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Caspase

Caspases are a family of cysteine-dependent aspartate-specific proteases that serve as critical regulators of programmed cell death, innate immunity, inflammation, and cellular homeostasis in multicellular organisms.^[1] These enzymes are synthesized as inactive zymogens, consisting of a prodomain, a large catalytic subunit (17–20 kDa), and a small catalytic subunit (10–12 kDa), which are activated through proteolytic processing into an active heterotetramer.^[1] Humans express 12 caspases, while mice have 10, with their catalytic activity relying on a histidine-cysteine dyad that hydrolyzes peptide bonds after aspartic acid residues.^[1] Caspases are broadly classified into three main groups based on function: inflammatory caspases (caspase-1, -4, and -5 in humans; caspase-11 in mice), which process pro-inflammatory cytokines like interleukin-1β (IL-1β) and interleukin-18 (IL-18) and trigger pyroptosis via gasdermin D cleavage; apoptotic initiator caspases (caspase-8, -9, and -10), which activate downstream effectors in response to extrinsic death receptor signals or intrinsic mitochondrial stress; and apoptotic effector caspases (caspase-3, -6, and -7), which dismantle cellular structures by cleaving hundreds of substrates, including DNA repair enzymes, cytoskeletal proteins, and nuclear lamins, leading to orderly cell demise without inflammation.^[1] Additional caspases, such as caspase-2, -10, -12, and -14, participate in non-apoptotic processes like cell differentiation, autophagy regulation, and tissue development.^[1] Beyond cell death, caspases contribute to physiological processes, including embryonic development, immune cell activation, and tissue remodeling, while dysregulation is implicated in diseases such as cancer, neurodegenerative disorders, and autoimmune conditions, making them promising therapeutic targets.^[1] The first caspase (caspase-1, originally termed interleukin-1β converting enzyme) was cloned in 1992, and its structure was solved in 1994, marking the beginning of extensive research into their mechanisms.^[1]

Overview and Structure

Definition and Properties

Caspases constitute a family of cysteine-dependent aspartate-specific proteases, commonly referred to as cysteine aspartases, that selectively cleave peptide bonds C-terminal to aspartic acid residues in their protein substrates.^[2] This enzymatic specificity distinguishes them from other protease families and is central to their biological roles. Caspases are evolutionarily conserved across metazoans, with homologs identified in organisms ranging from nematodes like Caenorhabditis elegans to mammals, underscoring their fundamental importance in multicellular life.^[2] These proteases are synthesized as inactive precursors known as zymogens or procaspases, which must undergo activation to exert their catalytic function.^[2] Activation typically involves proteolytic processing, ensuring tight regulation to prevent untimely cellular damage. A hallmark of their structure is the conserved catalytic dyad composed of a cysteine residue and a histidine residue within the active site, which facilitates efficient hydrolysis.^[2] In humans, 12 distinct caspases have been identified, reflecting their diverse yet coordinated contributions to cellular processes.^[1] The catalytic mechanism of caspases relies on the nucleophilic attack by the thiol group of the active-site cysteine on the carbonyl carbon of the peptide bond, with the adjacent histidine acting as a general base to deprotonate the cysteine and enhance its reactivity.^[2] This process exhibits stringent specificity for aspartic acid at the P1 position of the substrate, a feature that ensures precise targeting of caspase substrates during cellular signaling. Caspases play essential roles in maintaining programmed cell death and overall cellular homeostasis, processes critical for development, tissue remodeling, and immune responses in metazoans.^[2]

Molecular Architecture

Caspases are synthesized as inactive zymogens known as procaspases, which consist of an N-terminal prodomain of variable length, followed by a large catalytic subunit (approximately 20 kDa, or p20) and a small catalytic subunit (approximately 10 kDa, or p10).^[3] The prodomain, typically 20–120 amino acids long, is absent or short in effector caspases but extended in initiator caspases to include specific interaction motifs such as the caspase recruitment domain (CARD) or death effector domain (DED), which facilitate oligomerization and activation signaling.^[4] These domains enable homotypic interactions with adaptor proteins in activation platforms like the apoptosome or inflammasome.^[4] In their mature, active form, caspases assemble into a heterotetrameric structure comprising two p20 subunits and two p10 subunits, often denoted as (p20)₂(p10)₂.^[5] The active sites are located at the interfaces between the p20 and p10 subunits of adjacent monomers, where loops from both subunits contribute to substrate recognition and catalysis.^[6] This quaternary arrangement ensures that the enzyme's proteolytic activity is tightly regulated and only manifests upon proper dimerization.^[6] The catalytic domains of caspases exhibit a conserved beta-sheet-rich fold, featuring a central twisted six-stranded beta-sheet flanked by five alpha-helices on one side and additional helices and loops on the other, forming a compact globular structure.^[1] At the heart of the active site lies the highly conserved QACRG pentapeptide motif, where the cysteine residue (C) serves as the nucleophilic attacker in peptide bond hydrolysis, and the arginine (R) helps position the substrate.^[7] Caspases also harbor allosteric sites, often at dimer interfaces or remote loops, that modulate activity by stabilizing inactive conformations or enhancing catalytic efficiency upon ligand binding.^[8] Activation involves proteolytic processing that removes the prodomain and intersubunit linkers, inducing significant conformational changes, particularly in the active site loops (L1–L4).^[9] These rearrangements expose the substrate-binding cleft, which is occluded in the procaspase form, thereby enabling access for aspartate-containing substrates and full enzymatic competence.^[9] Dimerization further stabilizes this open conformation, linking architecture to function.^[9]

