Membranes Lecture Notes PDF

Membranes Carolyn K. Suzuki, Ph.D. Dept. of Microbiology, Biochemistry and Molecular Genetics January 31, 2024 Please see udated slides posted today 1 Membrane dynamics and phospholipids Red blood cell Platelet White blood cell Membrane systems within the cell Mitochondrion 3 Membrane bound systems...

Membranes Carolyn K. Suzuki, Ph.D. Dept. of Microbiology, Biochemistry and Molecular Genetics January 31, 2024 Please see udated slides posted today 1 Membrane dynamics and phospholipids Red blood cell Platelet White blood cell Membrane systems within the cell Mitochondrion 3 Membrane bound systems Intracellular compartment % total cell volume % of total cell volume Cytosol 54 Mitochondria 22 Rough ER cisternae 9 Smooth ER cisternae/ Golgi cisternae 6 Nucleus 6 Peroxisome 1 Lysosomes 1 Endosomes 1 4 Fluid mosaic model of membranes Phospholipid bilayers of cellular membranes are fluid and elastic Protein Phospholipid bilayer Hydrophobic tail Protein Hydrophilic head group 5 Different protein topologies in cellular phospholipid bilayers 6 Membranes are dynamic (not static) Endocytosis Exocytosis Extracellular Extracellular Intracellular Intracellular 7 Endoplasmic reticulum Immunofluorescence ER resident protein Video microscopy ER resident protein 8 Golgi complex Immunofluorescence Golgi resident protein Video microscopy Protein transport from ER to the Golgi 9 Retrograde protein transport from Golgi back to the ER Video microscopy 10 During mitosis, the nuclear envelope disassembles and reassembles Video microscopy of mitosis 11 Nuclear envelope is continuous with ER membrane system Nuclear pore complex (NPC) Nuclear Envelope (NE) NUCLEOPLASM inner nuclear envelope outer nuclear envelope 12 Lamin A and Lamin B Essential for structure of the nucleus Forms dense fibrous protein network under the inner nuclear envelope Plays a central role in the disassembly and reassembly of the nucleus during mitosis Important in chromosome localization and gene regulation nucleus lamins inner nuclear envelope outer nuclear envelope nuclear pore complex (NPC) Hutchinson Gilford Progeria Syndrome (HGPS) Genetic mutation in the gene encoding Lamin A (LMNA) Ultra-rare 1 in 4 million births 50 amino acid deletion Causes premature aging Progerin has been shown to impair DNA replication Regulation of transcription Cell cycle control Cell differentiation Average life expectancy 13 years old Cause of death usually heart problems or strokes Not cancer, neurodegeration HBO documentary 14 Abnormal Lamin A Progerin in HGPS Normal Lamin A protease cleavage site Lipid modification at C-terminus anchors protein to membrane proteolytic cleavage Mature Lamin A (72 kDa) Progerin mutant (67 kDa) Lipid anchor removed in mature protein Lipid anchor is NOT removed!!! 15 You don’t need to know these details Fluorescence microscopy of Lamin A and Progerin Normal lamin A normal nucleus Progerin abnormal nucleus PLoS Biol 3(11): e395, 2005. Membrane lipids Lipids are a class of molecules that have low solubility in water and high solubility in non-polar solvents. Three general classes of lipids compose cellular membranes 1. Phospholipids 2. Sphingolipids 3. Cholesterol Common featuresall are amphipathic (Greek ‘tolerant of both’) polar head group exposed to aqueous environment hydrophobic tail buried in n lipid environment Differenceschemical structure abundance in membrane leaflets abundance in different cellular compartments function R = different head groups Structure of phospholipids hydrophobic tail composed of 2 fatty acyl chains 3 carbon backbone derived from glycerol polar head group derived from an alcohol Name of phospholipid phosphatidylethanolamine phosphatidylcholine phosphatidylserine phosphatidylinositol 18 Phospholipids are primarily synthesized in the endoplasmic reticulum (ER) Phosphatidic acid is the simplest phospholipid, which is the precursor of other phospholipids Formed from glycerol-3-phosphate and glycerol glycerol-3-phosphate glycerol 19 Structure of the phospholipid cardiolipin Two molecules of phosphatidic