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In vivo Protein Modification Two Modes of Protein Modifications: 1. Reversible Reactions (on-off switch) 1. Phosphorylation of Ser/Thr/Tyr 2. Adenylation of Tyr 3. ADP-ribosylation of Arg 4. Methylation of the COOH groups 5. O-Glycosylation 6. Palmitoylation of Cys residues Reversible reactions can be regarded as the control of cellular activities. 2. Non-reversible reactions 1. Spontaneous reactions: Gln → Glu or Asn → Asp, Glycosylation of α- or ε-NH2 groups 2. Cross-links: Cys-Cys cross-linking in stabilizing protein structure, γGlu-εLys cross-linking in fibrin clots, -S-S- cross-linking, Alanino-His in hard tissue proteins, His-Cys thioether, Ser(Cys)γGlu esters 3. Covalently attached cofactors: Biotin to Lys, Heme to Cys, Pantothenyl phosphate to Ser, Flavin to Tyr, Cys or His 4. Membrane anchors: Myristate to Gly, Glycosyl Phosphatidyl Inositol to COOH-terminal, Isoprenyl to Cys 5. Ubiquitination for protein degradation 6. N-Glycosylation for many cellular functions Classical Protein Biosynthesis 1. Proteins are synthesized in ribosomes and one trinucleotide specifies one amino acid. 2. Codons are universal and the starting codon (AUG) specifies Met or fMet. 3. Every protein should start with Met or fMet at the NH2terminus. 4. Every protein should have no more than 20 amino acids. However, many exceptional amino acids were found in many naturally occurring proteins, therefore, proteins must be modified before or after ribosomal protein synthesis. Protein Biosynthesis at Three Levels of Modifications 20 Amino acids + 20 tRNA’s Pre-translational Modifications 20 aa-tRNA’s Co-translational Modifications Nascent polypeptide Post-translational Modifications Completed polypeptide ↓ ↓ ↓ Examples of Three Levels of Protein Modifications Levels 1. Pre-translational Examples a) Selenocysteine t-RNA b) Nonnatural amino acid t-RNA 2. Co-translational a) Signal sequence cleavage b) N-Glycosylation 3. Post-translational a) Glucosylation and O-Glycosylation b) Peptide bond cleavage c) Protein splicing d) Lipidation e) Disulfide bond formation f) Ubiquitination Selenocystyl Proteins Biosynthesis of Noncanonical Aminoacyl-tRNA: Selenocystyl-tRNA - H2O tRNASec + Serine Ser-tRNASec Aminoacrylyl-tRNASec +Selenophosphate Selenocysteyl-tRNASec (ATP + Se2- Selenophosphate + AMP + Pi) Incorporation of Selenocysteine into Proteins Gene sequences around the in-frame TGA codon for selenocysteine are normally coded for the stop codon. In the absence of selenium, the Se-Cys-containing proteins terminate at this codon. Proteins Containing Selenocysteine Designers Proteins Properties of Aminoacyl-tRNA Synthetases (AARSs) The 3’-terminal A is aminoacylated by the AARSs and the 20 amino acids are specifically joined to their cognate (同族) tRNAs. Thus, the aminoacyl-tRNAs (AA-tRNAs) are the main experimental efforts to incorporate nonnatural amino acids into proteins. Changing Relationships Between an Amino Acid and a tRNA 1. The concept of tRNA identity provided recognition of cognate tRNAs between species. This led to expression of heterologous AARSs in vivo as a means to incorporate the wrong amino acid into a protein at a specific position. 2. A heterologous synthetase-tRNA pair could be introduced into an organism and operate as an extra pair that was “orthogonal” (正規) to the existing homologous set of 20 AARSs. 3. Orthogonality means that the new AARS does not mischarge any of the tRNAs from the host organism and the new tRNA is not a substrate for any host AARS. 4. Thus, an organism engineered has 21 noncross-reacting AARSs that can be further manipulated so that the new twenty-first pair brings in a novel amino acid. Use of Amino Acid Auxotrophs 1. Incorporation of noncanonical (非典型) amino acids into proteins was discovered from the study of amino acid analogs synthesized by plants. These amino acids are toxic to microorganisms because they are misincorporated into proteins in place of a related canonical amino acid. 2. In strains auxotrophic for an encoded amino acid, high levels of substitution by an analog could be achieved. These analogs are only bacteriostatic and not bactericidal. 3. Thus, misincorporation of canavanine leads to cell death, but selenomethionine or trifluoroleucine which are well tolerated. 