Introductory Eukaryotic Cell Biology
Introductory Eukaryotic Cell Biology LIFE 210
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Date Created: 09/21/15
4 November Signaling between adjacent cells via Notch and Delta regulates cell fate choices Latent regulation cells signal their neighbors to become a certain cell type Notch and Delta are single transmembrane proteins Binding is modulated by Notch glycosylation Processing and activation of Notch by proteolytic cleavage Notch undergoes 3 successive changes the last 2 triggered by Delta binding ysecretase mediates the last cleavage The cytosolic fragment translocates to the nucleus 9 activation of transcription Once activated it cannot be used again The WntBcatenin signaling pathway Wnts are secreted proteins that act as local mediators involved in development No Wnt signaling 9 Bcatenin is degraded in proteasomes Binding of Wnt to its cell surface receptor 9 no Bcatenin degradation 9 it migrates to the nucleus and activates transcription 9 cell proliferates Colon cancer Hedgehog signaling Cubitusinterruptus Ci is normally cleaved 9 transcriptional repressor Hedgehog binding to surface receptor 9 no Ci cleavage 9trascrip activation Excessive Hedgehog signaling can lead to cancer skin basal cell carncinoma The tumor necrosis factor01 TNFotNFKB pathway Mediates many stressful inflammatory and innate immunity responses Excessive signaling can cause chronic inflammation and cancer NFKB is normally inhibited by KB9 degraded upon activation 9NFKBtranslocates to the nucleus Oscillation in NFKB activation by negative feedback NFKB activates transcription of the inhibitor IKB gene Short exposure to TNFot 9 single pulse of NFKB activation 9 turn on some genes Long exposure to TNFot 9 oscillatory NFKB activation 9 turn on additional genes 14 November Actinbased motor proteins are members of the Myosin superfamily 2 heavy chains and 4 light chains Heavy chain globular head domain long coiledcoil that mediates dimerization tail The tail bundles itself with the tails of other myosin molecules muscle myosin Myosin tailtail interactions form large bipolar llthick filaments Contains several hundred myosin heads that bind and hydrolyze ATP Good for sliding actin filaments of opposite orientation Basis of muscle contraction muscle myosin The myosin head alone can generate filament sliding in vitro The motor activity is contained within the head domain Members of the Myosin superfamily Not just in muscle All share a similar motor domain but the tails are very diverse Many form dimers like Myo One of them moves towards the minus end of the actin filament Yeast has Myo I II and V There are two types of microtubule motor proteins kinesins and dyneins Kinesins generally walk towards the plus end Structurally related to myosins Dyneins generally walk towards the minus end Major roles in cell division and organelle movement The structural similarity of myosin and kinesin indicates a common evolutionary origin Motor proteins generate force by coupling ATP hydrolysis to conformational changes Kinesin quotHandoverhandquot motion Adaptation to different functions Processive or not processive Variable speed 02 to 60 umsec Depends on ATPase rate of time bound to filament size or angle of the step Motor proteins mediate the intracellular transport of membrane enclosed organelles Myosin V was the first myosin shown to mediate organelle motility t associates with melanosomes and mediates their transfer form melanocytes to keratinocytes Cells regulate motor protein functions 5 Octo be r Electrochemical Gradient Produced by ETC ETC Uses AG of e transfer from NADH to O2 Transfers Hi out of matrix Electrochemical gradient across M Membrane 2 components Charge AV or NH 140 mV quot70 of AG pH ApH or AH 60 mV quot30 of AG AG AP or pmf ApH ALIJ 14 kcalmol 32 kcalmol Total AG 46 kcalmol Peter Mitchell amp the Chemiosmotic Theory In 1961 Radical idea at the time Energy from NADH amp FADHZ stored as an electrochemical Hi gradient Used to drive ATP synthesis Before then it was assumed that all ATP synthesis was through substratelevel phosphorylation Mitchell received the Nobel Prize in Chemistry in 1978 Mitochondrial nner Membrane Vesicles MIMs and AtP Synthase Mitochondrian mild detergent takes off outer membrane Left with inner mitochondrial membrane Sonicate breaks off bits of membranes and forms vesicles Some vesicles are inside out makes important parts accessible eg ATP synthase Have ETC amp ATP synthase Use to show Hi gradient is required Add dinitrophenol uncoupler Membrane soluble Hi carrier Uncouples ETC from ATP synthase Uncoupler shuttles protons out no gradient Just produces heat Brown adipose fall cells Found in infantshibernating mammals Functions to produce heat Nonshivering thermogenesis Too cold to shiver still produces heat Use to show Hi gradient is sufficient Soak MIMs at pH 4 Leads to high H inside Transfer to pH 8 Creates concentration gradient Add ADP and Pi 9 ATP created NADH amp Oz not required if there is another way to form an H gradient Use to show ATP synthesis and NADH Oz use are coupled Add m ADP 9 stop using NADH Oz No NADH or Oz used When AG of Hi pumping equals AG of e flow from NADH to Oz NADH and Oz use stop ATP Synthase Structure and Mechanism Function MIM vesicles treated with urea 9 peripheral membrane proteins dissociated Knob of ATP synthase dissociates Knob F1 peripheral membrane complex and ATPase Stalk left behind F0 integral membrane complex and Hi channel When F1 and F0 dissociated NADH and 02 use is decoupled from ADP and Pi use Add ATP to solution 9 F1 will hydrolyze it Structure and Mechanism of the ATP Synthase 5 subunits in F1 01 B v 6 E 3 subunits in F0 a b c a 2 HT channels one on each side of the membrane c wheel turned by Hi flow y crank or cam shaft couples Hi flow with ATP synthesis 01 and B form head 3 each hexagonal B ATP synthase works through conformational changes causing an increase in the affinity for ATP and a decrease in the affinity for ADP and Pi The H flow drives release of ATP decrease in affinity for ATP increase in affinity for ADP and Pi ATP Synthase can Make or Use ATP and a Proton Gradient Works in both directions Mitochondria use mostly in the direction of creating ATP The Proton Gradient Drives Other Functions in Mitochondria pH gradient drives phosphate import pH gradient drives pyruvate import Voltage gradient drives ADP ATP exchange Cancer Cell Metabolism The llWarburg Effectquot or llAerobic Glycolysisquot Warburg 1927 Cancer cells often revert back to the metabolism of early embryonic cells Astrocytes support cells for brain neurons also have aerobic glycolysis Form llCori cycle with neurons llCommensal metabolic relationship Aerobic Glycolysis should be called llanaerobic glycolysis in the presence of sufficient 02quot Also use glutamine Anapleurotic function for TCA cycle Biosynthetic precursor for N containing compounds May be an adaptation for tumor cells with inadequate Oz 2 November Like GPCRs enzyme linked receptors are transmembrane proteins but typically single pass The intracellular domain has enzymatic activity or associates directly with an enzyme Some subfamilies of receptor tyrosine kinase RTKs RTKs are the most numerous enzymecoupled receptors 60 genes Activation of receptor tyrosine kinases RTKs Ligand binding causes receptor dimerization bringing the kinase domains of two receptor chains together Transautophosphorylation on multiple tyrosine residues higher activity Phosphorylated RTKs serve as docking sites for intracellular signaling proteins 5H2 and PTB domains within signaling proteins recognize phospho tyrosine Formation of a signaling complex that relays the signal onward Some proteins bind activated RTKs to down regulate them by endocytosis Development of cancer when this is not effective Ras is a monomeric GTPase that relays signals from RTKs Has a covalently attached lipid for membrane anchoring Molecular switch GTPGDP Activated by GEFs inactivated by GAPs Can act as a signaling hub 30 of tumors have hyperactive mutant forms of Ras How an RTK can activate the GTPaseRas An example from the fly eye Adaptors and GEFs couple RTK activation to Ras activation Ras activates a mitogenactivated protein MAP kinase module Involved in cell proliferation and differentiation Activates transcription of specific genes Scaffold proteins help prevent crosstalk between parallel MAP kinase modules An example from budding yeast In humans different modules are activated by cell stress inflammation and signals from other cells The Rho family of GTPases couples cellsurface receptors to the actin and microtubule cytoskeletons Phosphoinositide 3kinase PI 3kinase produces lipid docking sites in the plasma membrane Pi 3 4 5 P3 remains in the membrane and is bound by an interaction module present in various signaling proteins PI 3kinase signals animal cells to survive and grow The TOR target of rapamycin protein stimulates nutrient uptake and protein synthesis Signal is terminated by phosphatases that remove the phosphate group Their mutation promotes uncontrolled cell growth Overlapping between downstream signaling pathways activated by RTKs and GPCRs Signaling by Janus kinase JAK signal transducer and activator of transcription STAT An example of tyrosinekinaseassociated receptor Signaling by transforming growth factorB TGFB proteins TGFBaredimeric proteins local mediators or hormones Stimulate receptor serinethreonine kinases Activate gene transcription through Smad proteins 31 October A Gprotein coupled receptor GPCR is a 7 pass transmembrane protein They use G proteins to relay signals into the cell interior GPCR pathways are targets of half of all known drugs Trimeric a 3 v Inactive when the ot subunit has GDP bound at and y attached by lipids GPCR activation induces the ot subunit to release GDP to bind GTP An active GPCR catalyzes the activation of many molecules of G protein Active G protein regulates enzymes or ion channels in the plasma membrane conformational change Some G proteins regulate production of cyclicAMP Synthesized by adenylyl cyclase and degraded by a phosphodiesterase Extracellular signals can increase cAMPZOx in a few seconds Different cell types respond differently to increases in cAMP One cell type often responds in the same way regardless of the extracellular signal that causes it This is because the receptors are different they bind the same signal but relay the message differently Cyclic AMPdependent protein kinase PKA mediates most of the effects of cyclic MMP t phosphorylates different target proteins according to the cell type this produces different effects Some responses mediated by cyclicAMP depend on gene transcription and take hours A short cyclic AMP signal can be transformed into a longterm change in the cell Ex learning and memory hormone production REB binds to the cyclic AMP site on a gene once activated Some G proteins regulate the plasma membranebound enzyme phospholipase 03 It secretes IP3 soluble and diacylglyercol remains in the plasma membrane Ca2 release from ER and activation of PKC at the plasma membrane nositol1 4 5 IP3 releases Ca2 from the ER Diacylglycerol activates protein kinase C Signal molecule 9 activated GPCR 9 activated G protein 9 activated phospholipase CB 9 BI3 diacylglycerol 2 Ca functions as an Intracellular medIator Ca2 can act as a signal because its concentration in the cytosol is low whereas outside the cell and in the ER is high 9 large gradient Ex in antiport Na and Ca2 exchange Cazcalmodolin Various proteins help relay the cytosolic Ca2 signal Calicalmodolinbinds to target proteins and alters their activities Calicalmodolinde endent rotein kinase CAMkinase P P CAMkinase is best known switch on by Calical t phosphorylates itself and becomes fully active and Ca2 independent 9 kinase remains active after the Ca2 activating signal It can phosphorylate itself Smell and vision depend on GPCRs that regulate cyclicnucleotidegated ion channels Olfactory GPCRs recognize odors and are present in the cilia ofolfactory neurons 9 including cAMP9 opens cAMPgated ion channels 9 nerve impulse Activation of rhodopsin in disc membranes is induced by light Activates phosphdiesterase9 decreases the cGMP and closes cation channels in the plasma membrane 9 hyperpolarization 9 less neurotransmitter release Dark less signaling light more signaling Amplification in the light induced catalytic cascade in rods GPCR desensitization depends on the receptor phosphorylation and arrestins Arrestins prevent interactions with G proteins Arrestins are adaptors that couple GPCRs to clathrinmediated endocytosis 7 December Cancer cells reproduce without restraint and colonize other tissues Cancer cells reproduce in defiance of normal restraints of cell growthdivision They invade and colonize territories normally occupied by other cells A tumor is benign if it is not invasive Tumor cancer when it is malignant invades surrounding tissue and forms metastases Common types of cancer in the United States Carcinomas from epithelial cells Sarcomas from connective tissue or muscle cells Leukemias and lymphomas from white blood cells