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'''Granzyme B''' is a [[serine protease]] most commonly found in the granules of cytotoxic lymphocytes (CTLs), [[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''' is a [[serine protease]] most commonly found in the granules of cytotoxic lymphocytes (CTLs), [[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 more recently found to be produced by a wide range of non-cytotoxic cells ranging from [[basophil granulocyte|basophils]] and [[mast cells]] to [[smooth muscle cells]].<ref name="Afonina2010">{{cite journal | title = Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B | author = Afonina IS | year = 2010 | pmid = 20536558 | doi=10.1111/j.0105-2896.2010.00908}}</ref> 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.
Granzyme B has also more recently found to be produced by a wide range of non-cytotoxic cells ranging from [[basophil granulocyte|basophils]] and [[mast cells]] to [[smooth muscle cells]].<ref name="Afonina2010">{{cite journal | title = Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B | author = Afonina IS | year = 2010 | pmid = 20536558 | doi=10.1111/j.0105-2896.2010.00908.x}}</ref> 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]].
Elevated levels of granzyme B are also implicated in a number of [[autoimmune diseases]], several skin diseases, and [[type 1 diabetes]].
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Granzyme B has many substrates located in the [[Cell nucleus|nucleus]]. Granzyme B can cleave [[Poly ADP ribose polymerase|PARP]] (poly ADP ribose polymerase) and [[DNA-PKcs|DNA PK]] (DNA protein kinase) to disrupt DNA repair and [[retrovirus|retroviral]] DNA integration. Granzyme B can also cleave [[nucleophosmin]], [[topoisomerase 1]] and [[nucleolin]] to prevent viral replication.
Granzyme B has many substrates located in the [[Cell nucleus|nucleus]]. Granzyme B can cleave [[Poly ADP ribose polymerase|PARP]] (poly ADP ribose polymerase) and [[DNA-PKcs|DNA PK]] (DNA protein kinase) to disrupt DNA repair and [[retrovirus|retroviral]] DNA integration. Granzyme B can also cleave [[nucleophosmin]], [[topoisomerase 1]] and [[nucleolin]] to prevent viral replication.


Graznyme B can cleave ICP4 from the [[Herpes simplex virus|HSV 1]] virus which is an essential protein used for gene [[transactivation]] and [[nuclear mitotic apparatus protein 1|NUMA]] (Nuclear mitotic apparatus protein) can be cleaved to prevent [[mitosis]].<ref name="Afonina2010">{{cite journal | title = Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B | author = Afonina IS | year = 2010 | pmid = 20536558 | doi=10.1111/j.0105-2896.2010.00908.x}}</ref>
Graznyme B can cleave ICP4 from the [[Herpes simplex virus|HSV 1]] virus which is an essential protein used for gene [[transactivation]] and [[nuclear mitotic apparatus protein 1|NUMA]] (Nuclear mitotic apparatus protein) can be cleaved to prevent [[mitosis]].<ref name="Afonina2010" />


Granzyme B can also cleave [[DNA-binding protein|DBP]] (DNA Binding Protein) into a 50 kDa fragment and then into an additional 60 kDa indirectly through the caspases it activates.<ref name="Waterhouse2007">{{ cite journal | title = H is for helper: granzyme H helps granzyme B kill adenovirus-infected cells | author = Waterhouse NJ | year = 2007 | pmid= 17766182}}</ref>
Granzyme B can also cleave [[DNA-binding protein|DBP]] (DNA Binding Protein) into a 50 kDa fragment and then into an additional 60 kDa indirectly through the caspases it activates.<ref name="Waterhouse2007">{{ cite journal | title = H is for helper: granzyme H helps granzyme B kill adenovirus-infected cells | author = Waterhouse NJ | year = 2007 | pmid= 17766182}}</ref>


===Extracellular Matrix===
===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.<ref name="Afonina2010">{{cite journal | title = Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B | author = Afonina IS | year = 2010 | pmid = 20536558 | doi=10.1111/j.0105-2896.2010.00908.x}}</ref> Fragments of fibronectin can attract [[neutrophils]] and stimulate [[Matrix metalloproteinase|MMP]] expression from [[chondrocytes]].<ref name="Boivin2009">{{cite journal| title = Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma | author= Boivin WA | year= 2009 | pmid= 19770840 | doi= 10.1038/labinvest.2009.91}}</ref> Basophils secrete granzyme B to degrade [[endothelium|endothelial]] cell-cell contacts allowing [[extravasation]] to sites of inflammation.<ref name="Cullen2010">{{ cite journal | title = Granzymes in cancer and immunity | author = Cullen SP | year = 2010 | pmid= 20075940 | doi= 10.1038/cdd.2009.206}}</ref>
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.<ref name="Afonina2010" /> Fragments of fibronectin can attract [[neutrophils]] and stimulate [[Matrix metalloproteinase|MMP]] expression from [[chondrocytes]].<ref name="Boivin2009">{{cite journal| title = Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma | author= Boivin WA | year= 2009 | pmid= 19770840 | doi= 10.1038/labinvest.2009.91}}</ref> Basophils secrete granzyme B to degrade [[endothelium|endothelial]] cell-cell contacts allowing [[extravasation]] to sites of inflammation.<ref name="Cullen2010">{{ cite journal | title = Granzymes in cancer and immunity | author = Cullen SP | year = 2010 | pmid= 20075940 | doi= 10.1038/cdd.2009.206}}</ref>


