The proteins function in said disease. Pyroptosis and

The role of the gasdermin protein family ininflammation Introduction  The gasdermin protein family is made up of 6 proteins in humans thatinclude Gasdermin A (GSDMA), Gasdermin B (GSDMB), Gasdermin C (GSDMC) GasderminD (GSDMD), Gasdermin E (DFNA5) and DFNB59. Almost all of the gasdermin familymembers are mediators of programmed cell death and bear a pore-forming abilityand cause pyroptosis once they have been cleaved. Our understanding of thisfield of innate immunity was altered in 2015 when two independent studiespublished findings, which identified Gasdermin D as the pyroptosis executioner.Pyroptosis for many years was considered to be a type of apoptosis as it was acaspase-1 programmed cell death.  Sincethe discovery of gasdermin D, we have now redefined pyroptosis as a highlyinflammatory, lytic form of programmed necrosis. Pyroptosis is a mechanism thatis heavily dependent on inflammatory caspases such as caspases-1 and caspases-11/4/5.

When the inflammatory caspases generating two significant terminalscleave pyroptosis regulator Gasdermin D – the pore forming N terminal and the Cterminal. It has been shown that other members of the gasdermin family have beenlinked to genetic diseases, however more research is needed to determine theiractivation mechanism to fully understand the proteins function in said disease. Pyroptosis and Apoptosis  Programmed cell death is an important feature of innate immunitythat involves the death of a cell due to the precise signaling events thatoccur inside a cell. Non-programmed cell death is referred to as necrosis andinvolves the deathof the cell occurring due to a physical change.

  There are two types of programmed cell death– apoptosis and pyroptosis. As many members of the Gasdermin protein family possesspore-forming abilities, which cause pyroptosis, it is important to understandthe difference between the two.  Apoptosis is a programmed form of nonlytic cell death that occurswhen a group of effector caspases cleave specific target sites in certaincellular proteins. In humans these effector or executioner proteins arecaspase-3,-6 and -7. Once these have been initiated a series of enzymatic andstructural changes occur which leads to both morphological and functionalalterations. These can include DNA cleavage and nuclear condensation. Characteristicof apoptosis, these cells are rapidly engulfed by surrounding macrophages oncethe cells have been shrunk and fragmented into apoptotic bodies. 1 The nature of thisdeath in non-inflammatory as the apoptotic bodies are targets for phagocytosismeaning there is no inflammation 2, 3.

Thischaracterizes the distinction between apoptosis and pyroptosis. Unlike apoptosis,pyroptosis is a pro-inflammatory form of cell death despite its dependency on acaspase.  4.

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Pyroptosis hasbeen recently defined as a lytic form of programmed cell death that isinitiated by inflammasomes and the death of the cell occurs due to membranerupture. Pyroptosis was long referred to as a caspases-1 mediated monocytedeath due to the involvement of Caspase-1, a member of the inflammatory caspasegroup, which is activated by inflammasomes. But is similarly activated bycaspase-11. This is activated by the inflammasomes is response to a cytosoliccontamination or perturbation. Gasdermin D is cleaved by caspase 1 andcaspase-11/4/5 resulting swelling and the eventual opening of a pore in themembrane leading to cell death. I will discuss this and the recent starringrole of Gasdermin D later on in the review.

Pyroptosis has been identified asan important mechanism involved in innate immunity. After the pore has formedas a result of the GSDMD being cleaved, swelling, cell lysis and water influxtend to follow *********     Gasdermin D Recently,Gasdermin D (GSDMD) has been discovered as the long awaited executioner ofpyroptosis. Gasdermin D acts as a substrate and is cleaved by inflammatorycaspase-1 or caspase-11/4/5 leading to the separation of the N-terminal fromthe C-terminal.

In the review I will refer to the N-terminal pore formingdomain as PFD and will refer to the C-terminal repressor domain as RD. GasderminD is highly expressed in gastrointestinal epithelium cells and demonstratesexpression in both the epidermis and in the upper gastrointestinal tract. In2015, two independent study groups led by Feng Shao and Vishva M Dixitidentified gasdermin D as the key substrate needed to cleave inflammatorycaspases and drive pyroptosis. In both groups it was found that when GSDMD wascleaved both an N-terminal (PFD) (~31Kda) and C terminal (RD) (~22kDa) were generated. Both groups alsonoted that the RD terminal has an auto inhibitory effect on the pyroptosisinducing activity of the PFD ******).  With regards to human cell lines, both groups usedCRISPR/Cas9 technology to delete gasdermin D and both were resistant to LPSinduced pyroptosis.