and precursors as well as from nervous system Most cancers derive from a single abnormal cell Cancer cells are a clone Can be demonstrated by molecular analysis of chromosomes same abnormality in all cells A single mutation is not enough to cause cancer Cancer occurs by progressive accumulation of random mutations in a single lineage of cells Relationship with age People exposed to carcinogens develop cancer many years later Tumor progression involves clonal evolution Development of cancer requires gradual accumulation of mutations Usually 5 or more genes need to be mutated for developed cancer Epigenetic changes contribute to tumor progression In addition to DNA mutations Also inherited from celltocell in somatic cells Packaging intoI 39 39 39 39 h t39 that39 39 genes Methylation of DNA also inactivates genes Cancer cells are genetically unstable Most cancer cells accumulate genetic changes at an abnormally rapid rate Could be point mutations fail to repair DNA damage or to maintain the number or integrity of chromosomes Both increased cell division and decreased apoptosis can contribute to tumorigenesis Cancers may arise from cancer stem cells Stem cells remain in the body and proliferate long enough to accumulate the number of mutations and epigenetic changes required to develop cancer Metastasis is the most deadly aspect of cancer Responsible for 90 of cancerrelated deaths Multistep process Tumor microenvironment influences cancer development Contain many cell types Stroma provides support to cancer cells remodel extracellular matrix secrete signals for cancer cell growth and division Ras the first human oncogene discovered is mutated in one in five cancers Oncogenes a gainof function mutation of the gene can drive cancer Tumor suppressor gene lossoffunction mutation can contribute to cancer Hyperactive Ras cannot hydrolyze GTP to shut itself off 22 August Tutorials On RamCT Quicktime movies course content gt learning modules Lecture objective Key terms and concepts course content Notes version of slides pdf Additional movies and links Quizzes Taken on RamCT Practice multiple choice questions to emphasize key points review course content Additional Study Questions Download from RamCT Course Content gt Learning Modules Help prepare you for essay questions on exams Will post answers a few days later Suggest practicing writing out the answers diagrams Keyword Glossaries are optional iClicker Questions in Class Register your iClicker Promote critical thinking in cell biology Lectures Covered by Five Exams Exam 1 will cover the material in lectures 1 9 The chemical components of cells chemical properties and protein structure and function The molecules that make up a cell and their chemistry Chemical bonds and interactions Biological order and energetics Molecular recognition Protein structure and folding Proteins as catalysts and enzyme regulation Exam 2 will cover the material in lectures 10 17 Membrane structure and function Membrane lipid structure and function Lipid bilayers and their properties Membrane protein structure and function Transport across membranes Cellular compartmentalization Protein trafficking between cellular compartments ER Golgi and membrane processing and trafficking Endocytosis Exam 3 will cover the material in lectures 18 25 Metabolism Catabolism and anabolism metabolic pathways Metabolic regulation Electron transport and oxidative phosphorylation in mitochondria Electron transport and oxidative phosphorylation in chloroplasts Exam 4 will cover the material in lectures 26 33 Cellular communication Signaling systems and their mechanisms Cytoskeleton Filament types composition and function Motor proteins Cytoskeleton dynamics Exam 5 will cover the material in lectures 34 40 More on cell shape and movement and cellular growth control Cytoskeleton in cellular behavior Cell cycle and its control and regulation Programmed cell death apoptosis and senescence Cell biology of cancer What is Life 1 Reproduction 2 Flow of matter and energy metabolism 3 Adaptation 4 Organization Membranes llEmergent properties context is as important or more important thatn the parts Cell Theory Cells Are the Basic Unit of All Life 1665 Robert Hooke quotCellulaequot little rooms in cork 1670 Anton van Leeuwenhoek Improved microscopes to look at live cells 1830s Better microscopes 1838 Robert Brown Cell quotkernelquot or nucleus 1839 Theodor Schwann amp Matthias Schleiden llCell theory All organisms are cells or are made up of cells Cells are the most basic unit of life 1858 Rudolfe Virchow All cells arise from another cell 1937 E Chatton Proka ryotes a nd euka ryotes Overview and Introduction to Cancer Cancer Cells generally Arise through genetic mutations and epigenetic changes that provide growth advantages Mutations and changes typically cause a breakdown in proliferative controls Cells form colonies tumors Cancers originate from a particular cell type single cell Two in five Americans will develop cancer about half of these will die from cancer Most cancers appear avoidable 80 90 Particular to locationlife style liver China lung US skin Australia Arise infrom somatic cells somatic mutationschanges A primary cause is cigarette smoking 30 of cancer deaths 500000 deathsyear in the US What is Cancer A malignant tumor of potentially unlimited growth that expands locally by invasion and systemically by metastasis An abnormal bodily state marked by such tumors First level of classification Benign grow but do not invade surrounding tissue Malignant invade surrounding tissue Metastasis colonization of other sites Second level of classification apparent tissue of origin Epithelial sheetlike tissues carcinomas Mouth to colon skin mammary pancreas lung liver ovary gall bladder urinary bladder 80 of deaths most common type in adult humans Nonepithelial Sarcoma Connective tissues fat cells bone cells muscle cells Bloodforming tissues Leukemias still circulating Lymphomas solid tumors Nervous system tissue Other Melanocytes melanomas Development of a Cancer Cell Requires Multiple Mutations and Changes Incidence of most cancers increase sharply with age Particularly carcinomas Some rarely even seen before 45 years At age 50 years 15000 20fold greater that at 25 y At age 65 11000 chance 100fold greater than at 25 y Tumor cell growth Scary part it takes 30 divisions to get to a billion cells the number needed to detect as a quotbumpquot 1 cm diameter What Causes Cancer Environmentlifestyle Risk factors carcinogens carcinogenic Chemical tobacco chimney soot coal tar Physical radiation xrays etc Lifestyle nuns no children The Human Genome Project The Human Genome 31 billion base pairs 25 30000 genes lOmics Chipbased tests Profiling which genes are expressed in normal cells and in specific cancer cells Will improve diagnosis and treatment of cancers Designer Anticancer Small Molecules Gleevec Developed to treat chronic myologenous leukemia Very specific cellular change Expression of fusion protein BCR ABLE Produces chromosomal change Forms Philadelphia chromosome Switches head and tail from 2 different chromosomes Kills CML cancer cells and not normal cells Fits into cleft on BCR ABLE enzyme Blocks activity Multidrug Treatments Treat with 2 drugs at the same time Low chance of cells becoming resistant to both 7 September Enzymes affect the kinetics of a reaction Can use enzyme kinetics to Compare chemical reactions Compare enzymes as catalysts Specificity and efficiency Study enzyme mechanisms Chemical Kinetics Experiments A P Rate or velocity v k S IltC l P at v0 only k1 is significant v0 k1 S 1 Compare rates or velocities v0 as it varies with S quotSimplequot case ESHESH EPH EP E S associate E catalyzes conversion of S to P E P dissociate Michaelis and Menton Used initial velocities v0 plot vs S Simplifies reaction to k1 k2 E S lt gt ES gt E P 1 Gives hyperbolic curve Michaelis Menton equation Vmax S V KM s KM Michaelis Constant S at V2 Vmax Briggs and Haldane Applied MampM Kinetics in terms and conditions useful for enzyme comparison ES in llsteady state Not changing K2 rate limiting k2ltlt k4 5 very high Enzyme is saturated E5 E1 E1 known V0 k2 ES So v0 k2 El Produces Terms Useful for Comparing Enzymes k1 k2 E S lt gt ES gt E P 1 71 Turnover Number V0 Vmax k2 El Vmax K2 kcal in T an Er 5 measures efficiency Michaelis Constant k71k2 KM k1 f k2ltlt k1 KM KD measure of E and S affinity M 1 Enzyme Rate Constant km KM both efficiency and specificity Comparing Enzymes is Useful for Determining the effect of changing active site amino acids Do they affect the Vmax km or KM NE S affinity Determine if an enzyme inhibitor anticancer drug Competes with S for binding active site quotCompetitivequot see higher apparent KM Binds elsewhere on enzyme not active site quotNoncompetitivequot see lower Vmax Enzyme allostery next lecture The KM for an enzyme catalyzed reaction consistently overestimates the E5 binding affinity Two enzyme preparations have the same KM but the bacterially expressed enzyme has a 10fold lower Vmax The most likely explanation for this is that 90 of the enzyme expressed in bacteria is inactive KM shows affinity If you were to design an enzyme for a cell you would give it a KM near the normal cellular substrate concentration to make the enzyme activity responsive to changes in cellular substrate concentrations Enzyme kinetic experiments are useful for comparing different enzymes that catalyze the same reaction studying the effect of amino acid changes on substrate binding and catalysis and determining if an enzyme inhibitor competes with the substrate for binding An anticancer drug that works through competing with a substrate for binding the active site will increase the apparent KM 12 September Anticancer Drug Synthesis and Testing Drug Design Overview Aspirin Has three functional groups The original source white willow tree bark tea New drugs derived by redesign of aspirin to form new analgesics Antibiotics Penicillin Original source fungus Penicilliumnotatatum Active agent identified and purified Penicillin G New variants ampicillin oxacillin cloxacillin penicillin O and amoxicillin Paul Ehrlich in early 1900 s Searched for a llmagic bulletquot to cure syphilis Systematically changed small molecules Assessed activity Approach now known as a llstructure activity relationship study Small Molecule Drugs llActive Area Bind through their specific shapefit and functional group non covalent interactions Function through binding in place of the normal ligand hormone ampc Example the opiate morphine Difficult to synthesize llActive area incorporate into a simpler more easily synthesized molecule Demerol Demerol still binds and has opiate activity Computer modeling is now used based on these same principles to design potential new drugs Combinatorial chemistry Synthesis of large quotlibrariesquot of molecules Use high throughput screening for most effective molecules Greatly reduced costs Cancer Drugs and Targets Drugs Low molecular weight Easier to synthesize More likely to enter tumors or cells Targets quotDruggablequot Proteins with enzymatic function Have defined actives site cleft for drug binding site Kinases are good candidates 518 in human kinome 90 are tyrosine kinases Hormone receptor binding sites also good candidates Tamoxifen for the estrogen receptor RasGTPase site is not a good candidate inhibitory drug would promote cancer Kinases Share similar structures Tyrosine kinases are very similar structurally and are particularly involved in cancer llRational drug design to target kinases malfunctioning in cancer cells Tarceva inhibitor of EGF receptor kinase activity 3D active structure of E6 FR known Design drug to quotfitquot well and form multiple non covalent bonds Still mostly using more empirical approach Testing compounds for specificity and effectiveness Kinases inhibition assays Binding affinity assayselection procedures Pretrials in cell culture Phase trials in humans Phase II trials in humans Age adjusted patient survival Is it really more effective Example EGF Receptor Directed Drugs Skeleton binds ATP binding sites Additional functional groups provide EG FR kinase binding specificity ressa has very high specificity and affinity A small proportion of nonsmallcell lung cancer patients respond to ressa treatment These patients have EG FR that is llstuck onquot Cancer cells have lloncogene addiction Dependent on oncogenes These cancers become resistant to ressa Summary Cancer may not be curable just controllable Cancer is minimally about 100 different diseases Cancer is primarily an old person s disease Measures of cancer incidence mortality and treatment effectiveness are still imprecise There are continual problems with the development of drug resistance multidrug therapies should improve this Targets for future drug development are plentiful Systems biology bioinformatics an omicsquot should provide a much more holistic understanding of individual cancers Exam 1 Covers Lectures 1 9 today 2 times amp places Thursday 15 September 6 8 PM Path 101 Friday 16 September noon 150 PM Aylesworth C 111 Review session Thursday 5 PM Path 101 The process of