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Advanced Cell Bio > Exam 3- Chapter 18- Necroptosis > Flashcards

Exam 3- Chapter 18- Necroptosis Flashcards

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1
Q

Purposes of necroptosis (3)

A
  1. Elimination of damaged cells
  2. Induction of inflammation
  3. Backup to apoptosis
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2
Q

Necroptosis

A

Another programmed cell death pathway that is a controlled “cellular explosion”. Necroptosis is perhaps more important to infection than apoptosis. It occurs in cases where an immune response is needed. Release of antimicrobial agents. Necroptosis can be referred to as the yang to apoptosis’ yin. The ultimate outcome is cell lysis

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3
Q

Defining features of necroptosis

A

Necroptosis is an inflammatory pathway that is caspase independent (does not require the signaling molecules used in apoptosis). The pathway can be damaging to neighboring cells due to the inflammation. It results in increased membrane permeability (so inflammatory mediators can leak out into the environment) and is associated with the release of damage associated molecular patterns (DAMPs). The outcome of this process is actual cell lysis, compared to the neat removal that occurs with apoptosis

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4
Q

Changes to the cell during necroptosis (7)

A
  1. Membrane vesiculation, vesicles form and pinch off, causes a loss of integrity
  2. Organelle membranes (nucleus, mitochondria) lose their integrity and become more permeable as well before they lyse
  3. Lysosome permeabilization- enzymes are released and add “fuel to the fire”
  4. DNA is damaged but differently from apoptosis
  5. Cell swells and bursts
  6. All cellular contents spill out- this is the height of the inflammatory component
  7. Orchestrated chaos
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5
Q

Importance of necroptosis (3)

A
  1. Cells are damaged due to overt insult- Ischemia-reperfusion, systemic inflammatory response syndrome (SIRS)
  2. Necessary during infection
  3. Resulting inflammation is meant to correct things, but sometimes this gets out of hand and can be damaging
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6
Q

Necroptosis vs. apoptosis

A

During apoptosis, the cell shrinks and the chromatin condenses. The organelles break down in a specific way, and the cell breaks down into apoptotic bodies. They are phagocytosed by macrophages and there is no inflammation. During necroptosis, the cell initially swells (due to increased membrane permeability and osmosis), then becomes leaky and blebs occur. Cellular and nuclear lysis causes inflammation

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7
Q

Ischemia-reperfusion

A

The paradoxical exacerbation of cellular dysfunction and death, following restoration of blood flow to previously ischemic tissues. Occurs with heart attacks and strokes

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8
Q

Systemic inflammatory response syndrome (SIRS)

A

An exaggerated defense response of the body to a noxious stressor (infection, trauma, surgery). Called sepsis when the source is an infection.

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9
Q

Necroptosis and apoptosis similarities (2)

A
  1. Induced by the same ligands/receptors: TNF-α, FasL, TRAIL
  2. Involves DISC & cytosolic complex formation
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10
Q

Necroptosis and apoptosis differences (3)

A
  1. Necroptosis is caspase-independent
  2. RIP1/RIP3-dependent (kinases)
  3. Cytosolic complex differs (necrosome)
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11
Q

Death-inducing signaling complexes (DISC)

A

The extrinsic pathway is induced by cytokines, especially TNF cytokines- TNF-α, Fas ligand (FasL), TRAIL. They create the DISC complex when they bind to their receptors- there can be one or two DISC complexes depending on the cytokine involved. For example, the Fas ligand only creates one death complex containing caspase 8. TNF creates 2 DISC complexes- one that is connected to the receptor, and one that is further internalized into the cell.

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12
Q

Necrosome

A

A protein complex that forms during necroptosis. It is considered the central cell signaling complex. Includes RIP1, RIP3, & MLKL. FADD & potentially caspase-8. Caspase-8 antagonizes necroptosis

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13
Q

What is the significance of necroptosis (3)

A
  1. Backup to apoptosis, used in situations where caspase 8 is inhibited
  2. Specific to certain situations: Ischemia-reperfusion, Infection, SIRS
  3. Some sort of molecular switch that decides apoptosis or necroptosis
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14
Q

Apoptosis pathway vs necroptosis pathway

A
  1. A cell death stimulus binds to its receptor- occurs with BOTH processes
  2. A point of divergence occurs in the pathway
  3. Then, the cell will undergo non-inflammatory apoptosis (caspases) or inflammatory necroptosis (RIP1)
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15
Q

