The Mitochondria
What is a caspase?
"C-ASP-ASE" "-ASE" = A type of enzyme that breaks down proteins "-ASP" = it specifically breaks down proteins AFTER the aspartate residue "C" = breaks down the proteins WITH a cystine residue
NADH
A coenzyme that has the ability to TRANSFER ELECTRONS -A "malate aspartate shuttle" -NADH is taken from the cytoplasm and transported into the mitochondria
FADH2
A coenzyme, like NADH, that assist in transferring electrons
Caspase 3
A cysteine protease that cleaves 200+ proteins and ultimately leads to cell death
How does Synta Pharmaceuticals treat BCL-2 over-expressing cancers?
A drug that trigger apoptosis through targeting the ETC in the cancer cell's mitochondria -Increases oxidative stress
HEPG2
A hepatoma cell line -Should NOT be used for in vitro toxicology because of their high glycolytic activity
BCL-2
A key ANTI-apoptic protein -Typically over-expressed in many cancers -Inhibits pro-apoptotic signals, allowing the cancer cell to survive under stressful conditions -Makes cancer cells chemo & radiation-resistant RESULT: Too much BCL-2 = No mitochondria-triggered apoptosis
PIGBOS
A microprotein that only has 54 amino acids -Plays a role in the Unfolded Protein Response (UPR) triggered by ER stress from the presence of unfolded proteins
F0F1-ATP Synthase
A multi-subunit protein complex on the inner mitochondrial membrane -Catalyzes the phosphorylation of ADP to ATP -Uses the H+ motive force, which was generated by an electron transport chain
Substrate Phosphorylation
A reaction that DIRECTLY transfers a phosphate group from a substrate to ADP to produce ATP -Much less efficient than oxidative phosphorylation at generating ATP
How does Obatoclax treat BCL-2 over-expressing cancers?
A small molecule that inhibits the BCL-2 protein
SGLT (Sodium-Dependent Glucose Transporter)
A symporter for glucose -Contributes to renal glucose reabsorption -Other glucose transporters: GLUT 1, etc.
BC1 Complex
A transmembrane protein on the INNER mitochondrial membrane -Reduces Cytochrome C -It is ANTI-apoptotic
Mitochondrial Permeability Transition Pore (MPTP)
A transmembrane protein residing in the mitochondrial INNER MEMBRANE and the OUTER MEMBRANE -When the MPTP is closed, apoptosis will not occur -If MPTP is open, it can trigger the mitochondrial apoptotic cascade.
Microtubules
A tubular structure in the cytoplasm that transport mitochondria and other organelles -Components of the cell skeleton that determines the shape of a cell. -Other Functions: a) Assembly of mitotic spindle b) Involved in dividing cells c) Axon extension of neurons
How are BCl-2 overexpressing cancers treated?
A) Genasense B) Obatoclax C) Venetoclax D) Synta Pharmaceuticals
Cytochrome C Function
A) On the electron transport chain, shuttling electrons from 3rd to 4th complex on the ETC B) When released from the inter-membrane space to the cytoplasm, it triggers APOPTOSIS.
Why Fission?
A) To distribute mitochondria into 2 daughter cells B) To eliminate damaged parts of the mitochondria (most likely reason)
Why Fusion?
A) To generate a more homogenous pool B) To restore damaged mitochondria C) Plays a part in mitochondria localization
What does Cytochrome C bind to once it's released from the mitochondria into the cytoplasm?
APAF1 (which is in the cytoplasm)
What triggers mitochondrial permeability transition pores (MPTP) to open?
Accumulation of Ca2+ in the matrix Increased oxidative stress
What does Cytochrome C activate in apoptosis?
Activates a family of enzymes in the cytoplasm called CASPASES
Intrinsic Apoptosis Pathway
Aka Mitochondrial Pathway A) Initiation for apoptosis via DNA lesions B) p53 protein C) Activates BAX in the cytosol D) Activated-BAX becomes mitochondrial membrane bound E) Opens the "VDAC" channels of the mitochondrial outer membrane F) Release of Cytochrome C into the cytosol G) Cytochrome C binds to APAF-1 H) Recruits Pro-Caspase 9 to activate Caspase 9 I) Caspase 3 is activated J) Cleavage of cytosolic and nuclear proteins K) Cell death
Colchicine
An anti-mitotic drug -Prevents the elongation of microtubules and STOPS the process of cell division
Cyclosporin A
An immunosuppressant -Protects stem cells during storage and transport -Blocks MPTP pores from opening -Treats organ rejection post-transplant
APAF1
Apoptotic protease activating factor 1 -Key molecule in the intrinsic mitochondrial pathway of apoptosis -Triggered by the release of Cytochrome C -Cytochrome C actually binds to APAF1 to form a large complex called apoptosome (mediates the activation of an initiator caspase at the onset of apoptosis)
How are the intrinsic and extrinsic pathways of apoptosis related?