anticancer drug discovery has begun to employ compound libraries through combinatorial chemistry KD V2 Vmax Drug resistance is a constant problem in anticancer therapy that can be addressed by giving patients multiple drugs that inhibit two or three oncoproteins found in a particular cancer 30 September Introduction to Metabolic Regulation Regulated steps at the beginning Catalyzed by enzymes that are regulated These enzymes Have sigmoidal responses to S Regulated hetereoallosterically by P P2 or purely regulatory molecules Regulated in response to ATPADP Catabolic vs Anabolic Pathways Catabolic produce ATP amp NADH Shut off by high ATP Activated by low ATP Anabolic use ATP and NADH Shut off by low ATP Activated by high ATP Have bypass reactions at key regulated steps catalyzed by different enzymes Avoid futile cycles catabolic and anabolic pathways running at the same rate just using up energy Glycolysis and Gluconeogenesis Takes about twice as much ATP to produce glucose than to break it down Energy flow diagram 2ADP 9 ATP AMP AMP used as effector indicative of low energy charge Summary of key regulated steps in glycolysis Step 1 Glucokinase is inhibited by GGP amp F6P in gycoysis GGP activates 66 Phosphatase in guconeogenesis Step 3 AMP and Fructose 263isP activate Pfructokinase in gycoysis ATP inhibits AMP and Fructose 263isP inhibit F16 Bisphosphatase in guconeogenesis AMP ratio of ATPADP energy charge Step 10 ATP and AcetylCoA inhibit Pyruvate kinase in gycoysis F163isP activates AcetylCoA activates Pyruvate Carboxylase in guconeogenesis ZADP 9 ATP AMP ATP 5 mM ADP 02 mM AMP 0004 mM Change ATP 6 lead to ADP 250 AMP 500 If AMP increases gycoysis increases guconeogenesis decreases AMP increase low ATPADP ratio Role of Fructose 263isP in Liver Cell Metabolism The liver produces glucose for the brain muscles etc Fructose 263isP F26bisP F26bisP is not a glycolytic pathway intermediate Regulates Phosphofructokinasel PFKl and Fructose 163isPhosphatase F16 BisPtse allosterically step 3 bypass reactions F26bisP levels are regulated by PFKZF26 BisPtse Activity Bifunctional enzyme Contains both kinase and phosphatase domains Both domainsactivities are in a single enzyme The enzyme is phosphorylated by CAMP dependent kinase PKA It is dephosphorylated by phosphoprotein phosphatase PKA activity is regulated allosterically by CAMP Glucagen in the blood regulates CAMP production in liver cells PKA is under hormonal control by glucagon produced when blood glucose is low PKA is activated by CAMP Overrides energy charge regulation Summary Low blood glucose 9 increase glucagon in blood 9 increased CAMP in liver cytoplasm 9 increased PKA activity 9 increased F26 BisPtse activity 9 decreased F26 BisP levelincreased F6P 9 increased F16 BisPtse activitydecreased PFKl activity 9 increased gluconeogenesisdecreased glycolysis 9 increased glucose in liver released into blood Tumor cells High levels of glycolysis 9 pyruvate 9 lactate Low 02 Lactate converted to glucose in cells w high Oz ATP production Glutamine 9 glutamate 9 ot ketaglutarate Produces ATP NADH amino acids Take home points Regulation of metabolic pathways Steps are catalyzed by enzymes Enzymes regulated Bypass reactions amp enzymes llEnergy charge ratio of ATP to ADP AMP Regulation of glycolysis vs gluconeogenesis 3 is key first committed step Step 3 F6P 9 F1GBisP Phosphorylation Phosphofructokinasel Fructose 163isPhosphatase works in other direction AMP activates PFKl inhibited by ATP F26P2 regulation of glycolysis vs gluconeogenesis in liver cells Glucagon 9 liver 9 PKA 9 phosphofructokinaseZ 3 Octo be r Introduction Cells produce ATP two ways Substrate level phosphorylation steps 6 amp 7 of glycolysis Oxidative phosphorylation ETC amp ATP synthase Oxidation of nutrients through glycolysis TCA cycle etc Produces NADH and FADHZ Does not use molecular Oz NADH and FADHZ pass e39s downhill to Oz through ETC Through series of e acceptorsdonors Uses molecular Oz Downhill e flow drives H pumping out of mitochondrial matrix Mitochondrial Structure Intermembrane space pH 7 Outer membrane porous nner membrane mpermeable ETC amp ATP synthase Matrix TCA cycle enzymes pH 8 Cytoplasm pH 7 Chemical Components of the ETC e carriers Go through redox cycles NADlt gt NADH 2 e39 1 HI FAD e FADHZ 2 e 2 HI FMN e FMNH2 2 e39 2 H CoQH CoQHz 2 e39 2 H Feig FeI2 1 e39 Cu29 Cu 1 e The Electron Transport Chain Complexes Overview of the Electron Transport Chain 4 complexes Electrons start from NADH succinate from TCA cycle Feed into ubiquinone pool Proton pumping in complexes I between II and III and IV Electrons from III pass to cytochrome C then complex IV then to Oz Complex I NADHCoQReductase Passes e from NADH to Coenzyme Q Passes through intermediate FMN flavine mononucleotide 9 FM NH2 9 iron sulfur complex Coenzyme Q picks up protons from matrix CoQH2 Protons are pumped Complex II Succinate Dehydrogenase Picks up e39 from succinate Puts through FAD 9 FADHZ Passes to iron sulfur complex No proton pumping Passes e to CoQ picks up protons 9 CoQHz Complex III Cytochrome c Reductase Passes e to cytochrome c CoQHz from complex I amp II and COO cycle feeds e into cytochrome b2 or bL Passes to cytochrome bH Iron sulfur complex cytochrome c1 Passes e to cytochrome c Cytochrome c Soluble heme protein Peripheral membrane protein Complex IV Cytochrome c Oxidase e from cyt c Passes to cyt a1a3 copper complex Holding place Pass to 02 picks up protons from matrix H20 Pumps proton from matrix to intermembrane space Complex IV Transfers 4 e39 to O2 Sequentially Intermediate steps are highly reactive Occurs in bimetallic center Copper complex ampheme hold e s Feeds into 029 H20 Energy of e passage drives proton pumping The Mechanism for Proton Pumping Proton binds Drives conformational change AG Coupling of energy drives additional change puts binding site on outside Release Resets conformation 12 October Overview Recap of the Warburg Effect First described by Otto Warburg and coworkers in 1927 Tumor and cancer cells Increased glucose uptake and lactate production relative to normal cells With normal Oz levels Termed quotaerobic glycolysis In the brain astrocytes and neuronal cells form a commensal metabolic relationship Astrocytes quotaerobic glycolysis Neurons import lactate use for oxidative phosphorylation Neurons also convert glutamine to glutamate release as neurotransmitter Astrocytes convert glutamate to glutamine toxic In early embryogenesis First three cell divisions quotaerobic glycolysis After that use both quotaerobic glycolysis and oxidative phosphorylation Embryonic cells also use glutamine to fill up the TCA cycle and produce amino acids etc Radiolabeled fluoro2deoxylgucose in positron emission tomography PET Shows high glucose uptake in cancer cells compared to normal cells Have found cancer cells also need a lot of glutamine Cancer cells have changes that short circuit the normal inhibition of glycolysis at normal Oz levels Tumor cells with poor blood supply and low 02 appear to have a commensal relationship with other cancer or normal cells with adequate Oz Cancer cells with quotaerobic glycolysis also have high oxidative phosphorylation levels Cancer cells rely on glutamine for filling up the TCA cycle and producing amino acids etc More research is needed to better understand the other metabolic changes that occur in cancer cells Targeting Cancer Cells Through the Warburg Effect Cancer cells vs normal cells have unregulated growth and cell division Independent of actual nutrient supply quotAddictedquot to nutrients If nutrients inadequate Autophagy Cell death So key metabolic enzyme inhibitors could be anticancer drugs When glucose first enters a cell phosphorylated by hexokinaseZ Forms glucose 6 phosphate keeps it in the cell HexokinaseZ inhibited by 3bromopyruvate Anticancer effects 3rd step Forms fructose 16bisphosphate Activated by fructose 26bisphosphate Decrease levels downregulate glycolysis 3P0 inhibits enzyme that products fructose 26bisphosphate phosphofructokinase 2 Pyruvate kinase Converts phosphoenol pyruvate to pyruvate Regulated by fructose 26bisphosphate Working on inhibiting Lactate dehydrogenase Converts pyruvate to lactate Also a target for drugs FXll inhibits Leads to death in cancer cells Decrease in NAD Buildup of NADH Probably activates ETC creating reactive oxygen species Pyruvate dehydrogenase kinase Phosphorylates pyruvate dehydrogenase converts pyruvate to acetylCoA Upregulated in cancer cells Inhibits anticancer effects Glutamine 9 glutamate 9aketoglutarate Glutaminase GLS Converts glutamine to glutamate 968 inhibits enzyme amp cancer cell development Glutamate dehydrogenase G LUD and glutamate pyruvate transaminase GPT Converts glutmate to otketoglutarate EGCG Component in green tea Inhibits GLUD AOA Inhibits GPT Isocitrate dehydrogenase IDH In TCA cycle Converts isocitrate to ot ketogluta rate Mutant versions convert otketoglutarate to 2hydroxyglutarate IfGLUD inhibited w BPTES inhibitors work better if IDH is mutant Summary Drugs that inhibit metabolic enzymes have promise as anticancer therapies Like other changes in cancer cells the metabolic changes in specific cancer types and individual cancers will vary Therefore characteristics of metabolic profiles of cancer types and individual cancers will inform more targeted and effective anticancer therapies using metabolic enzyme inhibitors etc perhaps in concert with other anticancer drugs 16 November Types of muscle Skeletal muscle Cardiac muscle and smooth muscle Muscle contraction depends on the ATPdriven sliding of highly organized arrays of actin filaments against myosin filaments Skeletal muscle cells also called muscle fibers A muscle fiber is a huge single cell formed during development by the fusion of many separate myoblasts Typically 50 pm in diameter and up to several centimeters long Contains the myofibrils A myofibril consists of a long repeated chain of sarcomeres The sarcomere is the contractile unit Partly overlapping array of parallel thin actin and thick Myo II filaments Electron micrograph of an insect flight muscle viewed in cross section Region of overlap between thin and thick filamentsHexagonal lattice The 300 myosin heads in a thick filament are not coordinated therefore low processivity is critical Organization of accessory proteins in a sarcomere Actin filament plus end binds to the 2 disc which is built by CapZ and ot actinin Nebulin is a large protein with an actinbinding repeat Molecular ruler Tropomodulin positions the thick filament midway between the 2 discs Molecular spring The force generated during contraction depends on the degree of overlap of the thick and thin filaments Muscle contraction is triggered by a nerve impulse that results in release of acetylcholine at the neuromuscular junction synapse A sudden rise in cytosolic Ca2 initiates muscle contraction Ca2 enters the cell and triggers more Ca2 release from the sarcoplasmic reticulum that surrounds the myofibrils Ca2 flooding into the cytosol initiates contraction of all myofibrils at once Ca2 is pumped back into the sarcoplasmic reticulum 9 myofibrils relax The control of skeletal muscle contraction by troponin Resting muscle troponin pulls tropomyosin to block the myosin binding site With high calcium troponin does not pull tropomyosin myosin heads can walk along the actin filaments Construction of the mitotic spindle requires microtubule dynamics and the interactions of many motor proteins Actin structures involved in cell crawling Protrusion at the leading edge New attachments made at the front and disassembly of old attachments at the back Contraction at the rear of the cell Different cell types generate different types of protrusive structures First step in locomotion they push the membrane outward Filopodia Lamellipodia actin organized in two dimensions best characterized Pseudopodia Actin filament nucleation and web formation in ameipodia ARP complex is highly concentrated near the front of the lamellipodium where actin polymerization is most active Treadmilling of the web as a whole drives protrusion of the leading edge Effect of the Rho protein family members on actin organization Rho stress fibers Rac lamellipodium Cdc42 filopodia Neutrophil polarization and chemotaxis Lamellipodium extends towards the source of chemoattractant Signaling involves Gprotein coupled receptors Rac activation at leading edge and Rho activation in opposite region gt actinmyosin contraction 2 September History and Overview Research on how sugar is digested and fermented 1850 s Pasteur a quotvitalistquot believed needed live cells 1897 Buchner a quotmechanistquot cell free extracts work Kiihne quotenzymequot Greek for quotin yeast 1926 Sumner 1st to purify an enzyme was a protein Enzymes Proteins Catalytic unchanged Named generally by ending in quotasequot Why special different from other catalysts Very specific for substrates and chemical pathway Can be regulated Can quotcouplequot reactions Business end the quotActive Site Cleft or crevice Substrate fits into active site cleft R groups provide interactions chemistry Multiple non covalent interactions Four main types