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Apoptosis - The Art of Dying

(15 May 2013)
by

Janske Nel

on 10 June 2015

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Transcript of Apoptosis - The Art of Dying

Apoptosis - Dying to Stay Alive
Definition
“Apoptosis” is Ancient Greek; the
Process of cellular self-destruction.
Cell death plays a crucially important part in animal and plant development, and
it usually continues into adulthood.
"falling
off
of petals from a flower"
Officially
; cytologically observable changes associated with a process of cellular self-destruction observed in all eukaryotes
The idea that animal cells have a built in
death program was proposed in the 1970s - first introduced by John Kerr in 1972 (as shrinkage necrosis) BUT first description in 1842 by Carl Vogt. General acceptance of apoptosis only occured 20 years later.
In a healthy adult human, billions of cells die in the bone marrow and intestine every hour.
By 2003: 13 000 articles published
annually
Why?
Too much
Too little
OR
Neurodegenerative diseases,
Parkinson's, Alzheimer's, spinal muscular atrophy, AIDS, etc.
Cancer [viral infection or DNA mutation or Bcl-2 or p53], or autoimmune diseases [diabetes type I]
PROBLEM
Homeostasis
Tissue homeostasis for development and maintenance via differentiation, proliferation, cell death, etc.
Apop. is tightly controlled via complex networks via balance of pro-survival signals
Deregulation = pathological defects =
INSUFFICIENT APOP vs. EXCESSIVE APOP
Apop. is also there for the control of tissue size and shape, & to downsize the # of specific immune-effector cells after pathogen eradication
So, what about immortality, eh? Why don't we just
STOP
cell death and live forever?
More than one type of CD
Apoptotic CD is of major importance during organ development, as well as in regulation of the immune system & in defense response to disease stimuli (e.g. viral diseases).
It is thus interconnected with cell survival & cell proliferation.
SEVERAL FORMS
of CD may typically be introduced
within the same tissue
.

NOTE: Apoptosis is 20X faster than mitosis.
Let's get a few things straight:
is apoptosis synonymous with programme cell death?
No.
Maybe?!
I don’t know; I’m sitting on the fence until someone else thinks of the answer, then I’m going to nod and agree with them.
Under certain conditions?
All thumbs are fingers
but not all fingers are thumbs?
(PCD)?
PCD
The term apop. was introduced as term for a specific morphology of cell death, it should not be used synonymously with the term PCD
PCD occurs
via
apoptosis
PCD refers to time- and position-programmed cell death during development of an organism
Apoptosis is considered a physiological form of CD
BIG DECIDING FACTOR:
Apop. can be induced with anti-cancer drugs: cell death program initiated but w/o this treatment the cells WOULD NOT DIE, thus, not PCD.
Other types of CD?
Necroptosis
Necrosis was long considered an accidental & uncontrolled, non-programmable CD
Has been discovered that necrotic CD is sometimes controlled & programmed - e.g. when a cell for some reason or other in unable to die via apop. (e.g. low ATP-level)
BONUS POINT: Do red blood cells have mitochondria?
Necroptosis is not generally associated with caspase activation instead two partly interconnected mechanisms exist:
1. caspase-dependant classical apop. &
2. caspase-independant programmed form of CD
Partial interconnection due to caspase leading to non-caspase protease activation
Connect with intrinsic pathway & its release of cytochrome c – an apoptosis-inducing factor & endonuclease-G protein also released from mitochondria, which migrates to the nucleus and condenses chromatin
Referred to as "programmed necrosis"
Autophagy
A highly conserved pathway for bulk cellular components;
Macroautophagy
Microautophagy
Chaperone-mediated autophagy
Major catabolic pathway
Related to numerous physiological & pathological processes, including cell survival, CD, cell metabolism, development, infection, immunity & aging
Predominantly serves as cell survival mech, via its suppressive role in necrotic CD (i.e. necroptosis & PARP-1-mediated CD)
Anti-necrosis function also NB for cancer & ischaemia/reperfusion injuries
Thus, autophagic CD occurs in the absence of chromatic condensation but accompanied by massive vacuolisation of cytoplasm
Definition:
membrane system that is used to sequester organelles, proteins, etc., into a membrane-bound structure. Structure is then targeted for degradation via lysosomes.
Autophagy, is "Janus-faced"
U
sually pro-survival response although the net outcome is far from certain
Some cancer cells die when autophagic genes are inhibited, whereas some die when there is an induction of the autophagic process
It all depends on the tumour!
Autophagy may be induced in cancer cells as an adaptation mechanism: offering resistance against chemo- and radio-therapy.
Inhibiting autophagy & thereby sensitising tumour cells to apoptosis - possible treatment for cancer.
Chloroquine & hydroxyl-chloroquine increases pH – inhibits acidification of lysosomes thus inhibiting autophagy.

