MCB 2210 Exam 1 and Exam 2 Notes
MCB 2210 Exam 1 and Exam 2 Notes MCB 2210 001
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Date Created: 03/07/16
1/20/16 Intro lecture Cell Theory All living things are made of cells The cell is the basic structural unit of living things Cells can only arise by division from preexisting cells o Core concepts that are still believed to be true today Basic prosperities of cells List is common in most cells but not all cells have all these properties There are many exceptions within our bodies Central Dogma How the cells exhibit all their properties Information that allows cells to do and express things are encoded in their genome Activation transcription Processing Translation More complex than what diagram shows Vast number and types of proteins encoded in the genomes of different eukaryotes Cells are dynamic Not all the info in a cell is encoded directly in DNA in a form we can read Cells are either methylated or acetylated 1/22/16 All cells have a common ancestry First eukaryotic ells are more related to archea than bacteria Prokaryotic cells arose first therefore they’re simpler cells but that doesn’t mean they’re inferior Single celled organisms are generalists therefore complicated and do many functions Core Tools of Cell Biology Type of cells cell biologists study Primary cells: cells that are isolate straight from a living organism that can be cultured to study cell specific functions. o Many are differentiated so they aren’t going to divide anymore/hang around in the dish longer o Can bring limited values bc we can’t understand their growth factors that keep them around longer. o Cells right out of the organ. Not many are differentiated so they don’t divide anymore Transformed cells: cells that have become cancerous because they divide continuously therefore easy to maintain in lab As technology increases, we can study cells in vivo/in their real environment instead of pulling them out in a dish bc you don’t know if how they act in a dish is relevant in their lives. Light microscopy: one of the biggest tools o Limited (around 200 nm) in contrast, magnification and resolving power, limiting what can be detected with them o The quality of microscopes have been improving but again, there are hard limitations o Cells are mostly water therefore they’re very transparent. Light passes through them bc they have poor contrast. Need to enhance their contrast (poor contrast due to water) Transmitted light microscopy: magnify a specimen using one or more lenses o White light passes thru the specimen before being collected o Cells are neither reflected or absorb much light so contrast is poor and little details can be made o Contrast is the difference in intensity between an object and its background o Bring out detail, we need to exploit changes in the phase of light or stain the object to make it darker o Phase contrast or differential interference contrast techniques for enhancing contrast Magnification vs. resolution Magnification is how much you blow up on image. Depends on lens. Resolution= how far apart two objects have to be to be seen as 2 different objects Depends on wavelength. Shorter wavelength = better resolution as well as how good ur lens are. Numerical aperture (how well lens can gather cone of light coming out of the specimen) Resolution of conventional light microscopes is ½ the wavelength of light being used 1/25/16 Ability to resolve doesn’t mean ability to detect. Just means you won’t be able to see if it’s smaller than a certain nm Anything smaller will look the default nm Epifluorescence microscopy improves contrast and allows specific structures to be labeled via staining. Smaller wavelength = higher energy Illuminates molecules w/ fluorophores via higher energy light Antibodies: immune proteins that bind to specific proteins. o To make, obtain protein, pure of interest and inject it to animal. o Monoclonal vs. polyclonal antibodies Immunocytochemistry only works for nonliving cells Western Blotting used to detect proteins with antibody detection. o Smallest proteins can elude through SDS PAGE gel faster o Gel separates protein by size of molecule o Use electric field to separate protein Fluorescent proteins can keep the cell in tack so we never kill the cell To get plasmid across membrane is called transfection o Infect cells with foreign DNA to cause a desired expression o Transfection can be transient or stable (expression from plasmid or DNA integrates into genome/heritable) o Transgenic lines of animals can be generated by stable transfection of germ cells o We can transfect mutant molecules to be active all the time by activating a protein to turn it on o Mutant molecules that are dominant negative don’t function right and block the function of the cell’s own version of the molecule Mutant form of enzyme can’t catalyze enzymatic reaction aka doesn’t work/non functional therefore causes the mutant form of the cell to not work as well. Has ability to even block function of normal enzymes 1/27/16 A multicolor fluorescent image uses different proteins and antibodies as well as DNA dyes to add color to a black and white microscopic image 200 nm is the lowest resolution of light microscopy you can see so any object smaller than 200 nm will always appear 200 nm, regardless of it’s size siRNA help transfect things into cells using new technologies that exploit RNAi mechanisms to allow us to knock out specific proteins of interest in cells by targeting mRNAs o **Don’t memorize image on this slide. Not a genetics class. Just understand generally what is going on. Two methods to deblur images (generally remove out of focus light from the focal plane) o Laser scanning confocal microscopy uses pinholes to deblur Pinholes can focus length Gives laser to stimulate fluorescent of