of catalysis Acidbase Covalent intermediate Reaction intermediate stabilization Orientationproximity Mechan Mechan isms of Catalysis kinetics vs thermodynamics Enzymes affect pathway and kinetics rate not thermodynamics Pathway independent Determines direction Enzymes can affect rate 103 to 1012 fold usually around 108 to 1012 fold Si is transition state AGi is activation energy AAGi is how much the enzyme changes the necessary activation energy Rate olt ii Enzymes affect AGi not AG of the reaction isms of Catalysis Chemistry AcidBase catalysis Makes acid and base more reactive Enzymes can do M at the same time Would make a reaction even faster Covalent Catalysis Amino acid R group forms transient covalent bond with S Transition State Stabilization Binds Si tighter than S Stabilizes Si lowers G Puts quotstrainquot in S pulling toward Si ProximityOrientation Brings reactants together Increases effective concentration Puts in proper orientation Functions of Apoptosis SculptingDeveloping Metamorphosis tadpole to frog ampc Nervous system development half die Classes of signals two leading to apoptosis Extracellular Death signals death receptor through FADDcaspase 8 Intracellular DNA damage and oncogene expression activatestabilize p53 to increase proapoptotic genes and initiate quotapoptosomequot formation Initiating effectors Caspase8 and Caspase9 Executing effectors Caspase3 6 7 Nuclear lamina nucleases Cytoskeleton accessory proteins Other cytoplasmic proteins Chews up cell parts Function through a proteolytic cascade 9 September General Enzyme Regulation Two main ways Expression protein level takes minutes to hours Enzyme activity takes seconds Conformational changes Affects ability to reduce AGIl kcal and KM In response to Substrate binding Effector binding bind site separate from active site Generally referred to as quotligandsquot Regulated enzymes often have sigmoidal kinetics Greek quotAllosquot other quotStereosquot shape Homoallostery substrate is effector Heteroallostery non substrate effector Hemoglobin T tight state binds BPG amp CO2 more tightly R relaxed state binds 02 more tightly Relaxed is higher affinity Hemoglobin vs myoglobin Myoglobin Mb monomer Hemoglobin Hb heterotetramer Hb also regulated by Lower pH stabilizes quotTquot C02 Cterminus modification stabilizes quotTquot BPG binds and stabilizes quotTquot Hb has cooperativity in binding since it has 4 monomers Positive linkage both substrates bind the same conformation more tightly cooperativity Negative linkage can occur as well Kinetics of Allosteric Enzymes Sigmoidal vs Hyperbolic M M Kinetics Sigmoidal kinetics is not Michaelis Menton kinetics Requires ltAS to go from 10 to 90 of Vmax Keeps S in tighter range Cooperativity multisubunit Still homoallostery Heteroallostery Second non substrate effector ligand Feedback Regulation and metabolic pathways Negative feedback usually occurs at first unique step Aspartate Transcarbamoylase ATCase quotTquot form has low affinity amp low activity quotRquot form has high affinity amp high activity CTP binds on nonregulatory ligands and stabilizes in T state Heteroallostery Kinetics Effect of Activators and Inhibitors on Enzyme Kinetics Binding of effectors Positive effector activator A Negative effector inhibitor I Additional Enzyme Regulatory Mechanisms Covalent modification posttranslational Phosphorylation dephosphorylation 30 of all proteins are phosphorylated 98 on SerThr 2 on Tyr Kinases and Phosphatases are also regulated Kinases add phosphate Phosphatases take off phosphate Phosphorylation state is in a lldynamic equilibrium or llsteady state Proteolytic cleavage Irreversible Example zymogens Expression or protein level Combinations Localiziation where enzymes are located in the cell 28 November Each cell cycle produces two genetically identical daughter cells The only way to make a new cell is to duplicate a cell that already exists Cell cycle the cycle of cell growth duplication of its contents and division The most fundamental task is the passing of its genetic information to the next generation of cells The major events of the cell cycle Chromosome duplication occurs during S phase 12 hours in mammalian cell Chromosome segregation and cell division occur in M phase 1 hour M phase 9 nuclear division cytoplasmic division The stages of nuclear division mitosis and cell division cytokinesis n prophase DNA molecules are U and 39 into sister Nuclear envelope disassemny 9 sister chromatids attach to the spindle giant array of microtubules Anaphase sister chromatids align at the spindle equator and separate Telophase packaging into separate nuclei The eukaryote cell cycle is divided into four phases Time to double the mass of proteins and organelles gt time to duplicate chromosomes and divide G1 phase S phase G2 phase interphase 23 hours The cell monitors conditions before committing to S phasemitosis GU phase Start point Cell cycle control is similar in all eukaryotes The organization of the cell cycle is essentially the same in all eukaryotic cells Yeast 9 genetic manipulation mutations growth in haploid state Many celldivisioncycle genes Cdc genes were discovered in yeast Cell cycle control can be studied by analysis of yeast temperaturesensitive mutants A mutant cell that cannot complete the cell cycle cannot be propagated Temperaturesensitive mutants have a conditional phenotype At the restrictive temperature cells halt at a specific point in which the mutant gene is needed Cell cycle control can be studied in animal embryos the frog egg 1 mm in diameter and contains 100000 times more cytoplasm than a human cell 9 easy to observe Upon fertilization the cell divides many times without growing 9 very fast no 61 or 62 and simplified Cell cycle control can be studied in cellfree systems the frog egg cytosol Xenopus eggs allow to prepare almost pure cytoplasm Reconstitution of many events of the cell cycle in the test tube Cell cycle control can be studied in cultured mammalian cells Problem tend to stop dividing replicative cell senescence quotImmortalizedquot cell lines are commonly used Cell cycle progression can be studied in various ways Measuring DNA content with a DNA binding dye Synchronization ofa cell population and follow up 11 November Nucleation is the ratelimiting step in polymer formation Formins nucleate the growth of straight unbranched filaments Remains associated with the growing plus end as the filament elongates Dimeric protein Proteins that bind to the free actin subunit modify filament elongation Thymosin binds actin subunits and prevents incorporation in the filament Profilin does the opposite Stathmin binds to tubulin dimers and inhibits polymerization Signals such as phosphorylation change the equilibrium Severing proteins regulate the length and kinetic behavior of actin filaments and microtubules Katanin severs microtubules Requires ATP Gelsolin severs actin filaments Proteins that bind along the sides of filaments can either stabilize or destabilize them Microtubuleassociated proteins MAPs stabilize microtubules Different lengths of projecting domain bundles microtubules at different density Tau axon MAPZ dendrites and cell body Tropomyosin stabilizes actin filaments Important for muscle contraction Cofilin forces the filament to twist more and become brittle It also speeds up ADPactin dissociation from the minus end Proteins that interact with filament ends can dramatically change filament dynamics Large effect on filament dynamics with low protein amounts Capping protein CapZ binds and stabilizes the actin filament plus end 9 only minus end elongates or shrinks slower dynamics MAPs and catastrophic factors compete to affect microtubule plus end dynamics Cell cycle regulation through phosphorylation TP proteins found at the growing plus ends of microtubules The plus ends of microtubules explore the entire cell space Plus end tracking proteins TPs associated with the plus end as passengers Some TPs control microtubule positioning and dynamic instability Filaments are organized into higher order structures in cells Individual filaments are linked to one another in various ways Example Plectin crosslinks microtubules with an intermediate filament MAPs bundle microtubules Crosslinking proteins organize different assemblies of actin filaments Bundles and weblike structures Modular structures of four actin crosslinking proteins Contain 2 actin binding sites Different spacer distance ot actinin has a 30nm spacer that allows myosin II to enter and contract the array for example in stress fibers Villin is a crosslinking protein in microvillus actin bundles Filamin crosslinks actin filaments into a web or gel rather than bundles Important for lamellipodia formation projection that helps cells crawl across surfaces Filamin is missing in most melanomas 21 September Introduction some definitions Lipid bilayersmonolayers are permeability barriers Molecules can cross by simple passive diffusion Molecules can cross through channel and carrier proteins Transport can be passive or active Passive drive by diffusion AG with concentration or charge gradient down hill Active AG against concentration or charge gradient up hill Primary active uses energy source directly Secondary active uses energy source indirectly AG of Concentration Gradients Across Membranes AGi lowest for nonpolar S highest for charged S AG mostly due to AS 5 IN 5 OUT AGRTln If sow gt 5m then M 1 50w Ln SIN 0 50w So AG lt 0 Simple Diffusion Passive Hydrophobic molecules free diffusion Small uncharged polar molecules slow diffusion Large uncharged polar molecules very slow diffusion Ions no diffusion Small uncharged polar molecules large uncharged polar molecules and ions all require transporters Passive Facilitated Diffusion and Transport Movement with concentration or charge gradient down hill electrochemical gradient Pores or channels amphipathic othelices Bbarrels Some are gated open and close in response to signal Carrier Proteins Proteins bind molecules Go through conformation change Catalyzed reaction enzyme kinetics Active Transport Movement against concentration or charge gradient up hill electrochemical gradient Primary Active Transport Uses energy source directly Energy sources ATP NADH light Example NaK ATPase or Na pump Na pumped against conc and charge gradient AG Ki pumped against conc gradient but with charge gradient gtAG ATP 9 ADP Pi gtAG Net AG NaiKATPase mechanism ElP to E2P transition has a large AG Used to pump Na ions against the Na gradient which has a smaller AG Digitalis inhibits drug for antiheart attack Secondary Active Transport Uses energy source indirectly Energy stored in an electrochemical gradient Uniport one molecule travels Cotransport Symport several molecules same direction Antiport several molecules opposite directions Example Naglucose transporter Glucose to inside Nai binds causes conformational change 9 high affinity for glucose Glucose binds conformation change flips binding site lower affinity for sodium Na leaves conformational change 9 low affinity for glucose falls off Flips back to starting position Glucose Transport Intestinal Lumen to Blood Starts with NaATPase pump from intestinal cells Na out of cell into blood Antiport primary active transport Naglucose transporter From intestinal lumen to intestinal epithelial cell Symport secondary active transport GLUTZ Uniport passive transport Glucose transported from cell to blood Tight junctions between cells prevent leakage Cancer cells can use transport to pump out anticancer drugs Takehome points Lipid Bilayers Permeability nonpolar gt polar gt charged small gt large Electrochemical gradients AG AGIl Membrane proteins Passive channelscarriers Active primarysecondary Active transport mechanisms 2 December Cells dying by apoptosis undergo characteristic morphological changes Cells shrink and condense cytoskeleton collapses nuclear envelope disassembly chromatin condenses and fragments Cells form blebs Necrosis cells die by acute insult they swell and burst Programmed cell death eliminates unwanted cells Crucial during development Half of the neurons normally die soon after they are formed Example 1 sculpting the digits in a developing mouse paw Example 2 metamorphosis ofa tadpole into a frog Quality control eliminate cells that are abnormal nonfunctional damaged DNA damage Apoptotic cells are biochemically recognizable Endonucleases cleave DNA Phosphatidylserine flips from inner to outer leaflet of plasma membrane Signals macrophages to phagocytose the dying cell Release of mitochondria proteins from the intermembrane space Cytochrome c Apoptosis depends on an intracellular protelytic cascade caspases Procaspases are activated by proteolysis forming heterotetramers Initiator and executioner caspases Amplification Targets nuclear lamin cytoskeletal proteins cellcell adhesions proteins Tissue specificity Not reversible Cell surface death receptors activate the extrinsic pathway of apoptosis Tumor necrosis factor TNF family of receptorsHomotrimers Killer lymphocyte best known example Fas ligand Death inducing signaling complex DISC Release of cytochrome c from mitochondria