Membrane derived caspase-3, compositions comprising the same and methods of use therefor

Abstract

Provided are isolated nucleic acids encoding a novel membrane derived caspase-3 and polypeptides expressed therefrom. In one embodiment, the nucleic acid expression vectors that produce membrane derived caspase-3 polypeptide may be introduced into host cells as a gene delivery vehicle. In other embodiments, methods are provided for treating pathological disorders caused by altered apoptosis, such as autoimmune disease, cancer, viral infections, and bacterial infections. Another aspect of the invention is the use of the isolated nucleic acid encoding membrane derived caspase-3 and polypeptides expressed therefrom as a means for promoting or inhibiting programmed cell death.

1 . An isolated nucleic acid molecule consisting essentially of a sequence encoding a membrane derived caspase-3 polypeptide of SEQ ID NO: 2.  
     
     
         2 . The nucleic acid molecule of  claim 1  wherein the membrane derived caspase-3 oligomerizes with a caspase.  
     
     
         3 . An isolated nucleic acid molecule encoding a membrane derived caspase-3 consisting essentially of a single stranded or double stranded polynucleotide sequence of SEQ ID NO: 2.  
     
     
         4 . A vector, comprising the nucleic acid molecule of any one of claims  1  or  3 .  
     
     
         5 . The vector of  claim 4  wherein the vector is a viral vector.  
     
     
         6 . A nucleic acid expression vector, comprising the nucleic acid molecule of any one of claims  1 - 3  wherein the nucleic acid molecule is operably linked to a promoter.  
     
     
         7 . The vector of  claim 6  wherein the promoter is an inducible promoter.  
     
     
         8 . A host cell containing the vector of  claim 6 .  
     
     
         9 . A host cell containing the vector of  claim 7 .  
     
     
         10 . The host cell of  claim 8  wherein the host cell is selected from the group consisting of a bacterium, a yeast cell, a nematode cell, an insect cell, and a mammalian cell.  
     
     
         11 . The host cell of  claim 9  wherein the host cell is selected from the group consisting of a bacterium, a yeast cell, a nematode cell, an insect cell, and a mammalian cell.  
     
     
         12 . An isolated membrane derived caspase-3 polypeptide consisting essentially of SEQ ID NO: 3.  
     
     
         13 . The polypeptide of  claim 12  wherein the polypeptide oligomerizes with a caspase.  
     
     
         14 . The polypeptide of  claim 13  wherein the caspase is selected from the group consisting of caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, and caspase-14.  
     
     
         15 . An antibody specific for a membrane derived caspase-3 polypeptide, the polypeptide consisting essentially of SEQ ID NO: 3.  
     
     
         16 . The antibody of  claim 15  wherein the antibody is a monoclonal antibody.  
     
     
         17 . A cell expressing the antibody of any one of claims  15  or  16 .  
     
     
         18 . An antibody that does not specifically recognize a cytoplasmic derived caspase-3 and does specifically recognize a membrane derived caspase-3 polypeptide, the membrane derived caspase-3 polypeptide consisting essentially of SEQ ID NO: 3.  
     
     
         19 . An antibody that specifically recognizes a cytoplasmic derived caspase-3 and does not specifically recognize a membrane derived caspase-3 polypeptide, the membrane derived caspase-3 polypeptide consisting essentially of SEQ ID NO: 3.  
     
     
         20 . A method of producing a membrane derived caspase-3 polypeptide, comprising culturing a host cell containing a nucleic acid expression vector comprising at least one promoter operaby linked to a nucleic acid molecule encoding a membrane derived caspase-3 polypeptide, the nucleic acid molecule consisting essentially of SEQ ID NO: 2, under conditions and for a time sufficient for expression of the polypeptide.  
     
     
         21 . The method of  claim 20  wherein the promoter is inducible.  
     
     
         22 . The method of any one of claims  20 - 21 , further comprising contacting the host cell with a caspase activator under conditions and for a time sufficient to activate the membrane derived caspase-3 polypeptide.  
     
     
         23 . The method of any one of claims  20 - 21 , further comprising the host cell expressing a caspase activator under conditions and for a time sufficient to activate the membrane derived caspase-3 polypeptide.  
     
     
         24 . The method of  claim 22  wherein the activator is selected from the group consisting of caspase-1, caspase-8, caspase-9, caspase-10, and Granzyme B.  
     
     
         25 . The method of  claim 23  wherein the activator is selected from the group consisting of caspase-1, caspase-8, caspase-9, caspase-10, and Granzyme B.  
     
     
         26 . A membrane derived caspase-3 polypeptide produced by any one of the methods of claims  20 - 21 .  
     
     
         27 . A method of inducing apoptosis in a cell, comprising delivering to a cell an effective amount of an isolated nucleic acid molecule encoding a membrane derived caspase-3 polypeptide, the nucleic acid molecule consisting essentially of SEQ ID NO: 2, under conditions and for a time sufficient for expression of the polypeptide and therefrom detecting apoptosis of the cell.  
     
     
         28 . The method of  claim 27  wherein the cell comprises a tissue culture cell.  
     
     
         29 . The method of  claim 28  wherein the tissue culture cell is selected from the group consisting of 697 lymphoblastoid cells, E15 primary brain cortical cells, MN9D cells, Jurkat T cells, THP-1 cells, and FL5. 12 cells.  
     
     
         30 . The method of  claim 27  wherein the step of delivering to the cell is selected from the group consisting of injection, transfection, transformation, electroporation, and receptor mediated endocytosis.  
     
