Syhthesi_PTM_MON 23_ΣΕΠΤ_2024.pptx

Protein synthesis and post translational modifications D. Mangoura 1/26  ~ 25000 genes ? Proteins ? D. Mangoura, M.D., Ph.D.  DNA 🡺 RNA 🡺 Protein 25000 The finding that the human g...

Protein synthesis and post translational modifications D. Mangoura 1/26  ~ 25000 genes ? Proteins ? D. Mangoura, M.D., Ph.D.  DNA 🡺 RNA 🡺 Protein 25000 The finding that the human genome contains genes fewer genes than previously predicted might be compensated for by combinatorial diversity generated at the level of postranslational modification of proteins J. C. Venter, et al., 2001, Science, 291:1304 D. Mangoura 3/26  1953: 1st sequence (bovine insulin) 1986: 4000 sequences 2006: 3.5 million sequences Where will it stop? 179'000'025'042 1st estimate: ~30 million species (1.5 million named) 2nd estimate: 20 million bacteria/archea x 4'000 genes 5 million protists x 6'000 genes 3 million insects x 14'000 genes 1 million fungi x 6'000 genes 0.6 million plants x 20'000 genes 0.2 million molluscs, worms, arachnids, etc. x 20'000 genes 0.2 million vertebrates x 25'000 genes D. Mangoura 4/26  Protein data banks UniProtKB/Swiss-Prot: (seqs are manually annotated and reviewed) – In July 2009: 500,000.00 entries; – In 2013: 1 million entries; – In 2026 (40th anniversary): 10 million entries; – In 2036 (50th anniversary): 100 million entries. UniProtKB/TrEMBL: – In May 2080 TrEMBL will have reached 10 billion entries – We can’t compute with Excel when we will reach 179 billion entries D. Mangoura 5/26  Πρωτεΐνες Proteins John J. Berzelius, 1838 Enzymatic catalysis Transport and storage Coordinated motion Mechanical support Immune protection Nerve impulses Control of growth and differentiation D. Mangoura 6/26  Proteins are designed, to bind every conceivable molecule – Simple ions – Fats – Sugars – Nucleic acids – Other proteins! Carry out their functions efficiently and under precise control – Evolution of their three-dimensional structure D. Mangoura, M.D., Ph.D.  Protein synthesis Protein synthesis is called translation because information present as a nucleic acid sequence is translated into a different language, the sequence of amino acids in a protein D. Mangoura, M.D., Ph.D.  Proteins facts Function is derived from its 3-dimensional structure and, this 3-D structure is specified by amino acid sequence Form and function are inseparable in protein architecture D. Mangoura, M.D., Ph.D.  Mature mRNA production nucleus DNA Transcription pre-mRNA x x x x mature mRNA cytosol translation amino acids protein Introns are removed by a process called splicing  Alternative splicing and alternative transcripts The exons in some genes are not utilized in the same way in every cell or stage of development; exons could be skipped or added = alternative splicing RBPs pre-mRNA Intron 2 Int3 Int4 Intron 1 exon 1 exon 2 exon 3 exon 4 exon 5 1+2+3+4+5… alternate alternate 1+2+3+5… Genetic Variation! 1+3+4+5… This means that variations of a protein (called isoforms) can be produced from the same gene One particular Drosophila gene (Dscam, the Drophila homolog of the human Down syndrome cell adhesion molecule DSCAM) can be alternatively spliced into 38,000 different mRNA.  For some genes splicing is very complex Fibrillin is a protein of the connective tissue. Mutations in its encoding gene FBN1 cause Marfan Syndrome: long limbs, crowned teeth elastic joints, heart problems and spinal column deformities. The protein is 3500 aa, and the gene is 110 kb long made up of 65 introns Titin has 175 introns With these large complex genes it is difficult to identify all of the exons and introns D. Mangoura, M.D., Ph.D. D. Mangoura, M.D., Ph.D.  Importance of protein synthesis: toxins I: Toxins that target the ribosome - Ricin, extremely potent protein toxin produced by the castor bean plant seed and no known antidote. A single molecule will inactivate almost 2000 ribosomes within a minute II: Toxins that target eukaryotic elongation factor 2 (It promotes the GTP-dependent translocation of the ribosome) – Diphtheria toxin and Pseudomonas exotoxin A Diphtheria (Corynebacterium diphtheriae) has a mortality rate of 5-10%. Diphtheria toxin (DT) is one of the most studied and best-understood bacterial toxins. This toxin and Pseudomonas exotoxin A target eukaryotic elongation factor 2 (eEF-2) by covalent modification of diphthamide, a D. Mangoura, M.D., Ph.D.  