MUNI SCI Bi4025en Molecular Biology Mgr. Jiří Kohoutek, Ph.D. 1 Department of Experimental Biology Lecture 6 • Posttranslational processing of proteins. 2 Department of Experimental Biology MUNI SCI Generation of maturated functuional protein Newly svnthetized propeptide chain Folding. o Noncovalent binding of cofactors. Covalent modifications o Glycolsylation, phosphorylation, acetylation, etc. Assembly o Noncovalent binding of other protein subunits/partners. o Maturated functional protein. 3 Department of Experimental Biology MUNI SCI Post-translation modifications are key to proteome diversity • Genome comprises 20,000 to 25,000 genes. • Changes at the transcriptional and mRNA levels increase the size of the transcriptome relative to the genome. Myriad of different post-translational modifications exponentially increases the complexity of the proteome relative to both the transcriptome and genome . The proteome is estimated to encompass over 1 million proteins. -2(}-25r0D0 genes AAA Alternative promoters Alternative Splicing mRNA editing Transcriptome -100,000 transcripts Post-transfational modifications Protecme si,000,000 proteins SP 4 Department of Experimental Biology https://www.thermofisher.com/cz/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/ove rview-post-translational-modification.html MUNI SCI Interactions in an Organism Compose the Interactome Proteome: • Complete set of proteins produced by genetic material of an organism. Interactome: • Complete set of protein interactions in an organism. : .....; ■ AN roArt W> J* 3" f. Vi * i UDP UDP and/or O-CMP CMP • The glucose residues are sequentially removed by two a-glucosidases (a-GIc l-ll) and an initial Man residue is removed by the ER a-mannosidase (ER a-Man). • After a quality-control checkpoint, the glycoprotein moves to the Golgi apparatus for additional trimming by a-mannosidase I and II (a-Man l-ll) and further glycan modifications. • A cis-to-trans distribution of glycosidases and transferases facilitates further processing by these carbohydrate-modifying enzymes to create a plethora of N-glycoforms that often terminate with sialic acid moieties. Nature Reviews Nephrology, 2019, volume 15, pages 346-366. Wl U 111 I 18 Department of Experimental Biology https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/N- O n T linked_Protein_Glycosylation_Begins_in_the_ER O L» 1 • Post-translational modifications MUNI 19 Department of Experimental Biology _ _ T O 0 J. Post-translational modifications - PTMs phosphorylation ubiquitiria'ron P ost-tra n slational modifications 5UMOyla1;on More than 300 PTMs are currently known. Addition of chemical groups (e.g. phosphate or acetate). Addition of complex molecules (e.g. carbohydrates or lipids). The covalent linkage of small proteins (like ubiquitin, ubiquitin-like proteins (UBLs), sumo). Cleavage and Splicing. Modification of specific amino acids (like deamidation or eliminylation). Department of Experimental Biology Journal of Experimental Botany, Volume 69, Issue 19, 31 August 2018, Pages 4499-4503 vbiquifiu-iifte proteolysis deamidation eliniiiiylation AMPytation ADP-riiWSViS'iori glycosyldtion scylfltion pre relation MUNI SCI Post-translational modification are carried out by enzymes Phosphorylation [X = Ser, Tyr, Thr) I Eliminylation {X = Thr) kinase ATP ADP phosphate (protein^ phosphatase phospholyase Pi J_ Acetylation {X = Lys, Ser, Thr) acetyltransferase acatyl-coA CPA acetate i X (protein j ( protein). (protein^ acetate H30 deacetylase AMPylation (X =Tyr, Thr) AMPylator amp „ ATP PP ' (protejnj A ^ ^ * (protein j AMP KjO AMP hydrolase ADP-ribosylation (X = Arg. Cys, Asn) ADP-ribgayltranataraae ADp.riDosa NAD nicotinamide ý Z^" (protein) A (protein). ADP-ribose H.O ADP-ribosylhydrolase Deamidatton 1 Polyamina deamidase NH. H,0 Glu polyamine transferase polyamine NH, (protein) polyamine Gin " Gin i (protein) —i^-i—♦ ^protein^ Glucosylation (X = Thr, Ser) glucosyltransferase a|UCOSe v UDP-glucose UDP ' (protein) « * f^protein^ glucose H.O glucosylhydrolase Ubiquitylation / UBL conjugation (X = Lys) Ubi/UBL conjugation machinery ykifUBL Ubi/UBL + ATP AMP+PP (protein) ^1 IT UbifUBL (protein) H,0 Ubi/UBL protease Proteolysis (protein) protease -I pro + 21 Department of Experimental Biology FEBS Letters, Volume 584, Issue 13, 2 July 2010, Pages 2748-2758 MUNI SCI Post-translational modification are carried out by enzymes 0 o-s-o" 1 o K n Tyrosine sulfation h\ 0 Lysine acetyiahon D \ HN , HO, 5f* Glycosylation