BOTH pathways converge at Caspase-3, leading to activation of other proteases. BOTH control apoptosis
Porin
Barrel proteins that cross a cellular membrane and acts as a pore through which molecules can diffuse through -Located on the outer mitochondrial membrane -Passes molecules up to 5,000 Daltons
How does Venetoclax treat BCL-2 over-expressing cancers?
Binds to the BCL-2 protein
Antisense Oligonucleotide
Binds to the target mRNA, inducing its degradation and preventing the mRNA from being translated into a detrimental protein product (e.g. BCL-2)
How is calcium involved in cellular respiration, which takes place in the mitochondria?
Ca2+ is required for multiple enzymes involved in cellular respiration and REGULATES the rate of ATP production.
What is "intrinsic" cell stress?
Can be caused by: -Increase in reactive oxidative species (ROS) -Chemotherapy & radiation -Too many mutations in nuclear DNA RESULT: Triggers the mitochondria to initiate apoptosis
Super Resolution Microscopy & Mitochondria
Can be used to image and create 3D models of mitochondria from cells
Warburg Effect
Cancer cells are "smart." -Increase the rate of glycolysis so that more ATP is generated compared to normal cells -Doesn't care what happens to the mitochondria because they are getting their energy from glycolysis (in an anaerobic environment)
Hepatocytes
Cells in the liver, involved in many functions (detoxification, carbohydrate metabolism, lipid metabolism, etc.)
Heme
Central to the ETC a) Synthesized in the mitochondria via mtDNA b) Modified in the cytoplasm c) Translocated back into the mitochondria
How do different intra-mitochondrial levels of Ca2+ affect the mitochondria?
Low = GOOD -Increases the rate of proton pumping and ATP synthesis Intermediate = NOT GOOD, BUT NOT BAD -Slight decrease in ATP synthesis -Still able to maintain membrane integrity and maintain a proton electrochemical gradient High = BAD -Dangerous and toxic; leads to mitochondrial permeability transition and membrane RUPTURE -Unable to maintain a proton electrochemical gradient and unable to generate ATP
BAD & BAX
Members of the BCL-2 family of proteins -The over-expression of BAD & BAX initiates and speeds up apoptosis.
Calcium-Phosphate Granules
Mitochondria can accumulate, store, and release a large amount of calcium ions.
Mitochondrial DNA
Mitochondria contain their own DNA; inside the matrix -Most of mitochondrial DNA is lost -There's 37 genes; total of 16,500 bases.
What is the key regulatory enzyme required for glycolysis?
Phosphofructokinase-1 (PFK) -Catalyzes the first step in glycolysis -Increases or decreases the rate of glycolysis in response to the cell's energy requirements (high ATP will inhibit PFK activity and glycolysis)
How is glucose phosphorylated when it enters the cell, in order to trap it in?
Phosphorylated by hexokinase to make glucose-6-phosphate
Why is glucose phosphorylated when it enters the cell?
Phosphorylation of glucose upon cell entry is to effectively TRAP it in the cell -Makes it very negatively charged (won't leak out of the cell; almost like a tracking mechanism)
Caspase 9
Precursor: Procaspase 9 The INITIATING CASPASE of apoptosis -Cleaves and activates downstream executioner caspases -Cleaves Caspase 3, 6, and 7
What is the ideal cell type for in vitro toxicology?
Primary human hepatocytes -But very expensive ALTERNATIVE: Trick the HEPG2 to acting like a primary human hepatocyte
How does prolonged UPR lead to cell apoptosis?
Prolonged UPR will induce ER stress-associated apoptosis because there is an overload of unfolded or misfolded proteins in the ER is not resolved.
Outer Mitochondrial Membrane
Separates the inter-membrane space from the cytosol
Where is heme synthesized? Where is heme modified?
Synthesized in the mitochondria Modified in the cytoplasm
How does mitochondria mediate Ca2+ homeostasis?
TAKES UP high levels of Ca2+ directly from the cytosol and STORES it
How does Genasense treat BCL-2 over-expressing cancers?
Targets the BCL-2 mRNA strand with a BCL-2 antisense oligonucleotide -Prevents the synthesis of BCL-2 protein
How does the outer mitochondrial membrane respond to apoptosis?
The outer mitochondrial membrane becomes MORE PERMEABLE. -This permeability is regulated by the BCL-2 family of proteins.
Mitochondria Fusion
The physical merging of the outer and then the inner membranes of 2 originally distinct mitochondria -Allows the transfer of gene products between mitochondria for optimal functioning -Necessary for cell survival and the movement of mitochondria within neurons
Procaspase 3
The precursor molecule to caspase 3 The conversion of procaspase-3 to caspase-3 is often considered as the "point of no return" for a cell.
