cell biology 5 Flashcards

Question

cytochrome C role in apoptosis

Answer 1

Cytochrome c is also an intermediate in apoptosis, a controlled form of cell death used to kill cells in the process of development or in response to infection or DNA damage. Cytochrome c binds to cardiolipin in the inner mitochondrial membrane, thus anchoring its presence and keeping it from releasing out of the mitochondria and initiating apoptosis. While the initial attraction between cardiolipin and cytochrome c is electrostatic due to the extreme positive charge on cytochrome c, the final interaction is hydrophobic, where a hydrophobic tail from cardiolipin inserts itself into the hydrophobic portion of cytochrome c. During the early phase of apoptosis, mitochondrial ROS production is stimulated, and cardiolipin is oxidized by a peroxidase function of the cardiolipin–cytochrome c complex. The hemoprotein is then detached from the mitochondrial inner membrane and can be extruded into the soluble cytoplasm through pores in the outer membrane. The sustained elevation in calcium levels precedes cyt c release from the mitochondria. The release of small amounts of cyt c leads to an interaction with the IP3 receptor (IP3R) on the endoplasmic reticulum (ER), causing ER calcium release. The overall increase in calcium triggers a massive release of cyt c, which then acts in the positive feedback loop to maintain ER calcium release through the IP3Rs. This explains how the ER calcium release can reach cytotoxic levels. This release of cytochrome c in turn activates caspase 9, a cysteine protease. Caspase 9 can then go on to activate caspase 3 and caspase 7, which are responsible for destroying the cell from within.

Answer 2

Caspases are essential in cells for apoptosis, or programmed cell death, in development and most other stages of adult life, and have been termed "executioner" proteins for their roles in the cell. Some caspases are also required in the immune system for the maturation of lymphocytes. Failure of apoptosis is one of the main contributions to tumour development and autoimmune diseases; this, coupled with the unwanted apoptosis that occurs with ischemia or Alzheimer's disease, has stimulated interest in caspases as potential therapeutic targets since they were discovered in the mid-1990s.

Answer 3

involves perturbation of mitochondrial outer membrane function, either spontaneously or following withdrawal of growth factors or some other physiological or pathological signal. Normally the mitochondrial membrane is guarded by “anti-apoptotic” members of the Bcl-2 protein family, Bcl-2 and Bcl- XL. When the cell receives the suicide signal, “pro-apoptotic” members of the family such as Bim and PUMA are made; they move to the mitochondrion and replace Bcl-2 and Bcl-XL. This allows other members of the same family, Bax and Bax, to act on the membrane, making it permeable so it releases cytochrome C into the cytoplasm. That activates a cytoplasmic protein called Apaf-1. Finally, activated Apaf-1 activates the protease caspase-9, and it activates caspase-3, that eventually result in the classic appearance of apoptosis. caspase-9 is a signal caspase, caspase-3 an executioner. The details here are not for memorizing, though the signaling philosophy—death controlled by pro- and anti-apoptotic Bcl-2 factors—is very important.

Answer 4

Cytotoxic (killer) T cells (CTL) are responsible for surveillance of the surfaces of all body cells. If a cytotoxic T cell recognizes that a cell is mutated or infected, it instructs the target cell to undergo apoptosis. Unneeded or undesirable lymphocytes are also eliminated by this mechanism. Different CTL seem to use one of two mechanisms to do this work. In one, the CTL upregulates expression of a surface molecule called Fas (or CD95) ligand (FasL, CD95L), which then engages and cross-links a corresponding molecule on the abnormal cell’s surface, Fas or CD95. CD95 transduces a signal into the cell’s interior, which recruits an adaptor molecule called FADD, which activates caspase-8. Like caspase-9 in the intrinsic pathway, caspase-8 then activates caspase-3. So the upstream process is different from that in the intrinsic pathway, but the downstream results are the same.

Answer 5

Fas forms the death-inducing signaling complex (DISC) upon ligand binding. Membrane-anchored Fas ligand trimer on the surface of an adjacent cell causes oligomerization of Fas. Recent studies which suggested the trimerization of Fas could not be validated. Other models suggested the oligomerization up to 5-7 Fas molecules in the DISC.[6] This event is also mimicked by binding of an agonistic Fas antibody, though some evidence suggests that the apoptotic signal induced by the antibody is unreliable in the study of Fas signaling. To this end, several clever ways of trimerizing the antibody for in vitro research have been employed. Upon ensuing death domain (DD) aggregation, the receptor complex is internalized via the cellular endosomal machinery. This allows the adaptor molecule FADD to bind the death domain of Fas through its own death domain.

Answer 6

an adaptor protein that bridges members of the tumor necrosis factor receptor superfamily, such as the Fas-receptor, to procaspases 8 and 10 to form the death-inducing signaling complex (DISC) during apoptosis. FADD also contains a death effector domain (DED) near its amino terminus,[8] which facilitates binding to the DED of caspase-8. Active caspase-8 is then released from the DISC into the cytosol, where it cleaves other effector caspases, eventually leading to DNA degradation, membrane blebbing, and other hallmarks of apoptosis.

Answer 7

It is expressed on cytotoxic T lymphocytes. Fas forms the death-inducing signaling complex (DISC) upon ligand binding. Membrane-anchored Fas ligand trimer on the surface of an adjacent cell causes trimerization of Fas receptor. Upon ensuing death domain (DD) aggregation, the receptor complex is internalized via the cellular endosomal machinery. This allows the adaptor molecule Fas-associated death domain (FADD) to bind the death domain of Fas through its own death domain. FADD also contains a death effector domain (DED) near its amino terminus, which facilitates binding to the DED of caspase-8. Active caspase-8 is then released from the DISC into the cytosol, where it cleaves other effector caspases, eventually leading to DNA degradation, membrane blebbing, and other hallmarks of apoptosis.

Answer 8

Recently, children with the condition named autoimmune lymphoproliferative syndrome (ALPS) have been identified. Their enormous lymphadenopathy suggests lymphoma or Hodgkin disease, although the pathogenesis is a failure of cells to die rather than uncontrolled proliferation; they have mutations in either Fas or FasL. Again it is worth considering that in every cell compartment of the adult at steady state, a cell must die for each one that divides. If proliferation exceeds death, the compartment grows; and this can happen because cells are dividing too fast, or not dying fast enough. This is a novel way of looking at malignancy.

Answer 9

There is a protein related to caspase-8, called FLIP, which is however proteolytically-inactive. It competes with caspase-8 for binding to FADD, and thus inhibits apoptosis signaling (Fig. 4). Amazingly, there are viral FLIPs (v-FLIPs), known from herpes viruses such as HHV-8, the Kaposi’s sarcoma virus. Clever pathogens will develop (or, in the case of viruses, steal) anti- apoptotic genes to keep the cell alive until they can finish their replicative cycle. The cell becomes, for practical purposes, a zombie.

