Exam 1 Notes!
Exam 1 Notes! Biol 3302
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Date Created: 02/01/16
Chapter 9: Culturing, Visualizing, and Perturbing Cells Culturing Plate – coated with AA and proteins that help the cells stick to the plate Average animal cell takes approx. 24 hours to divide Serum has all growth factors Visualization Techniques and Microscopy Fluorescence – absorbing light at one wavelength and emitting light at a specific and long wavelength Major function: To determine localization of specific cellular molecules (ex: proteins) Major advantages o Sensitivity: “glow” against dark background o Specificity: immunofluorescence (antibody specifically binds to one protein) o Cells may be fixed or living Flurochromes – fluorescent dyes or proteins o Flurochromes may be indirectly or directly associate with the cellular molecule o Multiple flurochromes may be used simultaneously Transformation – process of introducing rDNA into prokaryotes Transfection – process of introducing rDNA into eukaryotes Recombinant DNA technology (living cells) o Ex: Hydra expressing GFP Hydra transfected with plasmid (DNA) containing the GFP gene that is driven by the hydra betaactin promoter Early experiment that showed the usefulness of fluorescent proteins o Purpose: localization of protein X o 1) Find gene X o 2) Fuse gene X with GFP gene aka chimeric fusion protein or recombinant DNA construct or plasmid construct o 3) Transfect eukaryotic cells Only eukaryotic cells will have the machinery to recognize and translate o 4) Wait 2448 hours for plasmid to be translated o 5) Use fluorescence to find protein X Couldn’t do western blot because you don’t know anything about protein X – you don’t have an antibody that recognizes protein X Immunofluorescence (fixed cells) o Process of making antibodies: Inject epitope into rabbit/mouse Extract antibody – test specificity with ELISA – develop specific ab Add fluorescent tag to primary (not all because difficult/expensive) or use fluorescent secondary o Direct immunofluorescence fluorescentlytagged primary antibody o Indirect immunofluorescence – normal primary ab, fluorescentlytagged secondary o Tagged proteins myc or flag (be prepared to draw for exam) Scenario: looking for protein C but don’t have a primary antibody for it; gene sequence is know; can’t use live cell rDNA method because transgene is too large/won’t translate Alternate method – add myc or FLAG (approx. 12 AA) to the Nterm or C term Transfect cells, time, then use prim2+ ab to myc/FLAG o Fluorescent dye fura2 is used to monitor Ca concentrations within a cell Cannot be used for proteins; only detects calcium ions o Process of immunofluorescence: Prepare sample on slide, incubate with primary, wash, incubate with flurochromeconjugated (= fluorescentlytagged) secondary antibody, wash, microscope Doublelabel fluorescence microscopy o Can be used with fixed or living cells o Visualizes the relative distributions of two proteins o Overlay 2 channels to visualize both o Just because in overlay they’re in the same area doesn’t mean that they’re interacting because they could be in different focal planes o Ex: visualizing microtubules (indirect immunofluorescence) and actin filaments (with phalloidin – drug that specifically binds to actin) Fluorescent Microscopy SPED (stimulated emission depletion) fluorescent microscopy has a resolution of 20nm Confocal, deconvolution, and superresolution microscopy overcome the limitations of fluorescence microscopy (blurred images, thick specimens) Confocal o Laser as the energy source o Laser scans the specimen across and down to build an image o Uses a pinhole in front of the detector to block light from other focal planes o 2 types: Laserscanning and spinning disk Deconvolution o Takes image and uses a computer program to reconstruct a nicer image TIRF microscopy = Total internal reflection o Gives better resolution of the area that is closest to the cover slip o Restricted focal plane o Use regular + TIRF to create a combined image Superresolution Microscopy techniques FRAP = Fluorescence recovery after photobleaching o Gives information on the dynamics of a protein of interest Want to know if protein/phospholipid moves around in the phospholipid bilayer o Fluorescently tag protein or phospholipid o Bleach fluorescence in a specific ROI (region of interest) Bleaching = quenching o When/if region regains fluorescence it shows that the protein is moving because the fluorescent