The proteins function in said disease. Pyroptosis and

The role of the gasdermin protein family in
inflammation

 

Introduction

 

The gasdermin protein family is made up of 6 proteins in humans that
include Gasdermin A (GSDMA), Gasdermin B (GSDMB), Gasdermin C (GSDMC) Gasdermin
D (GSDMD), Gasdermin E (DFNA5) and DFNB59. Almost all of the gasdermin family
members are mediators of programmed cell death and bear a pore-forming ability
and cause pyroptosis once they have been cleaved. Our understanding of this
field of innate immunity was altered in 2015 when two independent studies
published findings, which identified Gasdermin D as the pyroptosis executioner.

Pyroptosis for many years was considered to be a type of apoptosis as it was a
caspase-1 programmed cell death.  Since
the discovery of gasdermin D, we have now redefined pyroptosis as a highly
inflammatory, lytic form of programmed necrosis. Pyroptosis is a mechanism that
is heavily dependent on inflammatory caspases such as caspases-1 and caspases
-11/4/5. When the inflammatory caspases generating two significant terminals
cleave pyroptosis regulator Gasdermin D – the pore forming N terminal and the C
terminal. It has been shown that other members of the gasdermin family have been
linked to genetic diseases, however more research is needed to determine their
activation mechanism to fully understand the proteins function in said disease.

 

Pyroptosis and Apoptosis

 

Programmed cell death is an important feature of innate immunity
that involves the death of a cell due to the precise signaling events that
occur inside a cell. Non-programmed cell death is referred to as necrosis and
involves 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 possess
pore-forming abilities, which cause pyroptosis, it is important to understand
the difference between the two.

 

Apoptosis is a programmed form of nonlytic cell death that occurs
when a group of effector caspases cleave specific target sites in certain
cellular proteins. In humans these effector or executioner proteins are
caspase-3,-6 and -7. Once these have been initiated a series of enzymatic and
structural changes occur which leads to both morphological and functional
alterations. These can include DNA cleavage and nuclear condensation. Characteristic
of apoptosis, these cells are rapidly engulfed by surrounding macrophages once
the cells have been shrunk and fragmented into apoptotic bodies. 1 The nature of this
death in non-inflammatory as the apoptotic bodies are targets for phagocytosis
meaning there is no inflammation 2, 3. This
characterizes the distinction between apoptosis and pyroptosis. Unlike apoptosis,
pyroptosis is a pro-inflammatory form of cell death despite its dependency on a
caspase.  4. Pyroptosis has
been recently defined as a lytic form of programmed cell death that is
initiated by inflammasomes and the death of the cell occurs due to membrane
rupture. Pyroptosis was long referred to as a caspases-1 mediated monocyte
death due to the involvement of Caspase-1, a member of the inflammatory caspase
group, which is activated by inflammasomes. But is similarly activated by
caspase-11. This is activated by the inflammasomes is response to a cytosolic
contamination or perturbation. Gasdermin D is cleaved by caspase 1 and
caspase-11/4/5 resulting swelling and the eventual opening of a pore in the
membrane leading to cell death. I will discuss this and the recent starring
role of Gasdermin D later on in the review. Pyroptosis has been identified as
an important mechanism involved in innate immunity. After the pore has formed
as a result of the GSDMD being cleaved, swelling, cell lysis and water influx
tend to follow *********

 

 

 

 

Gasdermin D

 

Recently,
Gasdermin D (GSDMD) has been discovered as the long awaited executioner of
pyroptosis. Gasdermin D acts as a substrate and is cleaved by inflammatory
caspase-1 or caspase-11/4/5 leading to the separation of the N-terminal from
the C-terminal. In the review I will refer to the N-terminal pore forming
domain as PFD and will refer to the C-terminal repressor domain as RD. Gasdermin
D is highly expressed in gastrointestinal epithelium cells and demonstrates
expression in both the epidermis and in the upper gastrointestinal tract. In
2015, two independent study groups led by Feng Shao and Vishva M Dixit
identified gasdermin D as the key substrate needed to cleave inflammatory
caspases and drive pyroptosis. In both groups it was found that when GSDMD was
cleaved both an N-terminal (PFD) 
(~31Kda) and C terminal (RD) (~22kDa) were generated. Both groups also
noted that the RD terminal has an auto inhibitory effect on the pyroptosis
inducing activity of the PFD ******).