4. The main mechanism for analogs to be incorporated into proteins is provided by AARSs. Thus, bypassing the synthetase’s specificity, and mischarging of an analog onto a tRNA can lead to insertion of the analog into a growing polypeptide chain. Selection for Replacement of an Amino Acid by an Analog 1. Selection methods have also been used to replace a canonical amino acid with an analog. 2. Cells can replace Leu with trifluoro-Leu with epigenetic adaptation. However, a low percentage ( 5%) of the natural amino acid was still present. 3. Total replacement of Trp with 4-fluoro-Trp could be achieved with a small number of genome-wide mutations in B. subtilis. Multisite Misincorporation in Overexpressed Proteins 1. Most amino acid analogs are too toxic to promote sustained exponential growth. However, modified proteins can be overexpressed in nondividing cells if enough biomass has been generated prior to induction. 2. By washing cells and replacing the exogenously added canonical amino acid with its analog just before inducing gene expression, high levels of misincorporation (80% to 99%) of nonnatural analogs into target proteins can be achieved, with good yields of the purified proteins (10–100 mg/L). Multi-site Incorporation of Analogs into E. coli Proteins Analog Target AARS Perthiaproline Norleucine ProRS MetRS Selenomethionine 4-Fluorotryptophan p-Fluorophenylalanine β-(Thienopyrrolyl)alanines Aminotryptophans 2-Methylhistidine 3-Fluorotyrosine O-Methylthreonine 3,4-Dehydroproline MetRS TrpRS PheRS TrpRS TrpRS HisRS TyrRS IleRS ProRS Yields 38% 100% 100% 75% 60% Applications Drug carrier Increased enzyme activity Crystallography NMR Tracer Chromophore PH sensors Expanding the Genetic Code: Use of the Stop Codon for Coding Unnatural Amino Acids 1. The unnatural amino acid is added to the cell growth medium, taken up by the cell. 2. It is specifically recognized by an ‘orthogonal’ aminoacyl-tRNA synthetase and attached to the orthogonal amber suppressor tRNA. 3. It is then decoded on the ribosome in response to an introduced amber codon (UAG), allowing its incorporation into the peptide. Selection of a New Orthogonal Synthetase-tRNA Pair A heterologous aminoacyl-tRNA synthetase–tRNA pair is imported into a host containing a set of natural synthetases and the subsequent selection of a mutated active site in the new orthogonal synthetase that recognizes an unnatural amino acid. Positive and Negative Selection Procedure 1. To generate a synthetase with this altered specificity, a large library of active-site variants of the synthetase is subject to positive selection for activity with either natural or unnatural amino acids, by virtue of their ability to suppress an introduced stop codon and so allow complete translation of a gene that is essential for survival. 2. The synthetases that use natural amino acids are subsequently removed by a negativeselection step, in which they use natural amino acids to suppress a stop codon introduced in a toxic gene, which leads to cell death. Application I: Site-Specific Modification at 53 Position of Sec Y A photocrosslinking reagent, p-benzoyl-l-phenylalanine (Bpa), provides a powerful tool to gain information about the interaction of a specific protein with another molecule. To analyse the basis of the interaction between SecA and SecY, Bpa was introduced at 53 positions in the cytoplasmic loops of SecY. The protein was then crosslinked in response to the light, forming covalent adducts with SecA. Covalently linked SecA–SecY complexes can then be isolated through cell lysis, and the complexes can be detected by SDS– PAGE and western blot analysis with antibodies against SecA. Application II: Genetically Encoded Post-Translational Modifications Genetically encoded Nε-Lys acetylation allows the role of acetylation in DNA ‘breathing’ to be assessed by single-molecule fluorescence resonance energy transfer (FRET). When FRET is measured between a donor and an acceptor fluorophore on DNA, a larger fraction of nucleosomes containing acetylated Lys56 on histone H3 are found with low FRET efficiency, suggesting that Lys56 acetylation favours DNA breathing and a more open conformation. Pharmaceutical Applications: The HIV Protease Inhibitors 1. Modified peptides are key pharmaceuticals for the treatment of diseases. A prominent class of compounds in this category are the protease inhibitors. 