acid esterified at their phosphate groups to glycerol Synthesized in mitochondria, component of mitochondrial membranes Is the only phospholipid that is antigenic (i.e., immunogenic) phosphatidic acid phosphatidic acid glycerol 20 Structure of sphingolipids All sphingolipids have a sphingosine backbone All sphingolipids are composed of ceramide as a common structural unit Ceramide = sphingosine attached by an amide linkage to a fatty acid Synthesized in the Golgi Primarily in cells of the nervous system Name Name sphingosineamino alcohol sphingosineamino alcohol with long hydrocarbon tailtail with long hydrocarbon phosphocholine phosphocholine head group head group sphingosine- amino alcohol with long hydrocarbon tail phosphocholine head group of of sphingolipid phospholipids phospholipids Name of phospholipids sphingomyelin sphingomyelin sphingomyelin acid various fatty acyl chains areare various fatty acyl chains attached to sphingosine by by amide bond attached to sphingosine amide bond glucosylcerebroside glucosylcerebroside various fatty acyl chains are attached to sphingosine by amide bond glucosylcerebroside glucose glucose head group head group glucose head group 21 Cellular membranes an have different percentages specific phospholipids 22 The inner and outer bilayer are asymmetricdifferent percentages of specific phospholipids 23 Lipid flippases in the ER and plasma membrane mediate asymmetric distribution of lipidS in the bilayer P-type ATPases move lipids to inner leaflet ABC transporters move lipids to outer leaflet Scramblase moves lipids bi-directionally Bi-directional flippase PS PE SP ATP-independent 24 The inner and outer bilayer are asymmetricdifferent percentages of specific phospholipids A uni-directional flippase that is ATP-dependent moves PS to the inner leaflet 25 Apoptosis (also known as programmed cell death) Different from necrosis, which is energy-independent and usually a consequence of cell injury Physiological mechanism in: organismal development and cellular homeostasis 105 cells are produced every second by mitosis 105 die every second by apoptosis in eliminating cells that are infected or mutated Hallmarks of apoptosis: fragmentation of the nucleus fragmentation of chromosomal DNA exposure of phosphatidylserine on the outer face of the plasma membrane ATP-dependent 26 Mitomycin Mitomycin-induced C an anti-cancer agent induces apoptosis apoptosis causes fragmentation of nucleus (-) MMC -MMC (+) MMC +MMC 20 hrs Blue stain binds DNA U2OS osteosarcoma cells incubated overnight with MMC 27 Mitomycin C an anti-cancer agent induces apoptosis causes fragmentation chromosomal DNA DNA marker 0 Mitomycin C (µM) U2OS cells incubated overnight with MMC 28 CORRECTION!!! Another hallmark of apoptosis is exposure of phosphatidylserine on the outer face of the plasma membrane During apoptosis, phosphatidylserine (PS) is transferred from the cytoplasmic face of the lipid bilayer to the extracellular face of the plasma membrance. An ATP-dependent scramblase moves PS to the outer leaflet of the plasma membrane. PS exposure functions in the recognition and the uptake of apoptotic cells by phagocytes. 29 Cell surface expression of phosphatidylserine during apoptosis early apoptosis late apoptosis apoptotic bodies Green: phosphatidylserine exposed on outer bilayer Phosphatidylserine on the surface of apoptotic bodies triggers engulfment by phagocytes Phagocyte Apoptotic bodies 30 Lipid rafts are cellular membrane microdomains Enriched with cholesterol, sphingolipids and phospholipids containing saturated fatty acids Ordered regions of membrane are distinct from the disordered non-raft regions of membranes Detergent-resistant as compared to detergent-sensitive non-raft regions Some proteins specifically partition to lipid rafts (e.g. proteins with lipid anchors or modifications) Function in protein trafficking, immune receptor signaling, virus entry and budding 31 HIV enters and exits cells at lipid raft microdomains HIV exits cells at lipid rafts HIV enters cells at lipid rafts 32 For review What is the difference between integral versus peripheral membrane proteins? What are the 3 general classes of membrane lipids? What are the structural features of phospholipids, sphingolipid and cholesterol? What makes them similar and different? What is the simplest phospholipid? What phospholipid is formed by 2 of these phospholipids? What are the functions and properties of different flippases? What are the biochemical properties of lipid rafts? What are the biochemical properties of lipid rafts? 