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1.
bioRxiv ; 2023 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-38014295

RESUMO

Age is a prominent risk factor for cardiometabolic disease, and often leads to heart structural and functional changes. However, precise molecular mechanisms underlying cardiac remodeling and dysfunction resulting from physiological aging per se remain elusive. Understanding these mechanisms requires biological models with optimal translation to humans. Previous research demonstrated that baboons undergo age-related reduction in ejection fraction and increased heart sphericity, mirroring changes observed in humans. The goal of this study was to identify early cardiac molecular alterations that precede functional adaptations, shedding light on the regulation of age-associated changes. We performed unbiased transcriptomics of left ventricle (LV) samples from female baboons aged 7.5-22.1 years (human equivalent ~30-88 years). Weighted-gene correlation network and pathway enrichment analyses were performed to identify potential age-associated mechanisms in LV, with histological validation. Myocardial modules of transcripts negatively associated with age were primarily enriched for cardiac metabolism, including oxidative phosphorylation, tricarboxylic acid cycle, glycolysis, and fatty-acid ß-oxidation. Transcripts positively correlated with age suggest upregulation of glucose uptake, pentose phosphate pathway, and hexosamine biosynthetic pathway (HBP), indicating a metabolic shift towards glucose-dependent anabolic pathways. Upregulation of HBP commonly results in increased glycosaminoglycan precursor synthesis. Transcripts involved in glycosaminoglycan synthesis, modification, and intermediate metabolism were also upregulated in older animals, while glycosaminoglycan degradation transcripts were downregulated with age. These alterations would promote glycosaminoglycan accumulation, which was verified histologically. Upregulation of extracellular matrix (ECM)-induced signaling pathways temporally coincided with glycosaminoglycan accumulation. We found a subsequent upregulation of cardiac hypertrophy-related pathways and an increase in cardiomyocyte width. Overall, our findings revealed a transcriptional shift in metabolism from catabolic to anabolic pathways that leads to ECM glycosaminoglycan accumulation through HBP prior to upregulation of transcripts of cardiac hypertrophy-related pathways. This study illuminates cellular mechanisms that precede development of cardiac hypertrophy, providing novel potential targets to remediate age-related cardiac diseases.

2.
Neurobiol Aging ; 132: 109-119, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37797463

RESUMO

The prefrontal cortex (PFC) has been implicated as a key brain region responsible for age-related cognitive decline. Little is known about aging-related molecular changes in PFC that may mediate these effects. To date, no studies have used untargeted discovery methods with integrated analyses to determine PFC molecular changes in healthy female primates. We quantified PFC changes associated with healthy aging in female baboons by integrating multiple omics data types (transcriptomics, proteomics, metabolomics) from samples across the adult age span. Our integrated omics approach using unbiased weighted gene co-expression network analysis to integrate data and treat age as a continuous variable, revealed highly interconnected known and novel pathways associated with PFC aging. We found Gamma-aminobutyric acid (GABA) tissue content associated with these signaling pathways, providing 1 potential biomarker to assess PFC changes with age. These highly coordinated pathway changes during aging may represent early steps for aging-related decline in PFC functions, such as learning and memory, and provide potential biomarkers to assess cognitive status in humans.


Assuntos
Disfunção Cognitiva , Multiômica , Humanos , Animais , Feminino , Envelhecimento/psicologia , Transdução de Sinais/genética , Córtex Pré-Frontal/metabolismo , Disfunção Cognitiva/genética , Disfunção Cognitiva/metabolismo
3.
Cancers (Basel) ; 15(19)2023 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-37835439

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with a mere 5-year survival of ~10%. This highlights the urgent need for innovative treatment options for PDAC patients. The nuclear factor κB (NF-κB) is a crucial transcription factor that is constitutively activated in PDAC. It mediates the transcription of oncogenic and inflammatory genes that facilitate multiple PDAC phenotypes. Thus, a better understanding of the mechanistic underpinnings of NF-κB activation holds great promise for PDAC diagnosis and effective therapeutics. Here, we report a novel finding that the p65 subunit of NF-κB is O-GlcNAcylated at serine 550 and 551 upon NF-κB activation. Importantly, the overexpression of either serine-to-alanine (S-A) single mutant (S550A or S551A) or double mutant (S550A/S551A) of p65 in PDAC cells impaired NF-κB nuclear translocation, p65 phosphorylation, and transcriptional activity, independent of IκBα degradation. Moreover, the p65 mutants downregulate a category of NF-κB-target genes, which play a role in perpetuating major cancer hallmarks. We further show that overexpression of the p65 mutants inhibited cellular proliferation, migration, and anchorage-independent growth of PDAC cells compared to WT-p65. Collectively, we discovered novel serine sites of p65 O-GlcNAcylation that drive NF-κB activation and PDAC phenotypes, thus opening new avenues by inhibiting the NF-κB O-GlcNAcylation enzyme, O-GlcNAc transferase (OGT), for PDAC treatment in the future.

