RESUMO
MicroRNAs (miRNAs) are small RNA molecules, typically 21â22 nucleotides in size, which play a crucial role in regulating gene expression in most eukaryotes. Their significance in various biological processes and disease pathogenesis has led to considerable interest in their potential as biomarkers for diagnosis and therapeutic applications. In this study, a novel method for sensing target miRNAs using Tailed-Hoogsteen triplex DNA-encapsulated Silver Nanoclusters (DNA/AgNCs) is introduced. Upon hybridization of a miRNA with the tail, the Tailed-Hoogsteen triplex DNA/AgNCs exhibit a pronounced red fluorescence, effectively turning on the signal. It is successfully demonstrated that this miRNA sensor not only recognized target miRNAs in total RNA extracted from cells but also visualized target miRNAs when introduced into live cells, highlighting the advantages of the turn-on mechanism. Furthermore, through gel-fluorescence assays and small-angle X-ray scattering (SAXS) analysis, the turn-on mechanism is elucidated, revealing that the Tailed-Hoogsteen triplex DNA/AgNCs undergo a structural transition from a monomer to a dimer upon sensing the target miRNA. Overall, the findings suggest that Tailed-Hoogsteen triplex DNA/AgNCs hold great promise as practical sensors for small RNAs in both in vitro and cell imaging applications.
Assuntos
Nanopartículas Metálicas , MicroRNAs , MicroRNAs/genética , MicroRNAs/análise , Prata/química , Espalhamento a Baixo Ângulo , Difração de Raios X , DNA/química , Espectrometria de Fluorescência/métodos , Nanopartículas Metálicas/químicaRESUMO
The redox regulation, maintaining a balance between oxidation and reduction in living cells, is vital for cellular homeostasis, intricate signaling networks, and appropriate responses to physiological and environmental cues. Here, a novel redox sensor, based on DNA-encapsulated silver nanoclusters (DNA/AgNCs) and well-defined chemical fluorophores, effectively illustrating cellular redox states in live cells is introduced. Among various i-motif DNAs, the photophysical property of poly-cytosines (C20)-encapsulated AgNCs that sense reactive oxygen species (ROS) is adopted. However, the sensitivity of C20/AgNCs is insufficient for evaluating ROS levels in live cells. To overcome this drawback, the ROS sensing mechanism of C20/AgNCs through gel electrophoresis, mass spectrometry, and small-angle X-ray scattering is primarily defined. Then, by tethering fluorescein amidite (FAM) and Cyanine 5 (Cy5) dyes to each end of the C20/AgNCs sensor, an Energy Transfer (ET) between AgNCs and FAM is achieved, resulting in intensified green fluorescence upon ROS detection. Taken together, the FAM-C20/AgNCs-Cy5 redox sensor enables dynamic visualization of intracellular redox states, yielding insights into oxidative stress-related processes in live cells.
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Intestinal inflammation is the underlying basis of colitis and the inflammatory bowel diseases. These syndromes originate from genetic and environmental factors that remain to be fully identified. Infections are possible disease triggers, including recurrent human food-poisoning by the common foodborne pathogen Salmonella enterica Typhimurium (ST), which in laboratory mice causes progressive intestinal inflammation leading to an enduring colitis. In this colitis model, disease onset has been linked to Toll-like receptor-4-dependent induction of intestinal neuraminidase activity, leading to the desialylation, reduced half-life, and acquired deficiency of anti-inflammatory intestinal alkaline phosphatase (IAP). Neuraminidase (Neu) inhibition protected against disease onset; however, the source and identity of the Neu enzyme(s) responsible remained unknown. Herein, we report that the mammalian Neu3 neuraminidase is responsible for intestinal IAP desialylation and deficiency. Absence of Neu3 thereby prevented the accumulation of lipopolysaccharide-phosphate and inflammatory cytokine expression in providing protection against the development of severe colitis.
