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Granzyme B
Granzyme B
Granzyme B
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Granzyme B
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EC no.3.4.21.79
CAS no.143180-74-9
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Granzyme B (GrB) is one of the serine protease granzymes most commonly found in the granules of natural killer cells (NK cells) and cytotoxic T cells. It is secreted by these cells along with the pore forming protein perforin to mediate apoptosis in target cells.

Granzyme B has also been found to be produced by a wide range of non-cytotoxic cells ranging from basophils and mast cells to smooth muscle cells.[1] The secondary functions of granzyme B are also numerous. Granzyme B has shown to be involved in inducing inflammation by stimulating cytokine release and is also involved in extracellular matrix remodelling.

Elevated levels of granzyme B are also implicated in a number of autoimmune diseases, several skin diseases, and type 1 diabetes.

Structure

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In humans, granzyme B is encoded by GZMB on chromosome 14q11.2, which is 3.2kb long and consists of 5 exons.[2] It is one of the most abundant granzymes of which there are 5 in humans and 10 in mice.[1] Granzyme B is thought to have evolved from a granzyme H related precursor and is more effective at lower concentrations than the other granzymes.[3]

The enzyme is initially in an inactive precursor zymogen form, with an additional amino terminal peptide sequence.[3] This sequence can be cleaved by cathepsin C, removing 2 amino acids.[4] Cathepsin H has also been reported to activate granzyme B.[2]

Granzyme B's structure consists of two six-stranded β sheets with three trans domain segments. In the granules of cytotoxic lymphocytes the enzyme can exist in two glycosylated forms. The high mannose form weighs 32kDa and the complex form, 35kDa.[2]

Granzyme B contains the catalytic triad histidine-aspartic acid-serine in its active site and preferentially cleaves after an aspartic acid residue situated in the P1 position. The aspartic acid residue to be cleaved associates with an arginine residue in the enzyme's binding pocket.[5] Granzyme B is active at a neutral pH and is therefore inactive in the acidic CTL granules. The enzyme is also rendered inactive when bound by serglycin in the granules to avoid apoptosis triggering inside the cytotoxic T cells themselves.[4]

Delivery

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Granzyme B is released with perforin which inserts into a target cell's plasma membrane forming a pore. Perforin has a radius of 5.5 nm and granzyme B has a stokes radius of 2.5 nm and can therefore pass through the perforin pore into the target to be destroyed.

Alternatively, once released, granzyme B can bind to negatively charged heparan sulfate containing receptors on a target cell and become endocytosed. The vesicles that carry the enzyme inside then burst, exposing granzyme b to the cytoplasm and its substrates.[3] Hsp-70 has also been linked to aiding granzyme B entry.[5][6]

Granzyme B has also been proposed to enter a target by first exchanging its bound serglycin for negative phospholipids in a target's plasma membrane. Entry then occurs by the less selective process of absorptive pinocytosis.[2]

Mediated apoptosis

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Once inside the target cell, granzyme B can cleave and activate initiator caspases 8 and 10, and executioner caspases 3 and 7 which trigger apoptosis.[1] Caspase 7 is the most sensitive to granzyme B and caspases 3, 8, and 10 are only cleaved to intermediate fragments and need further cleavage for full activation.[7]

Granzyme B can also cleave BID leading to BAX/BAK oligomerisation and cytochrome c release from the mitochondria. Granzyme B can cleave ICAD leading to DNA fragmentation and the laddering pattern associated with apoptosis.[1]

Granzyme B has a potential of over 300 substrates and can cleave Mcl-1 in the outer mitochondrial membrane relieving its inhibition of Bim. Bim stimulates BAX/BAK oligomerisation, mitochondrial membrane permeability and apoptosis. Granzyme B can also cleave HAX1 (Hs-1 associated protein X-1) to facilitate mitochondria polarisation.[2]

Granzyme B can also generate a cytotoxic level of mitochondrial reactive oxygen species (ROS) to mediate cell death.[8] The caspase independent pathways of cell death are thought to have arisen to overcome viruses that can inhibit caspases and prevent apoptosis.[4]

Targets

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Nucleus

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Granzyme B has many substrates located in the nucleus. Granzyme B can cleave PARP (poly ADP ribose polymerase) and DNA PK (DNA protein kinase) to disrupt DNA repair and retroviral DNA integration. Granzyme B can also cleave nucleophosmin, topoisomerase 1 and nucleolin to prevent viral replication.