4.
Biochem Biophys Res Commun ; 671: 343-349, 2023 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-37329657

RESUMO

Hydroxyl radical protein footprinting (HRPF) using synchrotron radiation is a well-validated method to assess protein structure in the native solution state. In this method, X-ray radiolysis of water generates hydroxyl radicals that can react with solvent accessible side chains of proteins, with mass spectrometry used to detect the resulting labeled products. An ideal footprinting dose provides sufficient labeling to measure the structure but not so much as to influence the results. The optimization of hydroxyl radical dose is typically performed using an indirect Alexa488 fluorescence assay sensitive to hydroxyl radical concentration, but full evaluation of the experiment's outcome relies upon bottom-up liquid chromatography mass spectrometry (LC-MS) measurements to directly determine sites and extent of oxidative labeling at the peptide and protein level. A direct evaluation of the extent of labeling to provide direct and absolute measurements of dose and "safe" dose ranges in terms of, for example, average numbers of labels per protein, would provide immediate feedback on experimental outcomes prior to embarking on detailed LC-MS analyses. To this end, we describe an approach to integrate intact MS screening of labeled samples immediately following exposure, along with metrics to quantify the extent of observed labeling from the intact mass spectra. Intact MS results on the model protein lysozyme were evaluated in the context of Alexa488 assay results and a bottom-up LC-MS analysis of the same samples. This approach provides a basis for placing delivered hydroxyl radical dose metrics on firmer technical grounds for synchrotron X-ray footprinting of proteins, with explicit parameters to increase the likelihood of a productive experimental outcome. Further, the method directs approaches to provide absolute and direct dosimetry for all types of labeling for protein footprinting.


Assuntos
Radical Hidroxila , Pegadas de Proteínas , Pegadas de Proteínas/métodos , Conformação Proteica , Proteínas/química , Espectrometria de Massas/métodos
5.
J Biol Chem ; 298(10): 102417, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36037967

RESUMO

Γ-Crystallins play a major role in age-related lens transparency. Their destabilization by mutations and physical chemical insults are associated with cataract formation. Therefore, drugs that increase their stability should have anticataract properties. To this end, we screened 2560 Federal Drug Agency-approved drugs and natural compounds for their ability to suppress or worsen H2O2 and/or heat-mediated aggregation of bovine γ-crystallins. The top two drugs, closantel (C), an antihelminthic drug, and gambogic acid (G), a xanthonoid, attenuated thermal-induced protein unfolding and aggregation as shown by turbidimetry fluorescence spectroscopy dynamic light scattering and electron microscopy of human or mouse recombinant crystallins. Furthermore, binding studies using fluorescence inhibition and hydrophobic pocket-binding molecule bis-8-anilino-1-naphthalene sulfonic acid revealed static binding of C and G to hydrophobic sites with medium-to-low affinity. Molecular docking to HγD and other γ-crystallins revealed two binding sites, one in the "NC pocket" (residues 50-150) of HγD and one spanning the "NC tail" (residues 56-61 to 168-174 in the C-terminal domain). Multiple binding sites overlap with those of the protective mini αA-crystallin chaperone MAC peptide. Mechanistic studies using bis-8-anilino-1-naphthalene sulfonic acid as a proxy drug showed that it bound to MAC sites, improved Tm of both H2O2 oxidized and native human gamma D, and suppressed turbidity of oxidized HγD, most likely by trapping exposed hydrophobic sites. The extent to which these drugs act as α-crystallin mimetics and reduce cataract progression remains to be demonstrated. This study provides initial insights into binding properties of C and G to γ-crystallins.