Assuntos
Colite/imunologia , Intestinos/imunologia , Neuraminidase/imunologia , Intoxicação Alimentar por Salmonella/imunologia , Animais , Colite/genética , Colite/microbiologia , Modelos Animais de Doenças , Feminino , Humanos , Intestinos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuraminidase/genética , Recidiva , Intoxicação Alimentar por Salmonella/genética , Intoxicação Alimentar por Salmonella/microbiologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/fisiologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
p21WAF1/Cip1 acts as a key negative regulator of cell cycle progression, which can form complexes with cyclin-dependent kinases together with specific cyclins to induce cell cycle arrest at specific stages. p21 protein levels have been shown to be regulated primarily through phosphorylation and ubiquitination during various stages of the cell cycle. Although phosphorylation and ubiquitin-dependent proteasomal degradation of p21 have been well established, other post-translational modifications that contribute to regulation of p21 stability and function remain to be further elucidated. Here, we show that p21 degradation and its function are controlled by tankyrases, which are members of the poly(ADP-ribose) polymerase (PARP) protein family. p21 interacts with tankyrases via newly defined tankyrase-binding motifs and is PARylated by tankyrases in vitro and in vivo, suggesting that PARylation is a new post-translational modification of p21. Up-regulation of tankyrases induces ubiquitin-dependent proteasomal degradation of p21 through an E3 ligase RNF146, thus promoting cell cycle progression in the G1/S phase transition. On the contrary, inhibition of tankyrases by knockdown or inhibitor treatment stabilizes p21 protein and leads to cell cycle arrest in the G1 phase. Together, our data demonstrate that tankyrase may function as a new molecular regulator that controls the protein levels of p21 through PARylation-dependent proteasomal degradation. Hence, a novel function of the tankyrase-p21 axis may represent a new avenue for regulating cell cycle progression.
Assuntos
Tanquirases , Tanquirases/química , Tanquirases/metabolismo , Poli ADP Ribosilação , Ubiquitinação , Ciclo Celular , Ubiquitinas/metabolismoRESUMO
The Hippo pathway controls organ size and tissue homeostasis by regulating cell proliferation and apoptosis. The LATS-mediated negative feedback loop prevents excessive activation of the effectors YAP/TAZ, maintaining homeostasis of the Hippo pathway. YAP and TAZ are hyperactivated in various cancer cells which lead to tumor growth. Aberrantly increased O-GlcNAcylation has recently emerged as a cause of hyperactivation of YAP in cancer cells. However, the mechanism, which induces hyperactivation of TAZ and blocks LATS-mediated negative feedback, remains to be elucidated in cancer cells. This study found that in breast cancer cells, abnormally increased O-GlcNAcylation hyperactivates YAP/TAZ and inhibits LATS2, a direct negative regulator of YAP/TAZ. LATS2 is one of the newly identified O-GlcNAcylated components in the MST-LATS kinase cascade. Here, we found that O-GlcNAcylation at LATS2 Thr436 interrupted its interaction with the MOB1 adaptor protein, which connects MST to LATS2, leading to activation of YAP/TAZ by suppressing LATS2 kinase activity. LATS2 is a core component in the LATS-mediated negative feedback loop. Thus, this study suggests that LATS2 O-GlcNAcylation is deeply involved in tumor growth by playing a critical role in dysregulation of the Hippo pathway in cancer cells.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Proliferação de Células , Células HEK293 , Via de Sinalização Hippo , Homeostase , Humanos , FosforilaçãoRESUMO
Unlike other types of glycosylation, O-GlcNAcylation is a single glycosylation which occurs exclusively in the nucleus and cytosol. O-GlcNAcylation underlie metabolic diseases, including diabetes and obesity. Furthermore, O-GlcNAcylation affects different oncogenic processes such as osteoblast differentiation, adipogenesis and hematopoiesis. Emerging evidence suggests that skeletal muscle differentiation is also regulated by O-GlcNAcylation, but the detailed molecular mechanism has not been fully elucidated. In this study, we showed that hyper-O-GlcNAcylation reduced the expression of myogenin, a transcription factor critical for terminal muscle development, in C2C12 myoblasts differentiation by O-GlcNAcylation on Thr9 of myocyte-specific enhancer factor 2c. Furthermore, we showed that O-GlcNAcylation on Mef2c inhibited its DNA binding affinity to myogenin promoter. Taken together, we demonstrated that hyper-O-GlcNAcylation attenuates skeletal muscle differentiation by increased O-GlcNAcylation on Mef2c, which downregulates its DNA binding affinity.
