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Unformatted text preview: Slides for Lectures 1 and 2 Welcome to BCH3120: to BCH3120: General Intermediary Metabolism Mondays 10:00-11:30am 10:0008:30Wednesdays 08:30- 10:00am Marion Auditorium Dr. Mary-Ellen Harper (Lecturer, Course Co-ordinator) Mary Harper (Lecturer Course Co Dr. Alfred Aziz (Lecturer) Dr. Amanda MacFarlane (Lecturer) Dr Zemin Yao (Lecturer) Dr. Zemin Yao (Lecturer) TAs: Brittany Beauchamp, Ariana Noel, and Renny Zhang Effects on metabolism of eating, Effects on metabolism of eating, fasting, fasting, and starvation Major Metabolic Diseases - Types 1 and 2 Diabetes Effects of exercise and training and training Intermittent Energy Energy Intake (Food) Continuous Energy Content of th the Body Energy Expenditure Avg. N.American Pro. 12% Pro. 25,000 kcal (15% of body E) Fat 30-45% 30- Fat 141,000 kcal (84% of body E) CHO 48-58% 48- CHO 1,800 kcal (1% of body E) Circulating: Glu 80 kcal FFA 3 kcal TG 30 kcal 30 kcal ATP content of body: 0.7 kcal 1. 2. Importance of dietary CHO intake, and stored body fat ENERGY CONTENT is only a small proportion of ENERGY FLOW A plethora of pathways! plethora Metabolism Metabolism the sum of biochemical processes involved in the synthesis, breakdown, and inter-conversion of constituents in cells and organisms Intermediary Intermediar metabolism is where anabolism and is anabolism and catabolism intersect, often sharing pathways and metabolites. Because the pathways of intermediary metabolism are essential for life, they are tightly regulated through a variety of mechanisms. Heterotrophs: require complex nutrient molecules, like glucose, as a source of energy. Autotrophs: can synthesize complex molecules from simple precursors like CO2 and ammonia or Body Stores of Energy Overview of interrelationships between major anabolic and catabolic pathways. Learning is easiest, when the information is perceived as relevant and/or interesting… Is intermediary metabolism relevant in the post-genomic era? How might you use the information from BCH3120? ‘Personal - Practical’: Effects of diet, exercise, disease on Personal Practical of diet exercise disease on characteristics of energy metabolism (body composition, types of fuels oxidized…) ‘Metabolism in the News’ and integrated examples in lectures th ‘Medical Implications’ examples ‘Current Research’ lectures, and relevant abstracts included in lectures Organisation of BCH3120 Purpose Content Evaluation Introduction to Fundamental Concepts Purpose Purpose of BCH3120: An integrated understanding of the major An integrated understanding of the major pathways pathways of carbohydrate, lipid and amino acid metabolism BCH3120 General Intermediary Metabolism Organisation of the course of the course Purpose Content Evaluation Introduction Few basic concepts BCH BCH 3120 Content (Continued) Introduction and Principles of Metabolism (Lectures 1, 2: Jan 4,10) Dr Harper Carbohydrate Metabolism Glycolysis, Fermentation and the Pentose Phosphate Pathway (Lectures 3 and 4; Jan 12 and Jan 17) Dr. Aziz Gluconeogenesis, Glycogenolysis and Glycogen synthesis (Lecture 5; Jan 19) Dr. Harper Tricarboxylic acid (TCA) cycle (Lecture 6; Jan 24) Dr. Harper Oxidative Phosphorylation (Electron transport chain and ATP synthase) (Lecture 7; Jan 26) Dr Harper 7; Jan 26 Research lecture: Costs and Benefits of Mitochondrial Uncoupling (Lecture 8; Jan 31) Dr Harper Midterm 1 (Feb 2) BCH 3120 Content (Continued) Metabolic Control Analysis & Introduction to Metabolomics (Lecture 9; Feb 7) Dr Harper Lipid Metabolism. Dr Yao Fatty acid catabolism (Lecture 10; Feb 9) acid catabolism 10; Feb Fatty acid and triglyceride synthesis (Lecture 11; Feb 14) Phospholipid metabolism (Lecture 12; Feb 16) Biosynthesis of cholesterol (Lecture 13, Feb 28) Introduction to lipoprotein metabolism (Lecture 14, March 2) Regulation of cholesterol homeostasis (Lecture 15, March 7) Amino Acid Metabolism- Dr Yao Acid Metabolism Yao Amino acid deamination and the urea cycle (Lecture 16, March 9) Biosynthesis of amino acids and related molecules (Lecture 17;March 14) Regulation of amino acid metabolism (Lecture 18; March 16) Midterm 2 (March 21) BCH 3120 Content (Continued) Purine and Pyrimidine Metabolism - Dr. MacFarlane Folate and ‘1-carbon’ metabolism and the ‘methyl-folate trap’ (Lecture 19; March 23) March 23) Pyrimidine biosynthesis, and cancer chemotherapeutic agents (Lecture 20; March 28) Metabolism: Toward an Integrated Understanding – Dr. Harper Nutrition and Intermediary Metabolism (Lecture 21; March 30) (L 21 30 Exercise and Intermediary Metabolism (Lecture 22; April 4) Course review class (Lecture 23; April 6) Dr. Harper BCH 3120 Content