519-2024 lecture #3

Caspases Overview

  • Definition: Caspases are a family of cysteine proteases that play essential roles in programmed cell death (apoptosis).

Caspase Activity Regulation

  • Regulation of caspases involves several mechanisms that determine when and where they become active.

Caspase Substrates

  • Caspases target specific substrates during apoptosis, leading to cleavage at precise sites mainly after aspartate residues.

Caspase Structure and Function

Caspase Characteristics

  • Cystein aspartate-specific proteases.

  • Contain an active cystein residue in the catalytic site.

  • Function in a specificity manner cleaving after the aspartate residue of the substrate.

  • Synthesized as inactive precursors known as zymogens.

  • Initiator Caspases activate Effector Caspases.

Types of Caspases

  • Divided into three groups:

    • Initiators: Trigger the apoptotic signaling cascade.

    • Effectors: Carry out the destruction of cellular components.

    • Inflammation-related: Involved in the inflammatory response.

Schematic Structure of Caspases

  • Zymogen: The proteolytically inactive precursor containing:

    • Prodomain.

    • Large catalytic subunit (p20).

    • Small catalytic subunit (p10).

  • Precursors activated by proteolytic cleavage.

Interaction Domains

  • DED: Death Effector Domain, mediates interactions for caspases 8 and 10.

  • CARD: Caspase Activation and Recruitment Domain, mediates interactions for caspases 2 and 9.

Caspase Activation on the Apoptosome

  • Interaction with proteins like Apaf-1 and cytochrome c leads to the formation of an apoptosome that activates procaspase 9.

Apaf-1 in Cancer

  • Apaf-1 plays a critical role in apoptosis; its loss in metastatic melanomas leads to chemoresistance.

  • Restoration of Apaf-1 enhances sensitivity to chemotherapy.

DNA Damage and Apoptosis

  • Apoptosis is initiated through various signals, including DNA damage pathways involving p53 and other proteins.

Molecular Mechanisms of Apoptosis

  • Caspases do not degrade proteins but cleave specific targets, leading to activation or inactivation.

  • About 150 known caspase targets exist, playing roles in cellular signaling.

Roles of IAPs (Inhibitors of Apoptosis)

  • IAPs inhibit caspases; their regulation is crucial in controlling cell death.

Apoptotic Defects and Restoration

  • Restoration of Apaf-1 expression can rescue apoptotic defects and enhance chemosensitivity against treatments like ADR.

Summary of Caspase Functions

  • Caspases target a variety of proteins involved in:

    • Regulation/execution of apoptosis.

    • Structural integrity of cells.

    • Cellular repair mechanisms.

Mechanisms of Inhibition

  • Various mechanisms, including viral inhibitors and peptide inhibitors, prevent caspase activity, illustrating the complexity of apoptotic regulation.

Caspases Overview

Definition

Caspases are a family of cysteine proteases that play essential roles in programmed cell death (apoptosis) and inflammation. They are crucial mediators that ensure the orderly execution of cellular demise, which is vital for normal development and maintenance of tissue homeostasis in multicellular organisms.

Caspase Activity Regulation

The regulation of caspases involves multiple mechanisms that determine not only when but also where they become active within the cell. These regulations are essential for preventing unintended cell death and ensuring proper cellular responses to various internal and external signals. Key regulatory factors include post-translational modifications such as phosphorylation, ubiquitination, and interactions with inhibitors of apoptosis proteins (IAPs).

Caspase Substrates

Caspases specifically target various substrates during apoptosis, leading to their cleavage at precise sites, predominantly after aspartate residues. This specificity ensures that the destruction of cellular components occurs with high fidelity, impacting various cellular pathways and functions, including cytoskeletal structure and DNA repair mechanisms.

Caspase Structure and Function

Caspase Characteristics

  • Cystein Aspartate-specific ProteasesCaspases belong to the cysteine peptidase family, characterized by their ability to cleave peptide bonds following aspartate residues.