2. The HIV protease inhibitors, containing a nonhydrolyzable peptide backbone at the site of proteolysis, are synthesized through incorporation of nonnatural amino acid. Biophysical Study Applications 1. The replacement of Met by Se-Met has been extensively used for phase determination in protein structure studies. 2. A spin-labeled nonnatural amino acid, containing nitroxyl 1-oxyl2,2,5,5-tetramethylpyrroline, was site-specifically incorporated into a T4 lysozyme using in vitro translation to yield a protein that had an electron paramagnetic resonance signal. 3. An E. coli strain that was auxotrophic for tryptophan was grown in the presence of 4-aminotryptophan. Incorporation of this amino acid into GFP created a new “Gold” fluorescent protein with a max 574 nm. Protein Glycosylation Sugar–Peptide Bonds Sugar–Amino Acid Linkages of Glycoproteins Type of bond N-glycosyl O-glycosyl C-mannosylation Phosphoglycosyl Glypiation Linkage Sugar Configuration Asn GlcNAc Asn Glc Asn GalNAc Asn Rha Arg Glc Ser/Thr GalNAc Ser/Thr GlcNAc Ser/Thr Gal Ser/Thr Man Ser/Thr Fuc Ser/Thr Pse Ser Glc Ser FucNAc Ser Xyl Ser Gal Thr Man Thr GlcNAc Thr Glc Thr Gal Hyli Gal Hyp Ara Hyp Gal Hyp GlcNAc Tyr Glc Tyr Glc Tyr Gal Trp Man Ser GlcNAc Ser Man Ser Fuc Ser Xyl Pr-C-(O)-EthN-6-P-Man β β * * β α β α α α α β β β α α α * * β β β * α β β α α-1-P α-1-P β-1-P *-1-P Examples Ovalbumin, fetuin, insulin receptor Laminin, H. halobium S-layer H. halobium S-layer S. sanguis cell wall Sweet corn amylogenin Mucins, fetuin, glycophorin Nuclear and cytoplasmic proteins Earthworm collagen, B. cellulosoleum Yeast mannoproteins Coagulation and fibrinolytic factors C. jejuni flagellins Coagulation factors P. aeruginosa pili Proteoglycans Cell walls of plants M. tuberculosis secreted glycoproteins Dictyosteliumh, T. cruzi Rho proteins (GTPases) H. halobium S-layer, vent worm collagen Collagen, C1q complement Potato lectin Wheat endosperm Dictyostelium cytoplasmic proteins Muscle and liver glycogenin C. thermohydrosulfuricum S-layer T. thermohydrosulfuricus S-layer RNase 2, interleukin 12, properdin Dictyostelium proteinases L. mexicana acid phosphatase Dictyostelium proteins T. cruzi cell surface T. brucei VSG, Thy-1, Sulfolobus proteins Consensus Squences or Glycosylation Motifs for the Formation of Glycopeptide Bonds Glycopeptide bond GlcNAc-β-Asn Glc-β-Asn GalNAc-α-Ser/Thr GlcNAc-α-Thr GlcNAc-β-Ser/Thr Man-α-Ser/Thr Fuc-α-Ser/Thr Glc-β-Ser Xyl-β-Ser Glc/GlcNAc-Thr Gal-Thr Gal-β-Hyl Ara-α-Hyp GlcNAc-Hyp Glc-α-Tyr GlcNAc-α-1-P-Ser Man-α-1-P-Ser Man-α-Trpf Man-6-P-EthN-C(O)-Pr Consensus sequence or peptide domain Asn-X-Ser/Thr (X = any amino acid except Pro) Asn-X-Ser/Thr Repeat domains rich in Ser, Thr, Pro, Gly, Ala in no special sequence Thr rich domain near Pro residues Ser/Thr rich domains near Pro, Val, Ala, Gly Ser/Thr rich domains EGF modules (Cys-X-X-Gly-Gly-Thr/Ser-Cys) EGF modules (Cys-X-Ser-X-Pro-Cys) Ser-Gly (Ala) (in the vicinity of one or more acidic residues) Rho: Thr-37d; Ras, Rac and Cdc42: Thr-35 Gly-X-Thr (X = Ala, Arg, Pro, Hyp, Ser) (vent worm) Collagen repeats (X-Hyl-Gly) Repetitive Hyp rich domains (e.g., Lys-Pro-Hyp-Hyp-Val) Skp1: Hyp-143d Glycogenin: Tyr-194d Ser rich domains (e.g., Ala-Ser-Ser-Ala) Ser rich repeat domains Trp-X-X-Trp GPI attached after cleavage of C-terminal peptide The Precursor of N-Glycosylation: In vivo Synthesis of Glc3Man9GlcNAc2-P-P-dolichol α; b; c, etc indicate the order of addition of the monosaccharide units for in vivo synthesis of Glc3Man9GlcNAc2-P-P-dolichol. N-Glycosylation 1. The GlcNAc-β-Asn bond is established through the cotranslational transfer of the dolichol-linked oligosaccharide which subsequently undergoes processing to the large array of N-linked carbohydrate units. 2. A consensus sequence, Asn-X-Ser/Thr, was postulated and supported by numerous studies employing structural, mutagenic, and in vitro approaches. 3. Although the Asn-X-Ser/Thr sequence occurs frequently in proteins, it does not necessarily indicate the actual presence of an N-glycosidic linkage, most probably due to conformational factors. 4. Replacement of Thr by Ser residues resulted in a pronounced decrease in glycosyl transfer. The Ser or Thr is required for a hydrogen-bond donor function in enzyme binding, although cysteine could take the place of the hydroxyamino acid. 5. The negative effect of Pro as the X amino acid has been attributed to its interference with the ability of the peptide chain to adopt a loop conformation. 