Prerequisite: BCH2333 or BCH2733 If you do not have this prerequisite you must obtain permission from the Department (contact Dr. Harper, provide reason and transcript). Lecture notes: Slide sets posted at least 24h prior to each lecture. Textbooks: Biochemistry, Third or Fourth Edition D. Voet and JG. Voet Available at: University Bookstore and the Agora Bookstore Also highly recommended: Lehninger: Principles of Biochemistry, Fourth or Fifth Edition Available at the University Bookstore (Text: $180 or Loose-leaf: $60) and the Agora Bookstore ($127 or $70, respectively) th ($127 $70 Lecture notes: Lecture notes: Will consist of slide sets, which will be posted at the course website 24h prior to each lecture. On occasion, relevant papers from the literature will be assigned for reading. The professor will tell you what information in the papers will be required for the purposes of exams purposes of exams. Tricarboxylic Tricarboxylic Acid Cycle: A Crucial Metabolic Interface Cornerstone of intermediary metabolism Cornerstone of intermediary metabolism – MEMORIZE ASAP Fig. 26-1 Email Dr. Harper prior to Jan 26th if you will submit an entry. SONG SONG CONTEST! with with prizes… prizes… Don Don’t miss it! End of class BCH3120 General Intermediary Metabolism Organisation of the course th Purpose Content Content Evaluation Introduction Few basic concepts BCH3120 Evaluation BCH3120 Evaluation Intended to assess your: to assess your: Fundamental comprehension Ability to integrate and apply the information Ability to integrate and apply the information BCH3120 Evaluation BCH3120 Evaluation 25% 50% 25% Midterm 1: 25% (Feb 2) Midterm 2: 25% (March 21) Final Exam: 50% (Exam Period) BCH3120 Evaluation BCH3120 Evaluation Mid-term exams Focus on new/untested material (e.g., MT#2 will focus on course material AFTER MT#1) Duration: Duration: 60 minutes Each worth 25% of final mark Format: Multiple choice questions True True or false questions Tables or pathways to complete (« fill in the blanks ») ** thi ** In this room; we will have ~8 proctors to make this possible. thi Leave your coats and notes in your locker/elsewhere if possible! BCH3120 Evaluation BCH3120 Evaluation Fi Final Exam Cumulative Worth 50% of final mark Increased emphasis on cross pathway interactions/ metabolic networking. i.e., your integrated understanding of the the material Format: similar to that of the mid-term exams **If your mark in the final exam is better than the average of your midterm marks, then the final exam mark will become your final course mark. Policy on Exams: Policy on Exams: If If you miss a mid-term exam due to illness, obtain a doctor’s midillness, note and forward it within 48h by Mme. Johanne Bouchard (Room 4107, Roger Guindon Hall) for departmental approval. Without Without a valid note from a doctor, you will get zero. With With an approved note, your final mark will be based on the note, performance overall in the exams completed over the term. If If you miss the final exam due to illness, contact the Faculty illness, of Sciences office immediately to inquire about current Faculty policies and possible deferred exam. If this is not approved by policies and a possible deferred exam. If this is not approved by Faculty a mark of zero will be assigned for the final exam. Questions about lecture materials: Email one of the two Teaching Assistants Email one of the two Teaching Assistants, Brittany Brittany Beauchamp (for students with last names starting with the letters names starting with the letters A – H) bbeau038@uottawa.ca or, Ariana Ariana Noel (last names starting with I – P) anoel017@uottawa.ca Renny Renny Zhang (last names starting with Q - Z) (last rzhan059@uottawa.ca Please Please be very specific, explaining what you do and don’t understand. Don’t leave Qs to the and don understand Don leave Qs to the last last hours prior to exams!!!! Learning is easiest, when the information is perceived as relevant information is perceived as relevant and/or interesting… How might you use information from BCH3120? Relevance Relevance to future potential careers in medical research (e.g., cell biology) di bi intermembrane space outer membrane inner matrix membrane Mitochondria are not static, oblong structures in cells! Relevance Relevance to future potential careers in industry (e.g., pharmaceutical or medical instrumentation) or medical instrumentation) Medtronic is the global leader in medical technology- alleviating pain, restoring health, and extending life for millions of people around the world. Medtronic develops and manufactures a wide range of products and therapies with emphasis on providing a complete continuum of care to diagnose, prevent