protein from other areas migrated into the ROI Know it’s moving and not just making more because not enough time (30 sec) o Compare to another control region that is not bleached FRET = fluorescence resonance energy transfer o CFP and YFP are used because emission wavelength of CFP = excitation wl of YFP o Measures the distance between proteins To find out if proteins are close enough to interact (using chromophores) Fuse gene X with CFP (cyan fluorescent protein) Fuse gene Y with YFP (yellow FP) Transfect into the same cell Shine light at CFP excitation wavelength (= 480nm) CFP will emit at 480 nm If CFP is close enough to YFP (excitation wavelength = 480nm) then YFP will emit (535nm) Yellow fluorescence = X and Y are close enough to interact Doesn’t necessarily mean that they are interacting o Measures conformational changes of a single protein Only 1 protein (ex: calmodulin) tagged to CFP and YFP at the same time Looking for conformational changes Ex: without calcium ions, CFP and BFP are not close enough to transfer energy but with calcium ions, protein bends and they become close enough Preparing samples for light and electron microscopy Fix with formaldehyde o Formaldehyde crosslinks amino groups on adjacent molecules o Dehydrates sample o Can also be glutaraldehyde (same family of molecules) Embed in paraffin for light or liquid plastic for electron Section 0.550 micrometers for light; 50100 nm for TEM o Do not section for SEM Stain with hematoxylin (binds to basic AA) and eosin (binds to acidic molecules) Electron microscopy Fixed cells only Transmission EM (TEM) o Used for clear 2D images Reveals surface details o Practical resolution: 0.1nm 1nm (2000x than light microscopy) o Highvelocity beam passes through the sample o 50100nm thick sections o Can “simulate” a 3D image by getting different sections from different areas and putting together using a computer program o Samples need to be fixed and stained Cannot image live cells o Stained with heavy metals such as uranium, lead, osmium tetroxide Stains the membrane Evaporate platinum (layers platinum on top) Then evaporate carbon (layers carbon on top) Creates a metal replica of the surface that is visualized Shows fine structural details o Detecting a protein using gold particles that are coated with protein A Use an antibody that binds specifically to the protein of interest (ex: catalase) Protein A that is attached to gold particles binds to a generic Fc domain in the antibody Results in a complex that is attached to gold, show up as black dots in TEM For catalase – show up only in peroxisomes Scanning EM (SEM) o Used for 3D images o Resolution about 10nm o Used to view unsectioned, metalcoated specimens Cryoelectron microscopy Allows visualization of specimens without fixation or staining Looks at sample in native hydrated state Useful when dehydrating the protein changes its structure 5nm resolution Method: an aqueous suspension of the sample is applied on a grid (frozen in liquid nitrogen) and held be a special mount Variation: Cryoelectron tomography o Allows determination of the 3D structure Purification of cell organelles Cell disruption (breaking open of cells) Should be done in isotonic sucrose o Isotonic – similar to cytoplasm environment (maintains ionic strength and pH) Can break open by o Sonication o Homogenization (similar to mortar/pestle) o Putting in hypotonic solution Separation of different organelles using centrifugation Differential centrifugation o Filter homogenate to remove unbroken cells etc. o Spin at 600g x 10 min Pellet = nuclei o Spin supernatant and spin at 100,000g x 60 min Pellet = mitochondria, chloroplasts, lysosomes, and peroxisomes o Spin supernatant 300,000g x 2 hours Pellet now = ribosomal subunits, small polyribosomes Supernatant = cytosol Density gradient centrifugation o Used after differential centrifugation Ex: if you want a mitochondrial protein, you would spin twice but then have to get mitochondria out of the pellet vs lysosomes, chloroplasts, etc. o Pour the gradient o Increasing density from top (1.09) to bottom (1.25) o Centrifuge, organelles will migrate to their density o When looking at the tube will see 3 fuzzy bands across o Top to bottom: Lysosomes – 1.12 g/cm3 Mitochondria – 1.18 g/cm3 Peroxisomes – 1.23 g/cm3 o To extract the bands – needle o After extraction: Lysosomes and mitochondria have similar densities so after removing, can’t be sure that you have 100% pure organelle o So, then have to run a