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 Glycosylation, 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. Gurion i ü -2G-25,0D0 genes Alternative promoters AftemativE splicing mflNA editing Transcriptome -IQG.MG transcripts Post-fransiätinnal modifi cations Protecme >l,O00,0Ö0 proteins 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 ÉŇmMttfeo «tww Av'ri'i. Tin* CMon Proteome: • Complete set of proteins produced by genetic material of an organism. Interactome: Complete set of protein interactions in an organism. 5 Department of Experimental Biology https://slideplayer.com/slide/5746723/ MUNI SCI Protein synthesis in three levels of modifications 20 Amino acids + 20 tRNAs Pre-translational Modifications I 20 aa - tRNAs Co-translational Modifications I Nascent polypeptide Post-translational Modifications I Complete polypeptide 6 Department of Experimental Biology _ _ T O U J. Co-translational and Post-translational modifications Post-translational modifications influence protein: o Charge o Conformation o Size Effects of post-translational modifications on protein: o Stability o Biochemical activity o Protein targeting (localization) o Protein signaling (protein-protein interaction) Localization Activity ( Signai input ) State'A' Enzymey. PTM crosstalk Conformation Binding 7 Department of Experimental Biology CELL, VOLUME 108, ISSUE 4, P557-572, FEBRUARY 22, 2002 MUNI SCI Protein synthesis in three levels of modifications Levels 1. Pre-translational 2. Co-translational 3. Post-translational 8 Department of Experimental Biology Modifications a) Selenocysteine tRNA b) Non-standard/natural amino acid tRNA - dansylalanine (fluorescent reporter) - phosphoserine, phosphothreonine, and phosphotyrosine (production of translational proteins in E coliwith Eukaryotic post-translational modifications ) a) Signal sequence celavage b) N-Glycosylation a) Phosphorylation b) Acetylation c) O-Glycosylation d) Methylation e) Lipidation f) Proteolytic cleavage g) Protein splicing h) Ubiquitination, Sumoylation https://slideplayer.com/slide/12777240/ MUNI SCI • Pre-translational modifications MUNI 9 Department of Experimental Biology _ _ T O 0 J. Pre-translational modifications Selenocysteine-tRNA is initially charged with serine. Then the attached serine is enzymatically modified to form selenocysteine. HSe- (Ser) + ATP tRNA!5")8"' AMP+PP] 10 Department of Experimental Biology HN 0=S=0O .NH; Adv Nutr. 2011 Mar; 2(2): 122-128. https://www.sigmaaldrich.com/CZ/en/product/sigma/d0125 H3C CH3 Alanine - conjugated to the fluorophore 5-(dimethylamino)naphthalene-l-sulfonyl is Dansyl-L-alanine. The unnatural amino acid is incorporated into the proteins by use of a mutated aminoacyl-tRNA synthetase specific for dansyl-L-alanine. MUNI SCI • Co-translational modifications MUNI 11 Department of Experimental Biology _ _ T O 0 J. N-terminal signal sequence N-terminal signal sequences • N-terminal signal sequence mediates targeting of nascent secretory and membrane proteins to the endoplasmic reticulum (ER) in a signal recognition particle (SRP)-dependent manner. • Signal sequences have a tripartite structure, consisting of a h-region (hydrophobic core region) flanked by an n-region and c-region. • The c-region contains the signal peptidase (SPase) consensus cleavage site. Usually, signal sequences are cleaved off co-translationally. SPase N-| || | I M*| MKWVTF1SLLF5SAYS-| albumin WDSKGS5CyGSRLLLLLWSNLLLCQGVV$-| prolactin""^ A^IVTA^LPHIIDEVINIWIIVLIirTSIKAVYNFATCGILALV MPhHQSGSPTGSSaLLSGKKQRPHLAiRRKRRRE^ 12 Department of Experimental Biology http://www.signalpeptide.de/ MUNI SCI N-terminal signal sequence Endoplasmic reticulum 13 Department of Experimental Biology http://www.signalpeptide.de/ MUNI SCI Protein glycosylation Protein glycosylation • One of the major post-translational modifications of many cell surface and secreted proteins. • Significant effects on protein folding, conformation, distribution, stability and activity. Selection of sugar ranges from simple monosaccharide to highly complex branched polysaccharide chains. Dol-PP • The sugar residues are often used as molecular flags or recognition signals to other cells that come in contact with them. Glucose Mannose N-acetylglucosamine 5' mRNA 14 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 Adv Neurobiol. 