L8 Post Translational Modification of Proteins PDF

L8 Post Translational Modification of Proteins Post-translational modification: a mechanism to regulate gene expression Professor Guy Tear [email protected]  Control of gene expression via post-translational modif...

L8 Post Translational Modification of Proteins Post-translational modification: a mechanism to regulate gene expression Professor Guy Tear [email protected]  Control of gene expression via post-translational modification (PTM) of proteins Learning objectives Understand what PTM means Understand the importance of PTMs to regulate protein activity Realise the diversity of PTMs Gain knowledge of how PTMs are used to regulate protein activity (gene expression) Control of Gene Expression DNA RNA Protein transcription translation Control of gene expression is complex Post translational modification (PTM) 2  Post-translational modifications modify the activity of proteins Covalent modification of proteins: Two broad classes : 1. Enzyme-assisted covalent addition (or elimination) of a chemical group 2. The covalent cleavage of peptide fragments in proteins driven by proteases or, less frequently, by autocatalytic cleavage Covalent changes often reversible Proteins can move from active to inactive form and vice versa. Cleavage of peptide fragments irreversible. 3  Post-translational modifications used to activate or repress protein activity Modifications can activate protein activity e.g. phosphorylation Modifications can inactivate protein activity e.g. phosphorylation Modifications can degrade proteins e.g. ubiquitination Modifications can change protein location in cell e.g. myristolation Some modifications have multiple roles e.g. phosphorylation. Function of post-translation modifications Contribute to control of gene activity by regulating: Protein folding Protein stability Protein activity Protein targeting to particular cell organelles or compartments Response to ligand binding PTM plays roles in regulating Signal transduction Cell division Cell differentiation Cell survival Gene expression 4  Proteins can be moved from one place in an inactive form to another place in an active form. Proteins can be made in an inactive form and then folded into an active form. Ubiquitination can stabilize proteins. Common types of post-translation modifications Addition of a phosphate group Phosphorylation Addition of –CH3 to lysines Addition of Methylation COCH3 Acetylation Modification of a protein to lysines Ubiquitination with a highly conserved 76 Cleavage of the Proteolysis AA polypeptide ubiquitin protein to remove Lipidation inhibitory regions Glycosylation Addition of lipids Addition of sugars Ubiquitin found in almost all cells Glycosylation often on receptor proteins. Understand examples, how their used and how they control protein/gene activity. 5  Variety of Enzymes regulate PTMs Enzymes involved in PTMs: Kinases, phosphatases, transferases and ligases, which add or remove functional groups, proteins, lipids or sugars to or from amino acid side chains, Proteases, which cleave peptide bonds to remove specific sequences or regulatory subunits. Kinases – add phosphates Transferases – transfer protein Ligases – ligate (add) other groups 6  Post-translational modifications: When can they occur? At any step in the "life cycle" of a protein. For example: Many proteins are modified shortly after translation to mediate proper protein folding or stability or to direct the nascent protein to distinct cellular compartments. To activate or inactivate catalytic activity or to otherwise influence the biological activity of the protein. Proteins can also be covalently linked to tags that target a protein for degradation. Besides single modifications, proteins are often modified through a combination of post-translational cleavage and the addition of functional groups through a step-wise mechanism of protein maturation or activation. Can always happen to a protein to make it active Can happen at any point when its needed. More than one modification can occur 7  Post-translational modifications also diversify the human proteome. Post translational modification increases complexity of proteins, meaning you can go from 25k genes to over 1m proteins. 8  Regulation by proteases Many proteins are produced in an initially inactive (but folded) form - often called a proprotein or proenzyme or zymogen The protein produced by translation includes amino acids in addition to those necessary for protein/enzyme activity. The inactive form can be converted to its active form by a protease that removes the additional amino-acids. This method of regulation does not require an energy source Therefore extracellular enzymes may be activated by this process This proteolysis is irreversible – once activated the protein remains in its activated state E.g. pepsinogen = zymogen, pepsin = active enzyme. Pepsinogen and pepsin 9  Masking sequence inhibits formation of the active site. Prothrombin and thrombin Cleaved by Factor Xa Cleaved by Factor Xa Factor Xa cleaves at Arg-320 and Arg-284. 