western blot using markers (enzyme markers) that are specific to the 3 organelles Catalase – peroxisomes Cyt C or cytochrome oxidase– mito Acid phosphatase – lysosomes Plasma membrane – amino acid permease Rough ER – ribosomal RNA Smooth ER – cytidylyl transferase o Example of western blot run from 3 samples Goal: to isolate and purify mitochondria Have 3 tubes (each band extracted from differential centrifugation) Run western blots From western blot 1 alone: Can NOT conclude that M is pure Can say that L and P are not contaminated with M From western blot 2 as well: M is contaminated with L L is not contaminated with M o If your mitochondrial fraction is contaminated you can purify by using differential centrifugation using a deeper/more spread out density gradient Rerun western blot Using antibodies to purify vesicles Cannot use density gradient centrifugation if the density of the vesicles are the same/similar Use antibody for protein that is unique to that vesicle (ex: clathrin) Then use a bacterial cell (which has protein A on the surface) Protein A binds to Fc domain in the antibody o A single bacterial cell can bind to multiple coated vesicle antibodies Bacterial cell is very heavy, quick spin will bring down the entire complex Easy to use with clathrin because it forms a cagelike structure on the outside/surface so it’s easy for the bacterial cell to bind to o Could not use with cytc to purify mitochondria because cytc is not a surface protein Screening for drugs that affect specific biological processes Ex: screening for drugs that specifically affect spindle morphology o Start with 16,320 chemical compounds o Screen for those that arrest cells in mitosis 139 compounds o Screen for those that do not affect microtubule formation 86 o Screen for those that specifically affect spindle morphology 5 siRNA (= small inhibitory RNA) Knockdown the expression of a specific protein Targets the degradation of specific mRNAs in cultured cells Can start with synthetic siRNA (antisense to target mRNA) o Directly introduced into cell goes to RISC complex Or DNA expressing shRNA (small heteroduplex RNA) o Integrated into cell as a DNA construct o Goes into nucleus (6,7,8,25) o shRNA is made and is a hairpin loop (useless bc double stranded) o Dicer (an RNA endonuclease) cleaves the loop region o Then goes to RISC complex RISC –RNA induced silencing complex then is introduced to the cell and allows the siRNA to bind to the target mRNA Target mRNA is degraded To look for effect of siRNA knockdown o Use an antibody that recognizes another gene in the intended area o Ex: knockdown of EBP50 (component of microvilli), then use ab against erzin (other protein in microvilli) Genomic screens use siRNA o RNAi screens explore the function of all the genes in cultured cells RNAi is used to suppress genes in specific tissues only o Parent A – introduce shRNA downstream of promoter o Parent B – introduce tissuespecific promoter upstream of promoter element o Progeny – have both constructs in all cells but shRNA only transcribed in that tissue because only in target tissue is the promoter made (ex: GAL4) that acts on the upstream promoter element to make the shRNA (RNAi) Animal/Plant Cell Organelles Plasma membrane – controls movement of molecules in and out; functions in cellcell signaling and cell adhesion Mitochondria – generate ATP by oxidation of glucose and fatty acids o Inner and outer membrane Intermembrane space in between the two Cristae – folded sections o Has own DNA but cannot make all the proteins it needs to function – has to “import” proteins Lysosomes – degrade material internalized by the cell and wornout cellular membranes and organelles o Acidic lumen; pH – 5.2 o Autophagy Digest cellular debris/damaged organelles o To digest: wraps membrane around damaged organelle, fuses with it, digests it using acidic hydrolases o Can bring soluble or insoluble material from outside (endocytosis) Soluble – pinocytosis Insoluble – phagocytosis Nuclear envelope – encloses the contents of the nucleus; the outer nuclear membrane is continuous with the rough ER Nucleolus site of most rRNA synthesis Nucleus – chromatin (DNA + proteins); site of mRNA and tRNA synthesis Smooth ER – synthesize lipids and detoxify certain hydrophobic compounds Rough ER – synthesize, process, and sort proteins Ribosomes Made of proteins and RNA; 2 subunits – large and small o There is an equilibrium between free large subunits, free small subunits, and ribosomes in