Apoptosis

extrinsic and intrinsic

plasma membrane rupture due to pathological conditions or death, it can release intracellular components, triggering inflammatory responses

conserved degradation a cell uses to remove unnecessary/damaged components through lysosome mediated pathway

vascular endothelial growth factor
it triggers endothelial cell (Go) proliferation
produced by cells that lack oxygen

-to form new blood vessels needed for repair and regrowth of damaged tissue

-platelet derived growth factor
-released at site of wound
-encourages fibroblasts in connective tissue (at Go) to proliferate quickly

-multipotent
-pluirpotent
-totipotent

-have the potential to produce a limited number of differentiated offspring cells

-have the potential to produce all the cells of an embryo

-can produce all embryonic and extra-embryonic cells

-stem cells of bone marrow
-stem cells of skin
-stem cells of GI tract
-stem cells of sperm

-divides and produces one daughter cell that remains a stem cell, the other divides and differentiates

proliferation capacity of multipotent stem cells?

-less differentiated
-self renewing
-present in most adult tissues

-in distinct microenvironments (niches)

-provide environmental signals that maintain stem cells throughout life
-they help control balance between self renewal and differentiation

-can grow indefinitely as pure stem cell pop
-have pluripotency

-influenced by addition of appropriate growth factors

-elliminates unwanted cells from a variety of tissues

balances cell proliferation

programed cell death, it is an active process

accidental cell death from acute injury or programmed respone

-DNA disruption (irregular breaking)
-Cell membrane disruption or rupture
-induced inflammatory response (release of cytokines)

nucleus present in apoptotic process, due to chromatin condensation and nuclear envelope being compromised due to caspase activity

cell fragmentation

-enzymatic cleavage of DNA
-chromatin and nuclear condensation (pyknotic nuclei)
-nuclear fragmentation
-increased mitochondria permeability
-enzymatic digestion of proteins and organelles
-changes to phospholipid distribution
-cell fragmentation (blebbing)

-phagocytes recognize the phosphatidylserine (PS) in outer leaflet of apoptotic cells

-normal: PS in inner cytosolic leaflet of cell membrane
-apoptotic: PS in outer leaflet of cell membrane

-phagocytes have PS receptors

PS= phosphotidylserine
normally in inner membrane leaflet
present in outer membrane leaflet in apoptotic cells
recognized by phagocytes

-protein that has affinity for PS
-it it tagged with fluorescent label to ID apoptotic cells

flourescent label (green) that tags Annexin-V, it is used to ID apoptotic cells in lab, because Annexin-V has an affinity for PS (tells us where it is)

PI- Propidium Iodide
-stains the nucleus of compromised cells

-assess the percentage of stained cells in a population that are dead or undergoing apoptosis (by imaging each cell and interpreting intensity of the flourescence.

activates apoptosome, released by inner mitochondrial membrane (ETC)

-C. elegans (nematode)
-development includes death of 131 specific cells
-total of 959 somatic cells
-experiments based on identifying mutant strains where cell death did not occur (figure out what was essential)

both genes required for developmental cell death in C. elegans model organism

-negative regulator of apoptosis

ced-3 and ced-4 (equiv to caspases)
ced-9 (equiv to bcl-2 inhibitor in mammalian cells)

bc1-2 (identified as oncogene)

-it is a protein
-inhibits apoptosis

-too much means that cells are unable to undergo apoptosis
could lead to cancer

-similliar to mammallian cells caspases

Apaf-1

-binds to caspases (caspase 9) and promotes their activation (protease activity)

proteases
Ced-3 family of proteases in c. elegans

cysteine (C) residues at active sites
they cleave after aspartic acid residues on protein substrates

-excecutioners of programmed cell death
-they celave 100 different target proteins
-activation of initiator caspase starts a chain reaciton of caspase activations (leads to cell death)

-synthesized as inactive precursor (procaspases)
-activated by proteolytic cleavage
-caspases are catalyzed by other caspases

-various signals
-they cleave and activate effector caspases (go on to digest cellular target proteins)

-apart of ETC
-it leaves mitochondria and associates with Apaf-1 and caspase 9

-association with Apaf1 in apoptosome complex
-when cytochrome C interact with it

-Apoptotic protease activating factor 1

when activated by cytochrome C in the cytosol, it activates caspase 9 which activates caspace 3 (effector caspase) which leads to apoptosis

-initiator caspase starts a chain reaction of caspase activation, they digest cellular protein targets, this leads to cell death

-they are cleaved, converted into active forms (procaspase to caspase)

-inhibitor of DNase
-protects the nuclear DNA degradation by DNAses
-when inactivated by caspase DNA fragmentation occurs

