Browsing by Author "Leandro, Paula"
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- Heterologous expression and purification of human carnitine acylcarnitine translocase (CACT)Publication . Ventura, Fátima V; Violante, Sara; Jorge, Ana Maria; Gaspar, Maria Manuela; Cruz, Maria Eugénia; Doveral, Graça; Leandro, Paula; Tavares de Almeida, Isabel
- Ichthyiological collection of the Museu Oceanográfico D. Carlos IPublication . Silva, Ana Serra; Groz, Maria Pitta; Leandro, Paula; Assis, Carlos A.; Figueira, RuiThe collection of the Museu Oceanográfico D. Carlos I is a historical specimen, instrument, and document collection that has been housed at the Aquário Vasco da Gama since 1935. The collection is largely the result of several scientific campaigns conducted by Dom Carlos de Bragança between 1896 and 1907. Specifically, the ichthyological collection consists of 675 surviving catalogue records of specimens caught, acquired or offered to D. Carlos I between 1892 to 1907, and includes the type specimen for Odontaspis nasutus Bragança, 1904 (junior synonym of Mitsukurina owstoni Jordan, 1898), along with several specimens of deep sea species. All specimens were captured in coastal Portuguese waters, and were preserved in alcohol, formalin, or mounted
- Ichthyological collection of the Museu Oceanográfico D. Carlos IPublication . Silva, Ana Serra; Pitta Groz, Maria; Leandro, Paula; Assis, Carlos; Figueira, RuiThe collection of the Museu Oceanográfico D. Carlos I is a historical specimen, instrument, and document collection that has been housed at the Aquário Vasco da Gama since 1935. The collection is largely the result of several scientific campaigns conducted by Dom Carlos de Bragança between 1896 and 1907. Specifically, the ichthyological collection consists of 675 surviving catalogue records of specimens caught, acquired or offered to D. Carlos I between 1892 to 1907, and includes the type specimen for Odontaspis nasutus Bragança, 1904 (junior synonym of Mitsukurina owstoni Jordan, 1898), along with several specimens of deep sea species. All specimens were captured in coastal Portuguese waters, and were preserved in alcohol, formalin, or mounted.
- In Silico and In Vitro Tailoring of a Chitosan Nanoformulation of a Human Metabolic EnzymePublication . Lino, Paulo Roque; Leandro, João; Amaro, Mariana; Gonçalves, Lídia; Leandro, Paula; Almeida, António JoséEnzyme nanoencapsulation holds an enormous potential to develop new therapeutic approaches to a large set of human pathologies including cancer, infectious diseases and inherited metabolic disorders. However, enzyme formulation has been limited by the need to maintain the catalytic function, which is governed by protein conformation. Herein we report the rational design of a delivery system based on chitosan for effective encapsulation of a functionally and structurally complex human metabolic enzyme through ionic gelation with tripolyphosphate. The rationale was to use a mild methodology to entrap the multimeric multidomain 200 kDa human phenylalanine hydroxylase (hPAH) in a polyol-like matrix that would allow an efficient maintenance of protein structure and function, avoiding formulation stress conditions. Through an in silico and in vitro based development, the particulate system was optimized with modulation of nanomaterials protonation status, polymer, counterion and protein ratios, taking into account particle size, polydispersity index, surface charge, particle yield production, protein free energy of folding, electrostatic surface potential, charge, encapsulation efficiency, loading capacity and transmission electron microscopy morphology. Evaluation of the thermal stability, substrate binding profile, relative enzymatic activity, and substrate activation ratio of the encapsulated hPAH suggests that the formulation procedure does not affect protein stability, allowing an effective maintenance of hPAH biological function. Hence, this study provides an important framework for an enzyme formulation process.
