Expert Answer:Mutation Of Platin Protein & Effects On Cells In S

  

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Mutation of plakin proteins
MUTATION OF PLAKIN PROTEINS AND THEIR EFFECT ON CELLS IN THE
STRATIFIED ENVELOPE AND OTHER TISSUES
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Mutation of plakin proteins
Abbreviations and Key Terms
SI units were used throughout the study.
DMEM Dulbecco’s Modified Eagle’s Medium
DP Desmoplakin
EB Epidermolysis Bullosa
EDTA Ethylenediaminetetraacetic acid
EVPL Envoplakin
FBS Foetal Bovine Serum
HA Haemagglutin
HRP Horseradish Peroxidase
IF Intermediate Filaments
IgG Immunoglobulin G
kDa Kilo-Daltons
PBS Phosphate Buffered Saline
PFA Paraformaldehyde
PG Plakoglobin
PPL Periplakin
PRD Plakin Repeat Domain
RPM Revolutions Per Minute
SDS PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
TTBS Tris-Tween Buffered Saline
UT Untransfected
WB Western Blot
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Mutation of plakin proteins
WT Wild Type
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Mutation of plakin proteins
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Table of Contents
1.0: Introduction. …………………………………………………………………………………………………………….. 7
1.0 Background. ……………………………………………………………………………………………………………… 7
1.2 Problem statement. …………………………………………………………………………………………………… 13
1.3 Research questions. ………………………………………………………………………………………………….. 15
1.4 Research aim. ………………………………………………………………………………………………………….. 15
1.5 Research Objectives. ………………………………………………………………………………………………… 15
1.6 Hypothesis………………………………………………………………………………………………………………. 16
1.7 Research Methodology. ……………………………………………………………………………………………. 16
1.8 Research organization. ……………………………………………………………………………………………… 16
1.9 Terminologies. ………………………………………………………………………………………………………… 16
2.1 Methodology Flowchart ……………………………………………………………………………………………. 17
2.2 Cell Culture …………………………………………………………………………………………………………….. 18
2.3 Cell Transfection ……………………………………………………………………………………………………… 19
2.3.1 Transfection Mixture …………………………………………………………………………………………….. 19
2.3.2 pcDNA Constructs ………………………………………………………………………………………………… 20
2.3.3 Transfection Protocol …………………………………………………………………………………………….. 21
2.4 Protein Gel (SDS PAGE) ………………………………………………………………………………………….. 21
2.4.1 Cell Lysis and Preparation ……………………………………………………………………………………… 21
2.4.2 SDS PAGE …………………………………………………………………………………………………………… 21
2.5 Protein Transfer, Antibody Staining, and Chemiluminescent detection (Western Blot) …….. 22
2.5.1 Antibodies ……………………………………………………………………………………………………………. 22
2.6 Pull-Down Assay …………………………………………………………………………………………………….. 23
2.6.1 Agarose Bead Equilibration ……………………………………………………………………………………. 23
2.6.2 Immunoprecipitation Process………………………………………………………………………………….. 23
2.7 Immunofluorescence Microscopy………………………………………………………………………………. 24
2.7.1 Permeabilising and Fixing ……………………………………………………………………………………… 24
2.7.2 Blocking ………………………………………………………………………………………………………………. 24
2.7.3 Immunofluorescence Antibody Incubation……………………………………………………………….. 25
2.7.4 Microscopy ………………………………………………………………………………………………………….. 25
3.1 Periplakin and Envoplakin ………………………………………………………………………………………… 26
3.1.1 Periplakin Substitution Mutation (WB Experiment 1) ……………………………………………….. 27
Mutation of plakin proteins
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3.1.2 Pull Down assay (WB Experiment 2) ………………………………………………………………………. 28
3.1.3 Periplakin Immunofluorescence ……………………………………………………………………………… 32
3.1.4 Periplakin and Envoplakin Immunofluorescence ………………………………………………………. 33
3.2 Desmoplakin Mutations ……………………………………………………………………………………………. 37
3.2.1 Desmoplakin Substitution Mutations (WB Experiment 3) ………………………………………….. 43
3.2.2 Desmoplakin PRD Deletions (WB Experiment 4) …………………………………………………….. 44
4.1 Discussion ……………………………………………………………………………………………………………… 48
4.2 Limitations ……………………………………………………………………………………………………………… 53
4.3 Further Research ……………………………………………………………………………………………………… 54
References ……………………………………………………………………………………………………………………. 55
Mutation of plakin proteins
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Abstract.