during apoptosis Initiate the intrinsic pathway of apoptosis Response to injury or stress such as DNA damage lack of oxygen or nutrients The intrinsic pathway of apoptosis Cytochrome c binds to apoptotic protease activating factor1 Apafl Procaspase9 activated by proximity in the apoptosome The three classes of Bcl2 proteins Antiapoptotic Bcl2 protein Proapoptotic BH123 protein Proapoptotic BH3only protein The intrinsic pathway of apoptosis role of BH123 proapoptotic Bcl2 proteins Apoptotic stimulus 9 BH123 aggregates on the outer mitochondrial membrane Mediate release of cytochrome c and other intermembrane proteins The intrinsic pathway ofapoptosis regulation by proapoptotic BH3only and antiapoptotic Bcl2 proteins Absence of apoptotic stimulus Bcl2 binds and inhibits BH123 on mitochondria Apoptotic stimulus BH3only binds Bcl2 so it can no longer inhibit BH123 Different stimuli activates different BH3only proteins The control of apoptosis in mammalian cells roles of IAPs and antiIAPs Inhibitors of apoptosis IAPs bind and inhibit caspases AntiIAP proteins reside in the mitochondria intermembrane space When BH123 is activated antiIAPs are released and bind IAPs blocking their ability to bind caspases Less important than Bcl2 regulation in mammals Extracellular survival factors inhibit apoptosis in various ways Most cells needs continuous survival factor signaling from other cells to avoid apoptosis Nerve cell example they compete for survival factors from target cells 31 August Overview Proteins Last step in gene expression are final product Constitute most of the dry mass of cells Carry out most of the cellular functions Structure dictates function Protein structure and function builds directly on the concepts and principles we covered in lectures 1 4 Building Blocks amino acids aa 20 common aa Convention for writingdrawing is N to C General aa structure 01 carbon central C of amino acid Carboxyl group on right Amino group on left R group differs between aa Positive charge on amine group end negative charge on carboxyl group end R Group Classifications Hydrophilic polar Charged Acidic Negative charge Form H amp ionic bonds Basic Positive charge Form H amp ionic bonds Uncharged Form H bonds Hydrophobic Form hydrophobic interactions amp van der Waals Special Are nonpoar Proton Basically no R group Glycene Form hydrophobic interactions and van der Waals Prolene Hydrocarbon chain bends back and bonds covalently to N ofamine group Forms hydrophobic interactions amp van der Waals Cystines Sulfhydryl group Forms disulfide bonds stabling structures for proteins Some polar R groups are charged at the appropriate pH Peptide bonds link amino acids dehydration synthesis Between C of carboxyl group amp N ofamine group Peptide Bonds in Protein Folding Have resonance Electrons jump back and forth CO or CN Partial double bond Planar structure Restricted rotation R groups and CO groups can clash Ste ric Protien Folding and Structure four levels Denatured unfolded Native renatured or properly folded Primary Structure 1 quotSequencequot of amino acids N to C MetAlaSeretc MASetc 20quot possible sequences where n aa Connected by peptide bonds covalent Secondary Structure 2 initial folding ot helix Stabilized by Hbonds between every 4 haa 36 aa turn R groups stick out Often drawn as cylinders Can be amphipathic hydrophilic on one side hydrophobic on the other B sheet made up of Bstrands extended polypeptides Stabilized by Hbonds R groups stick up and down Often drawn as strings of flat arrows Each called a Bstrandquot Loops or turns Between B strands amp sheets between 01 helices or between B sheets amp ot helices Induced bends by prolines Tertiary Structure 3 Folding of 2 structure into a stable 3D form Driven and stabilized by R group interactions Initially driven by hydrophobic groups entropy AS Hydrophobic inside Hydrophilic outside Stabilized by Hbonding ionic bonds van der Waals interactions Form from a single polypeptide From into subunits and domains Domains together make up subunits Quaternary Structure 4 Association of separate polypeptides or subunits Homodimer same 2 polypeptides Heterodimer different 2 polypeptides Trimer 3 polypeptides Tetramer 4 polypeptides Protein Structure Changes Leading to Cancer Tale of two proteins with two types of changes Gain of function changes mutations Ras Loss of function changes NFI Both changes lead to increased cell proliferation cancer Ras protein functions in signaling cell proliferation Cycles between inactive amp active form Inactive form bound by GDP guanine nucleotide diphosphate Active form bound by GTP Sends signals downstream Leads to cell proliferation GTPase cleaves 3rd phosphate group from GTP Returns Ras to inactive state Glycine 12 and Glutamine 61 in binding site for GTP most commonly change in cancer cells Blocks inactivation Stuck on GTPase inactivated Neurofibromin NFI protein changes lead to neurofibromatosis NFI functions to activate RasGTPase Changes in NH inactivate it loss of function Ras is stuck on 19 September Introduction Lipid bilayer Provides structure Acts as a permeability layer Membrane proteins provide functions Transport Signaling Cell recognition Photosynthesis electron transport chain oxidative phosphorylation Membrane protein content varies Three Classes of Membrane Proteins Classified by how the protein associates with the membrane Lipid anchored Peripheral Integral Lipid Anchored Proteins Intracellular side Stuck on fatty acids NterminalNHZ or serthr OH or prenyl group cys SH Extracellular side Stuck on membrane lipids C terminal COO Peripheral Membrane Proteins Bind anchored and integral membrane proteins and membrane lipids Bind through noncovalent bonds Can be removed by high salt washes Integral Membrane Proteins Inserted into lipid bilayer hydrophobic environment Transmembrane proteins Span lipid bilayer Single pass transmembrane protein Extracellular side Disulfide bonds sugars attached Hydrophobic ot helix 20 aa Hbonds between backbone Hydrophobic R groups Amphipathic proteins Hydropathy plots show which parts are hydrophilichydrophobic Multipasstransmembrane proteins Multiple othelices Forms channels Bbarrel Twisted curved Bsheet Amphipathic othelices amp Bsheets Detergents can be used to purify membrane proteins Nonionic detergents Triton X100 Easy on molecules not denaturing Mix w membranes Single tail conical form micelles Form micelles around proteins Experiments using mild detergents to study membrane proteins Replaces detergent w 39 39 39 known39 0 quot Membrane Protein and Lipid Glycosylation Sugars stuck on Glycosylation forms the llcell coat Protects exposed cells Functions in cell recognition Selectins recognize white blood cells through glycosylated membrane proteins Membrane Proteins Diffuse Freely in 2D Lateral diffusion can be restricted Usually anchored lipid raftscytoskeleton Examples RBC Cytoplasmic Membrane Proteins Simple cells Singlepass glycophorin A Multipass band 3 Peripheral spectrin Actin filaments stabilize cell membrane 9 December Cancer is caused by loss or gain of functions Tumor suppressor genes loss ofan inhibitory function recessive mechanism and requires loss of both copies Oncogenes gain of function ofa stimulatory protein act in a dominant fashion and require only one copy to be mutated Mutations can occur through large global chromosomal changes or small changes to nucleotides in DNA Multiple mutations are needed for a cell to become cancerous Cancer cells reproduce in defiance of normal restraints of cell growthdivision Mutations accumulate over time and as a result of previous mutations Mutations that cause cells to ignore signals are common in cancer Mutated proteins are generally in a signaling or repair pathways Myc Ras APC Rb and p53 mutations are common in cancers Mutations that deregulate cell division P16 is produced in response to cell stress to stop growthdivision Rb is a tumor suppressor and functions as a pocket protein Rb inhibits EZF a promoter for transcription factors required for entry into S phase Extracellular signals for growth and division A mitogen triggers cell division mitosis A growth factor can stimulate growth proliferation division and differentiation Mitogens growth factors and downstream signaling targets are often protooncogenes P53 is an oncogene that is mutated in many cancers Activate DNA repair proteins upon DNA damage Induce growth arrest on DNA damage recognition Initiate apoptosis p53 mutation allows for more downstream mutations and allows for genetic instability Genetic instability leads to additional mutations Removinginactivating p53 allows cells to divide with DNA damage Larger scale chromosome changes occur as a result Accumulation of mutations correlates with aggressive malignant tumors Viruses can also cause cancer Viruses hijack host proteins to reproduce viral genome DNA viruses Viral genomes are integrated into host genome Human papillomavirus HPV Human papillomavirus acts through Rb and p53 to cause cancer HPV early genes E6 and E7 act as oncogenes Responsible for 52 of new cases of cancer Higher risk for women cervical cancer cell type specific effect Metastasis is a bad sign Unknown what makes some cancers metastatic Tumor migration local invasion and metastasis Circulating cancers leukemia are more likely to be metastatic Once colonization occurs it makes treatment much more difficult Case study colorectal cancer Causes 11 of cancer deaths in US Tumors can be benign for 10 35 years before becoming malignant Tumor gets bigger and cells become more unusual develop more mutations Ras APC and p53 have important roles in colorectal cancer development Case study colorectal cancer subtypes Two main types familial adenomatous polyposis coli FAP and hereditary nonpolyposis colorectal cancer HNPCC FAP display a high degree of genetic instability HNPCC are caused by small quotinvisiblequot errors in key proteins small expansions and deletions Case study Colorectal cancer progression A progressive loss of tumor suppressors and gain of oncogenes correlates with advancing colorectal cancer FAP is detected at early stages as adenomas HNPCC isn t detected until larger tumors form Early detection is critical for treatment prognosis preloss of p53 Cancer treatment prevention Many cancers have known risk factors Behavioral smoking Age Environmental western diseases Genetic predisposition BRCA V2 mutations Cancer screening Cancer Treatment Shotgun approaches Chemotherapy cancer cells divide rapidly kill all rapidly dividing cells Radiation therapy damage DNA of cells with ionizing radiation cancer cells won t be able to repair themselves and will die Sniper approaches Target cells Using drugs cells or virus to attack cancer Target pathways Inhibitors to DNA repair pathways necessary for viability Target proteins Target oncogenes Cancer therapy targeting pathways Many tumor suppressors are involved in DNA damage repair p53 Cancer cells will attempt to divide even with DNA damage Damage cells so much they can t survive without repair Cancer therapy targeting oncogenes Specific oncogenes are present in cancer and never in normal cells BcrAbl is a chimeric oncogene Different structure than either Bcr or Abl BcrAbl is a hyperactive kinase that targets Ras Small molecule binds specifically to BcrAbl and blocks function Gleevec Specific nontoxic therapy Specific to certain cancer types Cancer therapy drug resistance Cancer cells are prone to mutation Drug resistance is common is cancer worse is multiple drug resistance MDR I I Can attack cancer with severa weakerquotmore genetic drugs that aren t strong enough alone 26 September Introduction Types of Metabolism metabolic pathways Catabolic Nutrient degradation oxidation Produce ATP Anabolic Biosynthesis reduction Requireuse ATP Amphibolic Pathways that can do both Oxidation and Reduction of Cellular Molecules Oxidation loss of e Reduction gain of e ATP Why is it a llhigh energy molecule ATP H20 9 ADP Pi H Intrinsic Property AG AG chemical standard conditions 1M everything 298 K K ADP Pi 11 9 39 ATP H20 AG 24 kcalmol AG biochemical std conditions HZO 56 M H 10 7 M or pH 7 310 K K ADP Pi 10 7 9 ATP AG 76 kcalmol System Property K AGrxn AG RT nK 10393 to 2 x 10395 ADP Pi K A TP RT an 43 to 67 kcalmole AGrxn 12 to 143 kcalmole I ADP 1 KIdney cell ATP 110 10 Muscle cell ADP 1500 2 x 10 3 ATP Pi 10392 M ATP hydrolysis Coupled Reactions Examples biosynthesis ADP Pi 9 ATP AA to proteins ATPs to DNA Cell organelle movement All have a AGrxn NADPH in reductionoxidation Used to drive things Electron transport chain Coenzyme A is another activated molecule Is an acetyl group carrier Coenzyme A Acetate 9 AcetylCoA AcetylCoA transfers Acetyl group for fatty acid biosynthesis and as a posttranslational modification on proteins Metabolism consists of pathways of reactions Catalyzed by enzymes Nutrients are used to obtain building blocks and to produce energy Reactant and products metabolites Glucose oxidation is coupled to ATP NADH production Metabolic pathways Linear cyclic spiral Enzymes are regulated 1st unique step Balance catabolism and metabolism Often compartmentalized