Answer 10

a T lymphocyte (a type of white blood cell) that kills cancer cells, cells that are infected (particularly with viruses), or cells that are damaged in other ways. When exposed to infected/dysfunctional somatic cells, TC cells release the cytotoxins perforin, granzymes, and granulysin. Through the action of perforin, granzymes enter the cytoplasm of the target cell and their serine protease function triggers the caspase cascade, which is a series of cysteine proteases that eventually lead to apoptosis (programmed cell death). A second way to induce apoptosis is via cell-surface interaction between the TC and the infected cell. When a TC is activated it starts to express the surface protein FAS ligand (FasL)(Apo1L)(CD95L), which can bind to Fas (Apo1)(CD95) molecules expressed on the target cell. However, this Fas-Fas ligand interaction is thought to be more important to the disposal of unwanted T lymphocytes during their development or to the lytic activity of certain TH cells than it is to the cytolytic activity of TC effector cells. Engagement of Fas with FasL allows for recruitment of the death-induced signaling complex (DISC). The Fas-associated death domain (FADD) translocates with the DISC, allowing recruitment of procaspases 8 and 10. These caspases then activate the effector caspases 3, 6, and 7, leading to cleavage of death substrates such as lamin A, lamin B1, lamin B2, PARP (poly ADP ribose polymerase), and DNAPK (DNA-activated protein kinase). The final result is apoptosis of the cell that expressed Fas.

Answer 11

The nastiest E. coli bacteria induce terrible diarrhea by changing the physiology of colonic epithelial cells. To grow effectively the bacteria must keep the epithelial cells alive. They do so by making an enzyme that specifically glycosylates FADD, making it unable to activate caspase 8 and thus blocking apoptosis.

Answer 12

As we noted, lymphocytes are more sensitive to radiation than any other cell. Why are lymphocytes so sensitive? It may be because they are so dangerous. A very minor change in the environment—the binding of antigen to the cell’s receptor—can drive a lymphocyte into rapid cycle, so that the one cell can become 64,000 cells within 4 days. If such a cell were to be damaged, perhaps mutated, the error would rapidly be locked into a substantial clone. This poses a risk of autoimmunity, or even lymphoma. So it seems reasonable that a damaged lymphocyte would respond not by repair, but by committing suicide. This is biologically sound, since the sole function of the body is to preserve and perpetuate gametes, and any single somatic cell may be sacrificed, if it presents a possible risk to the community of cells. We call this the “better dead than wrong” rule. Cells that are less risky, like fibroblasts, have the leisure to repair much more severe damage. So there is a continuum of response to injury: first, repair; if repair is impossible or unwise, apoptosis; if the damage is overwhelming, necrosis. For different cell types, the crossover to the next response will occur at different levels of damage. This could explain, for example, why certain toxins and chemicals are more harmful to specific tissues or cell types. At a more subtle level, if a population of cells were relatively resistant to apoptosis they might under some circumstances be more susceptible to malignant transformation—by their survival they would lock in mutations.

Answer 13

There are two major routes of endocytosis: (1) phagocytosis and (2) pinocytosis or small vesicle formation.

Answer 14

Phagocytosis in multicellular organisms is normally carried out by specialized cells in the blood, e.g., macrophages and neutrophils. These cells recognize foreign organisms like bacteria, engulf them, and deliver them to lysosomes for degradation. Macrophages and neutrophils also recognize apoptotic cells (one signal is negatively charged phosphatidylserine that moves from the inner to the outer leaflet) and aged cells (roughly 1011 of our red blood cells are phagocytosed every day).

Answer 15

Pinocytosis of vesicles involves small volumes, and usually is associated with specific uptake of ligands and receptors. Vesicles are typically formed by two mechanisms, either clathrin coat proteins or caveolae. Cargo molecules bind to a transmembrane receptor, which has a short motif on the cytoplasmic domain that is recognized and binds to an adaptor protein. An adaptor complex of proteins forms to enable a clathrin coat to assemble on a vesicle budding from the plasma membrane (or from the Golgi membrane in the secretory pathway). The vesicle remains attached to the membrane until dynamin pinches it off. The adaptor complex and clathrin rapidly dissociate from the endocytosed vesicle.

Answer 16

cholesterol uptake is dependent on the LDL receptor. Before examining the details of the pathway, note the general scheme: LDLR is reutilized - it cycles between the surface and the lysosome and brings LDL particles to the lysosome, where they get degraded. LDLRs get clustered in membrane pits because an adaptor protein complex AP2 binds the receptor and also binds clathrin. Clathrin assembles over the surface of the nascent vesicle, dynamin pinches off the neck of membrane, creating the clathrin-coated vesicle. Shortly thereafter the clathrin coat disassembles, and this vesicle (the early endosome) moves to the next compartment (late endosome) in which the pH is low. The early endosome fuses with the late endosome and exposes the LDL particle to the lysosomal lumen, which is acidic. LDL dissociates from its receptor in the low pH. The LDLR is recycled to the plasma membrane and the LDL is broken down into cholesterol, fatty acids, and amino acids. The lifetime of an LDLR is about 20 hours and it goes through the cycle shown in the figure about once every ten minutes.

Answer 17

Caveolae (little cavities) are small endocytic vesicles that form without coat proteins. They are found in most cells and are thought to be especially important for membrane domains known as lipid rafts (regions high in cholesterol and signaling molecules). Some animal viruses and cholera toxin enter cells specifically through caveolae. Caveolin is the structural protein that is required for caveolae formation. Each vesicle contains 144 caveolins. Caveolin is also a scaffolding protein for coordinating protein complexes (see cartoon to right). There are three caveolin genes in humans (caveolin-1, -2, and -3). Caveolin-3 is expressed in skeletal and cardiac muscle; mutations in this gene cause Limb Girdle disease and Rippling Muscle disease.

Answer 18

an autosomal class of muscular dystrophy that is similar but distinct from Duchenne muscular dystrophy and Becker's muscular dystrophy. Limb-girdle muscular dystrophy encompasses a large number of rare disorders. Currently, there is no cure and the disease inevitably worsens over time. LGMD is typically an inherited disorder, though it may be inherited as a dominant or recessive genetic defect. The result of the defect is that the muscles cannot properly form certain proteins needed for normal muscle function. Several different proteins can be affected, and the specific protein that is absent or defective identifies the specific type of muscular dystrophy. Among the proteins affected in LGMD are α, β, γ and δ sarcoglycans. The sarcoglycanopathies could be possibly amenable to gene therapy.