HN Lysine ubtqui'.iaaton -N-^V 'UN'Y' "^N^V Lysine metrtylafcon H^v^ HjAvnc -"NV^ HN HN Hf'. ""TIT ""«T "K o Argmine methytabon 22 Department of Experimental Biology Phospho kinase Tyrosine phosphatase Ubiquitin ligase Deubiquitylase/deneddylase AMPylator ADP-ribosyl transferase Acetyltransferase Deacetylases Methyl transferase Demethylase Etc... MUNI SCI Phosphorylation Principally on SERINE, THREONINE or TYROSINE residues. Also known as Phospho regulation. Critical role in cell cycle, growth, apoptosis and signal transduction pathways. O-phosphorylalion at serine residue .OH H 23 Department of Experimental Biology 0 0=P-0* ATP ADP ^0 \ / , H Non-Phosphorated Protein [ h ATP Ser-Thr Kinase ADP Pi Ser-Thr Phosphatase -P phosphorated Proton MUNI SCI Phosphorylation affects protein capability TRENDS rw Pharmacological Sciences 24 Department of Experimental Biology MUNI SCI Phosphorylation — y.-rl70##Thfl76 Cycürt H) tCAK1 phtuphalase 9 t Thi-161 TyilS ThTl&l $0 Inactive pnp-kinase ftclivE* kinase (MPFF O O Npwfjr made cvclln B IníiHvB Olui O o o o 00C J • As cyclin B is synthesized during S and G2 phases of the cell cycle, it associates with Cdc2. • Active CDK-activating kinase (CAK) phosphorylates Cdc2 at threonine 161, stabilizing its association with cyclin B • Wee1 and Myt1 phosphorylates inhibitory sites, threonine 14 and tyrosine 15. • Final activation is triggered by dephosphorylation of Thr14 and Tyr15 by CDC25 phosphatase. 25 Department of Experimental Biology Current Biology, Volume 5, Issue 1, January 1995, Pages 40-42 MUNI SCI Acetylation Acetylation is one of the major post-translational protein modifications in the cell, with manifold effects on the protein and the metabolome level. • Covalent attachment of an acetyl group eliminates the positive charge (+) of the amino group, thus affecting local electrostatic properties. • These reactions are catalyzed by various N-terminal and LYSINE acetyltransferases. • Involved in regulation of transcription factors, histones, effector proteins, molecular chaperons and cytoskeletal proteins. 26 Department of Experimental Biology Experimental & Molecular Medicine, 2018, volume 50, pages1-13. MUNI SCI Acetylation • N-terminal acetvltransferases (NAT) transfer an acetyl group (CH3CO) to an a-amino group of protein N-termini. • Acetvltransferases (KATs) catalyze the transfer of an acetyl group (CH3CO) to the £-amino group of LYSINE (K) side chains. • NATs and KATs use acetyl-CoA (Ac-CoA) as a donor of acetyl group. • In the case of lysine acetylation, the acetyl moiety may be removed by lysine deacetvltransferases (KDACs), making it a reversible protein modification - deacetylation. N-terminus Na Acetyl N-terminus (Nt) (AcNt) 27 Department of Experimental Biology Experimental & Molecular Medicine, 2018, volume 50, pages1-13. MUNI SCI N-terminal Acetylation • N-terminal acetylation (Nt-acetylation) is a common protein modification, affecting an estimated 80% of all human protein species to a varying extent. • Nt-acetylation has many functions in the cell, o Targets proteins for polyubiquitination and proteasomal degradation or protects against such degradation, o Proper folding of some proteins, o Protein-protein interactions, o Targets some proteins for membranes. 28 Department of Experimental Biology b Ai. Protein half-life At) Complex formation Folding. Membrane targeting MUNI SCI Lysine Acetylation Relaxed hromatin HDAC inhibitors (e>g-, vorinnstit, cntinostat) Altered gene lion Altered biological effects in malignant tells 1 Proliferation A Cell cycle 1 Migration 1 Cell death • Histone acetylation and deacetvlation are essential parts of gene regulation. • These reactions are typically catalyzed by enzymes with "histone acetvltransferase" (HAT) or "histone deacetvlase" (HDAC) activity. • Acetylation of histones alters accessibility of chromatin and allows DNA binding proteins to interact with exposed sites to activate gene transcription and downstream cellular functions. 29 Department of Experimental Biology https://www.wikiwand.com/en/Histone_acetylation_and_deacetylation MUNI SCI Lysine Acetylation Histories H2A, HS8, H4 Metabolic enzymes GAPDH, Enolase Transcription GAT A lint finger, bHLH Translation Initiation factor IF-3 Secondary metabolism Cytochrome P450 72 Al Signal transduction Lectin-líke receptur kinase 1 RNA processing ExonucJease Protein Stability £3SUMO)i_ase 51 zz Ceti death a Cell division Armadillo repeat Protein Transposan & Retrotrans-pos-an Mutator 6 En/Spm sub-classes Ty3-gypsy Tyl-copta 30 Department of Experimental Biology PLoS ONE 9(2): e89283. doi:10.1371/journal.pone.0089283 MUNI SCI Methylation Addition of methyl group to a protein to eliminate positive charge. Fr<"",5lno M"h""""" LysorK Kmel Kmo2 Kmo3 Usually at LYSINE or ARGININE sc,™ m~% residues, also HISTIDINE. Lysine contains a primary e-amine. spacefilling J 1 J Structures V R MNh ©N-^t ©."