19th Annual PKP Student Research and Fine Arts Conference: Oral Symposia V
Little did BRET know that his Studious Life in Immunoliposomes Would Never be the Same
Characterization of Serotonin Receptors in Response to Ligand Binding Using BRETG protein-coupled receptors (GPCRs) are receptors that act in signal transduction pathways via activation of guanosine nucleotide-binding proteins, known as G proteins. An extra-cellular signal (a ligand) activates a receptor from outside the cell and working through a G protein, the external signal is transmitted inside the cell. There are four main classes of G proteins: Gs, Gi, Gqand G12. When activated, eachof these G protein types is responsible for a specific intracellular event, often determined by measuring how the concentration of a second messenger changes as a function of the concentration of the external signal. This indirect approach limits our understanding of the role of each type of G protein in signaling pathways. Our group is currently using Bioluminescence Resonance Energy Transfer (BRET) to directly measure G protein activation by GPCRs in response to external stimuli (includingboth endogenous and synthetic ligands). We have generated recombinant DNA for nanoluciferase fused to GPCRs in the serotonin (5-hydroxytryptamine, 5-HT) receptor family. These genetic fusions, along with fusions of yellow fluorescent protein and various G proteins were co-transfected into HEK293 cells for BRET assays. Initial results show that activation of receptors 5-HT1D and 5-HT1F with serotonin are coupled to Gi. Future studies will include a G protein profile for all twelve receptors in the serotonin family.
Generation of immunoliposomes using microfluidic devicesImmunoliposomes, or antibody-conjugated liposomes, hold promise as an effective way to target drugs to specific tissues. Currently, we can find in the market immunoliposomes such as Doxil, an anti-cancer drug; Amphotec, an anti-fungal drug; and Allovectin-7, used for gene therapy. However the synthesis methods used areinefficient. The formation of liposomes is a multi-step process that requires sonication and filtering. In addition, its encapsulation efficiency is low, what leads to the waste, in many cases, of expensive drugs. We use microfluidic technology to solve these obstacles to efficient liposomal synthesis. We generated double emulsion drops (a drop inside another drop) where the inner liquid is the drug we want to encapsulate, the middle phase is a solution of lipids and the outer is an aqueous solution where our liposome will be dispersedand the conjugation with the anti-bodies will happen. The advantage of this method is its high encapsulating efficiency and the control of the size of the liposome. This techniquecouldpotentiallybe used to drastically reduce side effects and increase tissue-specific drug targeting for a wide variety of diseases.
THEORETICAL STUDY OF MOLECULAR ORGANIZATION IN CELL MEMBRANESCell is the fundamental building blocks of all living matter. The cell consists of cytoplasm enclosed within a membrane where the cell membrane is composed of lipids and protein molecules. Lipid molecules form bilayer structures in cell membrane when they are in aqueous environments. All membrane proteins carry out their cellular functions while they are sitting at membrane sites. Due to the collective behavior of these molecules, they undergo self-organization and form various structures, such as phase separation and domain formations. We use a thermodynamics approach to study three-component molecular organization by modeling the interaction between molecules using spin variables. Converting the interacting spins into an effectively non-interacting variables using a mean-field theory, we calculate the Helmholtz free energy (HFE). Then by investigating the HFE, we construct the phase diagram and study the molecular organization in cell membranes.