the cytoplasm o Ribosome assembly occurs during protein synthesis o Synthesize proteins in the nucleus Golgi – Process and sort proteins synthesized by rough ER o 3 parts – cis, medial, trans Secretory vesicles – store secreted proteins and fuse with the plasma membrane to release their contents Peroxisomes – detoxify molecules and break down fatty acids Cytoskeletal fiber – form networks and bundles that support cellular membranes, help organize organelles, and participate in cell movement Microvilli (animal only)– increase surface area for absorption of nutrients from surrounding medium Cell wall (plants only) – composed largely of cellulose (beta glucose units joined together); helps maintain the cell’s shape and provides protection against mechanical stress; allows higher pressure Vacuole (plants only)– stores water, ions, and nutrients, degrades macromolecules, and function in cell elongation during growth Chloroplasts (plants only) photosynthesis; proteins are separated by different membranes Mitochondria, nucleus, and chloroplasts have a double membrane Chapter 10: Biomembrane structure Membrane bilayer Fluid mosaic model o Fluid – elements in the phospholipid bilayer are dynamic o Mosaic – patterns of different proteins Phospholipid bilayers controls the movement of several molecules and protects the contents Integral membrane (or transmembrane) proteins – run through the membrane and stick out on both sides Peripheral membrane protein – no part of the protein is in the membrane but they are close to the membrane Lipidanchored protein – in the membrane but only sticking out of one side; can be out the outside or in the cytosol Phospholipids Synthesized in the ER Amphipathic molecules o Have a hydrophilic/polar (head) and a hydrophobic/nonpolar (tail) region o Hydrophobic tails try to get on the inside When mechanically dispersed in solution they form 1 of 3 structures… o Micelles Hydrophilic heads on outside, hydrophobic tails on inside o Liposomes Bilayer that has folded to form a sphere Core is hydrophilic Used in drug delivery; drug placed inside core When it comes into contact with the plasma membrane it fuses with it delivering its contents o Phospholipid bilayers Experiment: Treat phospholipid bilayer with organic solvent to remove proteins and oligosaccharides Put in solution then evaporate the solvent Disperse in… o Water – form liposome o OR solvent and apply to small hole in partition in water – forms bilayer Classes of lipids Phosphoglycerides o Glycerol 3phosphate backbone (3 carbons) + 2 fatty acids + 1 head group o Plasmalogen – same structure as phosphoglycerides except 1 carbon is attached by ether linkage instead of carboxyl/ester High levels of plasmalogens in brain and heart tissue Sphingolipids o Derived from sphingosine, an amino alcohol with a long hydrocarbon chain o Glycolipids most abundant in nervous tissue o Synthesized in the Golgi Steroids o Sterols: Cholesterol (animal), ergosterol (fungal), stigmasterol (plant) o Cholesterol is synthesized in the smooth ER o Cholesterol is a precursor for bile acids, steroid hormones and vitamin D (produced in the skin and kidneys) o 3 benzene rings (hydroxyl groups off of 1 benzene, hydrophilic), pentane ring, carbons off of pentane (hydrophobic) Properties of membranes Hydrophobic core (hydrocarbon tails) is an impermeable barrier Stability o Van der Waals interaction and hydrophobic interaction stabilize the fatty acyl groups o Ionic and hydrogen bonds stabilize the polar head groups Phospholipid bilayers spontaneously form closed sealed compartments because if left straight the water can touch the hydrophobic tails at either end Membrane budding and fusion Vesicles pinch off of and fuse with the lipid bilayer o Endocytosis – Taking things inside (pinching off) o Exocytosis – Taking things outside (fusing with membrane) Exoplasmic segment – the side that faces the outside Cytoplasmic segment – the side that faces the inside The membrane is always conserved which direction the vesicle pinches off determines which side (exoplasmic or cytosolic) will be on the outside o If it pinches off into the cell, the cytosolic segment is on the outside and exoplasmic faces lumen (cytoplasmic segment always facing cytosol) o Example problem: Want N terminus of transmembrane protein to face the cytoplasm (because catalytic subunit near the N terminus, substrate is anchored to the cell membrane) and C terminus of the protein to face the exoplasm Application of