Where are mitochondria usually found?
Typically localized where ATP is required the most
Oxidative Phosphorylation
a) Requires a functioning citric acid cycle b) Requires functioning F0F1 particles (part of ATP synthase) c) Requires a proton gradient across the inner mitochondrial membrane d) Requires a transmembrane voltage potential across the inner mitochondrial membrane
Matrix
Contains enzymes that are responsible for the citric acid cycle reactions Contains mitochondrial DNA, ribosomes, etc.
In apoptosis, what happens when the mitochondrial outer membrane becomes more permeable?
Cytochrome C, normally found within the intermembrane space, can EXIT the mitochondria and ENTER THE CYTOPLASM. -Cytochrome C is a member of the electron transport chain, and it helps shuttle electrons between the 3rd and 4th complexes of the ETC.
Extrinsic versus Intrinsic Apoptosis Pathway
Extrinsic Initiation: -Begins OUTSIDE the cell, when conditions of the environment determine that a cell must die Intrinsic Initiation: -Begins WITHIN the cell when an injury occurs; the resulting stress activates the apoptotic pathway
When and how was the first mitochondria seen?
First seen in the 19th century -Using the light microscope
Cristae
Folds on the inner membrane of the mitochondria -Increase the available surface area to enhance the productivity of cellular respiration -Extend deeply into the matrix -Contain most, if not all, of the fully assembled complexes of the ETC
Mitochondrial Contact Site and Cristae Organizing System (MICOS)
Forms contact sites between the mitochondrial inner and outer membranes
Where is Cytochrome C normally found?
Found within the inter-membrane space
Fission versus Fusion
2 process that counteract each other
Cardiolipin (CL)
A 4-legged phospholipid -Serves as the "glue" for the ETC -INCREASES the transmembrane potential -Located on the inner membrane (NOT on the outer membrane)
Tunneling Nanotubes
A BRIDGE between two adjacent cells -How mitochondria can travel between TWO different cells -Can exchange a lot of things: Ca2+, vesicles, mitochondria, etc.
Electron Transport Chain
Located on the inner mitochondrial membrane -Contains FOUR sets of enzyme complexes (Complex I to IV) -Electrons are donated by NADH and FADH2 to Complex I, and passed down the ETC -Movement of electrons generates energy for H+ ions to be pumped out into the inter-membrane space -High concentration of H+ ions in the inter-membrane space as compared to the matrix of the mitochondria -Electrochemical gradient formed, where H+ ions cannot pass the inner mitochondrial membrane -ATP synthase transports H+ into the matrix; uses the ENERGY from the FLOW to phosphorylate ADP into ATP
What happens if mitochondrial permeability transition pores (MPTP) are opened for too long?
Loss of mitochondrial membrane potential, leading to cessation of ATP synthesis RESULT: Cell death
Structure of Mitochondria
a) Outer membrane b) Inter-membrane space c) Inner membrane
Basic Characteristics of Mitochondria
a) Pleomorphic (several different shapes) b) Love to attach to microtubules
How does the mitochondria contribute to apoptosis?
-During development -DNA damage -External stress -ROS (reactive oxygen species) -Infection
HVEM
-Increased theoretical limit of resolution -1 million Volts -Visualizes WHOLE CELLS -You can also see the whole mitochondria
Describe the controlled cascade action that occurs between caspase enzymes
1) Cytochrome C in the cytoplasm 2) APAF1 3) Procaspase 9 = Caspase 9 4) Procaspase 3 = Caspase 3
What happens during chemiosmosis (aka the Electron Transport Chain)?
1) Electrons are transferred from NADH and FADH2 to protein complexes and electron carriers. 2) Electrons are used to generate a proton gradient. 3) Protons are pumped across to the inter-membrane space from the inner mitochondrial membrane. 4) An electrical + chemical gradient is formed. 5) Protons travel through ATP synthase. ATP synthase adds a phosphate group to ADP. 6) Oxygen is the final acceptor of the electrons. When oxygen combines with 2 H's, you get H2O (water).
Overview of Energy Production (aka Aerobic Cellular Respiration in Eukaryotes)
1) Glycolysis -Takes place inside the cytoplasm -No O2 required -Glucose is converted into pyruvate -NET YIELD = 2 pyruvate + 2 ATP + 2 NADH molecules 2) Pyruvate molecules are transported INTO the mitochondria via active transport 3) 2 pyruvate converted to 2 acetyl CoA, 2 CO2, and 2 NADH -In the matrix 4) Krebs Cycle (aka Citric Acid Cycle) -Still in the matrix -FADH2 is produced 5) Electron Transport Chain & Chemiosmosis
3 Types of Caspases
1) Initiator Caspases (e.g. Caspase 2, 8, 9) INITIATE the apoptosis signal 2) Executioner Caspases (e.g. Caspase 3, 6, 7) CAUSE mass proteolysis, leading to apoptosis 3) Inflammatory Caspases (e.g. Caspase 1, 4, 5, 11, 12) Not involved in apoptosis; involved in inflammatory cytokine signaling
Basics about mtDNA
1) mtDNA are self-replicating 2) mtNDA codes almost exclusively for hydrophobic proteins 3) Must be HIGHLY regulated by the mitochondria (might as well have them there in the organelle)
What does mitochondria fission require to occur?
