RESUMO
On a daily basis, the skin is exposed to many environmental stressors and insults. Over a 24-h natural cycle, during the day, the skin is focused on protection; while at night, the skin is focused on repairing damage that occurred during daytime and getting ready for the next morning. Circadian rhythm provides the precise timing mechanism for engaging those different pathways necessary to keep a healthy skin through clock genes that are present in all skin cells. The strongest clue for determining cellular functions timing is through sensing light or absence of light (darkness). Here, we asked the question if blue light could be a direct entrainment signal to skin cells and also disrupt their circadian rhythm at night. Through a reporter assay for per1 transcription, we demonstrate that blue light at 410 nm decreases per1 transcription in keratinocytes, showing that epidermal skin cells can sense light directly and control their own clock gene expression. This triggers cells to "think" it is daytime even at nighttime. Elsewhere, we measured different skin cell damage because of blue light exposure (at different doses and times of exposure) vs. cells that were kept in full darkness. We show an increase in ROS production, DNA damage and inflammatory mediators. These deleterious effects can potentially increase overall skin damage over time and ultimately accelerates ageing.
La peau est exposée chaque jour à de nombreux facteurs de stress et traumatismes environnementaux. Pendant un cycle naturel de 24 heures, dans la journée, la peau est axée sur sa protection, tandis que la nuit, elle se concentre sur la réparation des lésions survenues pendant la journée en préparation du lendemain matin. Le rythme circadien assure un mécanisme de cadencement temporel précis pour engager les différentes voies nécessaires au maintien d'une peau saine à travers les gènes de l'horloge interne qui sont présents dans toutes les cellules cutanées. La perception de la lumière ou l'absence de lumière (obscurité) est le plus fort indice pour déterminer le cadencement des fonctions cellulaires. Nous nous sommes ici posé la question de savoir si la lumière bleue serait un signal d'entraînement direct pour les cellules de la peau également capable de perturber leur rythme circadien la nuit. À travers un essai utilisant un gène rapporteur pour la transcription de per1, nous démontrons que la lumière bleue à 410 nm diminue la transcription de per1 dans les kératinocytes, montrant que les cellules épidermiques peuvent détecter directement la lumière et contrôler l'expression de leurs propres gènes horloges. Cela incite les cellules à « penser ¼ que la journée a commencé, même pendant la nuit. Par ailleurs, nous avons mesuré différentes lésions des cellules cutanées suite à l'exposition à la lumière bleue (à différentes doses et durées d'exposition) par rapport aux cellules qui étaient maintenues dans une obscurité complète. Nous montrons une augmentation de la production d'espèces réactives de l'oxygène (ROS), des lésions de l'ADN et des médiateurs inflammatoires. Ces effets délétères peuvent potentiellement augmenter les lésions cutanées globales au fil du temps et en accélérer ultérieurement le vieillissement.
Assuntos
Ritmo Circadiano/efeitos da radiação , Luz , Pele/efeitos dos fármacos , Células Cultivadas , HumanosRESUMO
SIRT6 is a member of the sirtuin family, which is involved in multiple cellular pathways related to aging, inflammation, epigenetics, and a variety of other cellular functions, including DNA repair (1). Multiple pathways involving different cellular functions are impacted by the deacetylase activity of SIRT6. Genomic integrity is maintained by the capacity of SIRT6 to modulate the accessibility of DNA repair proteins. Glucose metabolism is suppressed by SIRT6 via the deacetylation of histones located at the promoter regions of multiple glycolytic genes and the corepression of hypoxia-inducible factor-1α. SIRT6 is also a corepressor of nuclear factor (NF)-κB, silencing NF-κB target genes through the deacetylation of histones at their promoters' regions. We used SIRT6 small-interfering RNA as a tool to modulate residual DNA damage and NF-κB expression in human dermal fibroblasts. We measured NF-κB levels in the presence or the absence of ultraviolet B (UVB). The impact of SIRT6 knockdown as shown by a decrease in SIRT6 messenger RNA levels resulted in residual DNA damage as evaluated by the comet assay. Our results show that NF-κB was increased significantly (up to 400%) due to SIRT6 silencing in the absence of UVB, illustrating the master regulatory function of SIRT6 in inflammation. We also found a significant increase in DNA damage without UV exposure as a result of SIRT6 silencing, indicating the importance of SIRT6 in DNA repair pathways in cultured human dermal fibroblasts.