Classification

Initiator and Effector Types

Caspases are classified into initiator and effector types based on their structural features, activation mechanisms, and sequential roles within proteolytic cascades that drive programmed cell death.^[10] This dichotomy reflects their positions in signaling pathways, where initiators serve as sensors and amplifiers at the apex, while effectors act downstream to dismantle cellular components.^[11] In humans, twelve caspases are identified (numbered 1 through 10, 12, and 14), with most fitting neatly into these categories, though caspase-2 represents an atypical case that can function in both capacities depending on context.^[3] Initiator caspases, including caspase-8, caspase-9, and caspase-10, are characterized by long N-terminal prodomains that facilitate their recruitment to activation platforms.^[10] These prodomains typically contain death effector domains (DEDs) in caspase-8 and -10 or a caspase-recruitment domain (CARD) in caspase-9, enabling oligomerization and auto-activation through induced proximity upon receptor or sensor engagement.^[11] As the starting points of caspase cascades, initiator caspases amplify upstream death signals by undergoing conformational changes that enhance their catalytic activity, thereby processing downstream targets to propagate the response.^[10] In contrast, effector caspases, such as caspase-3, caspase-6, and caspase-7, feature short prodomains and exist predominantly as inactive zymogens until cleaved by active initiator caspases.^[3] This proteolytic activation removes the prodomain and separates the large and small subunits, forming the mature heterotetramer that executes the terminal phase of the cascade.^[11] Effector caspases specialize in the bulk proteolysis of cellular substrates, including structural proteins and regulatory factors, leading to systematic disassembly of the cell.^[10] The cascade model underscores this functional hierarchy: initiator caspases detect and integrate apoptotic stimuli to initiate signal amplification, while effector caspases, once activated, perform the widespread substrate cleavage that commits the cell to death.^[3] Caspase-2, with its long CARD-containing prodomain, blurs these lines as an atypical member capable of initiator-like auto-activation in some scenarios or effector-like roles in others, highlighting the nuanced diversity within the family.^[11]

Inflammatory Subtypes

Inflammatory caspases constitute a specialized subclass of the caspase family, distinct from those primarily involved in apoptosis, and are dedicated to orchestrating innate immune responses through cytokine maturation and, in some cases, lytic cell death. In humans, this group encompasses caspase-1, caspase-4, and caspase-5, while mice express caspase-11 as the ortholog to human caspase-4 and -5. These enzymes are characterized by an N-terminal caspase recruitment domain (CARD), which enables their assembly into multiprotein inflammasome complexes, facilitating proximity-induced activation via dimerization and autoproteolytic cleavage.^[12] Caspase-1 serves as the canonical inflammatory caspase, recruited to inflammasomes such as NLRP3 or AIM2, where it selectively cleaves pro-IL-1β and pro-IL-18 at specific aspartate residues (Asp116 for IL-1β and Asp36 for IL-18), yielding mature cytokines that amplify inflammation.^[13]^[14] Beyond cytokine processing, caspase-1 also targets gasdermin D (GSDMD) at Asp276, generating an N-terminal fragment that forms plasma membrane pores, which promotes IL-1 family cytokine release and executes pyroptosis—a proinflammatory form of cell death. In contrast, human caspase-4 and -5, along with murine caspase-11, operate in non-canonical pathways, directly binding lipopolysaccharide (LPS) via their CARD domains to sense cytosolic Gram-negative bacterial components; this leads to GSDMD cleavage and pyroptosis, often indirectly enhancing caspase-1 activity for cytokine production. These caspases exhibit substrate specificity favoring inflammatory mediators over the structural proteins dismantled by apoptotic executioners like caspase-3 and -7.^[13]^[12] Post-2020 research has illuminated expanded regulatory roles for these caspases within the NLRP3 inflammasome, highlighting mechanisms such as NEK7-mediated oligomerization for caspase-1 and cross-talk with non-canonical sensors like caspase-11 to integrate diverse danger signals. Structural studies have revealed how CARD-CARD interactions form filamentous assemblies, providing targets for therapeutic modulation in inflammatory diseases. These findings underscore the inflammatory caspases' pivotal position in bridging microbial detection to adaptive immune amplification, distinct from their apoptotic counterparts' focus on orderly cell demise.^[13]^[15]