Granzyme B can cleave ICP4 from the HSV 1 virus which is an essential protein used for gene transactivation and NUMA (Nuclear mitotic apparatus protein) can be cleaved to prevent mitosis.[1]

Granzyme B can also cleave DBP (DNA Binding Protein) into a 50 kDa fragment and then into an additional 60 kDa indirectly through the caspases it activates.[9]

Extracellular matrix

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Granzyme B can degrade many proteins in the extracellular matrix (ECM) including fibronectin, vitronectin and aggrecan. Cleavage can cause cell death by anoikis and release alarmins from the ECM inducing inflammation.[1] Fragments of fibronectin can attract neutrophils and stimulate MMP expression from chondrocytes.[5] Basophils secrete granzyme B to degrade endothelial cell-cell contacts allowing extravasation to sites of inflammation.[6]

Granzyme B can also induce inflammation by processing cytokines IL-1α and IL18. It can also trigger the release of IL6 and IL8 through activation of PAR1 (Protease activated receptor 1).[10]

Cleavage of vitronectin occurs at the RGD integrin binding site interrupting cell growth signalling pathways. Cleavage of laminin and fibronectin disrupts dermal-epidermal junction attachment and cross talk while decorin destruction by granzyme B causes collagen disorganisation, skin thinning and aging. Keratinocytes can express granzyme B after exposure to UVA and UVB which is linked to photoaging of the skin.[10]

Granzyme B can also impair wound healing. Cleavage of the von Willebrand factor inhibits platelet aggregation and of plasminogen produces an angiostatin fragment preventing angiogenesis. The cutting of fibronectins and vitronectins delays the formation of a provisional matrix impairing wound healing further.[10]

T cell regulation

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Granzyme B is secreted by regulatory T cells (tregs) to kill CD4+ T cells that have not been exposed to host cells that are restricted to the peripheral tissues and cannot reach the thymus. This activation-induced cell death (AICD) can be achieved without the Fas death pathway and prevents autoimmune reaction to self antigens.[1]

Inhibitors

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Granzyme B's most common inhibitor is SERPINB9 also known as proteinase inhibitor nine (PI-9) which is 376 amino acids long and found in the nucleus and cytoplasm.[2] It is produced by many types of cell to protect themselves from accidental granzyme B mediated cell death. PI-9 is metastable and forms an energetically favourable conformation when bound to granzyme B. The reactive loop centre (RCL) of the PI-9 molecule acts as a pseudosubstrate and initially forms a reversible Michaelis complex. Once the peptide bond of the RCL is cleaved between positions P1 and P1', granzyme B is permanently inhibited. However, if the RCL is cleaved efficiently, PI-9 does not act as a 1:1 suicide substrate and granzyme B is left uninhibited.[11] Granzyme M can also cleave PI-9 in the nucleus and cytoplasm to relieve granzyme B of inhibition.[2] Protein L4-100K from adenoviruses can also inhibit granzyme B by binding at exosites and specific binding pockets.[3] L4-100K is an assembly protein that can transport hexon capsomeres into the nucleus of an adenovirus. 100k can be cleaved to a 90kDa fragment by granzyme H to relieve this inhibition which is important in adenovirus 5 infected cells.[9]

Role in disease

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Granzyme B has a normal concentration of 20-40 pg/ml in the blood plasma while retaining 70% activity and elevated concentrations of granzyme B are found in a number of disease states.[5] Granzyme B can generate autoantigens by cleaving in disordered regions and linker regions of antigens exposing new epitopes and this can cause the development of autoimmune diseases.[5][12]

Granzyme B release with perforin from CD8+ T cells can cause heart and kidney transplant rejection through killing of allogeneic endothelial cells. The destruction of insulin producing β cells in pancreatic islets is mediated by T cells and granzyme B contributing to Type 1 Diabetes. Granzyme B can also mediate the death of cells after spinal cord injury and is found at elevated levels in rheumatoid arthritis.

Chronic obstructive pulmonary disease (COPD) has been attributed to granzyme B secreted from NK and T cells causing the apoptosis of bronchial epithelial cells. Matrix destabilisation and remodelling by granzyme B is also linked to asthma pathogenesis. Granzyme B can kill melanocytes causing the skin condition vitiligo and granzyme B overexpression is found in contact dermatitis, lichen sclerosus and lichen planus cases.

Cytotoxic cells expressing granzyme B have been identified close to hair follicles linking a possible role in hair loss.[5] The ECM remodelling properties of granzyme B have also implicated its involvement in left ventricular remodelling, which increases the subsequent chances of myocardial infarction. The weakening of the fibrous cap of atheromatous plaques by apoptosis of smooth muscle cells has also been linked to granzyme B.[13]

More recently, a key role for extracellular granzyme B has been forwarded for a number of autoimmune (eg. arthritis, autoimmune blistering, scleroderma, lupus)(Reviewed in [14]) and/or age-related chronic inflammatory disorders (Photoaging, aneurysm, atherosclerosis, COPD, macular degeneration, etc)(Reviewed in [15]). In many of these conditions, proof-of-concept has been demonstrated through the use of experimental models, genetic approaches and/or pharmacologic approaches.