 

 With regards to human cell lines, both groups used
CRISPR/Cas9 technology to delete gasdermin D and both were resistant to LPS
induced pyroptosis. The Shao group also used the CRISPR/Cas9 technology and
siRNA- mediated knockdown in an attempt to find the observe the relevance of
gasdermin d in LPS-induced pyroptosis in mouse cells. With regards to mice,  the Dixit group utilized a genetic based
approach. The Dixit group used a strain of mice, which had been treated with
mutagen ENU in order to screen for mutations which would alter the activation
of the caspse-11 stream. The mutation they were looking for was discovered in
the peritoneal macrophages that were harvested from a mouse strain which
possessed a mutation in the gene encoding for gasdermin D (subsequently named
GSDMD I105N/I105N ). These cells did not undergo pyroptosis nor did
they release IL-1Beta when transfected with LPS.  This presented evidence that gasdermin d was
involved in the mechanism of pyroptosis induction. The Shao group also went and
reported an important finding – that other members of the gasdermin protein
family (GSDMA,GSDMB,GSDMC,DFNA5 and DFNB59) were not cleaved by inflammatory
caspases 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 these
families?.

 

 

Both the Shao
and Dixit groups also went on to generate strains of mice containing a genomic
deletion of gasdermin d (gsdmd-/- ) to once again show how much
caspase-11 dependent pyroptosis relied on the protein. To further investigate
the role that gasdermin d plays in the caspase-11 mediated pathway. They found
that the bone marrow derived macrophages in this strain did not undergo
pyroptosis or secrete IL-1beta when LPS was administered intracellularlly and
the Dixit group went on to identify a lack of caspase 1 processing during the
same experiment. Many of these observations presented promising evidence that
gasdermin d was the executioner of pyroptosis which ultimately mediates the
release of matured IL-1B and ruptures the cell membrane and induces a pore. I
would say that the Shao group utilized the emerging CRISPR/Cas9 technology
throughout their work. The work done by the Shao and Dixit group also opened up
a 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 the
membrane in order to drive pyroptosis and pore formation? Or is it only
effective when it is used as a substrate for inflammatory caspases?

 

A closer
look at the pore

 

As we have
already established, Gasdermin D, when cleaved, is responsible for the
mechanism by which caspase-1 and caspase-11 trigger pyropotoic cell death. 5, 6 The N terminal domain (recently
referred 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 upon
cleavage but is thought to fold back on the N terminal and have an auto
inhibitory effect by monitoring the activity. When the PFD is expressed it is
highly toxic to E.Coli whereas when there was very little toxicity observed
when the RD or the full GSDMD is expressed. This once again demonstrates the
fundamental cytotoxicity of the PFD in mammalian cells. 7

 

In 2015 a
study by Ding et al showed the crystal structure of GSDMA3, which is the
predicted structure for the gasdermin family. From this was can predict the
auto inhibited two domain architecture of Gasdermin D. The structure is
separated into the PFD and the RD with approximately 480 amino acids split
between them. There is a 43-aa linker domain, which ties the PFD to the RD. The
alpha4 Helix from the PFD protrudes across to RD domain where it connects to a
looped pocket. Caspase-1 and caspase-11 cleave the GSDMD at an aspartate site
within this loop 7, after amino acid 275 at a conserved
(F/L)LTD motif 5, 6. It is then assumed that the
interface dissociates and the alpha helix is released from the looped pocket
and the PFD is liberated from the RD. In the PFD, caspase-3 has also been found
to cleave gasdermin D and inactivate the domain. This leads to the initiation
of tumor necrosis factor – alpha- induced apoptosis and the prevention of pyroptosis
8. Not only does this once again
provide us information on the types of caspases gasdermin D cleaves but it also
suggests that pyroptosis has faster kinetics and that apoptosis 6, 8, 9. A structural homology search was
carried out on this study and showed no similarities between the PFD and any
other 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-aa
linker is cleaved by the gasdermin d and the RD and PFD are separated and the
PFD goes into the cell membrane. A pore that is estimated to be around 10-15nm
in diameter is created when approximately 16 PFD monomers oligomerize During
experimentation the formation of these pores was observed via electron
microscopy and the following was identified. 7, 10.