-"Mt ©N Methyl donor is S-adenosvlmethionine ^ ^ ^ jA (SAM). Electrostatic .«> 9 i Potential Surface Enzyme for this is methyltransferase. 31 Department of Experimental Biology Chem. Rev. 2018, 118, 14, 6656-6705 MUNI SCI Methylation RNP granule ■ SMN • FU5 •FMR1 / •CAPRIN1 • C3BP Mitochondrion • NDUFS2 •TDRKH Focal adhesion •ACTN1 • EZR • ITCA2 • DOCK7 Ribosome Nuclear speckle Nucleolus • FMR1 •SRSF2 • FMR1 • RPS24 • RBM15 • PARP1 • LARP4B • SF3B1 •CAR1 • EIF4G1 •PRPF3 • NOP56 Cytoskeleton • CAMKII ACTN1 EZR COBL • RNA metabolic process • Cellular response to DNA damage • RNA processing • Protein folding • Chromosome organization • Gene expression • Aromatic, heterocycle and nucleic acid • Viral process metabolic process • Microtubule-based process • Co-translational protein targeting to membrane • Ribosome Protein, lysine and arginine, methylation function in: • Epigenetic regulation. • DNA damage response. • Signaling pathways. • Membrane less organelles by arginine methylation. 32 Department of Experimental Biology Nature Reviews Drug Discovery volume 20, pages509-530 (2021) MUNI SCI Methylation - Lysine H H \/ NH* I (CH2)4 Lysine H (ch) (CH^^) NH* I (9H2)4 KMT KDM KMT NH* I (CH2)4 Lys KDM Lys Mono-methyl lysine Di-methyl lysine KDM 33 Department of Experimental Biology @)-N+-@ (CH2)4 KMT KDM Lys Tri-methyl lysine Recruitment/ regulation Lysine can be methylated like mono-, di- or trimethylated (Kme1, 2 or 3) through the addition of a methyl group to its terminal side-chain £-amine. Lysine methylation dynamics are controlled by the regulated action of KMTs to add and KDMs to remove the methyl. Once the methyl modification has been made to the lysine, it can result in either the recruitment of binding proteins or direct effects to regulate protein function. FEBS Journal, 2017, Vol. 284, 2732-2744. MUNI SCI Methylation - Lysine Methylation of K310 leads to translational repression of NF-kB through the recruitment of the EHMT1 (Euchromatic Histone Lysine Methyltransferase 1). Recruitment of EHMT1 increases the localized methylation of H3(K9me2), resulting in the transcriptional repression of NF-kB target genes. Serine phosphorylation of S311 blocks the binding of EHMT1 to K310, relieving the methylation-induced translational repression. @3 SETD6 / \ Protein Kinase C MBD recruitment —I Target genes I Transcriptional repression Active transcription 34 Department of Experimental Biology FEBS Journal, 2017, Vol. 284, 2732-2744. MUNI SCI Methylation - Lysine - Epigenetic regulation K4 SET7/9 Hä mflj ftii lamily Meisetz Hs me3 5MYD family SMYDl Hs me1tf/3 5MYD3 Hs me2/3 SET2 family ASH1 Dm me3 ASH1 HS ms3 ASH iL Dm (ti83 ASH1L Hs ma3 SETI family Seil Sc mel/2/3 SMI Sp tt1&1 S2/3 Trithorax Dm mel TFR Dm me3 SET1A/B Hs me3 MLL1-4 Hs me3 ATX1 Al m&3 K99 flr me2/3 K125 srmel K127 ßftnel K36 SET2 family SET2 Sc 0161/2/3 SET2 SETD2 ASH1 ASH1L Sjo me 1/2/3 Hs me3 Dm meS Hs me 1/2 K43 Sf* mel K53 NC ms2 K56 We" me2/3 K69 NC me2 K96 We" meS/3 K111 71" mee/3 K118 Wc- rrtetffl K126 NC mel/2 K136 We- msl K37 SC m«2 Hs* me2 G$' mtó KS Sc" lMe K3 rr mei/2/3 Wc" mel/2/3 mel/2/3 NSD1/2 Hs mal/2 SMYD family SMYOř Hs meí SUV4-20 ramlly SET03 Or meí SUV39 family SET MAR Hs me2 K18 Hs*me1 KS Rlzlamily PRDM2 Hs me? SET2 family ASH1 0m me3 SUV39 family Ori Sp me273 Su(Vaj)3-9 Dm me3 SuV39H1/2 Dm rrwl GSa/QLP Dm me1/2 K23 Si* mel Hs' me2 Mm- me2 Gg' me3 Pb me3 öt) í1163 Hs met/2/3 Ce inu3 Af me3 G9a/GLP SETD81 SETDB1 Dim-S Hs melffi Mm rrteJ Hs me3 Nc rrve3 Kryptonyte A* me1/2 KÍTvSET EZ family EUDP UDP <1 O #-GDP GDP ? • © □ = N Acclylga actosaminc A = Galactose Q = Sialic acid =Fucosc https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/N-linked_Protein_Glycosylation_Begins_in_the_ER MUNI SCI Glycosyalation Heparan sulfate 6S NS NS 3S 6S 6S ■2S Proteoglycans SHK~ y^Tl 4S 4S 4S |S I Chondroitin or dermatan sulfate EGF or TSR c to u CT) Ó ★ ★• So- ♦ o • 4 s o ©-4-|5orT GPI-anchored glycoproteins Free GAG Hyaluronan Glycosphingolipids 3S 35 A Fuc • Glc ■ GlcNAc O Man O Gal ^GlcA r» t N-terminal myristoylation • P21-activated kinase 2 (PAK2) is