Answer 19

Rippling muscle disease is a condition in which the muscles are unusually sensitive to movement or pressure (irritable). The muscles near the center of the body (proximal muscles) are most affected, especially the thighs. In most people with this condition, stretching the muscle causes visible ripples to spread across the muscle, lasting 5 to 20 seconds. A bump or other sudden impact on the muscle causes it to bunch up (percussion-induced muscle mounding) or exhibit repetitive tensing (percussion-induced rapid contraction). The rapid contractions can continue for up to 30 seconds and may be painful. Rippling muscle disease can be caused by mutations in the CAV3 gene. Muscle conditions caused by CAV3 gene mutations are called caveolinopathies. The CAV3 gene provides instructions for making a protein called caveolin-3, which is found in the membrane surrounding muscle cells. This protein is the main component of caveolae, which are small pouches in the muscle cell membrane. Within the caveolae, the caveolin-3 protein acts as a scaffold to organize other molecules that are important for cell signaling and maintenance of the cell structure. It may also help regulate calcium levels in muscle cells, which play a role in controlling muscle contraction and relaxation.

Answer 20

Viruses and bacteria are two environmental problems for humans that illustrate the usefulness of knowing different ways that cells take up materials from the extracellular space. Multiple portals exist for virus and bacterial entry into mammalian cells. Some examples are (1) Clathrin-mediated entry (e.g. vesicular stomatitis virus). (2) Fusion-entry (e.g. HIV). (3) Macropinocytosis-mediated entry (e.g. vaccinia virus). (4) Phagocytosis-like-mediated entry (e.g. herpes simplex virus). (5) Phagocytosis-mediated entry (e.g. bacteria). (6) Caveolin-mediated entry (e.g. simian virus 40).

Answer 21

in the steady state, production and elimination are equal. All proteins have a characteristic lifetime, which for some proteins is minutes and for others can be days and weeks (even a lifetime for the lens of the eye). Moreover, even organelles have lifetimes and are being degraded and regenerated. Thus, protein and organelle degradation is vital for all cells. Two more examples emphasize the importance of degradation. A large fraction of proteins synthesized in the ER do not get properly folded and must be degraded to keep the cell healthy. Endocytosis brings both good and bad molecules and microbes into the cell and both need to be degraded (to utilize some components and destroy others).

Answer 22

an oligomeric complex made up of six protein subunits: a single copy of the A subunit (part A), and five copies of the B subunit (part B), connected by a disulfide bond. The five B subunits form a five-membered ring that binds to GM1 gangliosides on the surface of the intestinal epithelium cells. The A1 portion of the A subunit is an enzyme that ADP-ribosylates G proteins, while the A2 chain fits into the central pore of the B subunit ring. Upon binding, the complex is taken into the cell via receptor-mediated endocytosis. Once inside the cell, the disulfide bond is reduced, and the A1 subunit is freed to bind with a human partner protein called ADP-ribosylation factor 6 (Arf6).[13] Binding exposes its active site, allowing it to permanently ribosylate the Gs alpha subunit of the heterotrimeric G protein. This results in constitutive cAMP production, which in turn leads to secretion of H2O, Na+, K+, Cl−, and HCO3− into the lumen of the small intestine and rapid dehydration.

Answer 23

The cystic fibrosis genetic mutation in humans has been said to maintain a selective advantage: heterozygous carriers of the mutation (who are thus not affected by cystic fibrosis) are more resistant to V. cholerae infections. In this model, the genetic deficiency in the cystic fibrosis transmembrane conductance regulator channel proteins interferes with bacteria binding to the gastrointestinal epithelium, thus reducing the effects of an infection. The Cystic Fibrosis mouse model. Mice homozygous for the CFTR mutation (CF mouse) develop intestinal obstruction/ Cholera toxin cannot activate CFTR. Mice heterozygous for the CFTR mutation are protected from Cholera. They secrete ½ the amount of fluid compared to normal mice when infected with Cholera. However, CFTR mutations are less common in areas where cholera is endemic. Cholera affect Europe in the 19th century (too young)

Answer 24

Small molecule inhibitors for CFTR. Closed loop model of small intestine: Cholera toxin induces fluid secretion, CFTR inhibitor prevents fluid secretion. Potential therapeutic strategy for cholera and other secretory diarrhea conditions.

Answer 25

Inhibition of cAMP-activated intestinal chloride secretion. Regulation of tight junctions to decrease intestinal permeability

Answer 26

a deep and labored breathing pattern often associated with severe metabolic acidosis, particularly diabetic ketoacidosis (DKA) but also renal failure. It is a form of hyperventilation, which is any breathing pattern that reduces carbon dioxide in the blood due to increased rate or depth of respiration. In metabolic acidosis, breathing is first rapid and shallow but as acidosis worsens, breathing gradually becomes deep, labored and gasping. It is this latter type of breathing pattern that is referred to as Kussmaul breathing.

Answer 27

Three major protein degradation pathways exist in eukaryotic cells: the ubiquitin-proteasome system (UPS), the lysosome (mentioned above as part of the endocytosis pathway), and autophagy.

Answer 28

The UPS is responsible for the rapid degradation of proteins when fast adaptation is needed, and UPS protein makes up about 1% of the protein in a cell. Autophagy is mainly involved in the degradation of long-lived proteins and entire organelles; it plays an important role during development and is required for the adaptation to environmental stresses such as starvation.

Answer 29

Proteins with improper folding not only don’t work, but they also usually have hydrophobic domains now exposed to cytosol. These exposed hydrophobic domains often bind to hydrophobic domains of other misfolded proteins and form large aggregates inside the cell. This can be lethal to the cell. The cell makes many different proteins to help the folding, and these are generically called chaperone proteins. The two best examples of these are hsp70 and hsp60 (hsp stands for heat shock protein – these proteins are made in high abundance when cells are heat shocked).

Answer 30

helps fold a protein by binding to exposed hydrophobic patches in incompletely folded proteins and prevents aggregation. With the use of ATP, it will cause the protein to expand and relaxes to help the protein fin the correct folding.

Answer 31

forms an elaborate, large, barrel-shaped structure that acts as an isolation chamber. Misfolded proteins are fed into the chamber to prevent aggregation and to help it to refold. There are binding sites within the compartment that, with the use of ATP, helps proteins fold correctly.

Answer 32

LDL receptor complexes are present in clathrin-coated pits (or buds) on the cell surface, which when bound to LDL-cholesterol via adaptin, are pinched off to form clathrin-coated vesicles inside the cell. This allows LDL-cholesterol to be bound and internalized in a process known as endocytosis and prevents the LDL just diffusing around the membrane surface. This occurs in all nucleated cells (not erythrocytes), but mainly in the liver which removes ~70% of LDL from the circulation. Once the coated vesicle is internalized it will shed its clathrin coat and will fuse with an acidic late endosome. The change in pH causes a conformational change in the receptor that releases the bound LDL particle. The receptors are then either destroyed or they can be recycled via the endocytic cycle back to the surface of the cell where the neutral pH will cause the receptor to revert to its native conformation ready to receive another LDL particle. Synthesis of receptors in the cell is regulated by the level of free intracellular cholesterol; if it is in excess for the needs of the cell then the transcription of the receptor gene will be inhibited. LDL receptors are translated by ribosomes on the endoplasmic reticulum and are modified by the Golgi apparatus before travelling in vesicles to the cell surface.

