabolismo , Tirosina/metabolismo , Lipídeos , Regulação da Expressão Gênica de Plantas
8.
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
9.
Scand J Immunol ; 99(5): e13358, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38605535

RESUMO

Adapter proteins are flexible and dynamic modulators of cellular signalling that are important for immune cell function. One of these, the T-cell-specific adapter protein (TSAd), interacts with the non-receptor tyrosine kinases Src and Lck of the Src family kinases (SFKs) and Itk of the Tec family kinases (TFKs). Three tyrosine residues in the TSAd C-terminus are phosphorylated by Lck and serve as docking sites for the Src homology 2 (SH2) domains of Src and Lck. The TSAd proline-rich region (PRR) binds to the Src homology 3 (SH3) domains found in Lck, Src and Itk. Despite known interactors, the role TSAd plays in cellular signalling remains largely unknown. TSAd's ability to bind both SFKs and TFKs may point to its function as a general scaffold for both kinase families. Using GST-pulldown as well as peptide array experiments, we found that both the SH2 and SH3 domains of the SFKs Fyn and Hck, as well as the TFKs Tec and Txk, interact with TSAd. This contrasts with Itk, which interacts with TSAd only through its SH3 domain. Although our analysis showed that TSAd is both co-expressed and may interact with Fyn, we were unable to co-precipitate Fyn with TSAd from Jurkat cells, as detected by Western blotting and affinity purification mass spectrometry. This may suggest that TSAd-Fyn interaction in intact cells may be limited by other factors, such as the subcellular localization of the two molecules or the co-expression of competing binding partners.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Quinases da Família src , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Jurkat , Ligação Proteica , Domínios de Homologia de src , Quinases da Família src/metabolismo , Tirosina/metabolismo
10.
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
11.
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
12.
Sci Rep ; 14(1): 8695, 2024 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-38622194

RESUMO

AMPylation is a biologically significant yet understudied post-translational modification where an adenosine monophosphate (AMP) group is added to Tyrosine and Threonine residues primarily. While recent work has illuminated the prevalence and functional impacts of AMPylation, experimental identification of AMPylation sites remains challenging. Computational prediction techniques provide a faster alternative approach. The predictive performance of machine learning models is highly dependent on the features used to represent the raw amino acid sequences. In this work, we introduce a novel feature extraction pipeline to encode the key properties relevant to AMPylation site prediction. We utilize a recently published dataset of curated AMPylation sites to develop our feature generation framework. We demonstrate the utility of our extracted features by training various machine learning classifiers, on various numerical representations of the raw sequences extracted with the help of our framework. Tenfold cross-validation is used to evaluate the model's capability to distinguish between AMPylated and non-AMPylated sites. The top-performing set of features extracted achieved MCC score of 0.58, Accuracy of 0.8, AUC-ROC of 0.85 and F1 score of 0.73. Further, we elucidate the behaviour of the model on the set of features consisting of monogram and bigram counts for various representations using SHapley Additive exPlanations.


Assuntos
Processamento de Proteína Pós-Traducional , Tirosina , Tirosina/metabolismo , Sequência de Aminoácidos , Monofosfato de Adenosina/metabolismo , Treonina/metabolismo
13.
Environ Health Perspect ; 132(4): 47007, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38619879

