Lecture 10 - Introduction to Apoptosis (2) Flashcards
Apaf-1 Apoptosome
Cytochrome c activates an adaptor protein, known as apoptotic protease-activating factor-1 (Apaf-1). What is the structure of Apaf-1?
+ It is a multi-domain adapter protein containing an N-terminal caspase recruitment domain (CARD),
+ followed by a nucleotide-binding and oligomerization domain (NOD, also known as NB-ARC), and
+ a C-terminal regulatory Y-domain composed of 12—13 WD40 repeats (WDRs), which form seven- and six-blade beta-propellers.
Apaf-1 Apoptosome
How does cytochrome c affect Apaf-1?
+ Apaf-1 is normally present as a monomer in an inactive, locked conformation bound to dATP or ATP [(d)ATP]
+ Upon cytochrome c binding to its WDRs (most probably between its beta-propellers)
Apaf-1 is thought to undergo a conformational change, driven by (d)ATP hydrolysis.
Results in a semi-open conformation, where Apaf-1 is susceptible to unproductive aggregation.
But it normally undergoes nucleotide exchange, accelerated in vitro by a complex composed of Hsp70, the tumor suppressor PHAPI and cellular apoptosis susceptibility (CAS) protein.
Apaf-1 Apoptosome
What happens to the inactive Apaf-1 aggregates after nucleotide exchange?
Following nucleotide exchange, the CARD and ancillary helical domains Apaf-1 undergo additional conformational changes
- to expose its AAA+ ATPase domain allowing multiple Apaf-1 proteins to oligomerize into a circular heptameric apoptosome complex
- on top of NOD domains that also have oligomerized.
This forms what is a wheel-like particle in which seven bent spokes radiate outward from the central hub and procaspase-9 enzymes sit atop the complex.
How is procaspase-9 activated?
Apoptosome sequentially recruits and activates procaspase-9.
The prodomain in procaspase-9, in particular, contains a CARD that allows it to bind selectively to the CARD in the Apaf-l of the apoptosome.
The apoptosome serves primarily as a platform to either:
+ cause a conformational change and activation of procaspase-9 or
+ increase the local concentration of procaspase-9 and facilitate its dimer-driven activation.
Note that procaspase-9 is activated by both extrinsic and intrinsic stimuli.
What happens after the activation of initiator procaspase-9?
+ The apoptosome sequentially recruits and activates the initiator procaspase-9 then the effector procaspases-3 and -7.
+ Cleavage of procaspase-3 to to caspase-3
What is an example of a caspase-3 target?
Targets of caspase-3 include DNA fragementation factor (DFF)
made up of caspase-activated DNase
(CAD) and inhibitor of CAD(ICAD)
when caspase-3 is active, it cleaves off ICAD, releasing CAD as an active DNase.
+ CAD triggers high relative molecular mass DNA cleavage and results in oligonucleosomal DNA ladders.
What is the role of XIAP in apoptosis?
XIAP - X-Iinked inhibitor of apoptosis
member of IAP family of proteins
binds to caspase 9 (cleaved procaspase 9 due to autocatalysis by procaspase 9)
XIAP forms a complex with caspase-9 that prevents the caspase 9 from undergoing dimerization
+ Procaspase-9 autocatalysis - the idea is that the cleaved form caspase-9 is less active than procaspase-9 and procaspase-9 has a neo-epitope that is exposed upon cleavage - this exposed epitope is what binds to XIAP.
How is procaspase-9 regulated?
Other regulators of Procaspase-9 - complexity
some of these regulators act on procaspase-9 directly
+ e.g.Thr 125 is located in the hinge region near the N-terminus of the large subunit and thus might inhibit prodomain exposure and caspase- 9 recruitment to the apoptosome
+ But this has not been formally tested
others act via Apaf-1
How is Bcl-2 related to cancers?
BCL-2 was discovered owing to its frequent translocation and overexpression in B cell lymphomas, where BCL-2 is frequently grossly overexpressed.
BCL-2 is pro-survival so this could mean increased survival of mutant cells.
Note that normally there is high apoptotic sensitivity in peripheral resting B and T lymphocytes, which are extremely primed for apoptosis due to expression of high levels of pro-apoptotic molecules.
But when there are mutations, including those leading to increased cellular proliferation, and transformation, the increased pro-survival Bcl-2 proteins could potentially lead to the establishment of pre-malignant cells.
How may BH3 mimetics be used against cancers?
Modulating cell death in the treatment of many human diseases, especially cancers.
There is recent development of novel small-molecule inhibitors of pro-survival proteins from the BCL-2 family, called BH3 mimetics.
For the direct and selective activation of mitochondrial apoptosis in cells that are highly dependent on one or more pro-survival proteins.
A number of tumour cells are ‘primed to die’, that is, more sensitive than their normal counterparts. Why?
The basis of priming appears to be that the mutations and stresses suffered by a cell en route to malignancy upregulate BH3-only proteins such as BIM.
This imposes a selective pressure for elevated levels of pro-survival proteins.
+ Consequently, prosurvival proteins loaded with potent BH3-only proteins like BIM put the cells in many tumours on the brink of apoptosis.
+ This explains why many tumours with elevated levels of a pro-survival protein are actually sensitive to cytotoxic therapies, including BH3 mimetics.
How do BH3 mimetics work?
These agents work by inhibiting the activity of pro-survival proteins
+ resulting in the freeing of pro-apoptotic protein that is actively being sequestered.
+ The pro-apoptotic protein is then able to carry out its primary function in activating apoptosis