image causing it to focus on one specific point when it passes through keyhole When light comes back out, it passes through detector and another pinhole that focuses it as well o Digital deconvolution uses computational methods to deblur Superresolution microscopy techniques allows people to see below 200 nm If resolution limit is 200nm, how did we come up with all the textbook figures of organelles and cell structures that are smaller than that? o Electron microscopy Electron Microscopy Another means of getting around the resolution limits of light Electrons are used to illuminating specimen of interest TEM and SEM are two popular electron microscopy microscopes Sample Prep for TEM EM must be done in a vacuum for electron gun to work Sample must be dry so it’s low density to scatter electrons Need to stain the metal to bind to structures of the cell and stick there Immunoelectron microscopy Label structures in EM You can’t see antibodies in the EM, but you can attach dense particles to antibodies to make them visible in the EM (gold beads) Allows you to visualize the beads in your image/the fine details in structures Scanning electron microscope Coat sample with heavy metals Electron beams bounce off metals, collect rays with detector to give you a different type of image (see pictures on the slides) Differential centrifugation Take samples of cells and break them up in solution Put solution into ultracentrifuge tube and spin sample at different speeds and time Based on size or density of what is in the solution, more dense things go to bottom of tube in lower speeds and in less time compared to things that are less dense Gradient centrifugation Put sample on sucrose gradient Organelle moves through gradient until it reaches a sucrose level as the same density as itself Finer separation of organelles Membranes and Proteins Proteins specialize one organelle from another Each organelle contains its own subsets of proteins therefore differentiating their functions and structure Membranes are barrier between inside and outside worlds, and the barriers are selectively permeable Membranes are important for cellcell interactions and provide scaffold for biochemical activities glycolysis, electron transport chain, etc. 1/29/16 Lipid rafts: specialize regions w/ specialize proteins Three major lipid components of membrane: Amphipathic molecules (hydrophilic and hydrophobic ends) Level of saturations in fatty acid tails is important to consider. o Unsaturated creates spatial difference therefore kink in the tail. o Overall height dimensions of kinks are shorter o Taller vs. shorter phospholipids contribute to thicker or thinner membranes o Straight saturated chains = packed more and less fluid membrane compared to unsaturated chains o Double bonds creates a kink 1. Phospholipids o Cholesterol o Proteins that can be associated w/ or inserted in membrane to give it variety of functions o Phosphoglycerides: made up of hydrophilic head group and hydrophobic tail Glycerol group links chemical compounds of head to the tail Most abundant in membranes o Sphingomyelin: still a phospholipid but instead of glycerol, it has a sphingosine group 2. Glycolipids: head group linked by sphingosine group o head group is sugar group linked to FA hydrophobic tails 3. Sterols: made up of 4 fused rings o Cholesterol can alter quality of phospholipid membranes Lipids behave badly in membranes o Form either micelles or liposomes if you put them in water o Micelles: hydrophilic heads face aqueous solution and tails facing each other so they don’t face water Single phospholipids layers Form w/ lipids that have one phospholipid chain o Liposomes: formed in test tube with lipids Usually bilayers Bilayers can self assemble if there’s ever a tare Biological membranes are mixture of different types and amounts of different mixtures with proteins embedded and associated with them (different functions because of that) ** Don’t memorize the major lipid components of selected biomembranes table*** Composition can affect curvature of a membrane Variety of head groups on phospholipids can all be different sizes Phospholipids are not fixed in space. They float around in sea of phospholipids at different rates. Very dynamic o Lateral diffusion happens rapidly o Transverse diffusion/flip flop occurs rarely about 1 time a day because the phobic tail would be interacting with water and that’s not thermodynamically favorable. o Rotation o Flexion is also very rapid Method to measure mobility of phospholipids in membrane: o Generate liposomes in lab made up of phospholipid of interest. Conjugate fluorescence to head group of phospholipids to treat cells with that. o FRAP use photo bleaching technique. Irreversible killing of the fluorescence While things are mobile in membranes, things are not completely uniformed. o Two leaflets of membranes can have different compositions o Flippases are enzymes that can flip flop lipids together or bring leaflets back to their proper side Proteins are basic machinery of cells and important part of membranes because proteins allow things to come in and out of membrane if cells want that Side chains give amino acids different characteristics Hydrophilic = polar hydrophobic = non polar Peptide bond forms when amino and carboxyl sides interact with each other N terminus comes out of ribosome first aka made first Protein folding occurs cotranslationally (as protein undergoes translation) Protein needs to be folded in it’s native form for it to function properly o Chaperones and chaperonins help proteins fold properly o Heat shock family of proteins are proteins that act like chaperones o Regions on chaperones (hydrophobic regions) interact with hydrophobic interactions on unfolded protein to help them fold by hydrolyzing ATP. o Hydrolyzing energy allows chaperone to fold up protein as it undergoes conformational change Most membrane proteins almost always glycosylated 2/1/16 Proteins fold cotranslationally. Some can fold into proper shape on their own, some cannot. Disulfide bonds = covalent bonds. Occurs in oxidized environment to stabilize the protein Most membrane proteins are glycosylated sugar chains added onto proteins in ER as they’re being synthesized Many proteins have multiple functional domains which allows catalysis to occur better Many different proteins can share functional domains therefore causing similar protein structures Protein complexes can be stable, or can be triggered by a signal to assemble Proteinprotein interactions occur due to noncovalent interactions Quat structure: interactions of multiple subunits. 