cleaved by caspase 3 to produce caspase-truncated PAK2 (ctPAK2), which has a newly exposed glycine residue at the N-terminus. • Then, NMT catalyzes the covalent attachment of myristic acid to the glycine residue of ctPAK2. Post-translationally myristoylated ctPAK2 translocates to subcellular membrane compartments to induce apoptosis. Post-translational N-myristoylation in apoptosis ct-PAK2 translocates to the sub-cellular membrane compartments to affect apoptosis. MUNI 48 Department of Experimental Biology Cellular & Molecular Immunology, 2021, Vol. 18, pages 878-888. r> r» t S-palmitoylation S-CoA Paimitoyl Co-A Paimitoyl acyl transferase SH Cysteine residue Pülmitoyl (hio esiemíe Palmitoyhared cysteine residue Palmitoylation is post-translational attachment of the saturated 16-carbon palmitate from its lipid donor, palmitovl-coenzyme A ester, to a CYSTEINE. Paimitoyl S-acvltransferase (PATs) are enzymes responsible for catalyzing the addition of palmitate to the substrate. Removing of palmitol by acyl-protein thioesterase (APT). Palmitoylation is caried out on membranes, is a reversible process and several cellular proteins undergo dynamic palmitoylation. 49 Department of Experimental Biology https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/palmitoylation MUNI SCI PAT - palmitoyl S-acyltransferase S-palmitoylation DHHC PAT I II II II AUTOPALM ITOYLATION PALMITATE TRANSFER II II II II U II II II II II II II 11 II II II Substrate B APT - acyl-protein thioesterase 50 Department of Experimental Biology EMBO Reports (2018)19:e46666 MUNI SCI S-palmitoylation S-palmitoylation (Cys) N-palmitoylation HS. \ O-palmitoylation (Ser/Thr) O-palmitoleoylation (Ser/Thr) o r = k ch3 S-Palmitovlation occurs at CYSTEINE. N-palmitovlation occurs at AM I NO-TERMINAL CYSTEINE. O-Palmitovlation and O-palmitoleoylation occur at SERINE/THREONINE. Protein acyl transferases are located: • Endoplasmic reticulum • Golgi apparatus • Plasma membrane Cell Chem Biol, Volume 25, Issue 3, 15 March 2018, Pages 236-246. M U 111 51 Department of Experimental Biology _, _. _ ^ ay The FEBS Journal, 2022, Vol. 289,861-882. O p T S-prenylation • S-prenylation, similarly to S-palmitoylation, provides a hydrophobic, membrane attracted C terminus through the enzymatic addition of farnesyl (C15) or geranylgeranyl (C20) to a CYSTEINE residue. • Enzyme involved in this reaction is farnesyl transferase (FT) or geranylgeranyl transferases (GGTIand II). • Dysregulated S-prenylation is implicated in several diseases including cancer. o protein—MH-C myistoylated protein o protein—S—ü^ palmitoylated protein pren^lated protein protein—S- 52 Department of Experimental Biology https://www.mun.ca/biology/scarr/iGen3_06-08.html MUNI SCI Proteolytic cleavage • Proteolytic cleavage - very common irreversible post-translational modification of the protein's structure and biological function. • Also, first amino acid methionine of a newly synthesized polypeptide is very often cleaved off (also true for some prokaryotic f-Met). • The precursor protein is termed a proprotein, and the peptide that is cleaved off proprotein is called the propeptide. • Classical examples of proproteins are the hormone insulin, the cell death protein family of caspases, collagen and the Alzheimer-associated neural protein |3-amyloid. https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/N-Department of Experimental Biology iinked_Protein_Glycosylation_Begins_in_the_ER Chem. Rev. 2018, 118, 3, 1137-1168. Proteolytic cleavage In mammals - preproinsulin (inactive as a hormone) is first translated from the insulin mRNA. Proteolytic processing is necessary to make biologically active insulin. (A) The linear protein contains a signal sequence, which is cleaved after the protein enters the ER, an A chain, a B chain, and a C-peptide. (B) Inside the ER, the proinsulin (insulin precursor) folds and disulfide bonds form between cysteines. (C) Finally, two cleavages release the C peptide, which leaves the A and B chains attached by the disulfide bonds. This is now active insulin. B chain-__ C-peptide Achairk SH i-1 s s I-1-1-, K-R-^l-V-E-q-OC-T-S+C-S-L-Y-Q-L-E-N-Y-C-N -- s s I s s ,-1-1-, I F-VN-Q-H-LCG-SHL-V-E-A-L-Y-L-V-t-G-E-R-G-f-F-Y-T-P-K-T I 54 Department of Experimental Biology https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/N-linked_Protein_Glycosylation_Begins_in_the_ER MUNI SCI Proteolytic cleavage OH Procollagen H2N COOH OH OH Propeptide J Completed collagen molecule Propeptide Triple helix formation Secretion from cell Propeptides clipped Collagen is a very large secreted protein, twisted triple-helix of three subunit, that provides structure and shock absorbance for the extracellular matrix in animals. The collagen subunits are made as procollagen, and propeptides are lopped off of both N- and C-termini to generate the final protein. However, they are not cleaved off until after the three subunits assemble around one another. The propeptide sequences are clearly necessary for efficient assembly of the final protein complex. 