RESUMO
BACKGROUND: The rising prevalence of many chronic diseases related to gut barrier dysfunction coincides with the increased global usage of dietary emulsifiers in recent decades. We therefore investigated the effect of the frequently used food emulsifiers on cytotoxicity, barrier function, transcriptome alterations, and protein expression in gastrointestinal epithelial cells. METHODS: Human intestinal organoids originating from induced pluripotent stem cells, colon organoid organ-on-a-chip, and liquid-liquid interface cells were cultured in the presence of two common emulsifiers: polysorbate 20 (P20) and polysorbate 80 (P80). The cytotoxicity, transepithelial electrical resistance (TEER), and paracellular-flux were measured. Immunofluorescence staining of epithelial tight-junctions (TJ), RNA-seq transcriptome, and targeted proteomics were performed. RESULTS: Cells showed lysis in response to P20 and P80 exposure starting at a 0.1% (v/v) concentration across all models. Epithelial barrier disruption correlated with decreased TEER, increased paracellular-flux and irregular TJ immunostaining. RNA-seq and targeted proteomics analyses demonstrated upregulation of cell development, signaling, proliferation, apoptosis, inflammatory response, and response to stress at 0.05%, a concentration lower than direct cell toxicity. A proinflammatory response was characterized by the secretion of several cytokines and chemokines, interaction with their receptors, and PI3K-Akt and MAPK signaling pathways. CXCL5, CXCL10, and VEGFA were upregulated in response to P20 and CXCL1, CXCL8 (IL-8), CXCL10, LIF in response to P80. CONCLUSIONS: The present study provides direct evidence on the detrimental effects of food emulsifiers P20 and P80 on intestinal epithelial integrity. The underlying mechanism of epithelial barrier disruption was cell death at concentrations between 1% and 0.1%. Even at concentrations lower than 0.1%, these polysorbates induced a proinflammatory response suggesting a detrimental effect on gastrointestinal health.
Assuntos
Fosfatidilinositol 3-Quinases , Polissorbatos , Humanos , Polissorbatos/efeitos adversos , Polissorbatos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Células Epiteliais/metabolismo , Citocinas/metabolismo , Dieta , Mucosa Intestinal/metabolismoRESUMO
Antigen presentation by major histocompatibility complex (MHC) proteins to T-cell receptors (TCRs) plays a crucial role in triggering the adaptive immune response. Most of our knowledge on TCR-peptide-loaded major histocompatibility complex (pMHC) interaction stemmed from experiments yielding static structures, yet the dynamic aspects of this molecular interaction are equally important to understand the underlying molecular mechanisms and to develop treatment strategies against diseases such as cancer and autoimmune diseases. To this end, computational biophysics studies including all-atom molecular dynamics simulations have provided useful insights; however, we still lack a basic understanding of an overall allosteric mechanism that results in conformational changes in the TCR and subsequent T-cell activation. Previous hydrogen-deuterium exchange and nuclear magnetic resonance studies provided clues regarding these molecular mechanisms, including global rigidification and allosteric effects on the constant domain of TCRs away from the pMHC interaction site. Here, we show that molecular dynamics simulations can be used to identify how this overall rigidification may be related to the allosteric communication within TCRs upon pMHC interaction via essential dynamics and nonbonded residue-residue interaction energy analyses. The residues taking part in the rigidification effect are highlighted with an intricate analysis on residue interaction changes, which lead to a detailed outline of the complex formation event. Our results indicate that residues of the Cß domain of TCRs show significant differences in their nonbonded interactions upon complex formation. Moreover, the dynamic cross correlations between these residues are also increased, in line with their nonbonded interaction energy changes. Altogether, our approach may be valuable for elucidating intramolecular allosteric changes in the TCR structure upon pMHC interaction in molecular dynamics simulations.