- Lack of Aquaporin 3 in bovine erythrocyte membranes correlates with low glycerol permeationPublication . Campos, Elisa; Moura, Teresa F.; Oliva, Abel; Leandro, Paula; Soveral, GraçaIn general, erythrocytes are highly permeable to water, urea and glycerol. However, expression of aquaporin isoforms in erythrocytes appears to be species characteristic. In the present study, human (hRBC) and bovine (bRBC) erythrocytes were chosen for comparative studies due to their significant difference in membrane glycerol permeability. Osmotic water permeability (Pf) at 23 ºC was (2.89 ± 0.37) × 10-2 and (5.12 ± 0.61) × 10-2 cm s-1 for human and bovine cells respectively, with similar activation energies for water transport. Glycerol permeability (Pgly) for human ((1.37 ± 0.26) × 10-5 cm s-1) differed in three orders of magnitude from bovine erythrocytes ((5.82 ± 0.37) ×10-8 cm s-1) that also showed higher activation energy for glycerol transport. When compared to human, bovine erythrocytes showed a similar expression pattern of AQP1 glycosylated forms on immunoblot analysis, though in slight higher levels, which could be correlated with the 1.5-fold larger Pf found. However, AQP3 expression was not detectable. Immunofluorescence analysis confirmed the absence of AQP3 expression in bovine erythrocyte membranes. In conclusion, lack of AQP3 in bovine erythrocytes points to the lipid pathway as responsible for glycerol permeation and explains the low glycerol permeability and high Ea for transport observed in ruminants.
- Modulation of Human Phenylalanine Hydroxylase by 3-Hydroxyquinolin-2(1H)-One DerivativesPublication . Lopes, Raquel R.; Tomé, Catarina S.; Russo, Roberto; Paterna, Roberta; Leandro, João; Candeias, Nuno R.; Gonçalves, Lídia; Teixeira, Miguel; Sousa, Pedro M. F.; Guedes, R. C.; Vicente, João B.; Gois, Pedro M. P.; Leandro, PaulaPhenylketonuria (PKU) is a genetic disease caused by deficient activity of human phenylalanine hydroxylase (hPAH) that, when untreated, can lead to severe psychomotor impairment. Protein misfolding is recognized as the main underlying pathogenic mechanism of PKU. Therefore, the use of stabilizers of protein structure and/or activity is an attractive therapeutic strategy for this condition. Here, we report that 3-hydroxyquinolin-2(1H)-one derivatives can act as protectors of hPAH enzyme activity. Electron paramagnetic resonance spectroscopy demonstrated that the 3-hydroxyquinolin-2(1H)-one compounds affect the coordination of the non-heme ferric center at the enzyme active-site. Moreover, surface plasmon resonance studies showed that these stabilizing compounds can be outcompeted by the natural substrate l-phenylalanine. Two of the designed compounds functionally stabilized hPAH by maintaining protein activity. This effect was observed on the recombinant purified protein and in a cellular model. Besides interacting with the catalytic iron, one of the compounds also binds to the N-terminal regulatory domain, although to a different location from the allosteric l-Phe binding site, as supported by the solution structures obtained by small-angle X-ray scattering.
- Modulation of the Activity of Newly Synthesized Human Phenylalanine Hydroxylase Mutant Proteins by Low-Molecular-Weight CompoundsPublication . Nascimento, Catia; Leandro, Joao; de Almeida, Isabel Tavares; Leandro, PaulaPhenylketonuria, the most frequent disorder of amino acid metabolism, is caused by a deficient activity of human phenylalanine hydroxylase (hPAH). Rescue of the enzyme activity of several recombinant hPAH mutant forms (I65T, R261Q, R270K and V388M) by low molecular weight compounds namely glycerol, trimethylamine N-oxide (TMAO) and sodium 4-phenylbutyrate (4-PB) was investigated using a prokaryotic expression model. The studied compounds were added to the culture medium, in a concentration dependent manner, simultaneously to induction of protein expression. Among the tested molecules glycerol and TMAO were able to increase the enzyme activity of the studied mutant proteins. Furthermore, a decrease in aggregates and a recovery of the active tetrameric and dimeric forms were detected. Since the addition of the studied compounds to the medium did not change the expression level of E. Coli molecular chaperones we postulate that glycerol and TMAO rescue results from a direct stabilizing effect of the newly synthesized mutant hPAH enzymes.. - Fundacao para a Ciencia e Tecnologia, Portugal ; FEDER [MGI/POCTI/40844/2001, SFRH/BD/10807/2002, SFRH/BD/19024/2004]. - F. Ventura (iMed.UL) is gratefully acknowledged for critically reading of the manuscript. This work was supported by Fundacao para a Ciencia e Tecnologia, Portugal, and FEDER (MGI/POCTI/40844/2001, SFRH/BD/10807/2002 and SFRH/BD/19024/2004).