 

The PFD was
found to have affinity for cardiolipin (lipid containing two double phosphates
on the glycerol scaffold), which is found on the inner and outer leaflets of
bacterial membranes. It should be noted that there is still no conclusive
study, which indicates whether or not the gasdermin D has access to the inner
leaflet of the mitochondria. The PFD also binds to phosphatidylinositol
phosphates and phosphatidylserine (only present on cells inner membrane
leaflet). The PFD has been found to not bind to the positively charged head
groups (phosphatidylethanolamine and phosphatidylcholine) or the non charged
group lipid (phosphatidylinositol). This indicates that the gasdermin PFD has
an affinity for lipid species with negatively charged head groups 7, 10. Given that phosphatidylserine and
phosphatidylinositol phosphates are restricted to the cytosolic leaflet of the
membrane, it is clear that the pore can only form from the cytosolic face 10. This presented an important fact
about the Gasdermin PFD – that it kills from within the cell due to its
lipid-binding preferences it does not pose a risk when liberated during
pyroptosis to neighboring mammalian cells 10.  

 

Post Pore

 

So what
happens when the interaction between the PFD of gasdermin D and the membrane
interact? The pore is created when the ~16 monomers olgiomerize and the cell
membrane begins to endure disruptive events. The formation of the pore disrupts
the osmotic potential of the cell. This disruption is driven by ion-involved
osmotic pressure.41 In the absence of a pore or other disruption, the
extracellular fluid should have a high concentration of sodium and a low
concentration of potassium and in terms of the cytosol the reverse should be
observed. This creates two types of gradients – a concentration gradient and an
electrical gradient. The electrical gradient that exists across the membrane
causes positive ions to be pulled into the negative cytosol. The way these
gradients interact is relevant in terms of regulating the amount of potassium
in the cell. When a pore is opened there is minimal net potassium flux as the
electrical forces pulling potassium in to the cytosol offset the forces from
the concentration gradient driving the potassium out. Simultaneously, the
electrical gradient and the concentration gradient both drive sodium into the
cell 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 are
only a few pores, the cell will engage its compensatory mechanisms notably
regulatory volume decrease (RVD), an active process, in an effort to decrease
the volume. This is achieved by the extruding the major intracellular ions K+
and CI- and organic osmolytes. 41

 

If there are
several pores this can cause the membrane to rupture should the influx exceed
the volume capacity of the membrane and if the amount of pores overwhelm the
cells compensatory abilities as discussed above. For
example if there were only a small amount of pores an emergency exocytic
membrane fusion event similar to those seen in necroptosis,  should be able to patch up the membrane
22,23,24. If the volume is exceeded, membrane blebbing occurs and
pyroptic bodies (which are similar to apoptotic body like cell protrusions) are
produced directly before the membrane rupture event. 39

 

After this
the membrane rupture occurs and soluble cytosolic contents is released in a
non-explosion like way (pyroptosis tends to undergo cytoplasm flattening caused
by the leak rather than an explosion which tends to be seen in other forms of
cell death). The rupture tends to be larger than the gasdermin pore but equal
to or smaller than other organelles. The reason that pyropotoic cells do not
burst is due to the lack of selectivity of the pores formed from the PFD. As
the GSDMD pore lacks ion selectivity, there tends to be no increase in intracellular
osmolarity preventing a cell death that involves the contents bursting from the
membrane.39  Upon rupture, organelles
are retained but it is possible to dispel soluble proteins jorgeson ref. In
the aftermath of the rupture it is likely that the osmotic pressure equalizes.

 

Inteurlekin
(IL)-1beta and IL-18

 

By now we are
aware that pyroptosis occurs as a result of caspase-1 and caspase-11 activation
in inflammasomes and therefore plays an integral role in inflammation. GSDMD
has also been identified as a central part of the inflammasome, a multi protein
intracellular signaling complex of the innate immune system He. In a study by
He et al, it was found that GSDMD is required for IL-1beta secretion but is not
required for IL-1beta processing. The data revealed that after the gasdermin D
is cleaved by caspase-1 it both promotes pyroptosis as well as secreting
matured IL-1beta. The data from the He group reported that Gasdermin D is
essential for both non-canonical inflammasome pathways. Another independent
report published by Shi et al also came to the same conclusion.

