Compositions and methods for determining interactions of mitochondrial components, and for identifying agents that alter such interactions
AssigneeMitoKor
InventorsAnne N. Murphy, William Clevenger, Sandra Eileen Wiley, Alexander Y. Andreyev, Luciano G. Frigeri, Gonul Velicelebi, Robert E. Davis
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Abstract
Compositions and methods are provided for identifying agents that alter mitochondrial membrane permeability transition. The screening methods generally detect agents that alter the interaction between the mitochondrial adenine nucleotide translocator and cyclophilin D. Such agents may be used, for example, in the treatment of a variety of conditions associated with altered mitochondrial function.
Claims91
A nucleic acid expression construct comprising a promoter operably linked to a polynucleotide encoding a mitochondrial permeability transition pore component polypeptide fused to an energy transfer molecule polypeptide, or a variant thereof.
A nucleic acid expression construct comprising a promoter operably linked to a polynucleotide encoding a cyclophilin polypeptide fused to an energy transfer molecule polypeptide, or a variant thereof.
A polypeptide comprising a mitochondrial permeability transition pore component polypeptide fused to an energy transfer molecule polypeptide, or a derivative thereof.
A polypeptide comprising a cyclophilin polypeptide fused to an energy transfer molecule polypeptide, or a derivative thereof.
A host cell for identifying agents that alter mitochondrial permeability transition, comprising:
- (a) a first nucleic acid expression construct, comprising a promoter operably linked to a polynucleotide encoding a mitochondrial permeability transition pore component polypeptide fused to a polynucleotide encoding a first energy transfer molecule or a variant thereof;
- and
- (b) a second nucleic acid expression construct, comprising a promoter operably linked to a polynucleotide encoding a cyclophilin polypeptide fused to a polynucleotide encoding a second energy transfer molecule or a variant thereof, wherein binding of the mitochondrial permeability transition pore component polypeptide to the cyclophilin polypeptide results in detectable energy transfer between the first and second energy transfer molecules.
A host cell according to claim 39, wherein the eukaryotic cell is selected from the group consisting of 293, COS-7, Sf9, CHO, Hep-2, MDCK and Jurkat.
A method for detecting an agent that alters mitochondrial permeability transition (MPT), comprising the steps of:
- (a) contacting a cyclophilin D polypeptide with an adenine nucleotide translocator polypeptide and a candidate agent, under conditions and for a time sufficient to permit the cyclophilin D, adenine nucleotide translocator, and the candidate agent to interact;
- and
- (b) detecting a level of binding of cyclophilin D polypeptide to adenine nucleotide translocator polypeptide, relative to a level of binding detected in the absence of the candidate agent, and therefrom detecting an agent that alters MPT.
A method for preparing a mitochondrial permeability transition pore component polypeptide fused to an energy transfer molecule, comprising the steps of:
- (a) culturing a host cell comprising a nucleic acid expression construct that encodes a fusion protein comprising an adenine nucleotide translocator polypeptide or a derivative thereof fused to an energy transfer molecule polypeptide or a derivative thereof, under conditions that permit expression of the fusion protein;
- and
- (b) recovering fusion protein from the culture.
A method for preparing a cyclophilin polypeptide fused to an energy transfer molecule, comprising the steps of:
- (a) culturing a host cell comprising a nucleic acid expression construct that encodes a fusion protein comprising a cyclophilin polypeptide or a derivative thereof fused to an energy transfer molecule polypeptide or a derivative thereof, under conditions that permit expression of the fusion protein;
- and
- (b) recovering fusion protein from the culture.
A kit for screening for agents that alter mitochondrial permeability transition, comprising:
- (a) an isolated cyclophilin D polypeptide or a derivative thereof;
- (b) an isolated adenine nucleotide translocator polypeptide or a derivative thereof;
- and
- (c) a detection reagent that specifically binds to at least one of the foregoing polypeptides.
A kit for screening for agents that alter mitochondrial permeability transition (MPT), comprising:
- (a) a host cell;
- (b) a first nucleic acid expression construct, comprising a promoter operably linked to a polynucleotide encoding an adenine nucleotide translocator polypeptide fused to a first energy transfer molecule or a variant thereof;
- and
- (c) a second nucleic acid expression construct, comprising a promoter operably linked to a polynucleotide encoding a cyclophilin D polypeptide fused to a second energy transfer molecule or a variant thereof.
Dependencies are inferred from the claim text.