Catabolism example oxidation of sugars Glucose C5H1205 9 6 C02 quot 38 ADP 38 Pi 9 quot 38 ATP The four stages of glucose catabolism Glycolysis Get 2 pyruvates Acetyl CoA fed into next step Citric acid tricaryboxylic acid cycle Creates C02 NADH feeds electrons to next step Electron transport chain ATP synthase Substrate level 9 ATP made directly Step 1 Glycolysis Glucose 9 2 pyruvates C5H12059 2 C3H403 2 NAD9 2 NADH 2ADP2Pi9 ZATP llSubstrate level phosphorylation Ten reactionssteps ten enzymes In cytoplasm Aerobic or anaerobic Highly conserved First pathway worked out Step 2 the TCA Cycle 2 pyruvate 2 CoA 9 2 acetyl CoA 2 C02 2 NAD9 2 NADH Eight steps amp enzymes n mitochondria Only aerobic Two turns to oxidize 2 Ac CoA 9 4 CO2 Each turn produces 3 NADH 1 FADH 1 GTP ATP More Details for Glycolysis The quotStrategyquot of Glycolysis Investment amp payoff phases ATP is burned Payoff involves creation of NADH and ATP Net of both phases 2 NADH 2 ATP Thermodynamics coupled reactions Overall AG Larger AG of glucose ox coupled to NADH and ATP production Anaerobic ATP Generation Fermentation Aerobic glycolysis depends on the ETC to regenerate NAD Produces lactate or alcohol and C02 Cori cycle Liver Lactate produced 9 blood 9 liver 9 glucose 9 muscles Cancer cells divide rapidly provide a lot of lactate use a lot of glucose Warberg effect 19 October The ER is organized into a net of tubules and flattened sacs Constitutes about half of the total membranes in an animal cell It is the site of production of transmembrane proteins and lipids for most organelles Soluble proteins for secretion or lumen of ER Golgi apparatus lysosomes 2 Central role as Intracellular Ca store Ribosomes bind to the ER membrane during cotranslational translation The ER is structurally and functionally diverse Rough ER cotranslational protein synthesis Smooth ER Transitional ER Lipid synthesis Detoxification reactions Ca 2 storage Signal sequences were first discovered in proteins imported into the rough ER 1970 s The ER signal sequence varies greatly but each has 8 or more hydrophobic amino acids at its center The signal recognition particle SRP and its receptor SRP binds the signal sequence as it emerges from the ribosome and halts protein synthesis The pause gives the ribosome time to bind to the SRPreceptor Prevents formation of a folded protein in the cytosol The ribosome binds to a protein translocator that transfers the growing polypeptide chain across the membrane The protein passes through an aqueous pore in the translocator The translocator is sealed by a short othelix plug when closed It also opens laterally allowing access to the membrane hydrophobic core Translocation across the ER membrane is not always cotranslational Lateral gating of the translocator allows release of the signal peptide and integration of membrane proteins Single pass with internal signal sequence Also recognized by SRP 2 possible orientations depending on the charge surrounding the signal Multipassstransmembrane proteins use combinations of starttransfer and stoptransfer signals Protein glycosylation in the ER AsnXSer or AsnXThr Dolichol 14 sugars transferred en bloc Oligosaccharides are used as tags to mark the state of protein folding Export and degradation of misfolded ER proteins The Nlinked oligosaccharide serves as a timer that measures how long a protein has spent in the ER A slow mannosidase does the trimming The ER membrane synthesizes phospholipids and cholesterol to produce new cell membranes Phosphatidylcholine is the major phospholipid made Synthesis occurs in the cytosolic leaflet Scramblase equilibrates phospholipids between the 2 ER membrane leaflets 17 October Nuclear pore complexes perforate the nuclear envelope 125 million daltons composed of 30 different proteins which are present in multiple copies and arranged in octagonal symmetry Goes through outer and inner membrane The size cutoff to free diffusion through the NPC is 60000 daltons Proteins such as DNA polymerases cannot diffuse passively through the NPC Requirement for active transport The function of a nuclear localization signal Fully folded proteins can be transported Signal can be located almost anywhere in the protein Because nuclear localization signals are not cleaved off nuclear proteins can be imported repeatedly for example after mitosis when nucleus reassembles Nuclear import receptors There are several receptors that bind specific nuclear localization signals They bind both to the cargo protein and to NPC proteins FG repeats The Ran GTPase imposes directionality on transport through NPCs Nuclear import requires energy hydrolysis of GTP by the GTPase Ran There is a gradient of two conformational forms of Ran G DP or GTP bound Nuclear import receptor moves through nuclear pores bound to Ran Nuclear export Transport of proteins into mitochondria and chloroplasts Mitochondria are produced by growth and fission of preexisting organelles Most proteins reach the mitochondria by posttranslational transport from the cytosol Signal sequence targeting proteins to the mitochondrial matrix Best understood Form amphiphilic alphahelix Mitochondrial precursor proteins are imported as unfolded polypeptide chains Mitochondrial precursor proteins remain unfolded in the cytosol Chaperons prevent their aggregation before engaging the TOM complex The signal sequence Nterminus enters the translocation channel first TOM translocator of outer membrane and TM translocator of inner membrane usually work together ATP hydrolysis and a membrane potential drive protein import into the matrix space Routes for transport into the inner 39 la and iIILCI space Protein starts moving through membrane 9 signal sequence cut stoptransfer sequence stays in membrane Other methods 14 October The major functions of eukaryotic cells Intracellular compartments main difference with prokaryotes Cellular communication signaling Cell shape and movement cytoskeleton Cellular growth control cell cycle Eukaryotic cells can engulf other cells by phagocytosis Rapid change of shape Mediated by the cytoskeleton Example neutrophil engulfing a red blood cell Eukaryotic cells may have originated as predators The major intracellular compartments of a eukaryotic cell Eukaryotic cells have membrane enclosed organelles Provide more membrane and separate compartments for specialized functions Organelles have characteristic positions interaction with cytoskeleton Evolution of the nucleus and the endoplasmic reticulum ER lumen is continuous with the space between inner and outer nuclear membranes Modern eukaryotic cells evolved from a symbiosis Mitochondria has its own DNA ribosomes and transfer RNA DNA sequence similar to presentday bacteria Topological relationships between cellular compartments Nucleus and cytosol are topologically equivalent Endoplasmic reticulum golgi apparatus and 39 are Mitochondria and plastids constitute different types of compartments The 3 mechanisms of protein transport between compartments Gated transport Transmembrane transport Vesicular transport Signal sequences direct proteins to the correct cell address Signal sequences are both necessary and sufficient for protein targeting They are recognized by complementary sorting receptors that guide them to the correct destination in the cell Either linear or patches which form when protein folds 23 September IntroductionOverview Blood cancers and malignantmetastatic cancers 50 are treated with chemotherapy not localized Some patients are cured but some do not respond due to drug resistant cancers Drug resistant cancer arises through a range of mechanisms Drug target changes like BcrAbl protein catalytic site changes that affect Gleevec binding affinity More general multidrug resistant MDR conferring resistance to several drugs Impaired drug delivery concentration in blood and tumor Cellular changes include Increased DNA repair rate Blockage of programmed cell death Detoxifying system cyt P45 activation Decreased cell uptake by transporters polar drugs Increased cell export by ATPdependent pumps ABC family of transporters of nonpolar drugs ATPDependent Transporters ABC family Multidrug transporter Pglycoprotein PGP Encoded by MDR1 gene Binds hydrophobic neutral or positively charged drugs The binding site is in the lipid bilayer hydrophobic core Has broad nonpolar substrate specificity Its normal functions are protective keeping toxins out Bloodbrain barrier and bloodcerebrospinal fluid barrier Testicular tissues Developing fetus placenta Stem cells Liver intestinal tract and kidney Excrete toxins from body Stem Cells Exclude Fluorescent Dyes Replaces tissues that need to be replaced Stem cells express a lot of PGP Pumps in cell PGP would get rid of dye Cancer Cells PGP highlyover expressed in colon kidney adrenal liver and blood cancer cells PGP levels are increased in breast cancer cells after chemotherapy PGP levels are also high in putative quotcancer stem cells PGP inhibitors are being developed to reduce MDR Increases drug uptake from gut into blood Also inhibit cytochrome P450 breakdown of drugs So far have produced few side effects like brain toxicity May be more useful in combination with anticancer drugs before MDR is induced PGP Structure and Mechanism PGP structure Twelve transmembraneothelices Two folded ATPbinding domains cytoplasmic Forms an upsidedown V or cone shape The inner side of the cone In the hydrophobic membrane core Forms a binding site for pumped drugs Pumping mechanism Solute drug enters the membrane binds PGP at the base of the V opening ATP binds to the two globular domains The V shape reverses point now on cytoplasmic side Binding site facing out of cell membrane Solute is pumped back outside Binding Site Lined with hydrophobic amino acids Contains a broad range of nonpolar amino acids including aromatic ones Binds a large variety of solutes The Structure Has Been Used to Design New Drugs PGP inhibitors Anticancer drugs that do not bind PGP 26 August Gibbs Free Energy G of molecules available for work Each molecule has a G G depends on Temperature T Concentration Oxidation State Change ofG AG each reaction has a AG AG Gpmducls Greactants AH T AS H enthalpy potential and kinetic energy E S entropy randomness or disorder Related Laws of Thermodynamics 1 E H is conserved in the universe 2quotd the universe tends toward disorder Cells and the Second Law of Thermodynamics Erwin Schro39dinger in What is Life 1944 wondered quotHow can the events in time and space which take place within the spatial boundary of a living organism be accounted for by physics and chemistryquot Nutrients Cell 9 2 Cells Waste Nutrients 9 Cell Waste Less order 9More order Change in Free Energy AG in Cellular Reactions Will a reaction X 9 Y occur as written spontaneously on own Will it proceed X 9 Y orY 9 X How far will it proceed how much X converted to Y Spontaneous AG lt 0 Requires E input or spontaneous in reverse AG gt 0 Equilibrium AG 0 Is A H B reaction favorable or is B H A Determined by thermodynamics the change in free energy or AG Described mathematically by AG AH T AS T temperature in K Enthalpy AH Energy heat potential E Entropy AS Disorder degrees of freedom possibilities probability AG lt0 is favorable AG also determined by the concentration of reactants and products Aern GproduclsB GreaclanlsA Aern AG RTInK B K E R gas constant 2 calmole K T temperature in K At equilibrium AGrxn 0 AG RTnKeq Keq at equilibrium AG standard free energy intrinsic property Effect of the actual conditions for example in the cell Aern AG RTInK B K E K system property Free Energy and Cellular Reactions AGrxn AG RTInK X l E E A reactants B products A amp B concentrations At equilibrium AG 0 AG RTnKeq Keq at equilibrium AG standard free energy change Intrinsic property K system property Coupled Reaction Examples biosynthesis ADP Pi ATP AA to proteins NTPs to DNA Cell organelle movement All have a AGrxn AGrxn is the Energy Free to do Work Steady state homeostasis Replenishing system Using favorable reactions to drive unfavorable reactions AGrxn are additive in coupled reactions AGrxn overall AGrxnl AGrmz ATP coupled reactions Usually used to drive biosynthesis Through hydrolysis Synthesis of ATP used to drive nutrients being oxidized 26 October General model for a signaling pathway Communication between cells is mediated mainly by extracellular signaling molecules Extracellular signal molecule 9 receptor protein 9 intracellular signaling proteins 9 effector proteins Budding yeast Saccharomyces cerevisiae a wellstudied model system They are unicellular eukaryotes but they communicate extensively Example mating Animals have more complex signaling systems but they are similar between different species worms flies and mammals Extracellular signaling molecules bind to specific receptors Different kinds of signaling molecules proteins small peptides amino acids nucleotides steroids retinoids fatty acid derivatives ampc Exocytosis Receptors in the target cell bind the signal molecules and initiate a response Extracellular signal molecules can act over short or long distances Long range signaling by endocrine cells and neurons