BIO 421 Exam 2: Apoptosis

• development

• maintenance

• Cancer

Apoptosis: programmed cell death​

• Apoptosis is a highly regulated programmed cell death pathway​

• ​

• Has important roles in:​

• _____​

• tissue _____ ​

• prevention of ____​

Regulated cell death ​

Necurosis is not regulated ​

read and comprehend

Apoptosis is vital for control of cell number and growth

• Protective mechansim... however, its also important in development! ​

• Sculpts organs or gets rid of unnecessary tissue

• Webbing between toes is unregulated apoptosis

• Lots of apoptosis that happens within tissues that helps determine where cells are going to be (number of nerve cells) ​​

  ​​

Apoptosis provides a check on aberrant cell growth

• Cells that cannot apoptose have a high chance of of being cancerous ​

• DNA damage can be fixed by DNA repair mechanism... however, sometimes it does not ​

• Usually, when cells have unfixable DNA damage... they usually apoptose. However, if they do not, it can lead to cancer ​​​

• Necrotic

• Apoptosis

Extrnisic pathway and intrinsic pathway ​

Apoptosis: planned cell death

• _____ cell: it bursts; this can be dangerous to neighboring cells ​

• _____: is regulated and enzymes come in to degrade... also gets phagocytosed to facilitate removal ​​

Two pathways to induce apoptosis: _______​

procaspases

cleaved

Apoptosis is mediated by caspases​

• caspases are enzymes that cleave key intracellular proteins​

• They are ubiquitious in cells but are usually present in their inactive“procaspase” form​

• They become activated by cleavage​

Caspases: enzymes that break the peptide bonds of proteins, lipids, etc. (everything you do not want getting out!) ​

Pretty much always present in the cell... in an inactive form called _____ (have domains on the ends (amino and c terminus))​

To become active, both ends need to be _____ ​

• Intrinsic

Caspase activation​

• Now that they are cleaved, they rearrage into one caspase molecule ​

Caspase activation cascade​

• Activate one another in a cascade/chain reaction ​

• One gets activated initially... which then activates another type... which then activates another type... causing a rapidly spreading chain reaction ​

Intrinsic activation of apoptosis:

• Initial activation steps are different in extrinsic and intrinsic ​

• _____ is the more common one ​

​

read and comprehend

Mitochondria are the staging ground for the intrinsic apoptosis pathway​

• Intrinsic apoptosis begins with the release of cytochrome C from themitochondria​

• The Bcl2 family of proteins are the main intracellular apoptosis regulators​

• Bax or Bak directly promote apoptosis by releasing cytochromeC​

• Bcl2 inhibits cytochrome C release​

Bcl2 protiens get it out of the mitochondria

Bax or Bak releases it from the mitochondria ​​

Bcl2 blocks cytochrome C... regulating it ​

Cytochrome c is an electron carrier (ETC)

Cyt C is usually found in the intermembrane space ​

If it recieves a signal, then it goes through the channel ​​

read and comprehend

Release of cytochrome C regulates apoptosome formation:

• Apoptotic stimuluscauses a release of Cytochrome C through the Bax/Bak transporter. ​

• Cytosolic cytochrome c promotes the assembly of the apoptosome by activating Apaf-1. ​​

Apaf-1: usually inactive ​

Becomes activated by binding to cyt C ​

Once enough is bound, they oligomerize into a wheel structure (apoptosome) ​

​

Apoptosome activates the caspase cascade​

• Cyt C in the cytosol is a good indicactor that the cell will apoptose​

• Also formation of apoptosome is a godo indicator as well ​

Apoptosis is subject to multiple layers of regulation:

Before cyt C can be released... there are lots of layers of regulation ​

Bax and Bak ultimately regulated cyt C release ​

However, there are other protiens that regulate Bax and Bak ​

Apoptosis inhibitors can regulate them... and pro-apoptosis factors regulate the apoptosis inhibitors

Once you get to cyt C release... there is no going back! So theres a lot of regulation before then!​​

Once you get to cyt C release... there is no going back! So theres a lot of regulation before then! ​

Prevents healthy cells from dying​

You do not want to apoptose unless you absolutely have to ​

How to evolve different pathways: mechanisms involved include duplication events (since they have similar structure and function) ​

Why are there so many layers of regulation for apoptosis?​

G1-S

p53 and the apoptotic pathway:

P53 is important for ____ checkpoint ​

It can activate the checkpoint to repair DNA damage before replication ​

If its not repairable... then it can more the cell towards apoptosis

Without function p53, you can replicate damaged DNA and it will not apoptose... why it’s the most commonly mutated protein in cancers ​​​

Answer: transcription fcator

Answer: 2 and 4​

When DNA is good and happy... p53 gets ubiqutinated and degraded by proteasome​

Phosprylation blocks p53 from being degraded and activates it... it transcribes p21. P21 inhibits the cyclin-cdk complex... preventing it from moving forward into S phase​

1. P53 does not require p21... p53 transcribes p21. However, it is only expressed when the cell is damaged/stressed is true ​

3. P53 is always trasncribed and translated... its just usually being degraded normally ​

P53 also likes to transcrube apoptosis regulators (after p21). Therefore, if P53 is on for a little bit then p21 is only transcribed. If p53 is on for a while, then it can transcribe apoptosis regulators once p21 is full. ​

A TIMER FOR APOPTOSIS

What kind of protein is p53:

How is p53 activity controlled? ​​​

outside

Extrinsic activation of apoptosis:

Signal comes from ____ the cell that tells the cell to apoptose

Less common; happens more in development ​​​

• The extrinsic pathway is activated by an external signal. The external signal binds to Death Receptors found on the plasma membrane. ​

Activated when ligand binds to death receptors (when the ligand binds, it signals for cell death)

• The binding of the death receptors tothe ligand results in a clustering ofadaptor proteins. ​

• The adaptor proteins recruit a group ofcaspase proteins.​​

When the ligand and receptor binds... it clusters and adaptor proteins bind. They serve as a binding site for procaspase-8​

Once the procaspase-8 binds, the ligand can cleave procaspase-8 into its active form... and apoptosis occurs ​

EXTRINSIC ​

• Fas death ligand binds to death receptor (initating step) ​

• Structure that does cleavage: Disc ​

• What initiates: procaspase 8 ​

INTRINSIC ​

• Release of cyt C (initiating step) ​

• Structure that does cleavage: apoptosome ​

• What iniates: procaspase 9 ​

BOTH​

• Both have caspase cascade (actual effector in both cases... just different on how it gets there) ​

• Both use adaptor proteins (activated as a result of the trigger)

Most cells have apoptosis triggered through both pathways under different circumstances​

Which are shared and which are unique to each pathway?​

Answer: 2 and 3​

• Caspases are required for both ​

• Fas is required for extrinsic ​

Which of the following would be required for activation of apoptosis via the intrinsic pathway but not the extrnsic pathway? ​

read and comprehend

How were apoptosis genes identified?​

• Experiments in C.elegans first defined the apoptosis pathways​

• Apoptotic cells can be easily identified in C.elegans embryos and the same cells always undergo apoptosis(invariant lineage) ​

The same cells always apoptose (not true for all organisms... but for c. elegans its always the same amount in development) ​

C. elegans embryos are also transparent so it was clear to see which ones were about to apoptose (pointed out with arrows) ​

​

Apoptosis genes were identified in a screen​

• Randomly mutate genes (byexposing worms to chemicalmutagens)​

• Observe embryos for abnormal# of apoptotic cells​

• More apoptotic cells​

• Less apoptotic cells​​

Created random mutations and found the ones that caused an abormal amount of apoptosis ​

Answer: Forward genetics ​

• Phenotype --> gene (forward) ​

• Gene --> phenotype (reverse) ​

Is this screen an example of forward or reverse genetics?​

inhibits

promotes

Mutant genes identified:​

​

More apoptotic cells —> Gene normally ____ apoptosis​

​

Less apoptotic cells —> Gene normally ____ apoptosis​

read and comprehend

Many different signals can induce apoptosis​

DNA Damage​

Viral infection​

Growth factor depletion​

Cytoplasmic Stress​

Nutrient Starvation​

Senescence

_____: programmed cell quiescence

• Cells do not divide indefinitely​

• In the 1960s, Leonard Hayflick andPaul Moorhead found that humanfibroblasts in culture would divide amax of 50 times before they wouldstop​

• This phenomenon became known asthe Hayflick limit​

• ​

• Cells can enter a state of permanent and irreversible cell cycle arrest​

• After a certain # of cell divisions​

• Can be induced by DNA damage, stress, etc. ​

Also programmed ​

However, the cell does not die. It stops dividing ​

Features of senescent cells​:

• Cell cycle arrest;cells do not divide​

• Senescenceassociatedsecretory profile(SASP) – pro-inflammatory ​

• Resistance toapoptosis​

• Chromatinremodeling​

SASP activates the immune system ​

Becomes much harder to apoptose ​

Still have chromatin... but shifts which genes are open vs closed (euchromatin and heterchromatin) ​

Remain in this state until they die naturally ​

read and comprehend

Replicative senescence is triggered bytelomere shortening​:

• During chromosome replication, laggingstrands lose a bit of sequence every timethey are replicated​

• Telomeres are repetitive sequences found atthe ends of chromosomes, function as aprotective cap​

• When the telomeres become too short, ittriggers senescence and cells cease dividing​

• An enzyme called telomerase can add newtelomere sequence; most cells do not expressthis however​

Why they divide a specific number of times is because of telomere length

When the telomeres get too short, scenence gets triggered and they stop dividng (otherwise, the DNA would become damaged) ​

Telomerase get replenish telomeres... however, only germ cells have these (and sometimes cancer cells) ​

PROTECTIVE MECHANISM ​

Senescence is protective in the short term but can have deleterious long-term consequences​:

Different things can trigger scenesnce​

Its protective in the short term (stops tumors from growing, limits tissue damage by keeping tissue functional and not removing them, and in embryonic development) ​

However, it is harmful in the long term (has negatice consequences. Through inflammation, it can promote tumors in neighboring cells and contributes to aging) ​​

• more

• less

Senescence shares activationpathways with cell cycle arrestand apoptosis​:

• Senescence can also be triggered via p53​

• ​

• Cells will first arrest at the G1 checkpoint,and if damage is not resolved eithersenescence or apoptosis will be activated​

• ​

• Generally:​

• transient damage -> cell cycle arrest thenrecovery​

• ____ severe damage -> apoptosis​

• ___ severe damage -> senescence​

Signals that signal for apoptosis are also shared with scenscence (p53) ​

First step is cell cycle arrest (to see if problem can be solved) ​

If it cannot be resolved, then the cell makes a decision whether to do apoptosis or scenecnce ​