RIP1, RIP3, and MLKL

A

Ser/Thr kinases that are absolutely essential to necroptosis. RIP1 autophosphorylates, then p-RIP1 binds with and phosphorylates RIP3. p-RIP3 phosphorylates (activates) downstream targets, including: MLKL, Sphingomyelinases, glycolysis/metabolism, NADPH oxidase, calpains, cPLA2

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16
Q

Induction of apoptosis or necroptosis pathway (5)

A

The initial steps are the same for both pathways
1. TNF alpha and its receptor are both membrane proteins. TNF alpha will be released in membrane vesicles, and will then bind to its receptor
2. Then, a protein complex forms on the end of the TNF receptor in the cytoplasm. It contains TRADD, RIP1, and cIAP1/2. RIP1 is tagged with ubiquitin
3. If the ubiquitin stays on, we are committed to cell survival
4. Ubiquitin can be removed by an ubiquitylase enzyme called CYLD
5. The protein complex can then be endocytosed and the cell will be committed to cell death. At this point, it has not been decided whether the cell will undergo apoptosis or necroptosis

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17
Q

Apoptosis pathway (4)

A
  1. After the induction steps, a second protein complex forms (RIP1, TRADD, FADD). Caspase 8 joins and becomes activated by joining
  2. Caspase 8 cleaves and inactivates RIP1, preventing necroptosis
  3. It cleaves and activates its downstream executioner caspases (caspase 3, 6, 7)
  4. Promotes apoptosis
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18
Q

When is the point of divergence between apoptosis and necroptosis?

A

The protein complex is endocytosed, and the cell will be committed to cell death. At this point, it has not been decided whether the cell will undergo apoptosis or necroptosis. The induction steps are exactly the same until this step occurs. The complex is the “see-saw point”, and there are many different things that can tip the balance toward necroptosis or apoptosis. It is under active investigation

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19
Q

Necroptosis pathway (6)

A
  1. Necroptosis is initiated after the induction steps. It is unclear what exactly causes necroptosis, but we know that if caspase 8 is compromised, necroptosis will occur
  2. RIP1 is autophosphorylated and recruits and phosphorylates RIP3
  3. The phosphorylation of RIP3 recruits MLKL
  4. RIP3 phosphorylates MLKL
  5. The inclusion of all of those phosphokinases make up the necrosome
  6. The necrosome signals to downstream targets to cause all of the causes in the cell associated with apoptosis
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20
Q

RHIM domains

A

Domains found in the RIP1 and RIP3 proteins. This is how the 2 proteins bind to one another

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21
Q

Downstream effectors in necroptosis (7)

A
  1. MLKL itself
  2. Ceramide
  3. Sphingosine
  4. Ca2+-activated calpains
  5. Phospholipases
  6. ROS
  7. AGEs
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22
Q

Ceramide

A

A membrane lipid that can participate in lipid rafts. It is another downstream effector of necroptosis

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23
Q

Calpains

A

Enzymes that degrade the cytoskeleton. Calcium activated calpains are downstream effectors of necroptosis

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24
Q

Phospholipases

A

Enzymes that process the phospholipids of the membrane. They contribute to increased permeability and are downstream effectors of necroptosis

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25
Q

Domains of MLKL (2)

A
  1. N-terminus- coiled coil
  2. C-terminus- kinase-like domain- capable of phosphorylating other proteins
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26
Q

N-terminus of MLKL

A

The terminal domain which binds to other MLKL molecules and forms membrane pores once the molecules are oligomerized. Composed of 4-helices to form a bundle (4HB). MLKL associates with the membrane by binding to a specific phosphatidylinositide (PIP), which is called PI (4,5)P2. PI(4,5)P2 is located on the inner leaflet (cytoplasmic half) of the membrane. Phosphorylation is necessary for activation

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27
Q

Role of MLKL

A

MLKL is phosphorylated by RIP3 so it can be activated. It undergoes a conformational change when activated. The conformation changes allow it to bind to PIPs and then oligomerize with other MLKL molecules to form pores in the membrane. The pores are octameric, 8 MLKL molecules come together to form the pore.
Translocation to membrane after these form. Disulfide bond-based oligomers

28
Q

Role of MLKL- mechanism (4)

A
  1. PI (4,5)P2 are located on the cytoplasmic half of the cell membrane. Once activated the helix bundle binds to the PIP. A kinase (C-terminus) domain is bound to the helix bundle (N-terminus)
  2. Once at the membrane, the kinase domain associates with 7 other molecules of MLKL to form the octameric pore
  3. These pores form all around the membrane, which contributes to the increase in permeability
  4. MLKL may also be able to oligomerize and form pores in the outer and inner mitochondrial membranes, which could allow for the release of ROS
29
Q

MLKL translocation

A

MLKL translocates to the nucleus- it has a nuclear localization signal. It brings RIP3 with it, and RIP1 shuttles to the nucleus itself. This occurs before MLKL starts forming pores in the membrane. This is still an active area of activation- what are MLKL, RIP1, and RIP3 doing there?