2014; 9: 47-70. MUNI SCI Protein glycosylation • Two types of glycosylation: o N-linked - occurs on ASPARAGINE (N) residues within an X-N-X-S or X-N-X-T sequence - co-translational. o O-linked occurs on the side chain hydroxyl oxygen of either SERINE (S) or THREONINE (T) residues determined not by surrounding sequence, but by secondary and tertiary structure- post-translational. • N-linked glycosylation begins with a "tree" of 14 specific sugar residues that is then pruned and remodeled, but remains fairly large. • O-linked glycosylation is based on sequential addition of individual sugars and does not usually extend beyond a few residues. MUNI SCI Glycosylation Ribosome Asparagine - Asn • Glycosylation, both of which requires import of the target polypeptide into the ER first, but O-linked glycosylation is achieved in the Golgi apparatus. I/VA p Dolichol phosphate | NA(u;vIs!jCoi jminu (GkNAc) O r.'ar-csc (Mar. O GIucoí«(GIc) 16 Department of Experimental Biology https://pubmed.ncbi.nlm.nih.gov/23094261/ MUNI SCI N-glycosylation • N-glycosylation begins before a protein translation, as the dolichol diphosphate /pyrophosphate lipid, in the ER without being triggered by translation or protein entry. • Lipid-glycan are bound Asp by the multisubunit oligosaccharyltransferase (OST). • Glycosyltransferase catalyzes each step. • Sugar substrates are sugar nucleotides, not isolated sugar molecules. MUNI SCI N-glycosylation Asn-XSer/Thr <> O 0° • 111 Cytosol ^ - N-Ace:ylc.lucosamine # = Man nose Q = Glucose A = Galactose ® = Sialic add Transport to Golgi body -1111 l-I l-i t t-t i i riJ 11 ri i-t I *=t t «=t 11 i-l-t-i-M 1-1M t t-i t-1 t-M-i t * t tl«t ll-i liit-ttttltttiittl: Cc-:g lu-nen 7® <► *j\ t (5) 4~UDP UDP ♦ t>UDP UDP ond/or OCMP 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. Ml U 111 I 18 Department of Experimental Biology https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/N- POT 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 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). scetylation methväation m phosphorylation ubkjuitinatron SUMOyla1;on P ost-tra n si ational modifications ubiquitin-like proteolysis deamidation elimination AMPytation ADP-riiMSVlatjon glycosylation acylstion prenylatioit Department of Experimental Biology Journal of Experimental Botany, Volume 69, Issue 19, 31 August 2018, Pages 4499-4503 MUNI SCI Post-translational modification are carried out by enzymes Phosphorylation (X = Ser, Tyr, Thr) / Eliminylation (X = Thr) kinase phospholyase phosphate v ATP ADP ' Pi v ^[ orotein i ^^^h (protein) ^ ' 7 s P. H20 phosphatase *~ (jarotein) Acetylation (X = Lys, Ser, Thr) acetyltransferase ^ acetyl-coA coA (protein^ acetate ■ X ^(protein) acetate H20 deacetylase AMPylation (X = Tyr, Thr) AMPylator v ATP PP. (protein) AMP H20 AMP hydrolase AMP i X (protein) Glucosylation (X = Thr, Ser) glucosyltransferase g[ucose „ UDP-glucose UDP ' (protein)^ jr glucose glucosylhydrolase ^(protein) H20 MUNI 21 Department of Experimental Biology FEBS Letters, Volume 584, Issue 13, 2 July 2010, Pages 2748-2758 O 0 J. Post-translational modification are carried out by enzymes ADP-ribosylation (X = Arg, Cys, Asn) ADP-ribosyltransferase ADP.ribose NAD nicotinamide rote inj ADP-ribose H20 ADP-ribosylhydrolase ^ ^proteiřT) Deamidation / Polyamination deamidase polyamine transferase polyamine i Gin „ NH HO ^ polyamine NH ■ Glu * Gin 3 Gin (^protein^ ^protein^ nnm^^i^—^ (^protein^ Ubiquitylation / UBL conjugation (X = Lys) Ubi/UBL conjugation machinery ubi/UBL v