2 same subs = homodimer, 2 different subs = heterodimer More noncovalent interactions = stronger interactions therefore bind together tightly and stay bounded together longer before falling apart again. Weaker interactions bind and release rather quickly. Strength of interactions between proteins = affinity o Higher affinity interaction will have relatively small koff (kd) and off rate, complexes will stay bound longer, so there will be more of them at equilibrium o **Don’t go deeply into the equations o On rate depends on the concentrations of the reactants (high concentration = high interactions) and Kon (association) rate constant, which depends on size, rate of diffusion, and whether there is a favored orientation required for binding, etc. o Koff rate depends on the sum of the forces that will hold reactants together o Affinity controls what fraction of a molecule will be bound as the concentration of its binding partner is varied o Glutamate has higher binding affinity than gluazo lower kd = stronger affinity interaction If concentration is below kd value then low interactions occurs At equilibrium, there are more complexes High protein concentration than kd than you’ll see them complex Nano lower than micromole therefore AC will have a higher affinity interaction bc higher affinity has lower kd ACD complex decrease binding affinity bc kd increased Classes of Membrane Proteins Integral: AA sequence is tightly associated into the lipid bilayer o Transmembrane proteins span the bilayer Lipid anchored: AA sequence has undergone lipid modification which sticks it to hydrophobic region on the membrane o Different fatty acids used to attach to inner leaflet o GPI can be added to protein and insert themselves and become outer leaflet Peripheral: have proteinprotein interaction with either lipid anchored or integral membrane protein Immunofluorescence or immuneEM are two techniques used to show a protein associated w/ the membrane Proteases cleaves or digest accessible protein regions and can be used to deduce topology of a protein in the membrane o Topology = what kind of domain does the protein possess o Treat the cell with trypsin in culture medium to digest extracellular domains of protein while keeping intracellular domains in tact 1/3/16 One way to deduce topology of protein is to do an assay with proteases such as trypsin. Transmembrane region of proteins are amphipathic o If alpha helix based, it can be a single or multipass Transmembrane protein Neg = hydrophilic pos = hydrophobic on graphs Beta sheets can also interact w/ membranes o Amphipathic character o These sheets can role up to form a barrel Fluid Mosaic Model of Membranes The lipid bilayer is a flexible 2D fluid sheet Helps us picture the membrane of cells Membrane proteins float in this sheet Many proteins can and do move laterally in membrane but once a protein is inserted, it can’t easily leave the membrane Topology of protein is set once it’s inserted and synthesized Conformation can change, however. Not all proteins are mobile, but many are Figure 1043 shows what ways can prevent movement of proteins in the membrane Method 1 for measuring protein mobility: cell fusion Label proteins of one cell w/ red dye and second with green dye Fuse membrane of two cells to form heterokaryon with force bc usually membranes don’t like to fuse Watch what happens to the two dyes Result: overtime they become mixed Method 2: FRAP of fluorescently labeled protein CD2 is a membrane protein that links to yellow fluorescent proteins Take laser, photobleach a spot. Light is so intense it alters covalent bond of molecule so it can’t fluoresce again. Track over time does the fluorescence recover Recovery = mobile in membrane Method 3: single particle tracking Quantum dots are crystals that can fluoresce Can link to an antibody Track the dot in the membrane Lipid rafts Contain cholesterol, sphingolipids, and proteins Tend to accumulate diff proteins than nonraft areas Membrane Transport Lipid bilayer is impermeable to most things Small, nonpolar typically can diffuse right across membrane There are transporters in the membrane to control entry for those molecules that cannot easily cross the membrane Membrane transport proteins Passive Transport: allows net movement down a chemical concentration, electrical or electrochemical gradient; no energy beyond thermal motion needed (high areas of concentrations to low) o Facilitated diffusion by uniporter carrier proteins o Ion channels Active transport: move molecules against a chemical, electrical or electrochemical gradient; they require extra energy (low to high) o ATPdependent pumps o Symporters o Antiporters 1/8/15 Membrane Transport Make up lecture Membrane lipids and proteins associated with them create a selective permeability barrier Transporters decide what gets across the cell membrane o Required because not many molecules can diffuse across a lipid bilayer o This is due to hydrophobic tail thus creating hydrophobic barrier on either side of membrane and