References

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Granzyme B

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Granzyme B is a belonging to the granzyme family of cytotoxic granule-associated proteins, primarily responsible for inducing in target cells such as those infected by viruses or transformed into tumors. It functions as a key effector in the , where it is released by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells upon recognition of aberrant targets, working in concert with perforin to penetrate and disrupt the target cell's integrity. Structurally, granzyme B is a 32 kDa glycoprotein synthesized as a preproenzyme, featuring a catalytic triad (histidine 57, aspartic acid 102, serine 195) that confers caspase-like activity, preferentially cleaving substrates after aspartic acid residues. Once delivered into the cytosol via perforin-induced pores, it activates both caspase-dependent pathways—by processing initiator caspases like caspase-8 and executioner caspases such as caspase-3—and caspase-independent mechanisms, including the cleavage of Bid to initiate mitochondrial outer membrane permeabilization and the degradation of ICAD to facilitate DNA fragmentation. In addition to its canonical role in , granzyme B exhibits diverse non-canonical functions, such as extracellular proteolysis of components like and to promote immune , processing of pro-inflammatory cytokines, and direct effects against intracellular and parasites by disrupting their metabolic pathways. While essential for immune and host defense, dysregulated granzyme B activity contributes to pathological conditions, including autoimmune diseases like through excessive tissue damage, chronic inflammatory disorders such as , and paradoxical roles in cancer where it may facilitate tumor invasion or be suppressed by regulatory immune cells.

Molecular Structure and Biochemistry

Protein Structure

Granzyme B is a serine protease belonging to the granzyme family of proteins stored in the cytotoxic granules of lymphocytes. In humans, the mature enzyme has a molecular weight of approximately 32 kDa, reflecting its glycosylated form. The protein adopts a chymotrypsin-like fold characteristic of the S1 peptidase family, with a catalytic triad composed of histidine 57, aspartate 102, and serine 195 (numbered according to chymotrypsin homology). This triad enables nucleophilic attack on peptide bonds, and the enzyme exhibits stringent specificity for aspartic acid at the P1 position of substrates, distinguishing it from other granzymes. Granzyme B is initially synthesized as an inactive zymogen featuring an N-terminal pro-peptide of two residues (Gly-Glu), which is removed by dipeptidyl peptidase I (also known as cathepsin C) during maturation in cytotoxic granules. Additionally, the protein contains two N-linked glycosylation sites at asparagine residues 71 and 104, which enhance its stability and contribute to the observed mass discrepancy between the unglycosylated core (approximately 27 kDa) and the mature form. The three-dimensional structure of human granzyme B was elucidated through , as deposited in the (PDB entry 1IAU), in complex with a inhibitor. This structure highlights the S1 specificity pocket, a deep cleft where the of 226 forms a with the group of the P1 , ensuring selective substrate recognition. Sequence comparisons reveal that human GZMB shares approximately 70% identity with its ortholog Gzmb, particularly in conserved regions like the catalytic domain, though differences exist in extended substrate preferences.

Enzymatic Properties

Granzyme B functions as a with caspase-like activity, exhibiting a strong preference for cleaving on the carboxyl side of residues at the P1 position. This specificity contrasts with that of typical chymotrypsin-like granzymes, which favor aromatic or hydrophobic residues at P1. The enzyme's extended substrate recognition involves preferences at P2-P4 positions, such as small hydrophobic residues like or at P4, enabling efficient of sequences like IEPD. Kinetic parameters for Granzyme B have been characterized using fluorogenic model substrates such as Ac-IEPD-AMC. For recombinant rat Granzyme B, the Michaelis constant (Km) is approximately 160 μM, the turnover number (kcat) is 0.536 s⁻¹, and the catalytic efficiency (kcat/Km) is 3330 M⁻¹ s⁻¹, indicating moderate substrate affinity and turnover suitable for rapid proteolytic processing in cellular contexts. These values highlight Granzyme B's efficiency in cleaving aspartate-directed substrates under physiological conditions. Granzyme B is produced as an inactive , pro-granzyme B, featuring an N-terminal Gly-Glu that inhibits activity. Activation occurs within cytotoxic granules via sequential cleavage of this pro-domain by cathepsin C, a dipeptidyl peptidase that removes the to expose the mature . Optimal Granzyme B activity is observed at 7.4-8.0, aligning with the neutral cytosolic environment of target cells. The enzyme shows low dependence on calcium ions, with standard activity assays conducted in calcium-free buffers containing or Tris at physiological salt concentrations. Granzyme B's sensitivity to inhibitors includes members of the superfamily, which covalently trap the enzyme in an inactive complex, thereby modulating its proteolytic potential.