Ubi/UBL + ATP AMP+PP, £ ^protein^ ^ Ubi/UBL "•s- H20 Ubi/UBL protease ^ (protein^ Proteolysis protease H20 (protein^ •4 pro tein, 22 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 XT l 0 0-Š-0" 1 o K n Tyrosine sulfation mm 0 Lysine acetyiaton D Asa* \ HN , HO, K. \ 5f» Glycosylation HN Ä HN Lysine ubtqui'.inaton frV" '"n^v ^n^y1 Lysine methytabon HjN^N HN. Arg«nne methytabon 23 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. Non-Phosphorated Protein r~ioH ATP. Ser-Thr Kinase A DP ti Pi I Ser-Thr Phosphatase Hp O-phosphorylalion at serine residue .OH H 24 Department of Experimental Biology 0 0=P-0* ATP ADP ^0 \ /. H -P Phosphorated Preten MUNI SCI Phosphorylation affects protein capability TRENDS rw Phůrmacnkigicat Sciences 25 Department of Experimental Biology MUNI SCI Phosphorylation —h. CM015/CDK7") 4> plioiphalflse $0 => t Thf161 Ty<15 TT11I6I CDCiS ~ IV 1.1 ™ i ii'.-Hjhjr.i-j- _vg> =c>í_n=> Inactive pru?-kinase AéIíveí kinase (MPFr H if NpwFv marl* c^-flln B Inactive Ctkl o o 00 o0c J Cyttin Q j^rddHV:;-, • 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. 26 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. 27 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) Ac-CoA CoA Lysine Ne Acetyl-lysine (K) (AcK) 28 Department of Experimental Biology Experimental & Molecular Medicine, 2018, volume 50, pages1-13. MUNI SCI N-terminal Acetylation N-terminal acetylation (Nt-acetylation, AcNt) 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. b CSE" Protein half-life Folding Complex formation Membrane targeting 29 Department of Experimental Biology MUNI SCI Lysine Acetylation Cktóed Ahrain At in Nonhistone proteins p53 ,■: [tU 'I, i líh i Relaxed hromatjn HDAC inhibitors (e.g., voríníistít, cntinostat) Altered gene tion 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. 30 Department of Experimental Biology https://www.wikiwand.com/en/Histone_acetylation_and_deacetylation MUNI SCI Lysine Acetylation Histories H2A, H28H H4 Metabolic enzymes GAPDH, Enolase Transcription GATA lint finger, bHLH Translation Initiation factor Secondary metabolism Cytochrome P450 72 Al Signal transduction Leetin-like receptur kinase 1 RNA processing Exonudease Protein Stability £3SUMOli_ase sizz Ceti death a Cell dicier, Armadillo repeat Protein Transposang, Retrotrans-pos-an Mutator _ Ert/Spm sub-classes Ty3-gyp&y Tyl-copta 31 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 ££££ m~% residues, also HISTIDINE. Lysine contains a primary e-amine. spacefilling J 1 J Structures V R \r \r \r \r ■H~*h ©N-^t SN^n ett-m Methyl donor is S-adenosvlmethionine — ^ ^ (SAM). Electrostatic Potential Surface Enzyme for this is methyltransferase. Ml] ■ 32 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 1 CytoskeEeton • 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. 33 Department of Experimental Biology Nature Reviews Drug Discovery volume 20, pages509-530 (2021) MUNI SCI Methylation - Lysine H H \/ NH* I (CH2)4 Lys)^ Lysine H (ch) (ch^^) NH* I (CH2)4 KMT KDM KMT NH* I 3 <4f me3 K27v5ET EZ family EUDP UDP <1 O #-GDP GDP ? • □ = N Acctylgaactosamine A=Galictosc 0 = sialicaci 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 Pairnitoyl Co-A Pg I mi toy I acyl tränkte rasp SH Cysteine residue Pülmitoyl Ihio esterase pglmitoyrared cysteine residue Palmitoylation is post-translational attachment of the saturated 16-carbon palmitate from its lipid donor, palmitovl-coenzyme A ester, to a CYSTEINE. Palmitovl 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 S-palmitoylation DHHC PAT AUTOPALM ITOYLATION PALMITATE TRANSFER PAT - palmitoyl S-acyltransferase ! II II II II II II II II II II II II II li II II 11 II II II II II II I 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 \ 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 (1) 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 (2,3) release the C peptide, which leaves the A and B chains attached by the disulfide bonds. This is now active insulin. i—i s s I-1-1-, I KR-G-l-V-E-q-C-C-T-5+C-S L-Y-Q-L-E-N-Y-C-N I I F-V-N-q-H4.