RESUMEN
Currently, the extracellular matrix (ECM) is considered a pivotal complex meshwork of macromolecules playing a plethora of biomolecular functions in health and disease beyond its commonly known mechanical role. Only by unraveling its composition can we leverage related tissue engineering and pharmacological efforts. Nevertheless, its unbiased proteomic identification still encounters some limitations mainly due to partial ECM enrichment by precipitation, sequential fractionation using unfriendly-mass spectrometry (MS) detergents, and resuspension with harsh reagents that need to be entirely removed prior to analysis. These methods can be technically challenging and labor-intensive, which affects the reproducibility of ECM identification and induces protein loss. Here, we present a simple new method applicable to tissue fragments of 10 mg and more. The technique has been validated on human ovarian tissue and involves a standardized procedure for sample processing with an MS-compatible detergent and combined centrifugation. This two-step protocol eliminates the need for laborious sample clarification and divides our samples into 2 fractions, soluble and insoluble, successively enriched with matrisome-associated (ECM-interacting) and core matrisome (structural ECM) proteins.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Proteoma , Proteómica/métodos , Biología Computacional/métodos , Humanos , Espectrometría de Masas , Proteómica/normas , Reproducibilidad de los ResultadosRESUMEN
Species are chronically exposed to ionizing radiation, a natural phenomenon which can be enhanced by human activities. The induced toxicity mechanisms still remain unclear and seem depending on the mode of exposure, i.e. acute and chronic. To better understand these phenomena, studies need to be conducted both at the subcellular and individual levels. Proteins, functional molecules in organisms, are the targets of oxidative damage (especially via their carbonylation (PC)) and are likely to be relevant biomarkers. After exposure of Caenorhabditis elegans to either chronic or acute γ rays we showed that hatching success is impacted after acute but not after chronic irradiation. At the molecular level, the carbonylated protein level in relation with dose was slightly different between acute and chronic exposure whereas the proteolytic activity is drastically modified. Indeed, whereas the 20S proteasome activity is inhibited by acute irradiation from 0.5 Gy, it is activated after chronic irradiation from 1 Gy. As expected, the 20S proteasome activity is mainly modified by irradiation whereas the 26S and 30S activity are less changed. This study provides preliminaries clues to understand the role of protein oxidation and proteolytic activity in the radiation-induced molecular mechanisms after chronic versus acute irradiation in C. elegans.
Asunto(s)
Caenorhabditis elegans/efectos de la radiación , Rayos gamma , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Relación Dosis-Respuesta en la Radiación , Cinética , Carbonilación Proteica/efectos de la radiación , Proteolisis/efectos de la radiación , Reproducción/efectos de la radiaciónRESUMEN
Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that plays a major role in cellular adaptation to hypoxia. The mechanisms regulating HIF-1 activity occurs at multiple levels in vivo. The HIF-1alpha subunit is highly sensible to oxygen and is rapidly degraded by the proteasome 26S in normoxia. Activation in hypoxia occurs through a multistep process including inhibition of HIF-1alpha degradation, but also increase in the transactivation activity of HIF-1. Several data indicate that phosphorylation could play a role in this regulation. In this report, we investigated the role of casein kinase 2 (CK2), an ubiquitous serine/threonine kinase, in the regulation of HIF-1 activity. Hypoxia was capable of increasing the expression of the beta subunit of CK2, of inducing a relocalization of this subunit at the plasma membrane, of inducing nuclear translocation of the alpha subunit and of increasing CK2 activity. Three inhibitors of this kinase, DRB (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole), TBB (4,5,6,7-tetrabromotriazole) and apigenin, as well as overexpression of a partial dominant negative mutant of CK2alpha, were shown to inhibit HIF-1 activity as measured by a reporter assay and through hypoxia-induced VEGF and aldolase expression. This does not occur at the stabilization process since they did not affect HIF-1alpha protein level. DNA-binding activity was also not inhibited. We conclude that CK2 is an important regulator of HIF-1 transcriptional activity but the mechanism of this regulation remains to be determined. Since HIF-1 plays a major role in tumor angiogenesis and since CK2 has been described to be overexpressed in tumor cells, this new pathway of regulation can be one more way for tumor cells to survive.