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Projeto de investigação
Health Sciences Research Centre
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Enhanced stability of detergent-free human native STEAP1 protein from neoplastic prostate cancer cells upon an innovative isolation procedure
Publication . Barroca-Ferreira, Jorge; Cruz-Vicente, Pedro; Santos, Marino F. A.; Rocha, Sandra M.; Santos-Silva, Teresa; Maia, Cláudio J.; Passarinha, Luís A.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; MDPI - Multidisciplinary Digital Publishing Institute
Background: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (Tm) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism. Results: The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A Tm of ~55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein’s structural stability. Conclusions: A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment.
Unveiling the biopathway for the design of novel COMT inhibitors
Publication . Cruz-Vicente, Pedro; Gonçalves, Ana M.; Barroca-Ferreira, Jorge; Silvestre, Samuel M.; Romão, Maria J.; Queiroz, João A.; Gallardo, Eugénia; Passarinha, Luís A.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Elsevier
Catechol-O-methyltransferase (COMT) is an enzyme responsible for the O-methylation of biologically active catechol-based molecules. It has been associated with several neurological disorders, especially Parkinson's disease (PD), because of its involvement in catecholamine metabolism, and has been considered an important therapeutic target for central nervous system disorders. In this review, we summarize the biophysical, structural, and therapeutical relevance of COMT; the medicinal chemistry behind the development of COMT inhibitors and the application of computer-aided design to support the design of novel molecules; current methodologies for the biosynthesis, isolation, and purification of COMT; and revise existing bioanalytical approaches for the assessment of enzymatic activity in several biological matrices.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
6817 - DCRRNI ID
Número da atribuição
UID/Multi/00709/2013