10  Thrombin and blood clotting TPA TPA Proteins only become active when needed to prevent excess blood clotting. 11  Insulin is produced as a pre-pro-protein which is cleaved to produce the active protein Cleaved in the secretory granules of pancreatic beta cells To make pro: remove signal sequence, to make insulin remove chain C Regulation by phosphorylation Protein phosphorylation is the most abundant post translational modification in eukaryotes. Protein phosphorylation involves the addition of a phosphoryl group (PO32- ) to specific amino acids, usually serine, threonine or tyrosine Protein phosphorylation can cause the activation or inactivation of protein or enzyme This method of regulation requires ATP as an energy source Phosphorylation is reversible – protein kinases add phosphates, protein phosphatases remove phosphates. Phosphorylation often used to regulate protein activity in signal transduction pathways 12  Phosphorylation: a reversible PTM activation inhibition Protein Kinase O Protein OH + ATP Protein O P O- + ADP - O Pi H2O Protein Phosphatase Kinases remove gamma phosphate of ATP and add it to the target protein. 13  Example of phosphorylation I Regulation of glycogen metabolism glycogen phosphorylase Glycogen is phosphorylated to form glucose 1 phosphate. Example of phosphorylation II Receptor tyrosine kinases Receptor tyrosine kinases (RTKs) are transmembrane proteins that interact with extracellular signals (e.g. hormones). RTKs contain a tyrosine kinase domain in their intracellular region RTKs are inactive when not bound by a signal molecule. When a signal molecule binds a RTK the molecule forms a dimer which activates the tyrosine kinase domain. The kinase domain phosphorylates specific tyrosines in the intracellular domain. This activates the protein to bind intracellular proteins to activate further proteins in the cell. 14  ** Phosphorylation allows regulation of gene expression by controlling protein activity. Often used in signalling pathways to control cellular responses to extracellular signals. Often a series of proteins phosphorylate one another in a phosphorylation cascade – this allows signal amplification, each kinase phosphorylates many downstream target kinases, e.g. MAP kinase cascade Phosphorylation used to co-ordinately regulate many processes as some kinases can phosphorylate and activate multiple downstream proteins Insulin signalling P P P P P P P MAP kinase cascade Phosphorylation allows signal amplification. **signal amplification Cascade rapidly causes the activation of more and more proteins. PTMS – allow rapid responsive of some signals 15  Regulation by ubiquitination Protein ubiquitination is a common post translational modification in eukaryotes. Ubiquitination involves the addition of ubiquitin molecules to lysines Ubiquitin is a 8kD protein consisting of 76 amino acids Single ubiquitin molecules can be added (monoubiquitination) or a chain of multiple ubiquitin molecules (polyubiquitination) Polyubiqutination can cause the degradation of a protein or enzyme Monoubiquitination can cause the translocation of proteins in the cell Ubiquitination is reversible – ubiquitin ligases add ubiquitin, de- ubiquitinating enzymes remove ubiquitin. Ubiquitination often used to regulate protein activity during cell division Ubiquitination can result in translocation or degredationo. Ubiquitin ligase – adds ubiquitin. 16  Ubiquitination machinery Ubiquitin activating enzyme (E1) reacts with free ubiquitin (Ub) and transfers it to ubiquitin conjugation enzyme (E2). Ubiquitin ligases (E3) identify target proteins (substrate) and bring them to an E2 to catalyse the transfer of Ub from E2 to the target protein. Addition of multiple ubiquitin molecules to target protein directs the protein to the proteasome where it is degraded. Proteasome degrades unwanted proteins that have been tagged by polyubiquitination. 17  Multiple roles for ubiquitination Often: protein moves from cytoplasm to an organelle where it becomes activated. 18  Ubiquitination regulates cell division Progress through the cell cycle has to be tightly regulated, e.g cells only move into S phase if the environment is suitable. The cyclins are a group of proteins that control progression through the cell cycle. Different cyclins accumulate in different stages of the cell cycle. The cyclins control the decision to move from one stage of the cell cycle to the next. Once they have signalled that progression can take place they are quickly degraded through ubiquitination so the next cyclin can take over. Cyclins must be quickly removed to allow movement to the next stage of the cell cycle. Cyclins are removed by polyubiquitination. 