Methods for detecting membrane derived caspase activity and modulators thereof

Abstract

Provided are methods for detecting membrane derived apoptotic activity. In one embodiment, the present invention provides methods for identifying membrane derived caspase activity. In other embodiments, drug discovery methods are provided for screening compounds that inhibit or enhance membrane derived caspase activity. In the various embodiments, heavy membrane fractions are utilized for the screening methodologies described herein.

1 . A method for identifying membrane derived caspase activity, comprising incubating a membrane fraction comprising heavy or nuclear membranes under conditions and for a time sufficient to allow for the evolution of caspase activity, and subsequently detecting caspase activity.  
     
     
         2 . The method of  claim 1 , wherein caspase activity is detected by measuring caspase substrate turnover.  
     
     
         3 . The method of  claim 2 , wherein substrate turnover is measured by time course analysis.  
     
     
         4 . The method of  claim 2 , wherein substrate turnover is measured by endpoint analysis.  
     
     
         5 . The method of  claim 2 , wherein substrate turnover is detected 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.  
     
     
         6 . The method of  claim 1 , wherein caspase activity is detected by determining caspase processing said processing providing large and small caspase subunits.  
     
     
         7 . The method of  claim 6 , wherein large and small caspase subunits are detected 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.  
     
     
         8 . The method of any one of claims  1 - 7 , wherein the membrane fraction is derived from non-apoptotic cells.  
     
     
         9 . The method of any one of claims  1 - 7 , wherein the membrane fraction is derived from cells treated with a stimulator of apoptosis.  
     
     
         10 . The method of  claim 9 , wherein the stimulator of apoptosis is selected from the group consisting of deprivation of a growth factor, staurosporine, anti-fas antibody, ultraviolet irradiation, gamma irradiation, and Tumor Necrosis Factor.  
     
     
         11 . A method for identifying an inhibitor of the activity of a membrane derived caspase, comprising contacting a membrane fraction with a caspase substrate in the presence and absence of at least one candidate inhibitor; and comparing the levels of caspase substrate turnover, and therefrom identifying an inhibitor of the activity of a membrane derived caspase.  
     
     
         12 . The method of  claim 11 , 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.  
     
     
         13 . The method of  claim 12 , wherein the substrate comprises the peptide DEVD.  
     
     
         14 . The method of  claim 11 , wherein the membrane fraction is prepared from un-stimulated tissue culture cells selected from the group consisting of 697 lymphoblastoid cells, E15 primary brain cortical cells, MN9D cells, Jurkat T cells, and FL5.12 cells.  
     
     
         15 . The method of  claim 11 , wherein the membrane fraction comprises membranes selected from the group consisting of heavy membranes and nuclear membranes.  
     
     
         16 . The method of  claim 11 , wherein the membrane fraction comprises heavy membranes.  
     
     
         17 . The method of  claim 11 , wherein substrate turnover is detected by time course analysis.  
     
     
         18 . The method of  claim 11 , wherein substrate turnover is detected by endpoint analysis.  
     
     
         19 . The method of  claim 17  or  18 , 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.  
     
     
         20 . The method of  claim 11 , wherein the membrane fraction is derived from cells expressing pro-apoptotic polypeptides.  
     
     
         21 . The method of  claim 11 , further comprising incubating the membrane fraction with a caspase activator prior to or concurrent with the addition of the caspase substrate.  
     
     
         22 . The method of  claim 11 , wherein the membrane fraction is derived from non-apoptotic cells.  
     
     
         23 . The method of  claim 11 , wherein the membrane fraction is derived from cells treated with a stimulator of apoptosis.  
     
     
         24 . The method of  claim 23 , wherein the stimulator of apoptosis is selected from the group consisting of deprivation of a growth factor, staurosporine, anti-fas antibody, ultraviolet irradiation, gamma irradiation and Tumor Necrosis Factor.  
     
     
         25 . A method for identifying an enhancer of the activity of a membrane derived caspase, comprising contacting a membrane fraction with a caspase substrate in the presence and absence of at least one candidate enhancer; and comparing the levels of caspase substrate turnover, and therefrom identifying an enhancer of the activity of a membrane derived caspase.  
     
     
         26 . The method of  claim 25 , 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.  
     
     
         27 . The method of  claim 26 , wherein the substrate comprises the peptide DEVD.  
     
     
         28 . The method of  claim 25 , wherein the membrane fraction is prepared from un-stimulated tissue culture cells selected from the group consisting of 697 lymphoblastoid cells, E15 primary brain cortical cells, MN9D cells, Jurkat T cells and FL5.12 cells.  
     
     
         29 . The method of  claim 25 , wherein the membrane fraction comprises membranes selected from the group consisting of heavy membranes and nuclear membranes.  
     
     
         30 . The method of  claim 25 , wherein the membrane fraction comprises heavy membranes.  
     
     
         31 . The method of  claim 25 , wherein substrate turnover is detected by time course analysis.  
     
     
         32 . The method of  claim 25 , wherein substrate turnover is detected by endpoint analysis.  
     
     
         33 . The method of  claim 31  or  32 , 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.  
     
     
         34 . The method of  claim 25 , wherein the membrane fraction is derived from cells expressing an anti-apoptotic polypeptide.  
     
     
         35 . The method of  claim 34 , wherein the anti-apoptotic polypeptide is Bcl-2.  
     