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Acetilglucosamina/metabolismo , Diferenciação Celular , Desenvolvimento Muscular , Mioblastos/citologia , Acilação , Animais , Linhagem Celular , Glicosilação , Células HEK293 , Humanos , Fatores de Transcrição MEF2/metabolismo , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/metabolismoRESUMO
The composition and functions of the secreted proteome are controlled by the life spans of different proteins. However, unlike intracellular protein fate, intrinsic factors determining secreted protein aging and turnover have not been identified and characterized. Almost all secreted proteins are posttranslationally modified with the covalent attachment of N-glycans. We have discovered an intrinsic mechanism of secreted protein aging and turnover linked to the stepwise elimination of saccharides attached to the termini of N-glycans. Endogenous glycosidases, including neuraminidase 1 (Neu1), neuraminidase 3 (Neu3), beta-galactosidase 1 (Glb1), and hexosaminidase B (HexB), possess hydrolytic activities that temporally remodel N-glycan structures, progressively exposing different saccharides with increased protein age. Subsequently, endocytic lectins with distinct binding specificities, including the Ashwell-Morell receptor, integrin αM, and macrophage mannose receptor, are engaged in N-glycan ligand recognition and the turnover of secreted proteins. Glycosidase inhibition and lectin deficiencies increased protein life spans and abundance, and the basal rate of N-glycan remodeling varied among distinct proteins, accounting for differences in their life spans. This intrinsic multifactorial mechanism of secreted protein aging and turnover contributes to health and the outcomes of disease.
Assuntos
Proteínas/metabolismo , Glicosilação , Polissacarídeos/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Protein O-phosphorylation often occurs reciprocally with O-GlcNAc modification and represents a regulatory principle for proteins. O-phosphorylation of serine by glycogen synthase kinase-3ß on Snail1, a transcriptional repressor of E-cadherin and a key regulator of the epithelial-mesenchymal transition (EMT) programme, results in its proteasomal degradation. We show that by suppressing O-phosphorylation-mediated degradation, O-GlcNAc at serine112 stabilizes Snail1 and thus increases its repressor function, which in turn attenuates E-cadherin mRNA expression. Hyperglycaemic condition enhances O-GlcNAc modification and initiates EMT by transcriptional suppression of E-cadherin through Snail1. Thus, dynamic reciprocal O-phosphorylation and O-GlcNAc modification of Snail1 constitute a molecular link between cellular glucose metabolism and the control of EMT.
Assuntos
Acetilglucosamina/metabolismo , Hiperglicemia/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Regulação da Expressão Gênica , Glucose/metabolismo , Células HEK293 , Células HeLa , Humanos , Dados de Sequência Molecular , Fosforilação , Estabilidade Proteica , RNA Mensageiro/genética , Serina/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genéticaRESUMO
Binding of selectins to their glycan ligands is a prerequisite for successful leukocyte trafficking. During synthesis and transport through the secretory pathway, selectin ligands are constructed with the participation of one or more sialyltransferases of the ST3Gal subfamily. Previous studies established that ST3Gal-IV only partially contributes to selectin ligand formation, indicating that other ST3Gal-sialyltransferases are involved. By generating and analyzing St3gal6-null mice and St3gal4/St3gal6 double-deficient mice, in the present study, we found that binding of E- and P-selectin to neutrophils and L-selectin binding to lymph node high endothelial venules is reduced in the absence of ST3Gal-VI and to a greater extent in double-deficient mice. In an ex vivo flow chamber assay, P- and E-selectin-dependent leukocyte rolling was mildly reduced in St3gal6-null mice and more severely in double-deficient mice. In inflamed cremaster muscle venules of St3gal6-null mice, we found impaired P-selectin-dependent, but not E-selectin-dependent leukocyte rolling, whereas in double-deficient mice, E-selectin-dependent rolling was almost completely absent. Furthermore, neutrophil recruitment into the inflamed peritoneal cavity and lymphocyte homing to secondary lymphoid organs were impaired in St3gal6-null mice and more severely in double-deficient mice. The results of the present study demonstrate the coordinated participation of both ST3Gal-VI and ST3Gal-IV in the synthesis of functional selectin ligands.