Answer 33

a carrier protein for transferrin. It is needed for the import of iron into the cell and is regulated in response to intracellular iron concentration. It imports iron by internalizing the transferrin-iron complex through receptor-mediated endocytosis. Low iron concentrations promote increased levels of transferrin receptor, to increase iron intake into the cell. Thus, transferrin receptor maintains cellular iron homeostasis. TfR production in the cell is regulated according to iron levels by iron response/regulatory element binding protein (IRE-BP), also referred to as Iron Regulatory Protein (IRP). This protein binds to the hairpin like structure (IRE) that is in the 3' UTR of the TfR receptor. Once binding occurs, degradation of mRNA of IRE is inhibited.

Answer 34

Vesicles pass between the Golgi and endosomes in both directions. The GGAs and AP-1 clathrin-coated vesicle adaptors make vesicles at the Golgi that carry molecules to endosomes. In the opposite direction, retromer generates vesicles at early endosomes that carry molecules back to the Golgi.

Answer 35

Molecules are delivered from the plasma membrane to early endosomes in endocytic vesicles. Molecules can be internalized via receptor-mediated endocytosis in clathrin-coated vesicles. Other types of vesicles also form at the plasma membrane for this pathway, including ones utilising caveolin. Vesicles also transport molecules directly back to the plasma membrane, but many molecules are transported in vesicles that first fuse with recycling endosomes. Molecules following this recycling pathway are concentrated in the tubules of early endosomes. Molecules that follow these pathways include the receptors for LDL, the growth hormone EGF, and the iron transport protein transferrin. Internalization of these receptors from the plasma membrane occurs by receptor-mediated endocytosis. LDL is released in endosomes because of the lower pH, and the receptor is recycled to the cell surface. Cholesterol is carried in the blood primarily by (LDL), and transport by the LDL receptor is the main mechanism by which cholesterol is taken up by cells. EGFRs are activated when EGF binds. The activated receptors stimulate their own internalization and degradation in lysosomes. EGF remains bound to the EGFR once it is endocytosed to endosomes. The activated EGFRs stimulate their own ubiquitination, and this directs them to lumenal vesicles (see below) and so they are not recycled to the plasma membrane. This removes the signaling portion of the protein from the cytosol and thus prevents continued stimulation of growth - in cells not stimulated with EGF, EGFRs have no EGF bound to them and therefore recycle if they reach endosomes. Transferrin also remains associated with its receptor, but, in the acidic endosome, iron is released from the transferrin, and then the iron-free transferrin (still bound to the transferrin receptor) returns from the early endosome to the cell surface, both directly and via recycling endosomes.

Answer 36

Transport from late endosomes to lysosomes is, in essence, unidirectional, since a late endosome is "consumed" in the process of fusing with a lysosome. Hence, soluble molecules in the lumen of endosomes will tend to end up in lysosomes, unless they are retrieved in some way. Transmembrane proteins can be delivered to the perimeter membrane or the lumen of lysosomes. Transmembrane proteins destined for the lysosome lumen are sorted into the vesicles that bud from the perimeter membrane into endosomes, a process that begins in early endosomes. When the endosome has matured into a late endosome/MVB and fuses with a lysosome, the vesicles in the lumen are delivered to the lysosome lumen. Proteins are marked for this pathway by the addition of ubiquitin.

Answer 37

primarily degrade extracellular materials taken up by endocytosis and some intracellular components. Lysosomes contain many enzymes that together will degrade all classes of molecules – proteins, lipids and sugars. The lumen is acidic (pH 5) due to the activity of a proton pump. Monoubiquitinated plasma membrane proteins are targeted for endocytosis and are transferred via the late endosome/multivesicular body to the lysosome for degradation. The multivesicular body is a prelysosomal compartment; vesicles containing proteins slated for destruction bud into the lumen. When the multivesicular body fuses with the lysosome, these vesicles are delivered into the lysosome for degradation. Lysosomal membrane proteins, e.g., the proton pump and transporters, need to be protected from the degradation. They reach the lysosome by vesicles that bud from the Golgi and fuse with the lysosome. Defects in enzymes or membrane proteins can cause lysosomal storage diseases. These have diverse effects on many different organs and can be difficult to diagnose. The central and peripheral nervous systems are especially susceptible to lysosomal storage diseases

Answer 38

a special type of lipid raft, are small (50–100 nanometer) invaginations of the plasma membrane in many vertebrate cell types, especially in endothelial cells and adipocytes. Caveolae are one source of clathrin-independent raft-dependent endocytosis. The ability of caveolins to oligomerize due to their oligomerization domains is necessary for formation of caveolar endocytic vesicles. The oligomerization leads to formation of caveolin-rich microdomains in the plasma membrane. Increased levels of cholesterol and insertion of scaffolding domain of caveolins to the plasma membrane then lead to expansion of the caveolar invagination and to formation of endocytic vesicle. Fission of the vesicle from the plasma membrane is then mediated by GTPase dynamin II which is localized at the neck of the budding vesicle. The released caveolar vesicle can fuse with early endosome or caveosome. The caveosome is an endosomal compartment with neutral pH which does not have early endosomal markers, however, contains molecules internalized by the caveolar endocytosis.

Answer 39

a membrane-bounded compartment inside eukaryotic cells. It is a compartment of the endocytic membrane transport pathway from the plasma membrane to the lysosome. Molecules internalized from the plasma membrane can follow this pathway all the way to lysosomes for degradation, or they can be recycled back to the plasma membrane. Molecules are also transported to endosomes from the Golgi and either continue to lysosomes or recycle back to the Golgi. Furthermore, molecules can be directed into vesicles that bud from the perimeter membrane into the endosome lumen. Therefore, endosomes represent a major sorting compartment of the endomembrane system in cells.

Answer 40

the LDL dissociates from its receptors in the acidic environment of the endosome. After a number of steps, the LDL ends up in lysosomes, where it is degraded to release free cholesterol. In contrast, the LDL receptor proteins are returned to the plasma membrane via clathrin-coated transport vesicles that bud off from the tubular region of the early endosome. For simplicity, only one LDL receptor is shown entering the cell and returning to the plasma membrane. Whether it is occupied or not, an LDL receptor typically makes one round trip into the cell and back to the plasma membrane every 10 minutes, making a total of several hundred trips in its 20-hour life-span.