Activation Mechanisms

Dimerization Processes

Caspase activation frequently initiates through the dimerization of inactive procaspase monomers, a process that stabilizes the active conformation and facilitates subsequent proteolytic events. In this mechanism, homodimerization of the protease domains brings the catalytic sites into proximity, enabling low-level autoproteolytic activity that is amplified upon clustering. This step is essential for initiator caspases, such as caspase-8 and caspase-9, where the monomeric zymogens exhibit negligible activity due to disordered active site loops, but dimerization induces conformational changes that align these loops for catalysis.^[16] Adaptor proteins or scaffold complexes induce dimerization by promoting the oligomerization of procaspases, thereby increasing their local concentration and overcoming the intrinsically low affinity for self-association. For caspase-8, recruitment to the death-inducing signaling complex (DISC), formed by Fas-associated death domain (FADD) and death receptors like Fas/CD95, drives homodimerization through homophilic interactions between the protease domains. Similarly, caspase-9 dimerizes upon binding to the apoptosome, a heptameric platform assembled from apoptotic protease-activating factor 1 (Apaf-1) and cytochrome c released from mitochondria. These inducers create a high-density environment that shifts the equilibrium toward the dimeric state, essential for initiating the caspase cascade.^[16]^[17] The kinetics of dimerization follow the induced proximity model, in which adapter-mediated clustering compensates for the weak dimerization affinity (typically in the micromolar range) by elevating effective concentrations to nanomolar levels within the complex. This proximity-driven process results in a second-order reaction rate for activation, as demonstrated for caspase-9 on the apoptosome, where monomer-dimer equilibrium favors inactivity at physiological concentrations but shifts rapidly upon scaffolding. For caspase-8, interdimer processing within DISC oligomers further accelerates activation, with dimers cross-cleaving each other to expose processing sites. This model explains the rapid onset of activity without requiring high-affinity intrinsic interactions.^[16]00173-0)^[17] Structurally, the dimer interface involves conserved residues from β-strands and α-helices that mediate subunit contacts, allosterically regulating the active site. In caspase-9, key interactions occur at a seven-residue insertion loop between β-strands 3 and 3A (position 240), which spans the dimer interface and stabilizes the L1 and L2 loops critical for substrate binding and catalysis. Caspase-8 employs similar homophilic contacts in its protease domain, with residues in the death effector domains (DEDs) contributing to stable dimer formation upon DISC assembly. These interface residues not only promote dimer stability but also induce allosteric rearrangements that enhance catalytic efficiency by up to 100-fold compared to monomers. This structural basis underscores dimerization as a regulatory switch for caspase activation.^[17]^[18]

Proteolytic Cleavage

Caspases are synthesized as inactive zymogens known as procaspases, which require proteolytic processing to generate mature, active enzymes. This maturation involves cleavage at specific aspartic acid residues within the protein sequence, primarily following the consensus motif DXXD↓, where the arrow indicates the cleavage site after the terminal aspartate. These cleavage events occur in interdomain linkers: between the N-terminal prodomain and the large subunit (p20), and between the large and small subunits (p10), resulting in the heterotetrameric active form composed of two large and two small subunits.^[19] The proteolytic cascade amplifies the apoptotic signal through a hierarchical activation process. Initiator procaspases, such as caspase-8 and caspase-9, undergo initial auto-cleavage or trans-cleavage upon dimerization induced by upstream adaptors, generating partially active forms that then proteolytically process effector procaspases like caspase-3 and caspase-7. Effector caspases require cleavage at multiple sites for full activation but exhibit minimal auto-processing capability, relying predominantly on initiator caspases for their maturation; once activated, effectors further cleave downstream substrates to execute cell death. This sequential amplification ensures rapid and irreversible commitment to apoptosis.^[19]^[20] Regulation of proteolytic cleavage is tightly controlled by endogenous inhibitors, notably the X-linked inhibitor of apoptosis protein (XIAP). XIAP binds directly to the active sites of processed effector caspases (caspase-3 and -7) and the linker region of initiator caspase-9, preventing substrate access and further cleavage events. Relief of this inhibition occurs through mitochondrial release of second mitochondria-derived activator of caspases (Smac/DIABLO), which binds XIAP's BIR domains, displacing caspases and allowing progression of the proteolytic cascade.^[21]^[22] Recent studies have highlighted non-canonical proteolytic mechanisms in inflammatory contexts, particularly involving inflammatory caspases such as caspase-1, -4, -5, and -11. These caspases can cleave substrates like gasdermin D (GSDMD) at atypical sites deviating from the strict DXXD motif, facilitating pyroptosis without traditional inflammasome priming; for instance, caspase-11 autoprocessing enables direct recognition of bacterial lipopolysaccharide, leading to GSDMD pore formation and IL-1β/IL-18 processing. Such insights from 2024 analyses underscore the adaptability of caspase cleavage in innate immunity beyond canonical apoptosis.^[23]^[24]

Roles in Programmed Cell Death

Apoptosis Pathways

Apoptosis, a form of programmed cell death essential for development and homeostasis, is primarily executed through caspase-mediated proteolysis that systematically dismantles cellular structures. Caspases serve as central effectors in two major pathways: the extrinsic pathway, triggered by extracellular signals, and the intrinsic pathway, initiated by internal stresses such as DNA damage or mitochondrial dysfunction. In both, initiator caspases activate effector caspases, leading to irreversible cellular commitment to death while ensuring orderly fragmentation for efficient clearance by phagocytes.^[25] The extrinsic pathway is activated by ligand binding to death receptors, such as Fas (CD95) or tumor necrosis factor receptor 1 (TNFR1), recruiting the adaptor protein FADD and initiator caspase-8 to form the death-inducing signaling complex (DISC). Caspase-8 undergoes dimerization and autocleavage, becoming active and directly cleaving effector caspases like caspase-3 in type I cells, or indirectly amplifying the signal in type II cells by cleaving Bid to generate truncated Bid (tBid). tBid translocates to mitochondria, promoting Bax/Bak oligomerization and cytochrome c release, thereby linking the extrinsic pathway to the intrinsic amplification loop.^[26]^[27]^[28] In the intrinsic pathway, mitochondrial outer membrane permeabilization releases cytochrome c into the cytosol, where it binds Apaf-1 in the presence of dATP, inducing Apaf-1 oligomerization into a wheel-like heptameric apoptosome complex. This structure recruits and activates initiator caspase-9 through induced proximity and autocleavage, with caspase-9 remaining partially associated with the apoptosome to process effector caspases such as caspase-3 and -7. The pathway integrates diverse stressors, including genotoxic damage and growth factor deprivation, ensuring a robust response to cellular threats.^[29]^[30]^[25] Effector caspases, primarily caspase-3 and caspase-7, then cleave over 200 substrates to orchestrate apoptotic morphology and function. Caspase-3 cleaves poly(ADP-ribose) polymerase (PARP) at Asp214, abrogating DNA repair and conserving energy for dismantling; caspase-6 targets nuclear lamins A/C at Asp230, fragmenting the nuclear envelope to enable chromatin condensation.^[31] DNA fragmentation occurs via caspase-3-mediated cleavage of inhibitor of caspase-activated DNase (ICAD) at Asp117 and Asp224, liberating CAD to generate 180-bp nucleosomal ladders. Cytoskeletal changes drive membrane blebbing through caspase-3 cleavage of gelsolin at Asp352, promoting actin depolymerization, and Rho-associated kinase 1 (ROCK1) at Asp1113, enhancing actomyosin contraction. Phagocytosis is facilitated by caspase-7 cleavage of endonuclease G-like 1 (EMAP-II) and lipid scramblases like ATP11C at Asp439, exposing phosphatidylserine on the outer membrane to signal immune clearance without inflammation.^[32]^[33]^[34] Beyond full commitment to death, caspases can operate at sublethal thresholds, enabling partial activation for cellular remodeling without complete proteolysis. Low-level caspase-3 activity, for instance, supports differentiation in megakaryocytes and myoblasts by cleaving select substrates like actin regulators, facilitating morphological changes such as nuclear envelope adjustments during terminal maturation. This regulated, compartmentalized activation avoids widespread substrate degradation, highlighting caspases' dual roles in both demise and adaptation.^[35]^[36]^[37]

