BENG0004 Lecture 21.pdf

BENG0004 Biochemistry for Biochemical Engineers Jack Jeffries Lecture 21 Cell Biology  What is Cell Biology? The organisation and interaction of proteins, lipids, DNA and other components into bigger structures or more...

BENG0004 Biochemistry for Biochemical Engineers Jack Jeffries Lecture 21 Cell Biology  What is Cell Biology? The organisation and interaction of proteins, lipids, DNA and other components into bigger structures or more complex networks. Also the organisation of macromolecular structures (more than one protein subunit or component) into complexes that then do new or more complex things e.g.Pyruvate dehydrogenase complex For example: a protein, which on its own might have a property we can assay and a structure we can determine, can associate with itself or with others proteins to form macromolecular (big) complexes that now do quite different things e.g.. the formation of actin/myosin complexes which allow muscles and cells to move.  What is Cell Biology? Cell Biology is the study of these complexes at all the different levels of complexity and organisation - from the components they are made up of, to the macromolecular structures they make, to the organelles and sub-cellular structures they make, to the level of whole cell movement, activity and interactions. In one definition Cell Biology is how the components build the cell. How the individual elements make the living organism Cells interact with each other – e.g Bacteria can transfer DNA from one cell to another, eukaryotic cells can associate into multicellular structures called tissues and finally into whole organisms- metazoans.  Cell biology of Bacterial and Eukaryotic cells The differing cell biology of bacterial and eukaryotic cells has impacts on the process of life undertaken in these cell types mRNA is made in the nucleus but protein synthesis occurs in the cytoplasm. Necessitates transport of mRNA put of the nucleus In an analogous manner many proteins need to be transported to the final place where they are needed e.g. the mitochondrion, the nucleus, the endoplasmic reticulum, peroxisomes and secreted to the outside of the cell. Many therapeutic proteins such as immunoglobulins (IgG) are fully secreted to the outside of the cell. To get to these different destinations the proteins are made with signal sequences on the N- or C-terminus. The mitochondrial signal sequence (or targeting sequence) The first 18 amino acids of the precursor to subunit IV of Cytochrome oxidase serve as a signal sequence for import of the subunit into the mitochondrion. When the signal sequence is folded as an α helix, the positively charged residues (red) are seen to be clustered on one face of the helix, while the nonpolar residues (yellow) are clustered primarily on the opposite face. Mitochondrial matrix-targeting sequences always have the potential to form such an amphipathic α helix, which is recognized by specific receptor proteins on the mitochondrial surface. A mitochondrial signal peptidase removes the signal sequence during import.  Anatomy of a signal sequence full secretion to the external environment Signal peptide cleavage site  M A L K S L V L L S L L V L V L L L V R G Q P S L G K E T A Positive Hydrophobic region bend charge An integral membrane protein called the signal peptidase cuts off the N-terminal signal sequence from a secreted protein as it crosses the membrane. In bacterial cells this membrane is the cytoplasmic membrane. In mammalian cells it is the rough endoplasmic reticulum. Co-translational secretion. In bacterial cells a secreted protein is outside the cell once its secreted  Secretory pathway diagram, including nucleus, endoplasmic reticulum and Golgi apparatus. 1 Nuclear membrane 2 Nuclear pore 3 Rough endoplasmic reticulum (REM) 4 Smooth endoplasmic reticulum 5 Ribosome attached to REM 6 Macromolecules 7 Transport vesicles 8 Golgi apparatus 9 Cis face of Golgi apparatus 10 Trans face of Golgi apparatus 11 Cisternae of Golgi apparatus 12 Secretory vesicle 13 Cell membrane 14 Fused secretory vesicle releasing contents 15 Cell cytoplasm 16 Extracellular environment Proteins that have an N-terminal signal sequence for external secretion are made on the rough ER and get glycosylated in the rough ER and in the Golgi apparatus There is a huge amount of intracellular trafficking in eukaryotic cells. Proteins in membrane vesicles are constantly being actively moved to new locations, fused with other structures and moved again.  Protein secretion in Eukaryotic/ Mammalian cells ER = endoplasmic reticulum; GC = Golgi complex; TGN = trans golgi network; PM = plasma membrane Glycosylation will happen to proteins when they are secreted into the lumen of the ER and further glycosylation steps happen in the Golgi So, a protein destined for external secretion e.g. an IgG molecule has a long way to go before it reaches the outside of the cell.  Cell biology of bacteria Bacteria: The two major groups of bacteria are defined by a staining procedure – the Gram stain. Gram positive Gram negative Named after Christian Gram a Danish physician in 1884. It divides the bacteria into two main groups – those that take up the stain are Gram positive those that don’t are Gram negative. This is because of the structure of their cell envelope.  