-when ICAD is degraded
-Cad is activated and can act on nucleosomal linker regions
-yeilds 200 bp frgaments

-they degrade nuclear lamins (chromatin is no longer securly held to the nuclear envelop)
-the nucelus in destabilized, leads to nucelar envelope collapse

cytoskeleton proteins degradation: actin, myosin, a-acting, tubulin, vimentin
nuclear shrinkage and membrane blebbing visualized

-gogli is fragmented

CAD (Caspase-Activated DNase)
-A DNA-degrading enzyme, active during apoptosis.
-located in cytoplasm
-inactive, when bound to inhibitor ICAD
(when icad is degraded by executioner caspase, then cad is activated)

executionar caspase cleaves icad
icad releases from cad, now CAD is activated
-CAD now cuts in between nucleosomes (apart of apoptosis, intrinsic and extrinsic pathways)

-enable webbing to form limbs and digits

-death signal activates proapoptotic multidomain BCL-2 protein (BAX)
-BAX creates pores in outer mitochondrial membrane, leads to release of cytochrome C
-cytochrome C activates apoptosome and Caspase 9 activates pro-caspase 3 to be active caspase 3
-caspase 3 initiates apoptosis

-amount of proapoptotic and antiapoptotic Bcl-2 family members

-have about 20 related Bcl-2 proteins
-some inhibit apoptosis
-some induce caspase activation

-four domains (BH1-4)
Bcl-2
Bcl-xL

-three domains (BH1-3)
proapoptotic
Bax
Bak

just the BH3 domain
proapoptotic
Bid
Bad
Puma
Bim
Noxa

antiapoptotic (Bcl-2 and Bcl-xL)
proapoptotic multidomain (Bax and Bak)
Proapoptotic BH3 only (Bid, Bad, Puma, Bim Noxa)

Bcl2 proteins

DNA damage

-arrests the cell cycle
-activates transcription factor P53

-leads to apoptosis through expression of Bcl-2 family members (PUMA and NOXA)

BH3 only family members, work with p53

-nutrient deficiency
-hypoxia
-dna damage
-oncogenic signaling
-oxidative stress
-hormones
-physiological processes

p53's association with MDM2 inhibits it

E3 ubiquitin ligase
when it is in association with p53 transcription factor is polyubiquinates it and destroys it

ATM (it phosphorylates P53) leads to dissociation of MDM2 and p53 leading to activation of p53)

double stranded breaks in DNA

ATM phosphorylates CHK2 activating it's kinase function

-phosphorylates p53 (helps distrupt association with MDM2)
-phosphorylates CDC25A (leading to its degradation)

-phosphotase that activates cyclin dependent kinases (CDK) by removing a phosphate
impact: drives cells forward through G1/S and G2/M phases

-activates transription of p21 gene
p21 binds and inhibits CDK's (like CDK2-cyclin)

its polyubiquitinates it for destruction

-no more division, cell has been retired (remains in G1 phase)

-translocation domain (proline rich) (helps it cross membranes, get it where it needs to go)
-DNA binding domain
-tetramerization domain
-C-terminal domain

-by ER stress response (IRE1) receptor in ER detects misfolded proteins
-leads to pore formation in mitochondrial membrane by BIM
-leads to cytochrome c release, apoptosome activation, and caspase activation

-misfolded proteins
-reactive oxygen species

-Poly(ADP-ribose) polymerase 1
-nuclear enzyme
-One of the first responders to DNA single-strand breaks (SSBs)

-base excision

-when there is dna damage

caspases target Parp-1 for cleavage in apoptosis pathway (to prevent them from repairing DNA damage)

-IRE1
-PERK
-ATF-6

-misfolded protein detected by IRE1 receptor in ER membrane
-cross phosphorylation of receptor leads to cytosolic splicing of mrna (coding for specific transcription factors)
-these transcription factors go the nucleus and promote transcription of chaperone proteins (regulatory protein 1)
OR
-induction of intrinsic cellular apoptosis pathway
-leads to pore formation in mito membrane via Bim

-PERK (in ER membrane) cross phosphorylates when exposed to misfolded protein
-leads to reduced translation of mrna
-reduces amount of proteins entering ER
-initates translation of transcription factors to promote transcription of regulatory proteins

atf receptor in er that detects misfolded proteins
attached BiP (chaperone protein) detaches and binds to misfolded protein
ATF-6 goes to golgi apparatus (there it is cleaved by proteases)
>ATF6 transcription factor moves into nucleus to activate chaperon genes for the ER

-extrinsic cell death pathway

-TNF receptor found in cell membrane
-TNF binds to it
-initiates extrinsic cell death pathway

-caspase 8

-activates Bid (proapoptoic BH3-only Bcl-2 protein)
>which leads to activation of Bak and Bax (proapoptotic, multidomain Bcl-2 proteins)
extrinsic cell death pathway involving fasL, need disc formation

caspase 8 (apart of extrinsic death pathways, TNF) activates other effector caspases directly

-procaspases 3, 6, 7

-FasL

-immune cells (killer lymphocytes)
death-inducing protein expressed on the surface of killer cells such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells.

