abstract
Membrane-bound catechol-O-methyltransferase (MBCOMT), present in the brain and involved in the main pathway of the catechol neurotransmitter deactivation, is linked to several types of human dementia, which are relevant pharmacological targets for new potent and nontoxic inhibitors that have been developed, particularly for Parkinson's disease treatment. However, the inexistence of an MBCOMT 3D-structure presents a blockage in new drugs' design and clinical studies due to its instability. The enzyme has a clear tendency to lose its biological activity in a short period of time. To avoid the enzyme sequestering into a non-native state during the downstream processing, a multi-component buffer plays a major role, with the addition of additives such as cysteine, glycerol, and trehalose showing promising results towards minimizing hMBCOMT damage and enhancing its stability. In addition, ionic liquids, due to their virtually unlimited choices for cation/anion paring, are potential protein stabilizers for the process and storage buffers. Screening experiments were designed to evaluate the effect of distinct cation/anion ILs interaction in hMBCOMT enzymatic activity. The ionic liquids: choline glutamate [Ch][Glu], choline dihydrogen phosphate ([Ch][DHP]), choline chloride ([Ch]Cl), 1- dodecyl-3-methylimidazolium chloride ([C12mim]Cl), and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) were supplemented to hMBCOMT lysates in a concentration from 5 to 500 mM. A major potential stabilizing effect was obtained using [Ch][DHP] (10 and 50 mM). From the DoE 146% of hMBCOMT activity recovery was obtained with [Ch][DHP] optimal conditions (7.5 mM) at -80 degrees C during 32.4 h. These results are of crucial importance for further drug development once the enzyme can be stabilized for longer periods of time.
keywords
REFOLDING ADDITIVES; PROTEIN STABILITY; CYTOCHROME-C; STABILIZATION; PURIFICATION; SOLUBILITY; INHIBITORS; RESIDUES; SOLVENTS; ENZYMES
subject category
Biochemistry & Molecular Biology; Chemistry
authors
Goncalves, AM; Sousa, A; Pedro, AQ; Romao, MJ; Queiroz, JA; Gallardo, E; Passarinha, LA
our authors
Augusto Pedro
Junior ResearcherProjects
CICECO - Aveiro Institute of Materials (UIDB/50011/2020)
CICECO - Aveiro Institute of Materials (UIDP/50011/2020)
Associated Laboratory CICECO-Aveiro Institute of Materials (LA/P/0006/2020)
acknowledgements
The authors acknowledge the support from FEDER funds through the POCI-COMPETE 2020-Operational Program Competitiveness and Internationalization in Axis I-Strengthening research, technological development, and innovation (Project POCI-01-0145-FEDER-007491) and National Funds (Project UID/Multi/00709/2013). This work was developed within the scope of the CICS-UBI projects UIDB/00709/2020, UIDP/00709/2020, and CICECO-Aveiro Institute of Materials projects, UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020, financed by national funds through the Portuguese Foundation for Science and Technology/MCTES (PIDDAC). This work was also supported by the Applied Molecular Biosciences Unit UCIBIO (UIDB/04378/2020 and UIDP/04378/2020) and the Associate Laboratory Institute for Health and Bioeconomy-i4HB (project LA/P/0140/2020) which are financed by National Funds from FCT/MCTES. Researchers also acknowledge funding by FEDER through COMPETE 2020-Programa Operacional Competitividade e Internacionalizacao (POCI), and by national funds (OE), through FCT/MCTES from the project IL2BioPro-PTDC/BII-BBF/30840/2017. Ana M. Goncalves's individual PhD Fellowship (SFRH/BD/147519/2019), Augusto Q. Pedro research contract CEEC-IND/02599/2020 under the Scientific Stimulus-Individual Call, and Luis A. Passarinha's sabbatical fellowship (SFRH/BSAB/150376/2019) from the Portuguese Foundation for Science and Technology (FCT) within the scope of POCH-Advanced Formation programs co-funded by European Social Fund and MCTES.