therefore only smallest hydrophobic molecules can pass across the membrane o Charged, polar molecules can’t really get across Small ions with charges cannot move through bilayer bc of that charge If the cell wants to transport the biomolecules that can’t get across via passive diffusion, a solution are membrane transport proteins Transport proteins act enzymatically to move substances across membrane o Passive transport proteins allow net movement down an electrochemical gradient No extra energy besides thermal energy Carrier proteins or ion channels are the proteins that move molecules passively Facilitated diffusion = done by carrier proteins o Active transport when molecules move against a gradient therefore needs extra input of energy to make that happen Pumps directly use ATP to do this Symporters and Antiporters are carrier proteins that indirectly use ATP to move things against gradient Concentration + chemical = electrochemical gradient Carrier proteins vs. channels (passive) o Carrier proteins have binding site in 3 structure that interacts with solute (molecule being transported) Binds one side of membrane, conformational change in carrier protein tht opens it up on opposite side and releases molecule o Channel have little interaction w/ solute Either is in open or closed confo. When open, molecules move directly through an aq. Pore in center of channel. Diffusion doesn’t peak, no saturation. Transport peaks when binding sites on transporters are saturated. Due to binding of solute to carrier. Produces max rate of transport (vmax) Cells use channels to set unique ionic environment inside and outside the cell Osmolality refers to concentration of solute in solution o Water floats to higher solute concentration Cells use transport proteins to keep [solutes] on both side membrane are equal Hypertonic = cell shrinks bc water moves out to high solute outside the cell Charged molecules add another force o If the inside of the cell is net neg, an anion will be repelled and a cation will be attracted o Vise versa if inside cell is positively charged o The combo of the chemical concentration gradient and electrical gradient determines the rate and direction of transport of a charged molecule. Known as electrochemical gradient o Need to consider which way a charged molecule will move across a membrane Diffusion of ions gives rise to electrical potentials Lipid bilayer can separate charges (when you typically cannot) Only a small number of ions need to move across membrane to create a membrane potential Effective of charge > effect on concentration o Small number of molecules need to move to create the slightest charge o Nearly undetectable change of concentration will set up a membrane potential of 75 mV Measure membrane potential with an electrode Makeup lecture Part 2A & 2B Ion channels are proteins made up of multipass Transmembrane proteins to form a pore in center to allow passage of molecules Bidirectional ion channel can move in and out. Net direction is down combined electrochemical gradient Passive transport can go against concentration gradient If membrane potential changes, it can change position of proteins as well VoltageGated K+ Channel Made up of 4 subunits, each w/ multiple membrane spanning domains N and C termini are intracellular When subunits come together, they form a channel through the membrane Channel is opened by membrane depolarization Channel allows flux of >10^6 ions/sec Selective for K+ bc interactions with ions are limited 3 NA+/2 K+ using ATP for the pump Channels can be regulated (channel gating) Selectivity filter Important for allowing only passage of K+ through the channel When an ion is in the solution/water, water molecules can form H bonds around the ions in a hydration shell. Shell has molecular dimensions which make ion and water molecules happy. This helps them find an equilibrium. When the ion comes into channel and makes into vesibul into selectivity filter. Radius of sodium diff from K+ therefore diff size hydration shell. Sodium doesn’t match up well with potassium selectivity filter therefore that’s a poor interaction. Opening the channel requires a relatively small change in conformation Channel made up of proteins w/ alpha helices with various charges on them Some alpha helices sensitive to voltage therefore when it changes, conformation of helices change on one of subunit leading to conformation change to open the channel. K+ can pass through very rapidly and we can measure this via patch clamping o Current recordings when a current flows through an open channel o Inject current into cell = depolarize the membrane o Measure current flows through single channels Passive Transport 2: Uniporter Carriers Multipass Transmembrane proteins that act more like an enzyme than a pore. They bind substrate and undergo reversible conformational changes to bring about transport from one side of the membrane to the other; no energy other than random thermal fluctuation is necessary to drive the change Bidirectional movement can be in both directions but net passive movement is down a concentration gradient Slower than channels due to protein interaction Flickering back and forth randomly between open and closed state via thermal motion Concentration of solutes drives carrier to flip back and forth GLUT transporters o 12 transmembrane domains o intracellular N and C termini o looks a lot like a channel o millimolar affinity for glucose o One isoform is stored in vesicles and inserted into muscle or fat cell membrane in response to the hormone insulin Active Transporters Protein complexes that act as pumps to move molecules against a chemical, electrical or electrochemical gradient Requires extra energy input Primary active transporters: directly use ATP to generate energy to move molecules against