Inhibition of interleukin-1-beta secretion by card proteins

A method of identifying inhibitors or enhancers of Pseudo-ICE mediated inhibition of pro-caspase-1 activation, comprising:

• a. contacting a cell transfected with an expression vector encoding Pseudo-ICE having at least 80% amino acid identity to SEQ ID NO:
• 1 or a functional fragment of the Pseudo-ICE capable of inhibiting pro-caspase-1 activation with a candidate inhibitor or enhancer;
• and
• b. detecting an increase or decrease in pro-caspase-1 activation in the presence of the candidate inhibitor or enhancer, wherein a decrease in pro-caspase-1 activation indicates the presence of an enhancer of Pseudo-ICE mediated inhibition of pro-caspase-1 activation and an increase in pro-caspase-1 activation indicates the presence of an enhancer.

A method of identifying inhibitors or enhancers of Pseudo-ICE mediated inhibition of IL-1β secretion in response to an inflammatory stimulus, comprising:

• a. contacting a cell transfected with an expression vector encoding Pseudo-ICE having at least 80% amino acid identity to SEQ ID NO:
• 1 or a functional fragment of the Pseudo-ICE capable of inhibiting the IL-1β secretion with a candidate inhibitor or enhancer;
• b. subjecting the transfected cell to an inflammatory stimulus;
• and
• c. detecting an increase or decrease in the IL-1β secretion level induced by the inflammatory stimulus in the presence of the candidate inhibitor or enhancer, wherein a decrease in the induced IL-1β secretion indicates the presence of an enhancer of Pseudo-ICE mediated inhibition of the induced IL-1β secretion and an increase in the induced IL-1β secretion indicates the presence of an inhibitor of Pseudo-ICE mediated inhibition of the induced IL-1β secretion.

A method of identifying inhibitors or enhancers of Pseudo-ICE mediated NF-kB activation, comprising:

• a. contacting a cell transfected with an expression vector encoding Pseudo-ICE having at least 80% amino acid identity to SEQ ID NO:
• 1 or a functional fragment of the Pseudo-ICE capable of stimulating NF-κB activation with a candidate inhibitor or enhancer;
• and
• b. detecting an increase or decrease in NF-kB activation in the presence of the candidate inhibitor or enhancer, wherein a decrease in NF-kB activation indicates the presence of an inhibitor and an increase in NF-kB indicates the presence of an enhancer.

A method of identifying a polypeptide that specifically binds to a Pseudo-ICE having at least 80% amino acid identity to SEQ ID NO:

• 1 or a functional fragment of the Pseudo-ICE that has an activity selected from:
• specific binding to pro-caspase-1, specific binding to RICK, inhibiting induced IL-1β secretion, and stimulating NF-κB activation, comprising:
• a. contacting a sample with the Pseudo-ICE or the functional fragment under conditions that permit the formation of a complex between the Pseudo-ICE or the functional fragment thereof and the polypeptide;
• and
• b. detecting the complex and polypeptide in the complex.

A method for identifying a compound that increases the specific binding between a pro-caspase-1 or a fragment thereof that comprises a pro-domain of the pro-caspase-1 and a Pseudo-ICE having at least 80% amino acid identity to SEQ ID NO:

• 1 or a finctional fragment of Pseudo-ICE capable of binding to the pro-caspase-1 or the fragment thereof, comprising:
• a. combining the Pseudo-ICE or the functional fragment with the pro-caspase-1 or the fragment thereof in the absence of a candidate compound under conditions that allow specific binding between the Pseudo-ICE or the functional fragment and the pro-capase-1 or the fragment thereof;
• b. combining the Pseudo-ICE or the functional fragment with the pro-caspase-1 or the fragment thereof in the presence of the candidate compound under conditions of step (a);
• c. comparing the specific binding between the Pseudo-ICE or the functional fragment and the pro-caspase-1 or the fragment thereof of step (a) with that of step (b) to thereby determine whether the candidate compound is capable of increasing the specific binding between the Pseudo-ICE or the functional fragment and the pro-caspase-1 or the fragment thereof.

