HSS2305 Lecture 6 on Mitochondria PDF

What “breathes” in O2 and pyruvate, “spits” out CO2 and ATP, rhymes with hypochondria and Mitochondria! confuses my neighbour?  HSS2305: Molecular Mechanisms of Disease Lecture 6 – Mitochondria and Aerobic Metabolism – Today’s Outline Sample Questions Mitochondria and Aero...

What “breathes” in O2 and pyruvate, “spits” out CO2 and ATP, rhymes with hypochondria and Mitochondria! confuses my neighbour?  HSS2305: Molecular Mechanisms of Disease Lecture 6 – Mitochondria and Aerobic Metabolism – Today’s Outline Sample Questions Mitochondria and Aerobic Metabolism! Announcement Rare Disease Assignment Topics Emailed Out Please let me know if you did not receive an assignment topic! Deadline May 27 at midnight. Reminder: NO PLAGIARISM! Reference Managers: Save you time and improves accuracy! BUT don’t underestimate how much time it takes to put in references!!! https://uottawa.libguides.com /citation-management/home Announcement Test Respondus Lockdown Browser and Respondus Monitor Please let me know if you are unable to complete the quiz/want to use an alternative proctoring (i.e. zoom) other than Respondus Monitor as your Midterm is on May 19th (with zoom you still need to use Respondus lockdown and the zoom camera needs to be a separate webcam with a view of your screen, hands, head and desk space. If you use Respondus Lockdown Browser and Respondus Monitor then the camera can be your usual computer screen camera). Sample Questions 1. How many ATP molecules are used in the investment phase of glycolysis? 1. 0 2. 1 3. 36 4. 2 2. Oxidation is the: 1. Gain of electrons 2. Loss of electrons 3. Production of O2 4. Production of Oxygen Free Radicals 3. Glycolysis does not produce: 1. NADH 2. ATP 3. FADH2 4. Pyruvate 5. Glucose 4. What enzyme converts NADH to NADPH and vice versa? 1. Transhydrogenase 2. Hexokinase 3. NAD+ Kinase 4. NAMPT Mitochondria Singular: Mitochondrion Plural: Mitochondria Role: “Powerhouse of the Cell” Many others!!! Today’s Case Study Healthy baby born at term No complications in the pregnancy Met all developmental milestones up to 6 months 12 months of age Lost basic motor skills such as: Sucking Head control Walking Talking Irritable Appetite loss Seizures 18 months Vision loss Respiratory problems MRI found brain lesions, particularly in the midbrain and brainstem  Origin of Mitochondria The Origin of Mitochondria Endosymbiotic theory: Some organelles in eukaryotic cells were once prokaryotic microbes Examples: Mitochondria Believed to have developed from Alpha-proteobacteria Chloroplasts Believed to have developed from Cyanobacteria Evidence: Same size as prokaryotic cells Divide by binary fission https://mmegias.webs.uvigo.es/02-english/5-celulas/1- Contain DNA endosimbiosis.php  Mitochondria Structure 2 Membranes: 1. Outer mitochondrial membrane 2. Inner mitochondrial membrane 1. Inner boundary membrane 2. Cristae Contains Electron Transport Chain (ETC) - ATP synthesis machinery 2 Compartments: 1. Intermembrane space 2. Matrix Contains circular mitochondrial DNA, ribosomes, and enzymes (ex. TCA Cycle)  Mitochondria Structure - Membranes Mitochondria membranes are highly proteinaceous: Outer membrane ~50% protein Inner membrane ~75% protein Facilitates transport: Metabolites Ions Proteins Ex. Porin (a.k.a. voltage-dependent anion channel – VDAC) Large pore-forming protein a major metabolite channel Most abundant protein in outer mitochondrial membrane Maintains outer membrane permeability: the exchange of ions and small molecules, including NADH , and ATP, across the mitochondrial outer membrane Mitochondria Structure – Inner Mitochondrial Membrane Contains: ETC Cristae Invaginations/folds Inner membrane largely impermeable Except: O2 CO2 H2O Contains: Uniport Antiport systems for specific metabolites Regulated permeability key to bioenergetics of a cell, most importantly the electron transport