Posttranslational Protein Modifications PDF

POSTTRANSLATIONAL PROTEIN MODIFICATIONS (József Tőzsér, 2024) Protein synthesis As it has been described in the previous lectures, gene expression involves transcription and translation with multiple regulatory aspects, nevertheless, to obtain functional proteins, pos...

POSTTRANSLATIONAL PROTEIN MODIFICATIONS (József Tőzsér, 2024) Protein synthesis As it has been described in the previous lectures, gene expression involves transcription and translation with multiple regulatory aspects, nevertheless, to obtain functional proteins, posttranslational modifications are required. Posttranslational modifications may include addition of a small molecule to the proteins, while in proteolytic posttranslational modifications the protein undergoes a limited proteolytic fragmentation. Posttranslational modifications Posttranslational modifications (PTM) are covalent processing events that alter the characteristics of a given protein. In a typical addition-type posttranslational modification a reactive amino acid side chain or a protein N-terminal amino group reacts with an activated molecule (e.g. ATP, Acetyl-CoA) or with a reactive metabolite (e.g. glucose). A posttranslational modification may occur enzymatically (like phosphorylation, acetylation) or non- enzymatically (like protein glycations, amino acid side chain oxidations). They could be reversible (like phosphorylation-dephosphorylation) or irreversible (like proteolysis). Those modifications that require coenzymes, ATP or activated metabolites could only occur intracellularly, while others may also occur in the extracellular space or in the bloodstream. Many but not all posttranslational modifications have regulatory functions. Enzyme-catalyzed addition-type posttranslational modifications One of the most frequent posttranslational modifications is protein phosphorylation that requires ATP and can be reversed by the action of phosphatases. A good example of protein acetylation is the modification of histone proteins to relax chromatin structure. The result of protein acetylation is reversed by deacetylases. Ubiquitination is a very common posttranslational modification as described in the Protein Degradation chapter. Ubiquitin can be removed by deubiquitinating enzymes (Dub). Most of the protein methylases use S- adenosyl-methionine (SAM) as methyl donor, as described in the Amino Acid Metabolism chapter. Palmitoylation attaches a lipid moiety to the protein providing a hydrophobic tail for membrane interactions. While N-glycosylation is co-translational, O-glycosylation occurs posttranslationally in the Golgi apparatus. Non-enzymatic addition-type of posttranslational modifications. The spontaneous oxidation of proteins may occur on a variety of side chains. Succinylation occurs on cysteine residues. Glycation may occur on Lys and Arg side chains as well as at the amino-terminus of a given protein. Proteolysis Proteolysis is an enzymatic hydrolysis of peptide bonds. Enzymes specialized for this catalysis are named as proteases and they are widely distributed in nature, where they perform a variety of functions. If only one or a few peptide bonds are cleaved by the proteolytic enzyme, by a process called limited proteolysis, the product protein may acquire new functions. This type of limited proteolysis could be considered as a type of posttranslational modification. However, if a number of sites are cleaved, then the process yields degradation, and this process is rather part of the protein turnover (see later). Classification of proteolytic enzymes The classification of proteolytic enzymes (proteases, peptidases) is unique as it is not done based on the conventional approach referring to the catalyzed reaction as it is the same (peptide bond hydrolysis) in all cases. One way of the grouping is based on the region where the cleavage takes places. Exopeptidases cleave near the N- or C-termini of peptides and proteins. Among exopeptidase further subclassification is made: Aminopeptidases release an N-terminal amino acid of a protein. Dipeptidyl- or tripeptidyl-peptidases release di- or tripeptides from the N-terminus of a protein. Carboxypeptidases release a C-terminal amino acid of a protein. Peptidyl-dipeptidases release a dipeptide from the C-terminus of a protein substrate. Unlike exopeptidases, endopeptidases (proteinases) cleave internal peptide bonds in peptides and proteins. Biological roles of proteolytic processing Digestive enzymes (trypsinogen, chymotrypsinogen, proelastase, procarboxypeptidases) are synthesized in inactive precursor forms and are proteolytically activated after their secretion into the duodenum. Sequential activation of protease blood coagulation factors provides an enormous amplification, which results in the conversion of soluble fibrinogen into insoluble fibrin in its last step. Collagen is synthesized and secreted in the soluble procollagen form. It is proteolytically cleaved in the extracellular matrix by procollagen peptidase. Cells migrating through the basal membrane (macrophages, metastasizing tumor cells) synthesize and secrete proteolytic enzymes to degrade the proteins of the extracellular matrix. These cells contain protease receptors on their surface, which are able to bind the proteases produced by the cells. Most of the oligo- and polypeptides in the body are synthesized as part of larger proteins, and only a few are made by de novo synthesis. Several peptide hormones (e.g. ATCH) are produced as part of a larger protein. Proteolytic processing of larger viral proteins is an essential step in the life cycle of several pathogenic viruses. For example, functional proteins and the enzymes coded by the HIV, the virus causing AIDS, are synthesized in polyprotein forms and processed by the virus-encoded protease. Proteolysis in also involved in signal transduction. For example, the activity of calpains having calmodulin domains 2+ is regulated by Ca ions, while caspases, proteases capable of cleaving at aspartate residues, have a crucial role in programmed cell death and immune responses. The receptor of some proteolytic enzymes (e.g. thrombin) is proteolytically cleaved when the enzyme binds to it, triggering signal transduction. The action of proteolytic enzymes may take place in several cellular compartments as well as extracellularly. An important feature of the proteolytic activation is that it may occur outside the cells, since (the peptide bond hydrolysis) does not require ATP. Secreted Proteins An important pathway where proteolytic action is certainly involved is the secretory pathway of proteins. Secreted proteins are often synthesized in a prepro- form. The pre-part (signal peptide) targets these proteins to the endoplasmic reticulum (ER) and is cleaved off during transport to the rough ER. However, the form of these enzymes lacking the signal peptide part is still inactive, and called as zymogen (proenzyme). Its activation requires further proteolytic step(s) in which a proteolytic enzyme activates these proteins by cleaving them at one or a few places (limited proteolysis). These activating steps usually occur either in the Golgi or in the extracellular fluid. Protease inhibitors Tissues and cells producing proteases usually also synthesize protease inhibitors to keep the proteolytic enzymes under strict control. Protease inhibitors can be classified by various ways; the most generally used distinction is based on their specificity. Some protease inhibitors block the activity of only one specific, or a very few highly similar enzymes. An example for such inhibitors is the plasminogen activator inhibitor (PAI), which block the key enzymes (plasminogen activators) responsible for the initiation of fibrin dissolution. Other inhibitors, like α 2-macroglobulin of the blood or soybean trypsin inhibitor (SBTI) have broad specificity. Basis of inhibitor action is "trapping" of the enzyme. The structure of several protease-inhibitor complexes has been determined by X-ray crystallography. -13 Pancreatic trypsin inhibitor binds very strongly to trypsin (Kd = 10 M). The inhibitor is a very efficient substrate analog. It contains a Lys15-Ala16 sequence, and the side chain of this Lys fits to the substrate-binding pocket. The structure is essentially unchanged on binding to the enzyme. Members of the large family of serine proteinase inhibitors (serpins) in blood act as molecular mousetraps. Examples of serpins: antithrombin-III (AT-III), α1- antitrypsin. First, the exposed reactive center loop of the serpin is recognized by the protease, and an initial noncovalent complex is formed. Than attack by the protease active site Ser residue on the serpin “bait” peptide bond leads to an acyl-enzyme intermediate. This covalent complex becomes trapped by the release of the newly formed N-terminus, followed by an extensive conformational change and disruption of the protease structure..