Signal Transduction: Proteolytic Signalling by Inflammasomes

Introduction to Caspases and Programmed Cell Death

  • Apoptosis (Programmed Cell Death):

    • Defined as a "cellular suicide mechanism" designed for "neat and tidy" removal of cells by phagocytes.

    • It specifically avoids generating inflammation.

    • Immune Function: Utilised to eliminate infected or cancerous cells.

    • Physiological Role: Required for normal development and homeostasis.

    • Mechanism: The signal transduction involves "apoptotic caspases."

  • Pyroptosis (Programmed Necrosis):

    • Defined as cell lysis; it can be unplanned or programmed.

    • Pyroptosis is a form of programmed necrosis initiated by "inflammatory caspases" (not apoptotic caspases).

    • Messy Suicide: It is a deliberate form of cell suicide that activates the immune system.

    • Inflammatory Trigger: It recruits immune cells and activates antimicrobial functions.

    • Function: Critical for the clearance of intracellular pathogens.

Comparison of Pyroptosis vs. Apoptosis

  • Pyroptosis:

    • Mediator: Inflammatory caspases activated by an inflammasome.

    • Cellular Morphology: The cell cytosol expands and components are released as the cell bursts.

    • Immune Impact: Highly inflammatory; the release of contents alerts the immune system.

  • Apoptosis:

    • Mediator: Apoptotic caspases activated by death receptor signaling or the apoptosome.

    • Cellular Morphology: The cell shrinks; intracellular content is kept within "apoptotic bodies."

    • Immune Impact: Does not involve the immune system; bodies are consumed by phagocytes.

Mammalian Caspases: Structure and Function

  • Definition: Caspases are cysteine proteases that cleave specific sequences after an aspartic acid residue.

  • Zymogens: Caspases are expressed as inactive proteases known as zymogens.

  • Initiator Caspases:

    • Require clustering upon a signaling hub to dimerize and gain activity.

    • Often contain a CARD domain (Caspase Activation and Recruitment Domain), which facilitates interactions with other CARD-containing proteins.

  • Structure:

    • Includes a Card Linker (CDL) and an Interdomain Linker (IDL).

    • Composed of a Large subunit and a Small subunit.

Inflammasome Signaling Components

  • Definition: Inflammasomes are cytosolic signaling complexes or "molecular machines" that generate inflammatory responses.

  • Functions:

    1. Drive pyroptosis.

    2. Generate pro-inflammatory cytokines such as interleukin-1β\beta (IL-1β\beta) and interleukin-18 (IL-18).

    •     Pro-inflammatory Cytokines: Proteins released by one cell to trigger an inflammatory response in another cell.

  • Core Components:

    • Receptor: Example: NLRP3.

    • Adaptor: ASC.

    • Effector: Caspase-1 protease.

Signal Initiation: Receptor Activation

  • Mechanism: Signalling is initiated by receptor shape changes.

  • Interaction: The receptor, NLRP3, interacts with a "ligand" or regulator, such as NEK7.

  • Formation: This results in the formation of a large signalling complex "wheel" through an oligomerisation domain. (shrimp platter lecture)

Signal Relay and Amplification: ASC Polymerisation

  • Relay Mechanism: The receptor relays the signal to the adaptor protein, ASC.

  • ASC Structure:

    • Contains a Pyrin domain that interacts with the pyrin domain of the receptor (e.g., NLRP3).

    • Contains a CARD domain that interacts with the CARD domain of Caspase-1.

  • Signal Amplification: ASC has "prion-like" properties. It polymerises to form a giant signalling hub as it has increased affinity for itself and changes other ASC to prion-like conformation.

Effector Activation: Execution of Inflammation and Cell Death

  • Caspase-1 Recruitment: ASC recruits Caspase-1 monomers via CARD-CARD interactions, allowing them to cluster and dimerise. Increased Caspase-1 binding and dimersation causes protease domain to refold and become active.

  • Protease Activation:

    • Once dimerised, Caspase-1 undergoes self-cleavage at the interdomain linker.

    • This generates the full activeness of Caspase-1.

  • Downstream Substrates:

    • Gasdermin-D (GSDMD): Caspase-1 cleaves off the inhibitory domain of GSDMD. The remaining latent pore-forming protein oligomerises and forms pores in the plasma membrane. Water enters, causing the cell to swell and burst (pyroptosis).

    • Pro-cytokines: Caspase-1 cleaves pro-IL-1β\beta and pro-IL-18 into their active forms (IL-1β\text{IL-1}\beta and IL-18\text{IL-18}), which can then pass through membranes to recruit immune cells and induce antimicrobial responses like fever.

Signal Shutdown: The Intrinsic Proteolytic Timer

  • Necessity for Deactivation:

    • In apoptosis, IAPs (Inhibitor of Apoptosis Proteins) prevent accidental death.

    • In pyroptosis, shutdown is critical because some cells (e.g., neutrophils) activate Caspase-1 without undergoes lysis. Uncontrolled signalling leads to disease.

  • Self-Limiting Mechanism:

    • Inflammasome recruits and clusters caspase-1 monomers, promoting caspase-1 dimerisation, IDL cleavage and activation. Ejects protease from inflammasome that destabilises dimer and causes it to fall apart.

    • The inflammasome-caspase-1 complex acts as a holoenzyme.

    • Activity is governed by an intrinsic proteolytic timer, halts signalling after specific amount of time.

    • Stages of Activity:

    1. Basal Activity: Monomers are clustered.

    2. Full Activity: Dimerization and IDL cleavage (formation of p46 then p33/p10).

    3. Deactivation: Subsequent cleavage at the CDL (CARD linker) ejects the protease domain from the inflammasome, triggering the destabilization and deactivation of the dimer (formation of p20/p10).

Inflammasomes in Disease

  • New Therapeutic Targets:

    • NLRP3 inhibitors.

    • Caspase inhibitors.

    • Blocking IL-1 activity (which does not block IL-18).

Questions & Discussion

  • Q: How are initiator caspases activated?

  • A: They are activated by clustering upon a signaling hub (such as an inflammasome) which allows for dimerization.

  • Q: Why do initiator caspases require a signaling hub for their activation?

  • A: The proximity provided by the hub is necessary for the monomers to dimerize and gain proteolytic activity.

  • Q: What domain in initiator caspases allows their recruitment to a signaling hub?

  • A: The CARD (Caspase Activation and Recruitment Domain).

  • Q: What cellular response does caspase-1 trigger, and what mechanism is used to achieve this?

  • A: Caspase-1 triggers pyroptosis and inflammation. This is achieved by cleaving Gasdermin-D to form pores for cell lysis and cleaving pro-cytokines (IL-1β\beta, IL-18) for immune signaling.

  • Q: How is signal amplification achieved in the inflammasome signaling pathway?

  • A: Through the massive polymerization of the ASC adaptor protein, forming a giant prion-like signaling hub.

  • Q: How is the inflammasome signaling pathway turned off naturally?

  • A: Via an intrinsic proteolytic timer where Caspase-1 eventually cleaves its own CDL (CARD linker), causing the active protease dimer to dissociate from the signaling hub.

  • Q: What clinical approaches might be used to turn off inflammasome signaling in disease?

  • A: Use of NLRP3 inhibitors, Caspase inhibitors, or anti-IL-1 drugs/IL-1 receptor antagonists.

  • Q: What are the names of the proteins in the inflammasome signaling pathway?

  • A: Receptor (NLRP3), signal adaptor (ASC), effector (Caspase-1), and cell death executioner (Gasdermin D).