30
Q

Ceramide formation

A

Ceramide is a lipid that may form downstream in necroptosis. It is formed by processing sphingomyelin (a major lipid). Sphingomyelinase (SMase) chops the choline head group of sphingomyelin. Once the head group is removed, we are left with ceramide. This is important for lipid rafts

31
Q

Ceramide and lipid rafts

A

Lipid rafts aggregate and cluster together receptor signaling molecules. This creates a signaling platform. During necroptosis, sphingomyelinases can be activated to produce ceramide. Ceramide is an even better raft forming lipid that sphingomyelin or cholesterol. It causes greater aggregation of lipid rafts and greater clustering to create a stronger signal. This can promote the necroptosis pathway

32
Q

Sphingosine

A

Sphingomyelinase processes sphingomyelin to ceramide, but ceramide can be further processed by ceramidase to create sphingosine (a membrane lipid). One of the fatty acid tails of ceramide is removed to make sphingosine, which can be very damaging to the membrane. It contributes to a loss of integrity and increased permeability.

33
Q

AGEs and ROS

A

These are both side products of cellular metabolism. AGEs are produced as a side product during step 5 of glycolysis, and ROS are side products of the last step of the electron transport chain

34
Q

Advanced glycation end products (AGEs)

A

During glycolysis, glucose is broken down into sugar intermediates. During step 5, glucose takes the form of glyceraldehyde 3-phosphate or DHAP. These glucose intermediates are subject to losing phosphate groups from their structure. If they lose their phosphates, the sugar intermediates become highly reactive sugars called methylglyoxal. Once methylglyoxal attaches to proteins, they are called AGEs because their structure and function are impacted. AGEs are damaging to the cell

35
Q

Methylglyoxal

A

A highly reactive sugar intermediate of glycolysis, which attaches itself to proteins. These proteins are not supposed to be modified with sugar. They may create dimers or trimers that shouldn’t exist or heavily glycate molecules that are not supposed to have sugar as part of their structure.

36
Q

AGE formation (4)

A
  1. During step 5 of glycolysis, glyceraldehyde 3-phosphate forms from fructose 1,6-bisphosphate
  2. Triosephosphate isomerase can reversibly produce DHAP from glyceraldehyde 3-phosphate as well
  3. Glyceraldehyde 3-P and DHAP can both form methylglyoxal. Both of these molecules are susceptible to fragmentation and can lose phosphate groups to form MG
  4. Methylglyoxal is highly reactive/toxic and commonly reacts with lysines. It typically causes damage by forming an MOLD
37
Q

Methylglyoxal lysine dimer (MOLD)

A

Methylglyoxal predominantly interacts with lysine amino acids, so typical damage is the formation of a lysine dimer (MOLD). Methylglyoxal is the added sugar and contributes to an imidazolium crosslink. This creates a dimer where one shouldn’t have existed before. This is an unregulated process that compromises the structure and function of proteins

38
Q

Reactive oxygen species (ROS)

A

Species of oxygen that react with other molecules (proteins, lipids) and damage them. Includes hydrogen peroxide (H2O2), superoxide anion, and hydroxyl radical. Hydrogen peroxide is the least damaging of all the ROS because it can destabilize certain molecules. Free radicals are more dangerous

39
Q

What happens to a protein when it is damaged by ROS?

