Compositions and methods for quantitatively monitoring lipids

Abstract

Provided herein are fluorescent lipid binding proteins (FLBPs). The FLBPs comprise a lipid binding domain linked to a fluorophore, whereby the fluorophore's fluorescence emission undergoes a spectral change upon lipid binding.

1 . A fluorescent lipid-binding protein (FLBP), comprising a fluorophore and a lipid-binding protein. 
     
     
         2 . The FLBP of  claim 1 , wherein the fluorophore is selected from the group consisting of 2-dimethylamino-6-acyl-naphthalene (DAN) and RED fluorophore. 
     
     
         3 . The FLBP of  claim 1 , wherein the lipid binding protein is selected from the group consisting of ENTH domain of epsin 1, C2 domain of bovine lactadherin, C1B domain of protein kinase Cγ, C2 domain of cytosolic phospholipase A2β, and PH domain of Bruton's tyrosine kinase PH. 
     
     
         4 . The FLBP of  claim 3 , wherein the ENTH domain comprises SEQ ID NO:2, or a fragment thereof 
     
     
         5 . The FLBP of  claim 3 , wherein the C2 domain of bovine lactadherin comprises SEQ ID NO:4 or 5, or a fragment thereof. 
     
     
         6 . The FLBP of  claim 3 , wherein the C1B domain of protein kinase Cγ comprises SEQ ID NO:7. 
     
     
         7 . The FLBP of  claim 3 , wherein the C2 domain of cytosolic phospholipase A2β comprises SEQ ID NO:9. 
     
     
         8 . The FLBP of  claim 3 , wherein the PH domain of Bruton's tyrosine kinase comprises SEQ ID NO:11. 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . The FLBP of  claim 4 , wherein DAN is chemically linked to amino acid C10. 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . The FLBP of  claim 5 , wherein the C2 domain of bovine lactadherin is linked to DAN. 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . The FLBP of  claim 6 , wherein the C1B domain is linked to DAN. 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . The FLBP of  claim 7 , wherein the C2 domain is linked to DAN. 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . The FLBP of  claim 8 , wherein the PH domain is linked to DAN. 
     
     
         25 . A method of quantifying a specific lipid in a cell membrane, comprising
 (a) introducing the FLBP of  claim 1  to a cell;   (b) imaging the cell; and   (c) quantifying the lipid in the cell membrane.   
     
     
         26 . The method of  claim 25 , wherein the specific lipid is selected from the group consisting of: phosphatidic acid, phosphatidylserine, diacylglycerol, cardiolipin, and phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ). 
     
     
         27 - 40 . (canceled) 
     
     
         41 . A method of diagnosing a lipid-metabolism-related disorder comprising
 (a) introducing the FLBP of  claim 1  to a sample derived from a subject;   (b) imaging the cells in the sample;   (c) quantifying a target lipid in the cells; and   (d) comparing the quantity identified in (c) with a reference standard, wherein the quantity of the target lipid present in the reference standard is the quantity present in a comparable sample from an individual with or without the disorder.   
     
     
         42 . The method of  claim 41 , wherein the lipid-metabolism-related disorder is a PI-3 kinase-related disorder. 
     
     
         43 . The method of  claim 42 , wherein the FLBP comprises the C1B domain of protein kinase Cγ, and wherein the C1B domain comprises SEQ ID NO: 7. 
     
     
         44 . The method of  claim 43 , wherein the target lipid is PtdIns(4,5)P 2 .