Pyroptosis Mechanisms

Pyroptosis is a form of programmed lytic cell death characterized by the formation of plasma membrane pores, leading to osmotic cell lysis and the release of pro-inflammatory contents, primarily mediated by inflammatory caspases such as caspase-1, -4, -5, and -11 acting on gasdermin D (GSDMD).^[38] This process serves as a host defense mechanism against microbial infections but can contribute to pathological inflammation when dysregulated. Unlike apoptosis, pyroptosis amplifies immune responses through cytokine secretion and cell rupture.^[39] In the canonical pathway, pattern recognition receptors like NLRP3 or AIM2 detect pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), leading to inflammasome assembly with the adaptor protein ASC and pro-caspase-1.^[38] This complex autocleaves pro-caspase-1 into its active form, which then proteolytically cleaves GSDMD at Asp275 (in humans), separating the N-terminal fragment (GSDMD-N) from the inhibitory C-terminal domain. The GSDMD-N domain oligomerizes and inserts into the plasma membrane, forming pores approximately 10-20 nm in diameter that disrupt membrane integrity.^[40] Concurrently, active caspase-1 matures pro-interleukin-1β (pro-IL-1β) and pro-IL-18 into their bioactive forms, which are released through these pores to propagate inflammation.^[38]^[41] The non-canonical pathway operates independently of inflammasomes and is triggered by direct sensing of bacterial lipopolysaccharide (LPS) in the cytosol by caspase-4 and -5 in humans or caspase-11 in mice.^[42] These caspases bind LPS via their CARD domains, undergo autoprocessing, and cleave GSDMD at the same site as caspase-1, releasing GSDMD-N to form membrane pores.^[42] Pore formation induces potassium efflux, which secondarily activates the NLRP3 inflammasome and caspase-1, amplifying IL-1β and IL-18 release.^[38] This pathway is particularly relevant in Gram-negative bacterial infections, where it promotes rapid pyroptotic responses.^[42] The outcomes of both pathways include cell swelling, membrane rupture, and the extracellular release of inflammatory mediators, fostering immune cell recruitment and amplifying systemic inflammation.^[38] In recent studies, hyperactivation of pyroptosis via NLRP3 inflammasome and GSDMD has been implicated in metabolic diseases; for instance, in type 2 diabetes, excessive caspase-1-mediated GSDMD cleavage in pancreatic β-cells exacerbates insulin resistance and β-cell dysfunction.^[43] Similarly, in obesity and metabolic-associated fatty liver disease, adipocyte and hepatocyte pyroptosis drives chronic low-grade inflammation, linking pyroptosis to disease progression.^[43] These findings highlight pyroptosis as a potential therapeutic target in metabolic disorders.^[39]