Also using targeted removal of autophagy related genes (ATG-5)
BONUS POINT: This is called GENE THERAPY - what other disease has shown great success [and a very public failure] with this type of treatment?
Molecular charc. of Apop
Distinct set of biochemical and physical changes
Involving the cytoplasm, nucleus and plasma membrane
Cytoplasm
ER dilates & cisternae swell to form vesicles & vacuoles
Nucleus
Chromatin condenses & aggregates into dense compact masses, fragmented internucleosomally by endonucleases = diagnostic DNA ladder (total or cystosolic) is extracted from cells & run on agarose gel. Becomes convoluted and buds off: encapsulated within the forming apoptotic bodies
Plasma membr.
Cell junctions disintegrate, PM active & convoluted = blebbing
Cell breaks up in florid manner (i.e. away), packaged cellular content via apoptotic bodies
Modifications = recognition by phagocytes usually no inflammation w/ intact membranes
Thus, early in apoptosis - cells round up, lose contact with neighbouring cells, & shrink.

Like Edgar Allen Poe; their deaths' are quiet and intriguing affairs.
Blebs – (definition) progenitors of apoptotic bodies with small, nearly spherical cytoplamsic fragments encapsulated in cell membranes.
May contain functional organelles surrounded by intact PM
Phosphatidylserine, a phospholipid embedded in the PM, is exposed on the outer side of AB & act as “eat me” signals, thus attracting macrophages
Necrosis
Morphologically very different from apoptosis
In response to chemical or physical injury
Cell swells and disintegrates in an unordered manner, eventually leading to the destruction of cellular organelles and finally the rupture of the PM and leakage of the cell content
Numerous mediators, organelles and cellular processes have been implicated but it is still unclear how they relate to each other (2013)
Inflammatory response – cytosolic constituents pouring into intercellular space
BONUS POINT: Too much apop. for phagocytes to handle = necrosis. A good example of when this occurs is?
f
a
l
l
i
n
g
Genetic regulation of Apop.
Caenorhabditis elegans
Have sequenced its complete genome.
BONUS POINT: Since you've all done Advanced Techniques - how do we sequence genomes?
C. elegans has 1090 somatic cells - 131 will die whilst 959 live & develop into defined tissues.
116 of the 131 dying cells are mostly from the nervous system.
We know the exact genes needed for PCD of each of the 131 somatic cells, what inhibits and activates, which genes determine the death or survival of a cell, the engulfment of the cells, and which genes activate proteins for DNA degradation.
PCD is evolutionary conserved.
BONUS POINT: What does that mean?
Let's go down the rabbit hole.
The family of Bcl-2 proteins
Arise from developmental stages at which selection occurs (e.g. lymphocytes) are charac. by expression of low levels of Bcl-2 and/or Bcl-xL, whereas stages of proliferation are charac. by high expression levels of Bcl-2 and/or Bcl-xL
Control mitochondrial membrane permeability
Most important members of the Bcl-2 anti-apoptotic proteins Bcl-2 and/or Bcl-xL
What does Bcl-2 stand for?
Reported to act in an antioxidant manner
Contains four so-called Bcl-2 homology domains, BH1- BH4, which are absolutely required for its survival functions
Three groups of Bcl-2 family proteins can be distinguished:
Most of these proteins contain a C-terminal membrane anchor
Contain
all four BH domains
Includes Bcl-2 and Bcl-xL
Anti-apoptotic/Pro-survival
Pro-apop. proteins
Lack some of the four Bcl-2 BH domains
Includes Bax and Bak
BH3-only proteins
As the name suggests, only contains the 3rd BH domain
An amphipathic helical structure
All pro-apoptotic
Includes Bad, Bik, Bid, Bim
Cell susceptibility to apoptosis determined by relative levels of anti- and pro-apoptotic protein levels (Rheostat hypothesis)
Many death signals converge through BH3-only proteins at the mitochondria - NOT fully understood how, but evidence indicates involvement in pore formation
