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  • GSK2606414 synthesis br The classical ubiquitination pathway


    The classical ubiquitination pathway Ubiquitination is an enzymatic process that involves the addition of an ubiquitin protein to a substrate that usually becomes inactivated followed by degradation in the proteasome; however, several other functions have also been described. For the discovery of the ubiquitin-mediated protein degradation pathway, Aaron Ciechanover, Avram Hershko and Irwin Rose were awarded the Nobel prize in 2004 [7]. Over the last two decades, the molecular basis for how the individual components involved with protein ubiquitination interact with each other has been revealed, but it is still far from fully understood. Protein ubiquitination is initiated by an E1 ubiquitin-activating enzyme (E1) that requires ATP and Mg2+ to catalyse the C-terminal acyl-adenylation of ubiquitin, a reaction that results in an E1 non-covalently bound to adenylated ubiquitin. In the following second reaction step, the catalytic cysteine present in the E1, attacks the adenylated ubiquitin to form the activated E1-ubiquitin (E1∼Ub) thioester-bonded complex, with release of AMP [8]. Next, a second ubiquitin molecule is adenylated and bound by the E1∼Ub thioester-bonded complex. This is followed by ubiquitin-transfer of its thioester-bonded ubiquitin to an E2 conjugating enzyme (E2), again involving a cysteine residue, to yield a thioester-bonded E2∼Ubcomplex [9]. Finally, an E3 ubiquitin ligase (E3) ensures ubiquitin-coupling from the E2∼Ub to a primary amine (i.e. the ε‐amine of lysine or the N‐terminus) of substrate GSK2606414 synthesis resulting in a stable isopeptide bond linkage. E3 ligases are the final catalytic components in the E1-E2-E3 ubiquitination cascade, and are in most cases critical for ubiquitin transfer from the E2∼Ub complex to its specific substrate proteins. Mono-, multi- and poly-ubiquitination are signals used in protein sorting, signaling, DNA-repair, histone-regulation, and proteasomal/lysosomal degradation (extensively reviewed elsewhere [10], [11]). Most E3 ligases are grouped into the HECT-E3 (omologous to 6-AP arboxy erminus) or the larger RING-E3 (eally nteresting ew ene) ligase family [11]. RING-E3 ligases directly mediate ubiquitin transfer from an E2 to a specific substrate, while the HECT-E3s transfer ubiquitin via an E3∼Ub thioester intermediate [12]. The main functions of RING-E3s are to provide the scaffold that allows simultaneous binding of E2 and the substrate protein, and in addition to induce a “closed” E2∼Ub conformation that is primed for attack by the substrate nucleophile [13]. More than 600 RING-E3s have been found in humans and they outnumber the E1 and E2 proteins by far, with only two E1s GSK2606414 synthesis and nearly forty E2s in humans [13]. Thus, E3 ligases impose, to a large extent, specificity onto the ubiquitination pathway and are therefore considered prime drug targets [14]. Consistent with their fundamental role in orchestrating cellular trafficking of target proteins, malfunction and faulty regulation of E3s is often associated with human disease including viral infections [15], neurodegenerative disorders [16], [17] and cancer [14].
    MARCH E3 ligases A specific family of eukaryotic E3 ligases that has received additional attention recently consists of the embrane-ssociated ING- (MARCH) proteins. Initially, two viral MARCH-homologue gene products were discovered in Kaposi\'s sarcoma (KS)-associated herpesvirus (KSHV), and subsequently named K3 (kK3) and K5 (kK5). Later, kK3- and kK5-related E3s were found in poxviruses and a K3-related E3 ligase was discovered in myxomavirus (termed mK3) [18]. These viral MARCH E3s help escape from host defence mechanisms by down-regulating major histocompatibility complex (MHC) class I (MHC‐I) antigen presentation and are now thought to originate from the human MARCH proteins due to their overlapping substrate spectrum and structural similarity [18], [19], [20]. Interestingly, viral MARCHs are often able to ubiquinate their targets on non-lysine residues, such as cysteine, serine and threonine residues [19], [21], [22].