     
         31 . The method of  claim 27  wherein the step of delivering comprises administering the nucleic acid molecule to the circulatory system of a warm-blooded mammal in which the cell is located.  
     
     
         32 . A method of inducing apoptosis in a cell, comprising delivering to a cell an effective amount of a membrane derived caspase-3 polypeptide, the polypeptide consisting essentially of SEQ ID NO: 3, under conditions and for a time sufficient to detect therefrom the induction of apoptosis of the cell.  
     
     
         33 . The method of  claim 32  wherein the step of delivering to the cell comprises injecting the polypeptide.  
     
     
         34 . The method of any one of claims  27  or  32  wherein the step of apoptosis detection is selected from the group consisting of altered cellular morphology, DNA fragmentation, annexin binding, caspase activity, and mitochondrial release of cytochrome c.  
     
     
         35 . A gene delivery vehicle comprising a nucleic acid molecule according to any one of claims  1 - 3  wherein the nucleic acid molecule is operably linked to a promoter.  
     
     
         36 . The gene delivery vehicle of  claim 35  wherein the vehicle is a retrovirus or adenovirus.  
     
     
         37 . The gene delivery vehicle of  claim 35  wherein the nucleic acid molecule is associated with a polycation.  
     
     
         38 . The gene delivery vehicle of  claim 35 , further comprising a ligand that binds a cell surface receptor.  
     
     
         39 . A method of treating cancer, comprising administering to a patient a gene delivery vehicle according to any one of claims  35 - 38  wherein the gene delivery vehicle is internalized by tumor cells.  
     
     
         40 . A method of treating autoimmune disease, comprising administering to a patient a gene delivery vehicle according to any one of claims  35 - 38 , wherein the gene delivery vehicle is internalized by cells mediating autoimmune disease.  
     
     
         41 . A method of treating viral infections, comprising administering to a patient a gene delivery vehicle according to any one of claims  35 - 38 , wherein the gene delivery vehicle is internalized by virally-infected cells.  
     
     
         42 . A method of treating bacterial infections, comprising administering to a patient a gene delivery vehicle according to any one of claims  35 - 38 , wherein the gene delivery vehicle is internalized by bacterially-infected cells.  
     
     
         43 . A kit for screening for agents that alter apoptosis, comprising a host cell and an isolated nucleic acid molecule consisting essentially of a sequence encoding a membrane derived caspase-3 polypeptide of SEQ ID NO: 2.  
     
     
         44 . The kit according to  claim 43  wherein the host cell is a eukaryotic cell.  
     
     
         45 . The kit according to  claim 44  wherein the host cell is selected from the group consisting of 697 lymphoblastoid cells, E15 primary-brain cortical cells, MN9D cells, Jurkat T cells, THP-1 cells, and FL5. 12 cells.  
     
     
         46 . A kit for screening for agents that alter apoptosis, comprising a membrane derived caspase-3 polypeptide, the polypeptide consisting essentially of SEQ ID NO: 3 and a detection reagent that specifically binds to at least one of the foregoing polypeptides.  
     
     
         47 . A kit according to  claim 46  wherein the detection reagent is an antibody or antigen-binding fragment thereof.  
     
     
         48 . A composition, comprising a membrane derived caspase-3 polypeptide consisting essentially of SEQ ID NO: 3, and an excipient or diluent.  
     
     
         49 . A method for identifying an agent that alters the activity of a membrane derived caspase-3 polypeptide consisting essentially of SEQ ID NO: 3, comprising: 
 contacting the membrane derived caspase-3 polypeptide with a caspase substrate in the presence and absence of at least one candidate agent; and    comparing the levels of caspase substrate turnover and therefrom identifying an agent that alters the activity of the membrane derived caspase-3 polypeptide.    
     
     
         50 . The method of  claim 49  wherein the caspase substrate comprises a site cleaved by a caspase selected from the group consisting of a protein, a polypeptide, an oligopeptide, a peptide mimetic and a peptide.  
     
     
         51 . The method of  claim 50  wherein the substrate comprises the peptide DEVD.  
     
     
         52 . The method of  claim 49  wherein the membrane derived caspase-3 polypeptide is part of a membrane fraction.  
     
     
         53 . The method of  claim 52  wherein the membrane fraction comprises membranes selected from the group consisting of heavy membrane and nuclear membrane.  
     
     
         54 . The method of  claim 53  wherein the membrane fraction comprises heavy membrane.  
     
     
         55 . The method of  claim 49  wherein substrate turnover is detected by time course analysis.  
     
     
         56 . The method of  claim 49  wherein substrate turnover is detected by endpoint analysis.  
     
     
         57 . The method of  claim 55  or  56  wherein caspase substrate turnover detection is performed by a method selected from the group consisting of fluorescence spectroscopy, mass spectrometry, HPLC, colorimetry, fluorography, radiography, gel electrophoresis, chromatography and N-terminal peptide sequencing.  
     
     
         58 . The method of  claim 49 , further comprising incubating the membrane derived caspase-3 polypeptide with a caspase activator prior to or concurrent with the addition of the caspase substrate.  
     
     
         59 . An agent identified by any one of the methods of claims  49  or  58  that alters the activity of a membrane derived caspase-3 polypeptide consisting essentially of SEQ ID NO: 3.  
     
     
         60 . An agent of  claim 59  wherein the agent inhibits the activity of the membrane derived caspase-3 polypeptide.  
     
     
         61 . An agent of  claim 59  wherein the agent enhances the activity of the membrane derived caspase-3 polypeptide.