and monitor chronic conditions Every five seconds somewhere in the world person chronic conditions. Every five seconds, somewhere in the world, a person’s life is saved or improved by a Medtronic product or therapy. Revenue for the year ended April, 2009: $14.6 billion. New York Stock Exchange ticker: MDT. Key Businesses: Cardiac Rhythm Disease Management, Neuromodulation, Spinal and Biologics, Diabetes (develops advanced diabetes management solutions. We are the world leader in integrated diabetes management systems, insulin pump therapy, the world leader in integrated diabetes management systems, insulin pump therapy, continuous glucose monitoring systems and therapy management software), CardioVascular, and Others. Relevance Relevance to a better personal understanding of advances in medicine and life sciences lif To To know more about: Different types of diabetes types of diabetes Complex origins of obesity Genetic metabolic diseases Technologies used in these Technologies used in these and and other areas of metabolic research (e.g., PET scans) Crawford et al, 2010 Relevance Relevance to a better personal understanding of Nutrition and Health Energy, Nitrogen Nitrogen Nutrients Health www.enr.state.nc.us/DSWC/ pages/foodland.html www.patitucciphoto.com/htmls/ trailrunning06.html BCH3120 BCH3120 Metabolism in the News Weindruch R, et al. (1986). The retardation of aging in mice by dietary restriction: Weindruch R, et al. (1986). "The retardation of aging in mice by dietary restriction: Longevity, cancer, immunity and lifetime energy intake." Journal of Nutrition, 116(4) 641-54. An anti-aging method that works in virtually all species studied thus far… Caloric Restriction: but HOW does it work/ What are the mechanisms? What are the risks? BCH3120 BCH3120 Metabolism in the News Caloric Restriction the Traditional Okinawan Diet and Healthy Aging: Caloric Restriction, the Traditional Okinawan Diet, and Healthy Aging: The The Diet of the World's Longest-Lived People and Its Potential Impact on LongestMorbidity and Life Span Willcox et al. Ann.N.Y.Acad. Sci. 2007; 1114:434-55. 1114:434- ________________________________________________________________________________________________________________________________________________________ ________________________________________________________________________________________________________________________________________________________ • Long-term caloric restriction (CR) is a robust means of reducing ageLongagerelated diseases and extending life span in multiple species, but the effects in humans are unknown. The low caloric intake, long life expectancy, and the high prevalence of centenarians in Okinawa have been used as an argument to support the CR hypothesis in humans. •Therefore, we investigated six decades of archived population data on the elderly cohort of Okinawans (aged 65-plus) for evidence of CR. 65• Findings include low caloric intake and negative energy balance… with low risk for mortality from age-related diseases, and survival patterns ageconsistent with extended mean and maximum life span. This study lends support for benefits of CR in humans and is consistent with the literature support for benefits of CR in humans and is consistent with the literature on on animals with regard to CR phenotypes and healthy aging. Abridged abstract BCH3120 BCH3120 Metabolism in the News Aging, Adiposity, and Calorie Restriction Adiposity and Calorie Restriction Luigi Fontana; Samuel Klein JAMA. 2007;297:986-994. JAMA. 2007;297:986Excessive calorie intake and subsequent obesity increases the risk of developing Excessive chronic disease and decreases life expectancy. In rodent models, calorie restriction (CR) with adequate nutrient intake decreases the risk of developing chronic disease and extends maximum life span. Objective To evaluate the physiological and Objective clinical implications of CR with adequate nutrient intake Evidence Acquisition clinical implications of CR with adequate nutrient intake Evidence Acquisition Search of PubMed (1966-December 2006) using terms encompassing various aspects (1966of CR, dietary restriction, aging, longevity, life span, adiposity, and obesity; hand search of journals that focus on obesity, geriatrics, or aging; and search of reference lists lists of pertinent research and review articles and books… Evidence Synthesis CR Evidence in adult men and women causes many of the same metabolic adaptations that occur in CR rodents and monkeys, including decreased metabolic, hormonal, and inflammatory risk factors for diabetes cardiovascular disease and possibly cancer inflammatory risk factors for diabetes, cardiovascular disease, and possibly cancer. Excessive CR causes malnutrition... Conclusions CR in adult men and women Conclusions causes beneficial metabolic, hormonal, and functional changes, but the precise amount of calorie intake or body fat mass associated with optimal health and maximum longevity in humans is not known. In addition, it is possible that even it th moderate CR may be harmful in specific patient populations, such as lean persons who have minimal amounts of body fat. BCH3120 BCH3120 General General Intermediary Metabolism Organisation Organisation of the course Purpose Purpose Contents Contents Evaluation Evaluation Today 1. 