Synaptic signaling is fast and precise Molecules in local high concentration Endocrine signaling is relatively slow Molecules diluted Extracellular signals can act slowly or rapidly to change the behavior ofa target cell Changes in proteins already present can be fast Cells are programmed to respond to a combination of extracellular signals Each cell type displays a particular set of receptors to respond to the corresponding set of signals Different types of cells usually respond differently to the same extracellular signal molecule The type of response is determined by the receptors and intracellular machinery that interprets the signal The importance of rapid synthesis and turnover of an intracellular signaling molecule The signal withdrawal aspect Some signal molecules can diffuse into the cell and activate intracellular receptors Example the gas nitric oxide NO Guanylylcyclase activation Example ligands of nuclear receptors Travel in the blood bound to protein carriers Nuclear hormone receptors modulate transcription of specific genes primary response Three classes of cell surface receptors on channel coupled receptors Gprotein coupled receptors Enzyme coupled receptors Most cellsurface receptors relay signals via small molecules and a network of intracellularsignaling molecules Many intracellular signaling proteins function as molecular switches that are activated by phosphorylation or GTP binding Many intracellular signaling proteins form complexes using modular interaction domains Prolonged exposure to a stimulus causes desensitization 24 August Earth s crust is mainly oxygen silicon calcium Cells are mainly hydrogen calcium oxygen nitrogen potassium sulfur Valence electrons determine chemical properties Electronegativity O gt N gt C S gt H P Determines e39 sharing in covalent bonds Types of Chemical Bonds Covalent sharing onic give up or accept form ions form ionic compounds Polarity of Covalent Bonds Equal sharing non polar bond Unequal sharing polar bond Partial and If number of electrons associated with an atom is reduced it is oxidized If number is increased it is reduced Water Key Properties Polar 2 lone pairs dipolar Oxygen is more electronegative Hydrogen bond 801 O attracted to 6 of other molecule s H H switches from one molecule to another Hydronium H303 and hydroxyl OH formed Hydronium is a weak acid hydroxyl is conjugate base PH 39log I39ll H usually is actually H30 Scale from 1 to 14 Covalent Bonds and Carbon Valency of 4 Forms 4 covalent bonds Tetrahedral bonding geometry Bonding with other C 9 chains Side groups or functional groups Take up free electrons from C Branchingrings can form instead of chains All single bonds tetrahedral free rotation Double bond planar rotation restricted Resonance where single amp double bonds lie Homonuclear bonds C C same atoms Nonpolar Stronger Heteronuclear bonds Polar Weaker Between different atoms Functional groups Determine how molecule acts Hydrocarbons Just carbon and hydrogen Methyl group CH3 Nonpolar do not hydrogen bond with water Hydrophobic Carbon oxygen chemical groups Polar Alcohols are C bonded to hydroxyl Carbonyl C O Aldehydes have carbonyl at end Ketones have carbonyl in middle Carboxylic acid COOH is a carboxyl group H is lost in water Weak acid proton donor Carbon nitrogen groups Polar Amines Amides Amine carboxylic acid Sometimes in middle of ring structures or chains Also donate protons Hydrophilic carbon nitrogen and carbon oxygen groups Phosphates Polar Can replace protons on end of carboxyl group or alcohol Sulfhydryl groups Not polar Form disulfide bonds Linkages Connections between building blocks Ester connection to carbon w 0 in middle carbonyl off of one C Amide acid and amine Anhydride 2 carboxylic groups 0 in middle carbonyl group on either side Thioester acid and sulfhydryl Less sta ble Cancer 9 Cells 9 Organization Macromolecules proteins membranes Need to know the chemistry to understand how they work Bioenergetics how the elements come together chemistry of elements themselves Electronegativity of an atom refers to how strongly it attracts and holds electrons Carbon compound functional groups have properties largely determined by how they interact with water molecules 30 November The cell cycle control system Connected series of biochemical switches Onoff type 3 major checkpoints The cell cycle control system depends on cyclindependent protein kinases Cdks Cyclins undergo a cycle of synthesis and degradation in each cell cycle Cdk levels stay constant 3 cyclins are required in all eukaryotic cells Yeast 1 Cdk Vertebrate 4 Cdks Cdk activity can be suppressed by Cdk inhibitory proteins CKI I Help fine tune GlS and SCdks in addition to cyclin quot 39 g 39 main and Cdk phosphorylation The cellcycle control system depends on cyclical proteolysis Anaphasepromoting complex or cyclosome AFCC an ubiquitin ligase SCF is also an ubiquitin ligase The cellcycle control system function as a network of biochemical switches I 1 Each switch is influenced by various inhibitory about 39 cell damage incomplete cell cycle events etc SCdk initiates DNA replication once per cycle The genome must be copied only once to avoid gene amplification Origins of replication Cohesin holds sister chromatids together Chromatin DNA proteins histones that form nucleosomes Heterochromatin and euchromatin End of S phase the rquot 39 39 sister 39 39 39 are glued together along their length by cohesion MCdk drives entry into mitosis GzM checkpoint MCdk drives chromosome condensation and sister chromatid resolution The two sisters become separable units before they were too tangled and pulling them apart would cause breaks Condensin The late stages of M phase triggered by APCC The mitotic spindle is a microtubulebased machine Kinetochores attach sister chromatids to the spindle Sister pair biorientation is achieved by trial and error Cytokinesis Actin and Myosin II in the contractile ring generate the force Actin filaments formindependent Myosin filaments contraction Mitogens stimulate GlCdk and GlSCdk activities Meiosis is a special form of nuclear division involved in sexual reproduction 9 November Microtubule nucleation at the minus end by a protein complex containing ytubulin ytubulin ring complex yTuRC is a template that creates a microtubule with 13 protofilaments Formed by ytubulin and accessory proteins that stabilize ytubulin Microtubules emanate from the centrosome in animal cells Microtubules are generally nucleated from the microtubule organizing center MTOC Most cells have a single MTOC the centrosome near the nucleus Centrosome 9 many copies of the yTuRC and a centrosome matrix Embedded in the centrosome are the centrioles Cylindrical structures made of modified microtubules arranged at a right angle Organize the centrosome matrix Ensure centrosome duplication during cell division The organization of microtubules with minus ends collected at the cell center is very robust Experiment a single centrosome placed into a square well along with tubulin subunits Polymerization of microtubules push against the walls and stabilize the centrosome at the center of the well Even without a centrosome a microtubule array can find the cell center and form a new MTOC a general system Actin nucleation at the minus end by the ARP complex Actin related proteins are similar to actin and form the ARP complex Analogous to the yTuRC the ARP complex nucleates actin polymerization Arp 23 nucleates filaments most effectively when bound to the side of a preexistent filament formation ofa web New filaments grow at a 70 angle from the preexisting filament Important at the leading edge of a migrating cell and during endocytosis 29 August Intermolecular Recognition and Association Overview Mediated by quotweakquot noncovalent bonds and interactions k l CollIsIon between molecules energies of quot 1 a mo 9 Thermal motion or diffusion Brings molecules together Also knocks molecules apart kcal Covalent bonds reqUIre 90 to 100 male to break kcal Noncovalent bonds reqUIre 1 to 5 male to break So are more transient Require multiple bondsinteractions to be stable Provides specificity through quotfitquot Good quotfitquot provides for more bondsinteractions Collisions vs Associations Have to be correctly aligned for associating Poor vs Good fit binding specificity Four Types of Noncovalent Bonds and Interactions Van der Waals interactions Weakest kcal cal 01 02 strength Not affected by water Hydrogen bonds kcal 4 normally mole kcal 1 3 in water mole Ionic bonds kcal male 80 normally kcal mole 3 to 20 in water Hydrophobic forces Only occur in water Strength va ries Van der Waals hydrogen and ionic are electrostatic interactions Van der Waals InteractionsAttractions Induced dipoledipole Requires atoms to be very close together No room for H20 molecules so not affected by water Hydrogen H Bonds Partly electrostatic Partly covalent H shared Molecule that starts with H attached is donor weak acid Acceptor is a conjugate base Has directionality strongest w molecules line up 180 H20 competes with forms them a lot onic Bonds llsalt bridges electrostatic Electron transferred Coulomb s Law k ql 112 Force F 2 r D dielectric constant DVac 1 DHZO 80 because it likes to interact w ions Dorg 1 to 10 organic solvents Depends on polarity Q is charge Stroneg affected by water and ions Not directional Same charge results in repulsion Hydrophobic Forces and Interactions Require H20 ons strengthen Hydrocarbons ampc associate with each other Separates from water Measuring and Comparing Molecular Interactions Binding Strength or Affinity Function of fit Number of multiple weak noncovalent bonds and interactions Va Afree Bfree 9ABcompIex Vd Association rate v3 ka A B Dissociation rate vd kd AB At equilibrium v3 vd k A B kd AB AB ka kd AB Keq KA KA Association Constant Units M39l quotAffinityquot Range 1012 per M to 103 per M Dissociation Constant used more than KA Units M Range 103912 M to 10393 M AGAG RTan0 AG RT aneq Range 19 kcal to 4 krill mole mole All Intrinsic Properties Chronic Myelogenous Leukemia CML Caused by translocation between chromosomes 9 and 22 Forms Philadelphia Chromosomes Forms fused gene BcrAbl Results in a hyperactive kinase always on Kinases have similar structures with clefts for binding One inhibitor could affect them all Gleevec Small molecule Specifically designed to disable Abl Blocks activation loop so ATP can t bind to Abl Also kills gastrointestinal stromal tumor cells GIST Disables kit1 Some patients develop resistant CML cells Change in binding cleft Shape no longer complementary to gleevec New inhibitor AMN107 inhibits some gleevec insensitive BcrAbl 24 October Lysosomes contain acid hydrolases that are activated by proteolitic cleavage and require an acid environment Protection for cytosol content Lysosomal proteins are highly glycosylated Hi ATPase low pH Lysosomes are heterogeneous They have different sizes are according to the amount and nature of the material they are digesting A model for ysosome maturation Late endosomes contain endocytosed material and newly synthesized hydrolases Late endosome ysosome endolysosome Multiple pathways deliver material to lysosomes Digestive enzymes from the TGN Endocytosis Autophagy Phagocytosis Soluble lysosomal hydrolases carry a mannose 6phosphate M6P group The M6P receptor shuttles between the TGN and endosomes M6P receptor exits the TGN in clathrin coated vesicles M6P receptor releases the hydrolases in endosomes Empty M6P receptor in retrieved back to the TGN Phagocytosis Ingest invading microorganisms and senescent old cells Pinocytosis Constitutive process Most endocytic sites are cathrin coated There are also caveoin coated endocytic sites Receptormediated endocytosis The LDL receptor Increases selectivity and efficiency Example lowdensity lipoprotein LDL Atherosclerosis The LDL receptor recycles after dissociating from its ligand Tranferrin receptor also recycles to the plasma membrane Some receptors accumulate in cathrin sites only after binding their ligand and most of them do not recycle The constitutive and regulated secretory pathways All cells possess the constitutive secretory pathway quotdefault pathway Regulated secretory pathway is found in cells specialized for secreting products rapidly on demand Synaptic vesicles can form directly from endocytic vesicles 7 November The cytoskeleton Actin filaments microtubules and intermediate filaments Variety of functions shape and structure cell movement cell division intracellular transport muscle contraction axon extension etc Accessory proteins Cytoskeleton rearrangement can occur rapidly Example of a neutrophil in pursuit of bacteria They advance by extending protrusions at the leading edge filled with actin filaments The cytoskeleton can form stable structures Polarized epithelial cells actin bundles stabilize the microvilli that increases cell surface Each type of cytoskeletal filament is constructed from smaller protein subunits Small subunits can diffuse rapidly within the cytoplasm whereas assembled filaments cannot Filaments can be rapidly disassembled at one site and reassembled in another site far away Filaments