-C-G-S-H-l.-V-E-A-l.-Y-L-V<-G-E-R-G-f-F-Y-T-PK-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 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 pnotenri yri :ccr. protein N-extein N-extein N-extein intein C-exlein TranscrlptFon/tran^lahon intein C-extein Protein splicing C-extein 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-extain Homing Brxtonuds-ase lnletteirc2 TRANSGRI PTION TRANSCRI PTION L • Exon 1 Intron Exon 2 Extein 1 Intein Extein 2 g S PLIC IMG OF RNA mRNA Ej«jn 1 Euan 2 TRANSLATION TRANSLATION PROTEIN ^, Frcrii iíkíjii 2 Fi J1"! From exon 1 jnlejn From ^extein 1 From extein 2 SPLICING OF PROT ESN 57 Department of Experimental Biology Protein splicing The protein splicing is rare PMT 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 Chromosome with sptil dnsE gene UNA Extein 1 trite in 1 Intein 2 Extein 2 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 Intein 1 Intein 2 A \ Extein 2 I PROTEIN t ^Extein ^■Extern 1 Intein 1 Intein 2 A 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 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 A' RBX © c 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 [pub") _C—NH —(^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 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-linked polyubiquitin Lys63-linked polyubiquitin • DNA repair • Transcription • Trafficking • Endocytosis • 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 26S proteasome ATP +PPi_(fr4 (fcj ;i^|^;ĚT^F>ÍE2cT- 19S 20S 19S c O 03 T3 TO CD CD TD "rä E O 03 0 A—' 2 o. o TO CT _5 0 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 Substrate processing by the 26S proteasome polyubiquitin chain substrate <-> i-, 2-25 residue -|_ _f Uv^ peptides antigenic peptides amino acids 67 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 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 68 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. 69 Department of Experimental Biology MUNI SCI Sumolyation Deconjugation SUMO proteases c .o SUMO proteases (nine in human) SUMO precursor ATP T * + Uba2 Aos1 Uba2 Aos1 AMP +PPi 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. 70 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 72 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-m |J Conference: 2016 IEEE 2nd International Foru Industry Leveraging a better tomorrow (RTSI) 73 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 nonpolar side chains 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 74 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 75 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. o Quoase BiP A Ubiquitin Q Ritiosome V Tfunslocon O COPil Nature Reviews | Molecular Cell Biology 76 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 Chaperones Protein folding take place in the cytosol. Most proteins require the assistance of molecular chaperones. rriRNAQ Q (Hsp70) (Hsp40) 6 ^ Oilier chaperones GroEL + GrpE. ATP UJNEF) 20% + ATP Archaey TNCrrU^rTílť? Eukarya n ac- MAC /RAC Hsp70j HS^Oj syslem jgj -20 % NEFs ATP i + NEFs TFHC 1 77 Department of Experimental Biology Cold Spring Harb Perspect Biol 2010;2:a004390 MUNI SCI Chaperones • The heat-shock proteins (Hsp) are a family of molecular chaperones, which collectively form a network that is critical for the maintenance of protein homeostasis. • Most proteins require the assistance of molecular chaperones, like Hsp 70 & Hsp 60, to reach their final folded form. • Hsp 70 and Hsp 60 act sequentially on proteins to help them achieve their correct folded state. • Proteins that fail to be properly folded are ultimately targeted for destruction. 