Desmosomes are highly specialized ordered adhesive intercellular links mechanically integrating
the adjacent cells. Simply, they are highly organised specialised structures located in the plasma
membrane of cells that form extracellular connections with other desmosomes, as well as
intracellular connections with cytoskeletal components, to anchor cells together.
Through mediating cytoskeletal bonds and cell-to-cell adhesion, the desmosomes automatically
integrate the cells in between the tissues and hence work to resist the mechanical stress.
Plakins are a family of proteins associated with desmosomes. The family comprises of giant
proteins that are involved in the organization and cross-linking of the adhesion complexes and
the cytoskeleton.
Mutations in the three-desmosomal family of proteins is a cause of human disease. In
consideration of the prevalence of desmosomes in the heart and skin, patients with mutations of
the desmosomes often present with phenotypes of cardio-cutaneous systems. The DSP PRD
mutation experiment showed that these deletions still provide a functional variant of the protein;
i.e. the deletion does not affect the expression of the protein.
Key words: Plakin, desmosomes, mutations, western blot.
Mutation of plakin proteins
MUTATION OF PLAKIN PROTEINS AND THEIR EFFECT ON CELLS IN THE
STRATIFIED ENVELOPE AND OTHER TISSUES.
1.0: Introduction.
1.0 Background.
Desmosomes are highly specialized ordered adhesive intercellular links mechanically
integrating the adjacent cells. This occurs by coupling of the adhesive connections arbitrated by
the desmosomal cadherins into the intermediary cytoskeletal filament complex (Kowalczyk,
2013). Desmosomal cadherins are attached to intermediate filaments using a densely clustered
cytoplasmic plaque proteins that consist of members of the armadillo gene family. The family
includes plakoglobin and plakophilins. It also contains members of the plakin family of
cytolinkers, such as desmoplakin.
Desmosomes play a critical role in the integrity of the tissues in the human body. Their
importance is always depicted or highlighted in the case of a human disease that is often caused
by mutations in desmosomal genes, autoantibody attack of desmosomal cadherins, and bacterial
toxins that categorically select and target desmosomal cadherins.
The extracellular portion of the desmosome contains the single pass transmembrane
cadherin family of proteins: Desmoglein and Desmocollin, which interact with each other
heterogeneously. These cadherins are bound intracellularly via their C-terminals to the proteins
Plakophilins and Plakoglobin, which in turn are bound to Desmoplakin (DSP). Desmoplakin is
the bridge between the desmosome and the cytoskeleton – it binds to Intermediate Filaments
(IFs), such as Keratin and Vimentin (Kowalczyk, 2013).
Through mediating cytoskeletal bonds and cell-to-cell adhesion, the desmosomes
automatically integrate the cells in between the tissues and hence work to resist the mechanical
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Mutation of plakin proteins
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stress. Therefore, the desmosome is an intercellular adhesive junction crucial to all the tissues
that undergo mechanical stress. This occurs especially to the skin, myocardium, gastrointestinal
mucosa, and the bladder (Delva, 2009). Ultrastructurally, the appearance of desmosomes is as
dense discs roughly 0.2-0.5 micrometers in diameter. At cell-cell borders, they lump together
into mirror images organisation (Kowalczyk, 2013).
Thus, the desmosomes are integrated structures that comprise of adhesion fragments,
which fasten cells together, cytoskeletal chains that disband forces, and links the molecules at the
desmosome’s cytoplasmic plaques that convey mechanical load from adhesion molecules to the
cytoskeleton’s intermediate filament (Kowalczyk, 2013).
Giulio Bizzozero, an Italian pathologist (1846-1901) initially observed the desmosome in
the spinous layer of the epidermis. His observations of nodules that were small and dense led
him to a perceptive elucidation of these structures as cell-cell adhesive contact points. Josef
Schaffer later coined the term desmosome in 1920 (Delva, 2009).