Assuntos
Selectinas/biossíntese , Sialiltransferases/fisiologia , Animais , Capilares/metabolismo , Capilares/fisiologia , Selectina E/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Hemostasia/genética , Migração e Rolagem de Leucócitos/genética , Ligantes , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Selectina-P/metabolismo , Ligação Proteica , Fluxo Sanguíneo Regional/genética , Fluxo Sanguíneo Regional/fisiologia , Selectinas/metabolismo , Sialiltransferases/genética , Sialiltransferases/metabolismo , Distribuição Tecidual , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
We analyzed national data on blood lead levels (BLL) and blood cadmium levels (BCL) in residents living near 38 abandoned metal mining areas (n = 5,682, 18-96 years old) in Korea that were collected by the first Health Effect Surveillance for Residents in Abandoned Metal mines (HESRAM) from 2008 to 2011. The geometric mean BCL and BLL were 1.60 µg/L (95 % CI = 1.57-1.62 µg/L) and 2.87 µg/dL (95 % CI = 2.84-2.90 µg/dL), respectively, notably higher than levels in the general population in Korea and other countries. We found significantly higher BLL and BCL levels in people living within 2 km of an abandoned metal mine (n = 3,165, BCL = 1.87 µg/L, BLL = 2.91 µg/dL) compared to people living more than 2 km away (n = 2,517, BCL = 1.31 µg/L, BLL = 2.82 µg/dL; P < 0.0001) and to the general population values reported in the literature.
Assuntos
Cádmio/sangue , Exposição Ambiental/estatística & dados numéricos , Poluentes Ambientais/sangue , Chumbo/sangue , Mineração , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Monitoramento Ambiental , Feminino , Humanos , Masculino , Metais/sangue , Pessoa de Meia-Idade , República da Coreia , Adulto JovemRESUMO
Dysregulation of O-GlcNAcylation has emerged as a potential biomarker for several diseases, particularly cancer. The role of OGT (O-GlcNAc transferase) in maintaining O-GlcNAc homeostasis has been extensively studied; nevertheless, the regulation of OGA (O-GlcNAcase) in cancer remains elusive. Here, we demonstrated that the multifunctional protein RBM14 is a regulator of cellular O-GlcNAcylation. By investigating the correlation between elevated O-GlcNAcylation and increased RBM14 expression in lung cancer cells, we discovered that RBM14 promotes ubiquitin-dependent proteasomal degradation of OGA, ultimately mediating cellular O-GlcNAcylation levels. In addition, RBM14 itself is O-GlcNAcylated at serine 521, regulating its interaction with the E3 ligase TRIM33, consequently affecting OGA protein stability. Moreover, we demonstrated that mutation of serine 521 to alanine abrogated the oncogenic properties of RBM14. Collectively, our findings reveal a previously unknown mechanism for the regulation of OGA and suggest a potential therapeutic target for the treatment of cancers with dysregulated O-GlcNAcylation.
Assuntos
Estabilidade Proteica , Proteínas de Ligação a RNA , Humanos , Acetilglucosamina/metabolismo , Antígenos de Neoplasias , beta-N-Acetil-Hexosaminidases/metabolismo , Linhagem Celular Tumoral , Glicosilação , Células HEK293 , Histona Acetiltransferases , Hialuronoglucosaminidase , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , N-Acetilglucosaminiltransferases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Post-translational addition of O-linked N-acetylglucosamine (O-GlcNAc) to p53 is known to occur, but the site of O-GlcNAcylation and its effects on p53 are not understood. Here, we show that Ser 149 of p53 is O-GlcNAcylated and that this modification is associated with decreased phosphorylation of p53 at Thr 155, which is a site that is targeted by the COP9 signalosome, resulting in decreased p53 ubiquitination. Accordingly, O-GlcNAcylation at Ser 149 stabilizes p53 by blocking ubiquitin-dependent proteolysis. Our results indicate that the dynamic interplay between O-GlcNAc and O-phosphate modifications coordinately regulate p53 stability and activity.
Assuntos
Acetilglucosamina/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias Pulmonares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismo , Sobrevivência Celular , Eletroforese em Gel Bidimensional , Humanos , Imunoprecipitação , Fosfatos/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Tumorais CultivadasRESUMO
Necroptosis is a type of cell death with excessive inflammation and organ damage in various human diseases. Although abnormal necroptosis is common in patients with neurodegenerative, cardiovascular, and infectious diseases, the mechanisms by which O-GlcNAcylation contributes to the regulation of necroptotic cell death are poorly understood. In this study, we reveal that O-GlcNAcylation of RIPK1 (receptor-interacting protein kinase1) was decreased in erythrocytes of the mouse injected with lipopolysaccharide, resulting in the acceleration of erythrocyte necroptosis through increased formation of RIPK1-RIPK3 complex. Mechanistically, we discovered that O-GlcNAcylation of RIPK1 at serine 331 in human (corresponding to serine 332 in mouse) inhibits phosphorylation of RIPK1 at serine 166, which is necessary for the necroptotic activity of RIPK1 and suppresses the formation of the RIPK1-RIPK3 complex in Ripk1 -/- MEFs. Thus, our study demonstrates that RIPK1 O-GlcNAcylation serves as a checkpoint to suppress necroptotic signaling in erythrocytes.
