Granzyme B

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Granzyme B
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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.

Contents

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

In humans, granzyme B is encoded by GZMB on chromosome 14q.11.2, 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

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 sulphate 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

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

Nucleus

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

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

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

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

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.

Related Research Articles

<span class="mw-page-title-main">Apoptosis</span> Type of programmed cell death in multicellular organisms

Apoptosis is a form of programmed cell death that occurs in multicellular organisms and in some eukaryotic, single-celled microorganisms such as yeast. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, DNA fragmentation, and mRNA decay. The average adult human loses 50 to 70 billion cells each day due to apoptosis. For the average human child between 8 and 14 years old, each day the approximate loss is 20 to 30 billion cells.

<span class="mw-page-title-main">Cytotoxic T cell</span> T cell that kills infected, damaged or cancerous cells

A cytotoxic T cell (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T-cell or killer T cell) is a T lymphocyte (a type of white blood cell) that kills cancer cells, cells that are infected by intracellular pathogens (such as viruses or bacteria), or cells that are damaged in other ways.

<span class="mw-page-title-main">Tumor necrosis factor</span> Immune system messenger protein which induces inflammation

Tumor necrosis factor (TNF), formerly known as TNF-α, is a chemical messenger produced by the immune system that induces inflammation. TNF is produced primarily by activated macrophages, and induces inflammation by binding to its receptors on other cells. It is a member of the tumor necrosis factor superfamily, a family of transmembrane proteins that are cytokines, chemical messengers of the immune system. Excessive production of TNF plays a critical role in several inflammatory diseases, and TNF-blocking drugs are often employed to treat these diseases.

<span class="mw-page-title-main">Caspase</span> Family of cysteine proteases

Caspases are a family of protease enzymes playing essential roles in programmed cell death. They are named caspases due to their specific cysteine protease activity – a cysteine in its active site nucleophilically attacks and cleaves a target protein only after an aspartic acid residue. As of 2009, there are 12 confirmed caspases in humans and 10 in mice, carrying out a variety of cellular functions.

<span class="mw-page-title-main">Perforin-1</span> Mammalian protein found in Homo sapiens

Perforin-1 Perforin (PRF), encoded by the PRF1 gene, is a pore-forming toxic protein housed in the secretory granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Together, these cells are known as cytotoxic lymphocytes (CLs).

Granzymes are serine proteases released by cytoplasmic granules within cytotoxic T cells and natural killer (NK) cells. They induce programmed cell death (apoptosis) in the target cell, thus eliminating cells that have become cancerous or are infected with viruses or bacteria. Granzymes also kill bacteria and inhibit viral replication. In NK cells and T cells, granzymes are packaged in cytotoxic granules along with perforin. Granzymes can also be detected in the rough endoplasmic reticulum, golgi complex, and the trans-golgi reticulum. The contents of the cytotoxic granules function to permit entry of the granzymes into the target cell cytosol. The granules are released into an immune synapse formed with a target cell, where perforin mediates the delivery of the granzymes into endosomes in the target cell, and finally into the target cell cytosol. Granzymes are part of the serine esterase family. They are closely related to other immune serine proteases expressed by innate immune cells, such as neutrophil elastase and cathepsin G.

<span class="mw-page-title-main">Fas ligand</span> Protein-coding gene in the species Homo sapiens

Fas ligand is a type-II transmembrane protein expressed on various types of cells, including cytotoxic T lymphocytes, monocytes, neutrophils, breast epithelial cells, vascular endothelial cells and natural killer (NK) cells. It binds with its receptor, called FAS receptor and plays a crucial role in the regulation of the immune system and in induction of apoptosis, a programmed cell death.

<span class="mw-page-title-main">Caspase 1</span> Enzyme found in humans

Caspase-1/Interleukin-1 converting enzyme (ICE) is an evolutionarily conserved enzyme that proteolytically cleaves other proteins, such as the precursors of the inflammatory cytokines interleukin 1β and interleukin 18 as well as the pyroptosis inducer Gasdermin D, into active mature peptides. It plays a central role in cell immunity as an inflammatory response initiator. Once activated through formation of an inflammasome complex, it initiates a proinflammatory response through the cleavage and thus activation of the two inflammatory cytokines, interleukin 1β (IL-1β) and interleukin 18 (IL-18) as well as pyroptosis, a programmed lytic cell death pathway, through cleavage of Gasdermin D. The two inflammatory cytokines activated by Caspase-1 are excreted from the cell to further induce the inflammatory response in neighboring cells.