chain  Mitochondria Structure - MATRIX Mitochondrial matrix Inner most compartment TCA cycle occurs here Fat (β) -Oxidation Contains: Circular DNA molecule (mitochondrial DNA - mtDNA) Ribosomes Enzymes Can synthesize RNA and proteins  Mitochondria Mitochondrial DNA (mtDNA) Mitochondrial (mtDNA) Encodes 13 proteins All part of the ETC Numerous copies within cell 2 copies of nuclear genes >10 copies of mtDNA genes Inherited primarily from mothers via oocyte mitochondria Can be used to track human migration and evolution the mitochondrial genome mutates 5-10 times faster than nuclear DNA Single nucleotide polymorphisms used to track maternal ancestry Note: Most mitochondrial proteins (~1500) like Porin are not encoded by mtDNA but by the nuclear DNA  Mitochondria Dynamic Organelles Mitochondrial biogenesis: Creation of new mitochondria Mitophagy: Selective degradation of old/dysfunctional mitochondria via autophagy Autophagy – lysosomal recycling of cellular components Mitochondria fusion: Fuse with one another Mitochondrial fission: Split in two Evidence suggests this could be induced by endoplasmic reticulum (ER) Fusion and fission is likely a major determinant of Mitochondrial: Number Length Degree of interconnection. https://www.youtube.com/watch?v=CIXY-Ns5vks Mitochondria Roles and Importance of the Mitochondria for Human Health For example: 1. Cellular Respiration (ATP) → ENERGY 2. Cataplerosis → ANABOLISM 3. Calcium homeostasis → SIGNAL TRANSDUCTION 4. Apoptosis → CELL LIFE CYCLE  Roles Of Mitochondria: 1. Carbohydrate Metabolism in the Mitochondria 2  Glycolysis Glycolysis http://cfcc.edu/faculty/dnorris/pdf/glycolysis.jpg Investment Phase Metabolizes monosaccharides Primarily glucose Produces: Net 2 ATPs/glucose 2 NADH/glucose 2 Pyruvate/glucose Anaerobic pathway Or to fermentation Requires no O2 Pyruvate Catabolized aerobically or anaerobically Occurs in the cytosol Payoff Phase https://www.youtube.com/watch?v=8Kn6BVGqKd8 Or to fermentation  Roles Of Mitochondria: 1. Carbohydrate Metabolism in the Mitochondria Pyruvate Entry Glycolysis produces Pyruvate (Pyruvic acid) Pyruvate must be transformed and transported into mitochondrial matrix by Pyruvate Dehydrogenase (PDH) Pyruvate dehydrogenase (PDH) Multienzyme complex Highly regulated A switch regulating Carbohydrate vs Fat Metabolism Pyruvate transported and converted to Acetyl-CoA 1. Decarboxylated to form a 2 carbon acetyl group 2. Acetyl group transferred to coenzyme A (HS-COA) to produce Acetyl CoA Overall Reaction: Pyruvate + HS-CoA + NAD+ → acetyl CoA + CO2 + NADH + H+ PDH  Roles Of Mitochondria: 1. Carbohydrate (fatty acid) Metabolism in the Mitochondria TCA/Krebs/Citric Acid Cycle Glycolysis PDH β (Fat)-Oxidation In mitochondrial matrix Accepts: Acetyl-CoA From Pyruvate via PDH Fatty Acid Oxidation Per Acetyl-COA Produces: 1 ATP Reducing Equivalents: 3 NADH + 1 NADH from PDH for Pyruvate 1 FADH2  Roles Of Mitochondria: 1. Carbohydrate Metabolism in the Mitochondria TCA/Krebs/Citric Acid Cycle Glycolysis PDH β (Fat)-Oxidation Stepwise cycle 1. Acetyl-CoA (2 Carbons) is condensed with oxaloacetate (OAA; 4 C) to form citrate (citric acid; 6 C). 2. 2 Carbons lost to CO2, eventually regenerating the OAA 3. 4 pairs of electrons (carried via NADH and FADH2) are removed 4. NADH and FADH2 transported to the electron transport chain  Roles Of Mitochondria: 1. Carbohydrate (fatty acid) Metabolism in the Mitochondria Oxidative Phosphorylation Electron Transport Chain: Intermembrane Electrons from: space NADH (via Complex I) Matrix FADH2 (via Complex II) Pass through electron transport chain I Complexes pumps H+ in the matrix to the intermembrane space across the inner II membrane III Generates proton gradient and mitochondrial membrane potential Uses O2 at Complex IV IV ATP-synthase (Complex V) Potential Energy from H + Forms ATP Chemiosmosis NADH 2 or 3 ATP FADH2 2 ATP ATP SYNTHASE (V) Inner membrane 