Lecture 1 & 2: Introduction & Endoplasmic Reticulum & Golgi (PDF)

Lecture 1: Introduction & endoplasmic reticulum Prof. Mamoun Ahram School of Medicine Second year, First semester, 2024-2025 Me! Prof. Mamoun Ahram Office location: first floor, School of Medicine, Main building Office hours: By appointment; Tuesday 12-2 Come in groups  Course outline (1)...

Lecture 1: Introduction & endoplasmic reticulum Prof. Mamoun Ahram School of Medicine Second year, First semester, 2024-2025 Me! Prof. Mamoun Ahram Office location: first floor, School of Medicine, Main building Office hours: By appointment; Tuesday 12-2 Come in groups  Course outline (1) Introduction and biomembranes Endoplasmic reticulum and protein sorting Golgi apparatus Vesicular network Mitochondria and mitochondrial diseases Focus on Peroxisomes diseases The nucleus Cytoskeletal networks The extracellular network Cell signaling, proliferation, differentiation, and death Cancer cells  Course outline (2) Introduction and the central dogma of molecular biology Gel electrophoresis, restriction endonucleases, recombinant DNA technology, DNA cloning, and RFLP The utilization of denaturation/renaturation concepts Dot blotting and Southern blotting DNA replication Focus on PCR and DNA sequencing processes The human genome and Transcription, mechanisms of regulation, and epigenetics techniques Coding and non-coding RNAs RNA detection, quantification, and detection Translation Yeast two-hybrid system DNA mutations DNA repair and CRISPR-Cas9 The textbook The Cell: A Molecular Approach 8th Edition by Geoffrey Cooper, Sinauer Associates is an imprint of Oxford University Press. The cell What organisms do we use to study cells? Escherichia coli (E. coli) Yeast (Saccharomyces cerevisiae) Caenorhabditis elegans Drosophila melanogaster Mice Cultured cells and tissues Organelles Major molecular components of cells Nucleic acids Carbohydrates Proteins Lipids (50% of mass of plasma membranes, 30% of mitochondrial membranes) Molecules function by interacting with each non-covalently. How do we study cell components? Cell and protein detection Microscopy Light, fluorescence (immunofluorescence), electron, scanning electron Cell fractionation Biochemical composition of plasma membranes Lipid composition of organelles Cholesterol is an essential component of animal plasma membranes. It is not present in bacteria and plant cells, but the latter cells contain sterols.  Composition and properties of plasma membranes The phospholipids are asymmetrically distributed between the two halves of the membrane bilayer. The outer leaflet: choline, sphingomyelin The inner leaflet:ethanolamine, serine, inositol (minor) inositol has a role in cell signaling. Glycolipids are found exclusively on the outer membrane. Lipid rafts Specialized membrane regions with clusters of cholesterol and the sphingolipids (sphingomyelin and glycolipids). Rafts are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, and proteins involved in signal transduction and intracellular vesicular trafficking (transport). Caveolae (Latin for “little caves”) They are a subset of lipid rafts that require cholesterol for their formation. They are formed the membrane protein caveolin, which interacts with cholesterol and the cytoplasmic protein cavin. They are important for several cellular activities, including endocytosis, cell signaling, regulation of lipid transport, and protection of the plasma membrane against mechanical stress. Membrane proteins Types of membrane proteins Peripheral membrane proteins are indirectly and loosely associated with membranes through protein-protein interactions, mainly ionic bonds. Integral membrane proteins have some of their helical parts inserted into the lipid bilayer. Single-pass (type I or II) or multi-pass proteins. Lipid-anchored membrane proteins (myristoylation, palmitoylation , glycosyl- phosphatidylinositol)  Protein mobility Proteins and lipids are able to diffuse laterally through the membrane. The mobility of membrane proteins is restricted by Their association with the cytoskeleton Specific membrane domains, which maintain the specific distribution of apical and basolateral proteins Specific lipid composition (e.g. lipid rafts). Glycocalyx The surface of the cell is covered by a carbohydrate coat, known as the glycocalyx, formed by the oligosaccharides of glycolipids and glycoproteins. Functions: Cell-cell interactions such as immune cells Protection of cell surface from ionic and mechanical stress Formation of a barrier for microorganisms An overview Endoplasmic reticulum (ER) It is a network of membrane-enclosed tubules and sacs (cisternae) that extends from the nuclear membrane throughout the cytoplasm. It is the largest organelle of most eukaryotic cells. The rough ER: covered by ribosomes on its outer surface and