membrane conservation: mitochondria Mitochondria comes from long ago when eukaryotic cell engulfed prokaryotic cell o Mitochondria – double membrane o Ancient eukaryotes and prokaryotes both had a cytosolic leaflet (membrane) and exoplasmic leaflet – each had a single phospholipid bilayer membrane Lipid composition is different in the two leaflets The distribution of PC, PE + PS, SM + cholesterol vary among different membranes; they are moved around to different membranes in the cell o How the asymmetric distribution occurs is unknown Exoplasmic leaflet – less fluid layers formed from phosphatidyl choline and sphingomyelin (PC + SM) Cytosolic leaflet – more fluid bilayers formed from PE, PS, and PI Cholesterol is evenly distributed in both leaflets Enzymes called flippases powered by ATP move the phospholipids from one side of the membrane leaflets to the other o Enzyme necessary doesn’t flip spontaneously because it has a hydrophilic and hydrophobic component Section with cholesterol and sphingomyelin are more ordered and less fluid compared to the more fluid phosphoglycerides in the surrounding are o These areas are called lipid rafts o Lipid rafts are microdomains about 50nm in diameter Membrane fluidity depends on… Lipid composition o More than normal cholesterol content can decrease membrane fluidity Interaction of the steroid ring of cholesterol with the long hydrophobic tails tends to immobilize these lipids o Less than normal cholesterol content can increase membrane fluidity At lower than normal cholesterol concentrations, the steroid ring separates and causes the inner region of the phospholipid to become more fluid o Cholesterol increases membrane thickness in phosphoglyceride bilayers but not in sphingomyelin bilayers Membrane thickness affects membrane fluidity Structure of the hydrophobic tails o Saturated fatty acids aggregate forming a gellike state (no kink in structures, can be packed more tightly) o Short fatty acyl chains and cisunsaturated fatty acyl chains (“kink in structures”) result in less stable interactions and hence have more fluidity Temperature o Higher temperature = more fluid membrane Bilayer thickness of membranes Adding cholesterol to phosphoglycerides (ex: PC) increases the thickness o 2 PC in bilayer thickness is 3.5 nm o 2 PC with cholesterol in bilayer thickness in 4 nm Adding cholesterol to SM doesn’t increase the thickness o SM to SM + cholesterol – no big change; before and after 4.65.6nm These phospholipid stack very differently o PC together – cylinders side by side o PE together – Cone o Differences used to accommodate a phospholipid bilayer that needs to make a turn – PC in straight parts, PE on the insides of turns Lateral and rotational movement in the bilayer 7 A typical lipid molecule exchanges places about 10 times per second and also diffuses several micrometers per second at 37°C Increased temp = increased rate at which it’s going to move Exchanges places laterally (not flipping sides spontaneously; moving left to right) Gel like fluidlike consistency with heat FRAP experiments To detect the lateral movement of proteins and lipids within the plasma membranes Fluorescently label membrane protein; bleach ROI with laser – kills the fluorescence; monitor for fluorescence recovery Protein can also move back out – won’t see 100% recovery Integral membrane (transmembrane) proteins Contain 1 or multiple membrane spanning α helices 2025 hydrophobic uncharged amino acids o Total length is 3.75 nm = slightly greater thickness of bilayer Transmembrane segment can perpendicular to the membrane or at an oblique angle Hydrophilic amide peptide bonds in the interior of the αhelix Interactions o Hydrophobic side chains interact with fatty acyl groups by hydrophobic and Van der Waals interaction o Ionic interactions between the hydrophilic amino acids and the phospholipid polar head groups o These interactions serve to “anchor” the protein in the membrane Singlepass integral membrane protein o Ex: glycophorin A #7396 AA = hydrophobic Blue = charged = hydrophilic; interact with the hydrophilic head groups Functions as a dimer Multipass transmembrane protein o Ex: rhodopsin – bacterial protein – has 7 membrane spanning helices o Ex: Glycerol channel protein, Glpf belongs to the aquaporin family Allows you to transport glycerol from one side to the other The channel is lined by side groups of hydrophilic amino acids present in the alpha helix Transmembrane segments are arranged differently