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 multi-protein nanovesicular biomimetic to study the processes of initiation of skeletal mineralization.
ABSTRACT
Hypoxia (low oxygen tension) is a common feature of inflamed and infected tissues. The influence of hypoxia on macrophage responses to micro-organisms has only recently been studied. This study demonstrates that hypoxia induced macrophages to control Leishmania amazonensis, an intracellular parasite that causes cutaneous and cutaneous metastatic lesions. The mechanisms that contribute to the control of macrophages against L. amazonensis infection under a hypoxic microenvironment are not known. Nitric oxide, TNF-α, IL-10 or IL-12 is not responsible for the decrease in parasitism under hypoxia. Live L. amazonensis entry or exocytosis of internalized particles as well as energetic metabolism was not impaired in infected macrophages; no apoptosis-like death was detected in intracellular parasites. Reactive oxygen species (ROS) is likely to be involved, because treatment with antioxidants N-acetylcysteine (NAC) and ebselen inhibits the leishmanicidal effect of macrophages under hypoxia. Leishmania amazonensis infection induces macrophages to express hypoxia-inducible factor-1 (HIF-1α) and -2 (HIF-2α). Data indicate that hypoxia affects the microbial activities and protein expression of macrophages leading to a different phenotype from that of the normoxic counterpart and that it plays a role in modulating Leishmania infection.