E1/E2/E3
Ubiquitin is a 76-amino acid protein, highly conserved throughout evolution, which is found in all eukaryotic organisms and cell types. The C-terminus of ubiquitin is generally linked via an amide isopeptide bond to the e-amino group of an internal lysine residue of the substrate protein. Ubiquin is conjugated to the protein substrate by means of a three-step cascade mechanism. First, a ubiquitin-activating enzyme, E1, activates ubiquitin in an ATP-dependent reaction. Ubiquitin is then transferred to the active site cysteine of a ubiquitin-conjugating enzyme (E2). Finally, a ubiquitin protein ligase (E3) catalyzes the transfer of ubiquitin from E2 to the substrate. A number of families of E3s or E3 multiprotein complexes have been identified. RING-finger containing E3s catalyze direct transfer of the activated ubiquitin from E2 to the E3-bound substrate. With HECT-(homologous to E6-AP COOH terminus) domain E3 enzymes, the ubiquitin is transferred from E2 to the active site of E3, generating a high-energy intermediate, and is then transferred to the ligase-bound substrate [9]. E3s play a key role in protein ubiquitylation because they serve as the specific recognition factors of the system. Proteins may be modified by monoubiquitylation: the conjugation of a single ubiquitin to one or several lysines. Monoubiquitylation is involved in histone modification, and in several trafficking events. As ubiquitin itself carries several conserved acceptor lysines — notably Lys29, Lys48 and Lys63 — multiubiquitin chains can be generated, in some cases with the assistance of E4 enzymes. K48-linked (and sometimes K29-linked) multiubiquitin chains, at least four ubiquitin units long, are potent targeting signals leading to the recognition and subsequent degradation of target proteins by the 26S proteasome, a large multisubunit protease complex [10]. K63-linked ubiquitin chains are involved in other functions, including DNA-repair, the activation of translation, the activation of specific kinases, and endocytosis [11, 12]. Ubiquitylation is a dynamic process. Deubiquitylating enzymes (DUBs) cleave ubiquitin from proteins, and disassemble ubiquitin chains [13]. Several such enzymes are involved in endocytosis.
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