6. The oligosaccharyltransferase has been isolated and shown to be a heterooligomeric ER membrane complex. O-Glycosylation I: The GalNAc-α-Ser/Thr Bond 1. The enzymes for biosynthesis of the GalNAc-α-Ser/Thr bond are a family of at least nine GalNAc-transferases. 2. These enzymes work in concert in a hierarchical manner to form the clustered Ser/Thr-linked oligosaccharides that occur in the “mucin”-type of glycoprotein. 3. Several of these enzymes have been cloned and they are distinct gene products and distributed on different chromosomes. 4. Although these enzymes act on characteristic peptide regions, no specific consensus sequence has been identified. Because they are assayed without prior separation, overlapping distinct substrate specificities may be masked. 5. This linkage is found in clusters of Ser/Thr residues with a β-turn near Pro and at a distance from charged amino acids. 6. In vitro studies suggest that Thr is favored over Ser for α-GalNAc modification. 7. The α-GalNAc-transfer occurs in the cis-Golgi. O-Glycosylation II: The GlcNAc-β-Ser/Thr bond 1. Attachment of GlcNAc-β-Ser/Thr to eukaryotic nuclear and cytosolic proteins is as dynamic and possibly as abundant as Ser/Thr phosphorylation. 2. Known GlcNAc-β-Ser/Thr attached proteins include cytoskeletal proteins and their regulatory proteins; viral proteins; nuclear-pore, heat-shock, tumor-suppressor, and nuclearoncogene proteins; RNA polymerase II catalytic subunit; and a multitude of transcription factors. Although functionally diverse, all of these proteins are also phosphoproteins. 3. Most GlcNAc-β-Ser/Thr attached proteins form highly regulated multimeric associations. 4. GlcNAc-transferase is localized outside of the channels of the secretory apparatus and has been purified and cloned. 5. The Ser/Thr residues which GlcNAc-transferase glycosylates are identical to those that can undergo O-phosphorylation, suggesting that there is a reciprocal relationship between these two modifications in a potential regulatory cycle in which cytosolic βN-acetylglucosaminidase also plays a key role. 6. Although no specific amino acid consensus sequence has as yet been found, some information relating to the polypeptide domains that it favors has been obtained. The Role of Protein Glucosylation in Protein Folding If not properly folded, the glycoprotein is liberated by GII from the Cnx/Crt anchor and reglucosylated by GT to allow rebinding of the glycoprotein to the lectins. Unfolded glycoproteins with monoglucosylated oligosaccharides is recognized by two lectins, membrane-bound calnexin (Cnx) and its soluble homolog, calreticulin (Crt). Upon adoption of the native structure, the glycoprotein is released from Cnx/Crt by GII and not reglucosylated by GT. Protein-linked Glc3Man9GlcNAc2 is partially deglucosylated to the monoglucosylated derivative by GI and GII. The Importance of Protein Glucosylation in Protein Folding 1. The lectin-monoglucosylated oligosaccharide interaction is one of the alternative ways by which cells retain improperly folded glycoproteins in ER. 2. Although it decreases the folding rate, it 1) increases folding efficiency; 2) prevents premature glycoprotein oligomerization and degradation; 3) suppresses formation of nonnative disulfide bonds 3. This allows interaction of protein moieties of folding glycoproteins with chaperones that assists in further folding. Phosphoglycosylation 1. The attachment of a sugar to the polypeptide chain through a phosphodiester bridge has been termed phosphoglycosylation, 2. GlcNAc-1-phosphotransferase was partially purified and localized in the Golgi compartment. 3. GlcNAc-1-phosphotransferase recognizes Ser-containing peptides of various proteins among which cysteine proteinases are the most prominent. 4. No single specific motif was observed. However, the transfers occur in Ser-rich domains in which the flanking Ala residues preferentially influence phosphoglycosylation. 5. GlcNAc-1-phosphotransferase does not phosphoglycosylate Thr residues. 6. Man-1-phosphotransferase has been characterized and adds Man-α-1-phosphate to Ser residues in domains rich in Ser. It does not act on Thr and its action is promoted by flanking Asp and Glu residues. C-Mannosylation 1. The enzyme which links C-1 of mannose to the C-2 atom of the indole ring of Trp has been studied in rat liver microsomes. 