A method for identifying a compound that decreases the specific binding between a pro-caspase-1 or a fragment thereof that comprises a pro-domain of the pro-caspase-1 and a Pseudo-ICE having at least 80% amino acid identity to SEQ ID NO:

• 1 or a functional fragment of Pseudo-ICE capable of binding to a pro-caspase-1, comprising:
• a. combining the Pseudo-ICE or the functional fragment with the pro-caspase-1 in the absence of a candidate compound under conditions that allow specific binding between the Pseudo-ICE or the functional fragment and the pro-capase-1 or the fragment thereof,
• b. combining the Pseudo-ICE or the functional fragment with the pro-caspase-1 or the fragment thereof in the presence of the candidate compound under conditions of step (a);
• and
• c. comparing the specific binding between the Pseudo-ICE or the functional fragment and the pro-caspase-1 or the fragment thereof of step (a) with that of step (b) to thereby determine whether the candidate compound is capable of decreasing the specific binding between the Pseudo-ICE or the functional fragment and the pro-caspase-1.

A method for identifying a compound that disrupts the binding between a pro-caspase-1 or a fragment thereof that comprises a pro-domain of the pro-caspase-1 and a Pseudo-ICE having at least 80% amino acid identity to SEQ ID NO:

• 1 or a functional fragment of the Pseudo-ICE capable of binding to the pro-caspase-1 or the fragment thereof, comprising:
• a. contacting a candidate compound with a binding complex comprising the Pseudo-ICE or the functional fragment and the pro-caspase-1;
• and
• b. detecting the Pseudo-ICE, the functional fragment, the pro-caspase-1 or the fragment thereof that dissociates from the binding complex to thereby determine whether the candidate compound is capable of disrupting the binding between the pro-caspase-1 or the fragment thereof and the Pseudo-ICE or the functional fragment.

A method for identifying a compound that increases the specific binding between a RICK and a Pseudo-ICE having at least 80% amino acid identity to SEQ ID NO:

• 1 or a functional fragment of Pseudo-ICE capable of binding to the RICK, comprising:
• a. combining the Pseudo-ICE or the functional fragment with the RICK in the absence of a candidate compound under conditions that allow specific binding between the Pseudo-ICE or the functional fragment and the capase-1;
• b. combining the Pseudo-ICE or the functional fragment with the RICK in the presence of the candidate compound under conditions of step (a);
• c. comparing the specific binding between the Pseudo-ICE or the functional fragment and the RICK of step (a) with that of step (b) to thereby determine whether the candidate compound is capable of increasing the specific binding between the Pseudo-ICE or the functional fragment and the RICK.

A method for identifying a compound that decreases the specific binding between a RICK and a Pseudo-ICE having at least 80% amino acid identity to SEQ ID NO:

• 1 or a functional fragment of Pseudo-ICE capable of binding to a RICK, comprising:
• a. combining the Pseudo-ICE or the functional fragment with the RICK in the absence of a candidate compound under conditions that allow specific binding between the Pseudo-ICE or the functional fragment and the RICK;
• b. combining the Pseudo-ICE or the functional fragment with the RICK in the presence of the candidate compound under conditions of step (a);
• and
• c. comparing the specific binding between the Pseudo-ICE or the functional fragment and the RICK of step (a) with that of step (b) to thereby determine whether the candidate compound is capable of decreasing the specific binding between the Pseudo-ICE or the functional fragment and the RICK.

A method for identifying a compound that disrupts the binding between a RICK and a Pseudo-ICE having at least 80% amino acid identity to SEQ ID NO:

• 1 or a functional fragment of the Pseudo-ICE capable of binding to the RICK, comprising:
• a. contacting a candidate compound with a binding complex comprising the Pseudo-ICE or the functional fragment and the RICK;
• and
• b. detecting the Pseudo-ICE, the functional fragment, or the RICK that dissociates from the binding complex to thereby determine whether the candidate compound is capable of disrupting the binding between the RICK and the Pseudo-ICE or the functional fragment.

A method of identifying inhibitors or enhancers of ICE-Like mediated inhibition of pro-caspase-1 activation, comprising:

• a. contacting a cell transfected with an expression vector encoding ICE-Like having at least 80% amino acid identity to SEQ ID NO:
• 3 (ICE-Like amino acid sequence) or a functional fragment of the ICE-Like capable of inhibiting pro-caspase-1 activation with a candidate inhibitor or enhancer;
• and
• b. detecting an increase or decrease in pro-caspase-1 activation in the presence of the candidate inhibitor or enhancer, wherein a decrease in pro-caspase-1 activation indicates the presence of an enhancer of ICE-Like mediated inhibition of pro-caspase-1 activation and an increase in pro-caspase-1 activation indicates the presence of an inhibitor of ICE-Like mediated inhibition of pro-caspase-1 activation.