functions in protein processing. The smooth ER: lipid metabolism Transitional ER: exit of vesicles to Golgi apparatus  Protein sorting Proteins containing signal sequences are synthesized on membrane-bound ribosomes and translocated directly into the ER. These proteins may stay within the ER Proteins synthesized on free or transported to nuclear membranes, ribosomes either remain in the peroxisomal membranes, or the Golgi cytosol or are transported to the apparatus and, from there, to nucleus, mitochondria, or endosomes, lysosomes, the plasma peroxisomes. membrane, or outside the cell via secretory vesicles. In cell biology, a lumen is a membrane-defined space that is found inside several organelles, cellular components, or structures Signal sequence: a short sequence of amino acids of the polypeptide at the amino terminus. It is then cleaved from the polypeptide chain during its transfer into the ER lumen.  The signal sequence is recognized as the protein is synthesized and the ribosome is transported to the surface of the RER Translation resumes on the surface of RER, the peptide simultaneously translocates into the ER through the translocon, and the signal peptide is cleaved by signal peptidase The completed polypeptide chain is released within the ER lumen Pathways of protein sorting Secretory, ER, Golgi apparatus, and lysosomal proteins are released into the lumen of the ER. Membranous proteins are initially inserted into the ER membrane. Considerations Single vs. multiple membrane-spanning region Orientation of N- and C-termini The lumens of the ER and Golgi apparatus are topologically equivalent to the exterior of the cell.  Insertion of membrane proteins via internal transmembrane sequences Translocation of the polypeptide chain stops when the translocon recognizes a transmembrane sequence allowing the protein to become anchored in the ER Transmembrane membrane. Sequence The direction of the internal transmembrane sequence determines the direction of insertion and orientation of the protein ends. Transmembrane Sequence Multi-transmembrane domain proteins have multiple transmembrane sequences Once inside the ER, proteins are Chaperones Folded (with the help of chaperones) Complexed (quaternary structure) Disulfide bond formation by protein disulfide isomerase Glycosylated Anchored by lipids Sugars Protein folding and ER-associated degradation (ERAD) If correctly folded, proteins move on. If misfolded, proteins are sent to the cytosol, ubiquitylated (addition of small proteins called ubiquitins), and degraded in the proteasome.  Synthesis of phospholipids in ER The smooth ER is the major site of synthesis of: Membrane glycerophospholipids, which are then transported from the SER to other membranes. Sphingophospholipids (like ceramides and glycolipids) and steroids. Large amounts of smooth ER are found in steroid- producing cells, such as those in the testis and ovary. SER is abundant in the liver, which contains enzymes that metabolize various lipid- soluble compounds. ER-Golgi intermediate compartment (ERGIC) Proteins and lipids are carried from the ER to the Golgi in transport vesicles, which fuse with the ER– Golgi intermediate compartment (ERGIC), and are then carried to the Golgi. Retention of ER protein Many proteins with KDEL sequence (Lys-Asp-Glu-Leu) at C-terminus are retained in the ER lumen. If the sequence is deleted, the protein is transported to the Golgi and secreted from the cell. Addition of the sequence causes a protein to be retained in the ER. Lecture 2: Golgi apparatus and vesicular transport Prof. Mamoun Ahram School of Medicine Second year, First semester, 2024-2025 Functions of the Golgi apparatus Further protein processing and modification Further protein sorting Synthesis of glycolipids and sphingomyelin  Structure of the Golgi The Golgi apparatus consists of a stack of flattened sacs (cisternae) of four regions: cis, medial, and trans compartments and the trans-Golgi network. Proteins are carried through the Golgi apparatus in the cis-to-trans direction. Transport vesicles carry the Golgi proteins back to earlier compartments for reuse.  