MCDB 3145 Cell Biology PDF

MCDB 3145 Cell Biology Part I. Chemical Basis of Cell & Protein Folding Two basic classes of cells, prokaryotic & eukaryotic; 1) are distinguished by their size and the types of organelles. 2) share an identical genetic language, a common set of metabolic pathways, and many common struct...

MCDB 3145 Cell Biology Part I. Chemical Basis of Cell & Protein Folding Two basic classes of cells, prokaryotic & eukaryotic; 1) are distinguished by their size and the types of organelles. 2) share an identical genetic language, a common set of metabolic pathways, and many common structural features. © 2016 John Wiley & Sons, Inc. All rights reserved. Bacterial cell (prokaryote) Pro = before Karyon = nucleus (=All materials within a cell except nucleus)  eu = true Animal cell (eukaryote) karyon = nucleus  Can you find characteristics that are common vs. different between prokaryotic and eukaryotic cells? Pro = before eu = true Karyon = nucleus karyon = nucleus  Human proteins can be expressed in bacterial cells for commercial/therapeutic use. (e.g. insulin, interferon-b)  Many membrane-bound Animal Cell Organelles Organelles à functional compartmentalization Endosome/Lysosome/Vacuole a compartment for oxidation reactions, Peroxisome lipid biosynthesis (e.g. cholesterol) Breaks down all types of biological polymers (e.g. proteins, lipids) Mitochondria Nucleus a compartment for generation of metabolic energy (ATP!!) by oxidative phosphorylation ER Protein synthesis & processing (extension of Golgi nuclear membrane). Protein sorting center to 1) lysosomes, 2) plasma membrane 3) or secretion. Plasma membrane Cytoplasm separates the interior of all cells from the outside environment  Many cell types in the human body Cell differentiation Multicellular eukaryotes have different cell types for different functions. The numbers and arrangement of organelles relate to function & activity of cells. Figure 1. 17 Four major types of biological molecules #1 #2 #3 Nucleic acids #4 Major building blocks of the cytoplasm Material within a cell, excluding the nucleus Proteins (protein complexes) Lipids (membranes) Proteins (=polypeptides) All amino acids have a carboxyl and an amino group, separated by a single carbon atom, the α-carbon. R à any of a number of chemical groups -20 different amino acids construct proteins (=polypeptides). -Proteins are unique polymers made of amino acid monomers. -In the cell, this reaction is catalyzed by ribosome. -Amino acids sequence = ‘PRIMARY STRUCTURE’ of a protein  Amino acids sequence = ‘PRIMARY STRUCTURE’ of a protein 1 MATLEELDAQ TLPGDDELDQ EILNLSTQEL QTRAKLLDNE IRIFRSELQR LSHENNVMLE 61 KIKDNKEKIK NNRQLPYLVA NVVEVMDMNE IEDKENSEST TQGGNVNLDN TAVGKAAVVK 121 TSSRQTVFLP MVGLVDPDKL KPNDLVGVNK DSYLILDTLP SEFDSRVKAM EVDEKPTETY 181 SDVGGLDKQI EELVEAIVLP MKRADKFKDM GIRAPKGALM YGPPGTGKTL LARACAAQTN 241 ATFLKLAAPQ LVQMYIGEGA KLVRDAFALA KEKAPTIIFI DELDAIGTKR FDSEKSGDRE 301 VQRTMLELLN QLDGFSSDDR VKVLAATNRV DVLDPALLRS GRLDRKIEFP LPSEDSRAQI 361 LQIHSRKMTT DDDINWQELA RSTDEFNGAQ LKAVTVEAGM IALRNGQSSV KHEDFVEGIS 421 EVQARKSKSV SFYA* Is the primary structure alone sufficient to generate a functional protein? 13 The way a protein *FOLDs* generates its TERTIARY structure Biologically active form Structure = Function If the folding process goes awry, you can lose the the ENTIRE protein function of the PORTIONS of a protein folded protein. folded Formation of secondary structure or collapse driven by hydrophobic interactions Nobel Prize in Chemistry, 2024, goes to developers of AlphaFold AI that predicts protein structures David Baker Demis Hassabis John Jumper Univ. of Washington, Google DeepMind, London, UK USA, HHMI “for protein structure prediction” “for computational protein design” Since 2021 16  How do proteins fold? Christian Anfinsen 1972 Nobel Prize in Chemistry “for his work on ribonuclease, especially concerning the connection between the amino acid sequence & the biologically active conformation (=structure)”  Ribonuclease RNA-cleaving enzyme  Active ribonuclease Inactive 4 disulfide bonds ribonuclease --and-- Non-covalent bonds -Evidence that the primary sequence of a protein determines the final folded states (native conformations) break disulfide bonds -Mutation in a protein sequence can be detrimental Active ribonuclease  Noncovalent bonds Drive & stabilize tertiary structure Hydrophobic effect as well Noncovalent bonds A variety of weak linkages. à Mainly between atoms with an opposite charge. Large numbers of them act in concert, as between different parts of a large protein. à