than those in rhodopsin; many are at oblique angles Multi membranespanning beta strands o Can form a barrel with a hydrophilic interior and hydrophobic exterior o Ex: Porins Found in the outer membrane of Gramnegative bacteria, outer membrane of mitochondria and chloroplasts Porins provide channels for the movement of disaccharides, watersoluble molecules and ions Trimers of identical subunits Each subunit – 16 beta strands that twist to form a barrelshaped structure Lipidanchored membrane proteins Cytosolic anchors are different from exoplasmic anchors Blue beaded = protein Cytosolic anchors: o Acylation Glycine residue on the Nterminus of the protein attached to fatty acyl groups (either myristate/myristic acid or palmitate/palmitic acid) Example: vsrc o Prenylation Cysteine residue near the Cterminus attached to hydrocarbon chains These hydrocarbon chains are made from 5carbon isoprene units These are 15carbon farnesyl or 20carbon geranylgeranyl In some case a second geranylgeranyl group or plamitate is attached to a second cysteine Example: Ras Exoplasmic anchors: o GPI anchor = glycosylphosphatidyl inositol C terminus near membrane Add phosphatidyl inositol (red) then sugar residues (greens) then phosphoethanolamine (purple) Glycoproteins All humans have the enzymes for synthesizing the O antigen People with A blood group have the enzyme for synthesizing A antigen B blood group have the enzyme for synthesizing B antigen AB has enzymes to synthesize A and B antigens People are diploid – have 2 alleles o A is dominant over O, if you get one O and one A allele then A overrides o A and B are codominant Cannot receive blood from a type that you make antibodies against Blood Groups Antigens on RBCs Serum antibodies Can receive A A Anti B A and O B B Anti – A B and O AB A and B None All O O Anti – A and Anti B O Peripheral membrane proteins Enzymes cleave at specific spots of the phospholipid Phospholipase A2 is exoplasmic only Mechanism of phospholipase A2 o The enzyme has a calcium containing active site buried in a channel of hydrophobic amino acids o The enzyme contains a rim of positively charged amino acids that bind to the negatively charged phospholipids (eg. PS) o This binding induces a conformational change in the enzyme and it opens its hydrophobic channel o A phospholipid molecule moves from the bilayer to the channel o The enzyme bound calcium binds to the phosphate in the head group and positions the ester bond to be cleaved Detergents Detergents are amphipathic molecules that intercalate into the lipid bilayers and solubilize the membrane proteins and lipids o The hydrophobic part interacts with the hydrocarbons o The hydrophilic part interacts with water Critical micelle concentration (CMC) is characteristic for each detergent and depends on its hydrophobic and hydrophilic groups When separated from membranes, the hydrophobic regions of integral membrane proteins are exposed and they tend to interact with one another causing the formation of aggregates and precipitate from solution Nonionic detergent prevents this aggregation and aids in solubilization of proteins Ionic detergents (ex: SDS) o Denature proteins o Can break ionic and hydrogen bonds because of their charge o Bind to hydrophobic regions of membrane and watersoluble proteins o Useful in extracting membrane proteins before they are purified Nonionic (ex: Triton X100) o Lack a charged group unlike ionic detergents o Do not denature the protein; allow you to look at the protein in its natural state o Have to use at concentration less than the CMC At a concentration greater than the CMC the detergent tries to form micelles At a concentration below CMC the detergent dissolves/solubilizes without forming micelles Solubilization of peripheral membrane proteins Removed from membranes using high salt solutions – these break the ionic bonds These proteins are soluble in aqueous solutions and need not be solubilized using nonionic detergents Fatty acid synthesis Fatty acids are made from acetyl – CoA subunits Fatty acids have 14, 16, 18 or 20 carbons o 14 carbons = 7 acetyl CoA units o 16 carbons = 8 acetyl CoA subunits, etc. Fatty acyl CoAs are soluble in aqueous solutions Saturated fatty acids are made by acetyl CoA carboxylase and fatty acid synthase o Both are cytosolic enzymes Desaturase makes unsaturated fatty acids by introducing double bonds o Desaturase is present in the ER Palmitoyl CoA can be elongated to C18 or C24 in ER or mitochondria