Assuntos
Materiais Biomiméticos , Catarata , Cristalino , Chaperonas Moleculares , Agregação Patológica de Proteínas , Salicilanilidas , Xantonas , alfa-Cristalinas , gama-Cristalinas , Animais , Bovinos , Humanos , Camundongos , alfa-Cristalinas/metabolismo , Catarata/tratamento farmacológico , Catarata/prevenção & controle , Catarata/genética , gama-Cristalinas/metabolismo , Peróxido de Hidrogênio/metabolismo , Cristalino/metabolismo , Chaperonas Moleculares/metabolismo , Simulação de Acoplamento Molecular , Naftalenos/metabolismo , Ácidos Sulfônicos/metabolismo , Salicilanilidas/química , Salicilanilidas/farmacologia , Salicilanilidas/uso terapêutico , Xantonas/química , Xantonas/farmacologia , Xantonas/uso terapêutico , Agregação Patológica de Proteínas/tratamento farmacológico , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Materiais Biomiméticos/uso terapêutico
6.
Mol Cell ; 82(16): 3089-3102.e7, 2022 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-35931084

RESUMO

The ß2-adrenergic receptor (ß2AR), a prototypic G-protein-coupled receptor (GPCR), is a powerful driver of bronchorelaxation, but the effectiveness of ß-agonist drugs in asthma is limited by desensitization and tachyphylaxis. We find that during activation, the ß2AR is modified by S-nitrosylation, which is essential for both classic desensitization by PKA as well as desensitization of NO-based signaling that mediates bronchorelaxation. Strikingly, S-nitrosylation alone can drive ß2AR internalization in the absence of traditional agonist. Mutant ß2AR refractory to S-nitrosylation (Cys265Ser) exhibits reduced desensitization and internalization, thereby amplifying NO-based signaling, and mice with Cys265Ser mutation are resistant to bronchoconstriction, inflammation, and the development of asthma. S-nitrosylation is thus a central mechanism in ß2AR signaling that may be operative widely among GPCRs and targeted for therapeutic gain.


Assuntos
Asma , Animais , Asma/induzido quimicamente , Asma/genética , Camundongos , Transdução de Sinais
7.
Anal Chem ; 94(27): 9819-9825, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35763792

RESUMO

Protein footprinting with mass spectrometry is an established structural biology technique for mapping solvent accessibility and assessing molecular-level interactions of proteins. In hydroxyl radical protein footprinting (HRPF), hydroxyl (OH) radicals generated by water radiolysis or other methods covalently label protein side chains. Because of the wide dynamic range of OH reactivity, not all side chains are easily detected in a single experiment. Novel reagent development and the use of radical chain reactions for labeling, including trifluoromethyl radicals, is a potential approach to normalize the labeling across a diverse set of residues. HRPF in the presence of a trifluoromethylation reagent under the right conditions could provide a "one-pot" reaction for multiplex labeling of protein side chains. Toward this goal, we have systematically evaluated amino acid labeling with the recently investigated Langlois' reagent (LR) activated by X-ray-mediated water radiolysis, followed by three different mass spectrometry methods. We compared the reactivity of CF3 and OH radical labeling for all 20 protein side chains in a competition-free environment. We found that all 20 amino acids exhibited CF3 or OH labeling in LR. Our investigations provide the evidence and knowledge set to perfect hydroxyl radical-activated trifluoromethyl chemistry as "one-pot" reaction for multiplex labeling of protein side chains to achieve higher resolution in HRPF.


Assuntos
Aminoácidos , Pegadas de Proteínas , Aminoácidos/química , Radical Hidroxila/química , Oxirredução , Conformação Proteica , Pegadas de Proteínas/métodos , Proteínas/análise , Água
8.
mBio ; 13(3): e0179321, 2022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35612361