Answer 41

a thiol oxidoreductase that allows formation of disulfide bonds. Reside in the rough ER

Answer 42

About 30% of protein synthesis occurs in the rough ER; this is where transmembrane proteins are made as well as proteins destined for vesicular transport to other regions of the cell and for secreted proteins. The ER establishes an environment conducive for protein folding, for disulfide bond formation and to check on the quality of protein formation and folding. The ER contains folding enzymes such as ERp57. There are molecular chaperones in the ER, e.g. BiP. Finally, there are folding sensors that monitor unfolded proteins and hold them in the ER until they fold properly or are shuttled to a degradation pathway. One of these pathways involves a glucosyltransferase and two ER-resident proteins, calnexin and calreticulin.

Answer 43

an Hsp70-like protein that uses ATP to help proteins fold. Reside in the lumen of the rough ER

Answer 44

Calnexin (a transmembrane protein, CNX) and calreticulin (a soluble protein in the ER lumen) bind the oligosaccharide chain if there is a glucose. Proteins like these that bind sugars are called lectins, and both calnexin and calreticulin also bind calcium, as indicated by the first part of their names. When the glucose is removed by a glucosidase, the protein is released by calnexin and calreticulin. If the protein is correctly folded, it can exit the ER. If the protein is not correctly folded, it is recognized and bound by a glucosyltransferase (GT), which puts glucose back on the sugar chain and calnexin/calreticulin bind the protein again. Thus, a protein can go through many cycles of removal and addition of glucose until it (1) folds properly and exits or (2) “times out” and is retrotranslocated out of the ER and is destined for degradation. It is not known what controls the number of cycles that occur before the protein is deemed unfoldable.

Answer 45

Proteins retrotranslocated out of the ER are deglycosylated and have multiple ubiquitin molecules attached in order to target them to the proteosome. This is the ubiquitin-proteasome degradation system. The proteosome is a huge complex of proteins that unwinds the misfolded protein and feeds the protein strand into a compartment that cuts the protein into short (7-9 amino acids) peptides. The proteasome is dispersed throughout the cytoplasm and nucleus and constitutes about 1% of cellular protein.

Answer 46

The core is hollow and provides an enclosed cavity in which proteins are degraded; openings at the two ends of the core allow the target protein to enter. Each end of the core particle associates with a 19S regulatory subunit that contains multiple ATPase active sites and ubiquitin binding sites; it is this structure that recognizes polyubiquitinated proteins and transfers them to the catalytic core. The central cylinder of the proteasome is where the proteolytic cleavages take place. Each end of the cylinder has a cap, which recognizes the polyubiquitin and uses ATP to unfold the protein and feed it into the cylinder. The entire proteasome is ~ 2 million Daltons.

Answer 47

a 76 amino acid protein that is remarkably conserved in all cells. The sequence is the same in flies and humans, and 74 of 76 amino acids are identical between plants and animals. This strict conservation over hundreds of millions of years of evolution implies an essential function of the protein. Attachment of ubiquitin to a protein requires three ligase enzymes: E1, E2, and E3. E1 binds and activates ubiquitin; mammals have one gene for this function. Ubiquitin is then passed to an E2 enzyme; mammals have about 50 of these. E3 has the substrate specificity, and there are roughly 500 E3 enzymes in mammals. Ubiquitin is transferred from E2 by E3 to a lysine in the protein. E3 then attaches a string of additional ubiquitins to the first one, creating a chain of ubiquitins that is called polyubiquitin. A chain of at least four ubiquitins is required by the proteasome as a tag for degradation. The proteasome removes the ubiquitins for recycling and then chops up the protein. Ubiquitin has other purposes besides being a tag for destruction. Some proteins have a single ubiquitin attached, and some have single ubiquitins at multiple sites on a protein (multiubiquitination). These mono and multiubiquitins (as opposed to the polyubiquitins recognized by the proteasome) are used as regulatory signals, e.g. ubiquitination of histones and transcription factors can regulate transcription.

Answer 48

A growing polypeptide chain is shown acquiring its secondary and tertiary structure as it emerges from a ribosome. The N-terminal domain folds first, while the C-terminal domain is still being synthesized. In this case, the protein has not yet achieved its final conformation by the time it is released from the ribosome. Each domain of a newly synthesized protein rapidly attains a “molten globule” state. Subsequent folding occurs more slowly and by multiple pathways, often involving the help of a molecular chaperone. Some molecules may still fail to fold correctly; these are recognized and degraded by specific proteases.

Answer 49

a newly synthesized protein sometimes folds correctly and assembles with its partners on its own, in which case it is left alone. Incompletely folded proteins are helped to refold by molecular chaparones: first by a family of hsp70 proteins, and if this fails, then by hsp60-like proteins. In both cases the client proteins are recognized by an abnormally exposed patch of hydrophobic amino acids on their surface. These processes compete with a different system that recognizes an abnormally exposed patch and transfers the protein that contains it to a proteasome for complete destruction. The combination of all of these processes is needed to prevent massive protein aggregation in a cell, which can occur when many hydrophobic regions on proteins clump together and precipitate the entire mass out of solution.

Answer 50

(eating oneself) is typically described as a stress response to starvation; components of the cell are broken down to be reutilized for survival. However, it is also used throughout the life of the cell to degrade organelles that are damaged or undergo normal turnover. A double membrane forms around the organelle. The outer membrane fuses with the lysosome and the membrane enclosed organelle is delivered, like the vesicles in the multivesicular body, to the lysosome for degradation.

Answer 51

Glucosyl transferase binds unfolded proteins. Calnexin binds only those N-glycoproteins. Calnexin is a chaperone. After the last glucose is removed, if the protein is folded correctly, it exits the ER, usually to the Golgi complex. However, if the protein is still unfolded, then GT adds another glucose, and the protein gets bound again to calnexin and calreticulin for another chance to fold properly.

Answer 52

Lectins are carbohydrate-binding proteins, macromolecules that are highly specific for sugar moieties.

Answer 53

Ubiquitination is carried out in three main steps: activation, conjugation, and ligation, performed by ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s), respectively. The result of this sequential cascade binds ubiquitin to lysine residues on the protein substrate via an isopeptide bond or to the amino group of the protein's N-terminus via a peptide bond. At least four ubiquitin molecules must be attached to a lysine residue on the condemned protein in order for it to be recognised by the 26S proteasome. This is a barrel-shape structure comprising a central proteolytic core made of four ring structures, flanked by two cylinders that selectively allow entry of ubiquitinated proteins. Once inside, the proteins are rapidly degraded into small peptides (usually 3–25 amino acid residues in length). Ubiquitin molecules are cleaved off the protein immediately prior to destruction and are recycled for further use.

Answer 54

In structure, the proteasome is a cylindrical complex containing a "core" of four stacked rings forming a central pore. Each ring is composed of seven individual proteins. The inner two rings are made of seven β subunits that contain three to seven protease active sites. Substrate is “spiraled” through the chamber and cleaved by different activities associated with different b-subunits; ~7-9 amino acid peptides are released; cleavage does not require ATP; ATP required for unfolding and translocation. These sites are located on the interior surface of the rings, so that the target protein must enter the central pore before it is degraded. The outer two rings each contain seven α subunits whose function is to maintain a "gate" through which proteins enter the barrel. These α subunits are controlled by binding to "cap" structures or regulatory particles that recognize polyubiquitin tags attached to protein substrates and initiate the degradation process. The overall system of ubiquitination and proteasomal degradation is known as the ubiquitin-proteasome system.