Diacylglycerophospholipids (glycerol + 2 phospholipids) are synthesized in the smooth ER Sphingosine and Nacyl sphingosine (ceramide) are made in the ER Addition of a polar head group to ceramide occurs in the Golgi FABPs Very hard for fatty acids to move around because they so hydrophobic To move around, they are bound to fatty acid binding proteins (FABPs) o FABPs have a beta–sheet pocket that encompasses the fatty acid FABP expression is regulated by levels of fatty acid o More fatty acids = more FABP FABP levels are high in active muscles and in adipocytes (cells that store fat) Phospholipid biosynthesis and flippases In the cytoplasm: Acetyl CoA carboxylase and fatty acid synthase act on acetyl CoA to form a fatty acid Fatty acid fatty acyl CoA by adding an acetyl CoA 1 fatty acyl CoA then added to glycerol phosphate o This happens by GPAT (glycerol phosphate acyl transferase) o Enzyme is in the ER membrane o The entire process after this occurs in the ER membrane – important because the final destination of the phospholipid is in the membrane o After GPAT, you now have lysophosphatic acid Second fatty acyl CoA then added to lysophosphatidic acid by LPAAT (lysophosphatidic acid acyl transferase) which is an ER membrane enzyme o After LPAAT have phosphatidic acid (= phospholipid without a head group) in the ER membrane Phosphatidic acid – has to be in the membrane (cytosolic leaflet) because it now has nonpolar hydrocarbon chains Phosphatase then replaces the phosphate with an OH to form diacylglycerol Choline phosphotransferase adds PCholine by converting CDPcholine to CMP Phosphatidyl choline (PC) is then in the cytosolic leaflet o PC though is present in both cytosolic and exoplasmic so needs to be distributed Mechanism by which PC can go from cytosolic to exoplasmic – flippase Cholesterol biosynthesis Acetyl CoA (2 carbon) + acetoacetyl CoA (3 carbons) HMG CoA in the cytosol o HMGCoA = Hydroxy methyl glutaryl – CoA HMGCoA (HMG CoA reductase) mevalonate isopentenyl pyrophosphate (IPP) [5 carbons] farnesyl pyrophosphate squalene cholesterol HMGCoA reductase Ratelimiting enzyme Located in the ER membrane Has a watersoluble catalytic domain o Because HMGCoA is in the cytosol o Has to have a watersoluble domain so that it can accommodate HMGCoA Has 8 transmembrane helices 5 of the transmembrane helices form the sterolsensing domain (cholesterolsensing) Sensing capabilities are the deciding capabilities are the deciding factor as to whether cholesterol biosynthesis continues or stops o When cholesterol levels are high, cholesterol binds to the sterol sensing domain of HMG CoA reductase which cases the protein to bind 2 other membrane proteins (Insig1 and Insig2) o The binding of these 2 protein causes the enzyme to be ubiquitinated and degraded by the proteasome pathway Ubiquitination – tag that is added to a protein that marks it for degradation o Once HMGCoA is marked and degraded it cannot act on HMGCoA no more cholesterol made Statins drugs given to patients with high cholesterol Patients with high cholesterol have poor control of HMGCoA reductase o Statins inhibit HMGCoA reductase and thus shut off the cholesterol synthesis o Important that the process is stopped at the ratelimiting step for maximum control o Not only reduces LDL (bad) but also reduces HDL (good cholesterol) have to balance statins Want LDL less than 120 (old guidelines – less than 100) Want HDL greater than 50 o Several intermediates in the cholesterol biosynthesis pathway feed other pathways (IPP, farnesyl pyrophosphate, and cholesterol) Artherosclerosis = deposition of lipids, cholesterol and other extracellular material in the inner wall of arteries o Caused by high cholesterol This causes distortion or changes in arterial wall structure (reduction in diameter of artery) that may lead to a clot o Clots can break off but in other places it get stuck which can lead to heart attack or stroke (depending on what artery is blocked) Mechanisms of cholesterol and phospholipid transport between organelles 3 models 1) Transferred from one membrane to the other by means of vesicles o Same process of endocytosis 2) Hypothetical membranebound proteins (1 in each membrane) contact each other which brings the sections of the membranes together so that lipids can be transferred 3) Proteins moving around in cytosol (“carrier proteins”) bind phospholipids or cholesterol and carry/release them on the other membrane
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