RESUMO

ß-Lactamases hydrolyze ß-lactam antibiotics and are major determinants of antibiotic resistance in Gram-negative pathogens. Enmetazobactam (formerly AAI101) and tazobactam are penicillanic acid sulfone (PAS) ß-lactamase inhibitors that differ by an additional methyl group on the triazole ring of enmetazobactam, rendering it zwitterionic. In this study, ultrahigh-resolution X-ray crystal structures and mass spectrometry revealed the mechanism of PAS inhibition of CTX-M-15, an extended-spectrum ß-lactamase (ESBL) globally disseminated among Enterobacterales. CTX-M-15 crystals grown in the presence of enmetazobactam or tazobactam revealed loss of the Ser70 hydroxyl group and formation of a lysinoalanine cross-link between Lys73 and Ser70, two residues critical for catalysis. Moreover, the residue at position 70 undergoes epimerization, resulting in formation of a d-amino acid. Cocrystallization of enmetazobactam or tazobactam with CTX-M-15 with a Glu166Gln mutant revealed the same cross-link, indicating that this modification is not dependent on Glu166-catalyzed deacylation of the PAS-acylenzyme. A cocrystal structure of enmetazobactam with CTX-M-15 with a Lys73Ala mutation indicates that epimerization can occur without cross-link formation and positions the Ser70 Cß closer to Lys73, likely facilitating formation of the Ser70-Lys73 cross-link. A crystal structure of a tazobactam-derived imine intermediate covalently linked to Ser70, obtained after 30 min of exposure of CTX-M-15 crystals to tazobactam, supports formation of an initial acylenzyme by PAS inhibitors on reaction with CTX-M-15. These data rationalize earlier results showing CTX-M-15 deactivation by PAS inhibitors to involve loss of protein mass, and they identify a distinct mechanism of ß-lactamase inhibition by these agents. IMPORTANCE ß-Lactams are the most prescribed antibiotic class for treating bacterial diseases, but their continued efficacy is threatened by bacterial strains producing ß-lactamase enzymes that catalyze their inactivation. The CTX-M family of ESBLs are major contributors to ß-lactam resistance in Enterobacterales, preventing effective treatment with most penicillins and cephalosporins. Combining ß-lactams with ß-lactamase inhibitors (BLIs) is a validated route to overcome such resistance. Here, we describe how exposure to enmetazobactam and tazobactam, BLIs based on a penicillanic acid sulfone (PAS) scaffold, leads to a protein modification in CTX-M-15, resulting in irremediable inactivation of this most commonly encountered member of the CTX-M family. High-resolution X-ray crystal structures showed that PAS exposure induces formation of a cross-link between Ser70 and Lys73, two residues critical to ß-lactamase function. This previously undescribed mechanism of inhibition furthers our understanding of ß-lactamase inhibition by classical PAS inhibitors and provides a basis for further, rational inhibitor development.


Assuntos
Sulbactam , Inibidores de beta-Lactamases , Antibacterianos/farmacologia , Lisina , Testes de Sensibilidade Microbiana , Serina , Sulbactam/farmacologia , Tazobactam/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo
9.
Nat Commun ; 12(1): 5305, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489447

RESUMO

Mitochondrial dysfunction is a common hallmark of neurological disorders, and reducing mitochondrial damage is considered a promising neuroprotective therapeutic strategy. Here, we used high-throughput small molecule screening to identify CHIR99021 as a potent enhancer of mitochondrial function. CHIR99021 improved mitochondrial phenotypes and enhanced cell viability in several models of Huntington's disease (HD), a fatal inherited neurodegenerative disorder. Notably, CHIR99201 treatment reduced HD-associated neuropathology and behavioral defects in HD mice and improved mitochondrial function and cell survival in HD patient-derived neurons. Independent of its known inhibitory activity against glycogen synthase kinase 3 (GSK3), CHIR99021 treatment in HD models suppressed the proteasomal degradation of calpastatin (CAST), and subsequently inhibited calpain activation, a well-established effector of neural death, and Drp1, a driver of mitochondrial fragmentation. Our results established CAST-Drp1 as a druggable signaling axis in HD pathogenesis and highlighted CHIR99021 as a mitochondrial function enhancer and a potential lead for developing HD therapies.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Dinaminas/genética , Doença de Huntington/genética , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/genética , Calpaína/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Dinaminas/metabolismo , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Doença de Huntington/tratamento farmacológico , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Injeções Intraperitoneais , Masculino , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Transdução de Sinais
10.
PLoS Pathog ; 17(6): e1009642, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34138981

RESUMO

There is a limited understanding of structural attributes that encode the iatrogenic transmissibility and various phenotypes of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD). Here we report the detailed structural differences between major sCJD MM1, MM2, and VV2 prions determined with two complementary synchrotron hydroxyl radical footprinting techniques-mass spectrometry (MS) and conformation dependent immunoassay (CDI) with a panel of Europium-labeled antibodies. Both approaches clearly demonstrate that the phenotypically distant prions differ in a major way with regard to their structural organization, and synchrotron-generated hydroxyl radicals progressively inhibit their seeding potency in a strain and structure-specific manner. Moreover, the seeding rate of sCJD prions is primarily determined by strain-specific structural organization of solvent-exposed external domains of human prion particles that control the seeding activity. Structural characteristics of human prion strains suggest that subtle changes in the organization of surface domains play a critical role as a determinant of human prion infectivity, propagation rate, and targeting of specific brain structures.