A method of identifying inhibitors or enhancers of ICE-Like mediated inhibition of the IL-1β secretion in response to an inflammatory stimulus, comprising:

• a. contacting a cell transfected with an expression vector encoding ICE-Like having at least 80% amino acid identity to SEQ ID NO:
• 3 or a functional fragment of the ICE-Like capable of inhibiting IL-1β secretion with a candidate inhibitor or enhancer;
• b. subjecting the transfected cell to an inflammatory stimulus;
• and
• c. detecting an increase or decrease in the IL-1β secretion level induced by the inflammatory stimulus in the presence of the candidate inhibitor or enhancer, wherein a decrease in the induced IL-1β secretion indicates the presence of an enhancer of ICE-Like mediated inhibition of the induced IL-1β secretion and an increase in the induced IL-1β secretion indicates the presence of an inhibitor of ICE-Like mediated inhibition of the induced IL-1β secretion.

A method of identifying a polypeptide that specifically binds to an ICE-Like having at least 80% amino acid identity to SEQ ID NO:

• 3 or a functional fragment of the ICE-Like capable of specifically binding to pro-caspase-1 or inhibiting induced IL-1β secretion, comprising:
• a. contacting a cDNA expression library with the ICE-Like or the functional fragment under conditions that permit the formation of a complex between the ICE-Like or the functional fragment thereof and a polypeptide encoded by a clone of the cDNA expression library;
• and
• b. detecting the complex and the polypeptide in the complex.

A method for identifying a compound that increases the specific binding between a pro-caspase-1 or a fragment thereof that comprises a pro-domain of the pre-caspase-1 and an ICE-Like having at least 80% amino acid identity to SEQ ID NO:

• 3 or a functional fragment of ICE-Like capable of binding to the pro-caspase-1 or the fragment thereof, comprising:
• a. combining the ICE-Like or the functional fragment with the pro-caspase-1 or the fragment thereof in the absence of a candidate compound under conditions that allow specific binding between the ICE-Like or the functional fragment and the capase-1 or the fragment thereof;
• b. combining the ICE-Like or the functional fragment with the pro-caspase-1 or the fragment thereof in the presence of the candidate compound under conditions of step (a);
• and
• c. comparing the specific binding between the ICE-Like or the functional fragment and the pro-caspase-1 or the fragment thereof of step (a) with that of step (b) to thereby determine whether the candidate compound is capable of increasing the specific binding between the ICE-Like or the functional fragment and the pro-caspase-1.

A method for identifying a compound that decreases the specific binding between a pro-caspase-1 and an ICE-Like having at least 80% amino acid identity to SEQ ID NO:

• 3 or a fumctional fragment of ICE-Like capable of binding to a pro-caspase-1, comprising:
• a. combining the ICE-Like or the functional fragment with the pro-caspase-1 in the absence of a candidate compound under conditions that allow specific binding between the ICE-Like or the functional fragment and the capase-1;
• b. combining the ICE-Like or the functional fragment with the pro-caspase-1 in the presence of the candidate compound under conditions of step (a);
• c. comparing the specific binding between the ICE-Like or the functional fragment and the pro-caspase-1 of step (a) with that of step (b) to thereby determine whether the candidate compound is capable of decreasing the specific binding between the ICE-Like or the functional fragment and the pro-caspase-1.

A method for identifying a compound that disrupts the binding between a pro-caspase-1 or a fragment thereof that comprises a pro-domain of the pro-caspase-1 and an ICE-Like having at least 80% amino acid identity to SEQ ID NO:

• 3 or a functional fragment of the ICE-Like capable of binding to the pro-caspase-1 or the fragment thereof, comprising:
• a. contacting a candidate compound with a binding complex comprising the ICE-Like or the functional fragment and the pro-caspase-1 or the fragment thereof;
• and
• b. detecting the ICE-Like, the functional fragment, or the pro-caspase-1 or the fragment thereof that dissociates from the binding complex to thereby determine whether the candidate compound is capable of disrupting the binding between the pro-caspase-1 or the fragment thereof and the ICE-Like or the functional fragment.