1 molecule of glucose → 36 molecules of ATP Membrane Potential: -ve +ve (in theory)  Roles Of Mitochondria: 1. Carbohydrate (fatty acid) Metabolism in the Mitochondria Oxidative Phosphorylation *not every proton transported outside the mitochondrion necessarily makes it back into the matrix Net: 6 ATP Cytosolic NADH → 2 ATP/NADH * (actually between 1.5-2 ATP/NADH rather than 2.5-3 due to cost of going from cytosol to matrix) Mitochondrial NADH → 3 ATP/NADH Net: 30 ATP * (actually between 2.5-3 ATP/NADH) Mitochondrial FADH2 → 2 ATP/FADH2 * (actually between 1.5-2 ATP/FADH2) * (some reports indicate 32 ATP/Glucose) ATP Synthase http://youtu.be/xbJ0nbzt5Kw http://youtu.be/3y1dO4nNaKY  ATP: ENERGY CURRENCY Adenosine-5'-TriPhosphate (ATP) Molecular unit of currency → intracellular energy ATP + H2O → ADP + Pi (~7.3 kcal/mole free energy) Powers most of the energy-consuming activities of cells active transport of molecules and ions nerve impulses maintenance of cell volume by osmosis adding phosphate groups (phosphorylation) to many different proteins, e.g., to alter their activity in cell signaling. muscle contraction beating of cilia and flagella (including sperm) bioluminescence  ATP: OTHER FUNCTIONS Cell Signalling: Substrate in signal transduction pathways by kinases that phosphorylate proteins and lipids Used by adenylyl cyclase to produce the second messenger molecule cyclic AMP Anabolism: monomer used in the synthesis of RNA and, after conversion to deoxyATP (dATP), DNA Roles Of Mitochondria: 2. Cataplerosis Anabolic molecules produced in mitochondria Cataplerosis Specifically metabolic intermediates from TCA cycle used for anabolism Ex. Citrate For Fatty Acids, Lipids and Sterols ɑ-Ketogluterate For some amino acids Succinyl-CoA For Heme groups Malate For some amino acids  Roles Of Mitochondria: 3. Calcium regulation Free calcium (Ca2+) Intracellular signalling molecule Mitochondria can transiently store calcium “cytosolic buffers" for calcium 1. Porin/VDAC on the outer membrane allows Ca2+ into the intermembrane space 2. Calcium uniporter, in the inner membrane, transports Ca 2+ into the matrix Driven by the mitochondrial membrane potential Intermembrane space highly +ve Release of calcium back into cytosol occurs via a sodium- calcium (Na+-Ca2+) exchanger in the inner membrane Roles Of Mitochondria: 4. Apoptosis Reactive Oxygen Species (ROS) Impaired calcium homeostasis Abnormal fission/fusion Insufficient ATP production Release of mitochondrial ROS Roles Of Mitochondria: 4. Apoptosis Apoptosis = programmed cell death  Roles Of Mitochondria: 4. Apoptosis Intrinsic Apoptotic Pathway: De-phosphorylated Bad associates with the Bcl-2 proteins Bcl-2 dissociates from the Bax proteins Dissociation of Bax generated pores in the mitochondrial membrane which permit the influx of ions Cytochrome c is released through the pores and activates Apaf-1 Activated Apaf-1 activates the caspase pathway and initiates programmed cell death pathways Roles Of Mitochondria: 4. Apoptosis https://www.youtube.com/watch?v=hqhxnWty5jc Brief Review of Mitochondria Structure Roles (examples) 2 membranes 1. Carbohydrate Outer and Inner metabolism 2 compartments TCA Cycle (Matrix) Intermembrane space ETC (Inner Matrix mitochondrial Contains mtDNA membrane) Site of Aerobic 2. Cataplerosis Respiration Dynamic 3. Calcium Regulation Created, degraded, 4. Apoptosis fused, fizzed Case study Healthy baby born at term No complications in the pregnancy Met all developmental milestones up to 6 months 12 months of age Lost basic motor skills such as: Sucking Head control Walking Talking Irritable Appetite loss Seizures 18 months Vision loss Respiratory problems MRI found brain lesions, particularly in the midbrain and brainstem Case study What might be wrong with this child? What would be the molecular processes through which these symptoms manifested?  