55 Department of Experimental Biology https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/N-linked_Protein_Glycosylation_Begins_in_the_ER MUNI SCI DNA Precursor i proton Spliced protein N-extein N-extein intein C-exlein Transcrlptřon/translahon intein C-extein Protein splicing C-exiein intein Protein splicing Protein splicing is a post-translational process facilitated by an intervening polypeptide, called an intein (for internal protein). The intein interrupts flanking polypeptides called exteins. The intein is responsible for catalyzing its excision from the exteins, concomitant with extein ligation. N-axtoin Inlein frocKs: H Tis.'I Hciming Bndonudsase Irrinin _ Ď II i; 5.T C □ E F G • Inteins have four conserved sequence blocks. Block C, D and E where the split sites of naturally occurring split inteins and homing endonuclease domain (HED) are located. 56 Department of Experimental Biology Handbook of Proteolytic Enzymes, Volume 1, 2013, Pages 315-321 MUNI SCI DNA Exon 1 Intron Exon 2 Protein splicing TRANSCRIPTION RNA TRANSCRIPTION The protein splicing is rare PMT on 1 Intron Exon 2 Extein 1 Intein Extein 2 SPLICING OF RNA mRNA Exon 1 Exon 2 TRANSLATION TRANSLATION PROTEIN Fi J1"! Framexon 1 jnlejn From ^extein 1 From extein 2 SPLICING OF PROT ESN 57 Department of Experimental Biology • Inteins and exteins are the protein analogs of the introns and exons found in the DNA and RNA. In other words, inteins are intervening sequences in proteins that are present when the protein is first made, but are later spliced out. • The final protein is made of the exteins that are now joined together. • Inteins have been found in yeasts, algae, bacteria, and archaea (archaebacteria), such as VMA1 in a precursor of a vacuolar H+-ATPase enzyme. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/protein-splicing MUNI SCI Protein splicing Protein splicing is four-step process, which is achieved through structural conformational change and chemical bonds shifting on junction sites between intein and exteins. 1. A nucleophilic displacement converts the peptide bonding to an ester or thioester group. 2. Transesterification - transfers the to the first residue of C-extein, forming a branched intermediate. 3. The Asn cvclization leads to intein peptide bond cleavage and exteins ligation. 4. Rapid conversion from the ester bond to the amide bond occurs to form the final peptide. April 2013Molecules 18(1):440-65 Department of Experimental Biology Front Bjoeng Biotechno| 2022, Protein Splicing of Inteins: A Powerful Tool in Synthetic Biology, 10:810180 Protein splicing Chrůmosůme with spíil dnäE gene DNA Exiein 1 Intein 1 Mein 2 Extein The DNA coding for the DnaE protein of Synechocystis is transcribed and translated into two separate proteins, each containing an intein and an extein. The exteins of the two proteins are spliced together by the inteins. During splicing both inteins are lost. Also, DnaE, the catalytic subunit a of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c. TRANSCRIPTION AND TRANSLATION PRE-PROTEIN Xi^j/L Intein ^ i- -,-.-..1-, i PROTEIN Intein 1 Intein 2 * \^ Extein 1 Extein 2 I t *^ Extein Extein 1 Intein 1 intein 2 59 Department of Experimental Biology https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/protein-splicing MUNI SCI Post-translational Ubiquitination • Ubiquitin (Ub), a highly conserved regulatory protein containing 76 amino acids, can be covalently tagged to target proteins. • Ubiquitin is attached to the LYSINE residue in polypeptide. • Ubiquitin provides eight types of polyubiquitin linkages (K6, K11, K27, K29, K33, K48, K63 and Met1) with specific functions. • The ubiquitin ligase adds ubiquitin to the substrate and deubiquitilating enzyme removes Ub from the substrate. Thus, ubiquitination is reversible process. 