RESUMO

BACKGROUND: Environmental pollutants, including polychlorinated biphenyls (PCBs) have been implicated in the pathogenesis of liver disease. Our group recently demonstrated that PCB126 promoted steatosis, hepatomegaly, and modulated intermediary metabolism in a rodent model of alcohol-associated liver disease (ALD). OBJECTIVE: To better understand how PCB126 promoted ALD in our previous model, the current study adopts multiple omics approaches to elucidate potential mechanistic hypotheses. METHODS: Briefly, male C57BL/6J mice were exposed to 0.2mg/kg polychlorinated biphenyl (PCB) 126 or corn oil vehicle prior to ethanol (EtOH) or control diet feeding in the chronic-binge alcohol feeding model. Liver tissues were collected and prepared for mRNA sequencing, phosphoproteomics, and inductively coupled plasma mass spectrometry for metals quantification. RESULTS: Principal component analysis showed that PCB126 uniquely modified the transcriptome in EtOH-fed mice. EtOH feeding alone resulted in >4,000 differentially expressed genes (DEGs), and PCB126 exposure resulted in more DEGs in the EtOH-fed group (907 DEGs) in comparison with the pair-fed group (503 DEGs). Top 20 significant gene ontology (GO) biological processes included "peptidyl tyrosine modifications," whereas top 25 significantly decreasing GO molecular functions included "metal/ion/zinc binding." Quantitative, label-free phosphoproteomics and western blot analysis revealed no major significant PCB126 effects on total phosphorylated tyrosine residues in EtOH-fed mice. Quantified hepatic essential metal levels were primarily significantly lower in EtOH-fed mice. PCB126-exposed mice had significantly lower magnesium, cobalt, and zinc levels in EtOH-fed mice. DISCUSSION: Previous work has demonstrated that PCB126 is a modifying factor in metabolic dysfunction-associated steatotic liver disease (MASLD), and our current work suggests that pollutants also modify ALD. PCB126 may, in part, be contributing to the malnutrition aspect of ALD, where metal deficiency is known to contribute and worsen prognosis. https://doi.org/10.1289/EHP14132.


Assuntos
Poluentes Ambientais , Fígado Gorduroso , Hepatopatias Alcoólicas , Bifenilos Policlorados , Masculino , Camundongos , Animais , Multiômica , Camundongos Endogâmicos C57BL , Etanol/toxicidade , Etanol/metabolismo , Fígado/metabolismo , Bifenilos Policlorados/toxicidade , Bifenilos Policlorados/metabolismo , Hepatopatias Alcoólicas/etiologia , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Poluentes Ambientais/toxicidade , Poluentes Ambientais/metabolismo , Zinco/metabolismo , Tirosina/metabolismo
14.
Nat Commun ; 15(1): 3415, 2024 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-38649367

RESUMO

An important epigenetic component of tyrosine kinase signaling is the phosphorylation of histones, and epigenetic readers, writers, and erasers. Phosphorylation of protein arginine methyltransferases (PRMTs), have been shown to enhance and impair their enzymatic activity. In this study, we show that the hyperactivation of Janus kinase 2 (JAK2) by the V617F mutation phosphorylates tyrosine residues (Y149 and Y334) in coactivator-associated arginine methyltransferase 1 (CARM1), an important target in hematologic malignancies, increasing its methyltransferase activity and altering its target specificity. While non-phosphorylatable CARM1 methylates some established substrates (e.g. BAF155 and PABP1), only phospho-CARM1 methylates the RUNX1 transcription factor, on R223 and R319. Furthermore, cells expressing non-phosphorylatable CARM1 have impaired cell-cycle progression and increased apoptosis, compared to cells expressing phosphorylatable, wild-type CARM1, with reduced expression of genes associated with G2/M cell cycle progression and anti-apoptosis. The presence of the JAK2-V617F mutant kinase renders acute myeloid leukemia (AML) cells less sensitive to CARM1 inhibition, and we show that the dual targeting of JAK2 and CARM1 is more effective than monotherapy in AML cells expressing phospho-CARM1. Thus, the phosphorylation of CARM1 by hyperactivated JAK2 regulates its methyltransferase activity, helps select its substrates, and is required for the maximal proliferation of malignant myeloid cells.