33 Let’s take a break for 10 minutes Cholesterol Synthesis & Pharmacologic Regulation Carolyn K. Suzuki, Ph.D. [email protected] 35 Relevance and integration of lectures Cholesterol synthesis and regulation Cholesterol transport and uptake The biochemistry of cholesterol synthesis, elimination, transport and uptake is fundamentally relevant to managing and treating diseases linked to coronary artery disease, stroke, peripheral vascular disease, diabetes and high blood pressure. The biochemistry of cholesterol synthesis, transport and uptake is central to identifying new therapeutic targets for drug discovery, and developing new drugs for treating high cholesterol. Cholesterol in cellular membranes 37 Cholesterol is the precursor of steroid hormones, vitamin D and bile acids Cholesterol Steroid hormones (e.g., estrogen, glucocorticoids Vitamin D Bile acids (e.g. cholic acid) 38 Cholesterol 27 carbons all derived from acetate C-3 hydroxyl group C-17 side chain with 8 carbons cholesterol Sources in the body synthesized primarily in liver and intestine not required in diet intestinal uptake from diet hydrocarbon tail D C A steroid nucleus B Elimination converted into bile acids in liver stored in gall bladder, secreted into intestine small % excreted in feces, the remainder are re-absorbed in the intestine 21 Cholesterol esters esterification at C-3 with fatty acid primary form transported in plasma packaged in lipoprotein particles (e.g. LDL, HDL, focus of next lecture) 26 cholesterol ester C 3 A D B 39 fatty acid Cholesterol Endogenous de novo synthesis is primarily by liver (80%), also by intestine and other cells Exogenous uptake by intestine from diet, but no dietary requirement cytosol, mitochondria, peroxisomes cytosol, mitochondria, peroxisomes endoplasmic reticulum HMG CoA reductase- rate limiting enzyme, key drug target cytosol, mitochondria, peroxisomes peroxisomes Synthesizing 1 molecule of cholesterol requires ~36 ATPs endoplasmic reticulum, perroxisomes BottomlineCholesterol is synthesized when ATP is plentiful 40 Regulation of Cholesterol Synthesis intracellular cholesterol transcription of cholesterol synthesis proteins The transcription factor regulating many cholesterol synthesis genes is SREBP- sterol responsive element binding protein 41 Sterol-dependent regulation of cholesterol synthesis genes SREBPs- Sterol Regulatory Element Binding Proteins transmembrane proteins have a DNA binding domain have a SCAP interacting domain SCAP- SREBP Cleavage Activating Protein a transmembrane protein has a sterol sensing domain binds to SREBP in the ER when ER sterols are low, SCAP-SREBP move to the Golgi Protease 1 and Protease 2localized to the Golgi responsible for the two-step cleavage of SREBP resulting in soluble, cytosolic SREBP Mature, proteolytically-processed SREBP translocates from the Golgi to the nucleus activates the expression of cholesterol synthesis genes 42 ER Golgi nucleus 43 ER SCAP interacting domain SRE DNA binding domain sterol sensing domain cytosol ER membrane lumen SREBP SREBP SCAP SCAP ER step #1 Golgi when sterol levels are low SCAP and SREBP are transported to the Golgi 45 step #2 Golgi cytosol lumen Protease 1 SRE SREBP release Protease 2 SRE step #3 46 step #4 Golgi nucleus SREBP translocates SRE to nucleus SRE SRE SRE SRE SRE SRE transcriptional activation of sterol responsive element (SRE) controlled genes 47 When intracellular cholesterol is low ER Golgi nucleus SRE step #1 SCAP and SREBP translocate to the Golgi steps #2 and #3 SREBP is cleaved Protease 2 cytosol lumen Protease 1 step #4 cleaved