Lecture 1: Introduction & Endoplasmic Reticulum 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.