Involvement in Necroptosis and Ferroptosis

Caspases, particularly caspase-8, play a critical regulatory role in necroptosis by acting as inhibitors of the RIPK3-MLKL signaling pathway. In this lytic form of programmed cell death, caspase-8 normally cleaves receptor-interacting protein kinase 1 (RIPK1) at aspartate 325 (in humans) and RIPK3 at aspartate 328, thereby disrupting the formation of the RIPK1-RIPK3 necrosome complex that phosphorylates and activates mixed lineage kinase domain-like protein (MLKL).^[44]^[45] When caspase-8 is inhibited or absent, such as through viral infection or pharmacological blockade, RIPK1 and RIPK3 evade cleavage, undergo phosphorylation, and assemble the necrosome, leading to MLKL oligomerization, membrane translocation, and subsequent plasma membrane rupture characteristic of necroptotic cell lysis.^[46] This inhibitory function positions caspase-8 as a molecular switch, preventing necroptosis under conditions where apoptotic signaling predominates.^[47] In ferroptosis, an iron-dependent cell death driven by unchecked lipid peroxidation, caspases do not directly execute the process but exert indirect regulatory influence, with caspase-3 notably promoting susceptibility through modulation of lipid peroxidation regulators. Activation of caspase-3, often triggered by apoptotic stimuli, can sensitize cells to ferroptotic execution, shifting the balance toward ferroptotic outcomes in stressed cells without direct involvement in the core peroxidation cascade.^[48] Unlike necroptosis, where caspase inhibition unleashes the pathway, ferroptosis proceeds independently of caspases, but caspase-3's proteolytic activity indirectly amplifies lipid peroxidation in contexts where apoptotic pathways are engaged but incomplete.^[49] This non-executory role underscores caspases' broader influence on alternative death modalities when primary apoptotic pathways are engaged but incomplete.^[47] Crosstalk between caspase-mediated apoptosis and these alternative deaths emerges prominently when apoptotic execution is suppressed, redirecting cells toward necroptosis or ferroptosis in contexts like cancer and infection. In tumor microenvironments, inhibition of caspase-8 allows RIPK3-MLKL activation, promoting necroptotic lysis that enhances antitumor immunity, while in viral infections, caspase blockade similarly shifts to necroptosis for pathogen clearance.^[50] Similarly, apoptosis-resistant cancer cells, where caspase-3 activity is dampened, default to ferroptosis upon lipid peroxidation inducers, providing a therapeutic vulnerability exploited in infections where microbial evasion of caspases triggers ferroptotic host defense.^[51] This compensatory switching ensures cell elimination despite pathway blockade, with caspases serving as pivotal integrators.^[52] Recent reviews highlight caspases' potential in sensitizing ferroptosis for cancer therapy, particularly through combined modulation of apoptotic and ferroptotic axes to overcome resistance. For instance, caspase-3 activation via BH3-mimetics enhances lipid peroxidation in apoptosis-refractory tumors, amplifying ferroptosis inducers like RSL3 for synergistic cell killing.^[49] In 2025 analyses, caspase inhibition strategies are proposed to redirect suppressed apoptosis toward ferroptosis in immunotherapy-resistant cancers, improving outcomes in infection-associated malignancies by leveraging necroptotic-ferroptotic crosstalk.^[53] These findings emphasize caspases' therapeutic targeting to fine-tune cell death switches for enhanced efficacy.^[54]

Non-Apoptotic Functions

Regulation of Inflammation

Caspases, particularly caspase-1, play a central role in regulating inflammation by processing inactive precursor cytokines into their mature, bioactive forms, thereby amplifying innate immune responses. Caspase-1 specifically cleaves pro-interleukin-1β (pro-IL-1β) at the Asp116-Ala117 bond to generate the mature 17 kDa IL-1β protein, which acts as a potent mediator of fever, endothelial activation, and recruitment of immune cells. Similarly, caspase-1 processes pro-IL-18 into its 17.2 kDa mature form, promoting interferon-γ production and enhancing Th1 immune responses. These maturation events are essential for the cytokines' secretion and biological activity, enabling rapid propagation of inflammatory signals without requiring de novo protein synthesis.^[13]^[13]^[55] Inflammasomes serve as critical platforms integrating caspase activation with pathogen and damage sensing to fine-tune inflammatory outputs. Nucleotide-binding oligomerization domain-like receptors (NLRs), such as NLRP3, detect pathogen-associated molecular patterns (PAMPs) from microbes or damage-associated molecular patterns (DAMPs) from host cells, leading to NLR oligomerization. This recruits the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), which facilitates proximity-induced autocleavage and activation of pro-caspase-1 into its enzymatically active p20/p10 heterotetramer form. The resulting active caspase-1 then drives cytokine maturation as a primary signaling mechanism, distinct from other effector functions.^[56]^[56]^[56] Caspase activation establishes positive feedback loops that sustain pro-inflammatory signaling through crosstalk with transcription factors like NF-κB. Mature IL-1β and IL-18 secreted downstream of caspase-1 bind to their respective receptors (IL-1R and IL-18R), triggering intracellular cascades that activate the IκB kinase (IKK) complex and liberate NF-κB from inhibitory sequestration. Nuclear translocation of NF-κB then induces transcription of additional pro-inflammatory genes, including those encoding more pro-IL-1β, thereby amplifying and prolonging the inflammatory response in an autocrine and paracrine manner. This loop ensures robust innate immunity but requires tight regulation to prevent chronic inflammation.^[57]^[58]^[57] Dysregulated caspase overactivation contributes to pathological inflammation in conditions like sepsis and COVID-19, where unchecked cytokine release exacerbates tissue damage. In sepsis, sustained inflammasome-caspase-1 signaling, often via NLRP3 or NLRC4, drives excessive IL-1β production, fueling a systemic cytokine storm that correlates with multi-organ failure and high mortality rates exceeding 20% in severe cases. Recent analyses indicate that inhibiting this pathway reduces inflammatory burden in preclinical models. In COVID-19, SARS-CoV-2 infection hyperactivates NLRP3 inflammasomes, leading to elevated caspase-1 activity and IL-1β/IL-18 levels in severe patients, with studies showing caspase-1 activation in lung biopsies, linking this to immune dysregulation. Therapeutic targeting of caspase-1 has shown promise in mitigating these effects.^[59]^[59]^[60]^[60]