2. The glycosyl donor in this reaction is Dol-P-Man. 3. The dependence of the C-mannosylation on Dol-P-Man strongly suggests that it takes place in the ER, where all known Dol-P-Man-dependent reactions are localized. 4. The recognition signal for C-mannosylation has been assigned to a Trp-X-XTrp sequence in which the first Trp becomes glycosylated. 5. The Trp at position +3 is important for the glycosylation as the transfer activity was abolished when this amino acid was mutated to Ala and reduced to one-third when replaced by Phe. 6. A survey of protein databases has indicated that the Trp-X-X-Trp consensus sequence is present in 336 mammalian proteins, suggesting the possibility that C-mannosylation may occur quite frequently in higher eukaryotes. Protein Phosphorylation Historyic Events of Protein Phosphorylation 1. In the 1950s, it was discovered that the metabolic enzyme phosphorylase, responsible for the conversion of glycogen to glucose- 1-P, existed in an inactive (phosphorylase b) and an active (phosphorylase a) forms. 2. This conversion is made by phosphorylase kinase which catalyzes phosphorylation of phosphorylase and render it fully active. 3. Phosphorylase kinase was itself activated by protein kinase A (PKA) and the concept of protein phosphorylation as a key regulatory mark was born. Importance of Protein Phosphorylation 1. Most cellular processes and cell signaling pathways are regulated by protein phosphorylation catalyzed by protein kinases. 2. Protein kinases are regulated by inhibitory or activating protein partners, phosphorylation, cellular localization limiting availability of substrates and activators, protein degradation, and gene transcription. 3. Phosphorylation can result in enzyme activation, enzyme inhibition, the creation of recognition sites for recruitment of other proteins, and transitions in protein state from order to disorder or disorder to order. The Enzymes for Protein Phosphorylation: Protein Kinases 1. More than 518 human protein kinases were recognized through their conserved sequence motifs of which 478 protein kinases are typical and 40 are atypical. 2. The typical kinases are divided into those that phosphorylate serine or threonine residues (388 kinases) and those that phosphorylate tyrosine residues (90 kinases). 3. Atypical kinases have biochemical kinase activity but lack sequence similarity to the conventional kinases. 4. Unique kinase domain structures (170) from humans or closely related orthologs had been determined. 5. A characteristic feature of the protein kinase family is the different structures that they adopt between the active and inactive states. 6. Adoption of the active state occurs in response to specific signaling events, which are transduced via kinase associated regulatory domains and by phosphorylation of the kinase domain. Catalytic Mechanism of Protein Kinases Protein kinases catalyze the transfer of a phosphoryl group from the γ-phosphate of ATP to the hydroxyl group of serine, threonine, or tyrosine residues of proteins by the reaction scheme: Protein-OH + ATP4−.Mg2+ → Protein-O-PO3 2−+ ADP3− .Mg2+ + H+ Most protein kinases show specificity for the local region around the site of phosphorylation where certain residues are required for recognition. Protein Kinases and Their Preferred Substrate Specificities Substrate recognition at the catalytic site involves specific residues in the region near the site of phosphorylation. Association between kinase and substrate is often low affinity, and greater stability is achieved through docking sites that are remote from the catalytic site. Protein Kinase Catalysis 1. The kinase reaction proceeds with an inline mechanism in which the attacking group (serine, threonine, or tyrosine OH) comes in opposite to the leaving group (phosphate ester oxygen), leading to inversion of configuration at the phosphorus. 2. The catalytic step was fast (k3 ∼ 300–500 s−1), and the release of products relatively slow (k4 ∼ 20–30 s−1). 3. Thus, the rate-limiting step is the release of products, i.e., ADP and phosphorylated proteins. Protein Kinase Modulation 1. Phospho-signaling is a rapid action. Changes in specific phosphorylation targets can be detected within minutes after exposure stimuli. 