Assuntos
Dano ao DNA , Fibroblastos/metabolismo , NF-kappa B/biossíntese , Sirtuínas/genética , Pele/metabolismo , Células Cultivadas , Reparo do DNA , Fibroblastos/efeitos da radiação , Técnicas de Silenciamento de Genes , Humanos , RNA/biossíntese , RNA Interferente Pequeno/química , Transdução de Sinais , Pele/citologia , Pele/efeitos da radiação , Raios UltravioletaRESUMO
The molecular interactions between the CD8 co-receptor dependent N15 and N26 T cell receptors (TCRs) and their common ligand, the vesicular stomatitis virus octapeptide (VSV8) bound to H-2Kb, were studied to define the docking orientation(s) of MHC class I restricted TCRs during immune recognition. Guided by the molecular surfaces of the crystallographically defined peptide/MHC and modeled TCRs, a series of mutations in exposed residues likely contacting the TCR ligand were analyzed for their ability to alter peptide-triggered IL-2 production in T cell transfectants. Critical residues which diminished antigen recognition by 1000 to 10,000-fold in molar terms were identified in both N15 Valpha (alphaE94A or alphaE94R, Y98A and K99) and Vbeta (betaR96A, betaW97A and betaD99A) CDR3 loops. Mutational analysis indicated that the Rp1 residue of VSV8 is critical for antigen recognition of N15 TCR, but R62 of H-2Kb is less critical. More importantly, the alphaE94R mutant could be fully complemented by a reciprocal charge reversal at Kb R62 (R62E). This result suggests a direct interaction between N15 TCR Valpha E94R and Kb R62E residues. As Rp1 of VSV8 is adjacent to R62 in the VSV8/Kb complex and essential for T cell activation, this orientation implies that the N15 Valpha CDR3 loop interacts with the N-terminal residues of VSV8 with the Valpha domain docking to the Kb alpha2 helix while the N15 Vbeta CDR3 loop interacts with the more C-terminal peptide residues and the Vbeta domain overlies the Kb alpha1 helix. An equivalent orientation is suggested for N26, a second VSV8/Kb specific TCR. Given that genetic analysis of two different class II MHC-restricted TCRs and two crystallographic studies of class I restricted TCRs offers a similar overall orientation of V domains relative to alpha-helices, these data raise the possibility of a common docking mode between TCRs and their ligands regardless of MHC restriction.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Células Apresentadoras de Antígenos/imunologia , Sítios de Ligação , Linfócitos T CD8-Positivos/imunologia , Clonagem Molecular , Cristalografia por Raios X , Humanos , Interleucina-2/biossíntese , Linfoma de Células B , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção , Células Tumorais CultivadasRESUMO
To study how the T cell receptor interacts with its cognate ligand, the MHC/peptide complex, we used site directed mutagenesis to generate single point mutants that alter amino acids in the CDR3beta loop of a H-2Kb restricted TCR (N30.7) specific for an immunodominant peptide N52-N59 (VSV8) derived from the vesicular stomatitis virus nucleocapsid. The effect of each mutation on antigen recognition was analyzed using wild type H-2Kb and VSV8 peptide, as well as H-2Kb and VSV8 variants carrying single replacements at residues known to be exposed to the TCR. These analyses revealed that point mutations at some positions in the CDR3beta loop abrogated recognition entirely, while mutations at other CDR3beta positions caused an altered pattern of antigen recognition over a broad area on the MHC/peptide surface. This area included the N-terminus of the peptide, as well as residues of the MHC alpha1 and alpha2 helices flanking this region. Assuming that the N30 TCR docks on the MHC/peptide with an orientation similar to that recently observed in two different TCR-MHC/peptide crystal structures, our findings would suggest that single amino acid alterations within CDR3beta can affect the interaction of the TCR with an MHC surface region distal from the predicted CDR3beta-Kb/VSV8 interface. Such unique recognition capabilities are generated with minimal alterations in the CDR3 loops of the TCR. These observations suggest the hypothesis that extensive changes in the recognition pattern due to small perturbations in the CDR3 structure appears to be a structural strategy for generating a highly diversified TCR repertoire with specificity for a wide variety of antigens.