     
         36 . The method of  claim 34 , further comprising incubating the membrane fraction with a caspase activator prior to or concurrent with the addition of the caspase substrate.  
     
     
         37 . The method of  claim 25 , wherein the membrane fraction is derived from non-apoptotic cells.  
     
     
         38 . The method of  claim 25 , wherein the membrane fraction is derived from cells treated with a stimulator of apoptosis.  
     
     
         39 . The method of  claim 38 , wherein the stimulator of apoptosis is selected from the group consisting of deprivation of a growth factor, staurosporine, anti-fas antibody, ultraviolet irradiation, gamma irradiation and Tumor Necrosis Factor.  
     
     
         40 . The method of  claim 25 , wherein an exogenous anti-apoptotic polypeptide is added prior to or concurrently with the addition of the caspase substrate.  
     
     
         41 . A method for identifying an inhibitor or enhancer of the evolution of caspase processing within a membrane fraction, comprising contacting a membrane fraction with at least one candidate inhibitor or candidate enhancer; and detecting the presence of large and small caspase subunits, and therefrom determining the level of caspase processing, wherein a decrease in processing indicates the presence of a caspase processing inhibitor, and wherein an increase in processing indicates the presence of a caspase processing enhancer.  
     
     
         42 . The method of  claim 41 , wherein the membrane fraction is prepared from un-stimulated tissue culture cells selected from the group consisting of 697 lymphoblastoid cells, E15 primary brain cortical cells, MN9D cells, Jurkat T cells, and FL5.12 cells.  
     
     
         43 . The method of  claim 41 , wherein the membrane fraction comprises membranes selected from the group consisting of heavy membranes and nuclear membranes.  
     
     
         44 . The method of  claim 41 , wherein the membrane fraction comprises heavy membranes.  
     
     
         45 . The method of  claim 41 , wherein large and small caspase subunits are detected 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.  
     
     
         46 . The method of  claim 41 , wherein the membrane fraction is derived from cells expressing anti-apoptotic polypeptides.  
     
     
         47 . The method of  claim 46 , wherein the anti-apoptotic polypeptide is Bcl-2.  
     
     
         48 . The method of  claim 41 , wherein the membrane fraction is derived from non-apoptotic cells.  
     
     
         49 . The method of  claim 41 , wherein the membrane fraction is derived from cells treated with a stimulator of apoptosis.  
     
     
         50 . The method of  claim 49 , wherein the stimulator of apoptosis is selected from the group consisting of deprivation of a growth factor, staurosporine, anti-fas antibody, ultraviolet irradiation, gamma irradiation and Tumor Necrosis Factor.  
     
     
         51 . A method of identifying a compound that modulates membrane fraction derived caspase activity, comprising incubating a membrane fraction, an inhibitor of apoptosis, and a caspase substrate in the presence and absence of at least one candidate compound under conditions and for a time sufficient to allow for the evolution of caspase activity; and comparing the levels of caspase substrate turnover, thereby identifying a compound that modulates membrane derived caspase activity.  
     
     
         52 . The method of  claim 51 , wherein the caspase substrate comprises a site cleaved by a caspase and is selected from the group consisting of a protein, a polypeptide, an oligopeptide, a peptide mimetic, and a peptide.  
     
     
         53 . The method of  claim 52 , wherein the substrate comprises the peptide DEVD-amc.  
     
     
         54 . The method of  claim 51 , wherein the membrane fraction is prepared from un-stimulated tissue culture cells selected from the group consisting of 697 lymphoblastoid cells, E15 primary brain cortical cells, MN9D cells, Jurkat T cells and FL5.12 cells.  
     
     
         55 . The method of  claim 51 , wherein the membrane fraction comprises membranes selected from the group consisting of heavy membranes and nuclear membranes.  
     
     
         56 . The method of  claim 51 , wherein the membrane fraction comprises heavy membranes.  
     
     
         57 . The method of  claim 51 , wherein substrate turnover is detected by time course analysis.  
     
     
         58 . The method of  claim 51 , wherein substrate turnover is detected by endpoint analysis.  
     
     
         59 . The method of  claim 57  or  58 , 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.  
     
     
         60 . The method of  claim 51 , wherein the membrane fraction contains the inhibitor of apoptosis.  
     
     
         61 . The method of  claim 60 , wherein the membrane fraction is derived from cells expressing Bcl-2.  
     
     
         62 . The method of  claim 51 , wherein the inhibitor of apoptosis is a Bcl-2 polypeptide or a functional fragment thereof.  
     
     
         63 . The method of  claim 51 , further comprising incubating the membrane fraction with a caspase activator prior to or concurrent with the addition of the caspase substrate.  
     
     
         64 . The method of  claim 51 , wherein the membrane fraction is derived from non-apoptotic cells.  
     
     
         65 . The method of  claim 51 , wherein the membrane fraction is derived from cells treated with a stimulator of apoptosis.  
     
     
         66 . The method of  claim 65 , wherein the stimulator of apoptosis is selected from the group consisting of deprivation of a growth factor, staurosporine, anti-fas antibody, ultraviolet irradiation, gamma irradiation and Tumor Necrosis Factor.  
     
     
         67 . An inhibitor of the activity of a membrane derived caspase identified by any one of methods 11 and 41.  
     
     
         68 . An enhancer of the activity of a membrane derived caspase identified by any one of methods 25 and 41.