The Shao group also used the CRISPR/Cas9 technology andsiRNA- mediated knockdown in an attempt to find the observe the relevance ofgasdermin d in LPS-induced pyroptosis in mouse cells. With regards to mice,  the Dixit group utilized a genetic basedapproach. The Dixit group used a strain of mice, which had been treated withmutagen ENU in order to screen for mutations which would alter the activationof the caspse-11 stream. The mutation they were looking for was discovered inthe peritoneal macrophages that were harvested from a mouse strain whichpossessed a mutation in the gene encoding for gasdermin D (subsequently namedGSDMD I105N/I105N ). These cells did not undergo pyroptosis nor didthey release IL-1Beta when transfected with LPS.  This presented evidence that gasdermin d wasinvolved in the mechanism of pyroptosis induction. The Shao group also went andreported an important finding – that other members of the gasdermin proteinfamily (GSDMA,GSDMB,GSDMC,DFNA5 and DFNB59) were not cleaved by inflammatorycaspases and that caspase-1 cleaved gasdermin d at the same sit as caspase-11.

This poses an interesting question of what are the functions of the thesefamilies?.   Both the Shaoand Dixit groups also went on to generate strains of mice containing a genomicdeletion of gasdermin d (gsdmd-/- ) to once again show how muchcaspase-11 dependent pyroptosis relied on the protein. To further investigatethe role that gasdermin d plays in the caspase-11 mediated pathway. They foundthat the bone marrow derived macrophages in this strain did not undergopyroptosis or secrete IL-1beta when LPS was administered intracellularlly andthe Dixit group went on to identify a lack of caspase 1 processing during thesame experiment. Many of these observations presented promising evidence thatgasdermin d was the executioner of pyroptosis which ultimately mediates therelease of matured IL-1B and ruptures the cell membrane and induces a pore. Iwould say that the Shao group utilized the emerging CRISPR/Cas9 technologythroughout their work. The work done by the Shao and Dixit group also opened upa variety of other questions that will hopefully be investigated in the future.For example, would it be possible to directly insert the gasdermin d into themembrane in order to drive pyroptosis and pore formation? Or is it onlyeffective when it is used as a substrate for inflammatory caspases?  A closerlook at the pore As we havealready established, Gasdermin D, when cleaved, is responsible for themechanism by which caspase-1 and caspase-11 trigger pyropotoic cell death.

5, 6 The N terminal domain (recentlyreferred to as PFD in literature) leads to the formation of the gasdermin pore,and the C terminal (recently referred to as RD in literature) is removed uponcleavage but is thought to fold back on the N terminal and have an autoinhibitory effect by monitoring the activity. When the PFD is expressed it ishighly toxic to E.Coli whereas when there was very little toxicity observedwhen the RD or the full GSDMD is expressed. This once again demonstrates thefundamental cytotoxicity of the PFD in mammalian cells. 7  In 2015 astudy by Ding et al showed the crystal structure of GSDMA3, which is thepredicted structure for the gasdermin family.

From this was can predict theauto inhibited two domain architecture of Gasdermin D. The structure isseparated into the PFD and the RD with approximately 480 amino acids splitbetween them. There is a 43-aa linker domain, which ties the PFD to the RD.

Thealpha4 Helix from the PFD protrudes across to RD domain where it connects to alooped pocket. Caspase-1 and caspase-11 cleave the GSDMD at an aspartate sitewithin this loop 7, after amino acid 275 at a conserved(F/L)LTD motif 5, 6. It is then assumed that theinterface dissociates and the alpha helix is released from the looped pocketand the PFD is liberated from the RD. In the PFD, caspase-3 has also been foundto cleave gasdermin D and inactivate the domain. This leads to the initiationof tumor necrosis factor – alpha- induced apoptosis and the prevention of pyroptosis8. Not only does this once againprovide us information on the types of caspases gasdermin D cleaves but it alsosuggests that pyroptosis has faster kinetics and that apoptosis 6, 8, 9. A structural homology search wascarried out on this study and showed no similarities between the PFD and anyother know proteins suggesting that this is a new type of pore-forming protein.