Importance of protein synthesis: antibiotics Ribosomes are a major target for natural and synthetic antibiotics Tetracycline, a good example of drugs that bind directly to the prokaryotic ribosome (the 30S subunit) Chloramphenicol and other antibiotics target the 50S ribosomal subunit. Besides being of clinical importance, antibiotics are powerful probes to study ribosome structure and function for rational design of new antibiotics to overcome the increasing problem of resistance against currently used antibiotics Aminoglycoside antibiotics - affect translational fidelity. Many human diseases are caused by nonsense mutations (premature termination codons) that generate truncated, functionally inactive proteins. One example is Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene; the absence of dystrophin in striated muscle cells cause severe weakness of muscles. At present, there is no cure for DMD. D. Mangoura, M.D., Ph.D.  The Making of a protein hydrophobic cores salt bridges hydrogen bonds disulfide bonds Protein complex hydrogen bonds posttranslational modifications D. Mangoura, M.D., Ph.D. D. Mangoura, M.D., Ph.D.  Signal transduction pathways control all major cellular processes Extracellular space In Signal transduction Series of protein phosphorylations Protein mRNA gene translation transcription nucleus D. Mangoura 19/47  Same agonist, same receptor, different biological outcomes… Extracellular space Plasma membrane in Differentiation Cell cycle Proliferation + Dedifferentiation Apoptosis Differentiation Proliferation + Apoptosis + Differentiation Proliferation D. Mangoura 20/49  Signal transduction pathways change the proteotype of the cell Modification of existing protein species! x Signal transduction Series of protein phosphorylations Extracellular space In DNA Changes in protein Protein mRNA gene translation transcription dosage! nucleus D. Mangoura 21/47  Anatomy of proteins D. Mangoura, M.D., Ph.D.  Anatomy of an amino acid D. Mangoura, M.D., Ph.D.  aspartic Non-polar = R: either aliphatic or aromatic groups; glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; Hydrophobic! Polar No charge = R: range of functional groups with at least one atom (N, S, O) with electron pairs available for hydrogen bonding to water; serine, cysteine, threonine, tyrosine, asparagine, and glutamine; Hydrophilic! Negatively charged (acidic aa), polar = R: a carboxylic acid that gives it acidic (proton-donating) properties; aspartic acid and glutamic acid; Hydrophilic! Positively charged (basic a a), polar = R is basic (i.e., can accept a proton); arginine, histidine, and lysine, Hydrophilic! D. Mangoura, M.D., Ph.D.  Anatomy of a peptide Charged and polar R-groups tend to Non-polar R-groups tend to be map to protein surfaces buried in the cores of proteins Myoglobin Blue = non-polar group D. Mangoura, M.D., Ph.D. Red = Heme  Protein structure Post-translational modification Protein Function D. Mangoura, M.D., Ph.D.  Protein Modifications Why bother? Nearly every protein in a cell is chemically altered after its synthesis in the ribosomes Modifications affect: – Activity – Life span – Cellular localization – Function D. Mangoura, M.D., Ph.D.  Protein Modifications Translational and post-translational modification is the way of making a protein something other than what the wild-type gene specified chemical modification of proteins and site- directed mutagenesis Over 200 types of PTM have been identified!!! D. Mangoura, M.D., Ph.D.  