Assuntos
Apoptose , Subunidade alfa 2 de Fator de Ligação ao Core , Janus Quinase 2 , Proteína-Arginina N-Metiltransferases , Tirosina , Humanos , Fosforilação , Janus Quinase 2/metabolismo , Janus Quinase 2/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Tirosina/metabolismo , Linhagem Celular Tumoral , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Metilação , Especificidade por Substrato , Células HEK293 , Ciclo Celular , Mutação
15.
Sci Rep ; 14(1): 9198, 2024 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-38649417

RESUMO

Nitrosative stress promotes protein glycoxidation, and both processes can occur during an infection with the SARS-CoV-2 virus. Therefore, the aim of this study was to assess selected nitrosative stress parameters and protein glycoxidation products in COVID-19 patients and convalescents relative to healthy subjects, including in reference to the severity of COVID-19 symptoms. The diagnostic utility of nitrosative stress and protein glycoxidation biomarkers was also evaluated in COVID-19 patients. The study involved 218 patients with COVID-19, 69 convalescents, and 48 healthy subjects. Nitrosative stress parameters (NO, S-nitrosothiols, nitrotyrosine) and protein glycoxidation products (tryptophan, kynurenine, N-formylkynurenine, dityrosine, AGEs) were measured in the blood plasma or serum with the use of colorimetric/fluorometric methods. The levels of NO (p = 0.0480), S-nitrosothiols (p = 0.0004), nitrotyrosine (p = 0.0175), kynurenine (p < 0.0001), N-formylkynurenine (p < 0.0001), dityrosine (p < 0.0001), and AGEs (p < 0.0001) were significantly higher, whereas tryptophan fluorescence was significantly (p < 0.0001) lower in COVID-19 patients than in the control group. Significant differences in the analyzed parameters were observed in different stages of COVID-19. In turn, the concentrations of kynurenine (p < 0.0001), N-formylkynurenine (p < 0.0001), dityrosine (p < 0.0001), and AGEs (p < 0.0001) were significantly higher, whereas tryptophan levels were significantly (p < 0.0001) lower in convalescents than in healthy controls. The ROC analysis revealed that protein glycoxidation products can be useful for diagnosing infections with the SARS-CoV-2 virus because they differentiate COVID-19 patients (KN: sensitivity-91.20%, specificity-92.00%; NFK: sensitivity-92.37%, specificity-92.00%; AGEs: sensitivity-99,02%, specificity-100%) and convalescents (KN: sensitivity-82.22%, specificity-84.00%; NFK: sensitivity-82,86%, specificity-86,00%; DT: sensitivity-100%, specificity-100%; AGE: sensitivity-100%, specificity-100%) from healthy subjects with high sensitivity and specificity. Nitrosative stress and protein glycoxidation are intensified both during and after an infection with the SARS-CoV-2 virus. The levels of redox biomarkers fluctuate in different stages of the disease. Circulating biomarkers of nitrosative stress/protein glycoxidation have potential diagnostic utility in both COVID-19 patients and convalescents.


Assuntos
Biomarcadores , COVID-19 , Cinurenina/análogos & derivados , Estresse Nitrosativo , SARS-CoV-2 , Tirosina , Tirosina/análogos & derivados , Humanos , COVID-19/diagnóstico , COVID-19/sangue , COVID-19/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Biomarcadores/sangue , Adulto , Tirosina/sangue , Tirosina/metabolismo , Idoso , Cinurenina/sangue , Cinurenina/metabolismo , S-Nitrosotióis/sangue , S-Nitrosotióis/metabolismo , Óxido Nítrico/sangue , Óxido Nítrico/metabolismo , Triptofano/sangue , Triptofano/análogos & derivados , Triptofano/metabolismo , Produtos Finais de Glicação Avançada/sangue , Produtos Finais de Glicação Avançada/metabolismo , Curva ROC
16.
Proc Natl Acad Sci U S A ; 121(18): e2316819121, 2024 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-38657042

