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Percorrer por autor "Guerreiro, Marta Lúcia Amaro"

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  • Structural characterization and comparative analysis of human and piscine cartilage acidic protein (CRTAC1/CRTAC2)
    Publication . Guerreiro, Marta Lúcia Amaro; Power, Deborah; Anjos, Liliana; Morgado, Isabel
    CRTAC (Cartilage Acidic Protein) firstly identified as a chondrocyte marker in humans and implicated in a number of diseases. This ancient protein is present from prokaryotes to vertebrates and the teleost are the only group that contain duplicates (CRTAC1/CRTAC2). The structure of CRTACs is poorly characterized and was the starting point of the present study. To establish the molecular and structural characterization of CRTAC, three recombinant proteins [human (h) CRTAC1 and sea bass (Dicentrarchus labrax, dl) CRTAC1 and CRTAC2] were over-expressed in E.coli as inclusion bodies and their identity was confirmed by mass spectrometry. The resulting refolded recombinant proteins were obtained with a productivity of 11,51, 8,4 and 11,9 mg of protein per gram of biomass for hCRTAC1, dlCRTAC1 and dlCRTAC2 respectively and approximately 23,48%, 9,09% and 33,46% of, hCRTAC1, dlCRTAC1 and dlCRTAC2 were lost as insoluble aggregates. Size exclusion chromatography revealed the presence of mostly soluble aggregated forms of piscine CRTACs and a mixture of aggregates and monomeric form for hCRTAC1. Spectroscopic studies of human CRTAC1 and sea bass CRTAC1/CRTAC2 showed that all proteins possess secondary and tertiary structure and are particularly rich in β- pleated sheet structure (≈40%), have ≈10,3% alpha-helix and the remainder is disordered. The thermal stability of CRTAC´s structure was assessed considering heating (from 20-90ºC) and freezing (-80ºC). CRTACs retain their native secondary and tertiary structures upon heating, however a slight loss of structure was observed for hCRTAC1 at 60ºC. Freezing induced loss of secondary structure and conformational changes more pronounced for hCRTAC1 and third cycle of dlCRTAC2 and less perceptible for dlCRTAC1. Amyloid formation by CRTACs was assessed in Thioflavin-T assays and a decrease in fluorescence was observed after incubation. In summary, CRTAC´s have propensity to form soluble aggregates that are highly thermostable and these structural properties are conserved from teleosts to humans.
    2014Dissertação de mestrado Acesso aberto Ver mais