gradient ATP dependent pumps: ATP use as a direct energy source Light drive pumps: light used Also called ATP dependent pumps. Hydrolyze ATP and use the energy to move one or more molecules across the membrane P type: become Phosphorylated by the PO4 from ATP during transport. Plasma membrane Na/K ATPase V type: Vesicular H+ ATPases o Don’t become phosphorylated o Pump H+ into membrane compartments o Acidification of endocytic vesicles, lysosomes, golgi Secondary Active Transporters/coupled carriers Use an electrochemical gradient generated by primary active transporters to power movement of another molecule against a gradient Symporters/Cotransporters: move two molecules in same direction. One down and another up a gradient Antiporters/Exchangers: move two molecules in opposite direction; one down and another up gradient. The Na+/K+ ATPase Moves K+ (2) inward and 3 Na+ outward using energy from ATP Both are moving against electrochemical gradients Key role in maintaining the distribution of these ions in cells Mechanism involves conformational change in shape of protein drive by ATP and binding of ions o ATP cleavage is used to actually phosphorylate the transporter therefore P type Other ATP Dependent pumps and their functions *** Don’t memorize K+/K+ ATPases: stomach acidification and P tyle Ca++ ATPases: pumps calcium out of cell or into ER. P type ABC Transporters o Use ATP for transport but do not become phosphorylated but not V type either o 5% of bacterial genome is this type of gene o Some isoforms important in cancer. Cancer cells overproduce the transporter and can pump anti cancer drugs out of the cell making them resistant. Multidrug resistance transporters o Cystic fibrosis gene product is an ABC transporter for Cl that is found in lung, sweat glands and kidneys Secondary Active Transporters coupled transporters that run off ion gradients established by primary active transporters Transport two molecules 2Na+/1 Glucose symporter o Concentrates glucose from intestine into epithelium cells o Works against the glucose gradient using Na+ gradient o Can work against a 30,000 fold gradient ie aciculate glucose 30K fold 2/10/16 Membrane transporters in action 2: signaling by nerve cells Nerve cells generate, receive and transmit electrical signals throughout the body They can be up to a meter or more in length Action potentials are the solution Electrical signals rapid changes in Vm They are all or nothing effect Propagate actively own nerve axons Rapid, transient signals Neurons encode info b the frequency of action potentials **Understand graph and how the pump opens and closes How is action potential generated? Increases Na+ current membrane depolarizes opens V gated Na+ channels Ligand gated ion cannel A class of ligand binds and changes the conformation of the channel, opening or closing it Neurons talk to each other via chemical synapses electrical signal is transmitted won an axon to next neuron, turned into chemical signal, and then back to electrical signal when propagation occurs, it goes down the axon until t reaches the terminal where it synapses with dendrites of next neuron Special classes of calcium channel that are voltage gated Can you stimulate an action potential shortly after adding Ouabain? It’s a plant compound that can block K/Na ATPase If that’s blocked, you’re blocking cell’s ability to pump K/Na therefore overtime, it’s gong to lead up to build up of sodium and positive charges in the cell If pump isn’t working, the cell’s membrane potential will depolarize Osmotic effect also occurs bc more particles build up and water will want to come in to balance it out therefore cell will come in and burst Yes action potential shortly after adding Ouabain Cardiac action potentials bring calcium into the mix causes muscles to contract therefore beat 2/11/16 Cell Signaling Signal Transduction How signals are transmitted from the outside of the cell to the inside allowing cells to modify their behavior. Bilayer is both a physical barrier to substances and a barrier to many kinds of information How are signals transmitted across the membrane? Hydrophobic signaling molecules can cross the membrane DIRECTLY o Steroid hormones: receptor for this signal is in the cytoplasm or nucleus o Nitric Oxide/Carbon Monoxide: dissolved gas regulates many pathways Hydrophilic signaling molecules cannot cross the membrane o They need to indirectly signal across the membrane by binding to the extracellular domain of Transmembrane receptor proteins Types of signals Signaling via chemical messengers Free floating chemical messengers o Autocrine, paracrine, and endocrine signaling o Cell surface receptor binds a molecule secreted by itself (autocrine) a nearby cell (paracrine) or distant cell (endocrine) Contact dependent signaling Expresses and presents signal to neighboring cell upon direct cell contact Won’t influence each other unless they get close to each other o Cell surface receptor binds a signal on the surface of another cell or to extracellular matrix Basic scheme of signal transduction via membrane receptors The same signal can bind to multiple types of receptor or complex of protein in membrane o Some ligand may have one or multiple receptors The receptor can be coupled to multiple types of transducer upon binding o Can lead to association of other proteins that are enzymatic to produce second messengers Second messengers can provide high degree of signal amplification The same second messenger can be coupled to multiple effectors Types of plasma membrane receptors Ligand gated channels o Signal opens a channel/ion pore G protein coupled receptors o Signal activates a G protein o Transmembrane receptor Often 7 membrane spanning helices o Heterotrimeric G protein receives signal 3 diff subunits G alpha binds and interacts