Assuntos
Complexo Principal de Histocompatibilidade , Simulação de Dinâmica Molecular , Comunicação , Peptídeos , Ligação Proteica , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
The connection of Epstein Barr virus (EBV) with diseases such as Burkitt Lymphoma, Hodgkin disease, multiple sclerosis, systemic lupus erythematosus and various B-cell lymphomas made EBV glycoproteins one of the most popular vaccine immunogens. As a protein being encoded by EBV, the viral membrane envelope protein gp350 is studied extensively due to its abundancy on the surface and its interaction with complementary receptor, CR2. The binding of CR2 and gp350 not only leads to the entrance of the virus to the B-cells, but also prevents CR2 and C3d protein interactions that are required for immune response. Thus, understanding the inhibition of gp350 activity is crucial for vaccine development. Although, the active residues on gp350 structure were determined by several mutational studies, the exact mechanism of CR2 binding is still not clear. To this end, we have performed molecular docking followed by molecular dynamics simulations and MM-PBSA on wildtype and several mutated gp350 and CR2 structures. Apart from identifying crucial amino acids, the results of per-residue decomposition energy analysis clarified the individual energy contributions of amino acids and were also found to be accurate in differentiating the active site residues in CR2 binding. Here, we highlight the role of binding region residues (linker-1) but more interestingly, the dynamic relation between the distant sites of gp350 (linker-2 and D3 residues) and CR2. These findings can lead further vaccine development strategies by pointing to the importance of computationally found novel regions that can be potentially used to modulate gp350 activity.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Aminoácidos/metabolismo , Anticorpos Monoclonais , Glicoproteínas/metabolismo , Herpesvirus Humano 4/metabolismo , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Receptores de Complemento 3d/química , Receptores de Complemento 3d/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
Telomeres, and telomere length in particular, have broad significance for genome biology and thus are prime research targets for complex diseases such as cancers. In this context, BRCA1 and BRCA2 gene mutations have been implicated in relationship to telomere length, and breast cancer susceptibility. Yet, the linkages among human genetic variation and telomere length in persons with high hereditary cancer risk are inadequately mapped. We report here original findings in 113 unrelated women at high hereditary risk for breast cancer, who were characterized for the BRCA1 and BRCA2 mutations using next-generation sequencing. Thirty-one BRCA2 and 21 BRCA1 mutations were identified in 47 subjects (41.6%). The women with a mutation in BRCA1 and/or BRCA2 genes had, on average, 12% shorter telomere compared to women with no BRCA1 or BRCA2 mutation (p = 0.0139). Moreover, the association between telomere length and BRCA mutation status held up upon stratified analysis in those with or without a breast cancer diagnosis. We also indentified two rare mutations, c.536_537insT and c.10078A>G, and a novel mutation c.8680C>G in BRCA2 that was studied further by homology modeling of the DNA binding tower domain of BRCA2 and the structure of the protein. These data collectively lend evidence to the idea that BRCA1 and BRCA2 mutations play a role in telomere length in women at high hereditary risk for breast cancer. Further clinical and diagnostics discovery research on BRCA1 and BRCA2 variation, telomere length, and breast cancer mechanistic linkages are called for in larger study samples.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Predisposição Genética para Doença , Mutação , Encurtamento do Telômero , Alelos , Proteína BRCA1/química , Proteína BRCA2/química , Feminino , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Moleculares , Conformação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Medição de Risco , Fatores de Risco , Relação Estrutura-AtividadeRESUMO
Caspases are members of a highly regulated aspartate-cysteine protease family which have important roles in apoptosis. Pharmaceutical studies focused on these molecules since they are involved in diseases such as cancer and neurodegenerative disorders. A small molecule which binds to the dimeric interface away from the binding site induces a conformational change that resembles the pro-caspase form of the molecule by shifting loop positions. The fluctuation mechanisms caused by mutations or binding of a ligand can explain the key mechanism for the function of that molecule. In this study, we performed molecular dynamics simulations on wild-type and mutated structures (C290N, R187M, Y223A, G188L and G188P) as well as allosterically inhibited structure (DICA-bound caspase-7) to observe the effects of the single mutations on intrinsic dynamics. The results show that previously known changes in catalytic activity upon mutations or allosteric ligand binding are reflected in corresponding changes in the global dynamics of caspase-7. Communicated by Ramaswamy H. Sarma.