L5: Better phrasing for an essay Flashcards
Cell death purpose
Cell death is essential for maintaining health and normal body function. It plays a vital role in fighting infections, removing malfunctioning cells, and supporting physiological processes such as menstruation and immune system development. Apoptosis, a regulated form of cell death, is particularly important during development; it sculpts tissues and controls cell numbers. For instance, during mouse embryo development, apoptosis helps separate the fingers by clearing the cells between them. The protein PU.1, which is crucial for macrophage function, enables these immune cells to clean up the dying cells, ensuring proper tissue formation and preventing inflammation.
phosphotidylserine
During apoptosis, phosphatidylserine (PS), normally restricted to the inner leaflet of the plasma membrane, is externalised to the outer leaflet, serving as an “eat me” signal for phagocytes. This externalisation is driven by a Ca²⁺-activated scramblase that disrupts membrane asymmetry, while caspases inhibit flipase, an ATP-dependent enzyme that usually maintains PS on the inner side. The negative charge of PS attracts PS-binding proteins, aiding immune recognition. Membrane blebbing occurs due to cytoskeletal breakdown and osmotic shifts, but the plasma membrane remains sealed. The ESCRT machinery may contribute to membrane sealing during this stage, although the exact role of transglutaminase, an enzyme involved in crosslinking cytoskeletal proteins, is still debated. Nuclear changes include chromatin condensation (pyknosis) and DNA fragmentation by caspase-activated DNase (CAD), leading to the characteristic DNA ladder observed in apoptotic cells.
Phosphatidylserine externalizes during apoptosis, signaling phagocytes to engulf and digest the dying cell. ost-engulfment consequences: Postengulfmanent consequences: This also leads to the release of anti-inflammatory cytokines (TGFB, IL-10, PGE2) to prevent excessive immune activation. + cytosheletal rearrangement
‘find me’ signals released by nucleotides such as ATP or UTP released lipids such as lysophophatidylcholine or sphingosine-1-phosphate.
activation of apoptosis
Apoptosis can be triggered through two main pathways: intrinsic and extrinsic. The intrinsic pathway is activated by internal stress signals such as DNA damage, oxidative stress, or the withdrawal of growth (trophic) factors. These signals cause mitochondrial outer membrane permeabilisation and release of apoptogenic factors. In contrast, the extrinsic pathway is initiated by external signals, where immune cells detect abnormal or damaged cells and bind death ligands to their corresponding death receptors on the target cell’s surface. This direct signalling activates downstream caspases, leading to controlled cell death. Many different ways to trigger apoptosis but Final pathway and features the same. Once initiated, the process is irreversible – needs tight control.
regulatory cascade
The molecular components of apoptosis are highly conserved across species, from C. elegans to humans. These components are typically grouped into regulators, adaptors, and effectors, forming a regulatory cascade. Regulators act early to sense and control apoptotic signals, adaptors function in the middle to transmit the death signal, and effectors execute cell death, primarily through proteolytic pathways. In C. elegans, the cascade includes CED-9 (regulator), CED-4 (adaptor), and CED-3 (effector caspase), ultimately leading to apoptosis. In vertebrates, the equivalent cascade involves Bcl-2, Apaf-1, caspase-9, and caspase-3. Remarkably, the conservation is so strong that inserting the human Bcl-2 gene into worms lacking CED-9 can still suppress apoptosis, highlighting the deep evolutionary conservation of this pathway.
caspases
Caspases, the central regulators of apoptosis, are multi-domain proteins with a large and small subunit that are typically inactive as monomers. They require activation through cleavage and conformational changes during apoptosis. Caspases belong to the cysteine-aspartic acid protease family, cleaving proteins after aspartic acid residues, with cysteine in their active sites. Examples of caspase substrates include CAD, which cleaves DNA, the nuclear lamina (disrupting the nuclear membrane), and ROCK1, which induces membrane blebbing. Caspases are categorized into initiators (e.g., caspases 8, 9, 2, 10) and effectors (e.g., caspases 3, 6, 7). Initiators commit the cell to apoptosis, while effectors degrade cellular substrates.
Caspase activation during apoptosis. An initiator caspase contains a protease domain in its carboxy-terminal region and a small protein interaction domain near its amino terminus. It is initially made in an inactive, monomeric form, sometimes called procaspase. Apoptotic signals trigger the assembly of adaptor proteins carrying multiple binding sites for the caspase amino-terminal domain. Upon binding to the adaptor proteins, the initiator caspases dimerize and are thereby activated, leading to cleavage of a specific site in their protease domains. Each protease domain is then rearranged into a large and small subunit. In some cases (not shown), the adaptor-binding domain of the initiator caspase is also cleaved (see Figure 18– 5). Executioner caspases are initially formed as inactive dimers. Upon cleavage at a site in the protease domain by an initiator caspase, the executioner caspase dimer undergoes an activating conformational change. The executioner caspases then cleave a variety of key proteins, leading to the controlled death of the cell.
Bruce, Alberts, et al. Molecular Biology of the Cell, Taylor & Francis Group, 2014. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/kcl/detail.action?docID=5320520.
Created from kcl on 2025-04-29 22:21:38.
Caspase activation is irreversible, with the active caspase forming a hetero-tetramer, where two large and two small subunits form a 12-stranded beta sheet. This configuration ensures a one-way commitment to apoptosis.