with ATP or GTP and G protein has enzymatic activity to allow GTP to hydrolyze to GDP G beta and gamma have high affinity therefore they’re the betagamma unit Enzyme linked receptors o Signal activates the enzymatic activity of the receptor o Usually a kinase Opening of K channels by acetylcholine G protein coupled and stimulated channels Acetylcholine binds to receptor allow conformational change so GTP to hydrolyze and GDP to bind. This causes dissociation G betagamma binds to and causes opening of K channel Potassium leaves cell and inside becomes even more neg Adenyly cyclase: a key target of G protein signaling G alpha binds to adenylyl cyclase activating it Enzyme makes cAMP from ATP which is an important second messenger cAMP binds to Protein kinase A (PKA) regulatory subunit causing it to fall off Regulatory subunit blocks kinase activity and tethers the complex in the cytoplasm preventing the NLS from causing import Catalytic subunit of kinase now active and goes to nucleus PKS phosphorylates nuclear proteins to change gene expression Binding of a single ligand can lead to amplified signal because 1 more ligand can bind to a receptor that can interact and activate multiple heteroTransmembrane complexes o Each activated G alpha activates adenylyl cyclase, which makes a lot of cyclic AMP to interact w/ protein kinase A etc. 2/15/16 IP3 acts as a ligand in membrane Bursts of calcium into the cells when concentrations are low Calmodulin is a protein and a main transducer of Ca++ signals o Conformational change when calcium binds exposing hydrophobic residue and allows it to interact w/ amphipathic helices of target proteins Enzyme Linked Receptors Typically Transmembrane receptors, in response to ligand bonding results in an catalytic activity Involves receptor dimerization which brings receptors closer to each other Receptor Tyrosine Kinase = best example of enzyme linked receptor o Usually single pass Transmembrane protein that binds ligands also has intracellular catalytic domain o Catalytic domain that gets activated is a kinase which puts phosphates onto tyrosine molecules, hence the name o When the ligand is bound you get dimerization and cross phosphorylate one another Mitogens are factors that can help proliferation of cells and can change gene expression to help cells grow Ras – small G protein that brings another family of G proteins Small G proteins Relatively small proteins that binds GTP or GDP like G alpha When bound to GDP, G protein has intrinsic enzyme capability and is inactive when bound to GDP The release of GDP so that GTP can bind turns the small proteins on GTP will get cleaved to GDP to switch it back off over time Ability of G proteins to hydrolyze GTP back to GDP is very slow. Cells prefer a quicker dynamic ability rather than waiting for slow wait o Accessory proteins have for a quicker reaction GEF: causes exchange of GDP for GTP to bind by making GDP a lower affinity interaction GAP: GTPase activated so GTP is cleaved and activity turned off GDI: prevents GDP from coming off keeping it in the off state *** Know the diagram of how to get Ras in it’s activated state PI3 Kinase Signal Ligand stimulates RTK and cross phosphorylates so PI3 kinase gets activated. PI3 is a lipid kinase, a protein that is a kinase that puts phosphate groups onto a lipid Phosphorylated lipids by activated PI3 kinase can activate downstream proteins Akt is a serine kinase which is important for growth and proliferation signals Proteolytic cleavage of intracellular proteins Immune response in the diagram At the end of it all, the movement of NFkB goes to nucleus and turns on gene expression that’s important to immune responses in cell Signals from outside world tells cells a need for inflammation response and the NFkB complex gets in nucleus When there’s no signal, the complex is sequestered by IkB alpha so it can’t go to nucleus When there are upstream signals for an immune response, IkB kinase phosphorylates sequestered protein and bound to E3 ligase and poly=ubiquitin proteins bind to the complex. Polyubiquitin chain recognize by large protein machinery called proteasome and degrades the protein to free NFKB to elicit an immune response EXAM 2 NOTES 2/19/16 Protein Targeting How do proteins get localized to different places in the cell? 1. The Nucleus All proteins begin synthesis on cytosolic ribosomes All proteins need to get translated into the ER Topology of inside cellular compartments is similar to outside environment of cell Transport of proteins across organelle membranes Gated: whole folded protein is moved via aqueous pores Transmembrane: folded proteins or unfolded are moved across topologically distinct compartments o Moves via nonaqueous transport complexes Vesicular: protein is transported in small vesicles that fuse w/ target compartment Transport can occur: After while protein has been made/posttranslational Simultaneously w/ proteins synthesis /cotranslationally How are proteins targeted to specific places in cell? With amino acid sequences that act as an address labels It’s called targeting signal Can be detected w/ receptor proteins associated w/ departments the signal needs to get to Targeting signals can be a linear continuous sequence or a patch formed after the protein folds All proteins in nucleus must get transported Many eukaryotic cells, during each cell division, the nuclear envelope break down and all the proteins are free into cytoplasm. When daughter cells refold, the proteins needs to get back into cell’s nuclei and target proteins help do this job. Nuclear pore complex controls what gets into the cell Nuclear Pore complex huge multiprotein complex acts as selective barrier to movement in and out of complex Nuclear Localization Signal how the cell recognizes proteins to be transported through