2. When epidermal growth factor (EGF) is added to the media, greater than 500 proteins undergo phosphorylation changes by 5 min. 3. The specific kinetic details of these cellular phosphoryl transfer reactions are critical to their macroscopic effects on gene regulation, cell shape, and cell growth, which occur over longer timescales. Protein Methylation Methylase-Catalyzed Reactions Chromatin and Histone Modifications The eukaryotic DNA is compacted within the cell nucleus through its interactions with histone proteins, forming the nucleosomes. The histone N-terminal tails protrude outward beyond the gyres of DNA. Many of the amino acid residues within the histone tails can be posttranslationally modified, providing a landing pad for a diverse array of transcription factors, chromatin remodelers, and DNA-interacting proteins to regulate gene expression and other DNA-dependent processes. An Example of Protein Methylation: Histone H3 Lysine 4 (H3K4) Methylation 1. Methylation of histone Lys plays a crucial role in the regulation of key biological processes, such as cell cycle progression, transcription, and DNA repair . 2. In yeast, histone H3K4 methylation is carried out by SET [Su (var), Enhancer of Zeste, and Trithorax] domain-containing enzymes. The Set1 family protein forms a multiprotein complex named COMPASS (COMplex of Proteins ASsociated with Set1) in yeast. 3. Set1/COMPASS was the first identified histone H3K4 methylase capable of mono-, di-, and trimethylating H3K4. 4. In addition to the evolutionarily conserved SET domain located at the C terminus of Set1, most associating subunits are also conserved from yeast to human. 5. The histone H2B monoubiquitinase Rad6/Bre1 is required for proper H3K4 trimethylations. Protein Acetylation N-Acetylation Reactions N-Terminal Acetylation in Prokaryotes and Eukaryotes Acetylation Sites in Histones Hyperacetylated Chromatin Domains 1. In eukaryotes, the genome is packaged into two general types of chromatin: heterochromatin, which appears compact or condensed throughout the cell cycle, and euchromatin, which appears condensed only prior to mitosis. 2. A small number of loci that exhibit covalent histone modifications by histone acetyltransferases (HAT), such as hyperacetylation. 3. The hyperacetylated domains occur exclusively at loci containing highly expressed, tissue-specific genes, and that they are involved in the activation of these genes. Hperacetylated Domain of Heterochromatin A.A complex is nucleated at a regulatory sequence (blue line). B. This complex includes a HAT which modifies nearby nucleosomes. C. Modified nucleosomes in turn represent high affinity binding sites for a subset of the complex, resulting in the progressive spread of the complex. D.Additional sequences may be bound by factors that block the further spread of the complex and thus serve as boundaries (yellow oval). Protein Acetylation in Prokaryotes 1. Protein acetylation plays a critical regulatory role in eukaryotes but prokaryotes also have the capacity to acetylate both the N-terminal residues and the side chain of Lys and is widespread for regulation of fundamental cellular processes. 2. Lys acetylation in particular can occur in proteins involved in transcription, translation, pathways associated with central metabolism and stress responses. 3. Specific acetylated Lys residues map to critical regions in the 3-D of key proteins at active sites or surfaces that dock with other major cellular components. 4. Like phosphorylation, acetylation appears to be an ancient reversible modification that can be present at multiple sites in proteins, thereby potentially producing epigenetic combinatorial complexity. 5. Acetylation is particularly important in regulating central metabolism in prokaryotes due to the requirement for acetyl-CoA and NAD+ for HAT and HDAC, respectively. Signals and Combinatorial Functions of Histone Modifications 1. Alterations of chromatin structure are crucial for response to cell signaling and for programmed gene expression in development. 2. Posttranslational histone modifications influence changes in chromatin structure both directly and by targeting, or activating chromatin-remodeling complexes. 3. Histone modifications intersect with cell signaling pathways to control gene expression and can act combinatorially to enforce or reverse epigenetic marks in chromatin. 4. Through their recognition by protein complexes with enzymatic activities, cross talk is established between different modifications and with other epigenetic pathways, including noncoding RNAs (ncRNAs) and DNA methylation.