Post-translational Modifications and Protein Degradation PDF

Post-translational modifications and protein degradation Simon Alberti Center for Molecular and Cellular Bioengineering (CMCB) Biotechnology Center (BIOTEC) 1  What Are Post-Translational Modifications?...

Post-translational modifications and protein degradation Simon Alberti Center for Molecular and Cellular Bioengineering (CMCB) Biotechnology Center (BIOTEC) 1  What Are Post-Translational Modifications? Most proteins undergo chemical modifications before becoming functional The modifications are collectively known as post-translational modifications (PTMs) PTMs play a crucial role in generating heterogeneity and help in utilizing identical proteins for different cellular functions in different cell types 2 Post-Translational Modifications 3 PTM are Mediated by Enzymes PTMs occur at distinct amino acid side chains and are mediated by enzymatic activity 5% of the proteome comprises enzymes that perform more than 200 types of post- translational modifications These enzymes include kinases, phosphatases, transferases and ligases, which add or remove functional groups, proteins, lipids or sugars to or from amino acid side chains, and proteases Many proteins can also modify themselves using autocatalytic domains, such as autokinase and autoprotolytic domains 4  Purpose of PTMs Targeting (e.g., membrane targeting) Regulation of stability (e.g., secreted glycoproteins, ubiquitination) Structural role in proteins (e.g., surface glycoproteins) Control of activity (e.g., phosphorylation, caspases) 5  Different Types of PTMs Most PTMs are reversible; for example, kinases phosphorylate proteins at specific side chains; phosphatases hydrolyze the phosphate group to remove it In contrast, proteolytic cleavage is irreversible; cleavage of peptide bonds is a favorable reaction and is therefore permanent 6  Types of PTMs 1) Proteolytic processing: removal of domains or peptide sequences 2) Phosphorylation: addition of a phosphate, usually to serine, tyrosine, threonine or histidine 3) Glycosylation: addition of carbohydrates 4) Acetylation: addition of an acetyl group, usually at the N-terminus of the protein 5) Alkylation: addition of an alkyl group (e.g. methyl, ethyl) 6) Hydroxylation, oxidation, carboxylation of side chains; e.g. hydroxyproline in collagen 7) Biotinylation: acylation of lysine residues with a biotin appendage 8) Glutamylation: covalent linkage of glutamic acid to tubulin and other proteins 9) Glycylation: covalent linkage of one to more than 40 glycine residues to the C- terminal tail of tubulin 10)Isoprenylation: addition of an isoprenoid group (e.g. farnesol and geranylgeraniol). 11)Sulfation: addition of a sulfate group to a tyrosine 12)Selenation: Selenocysteine/selenomethionine modification 13)Amidation: usually at the C terminus 14)Many more… 7  Phosphorylation Reversible protein phosphorylation, principally on serine, threonine, tyrosine or histidine residues, is one of the most important and well-studied post- translational modifications Phosphorylation plays critical roles in the regulation of many cellular processes including cell cycle, growth, apoptosis and signal transduction pathways 8  Phosphorylation 9 Alberts, Molecular Biology of the Cell  Phosphorylation is Abundant Phosphorylation is the most common mechanism of regulating protein function and transmitting signals While phosphorylation has been observed for bacterial proteins, it is considerably more pervasive in eukaryotic cells It is estimated that one-third of the proteins in the human proteome are substrates for phosphorylation Phosphoproteomics is a branch of proteomics that focuses on the identification and characterization of phosphorylated proteins 10  Mechanism of Phosphorylation Phosphorylation only occurs at the side chains of three amino acids, serine, threonine and tyrosine, in eukaryotic cells These amino acids have a hydroxyl (–OH) group that attacks the terminal phosphate group (γ-PO32-) on adenosine triphosphate (ATP), resulting in the transfer of the phosphate group to the amino acid side chain This reaction is unidirectional because of the large amount of free energy that is released 11  Kinases and Phosphatases 12 Alberts, Molecular Biology of the Cell  Regulating Protein Activity Phosphorylation can affect proteins in two ways: first, they can directly regulate the catalytic activity of the protein; second, phosphorylated proteins recruit other proteins with conserved domains that recognize phospho-motifs These domains show binding specificity; for example, Src homology 2 (SH2) show specificity for phosphotyrosine; WW domain recognize phosphoserine; forkhead- associated (FHA) domains recognize phosphothreonine This is critical for signal transduction, in which downstream effector proteins are recruited to phosphorylated signaling proteins 13  Kinases and Phosphatases Protein phosphorylation is a reversible PTM that is mediated by kinases and phosphatases, which phosphorylate and dephosphorylate substrates, respectively Kinases and phosphatses facilitate the dynamic nature of phosphorylation The extent of the phosphoproteome in a given cell is dependent on the temporal and spatial balance of kinase and phosphatase