A representative Gram negative cell wall Cross section through the wall of a typical Gram-negative bacterium. The wall is a multilayered structure consisting of peptidoglycan (thinner than in Gram-positive bacteria) and an outer membrane with LPS (lipopolysaccharide).  The Gram negative cell wall There are two membranes. The outer membrane has unusual lipids in the outer leaflet - lipopolysaccharide. The peptidoglycan is also called murein and is the rigid cell wall that gives the bacterium osmotic strength and contributes to the overall shape. The lipoprotein is a very abundant, small protein which links the peptidoglycan to the outer membrane. To allow solutes (food molecules) through the outer membrane there are very abundant pores proteins call porins. The space between the inner (cytoplasmic) membrane and the outer membrane is called the periplasm or periplasmic space Peptidoglycan [murein] the rigid cell wall around all bacteria. The lipopolysaccharide (LPS) of a Gram negative bacterium There are 5 lipid chains on each LPS molecule. The core polysaccharide is very constant in a species and across genera. The O antigenic side chain is very changeable and leads to a major form of antigenic variation. Also called endotoxin. In infections by Gram negative bacteria the LPS can be much more toxic than other secreted toxins. Very small amount of LPS in the blood can lead to anaphylactic shock and can be lethal. A more realistic representation of a Gram negative cell wall  A representative Gram positive cell wall The peptidoglycan layer is much thicker than in Gram negative bacteria. There are other polymers such as Teichoic acid. No outer membrane. No need for porin type proteins as solutes can diffuse through the cell wall. There are usually proteins that are attached to the peptidoglycan through a covalent link and extend a long way into the environment. In pathogens (S. aureus) these are a cause of antigenic variation and evasion of the host immune response. Gram-positive cells have a much thicker peptidoglycan layer than Gram-negative cells. Gram-negative cells have an outer membrane. Functions of Prokaryotic Structures Plasma Membrane-Selectively permeable barrier, mechanical boundary of cell, nutrient and waste transport, location of many metabolic processes (respiration, photosynthesis, etc), detection of environmental cues for chemotaxis. Mesosome-Possibly cross-wall formation and the distribution of chromosomes during division. Gas vacuole-Buoyancy in bacteria for floating in aquatic environments Inclusion bodies-Storage of carbon, phosphate and other substances Periplasmic space-(in Gram negative bacteria) Contains hydrolytic enzymes and binding proteins for nutrient processing and uptake Cell wall-Gives bacteria shape and protection from lysis in dilute solutions Capsules and slime layers Resistance to phagocytosis, adherence to surfaces Fimbriae and pili-Attachment to surfaces, bacterial Mating Flagella-Movement  How do bacteria move? They can swim. They can glide over a solid surface. They can actively grow into things (penetrate) as filaments.  Bacterial movement: swimming with flagella Flagella can be single, polar as in (A), multiple polar as in (B) or all over as in (C)  Bacterial swimming with flagella Bacterium Forward movement Flagellum Tumble The surrounding water is more viscous than molasses to objects of bacterial size. The flagella rotates one way and the bacterium is propelled forwards. If the flagella rotates the other way the bacteria just tumbles. The flagella can push the bacterium at 20-90 mm/second. This is equivalent to 2 to 100 cell lengths per sec. Bacteria do not swim aimlessly. They are attracted to nutrients such as amino acids and sugars and repelled by many harmful substances and waste products. If a bacterium encounters an increasing concentration gradient of an attractant it continues swimming (the flagella continues rotating to push it forward), if a repelling substance is detected the flagella reverses and the bacterium tumbles.  Run and tumble When the flagella starts rotating again in the forward movement direction the cell can be facing in any direction. If it is now swimming up the concentration gradient of an attractant the flagella will keep rotating. If it happens to be now swimming down the concentration gradient the flagella reverses again, the bacterium tumbles for a bit and then resumes swimming. By this run and tumble mode of locomotion bacteria which are motile can easily migrate towards attractants (food) and move away from nasty chemicals. Tumble Run Increasing gradient of an attractant  Pili Pili are hair-like structures on the surface of bacteria. There are several types in E. coli from F pili small ones to the larger mating pili. The cell at the bottom of the picture is showing both the thin Type I pili which may be involved in attachment of E. coli to mammalian cells, and a much larger F plasmid mating pili. Type I pili Key Points to know Mechanisms of protein targeting for secretion and the pathways Structure of Gram Negative and Gram positive cell walls – Chemical composition etc How Bacterial cells move