pore protein was nucleoplasmin, a pentameric nuclear protein Conclusions from nucleoplasmin injection in experiments tail was necessary and sufficient for uptake. Head isn’t necessary or sufficient whole protein can be taken up into nuclei How are NLS’s and NES’s recognized? Proteins transported through the pore contain either NLS or NES Signals are recognized by proteins called karyopherins in the cytoplasm or nucleus that act as receptors and control movement through the nuclear pore complex Importins: import receptors. Help proteins get into nucleus Exportins: export receptors, help rpoteins get out of nucleus Small G protein Ran regulates both processes Import and export receptors have related sequences and work in similar ways but opposite directions 2/26/16 SRP can bind signal sequence and ribosome to halt translation 6 types of Transmembrane proteins (IIV and GPI linked proteins) Type 1 signal pass Transmembrane Protein Uses N terminal signal sequence to direct peptide to membrane Stop transfer sequence is made; transfer to lumen stops Type 2 and 3 No N terminal signal sequence First sequence is internal start transfer sequence aka signal anchor sequence It’s both the signal and Transmembrane anchor associated w/ the membrane and SRP is delayed Hydrophobic alpha helices as signals for protein transfer N terminal signal sequences, internal start transfer sequences, and internal stop signal sequences are all hydrophobic For multipass Transmembrane protein topology, the key is the order and orientation of these topogenic sequences Ultimate orientation all depends on order of various sequences Glycosylation of proteins Vast majority of proteins that are getting translocated into the ER are at the same time getting glycosylated Oligosaccharides are being covalently attached to the protein as it’s entering the ER lumens via oligosaccaryltransferases N linked Glycosylation because sugar trees are being covalently attached to Asn in the protein Source of Oligosaccharides Built initially n the cytoplasm on a lipid carrier (dolicol) Pathway through construction, it is moved across 2/29/16 GPI is a lipid anchoring mechanism are also being occurred in the lumen Protein folding in the ER Chaperone proteins in the ER lumen aid in folding o Heat shock proteins, the family of proteins that either aid in protein folding or protect the cell from unfolding in response to stress o Similar ER localized chaperones such as BiP, are constitutively expressed and bind unfolded or misfolded proteins in this compartment Protein disulfide isomerase o The destination of ER trafficked proteins is a much harsher environment than inside the cell. Disulfide bonds stabilize protein structure o Proteins that contain more than one cysteine can have covalent SS bonds form between the cysteines o PDI is a protein that drives cycle of formation and withdraw and reformation of disulfide bonds o Randomly allows diff sulfide bonds to form and ER will pick the best one for the protein’s conformation Quality Control Proteins that fold incorrectly are recognized as wrong ER associated protein degradation Misfolded proteins are dislocated back to the ER to the cytoplasm Dislocation is a poorly understood mechanism In the cytoplasm, they become tagged with polyubiqutin monomers of the small ubiquitin molecule added in chain A special protein degradation machine in the cytoplasm, Proteasome recognizes polyubiqutin and degrades the protein Proteasome also degrades cytoplasmic proteins Lectins = proteins that can bind to sugars Protein targeting to mitochondria, chloroplasts and peroxisomes Not apart of endomembrane system These organelles are unique membrane bound organelles Mitochondria and Chloroplast Both surrounded by a double membrane Each has its own DNA contained in a genome in the lumen inside the inner membrane Some proteins are made from own DNA on internal ribosomes and some are made in the cytoplasm from nuclear DNA and imported Both divide by fission like bacteria Both can only be generated from preexisting mitochondria or chloroplasts; but they to need import nuclear encoded proteins for proper function Chloroplasts Third compartment called granum with thylakoid spaces Small molecule transport The outer membrane is like the outer membrane of bacteria; it contains many types of Porinspore forming proteins which are beta barrel channels Small molecules and ions move freely btwn intermembrane space and cytoplasm The inner membrane has transporters that selectively move small molecules across the inner membrane into and out of the matrix ADP/ATP exchanger uses H+ voltage gradient Pyruvate/H+ cotransporter Phosphate/H+ cotransporter 3/2/16 Targeting proteins to the mitochondria usually at the N terminal with amphipathic alpha helix with positive charged amino acids Necessary and sufficient Posttranslational transmembrane transport All proteins that are destined to go to mitochondria and reside somewhere in the mitochondria, they all start their life by interacting with TOM complex Once exposed in the matrix, the targeting signal is cleaved off Protein being exported is held in unfolded state Secondary sequences react w/ a wide variety of TIM/TOM complexes and proteins Chloroplast Targeting Chaperones assist post translational import of unfolded proteins Stroma targeting signal directs them to the outer membrane where tey bind a receptor Transport occurs through TOC and TIC N terminal targeting signal is cleaved Secondary signals and complexes direct proteins to various areas Thylakoid introduces a further level of complexity Plants have both mitochondria and chloroplasts so the membrane receptors must be able to tell the signals apart Peroxisomes Produces h2o2 which is used in oxidative processes such as fatty acid oxidation They arise by budding off of the ER