Case study Leigh’s Syndrome/Disease Progressive neurometabolic disorder affects infants between the age of three months and two years 1 in 77,000 births At least 26 causative/contributory enzymatic defects have been identified Pyruvate dehydrogenase (PDH) deficiency, and respiratory chain enzyme (ETC) defects - Complexes I, II, IV, and V Deficient ATP production, severe mitochondrial defects, cellular apoptosis No cure, prognosis is poor → live anywhere from a few years to the mid- teens Mitochondrial Diseases https://www.youtube.com/watch?v=XX7irov3uKw Mitochondrial Diseases Refers to a group of disorders The result of a genetic mutation in the mitochondrial DNA or Nuclear-encoded mitochondrial proteins (nuclear DNA), resulting in mitochondrial failure Insufficient ATP production Impaired Ca2+ homeostasis Abnormal cell signalling and function Increased cell death ~ 1 in 6000 people have some form of mitochondrial disease Symptoms can range from mild to severe Currently no cure for these diseases**, research underway Mitochondrial Diseases Leigh Syndrome French Canadian Type First described in Saguenay-Lac- Saint-Jean Estimated ~ 1 in 2000 live births ~1 in 23 harbour mutation May be a founder effect Characterized by: Chronic metabolic acidosis Hypotonia Facial dysmorphism Delayed development Caused by: Mutation in LRPPRC Protein involved in the stability of mitochondrially-encoded mRNAs https://rarediseases.info.nih.gov/diseases/8370/leigh-syndrome-french-canadian-type Mitochondrial Diseases What influences severity of Mitochondrial Diseases? Heteroplasmy = presence of > 1 type of mtDNA within a cell or individual  Mitochondrial diseases: mtDNA 3 parent IVF http://www.wellcome.ac.uk/About-us/Policy/Spotlight-issues/Mitochondrial-diseases/  Mitochondrial diseases: mtDNA 3 parent IVF Successfully conducted in mice since 1990 2008 – Newcastle University, successfully transferred pronuclear DNA between day 1 single cell human embryo Embryos develop normally to blastocyst stage with minimal carryover of maternal mtDNA 2009 – successfully conducted on primates resulting in offspring with no detectable carryover of maternal mtDNA Feb 2015 – approved for use in UK, go into effect Oct 2015 2017 First baby was born from mother with mitochondrial disease http://www.geneticsandsociety.org/article.p hp?id=6527#1a  Mitochondrial diseases: mtDNA 3 parent IVF Mitochondrial Replacement Therapy (MRT) remains illegal in Canada Assisted Human Reproduction Act (2004) no person can knowingly "alter the genome of a cell of a human being or in vitro embryo such that the alteration is capable of being transmitted to descendants." fines of up to $500,000, or a jail sentence of up to 10 years, or both http://www.cbc.ca/news/health/why-canada-isn-t-ready-to-talk-about-3-parent- babies-1.2956257  Mitochondrial diseases: mtDNA 3 parent IVF Potential benefits: Parents can have children that are genetically related to them Children potentially born free from mitochondrial disorders This protection will be carried on in all future descendants  Mitochondrial diseases: mtDNA 3 parent IVF What are the ethical considerations of this treatment option? Identity of offspring? Designer babies? Slippery slope? Status/rights of donor mother? Long term adverse effects of treatment may not be immediately apparent. Is it OK to implement an experiment treatment on future generations?  Mitochondrial diseases: mtDNA 3 parent IVF - Unregulated MRT Dr. John Zhang 2016 Performed MRT with woman suffering from Leigh’s syndrome Mother previously had 4 miscarriages and 2 children die as a result of the disease Procedure illegal in US Performed procedure in Mexico https://www.washingtonpost.com/national/health-science/this-fertility-doctor-is-pushing- the-boundaries-of-human-reproduction-with-little-regulation/2018/05/11/ea9105dc-1831- 11e8-8b08-027a6ccb38eb_story.html Questions? Next Lecture Interactions Between Cells and Environment (Ch. 7)