SREBP translocates to nucleus activates genes mediating cholesterol synthesis Regulation of Cholesterol Synthesis (continued) 49 Regulation of Cholesterol Synthesis (continued) (AMP kinase) ATP AMP PP2A phosphatase cytosol ER lumen 50 Regulation of Cholesterol Synthesis (continued) glucagon inhibits HMGR expression and activity 51 Regulation of Cholesterol Synthesis (continued) glucagon inhibits HMGR expression and activity 52 Statins competitively inhibit HMGR Mevalonic acid Mevaldyl CoA HMG CoA CoASH Mevaldehyde NADPH NADPH Mevaldyl CoA Mevacor (Merck) Altocor (Canada) Pravachol (Bristol-Meyer Squibb) Lipitor (Park-Davis) Baycol (Bayer) Zocor (Merck) Lovastatin Lescol (Novartis) 53 Another drug mechanism for lowering blood cholesterol Zetia (ezetimibe) Mechanism of action acts at small intestine brush border inhibits absorption of cholesterol does not block absorption of fat-soluble vitamins or triglycerides advantage- does not enter the bloodstream, fewer side effects Vytorin (ezetimibe + simvastatin) Mechanism of action ezetimibe combined with simavastatin blocks cholesterol absorption in the intestine and cholesterol synthesis in the liver further reduces total cholesterol levels as compared to statin alone advantage- reduced cholesterol at lower doses of statins, which have side effects (e.g., muscle pain, liver damage) 54 Zetia combined with atorvastatin can decrease LDL-cholesterol at lower doses of the statin 55 Review- you tell me !!!! What is the transcription factor that activates the expression cholesterol synthesis genes? When cholesterol levels are low, where is SCAP-SREBP? What is the mechanism of action of statins? What is the mechanism of action of Zetia? What is the mechanism of action of Vytorin? 56 When intracellular cholesterol levels are low, is HMG CoA reductase phosphorylated or unphosphorylated? When is HMG CoA reductase likely to be degraded? What organ STORES bile acids and bile salts? What is the rate limiting step in bile acid synthesis? When is HMG CoA reductase likely to be degraded? 57 Let’s take a break for 10 minutes Lipoprotein Particle Transport and Uptake Carolyn K. Suzuki, PhD [email protected] Hawaii ~1930 60 Athersclerosis: Clinical significance LDL cholesterol and LDL receptor-mediated endocytosis Atherosclerosis, also referred to as hardening of the arteries, is caused by the buildup of “plaque” in the inner lining of arteries. Plaques are composed of deposited cholesterol, fatty substances, calcium, cellular waste products, and the protein fibrin that is involved in blood clotting. 61 Plaque build up in coronary artery Plaque build up in carotid artery \ coronary artery heart muscle dead heart muscle Over time, plaque causes narrowing of arteries, blocking blood flow Increases the risk for heart attack and stroke. 62 Signs of hyperlipidemia Normal Corneal arcus Cholesterol gallstones Xanthelasma cholesterol deposits around the eyes First Aid 2021 pages 94, 309, 715, 759 A. B. C. D. E. Cutaneous xanthomas Lipemic plasma (left), normal plasma (right) Lipemia retinalis Tuberous xanthomas, usually on extensor surfaces 63 Palmar crease xanthomas, associated with Type III Familial Hypercholesterolemia Lipoprotein particles LDL HDL Apo AI Cholesterol ester Triglyceride Cholesterol Apo B100 Phospholipid 64 Bad versus Good Diameter (nm) 1,000 70 40 20 10 65 Lipoprotein particles Lipoprotein particles- general characteristics and functions spherical particles with varying amounts of lipid and protein maintain solubility of constituent lipids transport of lipids in plasma throughout the body Major classes of lipoprotein particles Chylomicrons VLDLs very low-density lipoprotein particles intermediate density lipoprotein IDL low-density lipoprotein particles LDLs high-density lipoproteins HDLs Principal lipid components Triglycerides Cholesterol and cholesterol esters Phospholipids Principal protein components- Apolipoproteins Major forms are A, B, C and E (e.g., ApoAI, Apo B48, ApoB100, ApoCII, ApoE) Crucial for assembly and secretion of newly synthesized lipoprotein particles from