concentrations 14  Protein Kinases More than 500 kinases have been predicted in the human proteome; this subset of proteins comprises the human kinome Substrates for kinase activity are diverse and include lipids, carbohydrates, nucleotides and proteins ATP is the co-substrate for almost all protein kinases, although guanosine triphosphate is used by a small number of kinases While the substrate specificity of kinases varies, the ATP-binding site is generally conserved 15  3D Structure of Protein Kinases 16 Alberts, Molecular Biology of the Cell  Protein Kinases Protein kinases subfamilies show specificity and include tyrosine kinases or serine/threonine kinases Approximately 80% of the mammalian kinome comprises serine/threonine kinases, and >90% of the phosphoproteome consists of pS and pT Studies have shown that the relative abundance ratio of pS:pT:pY in a cell is 1800:200:1 Although pY is not as prevalent as pS and pT, tyrosine phosphorylation is important for biomedical research because of its relation to human disease, e.g., via the dysregulation of receptor tyrosine kinases (RTKs) 17  Protein Kinases Protein kinase substrate specificity is based not only on the target amino acid, but also on consensus sequences flanking it These consensus sequences allow some kinases to phosphorylate single proteins and others to phosphorylate multiple substrates (>300) Additionally, kinases can phosphorylate single or multiple amino acids on an individual protein if the kinase-specific consensus sequences are available 18  Protein Kinases Kinases have regulatory subunits that function as activating or auto-inhibitory domains; phosphorylation of these domains often regulates kinase activity Most protein kinases are dephosphorylated and inactive in the basal state and are activated by phosphorylation A small number of kinases are constitutively active and are made inactive when phosphorylated Some kinases, such as Src, require a combination of phosphorylation and dephosphorylation to become active 19  Src Kinase 20 Alberts, Molecular Biology of the Cell  Activation of Src Kinase: 1 st step 21 Alberts, Molecular Biology of the Cell  Activation of Src Kinase: 2 nd step 22 Alberts, Molecular Biology of the Cell  Activation of Src Kinase: 3rd step 23 Alberts, Molecular Biology of the Cell  Protein Phosphatases The intensity and duration of phosphorylation- dependent signaling is regulated by three mechanisms: 1) removal of the activating ligand; 2) kinase or substrate proteolysis; 3) phosphatase-dependent dephosphorylation The human proteome contains approximately 150 protein phosphatases with two types that show specificity for pS/pT or pY residues While dephosphorylation is the goal of these two groups of phosphatases, they do it through separate mechanisms 24  Signal Transduction Cascades The reversibility of protein phosphorylation makes this PTM ideal for signal transduction, which allows cells to rapidly respond to intracellular or extracellular stimuli Signal transduction cascades are characterized by one or more proteins sensing cues that then relay the signal to second messengers and signaling enzymes In the case of phosphorylation, these receptors activate downstream kinases, which then phosphorylate and activate their cognate downstream substrates, including additional kinases, until the specific response is achieved 25  Signal Transduction Cascades Signal transduction cascades can be linear, in which kinase A activates kinase B, which activates kinase C and so forth Signaling pathways have also been discovered that amplify the initial signal; kinase A activates multiple kinases, which in turn activates additional kinases With this type of signaling, a single molecule, such as a growth factor, can activate global cellular programs such as proliferation 26 Signal Transduction 27 MAP Kinase Cascade 28  Methylation SAM Methyltransferase Lysine Tri-methylated lysine 29  Methylation The transfer of methyl groups to nitrogen or oxygen (N- and O-methylation, respectively) increases the hydrophobicity of the protein and can neutralize a negative amino acid charge Methylation is mediated by methyltransferases, and S- adenosyl methionine (SAM) is the primary methyl donor SAM is the most-used substrate in enzymatic reactions after ATP A single or multiple methyl groups can be added Methylation is a mechanism of epigenetic regulation, as histone methylation and demethylation influence the availability of DNA for transcription 30 N-Acetylation on Lysines Lysine Acetylated lysine Acetylation by HATs De-acetylation by HDACs 31  N-Acetylation Acetylation at the ε-NH2 of lysine (termed lysine acetylation) on histones is a common method of regulating gene transcription The acetylation of lysine residues is regulated by transcription factors that contain histone acetyletransferase (HAT) activity Histone deacetylase (HDAC) enzymes reverse the effects of acetylation enzymes and are often co-repressors 32  Chromatin and PTMs 30 nm fiber 10 nm fiber Nucleosome core particle 30 nm DNA 10 nm 10 nm fiber 30 nm fiber 33 Alberts, Molecular Biology of the Cell  Nucleosome Core Particle 34 Alberts, Molecular Biology of the Cell  Chromatin and PTMs Nucleosomes not only condense DNA, but also provide additional