2. TCA Cycle Song Contest: Mon Jan 31 TCA Cycle Song Contest: Mon Jan 31 MT#1: Wed Feb 2 MT#2: Mon March 21 Introduction Introduction Fundamental Fundamental concepts Experimental approaches General principles of Regulatory Control of Metabolic Pathways principles of Reg Control of Metabolic Path Studying Studying Metabolism in the the Post Genomic Era ‘Big Science’ Science’ Ge seque Gene sequencing and Genome databases Ge da DNA DNA microarrays: mRNA expression levels and microRNAs Proteomics: Proteomics: protein expression levels, and post-translational modifications Metabolomics: Metabolomics: comprehensive analyses of metabolite levels Genome databases Genome databases Since Since July 2004, the full genomes of about 30 eukaryotic organisms and more than 170 microbes and 1,300 viruses, have been freely available and downloadable from NCBI. [take a look at this website!] il [t thi This This incredible source of information should be considered when studying intermediary metabolism studying intermediary metabolism. An An understanding of metabolic pathways and their control is a great advantage in researching genes of interest in genomic research. What is the metabolic impact of a mutation (‘variant’) in a specific gene? What is the metabolic impact of mutation in specific gene? • Gene sequence – variants? • mRNA expression levels ? expression levels • Protein levels and characteristics ? • Metabolite levels ? DNA microarrays DNA microarrays Purpose: To simultaneously assess expression levels of Purpose: To simultaneously assess expression levels of thousands thousands of genes. Check out, for example, the website of Affymetrix, which produces gene chips (http://www.affymetrix.com/index.affx ) To To assess the origins and development of disease, the subtypes of disease; disease prognosis (e.g., in cancer); treatment outcomes… (E.g., Ottawa Hospital Weight Management Clinic: Ott Cli Thousands of patients followed; Rate of weight loss and risk for diabetes etc. varies greatly and is related to unique gene expression profiles) expression profiles) To To clarify the specificity of pharmaceuticals. (E.g., hypohypocholesterolemic drugs, which represent > $20 billion market internationally. Microarray studies have demonstrated that some drugs affect metabolic pathways beyond those that were initially targeted.) ‘Cluster Analysis’ Analysis’ Many systems biology approaches for data analysis. Cluster-type systems biology approaches for data analysis Cluster approaches assess levels of transcripts, proteins or metabolites in relation to known pathways and biological functions. Proteomics Proteomics • • • • Gene sequence – variants? mRNA expression levels ? Protein levels and characteristics ? Metabolite levels ? Purpose: expression Purpose: expression levels and characteristics of proteins. E.g., within cells and tissues of an organism. Proteomic Proteomic approaches allow investigation into responses of a biological system to different stimuli, e,g, disease states, or e,g, following drug treatment. Recent Recent progress in analytical techniques for the study of proteins has led to significant improvements in the ability to identify peptides and their posttranslational modifications on a high throughput basis. E.g., “A novel proteomic approach for the discovery of chromatin associated protein networks” (By JP Lambert, L. Mitchell, A Rudner, K Baetz and D Figeys) Mol Cell Proteomics. 2008 Dec 22. [Epub ahead of print]. **From our Ottawa Institute of Systems Biology. Th The complexity of an organism is the result to only a small extent of gene expression th alone. In other words the one gene-one protein concept is incorrect (Grehlin and Obestatin example). The 2002 Nobel Prize in chemistry was awarded to Drs Fenn, Tanaka and Wuthrich for their contribution to the development of mass spectrometric and NMR methods for biomolecules, especially proteins. NMR bi Figures modified from Am J Physiol Lung 2004: 287:L1 and Nobel e-Museum. “Gut Feelings” Ghrelin and Obestatin are two hormones produced from one gene They have opposite effects! Grehlin stimulates hunger/food intake Obestatin inhibits hunger/food intake intake NB: Since its discovery in late 2005, there has been a substantial amount of controversy about obestatin; some researchers maintain that it is not a true hormone and does not have physiological effects. Science. 