formed by multiple protofilaments have advantages Protofilaments typically twist around each other forming lateral interactions Provides resistance to breakage while the ends are dynamic Nucleation is the ratelimiting step in polymer formation Short oligomers can assemble spontaneously but they are unstable Once a nucleus of several subunits is formed elongation proceeds rapidly The nucleation step can take a long time Regulation At equilibrium critical concentration CC The tubulin and actin subunits assemble headtotail to create a polar filament ot tubulin and Btubulin proteins form a unit and bind GTP t assembles headtotail generating structural polarity different ends The actin subunit is a single polypeptide chain monomer and binds ATP t assembles headtotail generating structural polarity different ends Microtubules and actin filaments have 2 distinct ends that grow at different rates The end has faster association and disassociation rates than the end At a given time filaments can elongate or shrink The quotTquot cap ATP GTP form and ADP GDP form have different CC Actin filament 39 quotquot for39 quot 39 39 of free actin the ADPform will u shrink while the ATPform will grow Actin filaments can treadmill and do work 21 October Endocytosis and Exocytosis Transport vesicles Communication with the surrounding environment Deliver new biosynthetic material to be secreted Uptake of nutrients Transport vesicles Vesicles are membraneenclosed transport containers Cargo soluble and membrane components Lumen components can be passed to another compartment without having to cross a membrane Budding and fusion The biosyntheticsecretory and endocytic pathways 2 major routes Balanced flow of membrane and proteins retrieval pathways Selectivity of cargo and target organelle Protein coats assemble on the cytosolic face of organelle membrane They concentrate specific membrane proteins in a specialized membrane patch that will form the vesicle The coat mechanism molds the forming vesicle Structure of a clathrin coat Triskelion 3 heavy chains 3 light chains Natural tendency to form cages pentagons hexagons Assembly and disassemny of a clathrin coat Adaptor proteins link clathrin to the membrane Adaptor proteins recruit selected transmembrane proteins such as receptors Recycling of coat compounds Not all coats form basketlike structures eg retromer Different types of phospholipids label the various organelles Monomeric GTPases control coat assembly and disassembly Arf proteins clathrin and COPI Sarl COPII Cytosolic proteins regulate pinching off and uncoating Dynamin HSC 70 and Availin Rab monomeric GTPases guide vesicle targeting Family with more than 60 members specific organellevesicle markers Rab effectors facilitate vesicle transport tethering andor fusion SNARE proteins mediate membrane fusion Complementary sets vSNARE and tSNARE TransSNARE complex fourhelix bundle Additional layer of specificity in the transport process The biosyntheticsecretory pathway involves transport from the ER to the Golgi apparatus Proteins leave the ER in COPllcoated transport vesicles COPII vesicles form in ER exit sites smooth ER Entry into COPII vesicles is a selective process signals The retrieval pathway to the ER uses sorting signals Transmembrane proteins KKXX COOH sequence for binding to COPI Soluble proteins KDEL COOH sequence for binding to KDEL receptor Necessary and sufficient KDEL receptor cycles between ER and Golgi The Golgi apparatus is usually located near the nucleus The Golgi apparatus consists of a collection of cisternae Each cisternae contains a characteristic set of enzymes Redesign of protein oligosacchardies cellcell recognition antigenicity protection against proteases The two main classes of asparaginelinked Nlinked oligosacchardies attached to mammalian glycoproteins Diversity The human genome encodes hundreds of Golgi glycosol transferases 10 October Introduction Overall reactions of photosynthesis 6C02 6HZO light 9 C5H1206 602 e s from H20 CO2 reduced to CHZO carbohydrates Oz released Photosynthesis lightdependent reactions ZHZO 2NADP 3ADP 3Pi light 9 Oz ZNADPH 3ATP use to fix C02 Two Photosystems PSII PSI Contain reaction centers and antenna complexes PSII oxidizes H20 to Oz PSII passes high energy e s to PSI H pumped on the way PSI reduces NADP to NADPH H gradient used for ATP synthesis Chloroplast three membranes Stroma enzymes for CO2 fixation pH 8 Thylakoid membrane contains PSII amp I antenna ATP synthase Thylakoid lumen pH 5 Chemical Components Chlorophylls Related to Hemes Hemes have porphorin ring Chlorophylls have photoporphorin Contain magnesium to carry e s Has hydrophobic tail to anchor in membrane Absorb light Remaining components NADP nicotinamide 2 e H FAD flavin flavoprotein FP 1 or 2 e amp H PQ plastoquinone 1 or 2 e amp H FeS ferredoxin FD Cyt b5 similar to cyth and bL Cyt bf in cyt bf complex functions like ETC complex I Cull plastocyanine PC functions like EI39C cyt c 4 Mn2 H 4 Mn3 water cleaving complex 4 e39 from ZHZO Chlorophylls are in the reaction centers of photosystems Energetics The Photosynthetic 2 Scheme AG coupled reaction Coupled to light energy Light energy hits reaction center chlorophyll in photosystem II Kicks e to higher energy 9 passes to plastoquinone Plastoquinone functions as lipidsoluble e39 carrier e carried to cytochrome b6 f complex Qcycle and proton pumping e passed to plastocyanin Plastocyanin is peripheral membrane protein e feed to photosystem I Photon absorbed Kicks e to even higher energy 9 passes to ferredoxin Passes to flavoprotein Passes e by reducing NADP to NADPH AG is Z shaped After PS passes e it needs another H20 cleaving complex w Mn feeds e to PS Proton gradient also forms The LightDependent Reactions Summary In thylakoid membrane Light enters 9 energy absorbed in PS 9 kicks e39 Feeds e into plastoquinone Q cycle before cytochrome b6f complex e pass to b6f complex Site of HT pumping Passes to plastocyanin Passes to PSI Absorbs light PS absorbs 680 PSI absorbs 700 e kicked passed to ferredoxin passed to flavoprotein Passed to NADP9 NADPH Used for biosynthesis ATP produced by H gradient The Two Non Chlorophyll Containing Complexes Water Cleaving Complexes Cytochrome bf Complex Plastacyanine Cub PC functions like Cyt c Ferredoxin FeS protein FD passes e s to FP Flavoprotein FP adapter passes e s to NADP Photosystem Chlorophylls Go Through Strong Electron ReducerAcceptor Cycles Driven by light PSII replenishes PSI via plastacyanine PSII replenished by MnHZO complex Chl cycles Excited good e donor strong reducer Good e39 acceptor strong oxidizer Ground Antenna Chlorophylls Antenna complex chlorophylls absorb light across the visible spectrum Reductive Pentose Cycle Light Independent Reactions Melvin Calvin early 1950s Glycerate3P is first to be labeled with 14C from 14C02 in algae 14C is in carboxyl group Rapid removal of CO2 results in 14C ribulose 1SBisP build up Combines with CO2 to form 2 molecules of glycerate 3P Reaction catalyzed by ribulose 1SBisP carboxylase No NADPH or ATP used in the actual C02 fixation reaction Carbon Fixation or Calvin Cycle Catalytic Ribulose 1SBisP used catalytically Used per 3 C02 9 ATP 6 NADPH Net yield 3 molecules C02 fixed give a net yield of 1 molecule of glyceraldehyde 3phosphate at a net cost of 9 molecules of ATP and 6 molecules of NADPH 28 September Substrate Level Phosphorylation Step 6 of glycolysis Step 7 Net AG 15 kcalmol First payoff step Start with glyceraldehyde 3phosphate Enzymeisg39 quot3939339 I39 quot390 Has sulfhydryl functional group Reacts through nucleophilic attack with carbonyl group Forms connection covalent catalysis Covalent bond with C Use NAD to oxidize glyceraldehyde 3phosphate Takes 2 e amp proton Forms NADH first product in payoff Highenergy bond between product and substrate Inorganic phosphate taken up Nucleophilic attack Displaces sulfur from enzyme breaks enzyme away Results in 13bisphosphoglycerate High energy Hydrolysis has AG Net AG 45 kcalmol Starts with 13bisphosphoglycerate Phosphoglycerate kinase transfers phosphate from 13biphosphoglycerate to ADP 9 ATP and 3phosphoglycerate ADP Pi 9 ATP AG 73 kcalmol 13BisPG9 3 PG AG 118 kcaImol net AG 45 kcalmol Coupling of the two reactions Steps 6 amp 7 AG 30 kcalmol The TCA Cycle Is the quothubquot for metabolism in the cell The TCA cycle works catalytically Acetyl CoA oxaloacetate 9 CoA leaves 9 citrate 9 2 C02 leaves 9 oxaloacetate Amphibolic Pathways Both catalytic and anabolic TCA cycle is amphibolic Amphibolic Functions of Glycolysis and the TCA Cycle Glycolysis catabolic Gluconeogenesis anabolic Pyruvate 9 oxaloacetate 9phosphoenolpyruvate9 3phosphoglycerate All the way to glucose Separate enzymes involved since there are bypass steps Anapleurotic llfilling upquot Intermediates in TCA Cycle can build up Regulation to avoid llfutile cycles Catabolism Oxidation of nutrients Production of NADH ATP Anabolism Reduction biosynthesis Use of NADH ATP Key regulated steps often first unique step Enzymes catalyze reactionssteps key enzymes regulated Often llbypass steps with separate enzymes Allows differential regulation of catabolic and anabolic reactions and enzymes Often compartmentalization to further separate Regulation functions to keep ATPADP ratio high in a llsteady state far from equilibrium Takes more ATP amp NADH to create something than is created in the opposite reaction High glucose metabolism in cancer cells Warberg effect Lactate produced in presence of Oz PET positron emission tomography scans High glucose by itself cannot support cells 5 December Telomerase replicates the ends of chromosomes Human telomeres 1000 repeats of the sequence GGGTI39A Needs to be replenished each time the cell divides Telomerase recognizes the tip of a teomere and extends it in 5 to 3 direction Uses RNA template that is part of the enzyme Telomere replication The 3 DNA end of each teomere is always longer than the 5 end This leaves a protruding singlestranded end that loops back forming a tloop A tloop at the end of a chromosome The protruding singlestranded end loops back and tucks its terminus into the duplex DNA Protects from degradation Telomeres grow and shrink in approximately balanced manner Telomere length is regulated by cells Cells that proliferate indefinitely maintain the number of repeats within a range Human cells use it as a counting mechanism to avoid unlimited proliferation Telomerase turns down 9 cells lose 100 200 telomerase nucleotides per division 9 withdraw from cell cycle senescence 14 September Introduction and Overview to Lipid Bilayers Membranes define cells and cell compartments Key properties Thin 50 atoms thick 1100 th to 11000 th thickness of a cell 10000 membranes sheet of paper Flexible Selfforming selfresealing mpermeable to polar molecules Membrane Lipids Iquot quot part3939 rquotquot part39I r39 quot Associate such that the nonpolar tails are away from the water Membrane Lipids Classification Backbone glycerol or sphingosine Head group has a net charge Contains sugars Contain phosphate Glycerophospholipids Glycerol backbone All have a phosphate which then bonds head group Fatty acyl tails 2 of them 14 24 C Saturated or unsaturated Head groups Serine negative charge Ethanoamine no net charge Choline no net charge Inositol negative charge Proton Phosphate has negative charge so phosphate head group together is what shows charge of entire head group Sphingolipids Sphingosine backbone 1 fatty acyl tail Head groups Sphingomyelin Phosphocholine no charge Cerebroside Simple sugar like galactose no charge Gangliosides Complex sugar contain sialic acid negative charge Cholesterol Steroid ring structure Hydroxyl for head group Nonpolar hydrocarbon tail Membrane Structure The lipid bilayer is asymmetrical Membrane Lipid Diffusion and Fluidity Lipid molecules in a membrane can diffuse freely Two dimensional fluid liquid Lateral diffusion Flexion Rotation Flipflop rarely occurs Fluidity depends on lipid composition and temperature Decreased temperature decreased fluidity quotparacrystallinquot Acyl tail saturation Unsaturated increased fluidity Cholesterol Decreased permeability Paracrystallin at lower temperature Exam 1 Preparation Aides Quizzes 1 9 open again Study guide answers on RamCT Key topics outline on RamCT Exam 1 Fall 2010 on RamCT Review session Thursday 5 6 PM Path 101 Office hours Practice on whiteboard Quizzes and study guide are the pool of questions for the exams Study guide free response 4 of 5 Quizzes and iClicker multiple choice 26 When a lipid bilayer is torn it does not seal by forming a hemimicelle cap because membrane lipids are cyclindrical Five students in this class always sit together in the front row f analogous to why lipid bilayers form the reason for this is nobody else in the class wants to sit next to them Although present in relatively small amounts in mammalian cell plasma membranes the phospholipid crucial in intracellular signaling is phosphatidylinositol Cholesterol is essential for lipid raft formation because sphingolipids have large head groups Cnacer cell membrane relative to normal cells may participate in cancer drug resistance