Answer 55

Misfolded soluble proteins in the ER lumen are translocated back into the cytosol, where they are deglycosylated, ubiquitylated, and degraded in proteasomes. Misfolded membrane proteins follow a similar pathway. Misfolded proteins are exported through the same type of translocator that mediated their import; accessory proteins that are associated with the translocator allow it to operate in the export direction.

Answer 56

Proteasome- degrades only proteins: Polyubiquibitinated proteins (tetra-ubiquitin minimal targeting signal). Lysosome- degrades all cellular components: Targeted via the endocytic pathway, Monoubiquitinated transmembrane proteins, Regulated at the Multivesicular Body. Autophagy: Direct transport into the lysosomal lumen from cytoplasm

Answer 57

1) sphingomyelinase, breaks down sphingomyelin in macrophages and 2) cholesterol transporter, moves cholesterol from the lysosome to the cytosol. Mutations causing loss of this transporter are one cause of Niemann-Pick disease (a lysosomal storage disease). Cells missing the transporter (from Niemann-Pick patients) are resistant to Ebola infection, and mice with this transporter missing are also Ebola resistant. Moreover, small molecules that block the interaction between Ebola viral GP and the cholesterol transporter block Ebola infection. These results reveal a step in the pathway of Ebola infection that could provide a target for therapeutic drugs.

Answer 58

Ebola virus has an envelope, is highly pathogenic, and causes a massive production of pro-inflammatory cytokines; mortality in some outbreaks exceeds 75-90 %. There is no therapy or effective vaccine and the disease progresses rapidly. The virus enters the cell by endocytosis when the viral spike glycoprotein (GP) binds to the cell surface; this is a macropinocytosis pathway that does not involve clathrin or caveolae. Heavily glycosylated domains of GP are cleaved in the endosome, and the remaining GP protein binds to a previously unknown membrane protein in the endosomal/lysosomal membrane. This binding induces fusion of the viral membrane and the endosome/lysosome membrane; the virus is released into the cell where it can replicate. These papers show that the mystery membrane protein in the endosome/lysosome is a cholesterol transporter (remember LDL from cholesterol regulation in the Membrane Structure lecture and the LDL receptor cycling at the beginning of this lecture). This cholesterol transporter is required to move cholesterol from the endosome/lysosome to the cytoplasm.

Answer 59

Phosphatidylserine(s) are actively held facing the cytosolic (inner) side of the cell membrane by the enzyme flippase. This is in contrast to normal behavior of phospholipids in the cell membrane which can freely flip their heads between the two faces of the membrane they comprise. However, when a cell undergoes apoptosis phosphatidylserine is no longer restricted to the cytosolic domain by flippase. When the phosphatidylserines naturally flip to the extracellular (outer) surface of the cell, they act as a signal for macrophages to engulf the cells.

Answer 60

In normal viable cells, phosphatidylserine (PS) is located on the cytoplasmic surface of the cell membrane. However, in the intermediate stages of apoptosis, PS is translocated from the inner to the outer leaflet of the membrane, exposing PS to the external cellular environment where it can be detected. Highly fluorescent annexin V conjugates provide quick and reliable detection methods for studying the externalization of phosphatidylserine.

Answer 61

shown to have the ability to inhibit transcription. Actinomycin D does this by binding DNA at the transcription initiation complex and preventing elongation of RNA chain by RNA polymerase.

Answer 62

This gene encodes a cytoplasmic protein that forms one of the central hubs in the apoptosis regulatory network. This protein contains (from the N terminal) a caspase recruitment domain (CARD), an ATPase domain (NB-ARC), few short helical domains and then several copies of the WD40 repeat domain. Upon binding cytochrome c and dATP, this protein forms an oligomeric apoptosome. The apoptosome binds and cleaves Procaspase 9 protein, releasing its mature, activated form.

Answer 63

is the main initiator caspase for the intrinsic pathway and activates the effector caspase 3

Answer 64

Apoptosis (or programmed cell death) is a controlled process leading to cell death characterized by chromatin condensation, DNA fragmentation, cell shrinkage, and compartimentalization of the dead cells to apoptotic bodies. The host immune system uses apoptosis to eliminate cells infected by pathogens. Apoptosis of infected cells is caused either by cytolytic cells activated during the anti-viral response, or directly by viral infection. Many viruses have evolved mechanisms to either inhibit or activate cell death depending on their needs. Inhibition of apoptosis allow viruses to optimize replication and progeny synthesis by prolonging the infected cell life. Alternatively, viruses may trigger host cell apoptsis to release progeny virions. Several large DNA viruses encode viral FLIP proteins, sharing strong sequence similarites with host c-FLIP, a protein that acts as an inhibitor of TNFRSF6 mediated apoptosis. Similar to c-FLIPs, the vFLIPs can block the interaction of the death receptor-adapter complex with the cellular effector FLICE (caspase-8), and this prevents the initiation of the downstream caspase cascade.

Answer 65

the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components through the actions of lysosomes. The breakdown of cellular components promotes cellular survival during starvation by maintaining cellular energy levels. Autophagy allows the degradation and recycling of cellular components. During this process, targeted cytoplasmic constituents are isolated from the rest of the cell within a double-membraned vesicle known as an autophagosome. The autophagosome then fuses with a lysosome and its cargo is degraded and recycled. There are three different forms of autophagy that are commonly described; macroautophagy, microautophagy and chaperone-mediated autophagy. In the context of disease, autophagy has been seen as an adaptive response to stress which promotes survival, whereas in other cases it appears to promote cell death and morbidity. autophagy is how stuff is delivered to the lysome. It is induced during time of nutrient stress. Autophagy signaling often converges on the mTOR pathway

Answer 66

the main pathway, occurring mainly to eradicate damaged cell organelles (only way) or unused proteins. This involves the formation of a double membrane around cytoplasmic substrates resulting in the organelle known as an autophagosome. In the canonical starvation-induced pathway, autophagosome formation is induced by class 3 phosphoinositide-3-kinase, the autophagy-related gene (Atg) 6 (also known as Beclin-1) and ubiquitin-like conjugation reactions. Non-canonical PI3K/beclin 1-independent induction pathways have been described for injury-induced autophagy and mitophagy.[16] Both canonical and non-canonical pathways converge on the covalent conjugation of Atg8 homologues to phosphatidylethanolamine at the phagophore expanding autophagic membranes. Other autophagy-related proteins such as Atg4, Atg12, Atg5, and Atg16 are also involved in the regulation of these pathways. The autophagosome travels through the cytoplasm of the cell to a lysosome, and the two organelles fuse; intersection with endosomal pathways also occurs. Within the lysosome, the contents of the autophagosome are degraded via acidic lysosomal hydrolases.