2005 Nov 11;310(5750):996-9. Metabolomics Metabolomics • Gene sequence – variants? • mRNA expression levels ? • Protein levels and characteristics ? • Metabolite levels ? A new investigative field that applies advanced new analytical techniques to study metabolite profiles in serum or tissue samples serum or tissue samples. Several hundreds of metabolites simultaneously! Several hundreds of metabolites simultaneously! By comparing metabolite levels in sequential By comparing metabolite levels in sequential samples samples and inferring metabolite flux, metabolomics can also identify rate-controlling steps – e.g., that ratecould could be targeted for the development of new drugs. Example: Example: Metabolite profiles of brain of individuals with schizophrenia vs vs healthy controls The results strongly suggest that markers of oxidative stress and mitochondrial perturbations provide a pathological signature for the disease. Figures Figures obtained from Mol Psychiatry 2004. An integrated understanding An integrated understanding of the pathways pathways of carbohydrate, lipid and amino acid metabolism li … IS FUNDAMENTALLY IMPORTANT! IS Useful Websites: Interactive Metabolic Pathways The ExPASy (Expert Protein Analysis System) proteomics proteomics server of the Swiss Institute of of the Swiss Institute of Bioinformatics (SIB): http://www.expasy.org/cgi-bin/show_thumbnails.pl IUBMB-Nicholson Metabolic Maps, Minimaps & Animaps: http://www.iubmb-nicholson.org/ Example: IUBMBNicholson Metabolic Maps Cell Cell biological and Biochemical Approaches to Assess Regulation and Control of Metabolic Pathways Control of Key Concepts: Concepts: Cell Cell-cell communication & compartmentalisation Thermodynamics Thermodynamics Inhibition Inhibition and allostery PostPost-translational modifications Enzyme Enzyme turnover CellCell-cell communication & compartmentalisation Procaryote unicellular organism; cells lack membrane bound Procaryote – unicellular organism; cells lack membrane bound nuclei; e.g., nuclei; e.g., bacteria, blue-green algae and mycoplasma blue- CellCell-cell communication & compartmentalisation Eukaryote – cell or organism having membrane-bound, structurally cell membrane-bound discrete nucleus other well-developed subcellular compartments. wellIncludes all organisms except viruses, bacteria, & cyanobacteria (blue-green (blue- algae) CellCell-cell communication & compartmentalisation Localization of distinct metabolic pathways: Examples of distinct metabolic pathways: Examples CellCell-cell communication Direct connection: Gap Junctions Direct connection: Gap Junctions Cell Mol Life Sci. 2010 Oct 21. [Epub ahead of print] Structure of the gap junction channel and its implications for its biological functions. Maeda S, Tsukihara T. Maeda Tsukihara Institute for Protein Research, Osaka University, OLABB, 6-2-3 Furuedai, Suita, 565-0874, Japan. Abstract Gap junctions consist of arrays of intercellular channels composed of integral membrane proteins called connexin in vertebrates. Gap junction channels regulate the passage of ions and biological molecules between adjacent cells and, th bi therefore, are critically important in many biological activities, including development, differentiation, neural activity, and immune response. Mutations in connexin genes are associated with several human diseases, such as neurodegenerative disease, skin disease, deafness, and developmental abnormalities. The activity of gap junction channels is regulated by the membrane voltage, intracellular microenvironment, interaction with other proteins, and phosphorylation. Each connexin channel has its own property for conductance and molecular permeability number of studies have own property for conductance and molecular permeability. A number of studies have tried to reveal the molecular architecture of the channel pore that should confer the connexin-specific permeability/selectivity properties and molecular basis for the gating and regulation. In this review, we give an overview of structural studies and describe the structural and functional relationship of gap junction channels. PMID: 20960023 [PubMed - as supplied by publisher] Cellular compartmentalisation Thermodynamics Inhibition and allostery Post-translational modifications Enzyme turnover A 1 B 2 C 3 D Thermodynamics The committing step catalyses a reaction that has a large free energy change (irreversible step) The other enzyme reactions are characterized by a relativelly lower free energy change The The Thermodynamics of Entropy: Th Ad The Advantages of Being Disorganized… Di The Oxidation of Glucose: • small number of relatively complex/ordered molecules to a larger number of simpler molecules (increased entropy) (i • energy released ! Creating Order Costs Energy, but Order has Potential (e.g., energy; information) “There is a tide in the affairs of men, Which, taken at the flood, leads on to fortune; Omitted, all the voyage of their life Is bound in shallows and in miseries.” Shakespeare Julius Caesar, Act IV, Scene 3 IV Energy released from an EXERGONIC PROCESS Can drive an ENDERGONIC PROCESS drive an ENDERGONIC PROCESS Chemical example of ENERGY COUPLING: ATP hydrolysis drives glucose phosphorylation ATP hydrolysis drives glucose phosphorylation Enzymes catalyze reactions by lowering the activation energy of the reaction process the activation energy of the reaction process Central Role of ATP in Metabolism ATP is: The chemical intermediate linking h li energy-releasing catabolic (exergonic) reactions with energy demanding anabolic (endergonic) reactions anabolic (endergonic) reactions Thermodynamics Thermodynamics and Control of Metabolic Pathways Committing/ Controlling step (1) Free energy A 2nd enzyme reaction 1 B 2 3rd enzyme reaction Carbon flux progression C 3 D Cellular compartmentalisation Thermodynamics Inhibition and allostery Post-translational modifications Enzyme turnover Regulatory Regulatory Control of Metabolic Pathways by Inhibition and Pathways by Inhibition and Allostery Allostery Inhibition: Common NonNon-reactant molecule that resembles the substrate interferes with access of the normal substrate to the substrate binding pocket Endogenous Endogenous inhibitor (end-products: “feedback inhibition” “negative effector”) (endinhibition” effector” Exogenous inhibitor (poisons) E1 = Threonine dehydratase E1 is specifically inhibited allosterically by the end-product, but by none of the four intermediates (B, C, D, or E). Inhibition Inhibition and Allostery Binding of a ligand at one site affects binding of another at another site of ligand at one site affects binding of another at another site Structural alterations interfere with the accessibility of other ligands, substrates or cofactors or may disturb the catalytic site substrates or cofactors, or may disturb the catalytic site. ‘Allosteric’ enzymes exist in at least two different states (controlled by the Allosteric’ regulatory substances): R state (R= relaxed): high affinity for substrate state (R= T state (T = tense): low or no affinity for substrate (T Allosteric activators and inhibitors induce conversion from the T to the R state, and the R to the T state, respectively. state, state, Allosteric Allosteric control of glycerol kinase: It’s smart/rational ! smart/rational Background: Both glycerol and glucose can replenish the Krebs cycle. A ‘smart’ process/pathway: The minor glycerol-dependent glycerolpathway is turned off when the usual substrate, glucose, is available. il Molecular mechanism: … 2nd next slide slide Allosteric Allosteric control of glycerol kinase by fructose 1,6-bisphosphate (FBP) 1,6- Molecular details corroborate the allosteric control of glycerol kinase : fructose 1,6-bisphosphate locks GK in its TETRAMERIC, INACTIVE state. “Smart House” House” (Cells, tissues and organisms are still smarter!) You You come home at the end of the day, and as you approach the house a retinal analyser recognises you, opens the door and greets you by turning on the th th lights. In the kitchen your fridge informs you that you are missing few ingredients for In the kitchen, your fridge informs you that you are missing a few ingredients for dinner. dinner. You breathe a sigh of relief, as the missing ingredients will be delivered within an hour. You enter the lounge, the lighting, temperature, and humidity are just as you like, th th li like, as you sit down to watch the 6 o’clock news. o’ Without you even knowing it, your dishwasher detects an internal technical fault Without you even knowing it, your dishwasher detects an internal technical fault and and has automatically requested maintenance assistance from its manufacturer... You are living in a smart house, a house which demonstrates interactivity and li reactivity reactivity - all elements are inter-connected and automatically regulated interModified from Agentland.com Control Control by Post-Translational PostModifications Modifications An important additional means of control i.e., i.e., beyond the ‘upstream’ control mechanisms such as: upstream’ Transcriptional control: controlling level of mRNA expression e.g., PPARs and their PPREs e.g., Translational control: at initiation, elongation and termination steps in conversion of mRNA to protein termination steps in conversion of mRNA to protein Post Post-translational Control To To modulate the activities of enzymes already synthesized th For For acute control of enzyme activities (milli(milliseconds to minutes) PostPost-Translational Modifications: Covalent Modifications E.g., Phosphorylation by PKs Other covalent modifications: covalent modifications: - Ca++ Ca++ - Methyl - Acetyl -- glutathionylation Induce conformational changes, changes, turning active site ON or OFF Reversible Antioxid Redox Signal. 