60 Department of Experimental Biology Mater methods, 2014;4:827 Physiological Reviews. Jul 2013, 93(3):1289-315. MUNI SCI (Ob) Ubiquitin ~ Thioester bond O AMP * Lys A Catalytic Cys ® Mg2\ ATP Post-translational Ubiquitination • Ubiquitin can be covalently tagged to target proteins via a cascade of enzymatic reactions. • 1. A ubiquitin-activating enzyme (E1) catalyzes binding to one ubiquitin by a thioester. • 2. E1 then binds a ubiquitin-conjugating enzyme (E2) and transfers ubiquitin from its catalytic cysteine to the catalytic cysteine of E2 to form E2—ubiquitin (~ indicates a thioester bond). • 3. A ubiquitin ligase (E3) recruits E2—ubiquitin and a substrate to catalyze ubiquitin transfer to a lysine on the substrate. 61 Department of Experimental Biology Nature Reviews Molecular Cell Biology, 2016, Volume 17, pages 626-642. MUNI SCI Post-translational Ubiquitination o li -c-o Ubiquitin activating enzyme ~2 Uba1 Ubae O -SH- ATP AMP+PPi Ubiquitin conjugating enzyme -20 Ubiquitin ligase -600 HECT E3 ligase RING E3 ligase e 3 F-box Three distinct classes of ubiquitin ligases. o HECT E3 o RING/U-box E3 o RBR E3. These ubiquitin ligases utilize different structural mechanisms for mediating the final transfer of ubiquitin onto substrates. IBR 62 Department of Experimental Biology [dub] _C—|NJH —(^SubJ Deubiquitinases-100 Signal Transduction and Targeted Therapy (2020) 5:11. MUNI SCI Post-translational Ubiquitination Protein could be: o Monoubiquitylated. o Multi-monoubiquitylated. o Polyubiquitylated. o Branched ubiquitin chain Monoubiquitylation Multi- monoubiquitylation Homotypic chains Mixed Branched K48 KG Heterotypic chains 63 Department of Experimental Biology Physiological Reviews. Jul 2013, 93(3):1289-315. Cell Discovery volume 7, Article number: 6 (2021) Post-translational Ubiquitination Branched ubiquitin chains of different topologies are specialized for different cellular functions and control the stability, activity, interaction properties, and localization of many different proteins. Branched ubiquitin chains regulate cell signaling and protein degradation pathways. Branched ubiquitin chains are remarkably diverse in terms of their chemical linkages, structures, and the biological information they transmit. Lys48 Monoubiquitylation or multi-mono ubiquitiylation Lys63 Polyubiquitylation chains Lys48-iinked polyubiquitin Lys63-linked polyubiquitin • DNA repair • Transcription • Trafficking • Endocytosis 1 26S proteasome • DNA repair, degradation • NFkB signalling • Trafficking • Endocytosis Jb) Ubiquitin ~ Thioester bond O AMP * Lys A Catalytic Cys Branched ubiquitin chain > APC/C-catalysed 26S proteasome degradation Nature Reviews | Molecular Cell Biology 64 Department of Experimental Biology Nature Reviews Molecular Cell Biology, 2016, Volume 17, pages 626-642. MUNI SCI Post-translational Ubiquitination Model for the role of branched K48/K63 chains in the activation of NF-kB signaling. Homotypic K63-linked chains are efficiently disassembled by CYLD, resulting in the removal of K63 linkages from TRAF6 and the termination of NF-kB signaling. Homotypic K63 chain ® ® © @ TRAF6 Termination of NF-kB signaling Branched K48/K63 chain 65 Department of Experimental Biology Sustained NF-kB signaling Branched K48/K63 chains are resistant to CYLD cleavage, resulting in the persistence of K63 linkages on TRAF6 and sustained activation of NF-kB signalling. Cell Discovery volume 7, Article number: 6 (2021) MUNI SCI Ubiquitin-proteasome system Ubiquitination ATP +PPj__(fr4 (^ 26S proteasome HECTE3 19S 20S 19S c O 03 TD CD CD "Ö "ČĎ E o CO en CD •4—' 2 Q_ O TO O" _5 CD Q Ubiquitin recycling 66 Department of Experimental Biology Lys11-Cell cycle regulation Lys27-Mitophagy Lys48-Proteasomal degradation Lys63-NFKB singnaling Endocytosis https://www.antibody-creativebiolabs.com/post-translational-modification-ptm.htm The overall system of ubiquitination and proteasomal degradation is known as the ubiquitin-proteasome system. Ubiquitination is covalently conjugated to a Lysine residue of the substrate proteins. Lys48-linked polyubiquitin chains usually target proteins for proteasomal degradation. MUNI SCI Ubiquitin-proteasome system The proteasome is a multi-catalytic molecular machine that plays a key role in the degradation of many cytoplasmic and nuclear proteins. The core of the proteasome consists of a symmetrical cylinder-shaped structure composed of four stacked rings, each containing 7 different subunits and is called the 20S proteasome. Gate opening of the 20S core occurs via capping by proteasome activators such as the 19S cap or PA28. The 19S cap is the most abundant activator and it forms the 26S proteasome together with the 20S core. Latent 20S Immunoproteasome Department of Experimental Biology Front. Mol. Biosci. 