Granzyme B can also induce inflammation by processing cytokines [[IL1B|IL-1β]] and [[Interleukin 18|IL18]] and by processing [[IL1A|IL-1α]] into a more inflammatory fragment. It can also trigger the release of [[Interleukin 6|IL6]] and [[Interleukin 8|IL8]] through activation of [[Coagulation factor II receptor|PAR1]] (Protease activated receptor 1).<ref name="Hiebert2012">{{ cite journal | title = Granzyme B in injury, inflammation, and repair | author = Hiebert PR | year = 2012 | pmid= 23099058 | doi= 10.1016/j.molmed.2012.09.009}}</ref>
Granzyme B can also induce inflammation by processing cytokines [[IL1B|IL-1β]] and [[Interleukin 18|IL18]] and by processing [[IL1A|IL-1α]] into a more inflammatory fragment. It can also trigger the release of [[Interleukin 6|IL6]] and [[Interleukin 8|IL8]] through activation of [[Coagulation factor II receptor|PAR1]] (Protease activated receptor 1).<ref name="Hiebert2012">{{ cite journal | title = Granzyme B in injury, inflammation, and repair | author = Hiebert PR | year = 2012 | pmid= 23099058 | doi= 10.1016/j.molmed.2012.09.009}}</ref>
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==T cell Regulation==
==T cell Regulation==
Granzyme B is secreted by [[regulatory T cells]] 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 receptor|Fas]] death pathway and prevents [[autoimmunity|autoimmune reaction]] to [[antigen|self antigens]].<ref name="Afonina2010">{{cite journal | title = Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B | author = Afonina IS | year = 2010 | pmid = 20536558 | doi=10.1111/j.0105-2896.2010.00908.x}}</ref>
Granzyme B is secreted by [[regulatory T cells]] 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 receptor|Fas]] death pathway and prevents [[autoimmunity|autoimmune reaction]] to [[antigen|self antigens]].<ref name="Afonina2010" />


==Inhibitors==
==Inhibitors==

Revision as of 15:22, 13 July 2014

Granzyme B
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CAS no.143180-74-9
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Granzyme B is a serine protease most commonly found in the granules of cytotoxic lymphocytes (CTLs), 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 more recently 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

Granzyme B is encoded by GZMB on chromosome 14q.11.2 which is 3.2kb long consisting of 4 introns and 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 6 stranded β sheets with 3 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]

Granzyme B 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.

Graznyme 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-1β and IL18 and by processing IL-1α into a more inflammatory fragment. 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 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 20-4-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.

COPD (Chronic Obstructive Pulmonary Disease) 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]

See also

References

  1. ^ a b c d e f g Afonina IS (2010). "Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B". doi:10.1111/j.0105-2896.2010.00908.x. PMID 20536558. {{cite journal}}: Cite journal requires |journal= (help)
  2. ^ a b c d e f g Rousalova I (2010). "Granzyme B-induced apoptosis in cancer cells and its regulation (review)". PMID 21042704. {{cite journal}}: Cite journal requires |journal= (help) Cite error: The named reference "Rousalova2010" was defined multiple times with different content (see the help page).
  3. ^ a b c d Kurschus FC (2010). "Delivery and therapeutic potential of human granzyme B". doi:10.1111/j.0105-2896.2010.00894. PMID 20536562. {{cite journal}}: Cite journal requires |journal= (help) Cite error: The named reference "Kurschus2012" was defined multiple times with different content (see the help page).
  4. ^ a b c Wowk ME (2004). "Cytotoxic activity of the lymphocyte toxin granzyme B". PMID 15207822. {{cite journal}}: Cite journal requires |journal= (help)
  5. ^ a b c d e f Boivin WA (2009). "Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma". doi:10.1038/labinvest.2009.91. PMID 19770840. {{cite journal}}: Cite journal requires |journal= (help) Cite error: The named reference "Boivin2009" was defined multiple times with different content (see the help page).
  6. ^ a b Cullen SP (2010). "Granzymes in cancer and immunity". doi:10.1038/cdd.2009.206. PMID 20075940. {{cite journal}}: Cite journal requires |journal= (help)
  7. ^ Waterhouse NJ (2006). "Role of Bid-induced mitochondrial outer membrane permeabilization granzyme B-induced apoptosis". PMID 16405654. {{cite journal}}: Cite journal requires |journal= (help)
  8. ^ Choy JC (2010). "Granzymes and perforin in solid organ transplant rejection". doi:10.1038/cdd.2009.161. PMID 19876069. {{cite journal}}: Cite journal requires |journal= (help)
  9. ^ a b Waterhouse NJ (2007). "H is for helper: granzyme H helps granzyme B kill adenovirus-infected cells". PMID 17766182. {{cite journal}}: Cite journal requires |journal= (help)
  10. ^ a b c Hiebert PR (2012). "Granzyme B in injury, inflammation, and repair". doi:10.1016/j.molmed.2012.09.009. PMID 23099058. {{cite journal}}: Cite journal requires |journal= (help)
  11. ^ Kaiserman D (2010). "Control of granzymes by serpins". doi:10.1038/cdd.2009.169. PMID 19893573. {{cite journal}}: Cite journal requires |journal= (help)
  12. ^ Darrah E (2010). "Granzyme B cleavage of autoantigens in autoimmunity". doi:10.1038/cdd.2009.197. PMID 20075942. {{cite journal}}: Cite journal requires |journal= (help)
  13. ^ Saito Y (2011). "Granzyme B as a noverl factor involed in cardiovascular diseases". doi:10.1016/j.jjcc.2010.10.001. PMID 21168312. {{cite journal}}: Cite journal requires |journal= (help)
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