From this we could predict the same regarding Gasdermin D. 7 The 43-aalinker is cleaved by the gasdermin d and the RD and PFD are separated and thePFD goes into the cell membrane. A pore that is estimated to be around 10-15nmin diameter is created when approximately 16 PFD monomers oligomerize Duringexperimentation the formation of these pores was observed via electronmicroscopy and the following was identified. 7, 10. The PFD wasfound to have affinity for cardiolipin (lipid containing two double phosphateson the glycerol scaffold), which is found on the inner and outer leaflets ofbacterial membranes. It should be noted that there is still no conclusivestudy, which indicates whether or not the gasdermin D has access to the innerleaflet of the mitochondria. The PFD also binds to phosphatidylinositolphosphates and phosphatidylserine (only present on cells inner membraneleaflet).

The PFD has been found to not bind to the positively charged headgroups (phosphatidylethanolamine and phosphatidylcholine) or the non chargedgroup lipid (phosphatidylinositol). This indicates that the gasdermin PFD hasan affinity for lipid species with negatively charged head groups 7, 10. Given that phosphatidylserine andphosphatidylinositol phosphates are restricted to the cytosolic leaflet of themembrane, it is clear that the pore can only form from the cytosolic face 10. This presented an important factabout the Gasdermin PFD – that it kills from within the cell due to itslipid-binding preferences it does not pose a risk when liberated duringpyroptosis to neighboring mammalian cells 10.

  Post Pore  So whathappens when the interaction between the PFD of gasdermin D and the membraneinteract? The pore is created when the ~16 monomers olgiomerize and the cellmembrane begins to endure disruptive events. The formation of the pore disruptsthe osmotic potential of the cell. This disruption is driven by ion-involvedosmotic pressure.41 In the absence of a pore or other disruption, theextracellular fluid should have a high concentration of sodium and a lowconcentration of potassium and in terms of the cytosol the reverse should beobserved. This creates two types of gradients – a concentration gradient and anelectrical gradient. The electrical gradient that exists across the membranecauses positive ions to be pulled into the negative cytosol. The way thesegradients interact is relevant in terms of regulating the amount of potassiumin the cell.

When a pore is opened there is minimal net potassium flux as theelectrical forces pulling potassium in to the cytosol offset the forces fromthe concentration gradient driving the potassium out. Simultaneously, theelectrical gradient and the concentration gradient both drive sodium into thecell creating a massive influx of sodium into the cell bringing water with it.This can result in an increase in cell volume. 21 If there areonly a few pores, the cell will engage its compensatory mechanisms notablyregulatory volume decrease (RVD), an active process, in an effort to decreasethe volume. This is achieved by the extruding the major intracellular ions K+and CI- and organic osmolytes. 41 If there areseveral pores this can cause the membrane to rupture should the influx exceedthe volume capacity of the membrane and if the amount of pores overwhelm thecells compensatory abilities as discussed above. Forexample if there were only a small amount of pores an emergency exocyticmembrane fusion event similar to those seen in necroptosis,  should be able to patch up the membrane22,23,24. If the volume is exceeded, membrane blebbing occurs andpyroptic bodies (which are similar to apoptotic body like cell protrusions) areproduced directly before the membrane rupture event.

39 After thisthe membrane rupture occurs and soluble cytosolic contents is released in anon-explosion like way (pyroptosis tends to undergo cytoplasm flattening causedby the leak rather than an explosion which tends to be seen in other forms ofcell death). The rupture tends to be larger than the gasdermin pore but equalto or smaller than other organelles. The reason that pyropotoic cells do notburst is due to the lack of selectivity of the pores formed from the PFD. Asthe GSDMD pore lacks ion selectivity, there tends to be no increase in intracellularosmolarity preventing a cell death that involves the contents bursting from themembrane.39  Upon rupture, organellesare retained but it is possible to dispel soluble proteins jorgeson ref. Inthe aftermath of the rupture it is likely that the osmotic pressure equalizes.

 Inteurlekin(IL)-1beta and IL-18  By now we areaware that pyroptosis occurs as a result of caspase-1 and caspase-11 activationin inflammasomes and therefore plays an integral role in inflammation. GSDMDhas also been identified as a central part of the inflammasome, a multi proteinintracellular signaling complex of the innate immune system He. In a study byHe et al, it was found that GSDMD is required for IL-1beta secretion but is notrequired for IL-1beta processing.