their attractive forces are additive. à they provide the structure with considerable stability. readily break & reform à allow conformational changes to regulate protein activities Example of a conformational change (proteasome) 21  Noncovalent bonds Hydrophobic effect as well - Two very close molecules with a weak attractive force (non-polar molecules) - operate at optimum distances and are maximized by complementary surfaces. - Hydrogen atom (partial positive charge) can approach a second electronegative atom. - Attraction between charged atoms Single amino acid change in primary structure in hemoglobin What this might be? Changes the shape of the cell => sickle-cell anemia (Glu to Val)  We will examine amino acid side chain. 2 questions 1. How do they affect protein folding? 2. How do mutations in amino acid sequence affect protein function? 24 Example: histones with arginine bind to the negatively charged phosphate DNA backbone.  These amino acids are often quite reactive. Potential phosphorylation sites Kinase phosphorylation à change protein folding to regulate protein activity  Cysteine: R group has reactive -SH which forms disulfide (-S-S-) bridge with other cysteines often at some distance away in polypeptide backbone (e.g. in experimental setting) Dr. Anfinsen’s experiment *mercaptoethanol* The way a protein folds generates its tertiary structure Portions of the protein folded  Linus Pauling Local Secondary structures - alpha helix structure The backbone: 1) lies on the inside of the helix and side chains project outward 2) assumes the form of a cylindrical, twisting spiral Stabilize the helical structure Local Secondary structures – beta sheet structure - several segments of a polypeptide lying side by side that form a folded or pleated conformation. - Hydrogen bonds are perpendicular to the long axis. β strands are extended and resist tensile forces. (e.g. Silk has an extensive amount of β sheet. 5 times stronger than steel of comparable weight) Additional secondary structures include hinges, turns, loops, or finger-like extensions. Often, secondary structures form: - in the most flexible portions of a polypeptide chain - In the sites of greatest biological activity. 36  Domains interact to make tertiary structure -- a region of the protein that folds Ribonuclease - the conformation of the entire polypeptide. independently from the rest. - Secondary structure is stabilized by hydrogen bonds, while tertiary structure is --responsible for a stabilized by **noncovalent bonds between particular function or the side chains** of the protein. interaction, contributing to the -Secondary structure is limited to a small overall function of a number of conformations, but tertiary protein. structure is virtually unlimited. -usually determined using the technique of X-ray crystallography.  Disposition of hydrophilic and hydrophobic amino acids in Cytochrome C (heme group in middle)) Hydrophilic side chains in green (on the surface à contact the surrounding aqueous medium) Hydrophobic side chains in red ((in the center) Myoglobin –first tertiary structure to be solved- no domains (John Kendrew at Cambridge) - no disulfide bonds; the tertiary structure is held together by noncovalent interactions. - Contain All of the noncovalent bonds thought to occur between side chains within proteins (hydrogen bonds, ionic bonds, and van der Waals).  Many proteins in eukaryotes have multiple domains. What do multi-domain proteins look like & How do they arise? 40 Shuffling of domains: --create proteins with combination of activities through evolution Catalytic domain Domains: -fold independently from the rest of a protein à functional and/or structural unit of a protein. -similar domains can be found in proteins with different functions. -Most eukaryotic proteins have two or more domains. PH domain  Quaternary structure of a protein complex (=more than 1 polypeptide) The Structure of Hemoglobin O2 binding to 1 polypeptide causes a conformational change in the other polypeptides à alters their affinity for O2. **Small changes in protein conformation enable complex function of a protein.** Red: Heme group à Binds to O2 2 α-globin chains & 2 β-globin chains (=4 proteins) joined by noncovalent bonds. Hub proteins protein-protein interactions form protein complexes Proteins w/ multiple RNA PolII binding partners Cdc28 -several different binding interfaces à a number of (kinase) different binding partners at the same time. -a single binding interface à can bind several different partners, but one at a time. more likely to be essential than non-hub proteins.  