Assuntos
Substituição de Aminoácidos , Antígenos Virais/imunologia , Antígenos H-2/imunologia , Proteínas do Nucleocapsídeo , Nucleocapsídeo/imunologia , Fragmentos de Peptídeos/imunologia , Mutação Puntual , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T Citotóxicos/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Antígenos Virais/química , Antígenos Virais/genética , Antígenos H-2/química , Antígenos H-2/genética , Hibridomas/imunologia , Interleucina-2/metabolismo , Ativação Linfocitária , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nucleocapsídeo/química , Nucleocapsídeo/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Relação Estrutura-Atividade , Linfócitos T Citotóxicos/metabolismo , Transfecção , Vírus da Estomatite Vesicular Indiana/genéticaRESUMO
Recently, a powerful approach for the detection of MHC/peptide-specific T cells has been made possible by the engineering of soluble-tetrameric MHC/peptide complexes, consisting of singly biotinylated MHC/peptide molecules bound to fluorescent-labeled streptavidin. These tetrameric molecules are thought to compensate for the low affinity and relative fast dissociation rate of the TCR/MHC-peptide interaction by increasing the avidity of this interaction, thus allowing the stable binding of MHC/peptide tetramers to TCR expressing cells. Here we describe a new more simplified procedure for obtaining MHC/peptide tetramers using the well-characterized H-2K(b)/VSV system. This procedure consists of the incorporation of an unpaired cysteine residue at the C-terminus of the H-2K(b) molecule, allowing site-specific biotinylation by a -SH-specific biotinylating reagent. The H-2K(b)/VSV tetramers bound only to hybridomas expressing H-2K(b)/VSV-specific TCRs. When coated on a plate, these tetramers were able to induce IL-2 release by those hybridomas. Furthermore, H-2K(b)/VSV tetramers bound to CTL populations obtained from mice immunized with VSV-peptide. The specificity of the binding was further refined by studying cross-recognition of VSV by CTL populations obtained from mice immunized with single amino acid substituted VSV peptide variants. H-2K(b)/VSV tetramers bound only to those CTL populations that cross-reacted with the wild-type VSV peptide. Our method provides a simple, efficient and inexpensive procedure for making MHC/peptide tetramers, a highly specific and very useful reagent with a number of important applications in basic and clinical T cell research.
Assuntos
Cisteína/genética , Antígenos H-2/genética , Proteínas do Nucleocapsídeo , Nucleocapsídeo/genética , Biotinilação , Reações Cruzadas , Cisteína/imunologia , Engenharia Genética , Antígenos H-2/imunologia , Interleucina-2/biossíntese , Ativação Linfocitária/imunologia , Nucleocapsídeo/imunologia , Peptídeos/genética , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Microglobulina beta-2/genética , Microglobulina beta-2/imunologiaRESUMO
As an approach to determine the structural basis of interactions between T cell receptors (TCRs) and MHC class I/peptide complexes, the fine specificities of a panel of vesicular stomatitis virus (VSV)-specific CTL clones recognizing the antigenic peptide (nucleoprotein 52-59) and the class I (Kb) molecule were correlated with the TCR primary structure. Each TCR showed a distinct interaction pattern with N52-59 and the Kb molecule. The large majority of the TCRs expressed by the panel of CTL clones used V beta 13 gene segments that had randomly recombined with D beta and J beta gene segments. The alpha chains were from randomly assorted V alpha and J alpha gene segments. Thus, the panel was found to be a highly heterogeneous set of TCRs, each member of which appeared to have an unique surface interface area, the recognition site, that interacted with a complementary surface formed by the single peptide bound in the class I antigenic groove.