Translational and post-translational modification Translational Co- and Post- translational Selenocysteine, the 21st Main chain modification: proteinogenic amino acid 1. proteolytic cleavage + rearrangement and inversion mutations 2. Intein splicing 3. N- and C-terminal modification 4. De/Re Tyrosination 5. Cholesterylation Side-chain modifications: 5. myristoylation, palmitoylation 6. Prenylation 7. Methylation 8. Acetylation 9. phosphorylation 10. O- and N-glycosidation 11. sulfation 12. hypusine, diphthamide, EPG 13. vitamin K- dependent carboxylation 14. Hydroxylation 15. Ubiquitination, sumoylation D. Mangoura, M.D., Ph.D.  Main chain modifications D. Mangoura, M.D., Ph.D.  I. Polypeptide chains are synthesized beginning with formylmethionine, but the formyl group always and indeed the methionine usually, is removed in the mature protein II. The cleavage of a precursor form, called a pro­protein, to yield the final active form, is the most common main chain modification anterior lobe intermediate lobe D. Mangoura, M.D., Ph.D.  III. Excision and splicing of internal protein sequences called inteins which result in proteins with sequence different from that coded for by the gene, PROTEIN SPLICING, PROTEIN INTRONS! Inteins are transcribed & translated together with their host protein Found in organisms of all 3 domains of life: Eucaryotes; Eubacteria; Archaea; and in viral and phage proteins Found in metabolic enzymes, DNA and RNA polymerases, proteases, ribonucleotide reductases… D. Mangoura, M.D., Ph.D.  IV. Tyrosination/detyrosination of the C-terminal tail of α-tubulin Possible deglutamylation Vasohibin (VASH) and small vasohibin- binding protein (SVBP) complex Mutations in SVBP have been linked to microcephaly and intellectual disability Polyglutamylation (or glycine, polyglycylation) D. Mangoura, M.D., Ph.D.  Polyglutamylation, polyglycylation are side chain modifications D. Mangoura, M.D., Ph.D.  V. Protein Cholesterylation A B C Post-translational processing of Sonic Hedgehog (Shh) protein in the ER A. cleavage of the signal peptide from the Shh protein precursor is followed by B. N-terminus palmitoylation by the membrane integral acyl transferase Hhat C. C-terminal autocleavage and cholesterylation to form a cholesteryl ester at the C-terminus D. Mangoura, M.D., Ph.D.  Side chain modifications A. Lipid modification Myristoylation Palmitoylation Prenylation D. Mangoura, M.D., Ph.D.  Lipid modifications – lipidation, acylation = protein + fatty acid D. Mangoura, M.D., Ph.D.  Protein Myristoylation 0.5% of all protein Is fairly specific addition of the C14 length-fatty acid myristate Consensus pattern on the protein sequence G-{EDRKHPFYW}-x(2)-[STAGCN]-{P} 1 2 3,4, 5 6 -The N-terminal residue must be glycine, is the N- myristoylation site - In position 2, uncharged residues are allowed - In positions 3 and 4, most residues are allowed - In position 5, only small uncharged residues - In position 6, proline is not allowed Mostly co-translational Many of the G proteins are myristoylated on the α subunit, and the modification is necessary for binding to the β and γ subunits Myristoylation has been characterized as an electrostatic switch – phosphorylation prevents myristoylated proteins going to their usual membrane site D. Mangoura, M.D., Ph.D.  Protein Palmitoylation Protein palmitoylation, or the addition of palmitic acid (a 16-carbon fatty acid) on sulfur atoms of the side chains of internal cysteine residues, is a reversible post-translational lipid modification A predictive sequence motif has now been identified Palmitoylated proteins include many membrane receptors, ankyrin and vinculin, and the α subunits of many G proteins. Cherezov et al, Science, 2007 D. Mangoura, M.D., Ph.D.  Protein Prenylation Attachment of the C15 isoprenoid farnesyl unit or the C20 isoprenoid geranylgeranyl unit to the sulfur atoms in the side chains of cysteine residues located near (within 5 residues of) the C terminus of the polypeptide chain Protein found to be prenylated, the nuclear envelope proteins lamin A and B, ras, the γ subunit of G-proteins Farnesyltransferase and geranylgeranyltransferase I modify proteins on the cysteine SH of a C terminal sequence CAAX, where A,A are neutral aliphatic amino acids and X is methionine, serine (rarely others); the result is not reversible Prenylation by FPTase or GGPTase I is followed by the removal of the 3 amino acids next to the modified cysteine; the COOH of the cysteine is then methylated. Th protein now has higher affinity for membranes, e.g., the photoreceptor G protein transducing or Ras D. Mangoura, M.D., Ph.D.  