Lecture 4: Cell Death Flashcards
It seems remarkably wasteful for so many cells to die, especially as the vast majority are perfectly healthy at the time they kill themselves. What purposes does this massive cell death serve?
They aid in morphology during development eg sculpting hands. Apoptosis also functions as a quality-control process in development, eliminating cells that are abnormal, misplaced, nonfunctional, or potentially dangerous to the animal.
How is apoptosis triggered?
Apoptosis is triggered by members of a family of specialised intracellular pro- teases, which cleave specific sequences in numerous proteins inside the cell, thereby bringing about the dramatic changes that lead to cell death and engulfment.
What are these proteases called and why?
These proteases have a cysteine at their active site and cleave their target proteins at specific aspartic acids; they are therefore called caspases (c for cysteine and asp for aspartic acid).
How do these caspases come about for apoptosis?
Caspases are synthesized in the cell as inactive precursors and are activated only during apoptosis.
What are the two major classes of caspases?
There are two major classes of apoptotic caspases: initiator caspases and executioner caspases.
Describe the function and procedure of initiator caspases
Initiator caspases, as their name implies, begin the apoptotic process. They normally exist as inactive, soluble monomers in the cytosol. An apoptotic signal triggers the assembly of large protein platforms that bring multiple initiator caspases together into large complexes. Within these complexes, pairs of caspases associate to form dimers, resulting in protease activation. Each caspase in the dimer then cleaves its partner at a specific site in the protease domain, which stabilises the active complex and is required for the proper function of the enzyme in the cell.
Describe the function and procedure of executioner caspases
The major function of the initiator caspases is to activate the executioner caspases. These normally exist as inactive dimers. When they are cleaved by an initiator caspase at a site in the protease domain, the active site is rearranged from an inactive to an active conformation. One initiator caspase complex can activate many executioner caspases, resulting in an amplifying proteolytic cascade. Once activated, executioner caspases catalyse the widespread protein cleavage events that kill the cell.
Various experimental approaches have led to the identification of over a thousand proteins that are cleaved by caspases during apoptosis. Only a few of these proteins have been studied in any detail. Describe some of these
These include the nuclear lamins, the cleavage of which causes the irreversible breakdown of the nuclear lamina
Another target is a protein that normally holds a DNA- degrading endonuclease in an inactive form; its cleavage frees the endonuclease to cut up the DNA in the cell nucleus
Other target proteins include components of the cytoskeleton and cell–cell adhesion proteins that attach cells to their neighbours; the cleavage of these proteins helps the apoptotic cell to round up and detach from its neighbours, making it easier for a neighbouring cell to engulf it, or, in the case of an epithelial cell, for the neighbors to extrude the apoptotic cell from the cell sheet.
How reversible is the process of apoptosis?
The caspase cascade is not only destructive and self-amplify- ing but also irreversible, so that once a cell starts out along the path to destruction, it cannot turn back.
How is the initiator caspase first activated in response to an apoptotic signal?
The two best-understood activation mechanisms in mammalian cells are called the extrinsic pathway and the intrinsic, or mitochondrial, pathway.
How is the extrinsic pathway triggered?
Extracellular signal proteins binding to cell-surface death receptors trigger the extrinsic pathway of apoptosis.
What are death receptors?
Death receptors are transmembrane proteins containing an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular death domain, which is required for the receptors to activate the apoptotic program. The receptors are homotrimers and belong to the tumor necrosis factor (TNF) receptor family, which includes a receptor for TNF itself and the Fas death receptor.
Describe a well-understood example of how death receptors trigger the extrinsic pathway of apoptosis
The activation of Fas on the surface of a target cell by Fas ligand on the surface of a killer (cytotoxic) lymphocyte. When activated by the binding of Fas ligand, the death domains on the cytosolic tails of the Fas death receptors bind intracellular adaptor proteins, which in turn bind initiator caspases (primarily caspase-8), forming a death-inducing signaling complex (DISC).
Once dimerized and activated in the DISC, the initiator caspases cleave their partners and then activate downstream executioner caspases to induce apoptosis. In some cells, the extrinsic pathway recruits the intrinsic apoptotic path- way to amplify the caspase cascade and kill the cell.
How may the extrinsic pathway be inhibited?
