RESUMO
Para-hydroxycinnamic acid (pHCA) is one of the most abundant naturally occurring hydroxycinnamic acids, a class of chemistries known for their antioxidant properties. In this study, we evaluated the impact of pHCA on different parameters of skin aging in in vitro skin models after H2O2 and UV exposure. These parameters include keratinocyte senescence and differentiation, inflammation, and energy metabolism, as well as the underlying molecular mechanisms. Here we demonstrate that pHCA prevents oxidative stress-induced premature senescence of human primary keratinocytes in both 2D and 3D skin models, while improving clonogenicity in 2D. As aging is linked to inflammation, referred to as inflammaging, we analyzed the release of IL-6, IL-8, and PGE2, known to be associated with senescence. All of them were downregulated by pHCA in both normal and oxidative stress conditions. Mechanistically, DNA damage induced by oxidative stress is prevented by pHCA, while pHCA also exerts a positive effect on the mitochondrial and glycolytic functions under stress. Altogether, these results highlight the protective effects of pHCA against inflammaging, and importantly, help to elucidate its potential mechanisms of action.
Assuntos
Senescência Celular , Ácidos Cumáricos , Queratinócitos , Estresse Oxidativo , Envelhecimento da Pele , Pele , Humanos , Ácidos Cumáricos/farmacologia , Senescência Celular/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pele/metabolismo , Pele/efeitos dos fármacos , Envelhecimento da Pele/efeitos dos fármacos , Inflamação/metabolismo , Dano ao DNA/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Raios Ultravioleta/efeitos adversos , Antioxidantes/farmacologia , Células Cultivadas , Interleucina-8/metabolismo , Interleucina-6/metabolismoRESUMO
Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related sensor proteins NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, their mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to assemble distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core of oligomerized CARD surrounded by an outer ring of FIINDUPA. Biochemically, self-assembled NLRP1-CARD filaments are sufficient to drive ASC speck formation in cultured human cells-a process that is greatly enhanced by NLRP1-FIINDUPA which forms oligomers in vitro. The cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments, solved here at 3.7 Å, uncover unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide structural insight into the mechanisms of activation for human NLRP1 and CARD8 and reveal how highly specific signaling can be achieved by heterotypic CARD interactions within the inflammasome complexes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Inflamassomos/química , Inflamassomos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Proteínas Adaptadoras de Sinalização CARD/genética , Caspase 1/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Humanos , Inflamassomos/genética , Inflamação , Simulação de Acoplamento Molecular , Mutação , Proteínas NLR , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Transdução de SinaisRESUMO
Superresolution microscopy offers the advantage of imaging biological structures within cells at the nano-scale. Here we apply two superresolution microscopy techniques, specifically 3D structured illumination microscopy (3D-SIM) and direct stochastic optical reconstruction microscopy (dSTORM), a type of single molecule localisation microscopy, to localise IRSp53 protein and its I-BAR domain in relation to F-actin within filopodia. IRSp53 generates dynamic (extending and retracting) filopodia 300 nm wide with a distinct gap between IRSp53 and F-actin. By contrast, protrusions induced by the I-BAR domain alone are non-dynamic measuring between 100-200 nm in width and exhibit a comparatively closer localisation of the I-BAR domain with the F-actin. The data suggest that IRSp53 membrane localisation is spatially segregated to the lateral edges of filopodia, in contrast to the I-BAR domain is uniformly distributed throughout the membranes of protrusions. Modeling of fluorescence recovery after photobleaching (FRAP) data suggests that a greater proportion of I-BAR domain is associated with membranes when compared to full length IRSp53. The significance of this new data relates to the role filopodia play in cell migration and its importance to cancer.