Roles in Differentiation and Stem Cells

Caspases, particularly caspase-3, exhibit sublethal activation that supports non-lethal cellular processes during development and homeostasis. In neuronal development, low-level caspase-3 activity facilitates synapse pruning by enabling activity-dependent elimination of synaptic connections, a process essential for circuit refinement without inducing cell death.^[61] This sublethal function extends to promoting neuronal migration, where caspase-3 enhances cytoskeletal dynamics and cell motility in non-apoptotic contexts, aiding proper positioning in the developing nervous system.^[62] In stem cell biology, caspase-3 plays a regulatory role in maintaining hematopoietic stem cell (HSC) quiescence, where its partial activation dampens responsiveness to extrinsic signals, thereby preserving the long-term repopulating potential of these cells.^[63] These non-apoptotic activities highlight caspase-3's contribution to stem cell durability and tissue regeneration. During cellular differentiation, caspases cleave key transcription factors to drive lineage commitment. In lens development, executioner caspases, including caspase-3, are activated in fiber cells to degrade nuclear components and facilitate differentiation, with caspase activity targeting regulatory proteins for precise organelle elimination.^[64] Similarly, in myogenesis, caspase-3 cleaves the transcription factor Pax7 in satellite cells, inhibiting self-renewal and promoting progression to myogenic differentiation by releasing cells from a stem-like state.^[65] Recent insights as of 2025 underscore caspase-3's involvement in embryonic stem cell (ESC) pluripotency, where sublethal activation cleaves Nanog to initiate differentiation while preserving cell viability, linking partial caspase signaling to the exit from naive pluripotency.^[66] In cancer stem cells, non-genetic inactivation of caspase-3 enhances survival and stemness, allowing tumor-initiating cells to evade apoptosis and promote resistance to therapies through sustained low-level activity that supports proliferation and metastasis.^[67] These findings emphasize caspase-3's paradoxical role in balancing survival and fate decisions in stem-like populations.

Historical Development

Discovery and Initial Characterization

In the 1980s, genetic screens conducted in the nematode Caenorhabditis elegans by H. Robert Horvitz and colleagues identified the gene ced-3 as essential for programmed cell death (apoptosis) during development.^[68] Mutations in ced-3 blocked nearly all cell deaths that occur in the wild-type worm, revealing it as a key executor of the apoptotic program. This discovery established ced-3 as a central component of the genetically defined apoptotic pathway in C. elegans, alongside genes like ced-4 and ced-9.^[69] In 1993, the ced-3 gene was cloned and found to encode a protein highly similar to the human interleukin-1β (IL-1β) converting enzyme (ICE), previously identified and cloned in 1992 as a cysteine protease responsible for processing the IL-1β precursor into its mature form.^[70] The ced-3 product shared about 30% sequence identity with ICE (now known as caspase-1), suggesting that ICE represented a mammalian homolog involved in apoptosis. Overexpression of either ced-3 or ICE in mammalian fibroblasts induced apoptotic cell death, linking the two proteins functionally across species.^[71] Early characterization of these proteases relied on cell-free systems, particularly in vitro reconstitution assays using extracts from Xenopus laevis eggs, which recapitulated key apoptotic events such as nuclear fragmentation and DNA laddering.^[72] These extracts demonstrated caspase-like proteolytic activity essential for apoptosis, allowing researchers to purify and study the enzymes' substrate specificities and activation mechanisms without the complexities of intact cells. The unified nomenclature "caspase" (for cysteine-dependent aspartate-specific proteases) was proposed in 1996 by an international committee to encompass ICE, CED-3, and related family members, standardizing the naming of this growing class of apoptosis regulators.^[73]

Key Milestones and Advances

In 1997, the identification of the apoptosome as a key activation platform for caspase-9 marked a pivotal advance in understanding the intrinsic pathway of apoptosis. This multiprotein complex, formed by Apaf-1 oligomerization in the presence of cytochrome c and dATP, recruits and activates procaspase-9, initiating the downstream caspase cascade that executes programmed cell death. The discovery elucidated how mitochondrial signals converge to amplify caspase activity, providing a mechanistic foundation for caspase regulation in apoptotic signaling.^[74] During the 2000s, the discovery of the inflammasome complex revolutionized insights into caspase-1's role beyond apoptosis, particularly in pyroptosis and innate immunity. In 2002, researchers identified the inflammasome as a molecular platform comprising NLR family proteins, ASC adaptor, and pro-caspase-1, which oligomerizes in response to microbial or danger signals to process pro-caspase-1 into its active form. This activation cleaves gasdermin D and pro-IL-1β, driving inflammatory cytokine release and lytic cell death in pyroptosis, thereby linking caspases to host defense against pathogens. Subsequent studies in the decade expanded this to multiple inflammasome sensors, highlighting caspase-1's central role in inflammation-related pathologies.^[75] The 2010s unveiled extensive non-apoptotic functions of caspases, particularly in stem cell biology and differentiation, broadening their physiological repertoire. For instance, low-level caspase-3 activity was shown to promote epigenetic reprogramming and pluripotency in induced pluripotent stem cells (iPSCs) without inducing cell death. This revealed caspases as regulators of stem cell maintenance, proliferation, and lineage commitment in tissues like the hematopoietic system and neural progenitors, where partial activation supports homeostasis rather than demise. These findings shifted paradigms, positioning caspases as versatile proteases in development and regeneration.^[76] In 2024, advances in cryo-electron microscopy (cryo-EM) provided high-resolution structures of caspase-involved complexes, enhancing therapeutic targeting for autoimmune diseases. Cryo-EM revealed detailed oligomerization states of NLRP3 inflammasomes bound to ASC and caspase-1, illustrating nucleotide-dependent activation mechanisms that drive pyroptosis in inflammatory contexts. Concurrently, preclinical studies of caspase-1 inhibitors like VX-765 for inflammatory and autoimmune conditions, such as osteoarthritis, have demonstrated reduced IL-1β production and inflammation in disease models by blocking inflammasome-driven cytokine storms.^[77]^[78]