DISC= death inducing signaling complex
-found in extrinsic cell death pathway (FasL)
-includes: Fas ligand, Fas receptor, death domain, death effector protein, and caspase 8

caspase 8 in FasL extrinsic cell death pathway

activates procaspases 3, 6, and 7 (in extrinsic pathway, triggered by external death signals, death ligands bind to death receptors on the cell surface)

-unwanted proteins and organelles

-nucleation (initation of autophagic membrane) and extension
-closure
-fusion with lysosomes
-digestion of engulfed material

-Nope (caspase independent)

-increased number of lysosomes

Autosis

autophagic cell death

-hypoxia
-starvation
-authophagy-inducing peptides
-ionic stresses (hypertonic environment)

major histocopatibility complex present on the surface of cells needing to undergo granzyme mediated cell death), presents antigens to T-cells

when T-cell recognizes ligand presented by cell's MHC receptor

-T-cell releases performins poke holes in cells membrane) then granzymes are secreted into target cell
apart of granzyme mediated cell death

-necessity of caspases

-perforin makes holes in cell membrane, granzyme A is released into cell, SET complex is made, all leads to DNA fragmentation and apoptosis

-perforin makes holes in cell membrane, granzyme B is released, caspase 9 and 3 are activated, reactive oxygen species accumulate in mitochondria leading to apoptosis

-heat
-trauma
-anoxia (lack of O2)
-infection

-leads to elevated levels of reactive oxygen species and ca2+
and depletion of ATP

-lysis of cell membrane and inflammation

-Pyronecrosis
-Pyroptosis

when it needs to induce inflammatory response

Pyroptosis

No apoptosome needed
use inflammasome!

leads to release of proinflammatory cytokines (leads to inflammation)

use in pyroptosis necrosis pathway (activated by inflammasome)

-30 minutes in some

extracellular signals
(growth factors/nutrient availability)
internal signals

Cyclically activated cyclin-dependent protein kinases (Cdks)

by phosphorylation

-when cyclin binds to it forming a t loop connection

-when adk bound to cyclin is phosphorylated

cyclin levels increase and decrease throughout cell cycle
-increases during interphase
-highest in mitosis then decreases

-G1 phase

-S phase

-G2 phase

Mitosis

phosphates can do both

E

D

B

A

Cyclin E-CDK2

Cyclin A-CDK1/CDK2

-M phase

-when we transcripte cyclins (presence of the proteins)
-how the cyclins are phosphorylated
-binding of inhibitory proteins
-destruction of cylins through polyubiqitination (E3 ubiquin ligase)

-inhibitory phosphorylation
-CDK inhibitor proteins (CKIs)

-regulated proteolysis

-cdk assoication with cyclin
-activating phosphorylation of threonine on CDK
-Inhibitory phosphorylation of threonine
-Assoication with CDK inhibitors (CKI's)

-phosphorylates CDK's (inhibiting their activity)
-inhibitory phosphates

it activates cdk by removing inhibitory phosphotase

CDK inhibitor, binds to cyclin-cdk complex (inactive)

E3 ubiquitin ligase, needs activating subunit (Cdc20)

(it polyubiquinates M-cyclin/CDK complex leading to its degradation of M-cyclin in proteasome

-AP/C is activated by CdC20, enabling it to polyubiquinate M-cyclin leading to its degradation and progression from metaphase to anaphase

-proteolysis by SCF

It's a multi-protein E3 ubiquitin ligase complex, it tags specific proteins with ubiquitin so they can be degraded by the proteasome.

-cyclin D and E

-the CKI is degraded inside a proteosome

inhibitor

-coordinated via cell cycle checkpoints

-DNA damage checkpoints (G1, S, and late G2) ensures DNA damage is not replicated and passed on to daughter cells
-Spindle assembly checkpoints (late M) ensures proper spindle, need all chromosomes aligned on metapheaseal plate

Start signal

external signals (nutrient avaliablity, cell size)

-progression from G1 to S phase

-restriction point

-extracellular growth factors

-analogous to growth factor in mammalian cells

transcription regulatory protein- initiates delayed response in gene expression