Ras 1. Farnesylati on 2. Proteolys is 3. Methylatio n D. Mangoura, M.D., Ph.D.  B. Protein Phosphorylation The most common protein modifications that occurs in animal cells. The vast majority of phosphorylations occur as a mechanism to regulate the biological activity of a protein and as such are transient The enzymes that phosphorylate proteins are termed kinases and those that remove phosphates are termed phosphatases: ATP + protein = phosphoprotein + ADP In animal cells serine, threonine and tyrosine are subjected to phosphorylation. The ratio of phosphorylation of the three different amino acids is approximately 1800/200/1 for serine/threonine/tyrosine. D. Mangoura, M.D., Ph.D.  more than 500! D. Mangoura, M.D., Ph.D.  Kinases and phosphorylation Changes instantly the conformation of the protein Adds negative cotranslationally myristoylated and charges posttranslationally palmitoylated in N-terminus which attract positively charged side chains of aminoacids D. Mangoura, M.D., Ph.D.  Phosphatases: PTP hereditary disease genes D. Mangoura, M.D., Ph.D. D. Mangoura, M.D., Ph.D.  Protein Methylation Post-translational methylation occurs on nitrogens and oxygens (lysine, arginine). The activated methyl donor is S- adenosylmethionine Methylation of lysine residues in histones in the nucleosome is an important regulator of chromatin structure and consequently of transcriptional activity and it is dynamically regulated by demethylases Humans express 27 lysine methyltransferases (KMT family) and 9 arginine methyltransferases. Numerous enzymes catalyze lysine demethylation reactions e.g., Jumonji C demethylases Other methylations of protein carboxyls include that on the imidazole ring of histidine, and the side chain amide of glutamine and asparagine Reversible! D. Mangoura, M.D., Ph.D.  Protein Acetylation Post-translational acetylation of proteins occurs on lysine residues Very often it occurs at the N-terminus, in most of the proteins that the initiator methione remains However, even when the initiator methionine is removed, as is the case for all secreted, transmembrane proteins, and glycoproteins, the protein can still be acetylated It is catalyzed by lysine (K) acetyltransferases (KAT family, 17 humans genes); the activated acetyl donor is acetyl-CoA Due to consumption of Ac-CoA for acetylation and NAD+ for deacetylation by specific KDACs this post-translational processing event is at the crossroads of metabolic regulation Examples, acetylation of histones typically promotes the recruitment of effector proteins, relaxation of chromatin conformation, and an increase in transcription D. Mangoura, M.D., Ph.D.  Hypusine, EPG, and Diphthamide Modification Only known modifications to occur on a single protein ethanolamine phosphoglycerol: eukaryotic elongation factor 1A (eEF1A) hypusine: initiation factor 5A(eIF5A) diphthamide: eukaryotic elongation factor 2 (eEF2) The unusual basic amino acid hypusine is a modified lysine with the addition of the 4-aminobutyl moiety from the polyamine spermidine Diphthamide is a unique amino acid formed by posttranslational modification of a strictly conserved histidine residue on eEF2 EPG protein modification was discovered when mammalian cells were labeled with [3H]ethanolamine, a precursor used to identify GPI-anchored proteins, and chemical and mass spectrometric analyses revealed that all the radioactivity was only associated with a previously unknown EPG moiety. D. Mangoura, M.D., Ph.D.  Sulfation Sulfate modification of proteins occurs at tyrosine residues such as in fibrinogen and in some secreted proteins (eg gastrin). The universal sulfate donor is 3'-phosphoadenosyl-5'- phosphosulphate (PAPS). Since sulfate is added permanently it is necessary for the biological activity, not regulatory Sulfation is central to intracellular signal transduction of chemokine receptors, sulfation modulates cell-cell and cell- matrix communication, blood clotting. D. Mangoura, M.D., Ph.D.  