19  Methylation and Acetylation Addition of methyl groups (CH3) to lysine or arginine. Addition of acetyl groups (CH3O) to lysine (or N terminus). Methylation of a protein can change its conformation and its function. Acetylation neutralizes positive charge of proteins to regulate activity. Histones are commonly regulated by methylation and acetylation–but can be found on other proteins. Both processes are reversible. Methylation and acetylation of histones regulate how compactly DNA is folded into chromatin. Acetylation loosens the chromatin allowing gene transcription while methylation tightens the chromatin causing silencing of genes. Methylation and acetylation of histones is the basis of epigenetics. Histones and other proteins are modified with these epigenetic mechanism. This is reversible. This is important in tightening and loosening the histones to determine if a gene is expressed. Methylation tightens and acetylation loosens. 20  Histone Methylation and Acetylation Changes accessibility of genes within chromatin. Regulation by lipidation Lipidation involves the addition of lipid or cholesterol molecules to proteins. Lipidation can direct targeting of proteins to specific locations in the cell or may be required for an enzyme to be active. Many proteins require lipidation since they are only active when in the correct location in the cell. Lipidation is reversible. Palmitoylation or myristoylation of cytoplasmic proteins can promote their association with the inner face of the plasma membrane, while the addition of a GPI-anchor may serve to anchor extracellular proteins to the outer face of the plasma membrane. myristoylation attachment of myristate, a C14 saturated acid to glycine palmitoylation attachment of palmitate, a C16 saturated acid to cysteine glycosylphosphatidylinositol (GPI) anchor formation via an amide bond to C- terminal tail 21  Normally takes proteins to the cell surface but can also target to mitochondria. Palmitoylation and myristolylation are two such mechanism, with palmitate (C16) being added or myristate (C14) being added. Examples of lipidation Lipidation of proteins allows them to be directed to specific locations in the cell. Lipidation of certain proteins also allows them to bind partner molecules. Directs to plasma membranes. Lipidation allows proteins to associate with the inner leaflet of the cell membrane. 22  Lipidation Glycosylation Glycosylation is the addition of sugars or glycans (chains of different sugars) to proteins. One of the most abundant and diverse PTM More than 50% of the human proteome is glycosylated Virtually all membrane proteins are glycosylated 23  Glycosylation is vital for the function of proteins, cells and organisms Glycans are involved in: Ensuring the correct folding of proteins Acquiring resistance to proteases Ensuring proteins interact with the appropriate partners Directing proteins to their correct location in the cell Cell: cell communication Cell: environment communication Cell trafficking Fertilisation Development Immune response Glycosylation is altered in cancers Types of glycans found in the cell Sugars can be added to intracellular and extracellular proteins. 24  Differing glycosylation occurs in different blood groups The most common types of glycosylation N-linked glycosylation O-linked glycosylation N-linked glycosylation: Initiated in the endoplasmic reticulum Initial sugars are added en bloc to the asparagine in the sequence Asp-N-Ser/Thr O-linked glycosylation: In most cases initiated in the Golgi Sugars are added singly and sequentially N linked – membrane proteins 25  Types of N-glycans Trimming of Mannose and addition of sugars Transferred en bloc to Asp in the sequence Asp-N-Ser/Thr GlcNAc Mannose Sialic acid Galactose Fucose Essentials of Glycobiology Second Edition Chapter 8, Figure 1 Big sugar structure added all at once Types of O-linked glycosylation Mannose Further extensions of cores GlcNAc GalNAc Galactose Fucose Sialic acid Xylose S/T S/T S/T K(OH) S/T S/T O-GlcNAc O-Man O-Fuc O-Gal O-Xyl O-GalNAc Mucin-type 26  Roles for glycosylation Glycosylation involves the addition of sugar molecules to proteins. Many cell surface proteins must be glycosylated to fold correctly The specific pattern of glycosylation of proteins changes the activity of proteins. Glycosylation of cell surface receptors is necessary to allow them to recognise signal molecules. A wide variety of glycosyltransferases catalyse the addition of different sugars to produce a large number of diverse and often highly complex pattern of glycosylation. Disorders of glycosylation can lead to cancer and immune disorders. Happens and allows large amplification of different protein fros Post-translational modifications are crucial for regulation of gene product activity And to increase diversity of the proteome 27  Further reading http://www.piercenet.com/method/overview-post-translational-modification Ardito et al., The crucial role of protein phosphorylation in cell signaling and its use in targeted therapy. Int. J. Mol. Med (2017) vol 40 pp271-280 Soltes et al Ubiquitin, ubiquitination and proteasomal degradation in the eukaryotic cell. Bios (2011) vol 82 pp64-71 Gree E.l. and Shi Y. Histone methylation: a dynamic mark in health , disease and inheritance. Nature Reviews Genetics (2012) vol 13 pp343-357 Casey, P.J. Protein lipidation in cell signaling. Science (1995) vol 268 pp221-225 Sachwarz F. A and Aebu M. Mechanisms and principles of N-linked protein glycosylation Current Opinion in Structural Biology (2011) vol 21 pp576-582 28