Cellular Sources and Delivery

Producing Cells

Granzyme B is primarily synthesized and stored by cytotoxic T lymphocytes (CD8+ T cells), natural killer (NK) cells, and natural killer T (NKT) cells, which constitute the core effector populations responsible for its production in the immune system. These cells package Granzyme B within specialized cytotoxic granules, enabling rapid deployment during immune responses against infected or malignant targets. Among these, CD8+ T cells express Granzyme B upon activation, while NK cells maintain constitutive expression even in resting states. Beyond the primary producers, Granzyme B is also expressed by gamma-delta (γδ) T cells, invariant NKT cells, and subsets of type 1 (ILC1), highlighting its broader distribution among innate-like and tissue-resident lymphocytes. Additionally, non-lymphocyte cells such as mast cells produce and store Granzyme B, releasing it upon activation to contribute to extracellular proteolytic activities in and defense. Certain populations, including those in , can also express Granzyme B under specific stimulatory conditions like IL-21 exposure. γδ T cells, for instance, upregulate Granzyme B in response to stress signals in mucosal and epithelial environments, contributing to early innate defense. Invariant NKT cells share transcriptional signatures with ILC1 that include Granzyme B production, particularly in contexts. The expression of Granzyme B is tightly regulated by cytokines and immune activation signals, with interleukin-2 (IL-2) and interferon-gamma (IFN-γ) playing key roles in its upregulation in + T cells and NK cells. IL-2 stimulation enhances Granzyme B transcription and cytotoxic capacity in both cell types, often in synergy with antigen recognition or other activators. IFN-γ signaling further amplifies this process during effector differentiation, promoting granule maturation. In contrast, resting NK cells exhibit constitutive Granzyme B expression in their granules, independent of immediate activation, ensuring preparedness for innate responses. Granzyme B is stored in the active conformation within cytotoxic granules of these producer cells, co-localized with perforin to form a potent lytic complex that remains inert until . These granules, akin to specialized lysosomes, contain high concentrations of Granzyme B alongside other serine proteases, with perforin facilitating its subsequent delivery. This storage strategy prevents autotoxicity while allowing swift release upon target engagement. Developmentally, Granzyme B expression emerges and peaks in mature effector cells following thymic selection and post-thymic maturation. In + T cells, recent thymic emigrants show lower Granzyme B levels compared to fully differentiated effectors, which acquire high expression during peripheral activation and expansion. Similarly, NK cell maturation in and secondary lymphoid tissues culminates in robust granule loading, with NKT and γδ T cells displaying effector-like profiles upon innate-like development. This progression ensures that Granzyme B is optimized in terminally differentiated states for efficient immune surveillance.

Delivery Mechanisms

Granzyme B (GzmB) is primarily delivered into target cells through perforin-induced pores formed on the plasma membrane during cytotoxic granule at the . Perforin oligomerizes to create transient transmembrane pores with an inner diameter of approximately 16 nm, enabling the direct cytosolic entry of GzmB, a 32 kDa with a of about 2.5 nm. This size-selective mechanism restricts delivery of larger cargos, such as GzmB fused to moieties exceeding a 5.0–5.5 nm , while allowing efficient translocation of wild-type GzmB independent of charge or binding. The pores facilitate rapid diffusion of GzmB-serglycin complexes from the synaptic cleft into the target cell , bypassing endosomal pathways in this context.00286-8) In perforin-deficient scenarios or for non-cytotoxic functions, GzmB uptake occurs via alternative pathways. The cation-independent mannose-6-phosphate receptor (CI-M6PR) serves as the primary surface receptor, binding the mannose-6-phosphate moiety on GzmB to internalize it into endosomes, where and support vesicle formation.00140-9) This pathway is augmented by cell surface , which enhances binding and uptake efficiency, particularly in tumor or inflamed cells expressing high CI-M6PR levels. Such mechanisms allow GzmB to exert effects in perforin-independent settings, though with lower cytosolic access compared to pore-mediated delivery. Extracellular release of GzmB occurs through degranulation of cytotoxic granules, primarily from natural killer cells and + T cells, at the or during non-contact . Upon target recognition via MHC-TCR or activating receptors, lytic granules polarize toward the synapse, enabling directional secretion of GzmB into the confined synaptic cleft to minimize dilution and maximize local concentration. In inflammatory contexts, such as mast cell activation, GzmB is released independently of cell-cell contact, contributing to soluble extracellular pools. Delivery efficiency is enhanced by polarized in effector cells, where granule docking and fusion at the ensure targeted release, with studies indicating that of as few as two to four granules suffices for effective in sensitive targets. This process is tightly regulated to prevent bystander damage, with rapid granule exocytosis occurring within minutes of conjugate formation. Several barriers limit GzmB delivery, including serum-circulating inhibitors and target cell resistance mechanisms. SERPINB9, the primary intracellular inhibitor, also complexes with GzmB in serum to neutralize its activity and prevent unintended uptake by non-target cells. Target cells expressing high levels of SERPINB9 or PI-9 confer resistance by inhibiting cytosolic GzmB, reducing apoptotic efficiency in contexts like cancer evasion.