Evolutionary Aspects

Origins and Conservation

Caspases belong to a superfamily of cysteine proteases that traces its origins to early eukaryotic evolution, with metacaspases identified in plants and fungi as distant relatives. These metacaspases, such as those in Arabidopsis thaliana and Saccharomyces cerevisiae, share a common ancestral fold with animal caspases but exhibit distinct substrate preferences. Paracaspases, another branch of this superfamily, are present in pre-metazoan organisms such as Dictyostelium discoideum, while metacaspases are found in choanoflagellates, the closest unicellular relatives to animals, indicating that caspase-like machinery evolved prior to the advent of multicellularity in the metazoan lineage.^[79]^[80] A key conserved feature across the caspase superfamily is the catalytic dyad composed of a histidine and cysteine residue, which is preserved from yeast metacaspases like Yca1 to human caspases, enabling nucleophilic attack during proteolysis. The aspartic acid (Asp) specificity at the P1 position of substrates, however, is a hallmark unique to animal caspases and remains highly conserved from early metazoans to humans, distinguishing them from metacaspases that prefer basic residues like arginine. This structural conservation highlights the fundamental mechanistic similarity despite functional divergence.^[81]^[82] In the vertebrate lineage, the caspase gene family underwent significant expansion through tandem and segmental duplications, resulting in specialized isoforms for apoptosis, inflammation, and non-death functions. For instance, early vertebrate genome duplications contributed to the diversification of initiator and effector caspases, with humans possessing around 12 active members compared to fewer in invertebrates, allowing fine-tuned responses to cellular signals. A 2025 study highlighted the evolution of caspase-16, where its prodomain arose from duplication of the caspase domain, with the human ortholog rendered a pseudogene by a frameshift mutation.^[83]^[84] The primordial function of these proteases likely centered on stress responses in unicellular eukaryotes, as evidenced by metacaspases in choanoflagellates and yeast mediating adaptation to oxidative and environmental stresses through lateral gene transfer from algae, predating their co-option into metazoan apoptosis pathways.^[80]

Phylogenetic Distribution

Caspases, as cysteine-dependent aspartate-specific proteases, are exclusively found in metazoans, while their homologs known as metacaspases are distributed in non-metazoan eukaryotes such as plants and fungi.^[81] In plants, metacaspases contribute to hypersensitive response-like cell death during pathogen defense, with multiple isoforms identified across species like Arabidopsis thaliana.^[85] Fungi also possess metacaspases, which are involved in developmental and stress-related processes, reflecting a broad presence in the fungal kingdom.^[86] True metacaspases and caspases are absent in bacteria, though scattered prokaryotic homologs with caspase-like domains exist in some lineages, indicating independent evolutionary origins rather than direct ancestry.^[87] Within metazoans, the caspase family is present across phyla, with the full complement of initiator, effector, and inflammatory subtypes evident in bilaterians such as arthropods, nematodes, and chordates.^[88] In contrast, non-bilaterian metazoans like cnidarians exhibit a caspase repertoire that is less diverse in terms of subgroup specialization, with genomes such as that of Hydra magnipapillata encoding around 15 genes but lacking certain bilaterian-specific expansions in apoptotic initiators.^[89] This reduction in cnidarian caspases highlights adaptations to simpler body plans, while core catalytic domains remain conserved from the metazoan ancestor.^[90] In vertebrates, the caspase family has undergone significant expansion through gene duplications, resulting in 12 distinct genes in humans (CASP1 through CASP10 and CASP14), which arose from tandem and whole-genome duplication events in early vertebrate evolution.^[84] Recent phylogenetic analyses in 2024 have revealed clade-specific innovations in inflammatory caspases, such as primate-specific duplications and sequence divergences in CASP1 and related genes, enabling specialized roles in innate immunity.^[91] These findings underscore the dynamic evolution of caspases across phylogenetic clades, building on their conserved ancestral features.^[92]