 

It has long
been discussed whether or not the rupture of the cell membrane was the release
mechanism for IL-Ibeta and IL-18. These cytokines have been referred to as
“leaderless proteins” due to their unusual method of secretion.43 They are
not secreted through the conventional ER-Golgi route like the majority of other
proteins.26 They are restricted to the cytosol until they encounter a signal
which is activated by cell stress or an infection43 A new study by Heilig et
al has shown using the murine system to investigate how the Gasdermin D pore
releases cytosolic proteins.

 

The study has
presented findings that show the gasdermin d pore is large enough to allow the
direct release of IL-1beta indicating that cell lysis is not mandatory. It also
presented findings that IL-18 and other small cytosolic proteins are released
in a gasdermin dependent event that is lysis independent and seems dependent on
size. This would indicate that cell lysis is not obligatory in order to release
these cytosolic proteins.

 

The findings
from this study showed the dual role of the GSDMD pore. It is essential for
cell lysis, which is accompanied by cytokine release and also allows the direct
release of IL-1beta and IL-18 regardless of whether or not lysis has occurred.

During live microcopy studies, glycine, a common cytoprotectant was added to
inhibit cell lysis. Markedly, the glycine only partially reduced the IL-1beta
from cells under these experimental conditions. This offers evidence that the
release of the cytokine is gasdermin dependent but lysis independent.H I
think it should be noted that mechanism by which glycine prevents cell lysis is
still unknown 34,36 and further investigation into this may offer a greater
insight into interpreting results obtained this way. 

 

The study
also offered evidence to suggest that gasdermin-d pores promote size-dependent
leakage 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 respectively
15,18. Knowing the diameter of the pore also tells us that the pore can
restrict larger proteins and organelles from passing through (eg a 25 nm
diameter would be too large to pass through) 2. Similarly in the study by
Heilig et al, their data supports the theory that GSDMD pores acts as channels
that release cytosolic proteins based on size. H

 

The
Gasdermin pore and bacteria

 

Pyroptosis
has also been shown to be able to defend against intracellular infections. If
the inflammasome detects the pathogen successfully, pyroptosis can attempt to
eradicate the cell by removing the protected intracellular niche. 2 With
certain bacteria, it has been shown that following the death of the infected
host cell, the intracellular bacterium has not been killed. 1. Following the
rupture, organelles and intracellular bacteria remain trapped in the corpse of
the cell who’s membrane is compromised but still largely intact. This structure
is defined as a pore-induced intracellular trap (PIT). The PIT increases the
amount of neutrophil chemo attractants (DAMPS and eicosanoids) 4 The
neutrophils then kill the PIT and its contents by efferocytosis. 2 This means
that the infection is actually cleared by the secondary phagocyte rather that
directly by the PIT itself. Due to the fact that pyroptosis can not kill
bacteria only damage it, the relevance of the gasdermin pore being able to
insert itself into the bacterial membranes remains unclear. 2.

 

The PFD from
the gasdermin d can insert itself into the cardiolipin in the inner and outer
leaflets of the bacterial membrane. (Dalebroux et al 2015)

 

There is
evidence to suggest a direct antibacterial defense triggered by  GSDMD cleavage. (book) It was found, in
vitro, that the PFD causes the death of gram negative E. coli and gram positive
S.Aureus bacteria at low nanomolar concentrations. It was also found that the
PFD was directly responsible for killing intracellular Listeria, as over
expression of the PFD in mammalian cells suppressed intracellular bacteria
growth (Liu). There is more work to be completed in this area, as the
protective nature of the PFD in terms of bacteria has only been witness in
vitro at this time (book). Difficulties arise due to the fact that there is
currently no method of separating host cell and bacterial membrane damage by
the PFD experimentally. If this is ever established, experiments could possibly
be carried out in mice that have been infected but have had the GSDMD knocked outGM1  to confirm the proteins function in
defending against pathogenic bacteria. (book)  

 GM1Check phrasing of this – may be called knock out mice