Apoptotic Mechanisms

Perforin-Dependent Apoptosis

Granzyme B induces in target cells through a perforin-dependent mechanism, where perforin forms pores in the target cell to facilitate the entry of granzyme B into the . Genetic studies using perforin-deficient mice have demonstrated that the absence of perforin abolishes granzyme B-mediated and in cytotoxic T assays against virus-infected or tumor targets. This dependency underscores perforin's essential role in enabling granzyme B access to intracellular substrates, as perforin knockout results in severely impaired killing without alternative pathways compensating effectively. Once in the , granzyme B activates the executioner cascade by cleaving Bid, a BH3-only protein, to generate truncated Bid (tBid), which translocates to the mitochondria and promotes -3 activation. This cleavage occurs rapidly and is essential for the granzyme B-initiated apoptotic pathway, as Bid-deficient cells exhibit reduced activation and upon granzyme B exposure. In parallel, granzyme B triggers mitochondrial outer membrane permeabilization (MOMP) by activating BAX and BAK, leading to release and amplification of the cascade. This MOMP induction is Bid-dependent and contributes to the core execution of , with granzyme B directly promoting BAX/BAK oligomerization on the mitochondrial membrane. Concurrently, a non- pathway involves granzyme B-mediated cleavage of DNA-dependent catalytic subunit (), resulting in direct DNA damage and fragmentation independent of activation. The perforin-dependent induced by granzyme B exhibits a rapid onset, with Bid cleavage and initial apoptotic events detectable within minutes to 30 minutes, accompanied by characteristic morphological changes such as condensation and nuclear fragmentation. This timeline reflects the efficient proteolytic action of granzyme B, leading to full apoptotic execution in 1-2 hours under physiological conditions.

Key Intracellular Targets

Granzyme B, a with caspase-like activity, primarily cleaves intracellular substrates at residues within DEVD-like motifs, distinguishing it from other granzymes that prefer basic residues. Proteomic studies have identified over 50 substrates, with some analyses revealing hundreds of cleavage sites, enabling Granzyme B to orchestrate rapid through multiple parallel pathways. These targets are non-redundant with other granzymes, as Granzyme B's unique specificity allows direct activation of apoptotic cascades without reliance on upstream in certain contexts. A pivotal mitochondrial target is Bid, a pro-apoptotic BH3-only protein, which Granzyme B cleaves at Asp75 to generate truncated Bid (tBid). This fragment translocates to mitochondria, promoting Bax/Bak oligomerization and outer membrane permeabilization, thereby releasing to amplify the apoptotic signal. Another anti-apoptotic regulator targeted is Mcl-1, cleaved by Granzyme B at multiple sites (Asp117, Asp127, Asp157), disrupting its inhibition of Bim and facilitating mitochondrial commitment to death. release subsequently recruits Apaf-1 to form the , which activates and downstream effectors. Granzyme B also directly activates effector , cleaving procaspase-3 at Asp175 and procaspase-7 to initiate a proteolytic cascade that amplifies , including DNA degradation and morphological changes. It further processes initiator procaspase-8 at multiple sites to activate it, contributing to the apoptotic cascade. Independently of full caspase activation, Granzyme B cleaves inhibitor of caspase-activated DNase (ICAD) at Asp117, liberating caspase-activated DNase (CAD) to induce condensation and internucleosomal DNA fragmentation. For cytoskeletal remodeling, Granzyme B targets Rho-associated coiled-coil containing protein kinase 2 (ROCK II), cleaving it at Asp1131 to generate a constitutively active fragment that drives membrane blebbing and cell detachment, hallmarks of apoptotic execution. This cleavage is specific to Granzyme B and complements caspase-mediated effects on ROCK I.