Processing of N-linked oligosaccharides in Golgi Proteins can also be modified by the addition of carbohydrates to the hydroxyl side chains of The N-linked oligosaccharides, which serine and threonine are added to asparagine residues of residues, hence called O- glycoproteins and transported from the linked sugars. ER, are further modified enzymatically in different compartments of the Golgi. Lipid and Polysaccharide Metabolism in the Golgi Ceramide is converted either to sphingomyelin (a phospholipid) or to glycolipids in the Golgi apparatus. Ceramide is synthesized in the ER  Protein Sorting and export In contrast to the ER, all of the proteins retained within the Golgi complex are associated with the Golgi membrane rather than being soluble proteins within the lumen Protein processing in Immature secretory vesicles Proteins can be targeted to late endosomes, which develop into lysosomes Regulated secretion after signaling from specialized vesicles Continuous, unregulated secretion Transport to the plasma membrane of polarized cells Proteins are selectively packaged into transport vesicles from the trans-Golgi or recycling endosomes. Targeting is determined by special sequences (basolateral) or GPI sugar modification (apical). Processing of lumenal lysosomal proteins Protein destined to lysosomes have a signal patch (a three- dimensional structural determinant), which is recognized by modifying enzymes that add mannose-6- phosphate to the proteins. Lumenal lysosomal proteins bind to a mannose- 6-phospahte receptor and are transported to late endosome, which mature into lysosomes. Formation and fusion of a transport vesicle Membrane proteins and lumenal secretory proteins with their receptors are grouped on the Golgi membrane before budding of a transport vesicle coated by a protein called clathrin. The clathrin-coated vesicle is then docks at its target membrane, gets uncoated, and fuses with the membrane. Delivery of vesicles: targeting and fusion Small G proteins called Rab determine the membrane targets of vesicles. There are over 60 Rab proteins where different combinations of these proteins mark different transport vesicles. v-SNAREs-t-SNAREs proteins are responsible for vesicular fusion with the target membranes.  The mechanism of fusion Rab protein binds to a tethering factor associated Closer with the target membrane. Interaction of SNAREs on the vesicle and vesicle-target effector proteins target membranes complex Fusion together. The SNAREs zip together, bringing the vesicle and target membranes into Tethering, close proximity, and the hydrolysis of GTP, Disassembly membranes fuse SNARE interactions of SNARE complex Griscelli syndrome (GS) A rare genetic condition Mutations in MYO5A (a motor protein), RAB27A and MLPH (a Rab effector protein) genes that encode the MyoVA-Rab27a-Mlph protein complex that function in melanosome transport and fusion. Pigmentary dilution of the skin, silver-grey hair, melanin clumps within hair shafts Structure Lysosomes are membrane-enclosed organelles that contain various enzymes that break down all types of biological macromolecules. Lysosomes degrade material taken up from outside and inside the cell. Lysosomal enzymes Lysosomes contain ~60 different acid hydrolases. The enzymes are active at the acidic pH (about 5) that is maintained within lysosomes. Levels of cell protection from these hydrolases: Containment Inactive if released A proton pump maintains the lysosomal pH. Lysosomal storage diseases Glycolipidoses (sphingolipidoses) Oligosaccharidoses Mucopolysaccharidoses: deficiencies in lysosomal hydrolases of glycosaminoglycans (heparan, keratan and dermatan sulfates, chondroitin sulfates. They are chronic progressively debilitating disorders that lead to severe psychomotor retardation and premature death. Glucocerebroside Glucocerebroside is a glycosphingolipids (a monosaccharide attached directly to a ceramide unit (a lipid) It is a byproduct of the normal recycling of red blood cells during, which are phagocytosed by macrophages, degraded and their contents recycled to make new cells. I-cell disease also called mucolipidosis IIA, or mucolipidosis II alpha/beta: ML-IIα/β Defective targeting of lysosomal enzymes from Golgi to the lysosomes A deficiency in tagging enzyme that phosphorylates mannose Features: severe psychomotor retardation that rapidly progresses leading to death between 5 and 8 years of age. Endocytosis Molecules are taken up from outside the cell in endocytic vesicles, which fuse with early endosomes. Early endosomes mature into late endosomes. Transport vesicles carrying acid hydrolases from the Golgi fuse with late endosomes, which mature into lysosomes. Note: the pH in endosomes is 6.0-6.5. Clathrin-dependent endocytosis Receptor-mediated endocytosis Ligands bind to their receptors stimulating endocytosis. In early endosomes, the acidic pH causes the release of ligands from their receptors. Membrane receptors are recycled via recycling endosomes and early endosomes mature into late endosomes. Transport vesicles carrying acid hydrolases from the Golgi fuse with late endosomes, which mature into lysosomes. Example: removal of plasma cholesterol by low-density lipoprotein (LDL) receptor  Phagocytosis Binding of a bacterium to the cell surface stimulates the extension of a pseudopodium, which eventually engulfs the bacterium. Fusion of the pseudopodium membranes then results in formation of a large intracellular vesicle (a phagosome). The phagosome fuses with lysosomes to form a phagolysosome