Bcl-2 family proteins involved in extrinsic and intrinsic pathway
Which brings us neatly to:
CASPASES
Cysteine aspartyl-specific proteases
Induction of apoptosis = internal or external stimuli
Involved in intrinsic or mitochondrial pathway
BOTH signalling pathways converge @ the level of the specific proteases – CASPASES
To date, 14 mammalian caspases
Synthesised by pro-enzymes, which usually undergo proteolysis & activation by other caspases via cascade
Peptide caspase inhibitors can inhib. down-stream caspase activation and thus apop.
Functionally, three classes of Caspases
Charac. by long pro-domains (>90 amino acids)
Containing either Death Effector Domain (DED) or caspase recruitment domain (CARD)
Initiator caspases
DED includes: Caspase 8 (C8) or Caspase 10 (C10)
CARD includes: C2, C9 & CED-3 (our old friend the worm)
Executioner caspases
Also known as effector caspases
Contains short prodomains
Includes C3, C6 and C7
Other caspases
All the remaining caspases & have CARD
Main role is cytokine maturation rather than apoptosis
Also includes inflammatory caspases (C1, 4, 5, 11, 12, 13, & 14)
How do Caspases work?
Activated via the cleavage of the pro-domains
The large and small subunits are therefore separated via caspase action (all cleavages occur after Asp residues) => NB remember what caspase name means??
Active site is formed by the interface of the two subunits by
1 Arg, 1 His and 1 Cys of the LARGE subunit
1 Arg of the SMALL subunit
Active caspases form heterotetramers
Initiator caspases cleave & activate effector caspases
These then cleave cellular substrates, which leads to the apop. morphological phenomenon
Structural motif
Large (α) and small (ß) subunit
Each heterodimer (α/ ß) comprises of six ß strands (a –f ), of which strands a – e is parallel & f is antiparellel
This 6-stranded ß-sheet forms a twisted struc. around 5 α-helics (H1-H5)
FOUR caspase
target categories
Mediators & Regulators of apoptosis:
BID, DFF45/ICAD
(DNA fragmentation factor 45)
Structural proteins:
Fodrin, gelsolin (actin filaments), cleavage of nuclear lamins (cell shrinkage & budding), keratins & vimentin (adherence junctions)
Cellular DNA repair proteins
DNA-PK (DNA prot. kinase), RAD51, ATM, PARP
Cell cycle related prot.
CDK1, WEEI, P21CIPI, KIPI (P21), Rb
Caspase inhibitors
Several cellular and viral proteins act as inhib.
Cells contain inhibitor of apop. proteins (AIPs) which can inhib. activated caspases.
All AIPS contain one or more BIR (baculovirus IAP repeat) which enables them to bind and inactivate caspases.
Neuronal cells typically contain such proteins (neural apop. inhib. prot.; NAIP) to protect from premature apop. (Parkinson’s)
Viruses contains viral IAPs (& Bcl-2 proteins) which protect infected cells from dying
Most powerful pan-caspase inhibitor is the baculoviral p53 protein
So, all very fascinating but how do we KNOW all this?
It all comes down to the Big C.
Cancer.

Successful tumour therapeutics in cancer relies on radiation, chemotherapy, thermal therapy, and photodynamic therapy, which requires the induction of apop.
Thus, given its central role – imaging of the process is desirable
Homework for tomorrow;
Please read up on one of the following,
and report your findings on the article tomorrow:

- Reactive Oxygen Species-Triggered Trophoblast Apoptosis is Initiated by Endoplasmic Reticulum Stress via Activation of Caspase-12, CHOP & the JNK pathway in T. gondii Infection in Mice
- Baicalin Selectively Induces Apoptosis in Activated Lymphocytes & Ameliorates Concanavalin A-Induced Hepatitis in Mice
- Survivin expression correlates with clinical stage, histological grade, invasive behavior & survival rates in endometrial carcinoma
- Novel target genes responsive to the anti-growth activity of triptolide in endometrial & ovarian cancer cells
- FAK mediates the activation of cardiac fibroblasts induced by mechanical stress through regulation of the mTOR complex
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