04_Protein_Mod_Glycoproteins_2024.07.18_Student.pptx

Protein Modification and Glycoproteins Steven J. Ontiveros, MBA, PhD  Objectives Describe chaperones and their function in protein folding. Describe the reactions catalyzed by protein disulfide isomerase. Summarize the modifications of proteins by a...

Protein Modification and Glycoproteins Steven J. Ontiveros, MBA, PhD  Objectives Describe chaperones and their function in protein folding. Describe the reactions catalyzed by protein disulfide isomerase. Summarize the modifications of proteins by additions of carbohydrates, lipidation, methylation, acetylation, and glycosylation. Explain protein regulation by protein-protein interactions, and the roles kinases and phosphatases play in regulating protein function. Explain the regulation by protein cleavage and degradation. Describe the properties of glycoproteins, glycosaminoglycans (GAG), and proteoglycans, and the biosynthesis. Protein Modification  Protein Synthesis & Post-translational Modification Protein biosynthesis Translation of protein by ribosomes 20-25K genes in the human genome Number of proteins is 1 million Post-translational modification Component of protein diversity & complexity Can occur at any step in the life of a protein Function Regulate activity Regulate localization Regulate interactions with other cellular molecules  Protein Synthesis and Folding Translation Nucleotides sequence converted into amino acid sequence Amino acid sequence provides the instructions for folding Molecular chaperones Proteins that assist in protein folding or unfolding Prevent misfolding and aggregation Bind and stabilize protein intermediates Found extensively in the ER Chaperonins Heat shock proteins (hsp70, hsp60) Bind and prevent premature folding Act as folding catalysts Bind target proteins, stabilize and prevent aggregation, Marks Basic Medical Biochemistry 5th Edition, 2018 and properly fold the protein  Protein Folding in the ER Calnexin (membrane) Calreticulin (lumen) ER chaperones (lectins) Direct proper folding Retain proteins in ER Prevent proteins from aggregating Harper’s Illustrated biochemistry 29th Edition, 2012  Enzymes that Catalyze Protein Folding Other enzymes help catalyze protein folding Protein Disulfide Isomerase (PDI) Found in the ER Catalyzes disulfide bond formations on cysteine residues Disulfide bonds generally form in the ER Important for mature insulin protein Disorders of Carbohydrate Metabolism. Clinical Chemistry, Ch. 13, 215-243  Glycosylation Glycosylation: Occurs in multiple subcellular locations ER, Golgi, cytosol, cell membranes Addition of carbohydrate chains (glycans) Significance of glycosylation: Prevents protein aggregation Protein folding Cell Biology 3rd Edition, Cell adhesion 2017 Cell-cell and cell-matrix interactions Protein activation Protein solubility Protein trafficking Glycoproteins: Most secretory, plasma membrane, and lysosomal proteins are glycosylated  Protein Misfolding in the ER – Unfolded Protein Response Unfolded Protein Response ER stress response ER-associated protein degradation (ERAD) Identify misfolded proteins Chaperones attempt refolding Distributed to cytoplasm Degraded by ubiquitin proteasome system Degraded material is recycled into cell  Ubiquitination & Protein Degradation Ubiquitination (ubiquitylation, ubiquitinylation) Addition of polyubiquitin chain Targets proteins for degradation by proteasome Can also regulate enzyme activities or localization Ubiquitin-Proteasome Pathway Proteins destined for degradation are tagged with ubiquitin Mediates regulated protein degradation Multistep process Histology and Cell Biology 5th Edition, Directed by ubiquitin enzymes (E1, E2, E3) Protein becomes polyubiquitinated (polyubiquitin chain) Proteasome Multisubunit protease complex 2021 Degrades ubiquitinated proteins  Protein Misfolding and Disease Protein Misfolding Diseases Defects in protein folding are responsible for a large number of diseases Cystic Fibrosis Cystic fibrosis transmembrane conductance regulator (CFTR) mutation Plasma membrane chloride transporter Deletion of PHE-508 disrupts chaperone binding Prevents appropriate protein folding Obstruction of the respiratory tract via thick mucus  Protein Cleavage Proteolysis: Cleavage of polypeptide chain Facilitates maturation of select proteins Insulin Caspases Caspases: Cell death protease enzymes Activated by protein cleavage Insulin: Synthesized as a precursor polypeptide (proinsulin) Cleavage results in mature insulin and c- peptide Other proteins/enzymes: Digestive