A

Most commonly, if a protein loses an electron, it will form a covalent bond to compensate. The bonds can be disulfide or other types of covalent bonds. This is compromises the structure and function of the protein because the bond is not supposed to be there- the protein is nonfunctional at this point. There are multiple complications of unregulated covalent bond formation- the protein can lose function, unfold, be cleaved, or aggregate with other proteins

40
Q

Free radicals

A

Superoxide anions and hydroxyl radicals are the most dangerous ROS. They are referred to as free radicals because they have an unpaired electron. Because they have an unpaired electron, they need to steal another electron from another molecule, regardless of where it is. Stealing the electron makes the target molecule into a free radical, which therefore creates a chain reaction of damage and radical formation. More commonly, if a protein loses an electron, it will form a covalent bond to compensate. This compromises the structure and function of the protein because the bond is not supposed to be there- the protein is nonfunctional at this point

41
Q

Formation of ROS

A

Side product of the ETC in oxidative phosphorylation. Cytochrome C oxidase, which has the final electron acceptor of oxygen gas, can release oxygen too early, before it has taken all 4 electrons. As a result, a superoxide anion can be formed- this is the main way that ROS form. A superoxide anion may also be formed when electrons leak from NADH dehydrogenase and are picked up by oxygen (reverse electron transport)

42
Q

Components of the ETC (3)

A
  1. NADH dehydrogenase
  2. Cytochrome bc1
  3. Cytochrome c oxidase
43
Q

Formation of ROS through NADPH oxidase

A

NADPH oxidase is a transmembrane protein complex. Can be activated by necroptosis. It transfers e- to O2 forming O2.- (an ROS). Primarily functions for defense in phagocytic cells, which is why it has functions in the respiratory burst when pathogens are destroyed. Glucose-6-P (1st intermediate) from glycolysis is broken down to ribulose-5-P in pentose phosphate pathway and forms NADPH along the way. This is what is powering NADPH oxidase

44
Q

Necroptosis- Glycolytic stimulation (4)

A
  1. The necrosome can stimulate glycogen phosphorylase (PYGL)
  2. PYGL begins to stimulate the breakdown of glucose from glycolysis
  3. Glycolysis produces methylglyoxal
  4. Methylglyoxal produces AGEs in the cell
    Therefore, necroptosis signaling ramps up glycolysis to produce AGEs
45
Q

Necroptosis and ROS (6)

A
  1. The necrosome can stimulate glycogen phosphorylase (PYGL)
  2. PYGL begins to stimulate the breakdown of glucose from glycolysis
  3. Glycolysis produces pyruvate, which is necessary for the citric acid cycle
  4. The citric acid cycle produces NADH
  5. NADH feeds into the ETC (oxidative phosphorylation)
  6. The ETC is the major producer of ROS in the cell
46
Q

3 other ways for the necrosome to ramp up cellular metabolism

A
  1. The necrosome can stimulate the enzyme pyruvate dehydrogenase (in the mitochondria). The enzyme converts pyruvate to acetyl CoA, which feeds into the citric acid cycle
  2. The citric acid cycle feeds into the ETC, which is the major production of ROS
  3. The necrosome can stimulate the respiratory protein PGC-1 (in the mitochondria) which is part of the ETC- a major producer of ROS
    Ultimately, necroptosis feeds into metabolism and increases the rate of metabolism, creating more toxic side products
47
Q

Fenton reaction

A

When hydrogen peroxide reacts with iron in the cell. It creates a hydroxyl radical, which the the most reactive ROS

48
Q

ROS- protein damage

A

ROS introduces disulfide and other covalent bonds in the protein. The protein may unfold, be cleaved, or aggregate with other proteins. The structure will be messed up and the protein will not function correctly

49
Q

Localization of the necrosome to the mitochondria (4)

A

The necrosome complex can localize to the mitochondria
1. RIP3 associates with a protein called PGAM5L. RIP3 then phosphorylates this protein
2. PGAM5L then goes to its partner protein (PGAM5S) located in the outer mitochondrial membrane
3. This brings the necrosome over to the mitochondria
4. Ramps up glycolysis, the citric acid cycle, and the electron transport chain

50
Q

How are ROS released from the mitochondria into the cytoplasm? (5)

A
  1. Pores form in both mitochondrial membranes. MLKL can form a pore in the inner mitochondrial membrane and Bak and Bax (BH123 proteins) can form pores in the outer mitochondrial membrane
  2. Once the pores form, ROS can be released, which contributes to necroptosis. OR:
  3. Dynamin related protein 1 (Drp1) works on the fission (division) of mitochondria. Drp1 is dephosphorylated
  4. Then, 2 molecules of Drp1 will dimerize. Just like dynamin, they will wrap around the mitochondria and pinch it apart into 2
  5. During this division process, ROS will be released into the cytoplasm and contribute to necroptosis
51
Q

How do BH123 proteins work?

