Week 11 damage response

• Extrinsic signals

  • Initiates from a different cell or tissue
  • Usually signals for a response of a cell that is part of a larger physiological process

  Intrinsic signals

  • Initiate from cues within the cell in question, and have a response in the same cell
  • Can indicate the following within a cell
  • Metabolic flux
  • Nutrient availability
  • Damage

  
• how signalling damage stress response usually happens:

  1. Cell stress or damage
  2. Sensor system picks up this stress
  3. Signal transduction moves it downstream allowing for a function to occur
  4. Then one of 2 things happen
  5. the cell tries to adapt and survive
  6. the cell goes through apoptosis

  
• Mechanistic Target of Rapamycin complex 1 (mTORC1)

  • Mitogenic (induces cell division) cues ---> growth factors
  • PI3K-Akt signals are able to activate mTORC1
  • Ras-Erk signals can activate mTORC1
  • Metabolic cues (induces breaking/making)
  • amino acid availability activates mTORC1
  • Glucose deprivation Inactivates mTORC1
  • other stress or damage Inactivates mTORC1
• activation of mTORC1 by mitogenic cues (growth factors)

  • Akt phosphorylates and inactivates TSC complexes (TSC2 directly)
  • Tuberous Sclerosis Complex (TSC) is a GTPase-activating protein that, when active, inactivates RHEB
  • RHEB is a GTPase that causes activation of mTORC1
  • mTORC1 is a serine/threonine kinase, a "master regulator
• When the TSC complex is OFF due to phosphorylation of TSC3, RHEB is not inhibited anymore and mTORC1 can turn on.

  
• Activation of mTORC1 by metabolic cues (amino acids)

  • Amino acids sensed by the V-ATPase and other mechanisms which lead to activation of Ragulator
  • Ragulator is a GEF for Rag A/B proteins (GTPases)
  • Once activated (GTP-bound), Rag A/B leads to mTORC1 binding to the lysosome and mTORC1 activation by Rheb

  
• mTORC1 activation requires:

  • Signalling by PI3K-Akt or Ras Erk (mitogenic signals)

  AND

  • Nutrient (amino acid) availability (metabolic signals)

  
• Once activated, mTORC1 promotes

  • biomass production (ribosome biogenesis, translation)
  • Lipid biosynthesis
  • Shift in glucose metabolism AWAY from oxidative phosphorylation (to maximize ATP production) and towards glycolysis

  
• AMP-activated protein kinase (AMPK) is made up of 3 subunits:

  • a - the catalytic subunit (gets phosphorylated)
  • B - regulatory subunit
  • y - regulatory subunit that binds to and senses AMP and ATP

  
• The y subunit of AMPK has 4 major sites

  • site 1 has a high affinity to ATP
  • Site 2 usually has nothing
  • site 3 has high affinity to ATP, ADP, and AMP
  • site 4 has high affinity with ADP and AMP

  
• depending on what type of energy variation we have (ATP, ADP, AMP) will determine what will bind to the site 3 competitive binding site.

  • when energy is high (high ATP) ATP binds to site 3
  • when energy is low (low ATP) AMP/ADP will bind to site 3
  • this will recruit LKB1 and CaMKKB to phosphorylate the a-subunit changing AMPK conformation
  • This turns on the AMPK kinase domain

  
• AMPK under different conditions:

  • under condition of energy sufficiency
  • Very low levels of AMP and high levels of ATP, so only ATP binds to AMPK
  • ATP-bound AMPK is a very good substrate for a phosphatase that de-phosphorylates AMPK
  • Non-phosphorylated AMPK is INACTIVE

  
• AMPK under different conditions:

  • Under conditions of energy deficiency/stress
  • Elevated levels of ADP and/or AMP, AMPK binds preferentially to AMP/ADP at site 3
  • High ADP levels are converted ATP and AMP by adenylyl kinase, to increase ATP levels
  • AMP-bound AMPK is not a good substrate for the phosphatase, retaining higher AMPK phosphorylation levels
  • Phosphorylated AMPK is ACTIVE

  
• AMPK function during metabolic stress
• AMPK function during metabolic stress

  • once active, AMPJ action has 2 major outcomes:
  • REDUCE (inhibit) ATP consumption
  • INCREASE catabolism (energy production)

  
• Adenylate kinase (AK) can turn ADP into ATP + AMP and vise versa

  • As ATP levels are starting to decline, ADP concentration will be higher
  • We can turn ADP into ATP and AMP --> This will increase ATP levels while decreasing ADP
  • Quick way to get ATP under ADP-rich conditions
  • AMP will also help to stimulate AMPK

  
• AMPK function during metabolic stress

  • AMPK controls many cellular processes.
  • Acetyl CoA carboxylase to control fatty acid metabolism
  • Glucose uptake into cells
  • cell migration

  
• AMPK favours catabolism and shuts off ATP consumption: Acetyl-CoA carboxylase (ACC) is inhibited in the process

  • the phosphorylation of ACC by AMPK inactivates it

  
• Acetyl-CoA carboxylase (ACC)

  • ACC with ATP makes Malonyl CoA
  • Malonyl CoA promotes fatty acid synthesis and inhibits B-oxidation
  • B-oxidation is a mechanism that makes Acetyl CoA
  • Acetyl CoA: cofactor used in many biological processes where it can be used for energy production

  
• when AMPK phosphorylates ACC this enhances B-oxidation --> Acetyl CoA is made --> more energy

