Biosynthesis and degradation of proteins Bruno Sopko

Content Proteosynthesis Post-translation processing of proteins Protein degradation

Proteosynthesis Aminoacyl-tRNA formation Iniciation Elongation Termination

Aminoacyl-tRNA formation Amino acid + ATP ↔ Aminoacyl-AMP + PPi Aminoacyl-AMP + tRNA ↔ Aminoacyl-tRNA + AMP Each amino acid has „its own“ tRNA and aminoacyl-tRNA synthetase (ARS) Reactions are cytosolic Errors are corrected by specific correcting enzymes ARS have other enzyme activity (other signaling molecule?)

Other ARS functions

Proteosynthesis Iniciation

Elongation

Termination

Post-translation processing of proteins Secondary structure and role of the chaperons Proteolytic modifications Glycosylation Other modifications (hydroxylation, phosphorylation, acetylation, methylation, carboxylation)

Protein transfer after translation

Contranslational translocation

Contranslational translocation –transmembrane proteins

Transmembrane proteins - examples Type I – glycophorin, LDL receptor, influenza HA protein, insulin receptor, growth hormone receptor … Type II – transferrin receptor, influenza HN protein, Golgi sialyltransferase, Golgi galactosyltranferase … Type III – cytochrome P450 … Type IV – G-protein, glucose receptors (GLUT 1 …), connexin, voltage gated Ca2+ channel …

Secondary structure

Secondary structure –HSP70 chaperon cycle

Secondary structure – GroEL/GroES system

Secondary structure – overview

Protein disulfid isomerase (PDI) and peptidyl prolyl cis-izomerase PPI:

Proteolytic modification - insulin

N-Glycosylation

Other modifications O-glycosylation Hydroxylation (hydroxyproline, hydroxylysine) Methylation (mono- , di- and even trimethyllysine) PHOSPHORYLATION Carboxylation (γ-carboxyglutamate, vitamin K, fibrinogen) Acetylation ……..

Protein degradation Proteases Protein degradation systems Ubiquitin and proteasome Activation of proteases Protease inhibitors

Proteases Serine proteases (trypsin, chymotrypsin, elastase ….) Aspartate proteases (pepsin, some proteases found in lysosomes, renin, HIV-protease …) Metalloproteases (carboxypeptidases, various matrix metalloproteases …) Cysteine proteases (papain, cathepsins, caspases, calpains …)

Protein degradation systems Vacuolar (lysosomes, endosomes, ER, …) Ubiquitin pathway (proteasome)

Ubiquitin pathway

Activation of proteases Most proteases are synthesized as larger pre-proteins. During activation, the pre-protein is cleaved to remove an inhibitory segment. In some cases activation involves dissociation of an inhibitory protein Activation may occur after a protease is delivered to a particular compartment within a cell or to the extracellular milieu. Caspases involved in initiation of apoptosis are activated by interaction with large complexes of scaffolding and activating proteins called apoptosomes.

Protease inhibitors IAPs are proteins that block apoptosis by binding to and inhibiting caspases. The apoptosis-stimulating protein Smac antagonizes the effect of IAPs on caspases. TIMPs are inhibitors of metalloproteases that are secreted by cells. A domain of the inhibitor protein interacts with the catalytic Zn2+.  Cystatins are inhibitors of lysosomal cathepsins. Some of these (also called stefins) are found in the cytosol and others in the extracellular space. Cystatins protect cells against cathepsins that may escape from lysosomes. Serpins are widely distributed proteins that utilize a unique suicide mechanism to inhibit serine or cysteine proteases. A large conformational change in the serpin accompanies cleavage of its substrate loop. This leads to disordering of the protease active site, preventing completion of the reaction. The serpin remains covalently linked to the protease as an acyl-enzyme intermediate. Non-specific: α2-macroglobulin

Fate of the protein

Literature Marks´ Basic Medical Biochemistry, A Clinical Approach, third edition, 2009 (M. Lieberman, A.D. Marks) B. Wilkinson, H.F. Gilbert / Biochimica et Biophysica Acta 1699 (2004) 35–44 F. Ulrich Hartl, Andreas Bracher & Manajit Hayer-Hartl, Molecular chaperones in protein folding and proteostasis, Nature 475 (2011)