The desmosomal cadherins; desmogleins and desmocollins are transmembrane
glycoproteins representing a distinct subfamily of the cadherin superfamily of consisting of
adhesion molecules which are calcium-dependent. The desmogleins and desmocollins
extracellular domains mediate the adhesion, whereas the cadherins’ cytoplasmic tails accompany
the plaque proteins of the desmosomes (Delva, 2009).
Mutation of plakin proteins
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Plakin Proteins
Plakins proteins are a family of proteins associated with desmosomes. They are a family
of cytolinker proteins that couple elements of the cell cytoskeleton to one another as well as
junctional complexes at the cell membrane. Plakin proteins are seven in the mammalian body
and they are attributed by presence of a plakin domain with or without a plakin repeat domain.
They include; plectin, BPAG1, desmoplakin, envoplakin, periplakin, microtubule–actin
crosslinking factor 1 and epiplakin.
The plakins proteins plays an important role in the mammalian body by conserving the
integrity of the tissue especially the skin and heart that are often subjected to mechanical stress.
The family comprises of giant proteins that are involved in the organization and cross-linking of
the adhesion complexes and the cytoskeleton. These proteins further modulate biological
processes including adhesion of cells, migration, and signaling pathways or polarization
(Bouameur, 2014).
The plakins were first identified as essential structural elements of the epidermis, later
they were also found to exert some other complex functions in various tissues, including the
Mutation of plakin proteins
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central nervous system and striated muscle. They include the likes of DSP, Envoplakin (EVPL)
and Periplakin (PPL). These three plakin proteins possess similar structures: a “plakin/head”
domain, and a “rod” domain, connected with “linker” domains. Present in DSP and EVPL, but
not PPL are Plakin repeat domains (PRDs) (Delva, 2009).
Mammalian plakins have a comparable structural organization and consist of seven
members: Desmoplakin, bullous pemphigoid antigen 1 (BPAG1), Envoplakin, microtubule-actin
cross-linking factor 1, epiplakin, Periplakin and plectin (Bouameur, 2014). Spectrin repeats
contained by the plakin domain display weak conservation when compared to those existing in
the domain of spectrin repeat, signifying that the spectrin repeats in the plakin domain functions
differently biologically (Goryunov, 2016).
Desmoplakin, a highly specialized complex ensuring adhesion of cell-to-cell is an
obligate constituent of desmosomes. It serves as an anchorage site for the networks of keratin.
Mutation of plakin proteins
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Different domains forming the two isoforms of Desmoplakin, DP I and DP II. Adapted from: The Desmosome-
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2742091/
Desmosomal junctions are of particular importance within tissues such as the intercalated
discs in cardiac muscle and the epidermis, where cells are constantly being renewed. There are
four distinct layers within the epidermis, with varying levels of cell-cell adhesion. As a result,
different isoforms of desmosomal and desmosomal-associated proteins have varying levels of
expression (Delva, 2009). As keratinocytes ascend through the epidermis, they differentiate;
changing their function, morphology, and connections. Once they arrive at the stratum corneum,
they form an epidermal barrier structure – the cornified envelope
Expression of desmosomal proteins in the epidermis.
Adapted from Current Biology-Desmosomes. https://doi.org/10.1016/j.cub.2011.04.035
Plakin proteins mutations increases the tendency of cardiomyopathy, skin blistering,
muscular dystrophy and autonomic neuropathy. Plakin proteins is associated with autoimmune
skin blistering diseases bullous pemphigoid and paraneoplastic pemphigus.
Mutation of plakin proteins
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Cornified Envelope.
The cornified envelope is a structure which is sculpted underneath the plasma membrane
in terminally differentiating stratified squamous epithelia. It acts as a vital physical barrier to
these tissues in mammals It is a 10 nm thick layer of highly crosslinked insoluble proteins. In
specialized cases the epidermis which is a 5nm thick layer of ceramide lipids is covalently bound
to the proteins. The organization of extracellular lipids in to and orderly lamellae, together with
the cell envelope and extracellular lipids forms an essential and effective physical and water
barrier.
The cornified envelope also acts a barrier preventing the entry of ultraviolet radiation,
bacteria, and other xenobiotics, as well as preventing the escape of water from the epidermis.
