Proteoliposomes in nanobiotechnology

Abstract

Proteoliposomes are systems that mimic lipid membranes (liposomes) to which a protein has been incorporated or inserted. During the last decade, these systems have gained prominence as tools for biophysical studies on lipid-protein interactions as well as for their biotechnological applications. Proteoliposomes have a major advantage when compared with natural membrane systems, since they can be obtained with a smaller number of lipidic (and protein) components, facilitating the design and interpretation of certain experiments. However, they have the disadvantage of requiring methodological standardization for incorporation of each specific protein, and the need to verify that the reconstitution procedure has yielded the correct orientation of the protein in the proteoliposome system with recovery of its functional activity. In this review, we chose two proteins under study in our laboratory to exemplify the steps necessary for the standardization of the reconstitution of membrane proteins in liposome systems: (1) alkaline phosphatase, a protein with a glycosylphosphatidylinositol anchor, and (2) Na,K-ATPase, an integral membrane protein. In these examples, we focus on the production of the specific proteoliposomes, as well as on their biochemical and biophysical characterization, with emphasis on studies of lipid-protein interactions. We conclude the chapter by highlighting current prospects of this technology for biotechnological applications, including the construction of nanosensors and of a multiprotein nanovesicular biomimetic to study the processes of initiation of skeletal mineralization.

Biophysical aspects of biomineralization

“…Garcia et al (2015) used electron spin resonance measurements along with spin labeled phospholipids to probe the possible dynamic changes prompted by the interaction of GPIanchored TNAP with model membranes. In this study, these authors showed that TNAP is probably close to the membrane surface, suggesting that this proximity can be related to the modulation of TNAP activity by the lipid composition of the vesicles, as previously reported (Bolean et al 2010(Bolean et al , 2011Ciancaglini et al 2012;Garcia et al 2015;Simao et al 2010a).…”

Liposomal systems as carriers for bioactive compounds

“…For example, MLVs may be sonicated or passed through a membrane with pores of nanometric dimensions (extrusion method) to form unilamellar vesicles that have properties very similar those of to natural membranes. Both the sonication and extrusion methods provide the energy required to break the various lamellae that initially formed and reorganize the system into uniform bilayers with controlled sizes of vesicles (Camolezi et al 2002;Ierardi et al 2002;Daghastanli et al 2004;Santos et al 2002Santos et al , 2005Rigos et al 2008;dos Santos et al 2009;Bolean et al 2010Bolean et al , 2011Simão et al 2010a, b;Ciancaglini et al 2012).…”

Reconstituição da Anexina V em sistemas de lipossomos: associação com a fosfatase alcalina e correlação com estudos de biomineralização

“…Recentes estudos têm demonstrado que a função da TNAP no tecido ósseo consiste em hidrolisar PP i para manter uma concentração adequada deste inibidor da mineralização, de modo a assegurar uma mineralização óssea normal Millán, 2006;Ciancaglini, et al, 2010Ciancaglini, et al, , 2012Simão et al, 2010).…”

“…Assim, diferentes formas da enzima (ligada à membrana, solubilizada com detergente ou tratada com PIPLC (fosfolipase C) apresentaram diferentes especificidades para os diversos substratos estudados, mostrando que a cinética da enzima é grandemente afetada pela presença tanto da âncora de fosfatidilinositol quanto de outros componentes da membrana celular (Ciancaglini et al, 2006;Simão et al, 2010;Bolean et al, 2010Bolean et al, , 2011Ciancaglini et al, 2012).…”

“…Em muitos estudos, as enzimas têm a função de serem liberadas das vesículas em sítios especificamente marcados e neste caso as vesículas servem como sistemas carreadores (Singh et al, 2010;Ciancaglini et al, 2012).…”