  
• AMPK controls membrane traffic to increase glucose transport into cells:

  • Glucose transport into cells occurs by the action of GLUT --> a family of glucose transporters at the cell surface
  • AMPK activation REDUCES the endocytosis of GLUT1 (every cell) and GLUT4 (muscle cells)
  • More glucose transport proteins at the cell surface = more glucose transport into cells = more ATP production

  
• GLUT is found on the surface of the cell

  • normally takes in glucose in the cell to make ATP

  GLUT normally has a set lifetime before they are sequestered in and recycled --> this controls blood glucose levels

  • internalized periodically so they are not always on the surface

  
• In a low ATP situation, --> GLUT endocytosis is inhibited

  • a-arrestins help sequester glucose --> When AMPK is activated it can phosphorylate a-arrestins which inactivates it --> This allows for more glucose transport since GLUT stays on the surface

  
• AMPK slows down cell migration:

  • Integrin proteins that link the cell to the extracellular matrix are moved around the cell by endocytosis
  • Cell migration requires the membrane traffic of integrins from the cell posterior to the cell anterior
  • AMPK activation causes a reduction in the rate of integrin membrane traffic and cell migration

  
• Trafficking of integrins from one face of a cell to another by endocytosis uses up energy --> when AMPK is turned ON it reduces the uptake and endocytosis of integrins --> therefore saving energy

  
• AMPK: is a possible therapeutic target for diabetes treatment

  • Diabetes: a defect in insulin production (type 1) or insulin action (type 2) that results in elevated blood glucose
  • AMPK: can increase glucose consumption and metabolism by skeletal muscle cells

  Diabetes increases glucose levels which is bad but AMPK increases the consumption of glucose which evens it out

  
• AMPK and cancer

  • when AMPK is ON
  • Pro-tumor: can generate ATP to help cancer survival
  • Anti-tumor: Can halt cancer growth by arresting cell cycle
  • when AMPK is OFF
  • Pro-tumor: Can generate biomass to help cancer cells grow
  • Anti-tumor: Prevents metabolic adaptation in cancer

  
• Oxygen levels are also important for metabolism

  • Aerobic respiration is required for glycolysis
  • We need a sensor system to monitor oxygen levels in cells
  • This is important so that cellular metabolism can switch if oxygen levels are too low

  
• Hypoxia-inducible factor 1a (HIF1a) helps yo check for O2 levels

  • when there is high O2 levels, there will be low HIF1a levels (OFF)
  • When there is low O2 levels, there will be high HIF1a levels. (ON)
  • cells switch to anaerobic respiration and secrete VEGF (growth factor) that stimulates angiogenesis

  
• low oxygen --> high HIF1a --> anaerobic respiration --> VEGF secretion --> angiogenesis stimulation

  angiogenesis: growth of new blood vessels

  • increase surface area for oxygen uptake
  • more O2 will be received in the cell
  • goes back to high O2 levels --> low HIF1a --> no more VEGF

  
• The mechanism for HIF1a (normoxia level) (high O2)

  • HIF1a gets some PTM form HPH
  • the proline residue gets hydroxylated
  • the lysine residue gets acetylated
  • This PTM allows the attachment of ubiquitin protein ligase complex
  • ubiquitination of HIF1a leads to degradation by proteasome.

  
• in hypoxia conditions (lots of HIF1a, little O2):

  • HIF1a is phosphorylated by ARNT
  • These 2 act together as a transcription factor that responds to HIF1a response elements (HRE)
  • DNA is regulated this way
  • can have positive/negative functions

  
• Hypoxia conditions of HIF1a:

  • can increase blood flow and ATP production (increase of GLUT1 and glycolytic enzymes) allowing for improved cell survival in times where O2 is scarce

  
• • under normal O2 (normoxia) levels, HIF1 is hydroxylated and acetylated, resulting in ubiquitination of HIF1 by ubiquitin protein ligase complex and degraded by proteosomes.
• • under conditions of reduced (O2) (hypoxia) levels, HIF1 is stabilized and phosphorylated by ARNT --> they become transcription factors to improve cell survival
• When present, HIF1 binds to the promoter region of target genes, leading to increased expression of:

  • vascular endothelial growth factor (VEGF) to promote angiogenesis
  • Inducible nitric oxide synthase (iNOS): vasodilation to increase blood flow
  • GLUT glucose transporters and glycolytic enzymes to make more ATP
• Reactive oxygen species (ROS) --> biproduct of enzymatic function by NADPH oxidase and electron transport chain (ETC) of the mitochondria during ATP production.

  • ROS is bad --> will damage protein, DNA, lipids
  • we have mechanisms that turn ROS into something that isn't as bad

  
• • ROS can contribute to cell signalling (E.g. phosphatases are inactivated by ROS)

  AND/OR

  • ROS can lead to protein/DNA/lipid damage.

  
• Sensing of reactive oxygen species by p38 MAPK (non-classical

  • Ask1 senses for High levels of ROS
  • Ask1 is a mitogen-activated protein kinase kinase kinase (MAP3K)
  • Ask1 phosphorylates and activates MKK3/4/6, which in turn phosphorylates and activated p38 (MAPK)
• NADPH oxidase -> mitochondrial enzyme that is part of the ETC that catalyzes oxygen into super oxide free radicals

  • highly reactive form of oxygen that interacts with proteins,DNA,etc in a damaging manner.