Assuntos
Apoptose , Necroptose , Humanos , Camundongos , Animais , Necrose , Apoptose/fisiologia , Eritrócitos/metabolismo , Serina , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
This study aims to propose an indoor air quality prediction method that can be easily utilized and reflects temporal characteristics using indoor and outdoor input data measured near the indoor target point as input to calculate indoor PM2.5 concentration through a multiple linear regression model. The atmospheric conditions and air pollution detected in one-minute intervals using sensor-based monitoring equipment (Dust Mon, Sentry Co Ltd., Seoul, Korea) inside and outside houses from May 2019 to April 2021 were used to develop the prediction model. By dividing the multiple linear regression model into one-hour increments, we attempted to overcome the limitation of not representing the multiple linear regression model's characteristics over time and limited input variables. The multiple linear regression (MLR) model classified by time unit showed an improvement in explanatory power by up to 9% compared to the existing model, and some hourly models had an explanatory power of 0.30. These results indicated that the model needs to be subdivided by time period to more accurately predict indoor PM2.5 concentrations.
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The colonic mucosal barrier protects against infection, inflammation, and tissue ulceration. Composed primarily of Mucin-2, proteolytic erosion of this barrier is an invariant feature of colitis; however, the molecular mechanisms are not well understood. We have applied a recurrent food poisoning model of acquired inflammatory bowel disease using Salmonella enterica Typhimurium to investigate mucosal barrier erosion. Our findings reveal an innate Toll-like receptor 4-dependent mechanism activated by previous infection that induces Neu3 neuraminidase among colonic epithelial cells concurrent with increased Cathepsin-G protease secretion by Paneth cells. These anatomically separated host responses merge with the desialylation of nascent colonic Mucin-2 by Neu3 rendering the mucosal barrier susceptible to increased proteolytic breakdown by Cathepsin-G. Depletion of Cathepsin-G or Neu3 function using pharmacological inhibitors or genetic-null alleles protected against Mucin-2 proteolysis and barrier erosion and reduced the frequency and severity of colitis, revealing approaches to preserve and potentially restore the mucosal barrier.
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Gangliosides-sialylated glycosphingolipids-are the major glycoconjugates of nerve cells. The same four structures-GM1, GD1a, GD1b and GT1b-comprise the great majority of gangliosides in mammalian brains. They share a common tetrasaccharide core (Galß1-3GalNAcß1-4Galß1-4Glcß1-1'Cer) with one or two sialic acids on the internal galactose and zero (GM1 and GD1b) or one (GD1a and GT1b) α2-3-linked sialic acid on the terminal galactose. Whereas the genes responsible for the sialylation of the internal galactose are known, those responsible for terminal sialylation have not been established in vivo. We report that St3gal2 and St3gal3 are responsible for nearly all the terminal sialylation of brain gangliosides in the mouse. When brain ganglioside expression was analyzed in adult St3gal1-, St3gal2-, St3gal3- and St3gal4-null mice, only St3gal2-null mice differed significantly from wild type, expressing half the normal amount of GD1a and GT1b. St3gal1/2-double-null mice were no different than St3gal2-single-null mice; however, St3gal2/3-double-null mice were >95% depleted in gangliosides GD1a and GT1b. Total ganglioside expression (lipid-bound sialic acid) in the brains of St3gal2/3-double-null mice was equivalent to that in wild-type mice, whereas total protein sialylation was reduced by half. St3gal2/3-double-null mice were small, weak and short lived. They were half the weight of wild-type mice at weaning and displayed early hindlimb dysreflexia. We conclude that the St3gal2 and St3gal3 gene products (ST3Gal-II and ST3Gal-III sialyltransferases) are largely responsible for ganglioside terminal α2-3 sialylation in the brain, synthesizing the major brain gangliosides GD1a and GT1b.