within which the ingested bacterium is digested. Macropinocytosis (clathrin-independent) is cell drinking via the formation of small vesicles. A pseudopodium is a temporary arm-like projection of a eukaryotic cell membrane Autophagy (self-eating) Regions of the cytoplasm or internal organelles (such as mitochondria) are enclosed by membranes derived from the endoplasmic reticulum, forming autophagosomes. Autophagosomes fuse with lysosomes to form large phagolysosomes in which their contents are digested. Purpose: removal of damaged organelles; survival during starvation; tissue remodeling during development Lecture 3: bioenergetics and metabolism (mitochondria and peroxisomes) Prof. Mamoun Ahram School of Medicine Second year, First semester, 2024-2025 What are the mitochondria? Mitochondria are thought to have evolved from bacteria via endosymbiosis. They play a critical role in the generation of metabolic energy in eukaryotic cells Generation of ATP from the breakdown of carbohydrates and fatty acids They contain their own DNA, which encodes tRNAs, rRNAs, and 13 mitochondrial proteins. But most mitochondrial proteins (~1500) are encoded by the nuclear genome. Most mitochondrial proteins are translated on free cytosolic ribosomes and imported into the organelle. Structure Outer membrane permeable to small molecules (~1000 Da) because of porins (channel proteins) Inner membrane contains a high percentage (>70%) of proteins Forms folds (cristae) to increase surface area Function; oxidative phosphorylation, ATP generation, transport of metabolites impermeable to most ions and small molecules Intermembrane space Composition is similar to the cytosol Matrix contains the mitochondrial genetic system and the enzymes responsible for the Krebs cycle Properties and features They are located in cells requiring high-energy use such as synapses. They are dynamic (fusion and division) Exchange genetic material Regulate autophagy Cell survival The Genetic System of Mitochondria Mitochondrial DNA (~16 Kb) is circular and exists in multiple copies per organelle. It encodes 13 proteins involved in electron transport and oxidative phosphorylation, rRNAs, and tRNAs. The oocytes are the main source of the mitochondria, meaning that mutations in the mitochondrial DNA are inherited from the mother. Mitochondrial proteins The nuclear genome encodes for most mitochondrial proteins including those required for DNA replication, transcription, translation, oxidative phosphorylation, and enzymes for mitochondrial metabolism. The proteins encoded by these genes (~99% of mitochondrial proteins) are synthesized on free cytosolic ribosomes and imported into the mitochondria as completed polypeptide chains.  Protein Import and Mitochondrial Assembly Proteins are targeted to the Tom complex in the mitochondrial outer membrane by N- terminal presequences. The protein passes through a channel ** (translocase) called the Tom complex on the Positively charged outer membrane followed by passing Amphipathic -helix through another channel (translocase) called the Tim complex in the inner membrane. The presequence is then removed and protein folding is completed. Some proteins with transmembrane domains exit The inner membrane channel laterally into the inner membrane. Targeting of inner membrane proteins Some inner membrane proteins encoded by the mitochondrial genome are inserted via Oxa translocase. Mitochondrial phospholipids Phosphatidyl… Phosphatidylcholine and phosphatidylethanolamine are synthesized in the ER and carried to mitochondria by proteins. Phosphatidylserine can then be synthesized from phosphatidylethanolamine in the mitochondria. Mitochondrial phospholipids Cardiolipin The unusual phospholipid, cardiolipin, which contains four fatty acid chains, is also synthesized in the mitochondria. This molecule improves the efficiency of oxidative phosphorylation by restricting proton flow across the membrane. General information The mammalian oocyte contains around 105-108 mitochondria and each one contains 2-10 copies of mitochondrial DNA, which are mainly inherited from the mother. If the mitochondrial genomes carry deleterious mutation, the embryo/fetus would generally not survive. Some mothers carry a mixed population of both mutant and normal mitochondrial genomes. Daughters and sons can inherit this mixture of normal and mutant mitochondrial DNAs and look healthy. In cases of mitochondrial defects, muscle and nervous tissues are most at risk, because of their need for particularly large amounts of ATP Mitochondria diseases can be classified according to their cause: genetic or biochemical. http://www.ncbi.nlm.nih.gov/books/NBK27914/ Defects of mitochondrial transport interfere with the movement of molecules across the inner mitochondrial membrane, which is tightly regulated by specific