Many cells produce inhibitory proteins that act to restrain the extrinsic pathway. For example, some cells produce the protein FLIP, which resembles an initiator caspase but has no protease activity because it lacks the key cysteine in its active site. FLIP dimerizes with caspase-8 in the DISC; although caspase-8 appears to be active in these heterodimers, it is not cleaved at the site required for its stable activation, and the apoptotic signal is blocked. Such inhibitory mechanisms help prevent the inappropriate activation of the extrinsic pathway of apoptosis.
When might the intrinsic pathway be activated?
Cells can also activate their apoptosis program from inside the cell, often in response to stresses, such as DNA damage, or in response to developmental signals.
What does this intrinsic patwhay of apoptosis depend on?
The release into the cytosol of mitochondrial proteins that normally reside in the intermembrane space of these organelles. Some of the released proteins activate a caspase proteolytic cascade in the cytoplasm, leading to apoptosis.
Describe a key protein in this intrinsic pathway, its normal function and its role in the intrinsic apoptosis pathway
A key protein in the intrinsic pathway is cytochrome c, a water-soluble component of the mitochondrial electron-transport chain. When released into the cytosol, it takes on a new function: it binds to an adaptor protein called Apaf1, causing the Apaf1 to oligomerize into a wheel-like heptamer called an apoptosome.
The Apaf1 proteins in the apoptosome then recruit initiator caspase-9 proteins, which are thought to be activated by proximity in the apoptosome, just as caspase-8 is activated in the DISC. The activated caspase-9 molecules then activate downstream executioner caspases to induce apoptosis
The intrinsic pathway of apoptosis is tightly regulated to ensure that cells kill themselves only when it is appropriate. What proteins help to regulate this?
A major class of intracellular regulators of the intrinsic pathway is the Bcl2 family of proteins, which, like the caspase family, has been conserved in evolution from worms to humans
What domains are contained in the bcl2 proteins?
The anti-apoptotic Bcl2 family proteins, including Bcl2 itself (the founding member of the Bcl2 family) and BclXL, share four distinc- tive Bcl2 homology (BH) domains (BH1–4). The pro-apoptotic Bcl2 family proteins consist of two subfamilies—the effector Bcl2 family proteins and the BH3-only pro- teins. The main effector proteins are Bax and Bak, which are structurally similar to Bcl2 but lack the BH4 domain. The BH3-only proteins share sequence homology with Bcl2 in only the BH3 domain.
What happens to the the pro-apoptotic effector Bcl2 family proteins in the case of apoptosis?
When an apoptotic stimulus triggers the intrinsic pathway, the pro-apoptotic effector Bcl2 family proteins become activated and aggregate to form oligomers in the mitochondrial outer membrane, inducing the release of cytochrome c and other intermembrane proteins by an unknown mechanism
What are the two best known pro-apoptotic effector Bcl2 family proteins and how essential are they?
In mammalian cells, Bax and Bak are the main effector Bcl2 family proteins, and at least one of them is required for the intrinsic pathway of apoptosis to operate: mutant mouse cells that lack both proteins are resistant to all pro-apoptotic signals that normally activate this pathway.
Where are bax and bak located when inactive?
Whereas Bak is bound to the mitochondrial outer membrane even in the absence of an apoptotic signal, Bax is mainly located in the cytosol and translocates to the mitochondria only after an apop- totic signal activates it. As we discuss below, the activation of Bax and Bak usually depends on activated pro-apoptotic BH3-only proteins.
Where are the anti-apoptotic Bcl2 family proteins located in an inactive state?
The anti-apoptotic Bcl2 family proteins such as Bcl2 itself and BclXL are also located on the cytosolic surface of the outer mitochondrial membrane, where they help prevent inappropriate release of intermembrane proteins
How do the Bcl2 proteins inhibit apoptosis?
The anti- apoptotic Bcl2 family proteins inhibit apoptosis mainly by binding to and inhibiting pro-apoptotic Bcl2 family proteins—either on the mitochondrial membrane or in the cytosol.
Give an example of how they inhibit bak
On the outer mitochondrial membrane, for example, they bind to Bak and prevent it from oligomerizing, thereby inhibiting the release of cyto- chrome c and other intermembrane proteins. There are at least five mammalian anti-apoptotic Bcl2 family proteins, and every mammalian cell requires at least one to survive.
