Post-Translational Modification of Proteins
Studying genes at the genetic level is generally relatively simple, but once we move up to the protein level, the difficulty increases significantly. This is due to the various post-translational modifications that proteins undergo. Post-translational protein modifications are covalent processes that proteins undergo during or after translation. These modifications involve adding functional groups to one or more amino acid residues or removing groups through protein hydrolysis, altering the properties of the protein. Phosphorylation, ubiquitination, acetylation, glycosylation, and others are all post-translational protein modifications.
Ubiquitin and Ubiquitin Chains
Ubiquitin (Ub) is a small protein consisting of 76 amino acids, with a molecular weight of approximately 8.5 kDa. It is highly conserved throughout evolution and contains seven lysine (Lys) residues (K6, K11, K27, K33, K48, and K63), one N-terminal methionine (Met) residue (M1), and one C-terminal glycine (Gly) residue (G76). Ubiquitin molecules can bind to each other through these residues to form ubiquitin chains. If ubiquitin molecules are linked in the same way, they form homotypic ubiquitin chains, while if they are linked differently, they form heterotypic ubiquitin chains. Ubiquitin chains can be linear or form branched structures. Additionally, ubiquitin itself can undergo various modifications.
Ubiquitination
Ubiquitination refers to the process in which ubiquitin is covalently attached to a target protein under the catalytic action of a series of enzymes. The series of enzymes here refers to three enzymes that work cooperatively in the ubiquitination cascade reaction: E1 Ubiquitin-activating enzyme, E2 Ubiquitin-conjugating enzymes, and E3 Ubiquitin-ligase enzymes.
There are primarily two major classes of E3 ubiquitin ligases: the HECT domain family and the RING domain family. Recently, a new E3 family called the U-box protein family has also been discovered. The HECT domain primarily functions by forming a catalytic thioester bond with ubiquitin, whereas the RING domain provides a docking site for both E2 and the substrate, facilitating the transfer of ubiquitin from E2 to the substrate.
The specific process of ubiquitination is shown in the following diagram. First, E1 activates the ubiquitin molecule using energy provided by ATP, forming the Ub-E1 complex. The Ub-E1 complex transfers ubiquitin to E2 through a transesterification reaction, forming the Ub-E2 complex. The Ub-E2 complex can transfer ubiquitin to the target protein in two ways: the first is by directly attaching the C-terminus of ubiquitin to the ε-amino group of a lysine residue on the target protein after E3 specifically recognizes the target protein. The second step involves the transfer of ubiquitin to E3, achieved through a specialized transesterification reaction. After this, E3 precisely identifies the intended protein target and binds the C-terminus of ubiquitin to the ε-amino group of a lysine residue on the target protein. To sum up, the process of attaching ubiquitin to the target protein or an existing ubiquitin chain on the target protein unfolds in a distinctive sequence catalyzed by E1, E2, and E3 enzymes. Notably, it is the E3 ubiquitin ligase that ultimately dictates the specificity in recognizing the target protein. Additionally, ubiquitination modifications can also occur at the N-terminus of proteins and on certain other amino acids (cysteine, serine, threonine).
Fig.1 Ubiquitination cascade (Salas-Lloret D., González-Prieto R. 2022).
Deubiquitination
Ubiquitination operates as a meticulously controlled reversible procedure, with deubiquitinating enzymes (DUBs) taking charge of reversing ubiquitination alterations by cleaving peptide or isopeptide bonds linking ubiquitin molecules or between ubiquitin and substrate proteins. Notably, these DUBs assume a dual role, not only impeding the ubiquitination process but also actively fostering it through various mechanisms, including disassembling ubiquitination inhibitors, restoring ubiquitin molecules, and overseeing the ubiquitination course. Consequently, the intricate interplay between deubiquitinating enzymes and the ubiquitination system forms an elaborate regulatory matrix.
The realms of ubiquitination and deubiquitination constitute vital, ever-evolving, and precise biological pathways. The equilibrium maintained between these processes significantly influences the destiny of a multitude of soluble proteins within cellular structures, steering an array of crucial biological operations. These encompass but are not confined to protein sorting, protein breakdown, DNA repair, activation of transcription, and orchestration of gene silencing.
Reference
- Salas-Lloret D.; González-Prieto R. Insights in post-translational modifications: ubiquitin and SUMO. International Journal of Molecular Sciences. 2022, 23(6): 3281.
.