Non-Apoptotic Functions

Extracellular Matrix Interactions

Granzyme B (GrB) exhibits proteolytic activity against several key extracellular matrix (ECM) proteins, contributing to matrix degradation independent of its intracellular apoptotic functions. Specifically, GrB cleaves fibronectin at multiple sites, including the cell-binding domain, which disrupts fibronectin-integrin interactions and promotes the solubilization of ECM structures. Similarly, GrB directly cleaves fibrillin-1, a microfibrillar component essential for elastic fiber assembly, leading to impaired tissue elasticity and structural integrity in conditions such as aortic aneurysms. Decorin, a proteoglycan that regulates collagen fibrillogenesis, is also a substrate for GrB, with cleavage occurring after specific aspartic acid residues that release bioactive fragments and facilitate further ECM remodeling by exposing sites for other proteases like matrix metalloproteinase-1. These proteolytic events have significant implications for tissue remodeling, particularly in promoting epithelial barrier dysfunction. In the skin, extracellular GrB contributes to barrier compromise by degrading ECM components, exacerbating conditions like where elevated GrB levels correlate with increased epidermal permeability and . Studies from 2022 highlight GrB's role in disrupting the epidermal barrier through ECM cleavage, leading to heightened susceptibility to environmental insults in psoriatic lesions. In the and , GrB-mediated ECM degradation impairs the structural support of the epithelial layer, fostering conditions such as dry eye disease and keratolysis by weakening adhesion and promoting inflammatory infiltration. GrB also interferes with clotting processes by degrading fibrinogen, cleaving it into fragments that inhibit polymerization and impair clot stability, thereby affecting dynamics. This antihemostatic activity occurs extracellularly and may limit excessive during immune responses but prolong bleeding in inflammatory contexts. Extracellular GrB concentrations in inflamed tissues typically range from 1 to several hundred ng/mL, with activity being concentration-dependent; low levels (around 1-3 ng/mL in plasma) initiate subtle remodeling, while higher levels (up to 3 ng/mL in local fluids such as ) drive pronounced ECM solubilization. Experimental evidence from assays demonstrates GrB's capacity to solubilize ECM without cellular entry, as recombinant GrB incubated with purified , fibrillin-1, or results in dose-dependent fragmentation detectable by and . These assays confirm that GrB's activity, inhibited by specific antagonists like 3,4-dichloroisocoumarin, directly mediates ECM breakdown, supporting its non-canonical role in barrier disruption during from immune cells.

Immune Regulation and Inflammation

Granzyme B (GrB) is expressed by regulatory T cells (Tregs), where it plays a key role in suppressing excessive immune responses and inflammation. In Tregs, GrB contributes to immune homeostasis by targeting effector cells and modulating inflammatory signals, independent of perforin-mediated cytotoxicity. For instance, during respiratory syncytial virus (RSV) infection, GrB-producing Tregs critically control lung inflammation by limiting effector T cell activity and cytokine production. This suppressive function is highlighted in studies showing that GrB-deficient Tregs fail to effectively resolve viral-induced inflammation in the lungs. Beyond cytotoxic effects, GrB exhibits non-cytotoxic roles in immune regulation, including the processing of pro-inflammatory cytokines to modulate signaling pathways. GrB cleaves the precursor form of interleukin-18 (pro-IL-18) into its mature, active form, which can amplify innate immune responses but, in the context of Treg activity, contributes to fine-tuning inflammation. Additionally, GrB has been implicated in dampening signaling through interactions with components of inflammatory pathways, though specific mechanisms like cleavage of receptor-interacting 2 (RIPK2) remain under investigation in regulatory contexts. These actions allow GrB to balance pro- and anti-inflammatory processes within immune microenvironments. GrB displays a pro-inflammatory duality, particularly in its extracellular form, where it can induce release from non-immune cells. Extracellular GrB stimulates endothelial cells to secrete pro-inflammatory s such as IL-6 and TNF-α, promoting and leukocyte recruitment during . This effect is mediated through GrB's proteolytic activity on components and cell surface receptors, exacerbating tissue in conditions like age-related . Recent post-2020 findings underscore GrB's role in and viral infections via regulatory mechanisms. In , GrB contributes to the dysregulated by processing cytokines and remodeling tissues, with extracellular forms driving both protective and pathological as reviewed in comprehensive analyses. During viral infections, GrB from Tregs and other cells modulates antiviral immunity without relying on , such as reducing in infected cells through direct activity. Furthermore, GrB links to barrier dysfunction by cleaving proteins like in epithelial cells, compromising mucosal integrity and facilitating inflammatory infiltration. This cleavage disrupts epithelial barriers in models of , highlighting GrB's contribution to permeability in inflamed tissues.

Inhibitors and Modulation

Endogenous Inhibitors

Endogenous inhibitors of granzyme B primarily consist of family proteins that regulate its proteolytic activity to maintain immune and prevent excessive tissue damage. The key intracellular inhibitor is proteinase inhibitor 9 (PI-9, also known as ), a cytoplasmic specifically expressed in immune cells such as cytotoxic T lymphocytes, natural killer cells, and dendritic cells. In mice, the homolog serine protease inhibitor 6 (SPI-6, also SERPINB9b) serves a similar role, protecting cytotoxic cells from self-inflicted injury during granule release. These employ a mechanism, where the reactive center loop (RCL) of the serpin acts as a pseudosubstrate for granzyme B. Upon cleavage at the P1-P1' site (Glu-Glu in PI-9), the RCL inserts into the serpin's β-sheet, distorting the of granzyme B and forming a stable covalent acyl-enzyme complex that inactivates the irreversibly. This exhibits high , with a second-order association rate constant (kassoc) of approximately 107 M-1 s-1, underscoring its physiological relevance in rapid inhibition. Extracellular regulation of granzyme B is less well-characterized but involves serum serpins such as alpha-1-antitrypsin (SERPINA1), which can modulate its activity in plasma and tissues. , an , has been shown to inhibit granzyme B-mediated in certain contexts, contributing to control of leaked enzyme during immune responses. Physiologically, these inhibitors safeguard bystander cells from unintended granzyme B-mediated during cytotoxic immune responses, such as in inflamed tissues or transplant settings. For instance, PI-9 expression in endothelial and epithelial cells limits collateral damage from perforin-delivered granzyme B. In pathological contexts, upregulation of PI-9 in tumor cells promotes immune evasion by blocking granzyme B-induced , correlating with poorer prognosis in cancers like and . Genetic variations in the SERPINB9 gene, such as the A329S polymorphism, impair PI-9 function and have been associated with autoinflammatory disorders, including enhanced susceptibility to immune dysregulation and chronic inflammation.