RESUMO

Posttranslational modifications regulate the properties and abundance of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that mediate fast excitatory synaptic transmission and synaptic plasticity in the central nervous system. During long-term depression (LTD), protein tyrosine phosphatases (PTPs) dephosphorylate tyrosine residues in the C-terminal tail of AMPA receptor GluA2 subunit, which is essential for GluA2 endocytosis and group I metabotropic glutamate receptor (mGluR)-dependent LTD. However, as a selective downstream effector of mGluRs, the mGluR-dependent PTP responsible for GluA2 tyrosine dephosphorylation remains elusive at Schaffer collateral (SC)-CA1 synapses. In the present study, we find that mGluR5 stimulation activates Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) by increasing phospho-Y542 levels in SHP2. Under steady-state conditions, SHP2 plays a protective role in stabilizing phospho-Y869 of GluA2 by directly interacting with GluA2 phosphorylated at Y869, without affecting GluA2 phospho-Y876 levels. Upon mGluR5 stimulation, SHP2 dephosphorylates GluA2 at Y869 and Y876, resulting in GluA2 endocytosis and mGluR-LTD. Our results establish SHP2 as a downstream effector of mGluR5 and indicate a dual action of SHP2 in regulating GluA2 tyrosine phosphorylation and function. Given the implications of mGluR5 and SHP2 in synaptic pathophysiology, we propose SHP2 as a promising therapeutic target for neurodevelopmental and autism spectrum disorders.


Assuntos
Endocitose , Depressão Sináptica de Longo Prazo , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Receptores de AMPA , Receptores de Glutamato Metabotrópico , Receptores de AMPA/metabolismo , Animais , Fosforilação , Endocitose/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Ratos , Tirosina/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Sinapses/metabolismo , Camundongos , Humanos , Neurônios/metabolismo
17.
J Am Chem Soc ; 146(17): 11944-11954, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38622919

RESUMO

Protein tyrosine nitration (PTN) by oxidative and nitrative stress is a well-known post-translational modification that plays a role in the initiation and progression of various diseases. Despite being recognized as a stable modification for decades, recent studies have suggested the existence of a reduction in PTN, leading to the formation of 3-aminotyrosine (3AT) and potential denitration processes. However, the vital functions of 3AT-containing proteins are still unclear due to the lack of selective probes that directly target the protein tyrosine amination. Here, we report a novel approach to label and enrich 3AT-containing proteins with synthetic salicylaldehyde (SAL)-based probes: SALc-FL with a fluorophore and SALc-Yn with an alkyne tag. These probes exhibit high selectivity and efficiency in labeling and can be used in cell lysates and live cells. More importantly, SALc-Yn offers versatility when integrated into multiple platforms by enabling proteome-wide quantitative profiling of cell nitration dynamics. Using SALc-Yn, 355 proteins were labeled, enriched, and identified to carry the 3AT modification in oxidatively stressed RAW264.7 cells. These findings provide compelling evidence supporting the involvement of 3AT as a critical intermediate in nitrated protein turnover. Moreover, our probes serve as powerful tools to investigate protein nitration and denitration processes, and the identification of 3AT-containing proteins contributes to our understanding of PTN dynamics and its implications in cellular redox biology.


Assuntos
Tirosina , Tirosina/análogos & derivados , Tirosina/química , Tirosina/metabolismo , Aminação , Humanos , Proteômica/métodos , Aldeídos/química , Aldeídos/síntese química , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Proteínas/química , Proteínas/metabolismo , Proteínas/análise , Camundongos , Animais
18.
J Am Chem Soc ; 146(17): 11726-11739, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38636166