Answer 67

involves the direct engulfment of cytoplasmic material into the lysosome. This occurs by invagination, meaning the inward folding of the lysosomal membrane, or cellular protrusion.

Answer 68

a very complex and specific pathway, which involves the recognition by the hsc70-containing complex. This means that a protein must contain the recognition site for this hsc70 complex which will allow it to bind to this chaperone, forming the CMA- substrate/chaperone complex. This complex then moves to the lysosomal membrane-bound protein that will recognise and bind with the CMA receptor, allowing it to enter the cell. Upon recognition, the substrate protein gets unfolded and it is translocated across the lysosome membrane with the assistance of the lysosomal hsc70 chaperone. CMA is significantly different from other types of autophagy because it translocates protein material in a one by one manner, and it is extremely selective about what material crosses the lysosomal barrier.

Answer 69

Re-cycle proteins and other macromolecules under conditions of nutrient deprivation- allow recycling of amino acids, nucleotides and fatty acids. Remove organelles- mitochondria and peroxisomes. Allow cell survival under stress conditions (growth factor deprivation, hypoxia etc.) Present antigens to MHC system. Neuro-protection- e.g. remove protein aggregates to prevent neuronal damage in e.g. Huntington’s, Parkinson’s disease etc. Remove intracellular pathogens. Aging- increased autophagy can extend life in worms and other model organisms. Tumor suppression- e.g. an autophagy gene (Beclin 1) is a haploinsufficient tumor suppressor in mice. Tumor promotion- e.g. required for progression and (perhaps) metastasis. Inhibits apoptosis/ cell death but also kills cells. Type II programmed cell death.

Answer 70

Macroautophagy is initiated by the formation of the phagophore (also called the isolation membrane). This membrane can both selectively and non-selectively engulf cytosolic components, grow and close around the sequestered components and then deliver them to a degradative organelle, the lysosome.

Answer 71

A cup-shaped protrusion from the endoplasmic reticulum that serves as a platform for autophagosome biogenesis in mammalian cells. After macroautophagy induction omegasomes are derived from ER and serve as intermediates for genesis of isolation membrane (also called autophagosome phagophore or preautophagosome) from ER cisternae membranes. They form a cradle for the formation of phagophore by membrane invagination. While the isolation membrane expands to engulf cytoplasmic components it is encircled or sandwiched by omegasome to the time the membrane is sealed and forms a double-membrane autophagosome.

Answer 72

The product of endosome–autophagosome fusion is known as an amphisome. The completed autophagosome or amphisome fuses with a lysosome, which supplies acid hydrolases. The enzymes in the resulting compartment, an autolysosome, break down the inner membrane from the autophagosome and degrade the cargo. The resulting macromolecules are released through permeases and recycled in the cytosol.

Answer 73

a spherical structure with double layer membranes. It is the key structure in macroautophagy, the intracellular degradation system for cytoplasmic contents (e.g., abnormal intracellular proteins, excess or damaged organelles) and also for invading microorganisms. After formation, autophagosomes deliver cytoplasmic components to the lysosomes. The outer membrane of an autophagosome fuses with a lysosome to form an autolysosome. The lysosome's hydrolases degrade the autophagosome-delivered contents and its inner membrane.

Answer 74

New developments in research have found that targeting autophagy may be a viable new therapeutic solution in fighting cancer. As discussed above, autophagy plays both a role in tumor suppression and tumor cell survival. Thus, these qualities about autophagy can be used and manipulated as a strategy for cancer prevention. The first strategy is to induce autophagy and enhance its tumor suppression attributes. The second strategy is to inhibit autophagy and thus induce apoptosis. The first strategy has been viewed and tested by looking at dose-response antitumor effects in autophagy-inducing therapies. These therapies have shown that autophagy extent increases in a dose-dependent manner. This is directly related to the growth of cancer cells in a dose-dependent manner as well. Therefore, this data supports the development of therapies that will encourage autophagy. Inhibiting the protein pathways directly known to induce autophagy may serve as another anticancer therapy. Lastly, overexpression of autophagy genes can be used.

Answer 75

Often times, cancer occurs when several different pathways that regulate cell differentiation are disturbed. Autophagy plays an important role in cancer – both in protecting against cancer as well as potentially contributing to the growth of cancer. Autophagy may protect against cancer by isolating damaged organelles, allowing cell differentiation, increasing protein catabolism, and even promoting cell death of cancerous cells. However, autophagy can also contribute to cancer by promoting survival of tumor cells that have been starved. In order to maintain homeostasis conditions, autophagy must not be disrupted. If this important mechanism is interrupted, tumor growth can likely occur. The main function of autophagy in tumor suppression is its ability to remove damaged proteins and organelles thus limiting any cell growth instability. Alternatively, autophagy has also been shown to play a huge role in tumor cell survival. In cancerous cells, autophagy is used as a way to deal with stress on the cell. Once these autophagy related genes were inhibited, cell death was potentiated. Tumor cells have high metabolic demands due to the increase in cell proliferation. The increase in metabolic energy is offset by autophagy functions. These metabolic stresses include hypoxia, nutrient deprivation, and an increase in proliferation. These stresses activate autophagy in order to recycle ATP and maintain survival of the cancerous cells. Autophagy has been shown to enable continued growth of tumor cells by maintaining cellular energy production. By inhibiting autophagy genes in these tumors cells, regression of the tumor and extended survival of the organs affected by the tumors were found. Furthermore, inhibition of autophagy has also been shown to enhance the effectiveness of anticancer therapies.

Answer 76

Autophagy is often identified morphologically. Assessing autophagy by following autophagosome associated proteins.

Answer 77

Regulate autophagy. Identified & characterized in yeast using “classical” genetic screens for genes affecting nutrient deprivation phenotypes. more than 20 gene products required for formation of autophagosomes. Different kinds of molecules- e.g. Atg 1- protein kinase; Atg12 Ubiquitin-like molecule; Atg6 (Beclin-1) “scaffold” that forms part of Type III PI3 Kinase complex. Mechanism in yeast and higher organisms conserved.

