Biosensors for monitoring biomolecule localization and trafficking in cells

Abstract

Bioluminescence resonance energy transfer (BRET) biosensors for assessing the intracellular localization, internalization and trafficking into cellular compartments of proteins such as receptors, and other biomolecules such as second messengers, are disclosed. These biosensors, which are dependent on the concentration/density of the BRET donor and acceptor in cellular compartments rather that specific protein-protein interactions, use a Renilla GFP/Luc BRET pair, which allows the robust and reproducible monitoring of protein trafficking/localization, with a sensitivity compatible with high-throughput screening (HTS). The use of these biosensors for various applications, including assessing/monitoring protein endocytosis, recycling and intracellular trafficking, receptor maturation/rescue by pharmacological chaperones, various endocytosis/exocytosis processes, activation/inhibition, as well as biomolecule concentration/density in different cellular compartments, is also disclosed.

1 - 73 . (canceled) 
     
     
         74 . A biosensor for assessing the trafficking and/or localization of a protein of interest comprising a cell comprising;
 a first fusion protein comprising said protein of interest tagged with a  Renilla  green fluorescent protein ( Renilla  GFP) or a  Renilla  luciferase protein ( Renilla  Luc);   a second fusion protein comprising a cellular compartment targeting moiety tagged with a  Renilla  GFP or a  Renilla  Luc;   
       wherein if said protein of interest is tagged with said  Renilla  GFP, said cellular compartment targeting moiety is tagged with said  Renilla  Luc, and if said protein of interest is tagged with said  Renilla  Luc, said cellular compartment targeting moiety is tagged with said  Renilla  GFP. 
     
     
         75 . The biosensor of  claim 74 , wherein said protein of interest is tagged with said  Renilla  Luc and said cellular compartment targeting moiety is tagged with said  Renilla  GFP. 
     
     
         76 . The biosensor of  claim 74 , wherein said protein of interest is i) a signalling polypeptide or a fragment thereof, ii) a protein recruited to the plasma membrane upon stimulation of a receptor, or a fragment thereof, iii) a protein sequestered away from the plasma membrane upon stimulation of a receptor, or a fragment thereof, or iv) a cell surface receptor or a fragment thereof. 
     
     
         77 . The biosensor of  claim 76 , wherein said cell surface receptor is a G protein-coupled receptor (GPCR) or a receptor tyrosine kinase (RTK). 
     
     
         78 . The biosensor of  claim 77 , wherein the GPCR is fused to the N-terminal of said  Renilla  Luc, and the cellular compartment targeting moiety is a plasma membrane (PM) targeting moiety or an endosomal targeting moiety, fused to the C-terminal of said  Renilla  GFP. 
     
     
         79 . The biosensor of  claim 74 , wherein said cellular compartment targeting moiety is a plasma membrane (PM) targeting moiety, an endosomal targeting moiety, a Golgi targeting moiety, a lysosomal targeting moiety, a peroxisomal targeting moiety, an autophagosomal targeting moiety, a ribosome targeting moiety, a mitochondrial targeting moiety, a cytoskeleton targeting moiety or a nuclear targeting moiety. 
     
     
         80 . The biosensor of  claim 79 , wherein said cellular compartment targeting moiety is a PM targeting moiety comprises (a) a palmitoylation, myristoylation, and/or prenylation signal sequence and/or (b) a polybasic sequence. 
     
     
         81 . The biosensor of  claim 80 , wherein said PM targeting moiety comprises a palmitoylation and/or myristoylation signal sequence from the human Src family kinase Lyn, and is fused to the N-terminal end of said  Renilla  Luc or said  Renilla  GFP or (ii) said PM targeting moiety comprises (a) a polybasic sequence and prenylation signal sequence from human KRAS splice variant b or HRAS; (b) a palmitoylation sequence from HRAS and prenylation signal sequence from Ral1; (c) Caveolin1α or a fragment thereof; or (d) a polybasic sequence from human GRK5, and is fused to the C-terminal end of said  Renilla  Luc or said  Renilla  GFP. 
     
     
         82 . The biosensor of  claim 79 , wherein said cellular compartment targeting moiety is an endosomal targeting moiety comprising a FYVE domain. 
     
     
         83 . The biosensor of  claim 79 , wherein said cellular compartment targeting moiety is an endosomal targeting moiety comprising a Rab protein or a fragment thereof. 
     
     
         84 . The biosensor of  claim 79 , wherein said cellular compartment targeting moiety is a Golgi targeting moiety comprising a Golgi protein or a fragment thereof that localizes to the Golgi. 
     
     
         85 . The biosensor of  claim 84 , wherein said Golgi targeting moiety comprises residues 1 to 73 of human eNOS1 (SEQ ID NO: 42). 
     
     
         86 . The biosensor of  claim 76 , wherein the signalling protein or fragment thereof is a β-arrestin polypeptide, or a fragment thereof, fused to the N-terminal of said  Renilla  Luc, and the cellular compartment targeting moiety is a plasma membrane (PM) targeting moiety or an endosomal targeting moiety, fused to the C-terminal of said  Renilla  GFP. 
     
     
         87 . The biosensor of  claim 74 , wherein said first and second component are covalently linked through a flexible linker. 
     
     
         88 . The biosensor of  claim 87 , wherein said flexible linker is a polypeptide of about 50 to about 500 amino acids. 
     
     
         89 . The biosensor of  claim 74 , wherein said  Renilla  Luc comprises a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:10. 
     
     
         90 . The biosensor of  claim 74 , wherein said  Renilla  GFP comprises a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 11. 
     
     
         91 . A method for assessing the trafficking of a protein of interest in a cell under a first condition and a second condition, said method comprising:
 measuring a BRET signal in the biosensor of  claim 74  under said first condition and said second condition;   
       wherein a difference in said BRET signal under said second condition relative to the first condition is indicative of the trafficking of said protein of interest in said cell. 
     
     
         92 . The method of  claim 91 , wherein said first condition is the presence of a test agent and said second condition is the absence of said test agent. 
     
     
         93 . The method of  claim 91 , wherein the BRET signal is measured using a plate reader or by microscopy.