<span class="mw-page-title-main">Fas receptor</span> Protein found in humans

The Fas receptor, also known as Fas, FasR, apoptosis antigen 1, cluster of differentiation 95 (CD95) or tumor necrosis factor receptor superfamily member 6 (TNFRSF6), is a protein that in humans is encoded by the FAS gene. Fas was first identified using a monoclonal antibody generated by immunizing mice with the FS-7 cell line. Thus, the name Fas is derived from FS-7-associated surface antigen.

<span class="mw-page-title-main">FADD</span> Human protein and coding gene

FAS-associated death domain protein, also called MORT1, is encoded by the FADD gene on the 11q13.3 region of chromosome 11 in humans.

<span class="mw-page-title-main">BH3 interacting-domain death agonist</span> Protein-coding gene in the species Homo sapiens

The BH3 interacting-domain death agonist, or BID, gene is a pro-apoptotic member of the Bcl-2 protein family. Bcl-2 family members share one or more of the four characteristic domains of homology entitled the Bcl-2 homology (BH) domains, and can form hetero- or homodimers. Bcl-2 proteins act as anti- or pro-apoptotic regulators that are involved in a wide variety of cellular activities.

<span class="mw-page-title-main">Caspase-9</span> Enzyme found in humans

Caspase-9 is an enzyme that in humans is encoded by the CASP9 gene. It is an initiator caspase, critical to the apoptotic pathway found in many tissues. Caspase-9 homologs have been identified in all mammals for which they are known to exist, such as Mus musculus and Pan troglodytes.

Pyroptosis is a highly inflammatory form of lytic programmed cell death that occurs most frequently upon infection with intracellular pathogens and is likely to form part of the antimicrobial response. This process promotes the rapid clearance of various bacterial, viral, fungal and protozoan infections by removing intracellular replication niches and enhancing the host's defensive responses. Pyroptosis can take place in immune cells and is also reported to occur in keratinocytes and some epithelial cells.

<span class="mw-page-title-main">Caspase 3</span> Protein found in humans

Caspase-3 is a caspase protein that interacts with caspase-8 and caspase-9. It is encoded by the CASP3 gene. CASP3 orthologs have been identified in numerous mammals for which complete genome data are available. Unique orthologs are also present in birds, lizards, lissamphibians, and teleosts.

<span class="mw-page-title-main">GNLY</span> Protein-coding gene in the species Homo sapiens

Granulysin (GNLY) is a protein expressed in most mammals which functions as an antimicrobial peptide released by killer lymphocytes in cytotoxic granules. It is a pore-forming peptide, as it can puncture a microbial cell wall, allowing for other death-inducing enzymes to enter the microbe and cause microptosis. GNLY is inhibited by cholesterol, and is most effective in helping to kill cholesterol-deficient microbes.

<span class="mw-page-title-main">NLRP1</span> Human protein-coding gene

NLRP1 encodes NACHT, LRR, FIIND, CARD domain and PYD domains-containing protein 1 in humans. NLRP1 was the first protein shown to form an inflammasome. NLRP1 is expressed by a variety of cell types, which are predominantly epithelial or hematopoietic. The expression is also seen within glandular epithelial structures including the lining of the small intestine, stomach, airway epithelia and in hairless or glabrous skin. NLRP1 polymorphisms are associated with skin extra-intestinal manifestations in CD. Its highest expression was detected in human skin, in psoriasis and in vitiligo. Polymorphisms of NLRP1 were found in lupus erythematosus and diabetes type 1. Variants of mouse NLRP1 were found to be activated upon N-terminal cleavage by the protease in anthrax lethal factor.

<span class="mw-page-title-main">GZMK</span> Protein-coding gene in the species Homo sapiens

Granzyme K (GrK) is a protein that is encoded by the GZMK gene on chromosome 5 in humans. Granzymes are a family of serine proteases which have various intracellular and extracellular roles. GrK is found in granules of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), and is traditionally described as being cytotoxic towards targeted foreign, infected, or cancerous cells. NK cells and CTLs can induce apoptosis through the granule secretory pathway, which involves the secretion of granzymes along with perforin at immunological synapses.

Within the scientific discipline of toxicology, Cytotoxic T lymphocytes (CTLs) are generated by immune activation of cytotoxic T cells (Tc cells). They are generally CD8+, which makes them MHC class I restricted. CTLs are able to eliminate most cells in the body since most nucleated cells express class I MHC molecules. The CTL-mediated immune system can be divided into two phases. In the first phase, functional effector CTLs are generated from naive Tc cells through activation and differentiation. In the second phase, affector CTLs destroy target cells by recognizing the antigen-MHC class I complex.