  • Active Cystein ResidueThey contain an active cysteine residue within their catalytic site, which is crucial for their proteolytic activity.

  • Zymogen ActivationCaspases are synthesized as inactive precursors known as zymogens, which require proteolytic cleavage to attain an active form.

  • Initiator and Effector CaspasesInitiator caspases, such as caspases 2, 8, and 9, activate effector caspases, such as caspases 3, 6, and 7, which execute the apoptotic program.

Types of Caspases

Caspases can be categorized into three primary groups based on their functions:

  1. Initiators: Trigger the apoptotic signaling cascade upon activation.

  2. Effectors: Carry out the destruction of various cellular components, including proteins associated with cell survival and homeostasis.

  3. Inflammation-related: Engage in the inflammatory response and contribute to the processing of pro-inflammatory cytokines.

Schematic Structure of Caspases

  • Zymogen StructureThe proteolytically inactive precursor (zymogen) consists of:

    • Prodomain: Functions in the regulation and activation of the enzyme.

    • Large Catalytic Subunit (p20): Contains the active site and is responsible for mediating the proteolytic activity.

    • Small Catalytic Subunit (p10): Complements the large subunit in forming the active enzyme.

  • Precursors are activated through specific proteolytic cleavage, allowing functional interactions with substrates.

Interaction Domains

  • DED (Death Effector Domain): Mediates interactions for initiator caspases like caspases 8 and 10, facilitating their activation in response to death signals.

  • CARD (Caspase Activation and Recruitment Domain): Mediates interactions for initiator caspases such as caspases 2 and 9, linking them to apoptosome formation during apoptotic signaling.

Caspase Activation on the Apoptosome

The interaction of procaspase 9 with proteins such as Apaf-1 and cytochrome c results in the formation of the apoptosome, which serves as a platform for the activation of procaspase 9 to its active form, leading to downstream effector caspase activation.

Apaf-1 in Cancer

Apaf-1 is integral to the apoptotic process, and its loss in certain cancers, such as metastatic melanomas, has been linked to increased drug resistance. Restoration of Apaf-1 expression can enhance chemosensitivity by reinstating apoptosis in cancer cells, making it a potential therapeutic target.

DNA Damage and Apoptosis

Multiple signals can initiate apoptosis, particularly in response to DNA damage. Key players such as the tumor suppressor protein p53 and other checkpoint proteins ensure that damaged cells are eliminated to maintain genomic integrity and prevent tumorigenesis.

Molecular Mechanisms of Apoptosis

Caspases are not merely non-selective proteases; instead, they cleave specific targets leading to either activation or inactivation of various signaling pathways. Approximately 150 known caspase targets exist, contributing to diverse cellular functions including apoptosis execution, cell cycle regulation, and stress responses.

Roles of IAPs (Inhibitors of Apoptosis)

Inhibitor of Apoptosis Proteins (IAPs) play a crucial regulatory role in apoptosis by inhibiting caspase activity. Their expression can be modulated in response to various stress signals, highlighting the complexity in the control of cell death. Dysregulation of IAPs is often implicated in cancer progression and treatment resistance.

Apoptotic Defects and Restoration

Restoring expression of Apaf-1 has been shown to rescue apoptotic defects in cancer cells, which enhances chemosensitivity against treatments such as doxorubicin (ADR) and signifies the importance of apoptotic pathways in effective cancer therapy.

Summary of Caspase Functions

Caspases are critical for the targeting of a wide variety of proteins involved in:

  • The regulation and execution of apoptosis.

  • Maintaining structural integrity of cells.

  • Engaging cellular repair mechanisms in response to damage.

Mechanisms of Inhibition

Caspase activity can be inhibited through various mechanisms, including viral proteins that counteract caspase activation and synthetic peptide inhibitors used in research and therapeutic contexts. This complexity reflects the tightly regulated nature of apoptotic processes and their significance in health and disease.