6:56., doi: 10.3389/fmolb.2019.00056 Ubiquitin-proteasome system Substrate processing by the 26S proteasome poly ubiquitin chain substrate <-> I-, 2-25 residue 1_ _f peptides / antigenic peptides amino acids 68 Department of Experimental Biology • The 26S proteasome is a 2.4-MDa molecular machine that makes up nearly 2% of total cellular protein. • It is composed of a 20S proteasome core particle capped on one or both ends by the 19S regulatory particle. • It degrades proteins by a multistep process; the 19S regulatory particle binds ubiquitinated substrates, opens a substrate entry gate in 20S and unfolds its substrates by linearly translocating them into the 20S catalytic chamber, where they are degraded to peptides. MUNI Pharmacological Reviews April 2019, 71 (2) 170-197 Post-translational Ubiquitination • The enhanced binding of branched chains to the 19S regulatory particle of proteasome as a result of an increase in the local concentration or "density" of ubiquitin subunits surrounding the substrate is illustrated by the multivalent-binding model. • Enhanced binding due to the recognition of novel interaction surfaces created by branching or recognition of the branch point itself is represented by the conformational recognition model. 19S regualtory particle 69 Department of Experimental Biology Cell Discovery volume 7, Article number: 6 (2021) MUNI SCI Sumolyation • Sumoylation is a post-translational modification, Small Ubiquitin-like Modifier (or SUMO) proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. • Sumoylation is reversed by the action of desumoylating enzymes. • Here are 4 confirmed SUMO isoforms in humans; SUMO-1, SUMO-2, SUMO-3 and SUMO-4. • SUMO proteins are small; most are around 100 amino acids in length and 12 kDa in mass. • SUMO protein has a unique N-terminal extension of 10-25 amino acids which other ubiquitin-like proteins do not have. MUNI 70 Department of Experimental Biology https://en.wikipedia.org/wiki/SUMO_protein O 0 J_ Sumolyation • First, SUMO (S) is matured by SUMO specific proteases (Prot), enabling it to become activated in an ATP-consuming reaction, to form a thioester bond (-S-) with the heterodimeric E1 (Aos1/Uba2). • SUMO is then transferred to the E2 (Ubc9), resulting in a thioester bond. • Finally, SUMO is conjugated directly or with the help of an E3 ligase to its substrate, forming an isopeptide bond. • Sumoylation is reversed by SUMO specific proteases that cleave SUMO from the substrate. 71 Department of Experimental Biology MUNI SCI Sumolyation Deconjugation SUMO proteases SUMO proteases (nine in human) c .0 CO 3 + - V w ATP SUMO ► + precursor 1 ik n AMP Uba2 +ppi Aos1 s Monosumoylation Polysumoylation Multisumoylation Substrates E2 conjugating (one) E1 E3 activating ligating (one) (ten in human) (thousands) Conjugation Enzymes in sumolyation o E1 - 1 o E2- 1 o E3-10 Substrates can be: o Monosumoylation o Multisumoylation o Polysumoylation. 72 Department of Experimental Biology BioMol Concepts 2017; 8(1): 13-36. MUNI SCI Sumolyation • Sumoylation is involved in various cellular processes, such as nuclear-cytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle by one of these mechanisms: Alters substrate Regulation of substrate Promotes Blocks conformation stability through recruitment of interactions interactions SUMO-targeted ubiquitin ligases Department of Experimental Biology Physiol Rev94: 1249-1285, 2014doi:10.1152/physrev.00008.2014 • Protein folding and Quality control MUNI 74 Department of Experimental Biology _ _ T O U J_ Protein folding • Protein folding is the physical process by which a linear polypeptide folds into its characteristic and functional three-dimensional structure. • The final folded configuration, or shape, of a protein is determined by its amino acid sequence. • Protein folding is also strongly influenced by the solubility of the AA R-groups in water. • Protein can reach its 3D conformation either alone, co-translational folding, or with help of other factors, chaperones. Unfolded Folded https://slidetodocxom/protein-folding-the-production-of-a-mature-protein/ |J Conference: 2016 IEEE 2nd International Foru Industry Leveraging a better tomorrow (RTSI) 75 Department of Experimental Biology Conference: 2016 IEEE 2nd International Forum on Research and Technologies for Society