cells Important for activating lipid-processing enzymes on endothelial cells and in plasma 66 Mediate uptake of lipoprotein particles into cells Chylomicrons Lippincott Illustrated Reviews: Biochemistry 8th Ed. (2022) https://bit.ly/3zO72Ns Figure 18.20 67 Major classes of lipoprotein particles 68 Uptake of exogenous cholesterol and other lipids Small intestine absorbs dietary triglycerides, cholesterol and cholesterol esters and assembles them into chylomicrons plasma before a fat-rich meal plasma after a fat-rich meal 69 Chylomicron metabolism begins in the small intestine (1) Chylomicrons are assembled in the intestine w/ Apo B48 Non-hepatic a cells (e.g., muscle, adipose, kidney, etc.) Dietary fat and cholesterol B Intestinal cell B B B B B B (2) Chylomicrons are released into lymph Liver chylomicron LPL HDL LDL LDL receptor 70 Chylomicron metabolism Non-hepatic peripheral cells (e.g., muscle, adipose, kidney, etc.) (1) Chylomicrons are assembled in the intestine w/ Apo B48 Dietary fat and cholesterol (3) Chylomicrons acquire Apo C-II and Apo E from HDL in plasma B Intestinal cell B B B B BCE BCE BCE B B CE (2) Chylomicrons are released into lymph B C B BCE EC E Liver chylomicron LPL HDL LDL LDL receptor 71 Chylomicron metabolism Non-hepatic endothelial cells (e.g., muscle, adipose, kidney, etc.) (1) Chylomicrons are assembled in the intestine w/ Apo B48 Dietary fat and cholesterol (3) Chylomicrons acquire Apo C-II and Apo E from HDL in plasma B Intestinal cell B B B B BCE BCE BCE B B (2) Chylomicrons are released into lymph FFA CE B C B BCE EC E FFA (4) Lipoprotein lipase (LPL) on endothelial cell surface hydrolyzes triglycerides, takes up fatty acids BC BC E E Liver chylomicron LPL HDL LDL LDL receptor 72 ApoCII activates lipoprotein lipase on the surface of endothelial cells Triglycerides are hydrolyzed to free fatty acids and glycerol Chylomicron ApoCII Lipoprotein lipase (LPL) non-hepatic endothelial cells Triglycerides (TG) Apo CII Apo B48 73 ApoCII activates lipoprotein lipase on the surface of endothelial cells Triglycerides are hydrolyzed to free fatty acids and glycerol Glycolysis Liver Gluconeogenesis Lipid synthesis Glycerol + Chylomicron ApoCII LPL Lipoprotein lipase (LPL) Free fatty acids non-hepatic endothelial cells free fatty acids are used for energy or stored in fat droplets Triglycerides (TG) Apo CII Apo B48 74 Chylomicron remnants are formed when triglycerides are depleted, transfer ApoCII back to HDL (4) Lipoprotein lipase (LPL) hydrolyzes triglycerides, and fatty acids are taken up by endothelial cells (1) Chylomicrons are assembled in the intestine w/ Apo B48 Dietary fat and cholesterol Intestinal cell chylomicron remnants chylomicron LPL HDL LDL LDL receptor (3) Chylomicrons acquire Apo C-II and Apo E from HDL in plasma BC E BC BC E E BCE BCE BCE BCE (5) Chylomicron remnants depleted of glycerol and FFA transfer Apo CII back to HDL (6) Remnants w/ ApoE and ApoB48, are taken up by the liver by binding to the ApoE receptor (also called LDL receptor, see slides 23-26) BC E BCE E C E EE E C E E C 75 VLDL and LDL metabolism starts in the liver (1) Assembly and export of nascent VLDL w/ ApoB100 LIVER non-hepatic tissue B100 non-hepatic tissue LPL HDL IDL LDL VLDL LDL receptor 76 VLDL and LDL metabolism starts in the liver (1) Assembly and export of nascent VLDL w/ ApoB100 LIVER (2) VLDL acquires ApoC-II and ApoE from HDL B100 C E CE CE B CE B non-hepatic tissue C E B non-hepatic tissue LPL HDL IDL LDL VLDL LDL receptor 77 VLDL and LDL metabolism starts in the liver (1) Assembly and export of nascent VLDL w/ ApoB100 LIVER (2) VLDL acquires ApoC-II and ApoE from HDL B100 C E CE CE B CE B C E B non-hepatic tissue C E B (3) Lipoprotein lipase (LPL) on endothelial cell surface hydrolyzes triglycerides C E B non-hepatic tissue LPL HDL IDL LDL VLDL LDL receptor 78 VLDL and LDL metabolism starts in the liver (1) Assembly and export of nascent VLDL w/ ApoB100 LIVER (2) VLDL acquires ApoC-II and ApoE from HDL B100 C E CE CE B CE B C E B non-hepatic tissue C E B CE B (4) Apo C-II and Apo-E on VLDL transfer