RESUMO

Lysine dioxygenase (KDO) is an important enzyme in human physiology involved in bioprocesses that trigger collagen cross-linking and blood pressure control. There are several KDOs in nature; however, little is known about the factors that govern the regio- and stereoselectivity of these enzymes. To understand how KDOs can selectively hydroxylate their substrate, we did a comprehensive computational study into the mechanisms and features of 4-lysine dioxygenase. In particular, we selected a snapshot from the MD simulation on KDO5 and created large QM cluster models (A, B, and C) containing 297, 312, and 407 atoms, respectively. The largest model predicts regioselectivity that matches experimental observation with rate-determining hydrogen atom abstraction from the C4-H position, followed by fast OH rebound to form 4-hydroxylysine products. The calculations show that in model C, the dipole moment is positioned along the C4-H bond of the substrate and, therefore, the electrostatic and electric field perturbations of the protein assist the enzyme in creating C4-H hydroxylation selectivity. Furthermore, an active site Tyr233 residue is identified that reacts through proton-coupled electron transfer akin to the axial Trp residue in cytochrome c peroxidase. Thus, upon formation of the iron(IV)-oxo species in the catalytic cycle, the Tyr233 phenol loses a proton to the nearby Asp179 residue, while at the same time, an electron is transferred to the iron to create an iron(III)-oxo active species. This charged tyrosyl residue directs the dipole moment along the C4-H bond of the substrate and guides the selectivity to the C4-hydroxylation of the substrate.


Assuntos
Domínio Catalítico , Lisina , Prótons , Hidroxilação , Lisina/metabolismo , Lisina/química , Transporte de Elétrons , Tirosina/química , Tirosina/metabolismo , Simulação de Dinâmica Molecular , Estereoisomerismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Humanos , Ferro/química , Ferro/metabolismo
19.
Sci Rep ; 14(1): 9923, 2024 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-38688959

RESUMO

Phosphorylation plays a crucial role in the regulation of many fundamental cellular processes. Phosphorylation levels are increased in many cancer cells where they may promote changes in mitochondrial homeostasis. Proteomic studies on various types of cancer identified 17 phosphorylation sites within the human ATP-dependent protease Lon, which degrades misfolded, unassembled and oxidatively damaged proteins in mitochondria. Most of these sites were found in Lon's N-terminal (NTD) and ATPase domains, though little is known about the effects on their function. By combining the biochemical and cryo-electron microscopy studies, we show the effect of Tyr186 and Tyr394 phosphorylations in Lon's NTD, which greatly reduce all Lon activities without affecting its ability to bind substrates or perturbing its tertiary structure. A substantial reduction in Lon's activities is also observed in the presence of polyphosphate, whose amount significantly increases in cancer cells. Our study thus provides an insight into the possible fine-tuning of Lon activities in human diseases, which highlights Lon's importance in maintaining proteostasis in mitochondria.


Assuntos
Mitocôndrias , Polifosfatos , Protease La , Tirosina , Humanos , Fosforilação , Protease La/metabolismo , Polifosfatos/metabolismo , Mitocôndrias/metabolismo , Tirosina/metabolismo , Microscopia Crioeletrônica , Domínios Proteicos
20.
J Cell Sci ; 137(8)2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38525600

RESUMO

In neurons, the microtubule (MT) cytoskeleton forms the basis for long-distance protein transport from the cell body into and out of dendrites and axons. To maintain neuronal polarity, the axon initial segment (AIS) serves as a physical barrier, separating the axon from the somatodendritic compartment and acting as a filter for axonal cargo. Selective trafficking is further instructed by axonal enrichment of MT post-translational modifications, which affect MT dynamics and the activity of motor proteins. Here, we compared two knockout mouse lines lacking the respective enzymes for MT tyrosination and detyrosination, and found that both knockouts led to a shortening of the AIS. Neurons from both lines also showed an increased immobile fraction of endolysosomes present in the axon, whereas mobile organelles displayed shortened run distances in the retrograde direction. Overall, our results highlight the importance of maintaining the balance of tyrosinated and detyrosinated MTs for proper AIS length and axonal transport processes.


Assuntos
Transporte Axonal , Lisossomos , Camundongos Knockout , Microtúbulos , Tirosina , Animais , Microtúbulos/metabolismo , Tirosina/metabolismo , Lisossomos/metabolismo , Camundongos , Axônios/metabolismo , Endossomos/metabolismo , Neurônios/metabolismo
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