Caspase targets include kinases, such as ROCK1, which, upon cleavage, induces cytoskeletal contraction and blebbing; the DNA fragmentation enzyme CAD, which is activated upon cleaving its inhibitor ICAD; and the nuclear lamina, which is disrupted by caspases to break down nuclear structure. Additionally, caspases act on the Golgi apparatus, cleaving GRASP proteins that hold the Golgi structure intact.
extrinsic pathway
The extrinsic pathway of apoptosis involves T cells and death receptors on target cells. T cells release perforin, which forms pores in the target cell membrane, allowing granzymes to enter and activate initiator caspases, thus starting the apoptotic cascade. A key interaction in the extrinsic pathway is between FasL (Fas ligand) on T cells and Fas, a death receptor on target cells. Binding of FasL to Fas trimerizes Fas, exposing its death domain, which recruits FADD (Fas-associated death domain protein) to activate caspase-8. Caspase-8 then activates effector caspases, triggering DNA fragmentation by releasing CAD from its inhibitor ICAD. This forms the Death-Induced Signaling Complex (DISC), which drives apoptosis.
Other receptors in the TNF receptor family, such as TNF, can also induce cell death or immune signaling. Binding of TNF to its receptor activates early caspases, initiating apoptosis. In some cells that lack FADD, alternate signaling molecules like TRAF-2 can be recruited, activating pathways like NF-κB, leading to immune responses rather than apoptosis. This illustrates how the TNF receptor family can either trigger apoptosis or other immune functions depending on the adaptor proteins expressed by the cell.
intrinsic pathway
Bcl-2 Family Proteins and the Regulation of Apoptosis:
The Bcl-2 family plays a pivotal role in regulating the intrinsic pathway of apoptosis by controlling mitochondrial outer membrane permeability. These proteins can either promote or prevent apoptosis. Anti-apoptotic proteins, such as Bcl-2, prevent apoptosis, offering cells resistance to death and contributing to their survival. In contrast, pro-apoptotic proteins like Bax and Bad promote apoptosis. There is a delicate balance between these opposing forces, which is often disrupted in cancer, leading to cell survival due to overexpression of pro-survival proteins and loss of pro-apoptotic proteins.
Cytochrome C Release and Apoptosome Formation:
Cytochrome c is released from the mitochondria when the intrinsic pathway is triggered. It binds to Apaf-1, leading to a conformational change in Apaf-1 that activates its ATPase activity. This allows Apaf-1 molecules to oligomerize into a heptameric complex that forms the apoptosome. The apoptosome provides a platform for the initiator caspase, caspase-9, to bind, cleave, and activate downstream effector caspases, such as caspase-3 and caspase-7. These effector caspases then cleave various cellular substrates, including ICAD, to drive the apoptosis process, leading to DNA fragmentation.
Regulation at the Mitochondrial Outer Membrane (MOM):
Apoptosis is tightly controlled at the MOM, where the pro-apoptotic Bcl-2 family members, like Bax, oligomerize and form pores. When pro-apoptotic BH3-only proteins, such as Bad or Bid, are activated, they bind to Bax, causing Bax to undergo a conformational change that allows it to insert into the MOM and oligomerize. This creates pores that increase the mitochondrial membrane’s permeability, allowing cytochrome c to leak out, initiating the intrinsic apoptotic pathway. Pro-survival Bcl-2 family proteins, such as Bcl-2, prevent Bax oligomerization, thus maintaining mitochondrial integrity and inhibiting apoptosis.
The interplay between the intrinsic and extrinsic pathways
The extrinsic pathway is typically activated by signals from outside the cell (e.g., death ligands binding to death receptors like Fas or TNF receptors).
This activation leads to the activation of caspase 8, which is an initiator caspase in the extrinsic pathway.
Caspase 8 can cleave and activate BID, a pro-apoptotic member of the Bcl-2 family.
The cleaved BID (now called tBID) then translocates to the mitochondria, where it promotes the release of cytochrome c into the cytoplasm, which is the key step in the intrinsic pathway.
The release of cytochrome c from the mitochondria activates the apoptosome and initiator caspases (like caspase 9), which then trigger the effector caspases that carry out apoptosis.
cell survival by PI-3 kinase signalling?
Bad, a pro-apoptotic BH3 only protein is sequestered by a 14-3-3 protein when phosphorylated by akt/PKB:
Without trophic factors: If trophic factors (signals promoting cell survival) are removed, pro-apoptotic proteins like BAD can promote apoptosis. BAD resides on the MOM and promotes permeability pores, which allows cytochrome c to leak out. This activates the apoptosome, which in turn activates initiator caspases like caspase 9, and these then activate effector caspases, leading to apoptosis.
With trophic factors: When trophic factors are present, they bind to their respective receptors on the cell membrane and activate a PI3-kinase (PI3K) signaling cascade. PI3K generates a lipid called PIP3 on the plasma membrane, which recruits and activates Protein Kinase B (Akt).
Akt’s role: Akt then phosphorylates BAD at a specific site. This phosphorylation prevents BAD from acting at the mitochondria.
14-3-3 proteins: After BAD is phosphorylated, it is recognized and bound by 14-3-3 proteins, which are a family of adaptor proteins in the cytosol. These 14-3-3 proteins sequester phosphorylated BAD, essentially “holding it in check” in the cytosol.
When BAD is phosphorylated by Akt, it binds to 14-3-3 proteins.
The 14-3-3 proteins sequester phosphorylated BAD in the cytosol, preventing it from interacting with the mitochondrial membrane.
When BAD is not at the mitochondria, it cannot promote pore formation in the mitochondrial membrane. As a result, cytochrome c is not released, and the intrinsic apoptotic pathway is inhibited.
So, in summary:
No trophic factors → BAD induces cytochrome c release, activating apoptosis.
With trophic factors → PI3K/Akt pathway phosphorylates BAD, sequestering it with 14-3-3, preventing apoptosis.
This is an example of post-translational regulation where the activity of BAD is controlled by its phosphorylation and sequestration, which ultimately determines whether the cell survives or undergoes apoptosis.