2008 Nov;10(11):1941-88. Molecular mechanisms and clinical implications of reversible protein Sglutathionylation. Mieyal JJ, Gallogly MM, Qanungo S, Sabens EA, Shelton MD. Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio Abstract Sulfhydryl chemistry plays a vital role iin normal biology and iin defense of cells against Sulfhydryl chemistry plays vital role n normal biology and n defense of cells against oxidants, free radicals, and electrophiles. Modification of critical cysteine residues is an important mechanism of signal transduction, and perturbation of thiol-disulfide homeostasis is an important consequence of many diseases. A prevalent form of cysteine modification is reversible formation of protein mixed disulfides (protein protein mixed disulfides (protein-SSG) with glutathione (GSH). The abundance of GSH in cells and with glutathione (GSH). The abundance of GSH in cells and the ready conversion of sulfenic acids and S-nitroso derivatives to S-glutathione mixed disulfides suggests that reversible S-glutathionylation may be a common feature of redox signal transduction and regulation of the activities of redox sensitive thiol-proteins. The glutaredoxin enzyme has served as focal point and important tool for evolution of this regulatory mechanism, because it is served as a focal point and important tool for evolution of this regulatory mechanism, because it is a specific and efficient catalyst of protein-SSG deglutathionylation. However, mechanisms of control of intracellular Grx activity in response to various stimuli are not well understood, and delineation of specific mechanisms and enzyme(s) involved in formation of protein-SSG intermediates requires further attention. large number of proteins have been identified as potentially regulated by further attention. A large number of proteins have been identified as potentially regulated by reversible S-glutathionylation, but only a few studies have documented glutathionylation-dependent changes in activity of specific proteins in a physiological context. Oxidative stress is a hallmark of many diseases which may interrupt or divert normal redox signaling and perturb protein-thiol homeostasis. Examples involving changes in homeostasis. Examples involving changes in S-glutathionylation of specific proteins are discussed in of specific proteins are discussed in the context of diabetes, cardiovascular and lung diseases, cancer, and neurodegenerative diseases. PMID: 18774901 [PubMed - indexed for MEDLINE] Control by Control by Enzyme Turnover Turnover The ubiquitin The ubiquitin pathway is an is an important important component for the control of enzyme turnover The PEST motif is a tag for the the ubiquitinization of proteins: Proteins with a N-terminal NPEST motif survive for about PEST motif “survive” for about 2-3 min Proteins lacking a N-terminal lacking terminal PEST PEST motif have half-lives of halfabout 10 hours Later Later this term, we will review METABOLIC METABOLIC CONTROL THEORY We We will see that Metabolic Control Analysis is an Metabolic approach that allows researchers to identify the major approach that allows researchers to identify the major controlling controlling steps. It It is no longer acceptable to say that there is one rate one limiting step in a metabolic pathway in Controlling Controlling steps can change based on changes in metabolic conditions, presence/absence of disease, etc. Review questions Review questions • List some of the different major pathways List some of the different major pathways related to intermediary metabolism. to intermediary metabolism • Why are metabolic controls necessary? • What are some different types of regulatory controls of enzyme activity? activity? • List some ways that results of DNA microarrays and proteomic arrays could be of use in metabolic studies be of use in metabolic studies • What is metabolomics? Why is it useful? metabolomics? • Wh What is a Metabolic Control Analysis? ...
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