A

They oligomerize and form pores in the mitochondrial outer membrane. This helps to induce the release of cytochrome C and other proteins into the cytoplasm. BH123 proteins include Bak and Bax- Bak is always in the outer membrane and Bax is recruited to the outer membrane from the cytoplasm so both of these proteins can oligomerize. BH123 proteins are pro-apoptotic. However, they are not specific to apoptosis and can be used for other pathways, including necroptosis

52
Q

Autophosphorylation of RIP1

A

ROS will steal electrons from RIP1. When RIP1 loses an electron, it will need to form a complex with other molecules of RIP1. Disulfide bonds will form between them, creating a RIP1 oligomer. When brought together in close proximity, RIP1 molecules can phosphorylate one another and therefore activate each other. This is the autophosphorylation process

53
Q

Why are ROS necessary to induce necroptosis signaling?

A

ROS are produced via metabolism, metabolism kicks off with glucose & glycolysis, and therefore glucose drives necroptosis. The more glucose you have, the more necroptosis you should have- hyperglycemia can actually induce necroptosis

54
Q

Downstream effectors of necroptosis that don’t always occur (3)

A
  1. Calcium dependent calpains- cause cytoskeletal damage
  2. Phospholipases (PLA)- causes membrane damage and lysosome permeabilization
  3. Lysosome permeabilization. Permeability is also the result of ROS, AGEs, sphingosine, PLA2-adds fuel to the fire
55
Q

Effectors of necroptosis

A

There are many effectors, and not all need to be involved to produce cell death by necroptosis (depends on cell type, perhaps the stimulus). ROS and MLKL are most often involved- MLKL is always involved and ROS is often involved

56
Q

Secondary effects of necroptosis (3)

A
  1. Bystander PCD
  2. Release of damage-associated molecular patterns (DAMPs)
  3. Release of damaging agents-AGEs and ROS, which may also cause bystander PCD
57
Q

Bystander programmed cell death (PCD)

A

Release of substances from the dying cell causes death of neighboring cells during necroptosis. Release of TNF, FasL, TRAIL in vesicles- this will induce necroptosis or apoptosis

58
Q

Release of damage-associated molecular patterns (DAMPs)

A

HMGB1- nuclear protein and AGEs are released among other inflammatory mediators. This induces inflammation

59
Q

Necroptosis- release of agents

A

Vesicles form and pinch off the cell membrane during necroptosis. This is one way in which TNF and FAS ligand (both membrane proteins) are released, and both can cause neighboring cell death. When the membrane is more permeable, DAMPs (ROS, AGEs) can leak out and damage neighboring cells or cause inflammation. Nuclear proteins (HMGB1) can also be released and cause inflammation during this process. As increased permeability occurs, osmosis occurs, and the cell eventually explodes. Only pieces of the cell remain, which can also cause secondary effects

60
Q

Endogenous inhibition of necroptosis (3)

A
  1. Caspase-8
  2. RIP1 ubiquitylation
  3. ESCRT complex
61
Q

Caspase 8

A

Endogenous inhibitor of necroptosis, inactivates RIP1

62
Q

RIP1 ubiquitylation

A

Endogenous inhibitor of necroptosis. RIP1 is tagged with ubiquitin, and when ubiquitin is present, we are not on a path to cell death. CYLD must deubiquitylate, A20 tags with ubiquitin for destruction by proteasome

63
Q

ESCRT complex

A

An endogenous inhibitor of MLKL

64
Q

ESCRT complex function

A

MLKL, once phosphorylated, forms pores in the membrane, and contributes to cell death. The ESCRT complex causes actin rearrangement such that vesicles will form and pinch off. A protein called ESCRT3 is what actually pinches off the vesicle. The idea is that we are shedding MLKL pores from the membrane. This does not inhibit necroptosis but it will delay for a time.

65
Q

Cellular changes that occur during apoptosis (5)

A
  1. DNA breaks down- the nucleus condenses and begins to fragment
  2. Organelles fragment
  3. Phosphatidylserine goes to the outer leaflet of the membrane
  4. The cell breaks apart into apoptotic bodies, which are then removed by macrophages with phosphatidylserine receptors
  5. The process results in little to no inflammation
66
Q

Cellular changes that occur during necroptosis (4)

A
  1. Membrane permeability increases, causing cellular expansion
  2. Organelles lose integrity and release all of their contents
  3. Osmosis continues to occur, the cell lyses and releases its intracellular contents
  4. This is a highly inflammatory process

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