translocation systems. Substrate utilization Pyruvate dehydrogenase (PDH) deficiency can cause alterations of pyruvate metabolism. The PDH complex (PDHC) catalyzes the irreversible conversion of pyruvate to acetyl-CoA. The most devastating phenotype of PDH deficiency presents in the newborn period. The majority of patients are male with severe metabolic acidosis, elevated lactate in blood or CSF, and associated elevations of pyruvate and alanine. Defects of the Krebs cycle Fumarase deficiency is reported in patients having mitochondrial encephalomyopathy. Features: excretion of large amounts of fumarate and, to a lesser extent, succinate in the urine. Abnormalities of the respiratory chain reaction Defect in any of the 4 electron chain complexes have been reported. Defects of oxidation-phosphorylation coupling The best-known example of such a defect is Luft's disease, or nonthyroidal hypermetabolism. Respiratory rate is at maximal rate even in the absence of ADP, an indication that respiratory control is lost. Respiration proceeds at a high rate independently of phosphorylation, and energy is lost as heat, causing hypermetabolism and hyperthermia. Defects of mitochondrial DNA (mtDNA) These disorders are associated with dysfunction of the respiratory chain because all 13 subunits encoded by mtDNA are subunits of respiratory chain complexes. Diseases due to point mutations are transmitted by maternal inheritance. MERRF and others One main syndrome is myoclonic epilepsy and ragged red fiber disease (MERRF), which can be caused by a mutation in one of the mitochondrial transfer RNA genes required for the synthesis of the mitochondrial proteins responsible for electron transport and production of ATP. Other syndromes include Lactic acidosis and stroke-like episodes (MELAS) Leber's hereditary optic neuropathy (LHON), Neurogenic atrophy, ataxia and retinitis pigmentosa (NARP)  Leber's hereditary optic neuropathy (LHON) Females (10%) are affected less frequently than males (50%), but males never transmit LHON to their offspring and not all individuals with mutations develop the disease. Inheritance is mitochondrial (cytoplasmic) not nuclear. The mutations reduce the efficiency of oxidative phosphorylation and ATP generation. A rare inherited disease that results in blindness because of degeneration of the optic nerve. Vision loss is only manifestation, occurs between 15-35. Defects of nuclear DNA The vast majority of mitochondrial proteins are encoded by nuclear DNA. All areas of mitochondrial metabolism can be affected. Mitochondrial Replacement Therapy The British-developed technique was performed in Mexico by a Chinese-American physician who worked in New York Structural features of peroxisomes Small, membrane-enclosed organelles They contain enzymes involved in a variety of metabolic reactions, including energy metabolism. They replicate by division. Most human cells contain 500 peroxisomes. Peroxins Peroxisomal proteins are called peroxins. There are 85 genes that encode peroxins, most of which are metabolic enzymes. Internal proteins are synthesized on free ribosomes and then imported into peroxisomes. Other membrane proteins act as receptors for the import of internal proteins. Function of peroxisomes Peroxisomes carry out oxidation reactions producing hydrogen peroxide, which is harmful to the cell But peroxisomes contain the enzyme catalase that concerts it to water and oxygen Substrates like uric acid, amino acids, and fatty acids are broken down by oxidative reactions in peroxisomes. Fatty acids are oxidized in both peroxisomes and mitochondria. Synthesis in peroxisomes Cholesterol Bile acids (liver) Plasmalogens important in membranes of the heart and brain Assembly of peroxisomes Peroxisomal transmembrane proteins are derived from the ER. A functional peroxisome is formed from carrying distinct transmembrane proteins. The internal peroxisomal proteins synthesized on cytosolic ribosomes can then be imported. Peroxisomal matrix proteins contain a targeting signal called PTS1. Different peroxins help in translocating the peroxisomal proteins into the peroxisomes by functioning in carrying and importing them. Formation of new peroxisomes New peroxisomes can be formed de novo by two mechanisms: The fusion of vesicles budding from the ER followed by the import of cytosolic proteins. New peroxisomes can be formed by the growth and division of old ones. Peroxisomal diseases Single peroxisomal enzyme deficiencies Defective specific peroxisomal enzymes Peroxisomal biogenesis disorders (PBDs). Mutations of PEX genes leading to deficiencies of multiple peroxisomal enzymes Example: Zellweger syndrome Lethal Due to mutations in at least 10 genes X-linked adrenoleukodystrophy (XALD). Defective transport of very long-chain fatty acid (VLCFA) across the peroxisomal membrane. 0