Therapeutic Inhibitors

Therapeutic inhibitors of Granzyme B (GzmB) represent a promising class of pharmacological agents aimed at mitigating its pathological roles in autoimmune and inflammatory conditions, particularly by blocking its activity. inhibitors have been developed to target the of GzmB, with examples including the aldehyde-based, reversible inhibitor Ac-IEPD-CHO, which exhibits a Ki of 80 nM and effectively suppresses GzmB-mediated in cytotoxic T assays. Another class involves fluoromethyl ketone-based compounds, such as diaryl fluoromethyl ketones (e.g., DFMD derivatives), which act as irreversible inhibitors by forming covalent bonds with the catalytic serine residue, demonstrating selectivity in preclinical models of immune-mediated tissue damage. These agents are designed for systemic or localized delivery to interrupt GzmB's cleavage of substrates like proteins. Serpin mimetics, engineered to emulate the inhibitory mechanism of endogenous serpins, offer an alternative approach by forming stable complexes with GzmB to prevent substrate access. For instance, serpina3n, a homolog of SERPINA3, has demonstrated neuroprotective effects by inhibiting granzyme B in models of , highlighting the potential of serpin-based inhibitors. These biomimetic inhibitors leverage the suicide inhibition strategy of natural , where the reactive center loop is cleaved by GzmB, leading to conformational change and trapping, and are particularly suited for conditions involving due to their biocompatibility. Recent preclinical studies highlight the efficacy of topical GzmB inhibitors in autoimmune blistering diseases. In a 2021 investigation using murine models of , daily topical administration of the inhibitor VTI-1002 (Ki ≈ 4.4 nM for human GzmB) significantly reduced dermal-epidermal separation by 80%, preserved hemidesmosomal proteins like COL17, and decreased infiltration by 30%, thereby alleviating blistering and fragility. This approach underscores GzmB's role in autoantibody-induced pathology and supports targeted inhibition as a disease-modifying strategy. A key challenge in developing GzmB inhibitors lies in achieving specificity to minimize off-target effects on other serine proteases, such as or cathepsins, which could disrupt normal immune or homeostatic functions. Early inhibitors like Ac-IEPD-CHO show some with , complicating systemic use, while advanced compounds like VTI-1002 demonstrate improved selectivity profiles in topical formulations to localize action at inflammatory sites. Delivery strategies, including encapsulation or gel-based vehicles, are being explored to enhance tissue penetration and reduce systemic exposure. As of November 2025, GzmB inhibitors remain in preclinical stages for autoimmune diseases, with no approved therapeutics available; ongoing studies focus on topical applications for conditions like and , emphasizing safety and efficacy in humanized models before advancing to clinical trials, alongside emerging research into plant-based inhibitors for applications.