78 Department of Experimental biology Molecular Biology, 430 (22), 4525-4546 https://slidetodoc.com/protein-folding-the-production-of-a-mature-protein/ MUNI SCI Chaperones • Molecular chaperones, like those in the Hsp 60 and Hsp 70 families, help guide the folding of most newly-synthesized proteins. • Hsp chaperones bind to hydrophobic patches that are exposed on incompletely folded proteins. • Repeated cycles of ATP binding and hydrolysis are generally required for the proper folding of a polypeptide chain. protein 79 Department of Experimental biology https://slidetodoc.com/protein-folding-the-production-of-a-mature-protein/ MUNI SCI Chaperones • Hsp 60 acts on fully-synthesized proteins that have not yet achieved their final folded form. • The presence of incompletely folded proteins can lead to the formation of aggregates that may have dangerous consequences for the cell. hip60like protein complex 80 Department of Experimental Biology https://slidetodoc.com/protein-folding-the-production-of-a-mature-protein/ MUNI SCI Chaperones Hsp70/40 mediates the initial stages of protein folding and acts as a central hub in the Hsp chaperone network. If Hsp70/40 is only able to partially fold the protein, it can be transferred to either the Hsp90 or Hsp60 systems in order to acquire a folded functional conformation. Cellular stress conditions can cause proteins to misfold and associate into insoluble, toxic aggregates. Physiological conditions Ribosome Cellular stress conditions Hsp70/40 81 Department of Experimental biology Molecular Biology, 430 (22), 4525-4546 MUNI SCI Quality control of protein synthesis • Because proteins are structurally dynamic, constant surveillance of the proteome by an integrated network of chaperones and protein degradation machineries is required to maintain protein homeostasis (proteostasis). newly synthesized protein protein aggregate correctly folded correctly folded incompletely without help with help of a folded forms molecular digested by the increasing time chaperone proteasome Figure 6-88 Molecular Biology of the Cell (© Garland Science 2008) 82 Department of Experimental biology https://slidetodoc.com/protein-folding-the-production-of-a-mature-protein/ MUNI SCI Quality control of protein synthesis • Chaperones also cooperate with other components of the proteostasis network, such as the proteasome system and autophagy, in the removal of terminally misfolded and aggregated proteins through proteolytic degradation. Promoted by chaperones Off-pathway interactions _II_II_II_ Folding Conformational Aggregate Clearance by maintenance formation degradation 83 Department of Experimental biology Science, Jul 2016 , Vol. 353, issue 6294. MUNI SCI Protein Misfolding Diseases Many inherited diseases result from mutant proteins that evade quality control processes, fold abnormally and ultimately form aggregates. The gradual decline of protein quality controls with age can also lead to disease by permitting normal proteins to form misfolded protein aggregates that can impair cellular functions. Protein misfolding diseases: Huntington's Alzheimer's Parkinson's aggregates in patients can be intracellular or extracellular. INI CI Huntingtons CI PdikinsorVs LB a d 1 INI Huntington's Alzheims i"s 84 Department of Experimental biology MUNI SCI Post-translational modifications in histones PTM events mediate diverse biological functions such as transcriptional activation and inactivation, chromosome packaging, and DNA damage and repair processes. 85 Department of Experimental biology ■e Acotyl.ition ^ Mothylation Q Phosphorylation O Ubtquitinatton 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 JL-6 IGF RAS RAF ERK1/2 I 6 + V PS3 4E-BP1 S6K1 <»«B™tUflion Micímí ffjfi fusion ami důgnitSňliůt? Transcription Protein synlTidsl: Tranacriptio i TranscrJptlor 1 ■.III^TTipl IÜ- Tranacrlptlon | Transcription 1 I T 86 Department of Experimental biology PROLIFERATION March 2017Pulmonary Circulation 7(2):204589321770143 MUNI SCI THANK YOU FOR YOUR ATTENTION 87 Department of Experimental biology https://www.redbubble.com/shop/biology+joke+poste