Corneodesmosomes are present here; as they degrade, cell adhesion weakens and a process
called desquamation occurs: the shedding of epidermal cells.
The cornified envelope is a transglutaminase layer of cross-linked proteins assembled
under the keratinocytes’ plasma membrane in the epidermis outermost layer. Its major role is
contributing to the function of the epidermis as a defensive barrier between the environment and
the body. The ε-(γ-glutamyl) lysine bonds crosslinking the precursor proteins in a reaction that is
calcium-dependent catalysed by the epidermal transglutaminases makes up the envelope
(Ruhrberg, 1997). The precursors for the cornified envelope include Loricrin, Involucrin,
Proelafin, Cystatin A, SPR, Profilaggrin, Type II keratins, Desmoplakin, Envoplakin, Periplakin,
Annexin I, Cornifelin, and S100A10 and S100A11.
Involucrin was discovered and cloned as the first cornified envelope precursor. Its
expression occurs all through the suprabasal layers in the stratified epithelium, especially of the
cervix, esophagus, epidermis, cornea, conjunctiva, and trachea. Fifteen percent of the protein is
Mutation of plakin proteins
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made of glutamine and a further 20% by glutamate. The α-helical structure is the predominant
one, however, other structural ancillary features have been suggested (Eckert, 2005).
Loricrin forms the major component of the cornified envelope layer in the epidermis. Its
expression is prompted by differentiating agents that include calcium. It is detected in the
granules within the granular layer. Due to its decreased solubility, it is likely that it is
requisitioned in granules (Eckert, 2005).
1.2 Problem statement.
The literature on mutations of plakin proteins is available; however, it is still not clear on
the effects these mutations have on cells in the stratified envelope and other tissues.
Mutations in all the three-desmosomal family of proteins have been described as a cause
of human disease. In consideration of the prevalence of desmosomes in the heart and skin,
patients with mutations of the desmosomes often present with phenotypes of cardio-cutaneous
systems (Adi D.Dubash, 2011)
The conformation of plakin proteins is designed to be complementary to their conjugates.
XXX has a string of negatively charged residues, which bind to XXX. Mutating these amino
acids can change the charge, and potentially the conformation of the protein, affecting its
binding.
The cornified envelope precursor proteins mutations, especially loricrin or the
keratinocyte-specific transglutaminase that is membrane bound will result in lethal perturbation
epidermal function and differentiation (Ruhrberg, 1997). In addition, mutations of loricrin
glycine-rich domain result in the formation of nuclear localization sequences that are rich in
arginine. It is thought that mutated involucrin accumulation in the nucleus is the underlying
cause of loricrin keratoderma (Eckert, 2005).
Mutation of plakin proteins
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Clinical correlation.
Desmosomes play a role in diseases. Most of the autoimmune dermatological conditions
have the desmosomes implicated. Dermatologists who were studying a family of autoimmune
diseases did initial studies that recognized a direct role of desmosomes in the causation of
disease. The dermatologists were studying pemphigus, which is associated with blistering and
fragility of the epidermal layer. The key defining factor in pemphigus is keratinocytes adhesion
loss (acantholysis) because of antibody generation against desmogleins (Kowalczyk, 2013).
Autoantibodies (IgG) that target Dsg1 are seen in pemphigus foliaceus, leading to blistering of
the epidermal granular layer. The activity of the pemphigus disease can be conveyed via the
introduction of a patient’s IgG into mice models or cell culture. This property has given
researchers a tool to investigate the path mechanisms of the disease and the basic cellular
mechanisms regulating the desmosome (Kowalczyk, 2013).
Desmosomes are also targets for certain pathogens and proteolysis. Although epidermal
desmogleins act as autoimmune antibodies targets in skin disease, Dsg2 has recently been
recognised as a receptor for adenoviruses subclass, serotypes 3, 7, 11 and 14. These serotypes
cause urinary tract and respiratory infections (Kowalczyk, 2013).
Another key area is the association of desmosomes as inherited disease targets in the skin
and heart. This section of mutations has been well covered on the desmosomal mutations.
In different cancers, loss of expression of these desmosomal proteins especially …
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