Assuntos
Síndrome de Creutzfeldt-Jakob , Proteínas PrPSc/química , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patologia , Humanos , Proteínas PrPSc/metabolismo , Conformação Proteica , Domínios Proteicos , Isoformas de Proteínas
11.
Sci Rep ; 10(1): 15934, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985589

RESUMO

The multifunctional protein Y-box binding protein 1 (YBX1), is a critical regulator of transcription and translation, and is widely recognized as an oncogenic driver in several solid tumors, including colorectal cancer (CRC). However, very little is known about the upstream or downstream factors that underlie YBX1's regulation and involvement in CRC. Previously, we demonstrated that YBX1 overexpression correlated with potent activation of nuclear factor κB (NF-κB), a well-known transcription factor believed to be crucial in CRC progression. Here, we report a novel interaction between NF-κB, YBX1 and protein arginine methyltransferase 5 (PRMT5). Our findings reveal for the first time that PRMT5 catalyzes methylation of YBX1 at arginine 205 (YBX1-R205me2), an event that is critical for YBX1-mediated NF-κB activation and its downstream target gene expression. Importantly, when WT-YBX1 is overexpressed, this methylation exists under basal (unstimulated) conditions and is further augmented upon interleukin-1ß (IL-1ß) stimulation. Mechanistically, co-immunoprecipitation studies reveal that the R205 to alanine (A) mutant of YBX1 (YBX1-R205A) interacted less well with the p65 subunit of NF-κB and attenuated the DNA binding ability of p65. Importantly, overexpression of YBX1-R205A significantly reduced cell growth, migration and anchorage-independent growth of CRC cells. Collectively, our findings shed important light on the regulation of a novel PRMT5/YBX1/NF-κB axis through PRMT5-mediated YBX1-R205 methylation. Given the fact that PRMT5, YBX1 and NF-κB are all among top crucial factors in cancer progression, pharmacological disruption of this pivotal axis could serve as the basis for new therapeutics for CRC and other PRMT5/YBX1/NF-κB-associated cancers.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , NF-kappa B/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Humanos , NF-kappa B/genética , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/genética , Células Tumorais Cultivadas , Proteína 1 de Ligação a Y-Box/genética
12.
FASEB J ; 34(10): 13918-13934, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32860273

RESUMO

Interphotoreceptor retinoid-binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo-electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N-linked carbohydrate post-translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three-dimensional architecture not seen among other retinoid-binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.


Assuntos
Proteínas do Olho/química , Proteínas de Ligação ao Retinol/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/química , Bovinos , Microscopia Crioeletrônica , Proteínas do Olho/imunologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação ao Retinol/imunologia , Imagem Individual de Molécula
13.
Aging Cell ; 19(7): e13176, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32567221

RESUMO

Oxoaldehyde stress has recently emerged as a major source of tissue damage in aging and age-related diseases. The prevailing mechanism involves methylglyoxal production during glycolysis and modification of arginine residues through the formation of methylglyoxal hydroimidazolones (MG-H1). We now tested the hypothesis that oxidation of vitamin C (ascorbic acid or ASA) contributes to this damage when the homeostatic redox balance is disrupted especially in ASA-rich tissues such as the eye lens and brain. MG-H1 measured by liquid chromatography mass spectrometry is several fold increased in the lens and brain from transgenic mice expressing human vitamin C transporter 2 (hSVCT2). Similarly, MG-H1 levels are increased two- to fourfold in hippocampus extracts from individuals with Alzheimer's disease (AD), and significantly higher levels are present in sarkosyl-insoluble tissue fractions from AD brain proteins than in the soluble fractions. Moreover, immunostaining with antibodies against methylglyoxal hydroimidazolones reveals similar increase in substantia nigra neurons from individuals with Parkinson's disease. Results from an in vitro incubation experiment suggest that accumulated catalytic metal ions in the hippocampus during aging could readily accelerate ASA oxidation and such acceleration was significantly enhanced in AD. Modeling studies and intraventricular injection of 13 C-labeled ASA revealed that ASA backbone carbons 4-6 are incorporated into MG-H1 both in vitro and in vivo, likely via a glyceraldehyde precursor. We propose that drugs that prevent oxoaldehyde stress or excessive ASA oxidation may protect against age-related cataract and neurodegenerative diseases.