Pathophysiological Roles

Autoimmune and Inflammatory Diseases

Granzyme B (GzmB) plays a significant role in the pathogenesis of , where GzmB-positive cytotoxic T cells and natural killer cells infiltrate the synovium, contributing to joint destruction. These cells express elevated levels of GzmB, which is detected at the invasive front of the synovium and in , promoting chronic inflammation through proteolytic activity. A key mechanism involves GzmB-mediated cleavage of aggrecan, a major in , leading to degradation of the and exacerbation of erosive joint damage. This process is supported by findings from a 2020 review highlighting elevated soluble GzmB levels in RA plasma and , correlating with disease progression. In inflammatory skin diseases such as (AD) and , GzmB contributes to epithelial barrier dysfunction by cleaving ECM components and proteins. In AD, GzmB secreted by immune cells disrupts the skin barrier through of E-cadherin and , impairing epidermal integrity and allowing allergen penetration that sustains inflammation. Similarly, in psoriasis, GzmB targets syndecan-1 on , promoting its shedding and further compromising the barrier while enhancing pro-inflammatory signaling. A 2022 study in the American Journal of Physiology - Cell Physiology underscores GzmB's accumulation in lesional skin of both conditions, linking its extracellular activity to persistent barrier defects and disease chronicity. GzmB also drives pathology in autoimmune blistering diseases, exemplified by acquisita (EBA), an antibody-mediated condition targeting type VII in the dermal-epidermal junction. In experimental models of EBA and other diseases, GzmB cleaves anchoring proteins like VII and α6 and β4, facilitating autoantibody-induced dermal-epidermal separation and formation. Genetic of GzmB or pharmacological inhibition with selective inhibitors significantly reduces blistering severity, total area, and inflammatory infiltration in murine models, indicating a direct pathogenic role. This 2021 study highlights GzmB as a therapeutic target in these disorders by mitigating tissue damage without broadly suppressing immunity. Extracellular GzmB is implicated in the progression of various autoimmune and inflammatory diseases, with elevated serum levels serving as a of disease activity. In RA, circulating GzmB concentrations positively correlate with disease activity scores (e.g., DAS28) and radiographic joint destruction, reflecting ongoing synovial inflammation and ECM remodeling. Similar elevations occur in autoimmune diseases, where serum GzmB levels align with severity and barrier impairment, suggesting its release from activated immune cells amplifies systemic . These findings from multiple cohort studies emphasize extracellular GzmB's contribution to chronic tissue injury beyond intracellular . GzmB exhibits a dual role in autoimmune diseases, acting protectively in some contexts while being detrimental in others. Protectively, GzmB expressed by regulatory T cells (Tregs) enables suppression of autoreactive effector T cells through targeted , helping maintain and limit excessive , as observed in models of where Treg-derived GzmB curbs pathogenic responses. Conversely, in disease states, extracellular GzmB promotes detriment by inhibiting Treg suppressive function—via cleavage of Treg surface molecules—and directly causing ECM degradation and tissue damage, thereby exacerbating . This duality, detailed in reviews of GzmB's non-apoptotic functions, underscores its context-dependent impact on inflammatory .

Cancer and Infections

Granzyme B plays a central role in anti-tumor immunity by enabling natural killer (NK) cells and + T cells to induce in malignant cells through perforin-dependent granule exocytosis. This mechanism is critical for eliminating tumor cells, as evidenced by studies showing that granzyme B-deficient cytotoxic lymphocytes exhibit impaired tumor clearance . In granzyme B models, tumor rejection is significantly reduced compared to wild-type controls, highlighting the protease's essential contribution to effective NK- and + T cell-mediated cytotoxicity against cancers such as and . Conversely, granzyme B can exert pro-tumor effects, particularly when secreted by regulatory T cells (Tregs), which suppress anti-tumor immune responses and promote tumor progression. Treg-derived granzyme B induces in effector T cells and NK cells within the , thereby dampening cytotoxic activity and facilitating immune evasion. A 2023 study demonstrated that murine Tregs utilize granzyme B to enhance metastatic burden in models, underscoring its role in Treg-mediated immunosuppression. Additionally, extracellular granzyme B contributes to tumor dissemination by remodeling the (ECM), cleaving components like and to create pathways for and . This non-canonical function has been observed in and colorectal cancers, where elevated extracellular granzyme B correlates with increased metastatic potential. In infectious contexts, granzyme B facilitates the clearance of virus-infected cells by cytotoxic lymphocytes, limiting and spread. For instance, during infection, + T cells upregulate granzyme B expression, which synergizes with perforin to lyse infected epithelial cells and resolve acute infection. However, dysregulated granzyme B activity contributes to pathological outcomes in severe infections, such as , where excessive release leads to immunoparalysis by depleting effector immune cells and promoting a hyporesponsive state. A 2020 review in detailed how granzyme B-driven of lymphocytes exacerbates during , increasing susceptibility to secondary infections. Recent research has implicated granzyme B in the hyperinflammatory response during , where elevated circulating levels in severe cases correlate with storms and tissue damage. In critically ill patients, granzyme B is markedly increased alongside TNF and other pro-inflammatory markers, contributing to and multi-organ failure. Bacterial pathogens also employ strategies to evade perforin-mediated granzyme B delivery, such as resisting membrane pore formation, which prevents intracellular granzyme B accumulation and allows survival within host cells. For example, species inhibit perforin activity, thereby limiting granzyme B's access to bacterial targets and promoting chronic infection. Therapeutically, enhancing granzyme B delivery holds promise for improving chimeric antigen receptor (CAR) T cell therapies against solid tumors and hematologic malignancies. Engineering CAR T cells to secrete granzyme B or express granzyme B-responsive payloads, such as IL-18, boosts cytotoxic efficacy and metabolic fitness while overcoming tumor microenvironment suppression. Preclinical models demonstrate that granzyme B-armed CAR T cells exhibit superior tumor infiltration and reduced metastasis, as seen in multiple myeloma xenografts and breast cancer models using granzyme B-responsive payloads. Nanoparticle-based systems for targeted granzyme B delivery further amplify this potential by mimicking natural cytotoxic granule release, enhancing apoptosis in antigen-positive cancer cells without systemic toxicity.

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