- Protein misfolding in conformational disordersPublication . Leandro, Paula; Gomes, Claudio M.Protein folding in the cell is a tightly regulated process, involving a series of proteins, from molecular chaperones to proteases that assist the folding process and monitor the quality of the final product. Despite this control, genetic or sporadic factors may compromise protein folding and the folded state resulting in the formation of non-native misfolded, destabilised, aggregated or fibrillar species. These are hallmarks of the so-called protein conformational disorders, in which the altered protein conformations result in cell toxicity, functional deficiency or lead to dominant negative effects. Examples of such pathologies include neurodegenerative and metabolic disorders. In recent years, it has become clear that several different small chemical compounds such as osmolytes, protein inhibitors, ligands and cofactors exert a chemical chaperoning effect and are able to rescue folding and trafficking defects, minimising or partly overcoming the pathological consequences of protein misfolding. Here we review the different types of chemical chaperones and provide a structural and energetic rationale for their action. Examples of chemical chaperoning are overviewed and discussed on the basis of the reported effects exerted by chemical compounds at different stages of the protein folding process and protein conformational states.. - Fundacao para a Ciencia e Tecnologia (FCT/MCTES). - C. Rodrigues-Pousada and T. Bandeiras (ITQB) are gratefully acknowledged for critically reading of the manuscript and for insightful comments. H. Botelho is gratefully acknowledged for data and assistance on (Fig. 5). Funding from the Fundacao para a Ciencia e Tecnologia (FCT/MCTES) is gratefully acknowledged.
- Systematic Modification and Evaluation of Enzyme-Loaded Chitosan NanoparticlesPublication . Lino, Paulo Roque; Leandro, João; Figueiredo, Lara; Amaro, Mariana P.; Gonçalves, Lídia; Leandro, Paula; Almeida, António JoséPolymeric-based nano drug delivery systems have been widely exploited to overcome protein instability during formulation. Presently, a diverse range of polymeric agents can be used, among which polysaccharides, such as chitosan (CS), hyaluronic acid (HA) and cyclodextrins (CDs), are included. Due to its unique biological and physicochemical properties, CS is one of the most used polysaccharides for development of protein delivery systems. However, CS has been described as potentially immunogenic. By envisaging a biosafe cytocompatible and haemocompatible profile, this paper reports the systematic development of a delivery system based on CS and derived with HA and CDs to nanoencapsulate the model human phenylalanine hydroxylase (hPAH) through ionotropic gelation with tripolyphosphate (TPP), while maintaining protein stability and enzyme activity. By merging the combined set of biopolymers, we were able to effectively entrap hPAH within CS nanoparticles with improvements in hPAH stability and the maintenance of functional activity, while simultaneously achieving strict control of the formulation process. Detailed characterization of the developed nanoparticulate systems showed that the lead formulations were internalized by hepatocytes (HepG2 cell line), did not reveal cell toxicity and presented a safe haemocompatible profile.
- The lytic cassette of mycobacteriophage Ms6 encodes an enzyme with lipolytic activityPublication . Gil, Filipa; Catalao, Maria Joao; Moniz-Pereira, Jose; Leandro, Paula; McNeil, Michael; Pimentel, MadalenadsDNA bacteriophages use the dual system endolysin-holin to achieve lysis of their bacterial host. In addition to these two essential genes, some bacteriophages encode additional proteins within their lysis module. In this report, we describe the activity of a protein encoded by gene lysB from the mycobacteriophage Ms6. lysB is localized within the lysis cassette, between the endolysin gene (lysA) and the holin gene (hol). Analysis of the deduced amino acid sequence of LysB revealed the presence of a conserved motif (Gly-Tyr-Ser-Gin-Gly) characteristic of enzymes with lipolytic activity. A BLAST search within the sequences of protein databases revealed significant similarities to other putative proteins that are encoded by mycobacteriophages only, indicating that LysB and those proteins may be specific to their mycobacterial hosts. A screening for His(6)-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. Examination of the kinetic parameters of recombinant His(6)-LysB for the hydrolysis of p-nitrophenyl esters indicated that although this protein could use a wide range of chain length substrates (C-4-C-18), it presents a higher affinity for p-nitrophenyl esters of longer chain length (C-16 and C-18). Using p-nitrophenyl butyrate as a substrate, the enzyme showed optimal activity at 23 degrees C and pH 7.5-8.0. Activity was increased in the presence of Ca2+ and Mn2+. To the best of our knowledge, this is the first description of a protein with lipolytic activity encoded within a bacteriophage.