Assuntos
Aldeídos/metabolismo , Ácido Ascórbico/uso terapêutico , Catarata/etiologia , Doenças Neurodegenerativas/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento , Animais , Ácido Ascórbico/farmacologia , Humanos , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade
14.
Int J Mol Sci ; 21(10)2020 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-32456215

RESUMO

The overexpression of PRMT5 is highly correlated to poor clinical outcomes for colorectal cancer (CRC) patients. Importantly, our previous work demonstrated that PRMT5 overexpression could substantially augment activation of the nuclear factor kappa B (NF-κB) via methylation of arginine 30 (R30) on its p65 subunit, while knockdown of PRMT5 showed the opposite effect. However, the precise mechanisms governing this PRMT5/NF-κB axis are still largely unknown. Here, we report a novel finding that PRMT5 is phosphorylated on serine 15 (S15) in response to interleukin-1ß (IL-1ß) stimulation. Interestingly, we identified for the first time that the oncogenic kinase, PKCι could catalyze this phosphorylation event. Overexpression of the serine-to-alanine mutant of PRMT5 (S15A), in either HEK293 cells or CRC cells HT29, DLD1, and HCT116 attenuated NF-κB transactivation compared to WT-PRMT5, confirming that S15 phosphorylation is critical for the activation of NF-κB by PRMT5. Furthermore, the S15A mutant when compared to WT-PRMT5, could downregulate a subset of IL-1ß-inducible NF-κB-target genes which correlated with attenuated promoter occupancy of p65 at its target genes. Additionally, the S15A mutant reduced IL-1ß-induced methyltransferase activity of PRMT5 and disrupted the interaction of PRMT5 with p65. Furthermore, our data indicate that blockade of PKCι-regulated PRMT5-mediated activation of NF-κB was likely through phosphorylation of PRMT5 at S15. Finally, inhibition of PKCι or overexpression of the S15A mutant attenuated the growth, migratory, and colony-forming abilities of CRC cells compared to the WT-PRMT5. Collectively, we have identified a novel PKCι/PRMT5/NF-κB signaling axis, suggesting that pharmacological disruption of this pivotal axis could serve as the basis for new anti-cancer therapeutics.


Assuntos
Neoplasias Colorretais/metabolismo , NF-kappa B/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Transdução de Sinais , Substituição de Aminoácidos , Proliferação de Células , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Fosforilação , Proteína Quinase C/metabolismo , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Serina/genética , Serina/metabolismo
15.
Mol Cell Proteomics ; 19(5): 852-870, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32132231

RESUMO

The redox-based modifications of cysteine residues in proteins regulate their function in many biological processes. The gas molecule H2S has been shown to persulfidate redox sensitive cysteine residues resulting in an H2S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent in detecting cysteine modifications. Here we developed a TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. We revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as a novel substrate in pancreatic beta cells. Moreover, we showed that under oxidative stress conditions, increased H2S can target enzymes involved in energy metabolism by switching specific cysteine modifications to persulfides. Specifically, we discovered a Redox Thiol Switch, from protein S-glutathioinylation to S-persulfidation (RTSGS). We propose that the RTSGS from S-glutathioinylation to S-persulfidation is a potential mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.


Assuntos
Metabolismo Energético , Compostos de Sulfidrila/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Animais , Bioensaio , Biotina/metabolismo , Linhagem Celular , Cisteína/metabolismo , Dissulfetos/metabolismo , Glicólise , Hepatócitos/metabolismo , Humanos , Sulfeto de Hidrogênio/metabolismo , Células Secretoras de Insulina/metabolismo , Espectrometria de Massas , Análise do Fluxo Metabólico , Mitocôndrias/metabolismo , Oxirredução , Proteoma/metabolismo , Proteômica , Ratos , Sulfetos/metabolismo
16.
Biochim Biophys Acta Mol Basis Dis ; 1864(11): 3595-3604, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30251679

RESUMO

Cysteine (Cys) residues are major causes of crystallin disulfide formation and aggregation in aging and cataractous human lenses. We recently found that disulfide linkages are highly and partly conserved in ß- and γ-crystallins, respectively, in human age-related nuclear cataract and glutathione depleted LEGSKO mouse lenses, and could be mimicked by in vitro oxidation. Here we determined which Cys residues are involved in disulfide-mediated crosslinking of recombinant human γD-crystallin (hγD). In vitro diamide oxidation revealed dimer formation by SDS-PAGE and LC-MS analysis with Cys 111-111 and C111-C19 as intermolecular disulfides and Cys 111-109 as intramolecular sites. Mutation of Cys111 to alanine completely abolished dimerization. Addition of αB-crystallin was unable to protect Cys 111 from dimerization. However, Cu2+-induced hγD-crystallin aggregation was suppressed up to 50% and 80% by mutants C109A and C111A, respectively, as well as by total glutathionylation. In contrast to our recently published results using ICAT-labeling method, manual mining of the same database confirmed the specific involvement of Cys111 in disulfides with no free Cys111 detectable in γD-crystallin from old and cataractous human lenses. Surface accessibility studies show that Cys111 in hγD is the most exposed Cys residue (29%), explaining thereby its high propensity toward oxidation and polymerization in the aging lens.