Assuntos
Antígenos Virais/imunologia , Antígenos H-2/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T Citotóxicos/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Capsídeo/imunologia , Células Clonais , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas do Core Viral/imunologiaRESUMO
The surface residues of the VSV8/Kb complex important for recognition by N15 and N26 alphabeta T cell receptors (TCR) were mapped by mutational analysis and compared to each other and with epitopes of well-characterized Kb specific monoclonal antibodies (mAb). Three features of immune receptor recognition emerge. First, the footprints of the two TCR on VSV8/Kb are similar with more than 80 % overlap between sites. Given that only 8 of 14 surface exposed VSV8/Kb residues identified as critical for TCR interaction are in common, the chemical basis of the N15 and N26 interactions is nevertheless distinct. Second, the cognate peptide is a major focus of TCR recognition: mutation at any of the three exposed side chains (at p1, p4 or p6) abrogates interaction of both TCR as measured by functional T cell activation. Third, in contrast to TCR, mAb bind to discrete segments on the periphery of the alpha1 and/or alpha2 helices without orientational restriction. These findings suggest that unlike soluble antibodies, surface membrane receptor-ligand interactions on opposing cells (i.e. TCR-peptide/ MHC, CD8-MHC) limit the orientational freedom of the TCR in the immune recognition process.
Assuntos
Antígenos Virais/imunologia , Antígenos H-2/fisiologia , Receptores de Antígenos de Linfócitos T/química , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Anticorpos Monoclonais/imunologia , Mapeamento de Epitopos , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Peptídeos/imunologia , Conformação Proteica , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Cytotoxic T cell (CTL) activation by antigen requires the specific detection of peptide-major histocompatibility class I (pMHC) molecules on the target-cell surface by the T cell receptor (TCR). We examined the effect of mutations in the antigen-binding site of a Kb-restricted TCR on T cell activation, antigen binding and dissociation from antigen.These parameters were also examined for variants derived from a Kd-restricted peptide that was recognized by a CTL clone. Using these two independent systems, we show that T cell activation can be impaired by mutations that either decrease or increase the binding half-life of the TCR-pMHC interaction. Our data indicate that efficient T cell activation occurs within an optimal dwell-time range of TCR-pMHC interaction. This restricted dwell-time range is consistent with the exclusion of either extremely low or high affinity T cells from the expanded population during immune responses.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Ativação Linfocitária , Proteínas do Nucleocapsídeo , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos/genética , Antígenos/imunologia , Citocinas/biossíntese , Meia-Vida , Antígenos de Histocompatibilidade Classe I/genética , Hibridomas , Cinética , Mutagênese Sítio-Dirigida , Nucleocapsídeo/genética , Nucleocapsídeo/imunologia , Peptídeos/genética , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
Generation of soluble T-cell receptor (TCR) molecules by a variety of genetic engineering methods has been hampered by inefficient pairing of alpha and beta subunits in the absence of their respective transmembrane regions and associated CD3 components. To overcome this obstacle, we have added 30-amino acid-long segments to the carboxyl termini of alpha and beta extracellular domains via a cleavable flexible linker. These peptide segments (BASE-p1 for alpha and ACID-p1 for beta) have been previously shown to selectively associate to form a stable heterodimeric coiled coil termed a leucine zipper. Homodimeric structures are not permitted due to electrostatic repulsion among amino acid side chains. Expression of a representative TCR-leucine zipper fusion protein in a baculovirus expression system results in production of alpha beta TCR heterodimer at 0.6-1.4 mg/liter. This yield is 5- to 10-fold greater than that of the TCR expressed in the absence of the synthetic leucine zipper sequence. The structure of the TCR component of the fusion heterodimer was judged to be native when probed with a panel of 17 mAbs specific for alpha and beta constant and variable domains. A mAb specific for the isolated BASE-p1/ACID-p1 coiled coil was also generated and shown to react with the TCR fusion protein. The above technology should be broadly useful in the efficient production and purification of TCRs as well as other heterodimeric proteins.