Chaperones assist protein folding in the cell On nascent polypeptide, *hydrophobic patches* are exposed àthey can aggregate or misfold by random interaction w/ other molecules. à chaperones help prevent undesirable interactions & assist protein folding. chaperone protein complex with a *chamber* for protein folding) Chaperonin: Chambered chaperone protein complex A model of the GroEL complex built according to data from electron microscopy and molecular-weight determination. The complex is seen to consist of two disks, each composed of seven identical subunits arranged symmetrically around a central axis. Subsequent studies showed the complex contains two internal chambers.  When the GroES cap binds, hydrophobic The chaperonin residues in the chamber wall retract & hydrophilic residues are exposed. system à this releases the protein, allowing for its folding in the chamber. ATP hydrolysis nascent = a “timer” polypeptides have exposed hydrophobic residues. hydrophobic residues line the GroEL chamber wall. The chaperonin GroEL [GroEL + GroES] system the binding of the GroES is accompanied by a marked change in conformation of high-resolution the proteins in the top GroEL ring (arrow) electron à **enlargement of the upper chamber**. micrographs.  Conformational change in GroEL GroES GroEL GroEL the binding of the GroES is accompanied by a marked change in conformation of the proteins in the top GroEL ring. à results in a marked enlargement of the upper chamber. How does the cell deal with the proteins, if they fail to fold properly? 49 The Proteasome It degrades most proteins in the cell in a selective manner. (e.g. misfolded proteins & many short-lived proteins) **chambered** protease à conceals protease activity within a chamber (inner b rings) to prevent nonselective protein degradation in the cell environment  Ubiquitin a “degradation signal” Ubiquitin signal is recognized by the cap selectively marks a protein. in the proteasome. Mutations in the ubiquitin & proteasome system are related to protein misfolding disease The cap unfolds the protein & (=abnormal proteins concurrently cannot be removed) removes the ubiquitin signal. Protein is destroyed within the b ring chamber.  Protein Misfolding Diseases Disease Misfolded Protein Alzheimer's disease Abb peptides (plaques); tau protein (tangles) Spongiform encephalopathies Prion (whole or fragments) Parkinson's disease α-synuclein (wt or mutant) Primary systemic amyloidosis Ig light chains (whole or fragments) Secondary systemic amyloidosis Serum amyloid A (whole or 76-residue fragment) Fronto-temporal dementias Tau (wt or mutant) Senile systemic amyloidosis Transthyretin (whole or fragments) Familial amyloid polyneuropathy I Transthyretin (over 45 mutants) Hereditary cerebral amyloid angiopathy Cystatin C (minus a 10-residue fragment) Haemodialysis-related amyloidosis 2-microglobulin Familial amyloid polyneuropathy III Apolipoprotein AI (fragments) Finnish hereditary systemic amyloidosis Gelsolin (71 amino acid fragment) Type II diabetes Amylin (fragment) Medullary carcinoma of the thyroid Calcitonin (fragment) Atrial amyloidosis Atrial natriuretic factor Hereditary non-neuropathic systemic Lysozyme (whole or fragments) amyloidosis Injection-localised amyloidosis Insulin Hereditary renal amyloidosis Fibrinogen -A chain, transthyretin, apolipoprotein AI, apolipoprotein AII, lysozyme, gelsolin, cystatin CAfter Stefani and Dobson ‘03 Amyotrophic lateral sclerosis Superoxide dismutase 1 (wt or mutant) Huntington's disease Huntingtin Spinal and bulbar muscular atrophy Androgen receptor [whole or poly(Q) fragments] Spinocerebellar ataxias Ataxins [whole or poly(Q) fragments] Spinocerebellar ataxia 17 TATA box-binding protein [whole or poly(Q) fragments] Hallmark of Protein misfolding disease  AD patient’s brain specimen Alzheimerʼs disease (AD) -memory loss, confusion, and loss of reasoning ability. -strikes >10% of individuals at >65 yo. (Both protein folding & degradation machineries decline with aging) - fibrillar deposits of an insoluble material referred to as amyloid.  -a common pathogenetic mechanism - a normal protein becomes amyloid deposit. How could a normal protein convert into TEM image of amyloid deposit? amyloid fibrils (slide by T. Serio)  Amyloid hypothesis nerve cell in brain the disease is caused by Ab production (part of the APP). The secretases cut APP àproduces Aβ40 or Aβ42. à Both can exist in a soluble form (alpha helices). *harmless* Aβ42 overproduction: -duplication of the APP gene à Aβ42 tends to refold into -mutations in the APP gene a conformation with β -mutations in γ-secretase sheets & self-associate to form complexes (*oligomers, toxic*) Figure 3 (p65)  Protein Misfolding Diseases Disease Misfolded Protein Alzheimer's disease Abb peptides (plaques); tau protein (tangles) Spongiform encephalopathies Prion (whole or fragments) Parkinson's disease α-synuclein (wt or mutant) Primary systemic amyloidosis Ig light chains (whole or fragments) Secondary systemic amyloidosis Serum amyloid A (whole or 76-residue fragment) Fronto-temporal dementias Tau (wt or mutant) Senile systemic amyloidosis Transthyretin (whole or fragments) Familial amyloid polyneuropathy I Transthyretin (over 45 mutants) Hereditary cerebral amyloid angiopathy Cystatin C (minus a 10-residue fragment) Haemodialysis-related amyloidosis 2-microglobulin Familial amyloid polyneuropathy III Apolipoprotein AI (fragments) Finnish hereditary systemic amyloidosis Gelsolin (71 amino acid fragment) Type II diabetes Amylin (fragment) Medullary carcinoma of the thyroid Calcitonin (fragment) Atrial amyloidosis Atrial natriuretic factor Hereditary non-neuropathic systemic Lysozyme (whole or fragments) amyloidosis Injection-localised amyloidosis Insulin Hereditary renal amyloidosis Fibrinogen -A chain, transthyretin, apolipoprotein AI, apolipoprotein AII, lysozyme, gelsolin, cystatin CAfter Stefani and Dobson ‘03 Amyotrophic lateral sclerosis Superoxide dismutase 1 (wt or mutant) Huntington's disease Huntingtin Spinal and bulbar muscular atrophy Androgen receptor [whole or poly(Q) fragments] Spinocerebellar ataxias Ataxins [whole or poly(Q) fragments] Spinocerebellar ataxia 17 TATA box-binding protein [whole or poly(Q) fragments]  Links Between Mammalian Neurodegenerative Disorders Mid ‘50’s - ‘60s Gajdusek Hadlow Kuru ~15% ~85% -inherited/sporadic -loss of motor coordination -dementia. Scrapie Creutzfeldt-Jakob disease (CJD)  Kuru: “Trembling” Fatal neurodegenerative disease Progressive dementia and ataxia Fore Tribe Islanders of Papua, New Guinea contract “kuru,” a spongiform encephalopathy, from eating brain tissue of a recently deceased relative. à causes them to acquire CJD Disease incidence: 1% of population & tended to run in families Transmissible Spongiform Encephalopathies (TSEs) brain homogenates of kuru, CJD patients intracerebral inoculation of chimpanzees kuru, CJD in chimpanzees D. Carleton Gajdusek Nobel Prize in Physiology or Medicine 1976 “discoveries concerning new mechanisms for the origin and dissemination of infectious diseases” (slide by T. Serio)  Prions Science 216:136-44 (1982) A new term ”prion” is proposed to denote a small proteinaceous infectious particle which is resistant to inactivation by most procedures that modify nucleic acids. Stanley Prusiner Nobel Prize in Physiology or Medicine 1997 “For his discovery of prions - a new biological principle of infection” (slide by T. Serio) Prions cannot be destroyed by boiling, alcohol, acid, standard autoclaving methods, or radiation. In fact, infected brains that have been sitting in formaldehyde for decades can still transmit spongiform disease. Cooking your burger 'til it's well done won't destroy the prions! 62  PrP - A Host Encoded Protein PrP (or PRNP) = Pr for prion, and P for protein 1985: isolation of PrP cDNA (Prusiner, Weissman, Cheesebro) 1986: cloning of PRNP gene (Weissman) If the PRNP gene causes scrapie, it should only be isolated from individuals with disease, but PRNP was present in both healthy and diseased individuals (slide by T. Serio) Prion Hypothesis Healthy Are normal and pathogenic PrP different? Disease (slide by T. Serio)  Prion protein (PrP) A contrast in structure between normal and infectious prion protein consists largely Alpha helices of beta-sheets PrPC (normal) PrPSc (abnormal, infectious) (c =cellular) (Sc =scrapie) soluble & protease-sensitive -insoluble & protease-resistant -can bind to PrPC & cause it to fold into the abnormal, PrPSc.  Normal and Pathogenic PrP Are Conformers PrPC PrPSc H. Sabil  **basis of prion infection** How does PrPSc convert PrPc into misfolded conformation?  Low sporadic rate of disease: ~1/million/year  Self-Templated Protein State Replication Protein states replicate by nucleated polymerization Progressive nature of disease Accelerated rate of disease due to mutation e.g. prion-contaminated beef (mad cow disease) Aggregates cause disease PrPSc!!!  Self-Templated Protein State Replication Are synthetic prions infectious? + = ? recMoPrP(89-230) (slide by T. Serio) PrP/TSE Connections Transmissible spongiform encephalopathies (slide by T. Serio)