Answer 78

The result of the fusion of a autophagosome and a lysosome

Answer 79

1. induction by nutrient starvation, growth factor-mediated starvation, exposure to chemo durgs, rapamycin etc. 2. vesicle nucleation (phagophore) 3. vesicle expansion (omegasome) 4. cargo targeting (LC3II and p62). 5. vesicle closure (autophagosome) 6. vesicle fusion with endosome (amphisome) 7. vesicle fusion with lysosome (autolysosome). The Atg genes regulate different steps in the process

Answer 80

a protein complex associated with the nuclear envelope. The p62 protein remains associated with the nuclear pore complex-lamina fraction. p62 is synthesized as a soluble cytoplasmic precursor of 61 kDa followed by modification that involve addition of N-acetylglucosamine residues, followed by association with other complex proteins. P62 appears to interact with mRNA during transport out of the nucleus. P62 also interacts with a nuclear transport factor (NTF2) protein that is involved in trafficking proteins between cytoplasm and nucleus. Another protein, importin (beta) binds to the helical rod section of p62, which also binds NTF2 suggesting the formation of a higher order gating complex. Karyopherin beta2 (transportin), a riboprotein transporter also interacts with p62. P62 also interacts with nucleoporin-93kDa, and when Nup98 is depleted p62 fails to assemble with nuclear pore complexes. Mutant pores could not dock/transport proteins with nuclear localization signals or M9 import signals.

Answer 81

Microtubule-associated protein 1A/1B-light chain 3 (LC3) is a soluble protein with a molecular mass of approximately 17 kDa that is distributed ubiquitously in mammalian tissues and cultured cells. During autophagy, autophagosomes engulf cytoplasmic components, including cytosolic proteins and organelles. Concomitantly, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes. Autophagosomes fuse with lysosomes to form autolysosomes, and intra-autophagosomal components are degraded by lysosomal hydrolases. At the same time, LC3-II in autolysosomal lumen is degraded. Thus, lysosomal turnover of the autophagosomal marker LC3-II reflects starvation-induced autophagic activity, and detecting LC3 by immunoblotting or immunofluorescence has become a reliable method for monitoring autophagy and autophagy-related processes, including autophagic cell death.

Answer 82

Amino acids and other nutrients. Growth factors. Lipids. Everything that regulates the PI3 kinase pathway. Multiple protein kinases. Tumor suppressor proteins e.g. p53 (positive and negative regulation). Mutated oncogenes e.g. Ras (positive and negative regulation). Many drugs.

Answer 83

One of the mechanisms of programmed cell death (PCD) is associated with the appearance of autophagosomes and depends on autophagy proteins. This form of cell death most likely corresponds to a process that has been morphologically defined as autophagic PCD. An example: How apoptosis regulates autophagy-Caspase cleavage of Beclin amplifies apoptosis. One question that constantly arises, however, is whether autophagic activity in dying cells is the cause of death or is actually an attempt to prevent it. Morphological and histochemical studies so far did not prove a causative relationship between the autophagic process and cell death. In fact, there have recently been strong arguments that autophagic activity in dying cells might actually be a survival mechanism. Studies of the metamorphosis of insects have shown cells undergoing a form of PCD that appears distinct from other forms; these have been proposed as examples of autophagic cell death. Recent pharmacological and biochemical studies have proposed that survival and lethal autophagy can be distinguished by the type and degree of regulatory signaling during stress particularly after viral infection.

Answer 84

Inhibitors of Bcl-XL/Bcl-2 can induce autophagy by releasing the autophagic protein Beclin 1 from its complexes with these proteins. Here we report a novel compound targeting the BH3 binding groove of Bcl-XL/Bcl-2, Z18, which efficiently induces autophagy-associated cell death in HeLa cells, without apparent apoptosis.

Answer 85

BH3 domains were originally discovered in the context of apoptosis regulators and they the mediate binding of proapoptotic Bcl-2 family members to antiapoptotic Bcl-2 family members. Yet, recent studies indicate that BH3 domains do not function uniquely in apoptosis regulation; they also function in the regulation of another critical pathway involved in cellular and tissue homeostasis called autophagy. Antiapoptotic Bcl-2 homologs downregulate autophagy through interactions with the essential autophagy effector and haploinsufficient tumor suppressor, Beclin 1. Beclin 1 contains a BH3 domain, similar to that of Bcl-2 proteins, which is necessary and sufficient for binding to antiapoptotic Bcl-2 homologs and required for Bcl-2-mediated inhibition of autophagy.

Answer 86

Associated with formation of autophagic vesicles. Requires autophagic machinery- death is inhibited by knockdown of Beclin-1/Atg6 and other Atg genes AND allows continued growth of cells. Occurs in cells where apoptosis is not possible? Cells prefer to die by apoptosis if they can? But little clear evidence that true “cell death by autophagy” exists in normal physiological circumstances, except perhaps during drosophila development. There may however be specific cases where autophagy promotes death by making other death mechanisms (i.e. apoptosis or necrosis) easier.

Answer 87

Diseases caused by aggregate -prone Poly Q, Poly-A proteins (Huntington’s, Ataxia’s, etc.), other aggregate-prone proteins e.g. Tau (Alzheimers). Proteins cleared by autophagy. Clearance delayed by autophagy inhibition. Increase autophagy - protect in vitro against cell death & in vivo (flies and mouse models) against tissue damage. Use rapamycin and derivatives or other autophagy inducers as chemoprevention/ treatment for neurodegenerative diseases

Answer 88

Autophagy is a highly regulated process and likely important in many physiological and pathological processes. Intimately associated with apoptosis & cell death. Can kill cells- may involve components of apoptosis machinery. Can protect cells against nutrient deprivation-induced stress, neurodegeneration, and anti-cancer agents. What determines which response occurs? Can we manipulate these pathways therapeutically, but what direction should we try to do it?

Answer 89

Macroautophagy (best studied)- form a double membrane vesicle that captures cytosolic components/organelles. Then fuse with lysosome where hydrolases degrade contents of autophagosome. Chaperone-mediated--recognition of specific proteins that contain a specific recognition sequence (based on amino acid sequence KFERQ). Direct binding and delivery to lysosome.

Answer 90

Activate a PI3K complex that allows nucleation of a membrane that will eventually form autophagosome. Regulation of protein conjugation events to extend membrane. Randomly capture, or specifically deliver cargo to the extending autophagosome, then join the membranes to close the vesicle. Fuse with lysosome. Recycle amino acids and other macromolecular precursors.

Answer 91

Aggregate-prone proteins (e.g. those with expanded stretches of glutamine residues in diseases like Huntington’s disease) will cause neuronal cell death. Autophagy degrades the aggregate-prone proteins (perhaps after they have started to form small aggregates). No toxic stimulus, no neuronal cell death.

Answer 92

Many proteins - e.g. Bcl-2 - that regulate apoptosis/ cell death, also control autophagy--remember, this could create problems in interpreting results of therapeutic interventions designed to target these proteins. Apoptotic proteases (caspases) can cleave essential autophagy regulators inactivating them and therefore blocking the process of autophagy. In some cases, -e.g. with starvation-induced cell death, it is easy to see why autophagy would protect– it provides essential nutrients at least in the short term. In others, e.g. stress-induced cell death from chemotherapy etc. its less clear why autophagy would be protective.

cell biology 5 Flashcards

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