Assuntos
Encéfalo/metabolismo , Gangliosídeos/biossíntese , Animais , Camundongos , Camundongos Knockout , Sialiltransferases/deficiência , Sialiltransferases/metabolismo , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
The balance between cellular proliferation and apoptosis and the regulation of cell differentiation must be established to maintain tissue homeostasis. These cellular responses involve the kinase cascade-mediated Hippo pathway as a crucial regulator. Hence, Hippo pathway dysregulation is implicated in diverse diseases, including cancer. O-GlcNAcylation is a non-canonical glycosylation that affects multiple signaling pathways through its interplay with phosphorylation in the nucleus and cytoplasm. An abnormal increase in the O-GlcNAcylation levels in various cancer cells is a potent factor in Hippo pathway dysregulation. Intriguingly, Hippo pathway dysregulation also disrupts O-GlcNAc homeostasis, leading to a persistent elevation of O-GlcNAcylation levels, which is potentially pathogenic in several diseases. Therefore, O-GlcNAcylation is gaining attention as a protein modification that regulates the Hippo pathway. This review presents a framework on how O-GlcNAcylation regulates the Hippo pathway and forms a self-perpetuating cycle with it. The pathological significance of this self-perpetuating cycle and clinical strategies for targeting O-GlcNAcylation that causes Hippo pathway dysregulation are also discussed.
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BACKGROUND: Although sepsis accounts for 1 in 5 deaths globally, few molecular therapies exist for this condition. The development of effective biomarkers and treatments for sepsis requires a more complete understanding of host responses and pathogenic mechanisms at early stages of disease to minimize host-driven pathology. METHODS: An alternative to the current symptom-based approach used to diagnose sepsis is a precise assessment of blood proteomic changes during the onset and progression of Salmonella Typhimurium (ST) murine sepsis. FINDINGS: A distinct pattern of coagulation factor protein abundance was identified in the pre-septic state- prior to overt disease symptoms or bacteremia- that was predictive of the dysregulation of fibrinolytic and anti-coagulant activities and resultant consumptive coagulopathy during ST murine sepsis. Moreover, the changes in protein abundance observed generally have the same directionality (increased or decreased abundance) reported for human sepsis. Significant overlap of ST coagulopathic activities was observed in Gram-negative Escherichia coli- but not in Gram-positive staphylococcal or pneumococcal murine sepsis models. Treatment with matrix metalloprotease inhibitors prevented aberrant inflammatory and coagulopathic activities post-ST infection and increased survival. Antibiotic treatment regimens initiated after specific changes arise in the plasma proteome post-ST infection were predictive of an increase in disease relapse and death after cessation of antibiotic treatment. INTERPRETATION: Altered blood proteomics provides a platform to develop rapid and easy-to-perform tests to predict sepsis for early intervention via biomarker incorporation into existing blood tests prompted by patient presentation with general malaise, and to stratify Gram-negative and Gram-positive infections for appropriate treatment. Antibiotics are less effective in microbial clearance when initiated after the onset of altered blood proteomics as evidenced by increased disease relapse and death after termination of antibiotic therapy. Treatment failure is potentially due to altered bacterial / host-responses and associated increased host-driven pathology, providing insight into why delays in antibiotic administration in human sepsis are associated with increased risk for death. Delayed treatment may thus require prolonged therapy for microbial clearance despite the prevailing notion of antibiotic de-escalation and shortened courses of antibiotics to improve drug stewardship. FUNDING: National Institutes of Health, U.S. Army.
Assuntos
Bacteriemia , Infecções Pneumocócicas , Sepse , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bacteriemia/microbiologia , Biomarcadores , Fatores de Coagulação Sanguínea/uso terapêutico , Humanos , Camundongos , Infecções Pneumocócicas/tratamento farmacológico , Proteômica , Recidiva , Sepse/complicações , Sepse/tratamento farmacológicoRESUMO
The transcription factor NFkappaB is activated by phosphorylation and acetylation and plays important roles in inflammatory and immune responses in the cell. Additionally, posttranslational modification of the NFkappaB p65 subunit by O-linked N-acetylglucosamine (O-GlcNAc) has been reported, but the modification site of O-GlcNAc on NFkappaB p65 and its exact function have not been elucidated. In this work, we show that O-GlcNAcylation of NFkappaB p65 decreases binding to IkappaB alpha and increases transcriptional activity under hyperglycemic conditions. Also, we demonstrate that both Thr-322 and Thr-352 of NFkappaB p65 can be modified with O-GlcNAc, but modification on Thr-352, not Thr-322, is important for transcriptional activation. Our findings suggest that site-specific O-GlcNAcylation may be a reason why NFkappaB activity increases continuously under hyperglycemic conditions.