enzymes Blood clotting proteins  Acetylation/ Cell Biology 3rd Edition, Deacetylation 2017 Function Chromatin stability and gene expression Protein-protein interactions Cell cycle control Nuclear transport Histone Acetylation Acetylation of histones plays roles in gene expression Histone acetyltransferases (HATS) transfer Marks Basic Medical Biochemistry 5th Edition, 2018 acetyl groups from acetyl coenzyme A (Acetyl CoA) to lysine residues Removes positive charge from amino group on lysine residue Disrupts the electrostatic charge attraction between (+) lysine and (-) DNA Causes DNA to unwind Posttranslational - Protein Methylation (Protein & DNA) Protein Methyl transferases: Transfer of one-carbon methyl (-CH3) group Occurs on lysine or arginine residues Increases the hydrophobicity of the protein Affects protein-protein interactions Epigenetic - DNA DNA-methyl transferases (DNMT): Transfer of a methyl group to cytosine residues S-Adenosyl methionine (SAM): Primary methyl group donor Significance: Gene regulation Adaptation to environment conditions DNA methylation – inactive chromatin  Lipid Additions (Lipidation) Lipid modification Addition of lipid (hydrophobic) moiety to a peptide chain Additions generally occur on: N-terminal glycine residues C-terminal and internal cysteine residues Types of additions N-myristoylation, prenylation, palmitoylation, cholesterylation Significance Protein targeting: Commonly found in membrane-associated proteins  Protein Structure/Function can be Regulated by Protein-Protein Interactions Inactiv Binding of regulatory molecule e Alters protein conformation PKA Binds chains or subunits that regulate protein activity Protein Kinase A (PKA) cAMP-dependent protein Kinase Comprises multiple subunits (regulatory and catalytic subunits) Active cAMP PKA Binding causes conformation change in PKA regulatory subunits  Protein Phosphorylation Protein phosphorylation Marks Basic Medical Biochemistry 5th Edition, 2018 Addition of phosphate groups to protein targets Hydrolysis of ATP Catalyzed by protein kinases Types of protein kinases Serine/Threonine & Tyrosine kinases Protein phosphatases Catalyze the removal of phosphate group Function Turn proteins on or off Regulate protein targeting Phosphorylation & Glycogenolysis Glycogenolysis Glycogen breakdown is regulated by phosphorylation Phosphorylation by PKA Activation of glycogen phosphorylase Promotion of glycogenolysis Inactivation of glycogen synthase Inhibition of glycogenesis Glycoproteins  Glycoproteins Glycoproteins (via glycosylation) Proteins with short carbohydrate chains (glycan chains) attached to an amino acid side chain Highly found on cellular membranes 2 common types N- and O-linked Lippincott Illustrated Reviews Biochemistry 7th, 2017 Depends on site of attachment of carbohydrate moiety Function Cell-surface recognition Cell-cell contacts Cell-surface antigenicity For blood group recognition Component of extracellular matrix (ECM) Provide structure to ECM Biological lubricants Mucins of the GI and urogenital tract  Carbohydrates Chains N-linked Carbohydrate is attached to amino (-NH2) group of asparagine residues Occurs in the ER O-linked Carbohydrate is attached to Hydroxyl (-OH) group of serine or threonine residues Occurs in the ER, Golgi, & cytoplasm Most commonly found in mucus proteins  Proteoglycans Proteoglycans A type of glycoprotein Proteins that are significantly glycosylated Major component of the ECM Found in connective tissues Cartilage Loose connective tissue Made up of glycosaminoglycans (GAGs) Function Bind cations and water Serve as lubricants Marks Basic Medical Biochemistry 5th Edition, 2018  Lippincott Illustrated Review 7th Edition, 2017 Glycosaminoglycans (GAGs) GAGs Polysaccharides chains Repeating disaccharide units Acid sugar + amino sugar Classification Major types of GAGs Chondroitin-4 & 6 sulfates Keratan sulfates Hyaluronan (Hyaluronic acid) Marks Basic Medical Biochemistry 5th Edition, 2018 Heparin Haparan sulfate Dermatan sulfate Significance Have a large capacity to bind water Can form a gel-like matrix  Structure & Function of Lippincott Illustrated Review 7th Edition, Proteoglycans Structure Contain chains of repeating GAGs 2017 Hyaluronan linked to core protein Core protein structure is linked long chains of GAGs Linked via a trihexoside (Xylose-Gal- Gal) + Serine residues Function Form large complexes with other Marks Basic Medical Biochemistry 5th Edition, 2018 molecules (eg. collagen) in extracellular matrix Lubricant Heavily hydrated (large water-binding capacity)