They are bounded by a single membrane Nearly all proteins must be imported; a few import proteins are added in the ER Import is posttranslational and proteins are fully folded N term or C term signal sequences with unique cytoplasmic receptors for each Use same pore for transport Signal sequence NOT cleaved Mutation in Pex5 leads to accumulation of long chain fatty acids in peroxisomes ER to the Golgi body Vesicle Transport o Membrane enclosed vesicles bud and fuse to move proteins from compartment to compartment o ER to Golgi and beyond, endocytosis Proteins being trafficked to the ER go through the Cis body network and trans body network (anterograde transport) 3/4/16 Mannose trimming happens in the Cis golgi When a protein has a mannose trimmed, it makes the sugar tree vulnerable to endoglycosidase D so it can chop the whole protein Proteins on the cytoplasmic face of vesicles regulate budding and fusion; 1 Budding Mechanical process of membrane budding is driven by a complex of cytoplasmic proteins that assembles on the outside of budding vesicles Different vesicle types have diff coat proteins o COPII: coats vesicles moving ER to golgi o COPI: coats vesicles moving Golgi to ER and from more trans Golgi to more cis Golgi o Clathrin: Coats vesicles moving from TGN to surface, and from surface to endosomes, lysosomes Contents of vesicles apparently play no direct role in triggering budding A class of small G proteins regulates budding Cytoplasmic G proteins associate with membranes GTP bound: budding initated GDP Bound: no budding or removal of coat Different types of membrane budding use a different G protein COPII use Sar 1 COPI and Clathrin use Arf These G proteins have a covalent lipid anchor that can be exposed or hidden allowing them to reversibly associate w/ the cytoplasmic surface of the membrane The vesicle cargo Some soluble proteins seem to be carried along nonspecifically by bulk flow Other soluble proteins seem to bind to cargo receptors which concentrate them in vesicles; these Transmembrane receptors interact via cytoplasmic domain w/ coat proteins Docking and Fusion regulated by the Rab family of small G proteins Different routes of vesicle trafficking appear to be controlled by different Rab members Together with SNARES they control the specificity of vesicular traffic GTP bound state is active; tightly associated w/ members or vesicles; mediate tethering and docking of vesicles via a variety of effector proteins in target membrane Hydrolysis to GDP allows release of inactive Rab to cytosol Together, Rabs and SNARES control the specificity and directionality of vesicular delivery and target 3/7/16 Coat protein provide a mechanical force to create budding COP II takes materials from ER to the Golgi Uses Sar1 to hydrolyze GDP so it can stick to the membrane of the ER Vesicular transport: anterograde way with vesicles but there are complications because COP II retrograde vesicles couldn’t transport through the Golgi Cisternal maturation model: protein stays in compartment once it’s reached and specialized enzymes change because retrograde transport is bringing back enzymes from trans to cis. Accumulates more trans enzymes over time therefore matures to a more trans model TGN sorting pathways 1. Signal mediated diversion to lysosomes 2. Signal mediated diversion to secretory vesicles for regulated secretion 3. Constitutive secretory pathway Lysosomes Vesicles that take part in degradation of macromolecules Internal environment is acidic due to membrane pump that transports H+ in using ATP as an energy source Contents are hydrolytic enzymes that work in an acidic environment Component containing vesicles from the Golgi fuse w/ late endosomes that mature into lysosomes Degradation takes place in both How are Lysosomal proteins targeted to lysosomes? Stretch of AA acts as a signal Signal patch multiple stretches of AA in primary sequence Signal patch recognized by enzyme that can put phosphate group on it’s sugar tree Creates a Man6P group and acts as a secondary signal Clathrin First coat protein discovered Forms triskelions three legged structure Can self assemble in test tube into polyhedral cage This assembly presumably drives membrane budding (Arf regulated) Various adaptor proteins control cargo specificity Human Lysosomal Storage Diseases Rare human genetic diseases lead to formation of inclusion bodies in cells In some diseases, a single type of enzyme is missing and accumulates certain types of macromolecules In some, none of the lysomsomal enzymes are packaged correctly I cell diseases; inclusion body diseases Man6P receptor mutation: Man6P receptor won’t recognize this enzyme anymore Man6P transferase mutations Endocytosis: Movement of membranes vesicles into the cell Pinocytosis: uptake of soluble fluid phase material from outside the cell o Pinching mechanism o Small molecules get taken up by vesicles o Some is constitutive; takes place all the time o Brings in nutrients and other soluble materials o Balances the outward flow of membrane in secretion o Some is regulated/receptor mediated endocytosis o Two main modes of entry: Caveolae small materials Contains lipid rafts Self associates to form a striated coat on the cytoplasmic surface Also called uncoated pits b/c not visible by EM Caveolin is immobilized in caeolae and doesn’t diffuse laterally in the membrane. This contrasts w/ the transient recruitment and regulated assembly of coat proteins involved in the formation of clathrin coated pits Clathrin coated pits larger materials Phagocytosis: engulfment of solid material from outside by direct attachment o Outward protrusion of membrane o Don’t need to know the little details Materials from both can end up in lysosomes Receptor Mediated Endocytosis
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