Assuntos
Catarata/patologia , Cisteína/metabolismo , Agregação Patológica de Proteínas/patologia , Multimerização Proteica/genética , gama-Cristalinas/metabolismo , Adolescente , Fatores Etários , Idoso , Catarata/genética , Cátions Bivalentes/toxicidade , Criança , Pré-Escolar , Cobre/toxicidade , Dissulfetos/metabolismo , Glutationa/farmacologia , Humanos , Cristalino/efeitos dos fármacos , Cristalino/metabolismo , Cristalino/patologia , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , Estresse Oxidativo/efeitos dos fármacos , Agregação Patológica de Proteínas/induzido quimicamente , Agregação Patológica de Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , gama-Cristalinas/química , gama-Cristalinas/genética
17.
J Biol Chem ; 293(26): 10009-10025, 2018 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-29743242

RESUMO

The HIV trans-activator Tat recruits the host transcription elongation factor P-TEFb to stimulate proviral transcription. Phosphorylation of Thr-186 on the activation loop (T-loop) of cyclin-dependent kinase 9 (CDK9) is essential for its kinase activity and assembly of CDK9 and cyclin T1 (CycT1) to form functional P-TEFb. Phosphorylation of a second highly conserved T-loop site, Ser-175, alters the competitive binding of Tat and the host recruitment factor bromodomain containing 4 (BRD4) to P-TEFb. Here, we investigated the intracellular mechanisms that regulate these key phosphorylation events required for HIV transcription. Molecular dynamics simulations revealed that the CDK9/CycT1 interface is stabilized by intramolecular hydrogen bonding of pThr-186 by an arginine triad and Glu-96 of CycT1. Arginine triad substitutions that disrupted CDK9/CycT1 assembly accumulated Thr-186-dephosphorylated CDK9 associated with the cytoplasmic Hsp90/Cdc37 chaperone. The Hsp90/Cdc37/CDK9 complex was also present in resting T cells, which lack CycT1. Hsp90 inhibition in primary T cells blocked P-TEFb assembly, disrupted Thr-186 phosphorylation, and suppressed proviral reactivation. The selective CDK7 inhibitor THZ1 blocked CDK9 phosphorylation at Ser-175, and in vitro kinase assays confirmed that CDK7 activity is principally responsible for Ser-175 phosphorylation. Mutation of Ser-175 to Lys had no effect on CDK9 kinase activity or P-TEFb assembly but strongly suppressed both HIV expression and BRD4 binding. We conclude that the transfer of CDK9 from the Hsp90/Cdc37 complex induced by Thr-186 phosphorylation is a key step in P-TEFb biogenesis. Furthermore, we demonstrate that CDK7-mediated Ser-175 phosphorylation is a downstream nuclear event essential for facilitating CDK9 T-loop interactions with Tat.


Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , HIV-1/fisiologia , Fator B de Elongação Transcricional Positiva/metabolismo , Ativação Viral , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Quinase 9 Dependente de Ciclina/química , Ativação Enzimática , HIV-1/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Células Jurkat , Fosforilação , Ligação Proteica , Serina/metabolismo , Treonina/metabolismo
18.
Cell Rep ; 21(11): 3129-3140, 2017 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-29241541

RESUMO

Adipose tissue stores energy in the form of triglycerides. The ability to regulate triglyceride synthesis and breakdown based on nutrient status (e.g., fed versus fasted) is critical for physiological homeostasis and dysregulation of this process can contribute to metabolic disease. Whereas much is known about hormonal control of this cycle, transcriptional regulation is not well understood. Here, we show that the transcription factor Kruppel-like factor 15 (KLF15) is critical for the control of adipocyte lipid turnover. Mice lacking Klf15 in adipose tissue (AK15KO) display decreased adiposity and are protected from diet-induced obesity. Mechanistic studies suggest that adipose KLF15 regulates key genes of triglyceride synthesis and inhibits lipolytic action, thereby promoting lipid storage in an insulin-dependent manner. Finally, AK15KO mice demonstrate accelerated lipolysis and altered systemic energetics (e.g., locomotion, ketogenesis) during fasting conditions. Our study identifies adipose KLF15 as an essential regulator of adipocyte lipid metabolism and systemic energy balance.


Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Proteínas de Ligação a DNA/genética , Glucose/metabolismo , Lipogênese/genética , Lipólise/genética , Fatores de Transcrição/genética , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/deficiência , Jejum/fisiologia , Regulação da Expressão Gênica , Glucose/farmacologia , Humanos , Insulina/metabolismo , Insulina/farmacologia , Fatores de Transcrição Kruppel-Like , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais , Fatores de Transcrição/deficiência , Triglicerídeos/metabolismo
19.
PLoS One ; 12(12): e0188614, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29228007

RESUMO

BACKGROUND: An association of acetaminophen use and asthma was observed in the International Study of Asthma and Allergies in Childhood study. However there are no clear mechanisms to explain an association between acetaminophen use and immunologic pathology. In acidic conditions like those in the stomach and inflamed airway, tyrosine residues are nitrated by nitrous and peroxynitrous acids. The resulting nitrotyrosine is structurally similar to 2,4-dinitrophenol and 2,4-dinitrochlorobenzene, known haptens that enhance immune responses by covalently binding proteins. Nitrated acetaminophen shares similar molecular structure. OBJECTIVE: We hypothesized the acetaminophen phenol ring undergoes nitration under acidic conditions, producing 3-nitro-acetaminophen which augments allergic responses by acting as a hapten for environmental allergens. METHODS: 3-nitro-acetaminophen was formed from acetaminophen in the presence of acidified nitrite, purified by high performance liquid chromatography, and assayed by gas-chromatography mass spectrometry. Purified 3-nitro-acetaminophen was reacted with Dermatophagoides pteronyssinus (Der p1) and analyzed by mass spectrometry to identify the modification site. Human peripheral blood mononuclear cells proliferation response was measured in response to 3-nitro-acetaminophen and to 3-nitro-acetaminophen-modified Der p1. RESULTS: Acetaminophen was modified by nitrous acid forming 3-nitro-acetaminophen over a range of different acidic conditions consistent with airway inflammation and stomach acidity. The Der p1 protein-hapten adduct creation was confirmed by liquid chromatography-mass spectrometry proteomics modifying cysteine 132. Peripheral blood mononuclear cells exposed to 3-nitro-acetaminophen-modified Der p1 had increased proliferation and cytokine production compared to acetaminophen and Der p1 alone (n = 7; p < 0.05). CONCLUSION: These data suggests 3-nitro-acetaminophen formation and reaction with Der p1 provides a mechanism by which stomach acid or infection-induced low airway pH in patients could enhance the allergic response to proteins such as Der p1.


Assuntos
Acetaminofen/química , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Cisteína Endopeptidases/imunologia , Monócitos/imunologia , Nitratos/química , Animais , Antígenos de Dermatophagoides/química , Proteínas de Artrópodes/química , Asma/imunologia , Cisteína Endopeptidases/química , Dermatophagoides pteronyssinus/imunologia , Humanos
20.
Proc Natl Acad Sci U S A ; 114(33): E6912-E6921, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28760953

RESUMO

It has been proposed that CD6, an important regulator of T cells, functions by interacting with its currently identified ligand, CD166, but studies performed during the treatment of autoimmune conditions suggest that the CD6-CD166 interaction might not account for important functions of CD6 in autoimmune diseases. The antigen recognized by mAb 3A11 has been proposed as a new CD6 ligand distinct from CD166, yet the identity of it is hitherto unknown. We have identified this CD6 ligand as CD318, a cell surface protein previously found to be present on various epithelial cells and many tumor cells. We found that, like CD6 knockout (KO) mice, CD318 KO mice are also protected in experimental autoimmune encephalomyelitis. In humans, we found that CD318 is highly expressed in synovial tissues and participates in CD6-dependent adhesion of T cells to synovial fibroblasts. In addition, soluble CD318 is chemoattractive to T cells and levels of soluble CD318 are selectively and significantly elevated in the synovial fluid from patients with rheumatoid arthritis and juvenile inflammatory arthritis. These results establish CD318 as a ligand of CD6 and a potential target for the diagnosis and treatment of autoimmune diseases such as multiple sclerosis and inflammatory arthritis.


Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Neoplasias/imunologia , Encefalomielite Autoimune Experimental/imunologia , Glicoproteínas de Membrana/imunologia , Células A549 , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Humanos , Ligantes , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
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