and g Q J Protein folding Hydrophobic versus hydrophilic characteristic of AA. Three types of noncovalent bonds help proteins to fold. polar side chains nonpolar side chains hydrophobic core region contains nonpolar side chains polar side chains on the outside of the molecule can form hydrogen bonds to water unfolded polypeptide folded conformation in aqueous environment hydrogen bond 76 Department of Experimental Biology https://slidetodoc.com/protein-folding-the-production-of-a-mature-protein/ MUNI SCI Co-translational protein folding • Protein domains can fold into stable tertiary structures while they are synthesized by the ribosome in a process known as co-translational folding. Folding begins early inside the polypeptide exit tunnel. • The nascent chain (NC) emerging from the ribosome can interact with chaperones, biogenesis factors, or other proteins. NC starts to fold vectorially Secondary structures can form NC interacts with the exit tunnel NC can compact to tertiary structures Ribosome surface destabilizes folding intermediates and ensures correct timing of folding for single- and multi-domain proteins NC can interact with its partner subunits of an oligomeric complex, chaperones or biogenesis factors 77 Department of Experimental Biology Biomolecules 2020, Cotranslational Folding of Proteins on the Ribosome, 10(1), 97. MUNI SCI Protein folding * The ER lumen plays four major protein processing roles: o folding/refolding of the polypeptide, o glycosylation of the protein, o assembly of multi-subunit proteins o packaging of proteins into vesicles. CS3' Q Glucose <^ BiP A Ubiquitin Ö Ribosome V Transtocon O COPil Nature Reviews | Molecular Cell Biology 78 Department of Experimental Biology https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/N- linked_Protein_Glycosylation_Begins_in_the_ER https://www.sciencedirect.com/science/article/pii/S1568163709000178 MUNI SCI Protein folding The internal redox environment of the endoplasmic reticulum, is significantly more oxidative than that in the cytoplasm. • This is largely determined by glutathione, which is found in a 30:1 GSH:GSSG ratio or higher in the cytoplasm but at nearly 1:1 ratio in the ER lumen. Reduced (2 GSH) COOH O SH COOH O H2N ^~*^~^Y N Y^"N ^COOH 0 v .N^COOH COOH O Oxidized (GSS6J Glutamate cysteine ligase [GSH]^ > [GŠH] synthetase GSH GSH Reductase ( «» -3- Cytosol 79 Department of Experimental Biology Antioxid Redox Signal. 2017 Nov20;27(15):1178-1199. doi:10.1089/ars.2017.7148. Other compartment MUNI SCI Protein folding • The two rate-limiting reactions influence folding of newly synthesized proteins: o formation of disulfide bonds (S-S), catalyzed by disulfide isomerases o isomerization of prolyl-peptide bonds, catalyzed by peptidyl—proline isomerases 80 Department of Experimental Biology https://www.nejm.org/doi/full/10.1056/nejm199812033392307 Protein folding • If there is incorrect bound between cysteines, and more stable cysteines bond in the context of the whole protein is available, than the exchange of disulfide bonding is catalyzed by protein disulfide isomerase (PDI). • This enzyme uses a sulfhydryl group of a cysteine residue as temporary bonding partner in order to break disulfide bonds on the target protein and allow for new ones to form. • Note that the formation of a new bond is not directed by PDI, but is instead a stochastic process in which a stronger binding partner displaces the PDI —SH. A B C D 81 Department of Experimental Biology MUNI SCI Chaperones Protein folding take place in the cytosol. Most proteins require the assistance of molecular chaperones. Bacteria (Hsp70) (Hsp40) Oltet chapflfofws GroEL + GrpE. ATP * LJNEF) 20 + ATP Althaea i TlHjfmůsofne Eukarya NAC MAC RAC Hsp40ťIL FtAC Hep40 HSp70| hrS070| syslem SC H2B 5^©-o-o-©^f « • » H4 91 Department of Experimental biology https://www.thermofisher.com/cz/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-post-translational-modification.html MUNI SCI Post-translational modifications in cell signaling PDGF FGF2 IGF HTK HTK RAS RAF i o* I PI3K PIPS —i. POKt W JAK MSTItt / Calcineurin Nucleaf exc fusion 4E-BP1 S6K1 <»«B™tU«ion 4 I Transcription Protein synlTidsl: Tranacrrplio i TranscrJptlor Transcription Transcription | Transcription 1 I T 92 Department of Experimental biology PROLIFERATION March 2017Pulmonary Circulation 7(2):204589321770143 MUNI SCI THANK YOU FOR YOUR ATTENTION 93 Department of Experimental biology https://www.redbubble.com/shop/biology+joke+poste