DRP1
How does the excessive release of Ca2+ from the ER affect the cell overall?
Excessive Ca2+ released by the ER will increase the Ca2+ in the mitochondria -Release of signaling molecules such as Cytochrome C and caspases
Where are most mitochondrial lipids synthesized?
In the ER and transported to the mitochondria via the ER-associated MAMs
Where are most mitochondrial proteins synthesized?
In the cytosol
What does the loss of PIGBOS lead to?
Increases the Unfolded Protein Pathway (UPR), leading to increased cell death
How are caspases initially synthesized?
Initially synthesized as INACTIVE PROCASPASES (the precursor of caspase) -Once activated during apoptosis, these procaspases are converted to caspases via rapid cleavage.
What is Na+/K+ ATPase?
Na+ and K+ move against the concentration gradient. -Pumps 2 K+ ions INTO the cell (where K+ levels are high) -Pumps 3 Na+ ions OUT of the cell and into the extracellular fluid -Depends on ATP RESULT: A transmembrane Na+ gradient across the plasma membrane.
BCL-2 Family of Proteins
Named because it was first discovered in a B cell lymphoma TWO Types of Proteins in this Family: A) Pro-apoptotic (push the cell towards apoptosis) B) Anti-apoptotic (oppose apoptosis)
Crista Junction
Narrow openings that connect the cristae membranes to the inner boundary membrane
Glycolysis
Net Products = 2 Pyruvate molecules (or lactic acid), 2 NADH, and 2 ATP -Anaerobic -Utilizes substrate phosphorylation -Done in the cytoplasm, outside of the mitochondria
Why are mitochondria so attached and dependent on microtubules?
OBSERVATION: Mitochondria are always moving on microtubules (which serve as tracks or molecular motors) via the activity of motor proteins kinesin-1 and dynein A
How are the mitochondria and the ER involved in calcium homeostasis?
The ER has the main STORAGE of Ca2+ ions. The mitochondria is the main EFFECTOR of Ca2+ ions (needs it for ATP production and apoptosis). CONCLUSION: The ER can alter the production of ATP by altering the release of Ca2+ at the mitochondria-associated membranes.
How does the UPR pathway affect calcium homeostasis and the mitochondria's response to it?
The UPR response (unfolded proteins accumulate) triggers the MORE Ca2+ to be released from the ER to the mitochondria. -Increases ATP production -Increases the cell's ability to properly fold protein using the newly-generated ATP
Procaspases
The inactive precursors to caspases -Activated by cleaving one or two specific aspartic acids
Structure of the Inner Mitochondrial Membrane
The inner membrane is SUBDIVIDED into: a) The inner boundary membrane, which runs parallel to the outer membrane b) Crista membrane BOTH are LINKED by "crista junctions"
Apoptosome
The protein complex (Cytochrome C bound with APAF1) that mediates the ACTIVATION of an INITIATOR caspase at the onset of apoptosis -Triggered by the release of Cytochrome C from the mitochondria
Inter-Membrane Space
The region between the inner and outer membranes
How are caspases activated?
Via cleavage
What can be used to explore the UPR and MPTP pathway?
Western Blotting
Mitochondria Fission
When mitochondria divide or segregate into 2 separate mitochondrial organelles -Involves constriction -Requires DRP1
During apoptosis initiation, what is considered the "point of NO return" for the cell?
When procaspase 3 is converted to caspase 3
Inner Mitochondrial Membrane
Where electron transport chain (ETC) is located -Where ATP is generated (where ATP synthase is located) -Where H+ is pump out into the inter-membrane space -Has 5-15x MORE "membrane" than the outer mitochondrial membrane due to oxidative phosphorylation and the ETC A VERY BUSY place
ER-associated-Mitochondria-associated Membranes (ER-Associated MAMs)
Where the mitochondrial membranes are associated with the ER -Tethered together -Proteins regulate Ca2+ homeostasis, synthesis of lipids, and triggering of apoptosis -Many of the proteins function in LIPID METABOLISM.
Peter Mitchel & the Chemiosmotic Model of Mitochondrial Function
Won the 1978 Nobel Prize States that the TRANSFER OF ELECTRONS down an electron transport system through a series of redox reactions RELEASES ENERGY that is used to generate a H+ electrochemical gradient