Vitamin K-Dependent Modifications Vitamin K is a cofactor in the carboxylation of glutamic acid residues. The result of this type of reaction is the formation of a g-carboxyglutamate (gamma-carboxyglutamate), referred to as a gla residue Structure of a gla residue The presence of gla residues allows the protein to chelate calcium ions: the formation of gla residues within proteins of the blood clotting cascade is critical for their normal function D. Mangoura, M.D., Ph.D.  Other Protein Modifications Ubiquitination: formation of a thioester bond between the C- terminal glycine of ubiquitin and a cysteine of a protein called E1. It is then passed to a cysteine on another protein, E2 or UBC, and then to a lysine residue of a target protein, a reaction catalyzed by one of a number of ligating enzymes E3. A protein can have many ubiquitins attached; a polyubiquitinated protein is then recognized by a large complex protease, called a proteasome, and degraded (Alberts Fig. 6-87) SUMOylation: SUMO, short for short ubiquitin-like modifier, also called sentrin, attached to proteins like ubiquitin, but it does not lead to proteolysis – indeed SUMOylation can block ubiquitination. D. Mangoura, M.D., Ph.D.  Glycosylation Glycosylation is the enzymatic addition of saccharides to proteins from donor nucleotide sugars. N-Linked glycans are attached in the ER to the nitrogen (N) in the side chain of asparagine in the sequon (an Asn-X-Ser or Asn-X-Thr sequence); the glycan may be composed of N-acetyl galactosamine, galactose, neuraminic acid, N- acetylglucosamine, fucose, mannose, and other monosaccharides. O-Linked glycans are assembled one sugar at a time on the hydroxyl oxygen of a serine or threonine residue of a peptide chain in the Golgi apparatus (no known template sequence yet) Only the correctly folded, get packaged into ER-to-Golgi transport vesicles… D. Mangoura, M.D., Ph.D. D. Mangoura, M.D., Ph.D.  Enzyme availability (genetically, pH, temperature, ammonia, O2…) Glycosyltransferas es … for further processing and addition of GlcNac's to form branched structures in the cis Golgi, and for addition of more sugar residues in the trans-Golgi (I.e. fucose and sialic acid) to produce the diversity that is seen in mature glycans D. Mangoura, M.D., Ph.D.  Branching of complex N-glycans Enzyme availability (genetically+pH, temperature, ammonia, O2…) D. Essentials of Glycobiology 56/47 Genes and biosynthesis, activation, transfer, and recycling pathways involved in the biology of animal sialic acids Essentials of Glycobiology D. 57/47  Taxa, species differences in N-glycan processing pathways Eukaryotes Essentials of Glycobiology D. Mangoura, M.D., Ph.D.  Glycosylphosphatidylinositol (GPI Anchored Protein), or glycophosphatidylinositol, or GPI Neural cell adhesion molecule 120, NCAM120 Neural cell adhesion molecule TAG-1 Ciliary neurotrophic factor receptor (CNTFR) Glial-cell-derived neurotrophic factor receptor (GDNFR) The GPI anchor is assembled on a phosphatidylinositol lipid in the ER by a series of enzymatic reactions and then is covalently attached to the carboxyl terminus of proteins – presence of a hydrophobic signal sequence that triggers the addition of the GPI anchor D. 59/47  The A and B alleles of the ABO gene express enzymes with glycosyltransferase activities that differ, adding either N-acetyl galactosamine or glucosamine only to the H antigen D. Mangoura 60/47  Glycosylation versus Glycation, Advanced glycation end products (AGEs) D. Mangoura, M.D., Ph.D. D. Mangoura, M.D., Ph.D.  Diseases caused by mutations of enzymes for glycosylation D. Mangoura, M.D., Ph.D. D. Mangoura, M.D., Ph.D. D. Mangoura, M.D., Ph.D. D. Mangoura, M.D., Ph.D. D. Mangoura, M.D., Ph.D. D. Mangoura, M.D., Ph.D.  