information for DNA replication, repair and transcription This information is conveyed through numerous histone post-translational modifications A variety of enzymes reversibly modify nucleosomes and nucleosome-remodeling complexes, such as histone kinases, methylases, acetylases, histone deacetylases The histone code hypothesis proposes that specific PTMs regulate gene expression by two mechanisms: 1. Changing the chromatin structure into activated or repressed transcriptional state 2. Acting as a docking site for transcriptional regulators 35  PTMs on Histones 36 Alberts, Molecular Biology of the Cell  Histone Tails 37 Alberts, Molecular Biology of the Cell  PTMs on Histone Tails 38 Alberts, Molecular Biology of the Cell  Meaning Through PTMs 39 Alberts, Molecular Biology of the Cell Active and Repressed Genes 40  Proteolysis Peptide bonds are stable under physiological conditions, and therefore cells require a mechanism to break these bonds Proteases comprise a family of enzymes that cleave the peptide bonds of proteins The family of over 11,000 proteases varies in substrate specificity, mechanism of peptide cleavage, location in the cell and the length of activity 41  Proteolysis Degradative proteolysis is critical to remove unassembled protein subunits and misfolded proteins and to maintain protein concentrations at homeostatic concentrations by degrading a given protein to small peptides and single amino acids Proteases also cleave signal peptides from nascent proteins and activating zymogens; in this respect, proteases act as molecular switches to regulate enzyme activity Proteolysis is a thermodynamically favorable and irreversible reaction; therefore, protease activity is tightly regulated to avoid uncontrolled proteolysis through temporal and/or spatial control mechanisms and compartmentalization (e.g. proteasomes, lysosomes) 42 Degradative Proteolysis: Coupled Unfolding and Degradation Doyle et al., Nature Rev. Mol. Cell Biol., 2013.  Degradation in Eukaryotes Lysosomal (extracellular) protein degradation – Protein degraded by lysosomal enzymes Cytosolic (intracellular) protein degradation – The ubiquitin proteasome pathway 44  2004 Noble Prize Chemistry Avram Hershko Aaron Ciechanover Irwin Rose For the discovery of ubiquitin-mediated protein degradation. 45  Ubiquitin-Mediated Degradation A selective, ATP-dependent pathway for degradation: the ubiquitin-mediated pathway Ubiquitin is a highly-conserved, 76 residue protein in all eukaryotes Proteins are committed to degradation by conjugation with ubiquitin 46 Ubiquitin-Proteasome System 47  Ubiquitin 76 amino acids, 8.5 kDa protein Very stable Folds into a compact globular structure Found in all eukaryotic cells Human and yeast ubiquitin share 96% sequence identity Involved in many cellular processes 48  Ubiquitin and Degradation Three proteins involved: E1, E2 and E3 E1 is the ubiquitin-activating enzyme - it forms a thioester bond with C-terminal Gly of ubiquitin Ubiquitin is then transferred to a Cys of an E2, the ubiquitin-carrier (ubiquitin-conjugating) protein Ubiquitin ligase (E3) selects proteins for degradation; the E2-S~ubiquitin complex transfers ubiquitin to these selected proteins More than one ubiquitin may be attached to a protein target: polyubiquitination 49 The Ubiquitination Cascade 50  E1 Activating Enzyme Two step catalysis First: E1 activates C-terminus of Ub by forming acyl-adenylate intermediate (ATP dependent) Second: catalytic Cys residue then forms thio-ester with Ub Then transfer of activated Ub to E2 51 E2 Ubiquitin Conjugating Enzyme Accepts activated Ub from E1 and carries it to the substrate Also forms a thioester with Ub There are many more E2s than E1s Each E2 pairs with a specific set of E3s 52  E3 Ubiquitin Ligases O ubiquitin C S Cys E2 + H 2N Lys protein to be degraded E3 (Ubiquitin-Protein Ligase) O ubiquitin C N Lys protein to be degraded + HS Cys E2 H Final target selection and specificity Position an activated Ub near Lys of a substrate A Ubiquitin-Protein Ligase (E3) promotes transfer of ubiquitin from E2 to the є-NH2 group of a Lys residue of a protein recognized by that E3, forming an isopeptide bond There are many distinct ubiquitin ligases with differing substrate specificity 53 26S Proteasome: a Destruction Machine Regulatory particle (19S) Core particle (20S) Regulatory particle (19S)  The Proteasome Dedicated protein degrading machine ATP-dependent protease Constitutes nearly 1% of cellular protein Present in the cytosol and the nucleus Consists of a central hollow cylinder (20S) that harbors the catalytic sites Ends of the cylinder are associated with the 19S cap Function of the 19S cap: substrate recognition, unfolding, deubiquitination 55  Proteasomal Degradation Proteasomal degradation of particular proteins is an essential mechanism by which cellular processes are regulated, such as cell division, apoptosis, differentiation and development E.g., progression through the cell cycle is controlled in part through regulated degradation of proteins called cyclins that activate cyclin-dependent kinases Proteasome inhibitors cause cell cycle arrest when added to rapidly dividing cells The potential use of proteasome inhibitors in treating cancer is being investigated 56