A-Z of Post-Translational Modifications
We all know the two stages of information flow from nucleic acid to protein – transcription and translation. But it should be three stages, really where the last stage is post-translational modification (PTM) of proteins – covalent and usually enzymatic modification of proteins following protein biosynthesis. In fact, there is hardly any protein that doesn’t have a PTM, so though DNA encodes 20 primary amino acids, proteins contain more than 140 different residues.
One of the most common modifications is glycosylation – the addition of sugar monomers and polysaccharide chains to proteins. The most dramatic is probably phosphorylation, attachment of the phosphate groups that makes kinases active, triggering regulatory cascades in the cells. [1]
The Importance of Post-translational Modification
For many proteins, especially eukaryotic, the absence of PTM makes them as useless as a Christmas tree without decorations. The absence of many types of PTMs characteristic to eukaryotic cells makes the production of functional eukaryotic proteins in E.coli difficult.
For example, the first human hormone produced using recombinant technology, insulin, consists of two polypeptide chains connected by another common PTM, a disulfide bond that forms between two methionines. The chains are produced separately in E.coli, and after the purification, the disulfide bonds have to be chemically introduced [2].
PTMs occur on the amino acid side chains or at the protein’s C- or N- termini. Amino acids that receive post-translational modification usually have a functional group that can serve as a nucleophile in the reaction:
- N- and C-termini that have free amine or carboxyl groups;
- the amine forms of arginine, histidine, and lysine;
- the hydroxyl groups of serine, threonine, and tyrosine;
- the thiolate anion of cysteine or the carboxylates of aspartate and glutamate.
In addition, despite being a weak nucleophile, the amide of asparagine can act as an attachment point for glycans – an oligo or polysaccharide. Rarer PTMs take place at oxidized methionines and some methylenes in side chains. [3]
A-Z of Post-translational Modifications
Let’s walk through the alphabet of the post-translational modifications. You can award yourself a point for each one you know.
| PTM | Details | Frequency of modification (from experimental data)[4] | |
|---|---|---|---|
| A | |||
| Acetylation | The addition of an acetyl group. This can occur at lysine residues or at the protein’s N-terminus. The reverse is called deacetylation. Lysine acetylation of histones neutralizes lysine electrical charge, loosening the chromatin | 6,775 | |
| Acylation | O-acylation (esters), N-acylation (amides), S-acylation (thioesters). | NR | |
| Alkylation | The addition of an alkyl group such as methyl, ethyl | NR | |
| Amino acid addition (not mediated by the ribosome) | |||
| •Arginylation | A tRNA-mediation addition | NR | |
| •Polyglutamylation | The addition of multiple glutamic acid residues to certain proteins (e.g. tubulin) via the N-terminus | NR | |
| •Polyglycylation | The addition of one or more glycine residues to the C-terminal tail of alpha or beta tubulin | NR | |
| Amide bond formation | Also known as amidation. at protein C-terminus. Formed by oxidative dissociation of a C-terminal glycine residue | 2,852 | |
| AMPylation | Also, known as Adenylylation. The covalent attachment of adenosine monophosphate (AMP) molecule to tyrosine, threonine, or serine residues. Common in GTPases | NR | |
| B | |||
| Butyrylation | Adding butyric acid residue or one of its derivatives | NR | |
| C | |||
| Carbonylation | Modification of histidine, cysteine, and lysine in proteins to aldehydes and ketones. While most of the rest of PTMs are “by design” to enable the protein function, carbonylation is a result of oxidative stress. It damages the proteins | NR | |
| D | |||
| Disulfide bond formation | Formation of the covalent bond between two cysteines. Usually stabilizes the 3D structure of the protein. Unlike most of the covalent bonds, it is reversible by oxidoreductases | NR | |
| F | |||
| Flavin moiety (FMN or FAD) attachment | (FMN or FAD) attachment that is the catalytic center of the oxidoreductases, components of the succinate dehydrogenase complex, correspondingly. | NR | |
| G | |||
| Gamma-carboxylation | Of glutamic acid residues. Found in clotting factors and other proteins of the coagulation cascade and other proteins | 450 | |
| Glycosylation | The addition of a glucose derivative, glycosyl group to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, or tryptophan resulting in a glycoprotein. One of the most abundant post-translational modifications | 5,593 -N-linked glycosylation 1,189 - O-linked glycosylation 148 - C-linked glycosylation | |
| Glycation | Non-enzymatic attachment of sugars | NR | |
| H | |||
| Heme attachment | The significance of heme as a protein cofactor goes far beyond the most famous heme-containing protein, hemoglobin. Hemes are found in a number of other important hemoproteins such as myoglobin, cytochromes, catalases, heme peroxidase | NR | |
| I | |||
| (Iso)prenylation | The addition of an isoprenoid group farnesol or geranylgeraniol to C-terminal cysteine(s) of the target protein. | NR | |
| Isopeptide bond formation | This amide bond involves at least one amino acid side chain and can result in stably linked protein dimers/mulitmers. Common in surface proteins of Gram-positive bacteria | NR | |