Prions: Proteinaceous Infectious Particles 1996 Nobel Prize in Medicine & Physiology – Stanley Pruisner Prion Diseases – Spongiform Encephalopathies Scrapie:sheep, goats Transmissible mink encephalopathy Chronic wasting disease of mule deer and elk Feline spongiform encephalopathy Bovine spongiform encephalopathy D. Mangoura, M.D., Ph.D.  Human Prion Diseases Kuru Creutzfeldt-Jacob disease Gerstmann Straussler-Sheinker disease Familial fatal insomnia Prion Properties Transmissible No nucleic acid component Species barriers Protein present in normal cells D. Mangoura, M.D., Ph.D.  Protein conformation changes underlie prion formation Prions, Commonly denoted PrPSc , can template their conformation to cellular PrP D. Mangoura, M.D., Ph.D. D. Mangoura, M.D., Ph.D.  Why purify a protein? To study its function To analyze its physical properties To determine its sequence For industrial or therapeutic applications Proteins vary in size, charge and water solubility, therefore no single method can be used 300,000 – 3,000,000,000 proteins (estimate) in the cell D. Mangoura, M.D., Ph.D.  Key approaches to purifying and analyzing proteins Centrifugation/Ultracentigugation Electrophoresis (one- two-dimensional electrophoresis) Liquid Chromatography (gel filtration, ion-exhange, affinity) In vivo localization: antibodies, fusion proteins, fluorescent probes Recombinant DNA technology D. Mangoura, M.D., Ph.D.  Cell lysis Cell lysis: rupture cell wall / plasma membrane, --> release contents (organelles, proteins…) 1. Physical means 2. Sonication 3. Osmotic shock D. Mangoura, M.D., Ph.D.  Differential Centrifugation Cell fractionation by centrifugation. 1000 Repeated centrifugation at progressively higher speeds will fractionate homogenates of cells into their components. 10,000 In general, the smaller the subcellular component, the greater is the centrifugal force required to sediment it. 20,000 100,000 D. Mangoura, M.D., Ph.D.  Zonal centrifugation D. Mangoura, M.D., Ph.D.  Protein purification – column chromatography - Protein mixture applied to column - Solvent (buffer) applied to top, flowed through column - Different proteins interact with matrix to different extents, flow at different rates - Proteins collected separately in different fractions D. Mangoura, M.D., Ph.D.  Three types of matrices used for chromatography Charge size specific binding (A) ion-exchange chromatography the insoluble matrix carries ionic charges that retard the movement of molecules of opposite charge. Matrices: (+) diethylaminoethylcellulose (DEAE- cellulose); (-) carboxymethylcellulose (CM-cellulose) and phosphocellulose. (B) In gel-filtration chromatography, the matrix is inert but porous. Molecules that are small enough to penetrate into the matrix are thereby delayed and travel more slowly through the column. Beads of cross-linked dextran, agarose, or acrylamide are available in a wide range of pore sizes, making possible the fractionation of molecules of Mr of 500 to more than 5 × 106. (C) Affinity chromatography uses an insoluble matrix that is covalently linked to a specific ligand, such as an antibody, that will bind a specific protein; immobilized substrates on such columns can be eluted with a concentrated solution of the free form of the substrate molecule, while molecules that bind to immobilized antibodies can be eluted by dissociating the antibody-antigen complex with concentrated salt solutions or solutions of high or low pH. Figure 8-11. D. Mangoura, M.D., Ph.D.  Protein isolation by Immunoprecipitation D. Mangoura, M.D., Ph.D.  Protein analysis, SDS polyacrylamide-gel electrophoresis (SDS-PAGE) Figure 8-14. (A) An electrophoresis apparatus. (B) Individual polypeptide chains form a complex with negatively charged molecules of sodium dodecyl sulfate (SDS) and therefore migrate as a negatively charged SDS-protein complex through a porous gel of polyacrylamide. Because the speed of migration under these conditions is greater the smaller the polypeptide, this technique can be used to determine the approximate molecular weight of a polypeptide chain as well as the subunit composition of a protein. D. Mangoura, M.D., Ph.D.  