The data revealed that after the gasdermin Dis cleaved by caspase-1 it both promotes pyroptosis as well as secretingmatured IL-1beta. The data from the He group reported that Gasdermin D isessential for both non-canonical inflammasome pathways. Another independentreport published by Shi et al also came to the same conclusion.  It has longbeen discussed whether or not the rupture of the cell membrane was the releasemechanism for IL-Ibeta and IL-18. These cytokines have been referred to as”leaderless proteins” due to their unusual method of secretion.

43 They arenot secreted through the conventional ER-Golgi route like the majority of otherproteins.26 They are restricted to the cytosol until they encounter a signalwhich is activated by cell stress or an infection43 A new study by Heilig etal has shown using the murine system to investigate how the Gasdermin D porereleases cytosolic proteins. The study haspresented findings that show the gasdermin d pore is large enough to allow thedirect release of IL-1beta indicating that cell lysis is not mandatory. It alsopresented findings that IL-18 and other small cytosolic proteins are releasedin a gasdermin dependent event that is lysis independent and seems dependent onsize. This would indicate that cell lysis is not obligatory in order to releasethese cytosolic proteins.

 The findingsfrom this study showed the dual role of the GSDMD pore. It is essential forcell lysis, which is accompanied by cytokine release and also allows the directrelease of IL-1beta and IL-18 regardless of whether or not lysis has occurred.During live microcopy studies, glycine, a common cytoprotectant was added toinhibit cell lysis. Markedly, the glycine only partially reduced the IL-1betafrom cells under these experimental conditions. This offers evidence that therelease of the cytokine is gasdermin dependent but lysis independent.H Ithink it should be noted that mechanism by which glycine prevents cell lysis isstill unknown 34,36 and further investigation into this may offer a greaterinsight into interpreting results obtained this way.

   The studyalso offered evidence to suggest that gasdermin-d pores promote size-dependentleakage of cytosolic proteins. The diameter of the gasdermin pore is 10-15 nm.The diameter of mature IL-1beta and IL-18 is 4.

4nm and 5nm respectively15,18. Knowing the diameter of the pore also tells us that the pore canrestrict larger proteins and organelles from passing through (eg a 25 nmdiameter would be too large to pass through) 2. Similarly in the study byHeilig et al, their data supports the theory that GSDMD pores acts as channelsthat release cytosolic proteins based on size. H  TheGasdermin pore and bacteria Pyroptosishas also been shown to be able to defend against intracellular infections. Ifthe inflammasome detects the pathogen successfully, pyroptosis can attempt toeradicate the cell by removing the protected intracellular niche. 2 Withcertain bacteria, it has been shown that following the death of the infectedhost cell, the intracellular bacterium has not been killed. 1.

Following therupture, organelles and intracellular bacteria remain trapped in the corpse ofthe cell who’s membrane is compromised but still largely intact. This structureis defined as a pore-induced intracellular trap (PIT). The PIT increases theamount of neutrophil chemo attractants (DAMPS and eicosanoids) 4 Theneutrophils then kill the PIT and its contents by efferocytosis. 2 This meansthat the infection is actually cleared by the secondary phagocyte rather thatdirectly by the PIT itself.

Due to the fact that pyroptosis can not killbacteria only damage it, the relevance of the gasdermin pore being able toinsert itself into the bacterial membranes remains unclear. 2.  The PFD fromthe gasdermin d can insert itself into the cardiolipin in the inner and outerleaflets of the bacterial membrane. (Dalebroux et al 2015)  There isevidence to suggest a direct antibacterial defense triggered by  GSDMD cleavage.

(book) It was found, invitro, that the PFD causes the death of gram negative E. coli and gram positiveS.Aureus bacteria at low nanomolar concentrations. It was also found that thePFD was directly responsible for killing intracellular Listeria, as overexpression of the PFD in mammalian cells suppressed intracellular bacteriagrowth (Liu). There is more work to be completed in this area, as theprotective nature of the PFD in terms of bacteria has only been witness invitro at this time (book).

Difficulties arise due to the fact that there iscurrently no method of separating host cell and bacterial membrane damage bythe PFD experimentally. If this is ever established, experiments could possiblybe carried out in mice that have been infected but have had the GSDMD knocked outGM1  to confirm the proteins function indefending against pathogenic bacteria. (book)   GM1Check phrasing of this – may be called knock out mice