| L | |||
| Lipidation | Adding lipids to the proteins, for example, lipid-anchored proteins - prenylated protein (see above), fatty acylated proteins and glycosylphosphatidylinositol-linked proteins | NR | |
| Lipoylation | Attachment of a lipoate (C8) | NR | |
| M | |||
| Malonylation | Adding malonyl group to the amino acid lysine, changing its charge from +1 to −1 | NR | |
| Methylation | Involved the addition of a methyl group, at various residues (commonly lysine or arginine). The reverse reaction is called demethylation. This is another important modification of chromatin remodeling | 1,545 | |
| Myristoylation | Attachment of myristate, a C14 saturated acid | 179 | |
| N | |||
| Nucleotide addition | Such as ADP-ribosylation | NR | |
| O | |||
| O-linked β-N-acetylglucosamination | The addition of N-acetylglucosamine to serine or threonine residues of nucleocytoplasmic proteins | NR | |
| Palmitoylation | Attachment of palmitate, a C16 saturated acid | 314 | |
| Phosphorylation | Addition of a phosphate group to a residue. The main residues for phosphorylation are serine, threonine, and tyrosine (O-linked), or histidine (N-linked). Possibly the most well known PTM. It sometimes seems that half of the cell proteins phosphorylate and dephosphorylate each other | 58,525 | |
| Phosphate ester | (O-linked) or phosphoramidate (N-linked) formation | NR | |
| Propionylation | Adding the propyl group, for example, lysine in histones | NR | |
| Pyroglutamate formation | Formation of pyroglutamate (a cyclic amino acid) from either glutamic acid or glutamine – can occur spontaneously or catalyzed by glutaminyl cyclase | NR | |
| Polysialylation | The addition of polysialic acid, PSA | NR | |
| S | |||
| Signal sequence processing | Signal sequences are N-terminal extensions of newly synthesized secretory and membrane proteins from 16 up to 50 amino acid residues and have C-terminal region with the cleavage site for signal peptidase. In eukaryotes, signal sequences direct the insertion of proteins into the membrane of the endoplasmic reticulum and are usually cleaved off by signal peptidase | NR | |
| S-modification of cysteine residue | |||
| •S-glutathionylation | Addition of glutathione, a tripeptide with a gamma peptide linkage between the carboxyl group of the glutamate side chain and cysteine. Glutathione is a major antioxidant | NR | |
| •S-nitrosylation | Formation of S-nitrosothiol (SNO). Occurs when a nitric oxide group is attached to a cysteine thiol. S-nitrosylation has diverse regulatory roles in bacteria, yeast, and plants and in all mammalian cells | 63 | |
| •S-sulfenylation | Reversible covalent addition of an oxygen atom to the thiol group of a cysteine | NR | |
| •S-sulfinylation | Covalent attachment of two oxygen atoms to the thiol group of a cysteine | NR | |
| •S-sulfonylation | Covalent attachment of three oxygen atoms to the thiol group of a cysteine, leading to the formation of a cysteic acid | NR | |
| Succinylation | Attachment of a succinyl group to lysin | NR | |
| Sulfation | The addition of a sulfate group to a tyrosine | 523 | |
| Sumoylation | Attachment of SUMO (small ubiquitin-like protein modifiers). Sumoylation can affect a protein’s structure and subcellular localization | 422 | |
| U | |||
| Ubiquitylation/ ubiquitination | Attachment of a 8.6K protein, ubiquitin (from Latin ubique - “everywhere”). The ubiquitination can either consist of a single ubiquitin protein or a chain of ubiquitin (polyubiquitylation). The first ubiquitin molecule is covalently bound through its C-terminal carboxylate group to a particular lysine, cysteine, serine, threonine or N-terminus of the target protein. Ubiquitination can direct proteins for degradation via the proteasome, alter their cellular localization, affect their activity, and promote or prevent protein-protein interactions | 905 | |
| Uridylylation | The addition of a uridylyl-group (i.e. uridine monophosphate, UMP), usually to tyrosine | NR | |
Summary of Post-translational Modifications
I bet that’s more post-translational modifications than you knew about. Being aware of all these “decorations” will help you in appreciating any unexpected traits of your proteins.
For example, if your protein on western blot shows as multiple bands and you are sure it’s not degradation products or non-specific binding in your western blots, this might be an indication of post-translational modifications that change the molecular mass or charge of your protein, for example, glycosylation. These modifications are usually biologically relevant and point out research avenues to explore.
Do let us know if we missed some important PTMs or need to update the article, by leaving a comment below.
References
- Uversky, V.N. (2013), Posttranslational Modification. in Brenner’s Encyclopedia of Genetics (Second Edition). Editors Maloy, S and Hughes K. pp. 425-30. doi: 10.1016/B978-0-12-374984-0.01203-1
- Kjeldsen T. Yeast secretory expression of insulin precursors. Appl Microbiol Biotechnol. 2000;54(3):277-286. doi:10.1007/s002530000402
- Walsh, C.T. (2006). Post-translational modification of proteins: expanding nature’s inventory. Englewood: Roberts and Co. pp. 12–14. doi: 10.1002/anie.200585363
- Khoury, G. A., et al. Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database. Sci. Rep. 1, 90; doi: 10.1038/srep00090 (2011).