SDS polyacrylamide-gel electrophoresis and Coomassie-staining gel D. Mangoura, M.D., Ph.D.  after SDS-PAGE, 1, Western bloting D. Mangoura, M.D., Ph.D.  Cortactin’s developmental regulation is retained during differentiation of telencephalic neurons in culture Embryonic 6 8 10 1 2 13 14 15 18 day 20 Day1 p85 cortactin p80 p80 cortactin cortactin MW M vinculi n p80 p85 cortactin cortactin p80 Culture cortactin day 1 2 3 5 Neuronal lysate were collected at indicated days in culture and analyzed by Western blotting with the monoclonal 4F11 against cortactin. Vinculin expression was developmentally regulated (arrowhead) Cheng et al., J. Cell Sci. 2000 D. Mangoura, M.D., Ph.D.  Example: EGF transiently activates Raf-B and ERK PC12 cells were solubilized in RIPA; Raf-B abundance and phosphorylation was analyzed by Western blotting with a polyclonal antibody to Raf-B and ECL. Phosphorylation of Raf-B is evident from the retarded in gel mobility. ERK activity was analyzed by Western blotting with a monoclonal antibody against ERK1/2 and ECL. Cheng et al., J. Biol. Chem. 2000 D. Mangoura, M.D., Ph.D.  after SDS-PAGE, 2, autoradiography D. Mangoura, M.D., Ph.D.  Example: PKC phosphorylates Neurofibromin in vivo PKCalpha PKCepsilo n Mangoura et al., 2006 Oncogene D. Mangoura, M.D., Ph.D.  Protein imaging by Immunocytochemistry D. Mangoura, M.D., Ph.D.  CTD-GFP localizes in the nucleus Endogenous CTD-GFP D. Mangoura, M.D., Ph.D.  Antibodies in the study of proteins Westen Blotting Immunoprecipitation Immunocytochemistry ELISA Flow cytometry D. Mangoura, M.D., Ph.D.  Ta eipa ola ektos ta prion kai tis methodous! D. Mangoura, M.D., Ph.D.  Complex N-glycans size rivals the size of the polypeptide Fc Stabilization of the CDR (complimentarity- determining region) surface D. Mangoura, M.D., Ph.D.  Importantly, the populations of sugars attached to each glycosylated asparagine in a mature IgG will depend on Enzyme availability (genetically+pH, temperature, culture duration [for cells in culture], ammonia, O2…) in other words on, the genotype and the physiological status of the cell D. Mangoura, M.D., Ph.D.  Structure-function of the LA5 neutralizing Fab and the surface antigens D8 LA 5 Footprint of LA5 on the D8 epitope of the vaccinia virus D Matho et al J. Virol, D. Mangoura, M.D., Ph.D. 2012  IgG, co-translational and post-translational modifications, continued IgG Modificatio regulate: ns Glycosylation ADCC, CDC, PK, immunogenicity Mostly of methionine, - Oxidation alters surface charge Asn🡪IsoAsp+Asp (3:1) - Deamidation impacts antigen binding (potency) and degradation Inter-chain disulfide bonds - scrambling: changes in hydrodynamic may Disulfide size (CDR) reduction: half IgG body (S- occur modification H), fragmentation (S-R)(potency, in vivo clearance), and aggregation (free SH) in culture Non-enzymatic addition of sugars 🡪 AGE- in vitro interdepende Glycation IgG- ntly! 🡩🡩🡩 immunogenicity D. Mangoura, M.D., Ph.D. Manufacturing an IgG Post-translational modifications of Enzyme Propagat IgGs dependavailability on (genotype) pH, temperature, culture duration e ammonia, O2, nucleotide-sugars r t se availability In Pu fy ri plasmid vector transfect E.Coli to bacteria mammalia IgG encoding nSccells DNA a cu up le ltu re a s ul o rm F te Puri Charact fy erize D. Mangoura, M.D., Ph.D.  Thus, a biological drug = protein + process D. Mangoura, M.D., Ph.D. Manufacturing a biosimilarPropagat e r t se In Pu fy ri Plasmid vector? Strain? CHO Karyotype Same aa sequence, ? but , same genetic sequence? D. Mangoura, M.D., Ph.D. Manufacturing a biosimilarPropagat e r t se In Pu fy ri Plasmid vector? Strain? CHO Karyotype Same aa sequence, Sc ? but , a Cu up le same genetic co lt , sequence? a nd u r e ul ns iti rm ? ? o o F ion t Purificatio Characterizat n? ion? D. Mangoura, M.D., Ph.D.  Recombinant proteins D. Mangoura, M.D., Ph.D.  Complex N-glycans size rivals the size of the polypeptide Fc Stabilization of the CDR (complimentarity- determining region) surface D. Mangoura, M.D., Ph.D. D. Mangoura 102/26 D. Mangoura, M.D., Ph.D. D. Mangoura, M.D., Ph.D.