Detection and Analysis

Biochemical Assays

Biochemical assays for caspases primarily involve in vitro and ex vivo measurements of proteolytic activity using synthetic peptide substrates that mimic natural cleavage sites. These methods quantify enzyme kinetics through the release of detectable reporter groups, enabling precise evaluation of activation, inhibition, and specificity in recombinant proteins or cellular extracts.39097-5/fulltext) Fluorogenic substrates are widely used due to their sensitivity in detecting low levels of caspase activity. For instance, Ac-DEVD-AMC serves as a selective substrate for caspase-3, where cleavage at the aspartic acid residue liberates 7-amino-4-methylcoumarin (AMC), producing fluorescence with excitation at approximately 380 nm and emission at 460 nm.39097-5/fulltext) Similarly, Ac-YVAD-AMC is specific for caspase-1, undergoing hydrolysis to release fluorescent AMC under analogous conditions, allowing real-time monitoring of fluorescence kinetics to determine reaction rates via Michaelis-Menten parameters such as Km values around 10-20 μM for these substrates.39097-5/fulltext) These assays are typically performed in 96-well plates with cell lysates or purified enzymes, providing linear fluorescence increases over time that correlate with caspase concentration.^[93] Caspase activity assays often employ recombinant enzymes or lysates from treated cells to assess activation and inhibitor efficacy. In these setups, substrates like DEVD-AMC are incubated with samples, and activity is quantified by the rate of fluorophore release, often normalized to protein content. Inhibitors such as z-VAD-fmk, a cell-permeable pan-caspase peptide fluoromethylketone, are evaluated for potency, with IC50 values typically in the low nanomolar to micromolar range (e.g., 1-50 nM for caspase-3), determined by dose-response curves monitoring substrate cleavage inhibition.^[94] These assays confirm specificity and are essential for validating caspase involvement in apoptotic pathways using controls like untreated lysates.^[93] Specificity profiling of caspases utilizes positional scanning synthetic combinatorial libraries (PS-SCLs) to identify optimal substrate motifs. These libraries systematically vary amino acids at P4-P1 positions upstream of the cleavage site, revealing preferences such as DEVD for executioner caspases like caspase-3 and YVAD/WEHD for initiator caspases like caspase-1.39097-5/fulltext) By measuring cleavage rates of library subsets with fluorogenic reporters, researchers derive consensus sequences that guide inhibitor design and distinguish caspase subtypes based on extended subsite requirements.39097-5/fulltext) High-throughput screening adaptations enhance these assays for drug discovery, incorporating fluorescence resonance energy transfer (FRET)-based probes or luminescent detection. FRET substrates, such as those linking donor-acceptor fluorophores across a caspase-cleavable linker (e.g., DEVD), exhibit decreased energy transfer and increased donor emission upon proteolysis, enabling 384-well format readouts with Z' factors >0.7 for robust screening.^[95] Luminescent assays, like the Caspase-Glo system, use proluminogenic DEVD analogs that generate light via luciferase upon cleavage, offering superior signal-to-noise ratios (>100-fold) and automation compatibility for evaluating thousands of compounds against caspase activity in lysates.

Imaging Techniques

Fluorescent probes have been instrumental in visualizing caspase activation at the cellular level, particularly for effector caspases like caspase-3. PhiPhiLux represents a prominent class of cell-permeable fluorogenic substrates designed specifically for caspase-3 and -7 detection; upon cleavage by active caspase-3, the probe releases a fluorescent moiety, such as in the green-emitting G1D2 or red-emitting G2D2 variants, enabling real-time imaging of apoptosis in live cells via confocal or widefield microscopy.^[96] These probes offer high specificity and low background fluorescence, allowing localization of caspase activity within subcellular compartments like the cytosol or nucleus.^[97] Activatable cell-penetrating peptides (ACPPs) extend this capability by incorporating a polycationic cell-penetrating domain masked by a caspase-cleavable linker, which, upon activation, permits peptide entry into cells and subsequent fluorescence or near-infrared signal generation. For instance, the KcapQ probe, an improved near-infrared fluorescent ACPP, targets effector caspase activity in whole cells and live animals, providing enhanced signal-to-noise ratios for non-invasive apoptosis imaging compared to non-activatable counterparts.^[98] This approach has been particularly useful in tracking caspase-mediated processes in tissues with limited accessibility, such as the retina or brain.^[99] Förster resonance energy transfer (FRET)-based sensors provide dynamic, real-time monitoring of caspase activation through intramolecular quenching that is relieved upon proteolysis. The SCAT3 sensor, specific for caspase-3, consists of enhanced cyan fluorescent protein (ECFP) and Venus yellow fluorescent protein (YFP) flanking a DEVD cleavage sequence; in the intact state, FRET occurs between the donor (ECFP) and acceptor (YFP), but caspase-3 cleavage separates them, shifting emission from yellow to cyan for ratiometric imaging of activation kinetics in living cells.^[100] This biosensor has revealed spatial and temporal heterogeneity in caspase-3 onset during apoptosis, with applications in high-resolution confocal setups to map activation within cellular structures.^[101] In vivo imaging techniques extend caspase visualization to whole organisms, facilitating longitudinal studies of inflammation and apoptosis. Bioluminescent reporters, such as those fusing luciferase to a caspase-3-cleavable linker (e.g., pro-luciferin-DEVD), enable non-invasive detection in mouse models; substrate administration leads to light emission upon cleavage, quantifying caspase activity in tissues like tumors or inflamed organs with high sensitivity. For instance, this approach has monitored caspase-3 in hepatic apoptosis following hydrodynamic gene delivery, correlating bioluminescence intensity with disease progression.^[102] Positron emission tomography (PET) tracers target caspase activation in inflammatory contexts, with [18F]ICMT-11 serving as a selective inhibitor-based probe for caspase-3/7; it accumulates in apoptotic cells via irreversible binding, allowing quantification of inflammation in murine models of acute lung injury or tumors.^[103] Recent developments include 18F-labeled activity-based probes that enhance specificity for treatment response monitoring in vivo.^[104] Advances as of 2025 have refined the spatial resolution of caspase-related structures, particularly the apoptosome, through live-cell imaging techniques. Studies employing GFP-tagged Apaf-1 in human cell lines have demonstrated that apoptosomes form as large, transient assemblies of Apaf-1 oligomers upon cytochrome c release, visualized via time-lapse confocal microscopy to capture their dynamic assembly and disassembly during mitochondrial apoptosis.^[105] While super-resolution methods like STED have been applied to related apoptotic complexes, ongoing integration with these approaches promises sub-diffraction limit insights into caspase-9 recruitment and activation on the apoptosome platform.^[106] These techniques complement biochemical assays by providing spatial context for caspase localization without disrupting cellular integrity.

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