back to HDL resulting in IDL (5) IDL triglycerides are further removed thereby forming LDL LPL HDL IDL LDL VLDL LDL receptor (3) Lipoprotein lipase (LPL) on endothelial cell surface hydrolyzes triglycerides B C E B CE B B B B B non-hepatic tissue 79 VLDL and LDL metabolism starts in the liver (1) Assembly and export of nascent VLDL w/ ApoB100 LIVER (2) VLDL acquires ApoC-II and ApoE from HDL B100 C E CE CE B CE B C E B non-hepatic tissue C E B CE B (4) Apo C-II and Apo-E on VLDL transfer back to HDL resulting in IDL (5) IDL triglycerides are further removed thereby forming LDL LPL HDL IDL LDL VLDL LDL receptor (3) Lipoprotein lipase (LPL) on endothelial cell surface hydrolyzes triglycerides B C E B CE B B B B B (6) LDL binds non-hepatic LDL receptors tissue on cells increasing intracellular cholesterol 80 VLDL and LDL metabolism starts in the liver (1) Assembly and export of nascent VLDL w/ ApoB100 (2) VLDL acquires ApoC-II and ApoE from HDL LIVER B100 C E CE B non-hepatic tissue C E B C E B CE B B B (5) IDL triglycerides are further removed thereby forming LDL B B B LPL HDL IDL LDL VLDL LDL receptor CE B (4) Apo C-II and Apo-E on VLDL transfer back to HDL resulting in IDL B (7) LDL and HDL bind their respective receptors on liver and are taken up CE B (3) Lipoprotein lipase (LPL) on endothelial cell surface hydrolyzes triglycerides B B B C E B CE B B B B B (6) LDL binds non-hepatic LDL receptors tissue on cells increasing intracellular cholesterol B 81 LDL receptor (LDLR) Binds ApoB100 on VLDLs, IDLs, LDLs Binds ApoE on chylomicron remnants and IDLs Mediates uptake of particles into cells by “receptor mediated endocytosis” Intracellular LDL particles are broken down and cholesterol esters are stored or used Increased intracellular cholesterol down-regulates cholesterol synthesis LDL ligand binding domain EGF precursor homology domain Transmembrane domain Cytosolic domain 82 Receptor-mediated endocytosis of LDL Lippincott Illustrated Reviews: Biochemistry 8th Ed. (2022) Fatty Acid and Triacylglycerol Metabolism https://bit.ly/3zO72Ns 83 Receptor-mediated endocytosis of LDL LDL particle ApoB100 endocytosis of LDL bound receptor into cell LDL receptors recycle to plasma membrane 1 2 LDL dissociates from receptor in endosome 5 3 4 84 endosome endosome fuses w/ lysosome lysosome breaks down LDL amino acids fatty acids Excess cholesterol is stored as cholesterol esters in lipid droplets by the enzymes: LCAT in liver cells ACAT in non-hepatic cells cholesterol ACATACAT or LCAT STORAGE OF CHOLESTEROL ESTERS 85 Immunotherapy for lowering LDL cholesterol Prescribed: When statins, ezetimbe and other approaches fail to lower LDL cholesterol For individuals who experience severe/intolerable side effects of statins Individuals must have normal LDL receptor function Mechanism: Antibody targeting a protein called PCSK9, which is secreted from cells and binds to the LDL receptor at the EGFR precursor homology domain (not LDL binding domain) FDA approved in 2015 86 Normal function of PCSK9 PCSK9 is a normal human protein that binds the LDL receptor and stimulates LDL receptor endocytosis plasma membrane endosomes Endosomes containing the LDL receptor fuse with lysosomes, resulting in the degradation of the LDL receptor in lysosomes Result: less LDL rceptor on surface, more LDL particles in blood 87 Anti-PCSK9 antibody antibody binds PCKS9 and prevents lysosomal degradation of LDLR PCSK9 bound to antibody Outcome: increased LDLR on the cell surface, increased LDL particls uptake and increased LDLR recycling to the surface, reducing LDL cholesterol in blood 88 Review- you tell me !!!! Which lipoprotein particle has the highest percentage of triglycerides & cholesterol esters? Where are chylomicrons, VLDL and LDL particles formed? List 4 properties that distinguish chylomicrons, LDLs and HDLs Non-functional LDL receptors result in: Lower or higher plasma levels of cholesterol? Lower or higher intracellular levels of cholesterol? Do anti-PCSK9 antibodies increase or decrease the levels of LDL receptor at the plasma membrane? Why? 89