<span class="mw-page-title-main">Death regulator Nedd2-like caspase</span> Type of cysteine protease

Death regulator Nedd2-like caspase was firstly identified and characterised in Drosophila in 1999 as a cysteine protease containing an amino-terminal caspase recruitment domain. At first, it was thought of as an effector caspase involved in apoptosis, but subsequent findings have proved that it is, in fact, an initiator caspase with a crucial role in said type of programmed cell death.

Jürg Tschopp was a Swiss biochemist, known for his research on apoptosis and the immunology of inflammation. His greatest achievement was perhaps his team's discovery and scientific description of the inflammasome.

References

  1. 1 2 3 4 5 6 7 Afonina IS, Cullen SP, Martin SJ (May 2010). "Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B". Immunological Reviews . 235 (1): 105–16. doi:10.1111/j.0105-2896.2010.00908.x. PMID   20536558.
  2. 1 2 3 4 5 6 7 Rousalova I, Krepela E (December 2010). "Granzyme B-induced apoptosis in cancer cells and its regulation (review)". International Journal of Oncology . 37 (6): 1361–78. doi: 10.3892/ijo_00000788 . PMID   21042704.
  3. 1 2 3 4 Kurschus FC, Jenne DE (May 2010). "Delivery and therapeutic potential of human granzyme B". Immunological Reviews. 235 (1): 159–71. doi:10.1111/j.0105-2896.2010.00894.x. PMID   20536562.
  4. 1 2 3 Wowk ME, Trapani JA (July 2004). "Cytotoxic activity of the lymphocyte toxin granzyme B". Microbes and Infection . 6 (8): 752–8. doi: 10.1016/j.micinf.2004.03.008 . PMID   15207822.
  5. 1 2 3 4 5 6 Boivin WA, Cooper DM, Hiebert PR, Granville DJ (November 2009). "Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma". Laboratory Investigation; A Journal of Technical Methods and Pathology. 89 (11): 1195–220. doi: 10.1038/labinvest.2009.91 . PMC   7102238 . PMID   19770840.
  6. 1 2 Cullen SP, Brunet M, Martin SJ (April 2010). "Granzymes in cancer and immunity". Cell Death and Differentiation . 17 (4): 616–23. doi: 10.1038/cdd.2009.206 . PMID   20075940.
  7. ↑ Waterhouse NJ, Sedelies KA, Trapani JA (February 2006). "Role of Bid-induced mitochondrial outer membrane permeabilization in granzyme B-induced apoptosis". Immunology and Cell Biology . 84 (1): 72–8. doi:10.1111/j.1440-1711.2005.01416.x. PMID   16405654.
  8. ↑ Choy JC (April 2010). "Granzymes and perforin in solid organ transplant rejection". Cell Death and Differentiation. 17 (4): 567–76. doi: 10.1038/cdd.2009.161 . PMID   19876069.
  9. 1 2 Waterhouse NJ, Trapani JA (September 2007). "H is for helper: granzyme H helps granzyme B kill adenovirus-infected cells". Trends in Immunology . 28 (9): 373–5. doi:10.1016/j.it.2007.08.001. PMID   17766182.
  10. 1 2 3 Hiebert PR, Granville DJ (December 2012). "Granzyme B in injury, inflammation, and repair". Trends in Molecular Medicine . 18 (12): 732–41. doi:10.1016/j.molmed.2012.09.009. PMID   23099058.
  11. ↑ Kaiserman D, Bird PI (April 2010). "Control of granzymes by serpins". Cell Death and Differentiation. 17 (4): 586–95. doi: 10.1038/cdd.2009.169 . PMID   19893573.
  12. ↑ Darrah E, Rosen A (April 2010). "Granzyme B cleavage of autoantigens in autoimmunity". Cell Death and Differentiation. 17 (4): 624–32. doi:10.1038/cdd.2009.197. PMC   3136751 . PMID   20075942.
  13. ↑ Saito Y, Kondo H, Hojo Y (March 2011). "Granzyme B as a novel factor involved in cardiovascular diseases". Journal of Cardiology . 57 (2): 141–7. doi: 10.1016/j.jjcc.2010.10.001 . PMID   21168312.
  14. ↑ Aubert et al, Granzyme serine proteases in inflammation and rheumatic diseases. Nature Rev. Rheumatol. 2024 20(6):361-376. doi: 10.1038/s41584-024-01109-5.
  15. ↑ Richardson et al. Granzyme B in aging and age-related pathologies. Trends Mol Med. 2024 Aug 23:S1471-4914(24)00204-1. doi: 10.1016/j.molmed.2024.07.010.
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