Groll M., Ditzel L., L?we J., Stock D., Bochtler M., Bartunik H. misfolded proteins, nascent prematurely terminated polypeptides, or proteins that fail to assemble into complexes. These diverse activities and its central role in apoptosis have made the proteasome an important target for drug development, in particular to combat malignancies. Marking Proteins for Degradation Targeting of most substrates to the 26 S proteasome requires their prior marking by a covalently linked polyubiquitin chain(s). During association with the proteasome, the substrate is directed into the catalytic core, where it is digested, whereas most of the ubiquitin molecules are recycled. Protein ubiquitination is a multistep process orchestrated by the concerted action of three enzymes. The reaction begins with E1,2 which initially adenylates the C-terminal glycine of ubiquitin and then forms a thioester bond between the activated glycine residue and a cysteine residue on the E1 catalytic site. Next, E2 acquires the activated ubiquitin through a transthioesterification reaction to form a similar thioester bond between the E2 active-site cysteine and the activated ubiquitin. Finally, E3 recruits the target protein and guides the transfer of the activated ubiquitin from the E2 enzyme to the substrate. In Rabbit Polyclonal to OR2T2 most cases, an ?-NH2 group of a lysine residue on the substrate attacks the thioester bond between the ubiquitin and E2, and an isopeptide bond is formed, linking the activated C-terminal glycine of ubiquitin to the amino group in the attacking lysine of the target substrate (1). Ubiquitin transfer from the E2 enzyme to the substrate is SR 3677 dihydrochloride catalyzed directly by RING (really interesting new gene) finger-containing E3 enzymes and indirectly when a HECT (homologous to E6-AP carboxyl terminus) domain-containing E3 is mediating the transfer. The process is repeated in a cyclic manner where, in each step, a new moiety of ubiquitin is conjugated to an internal lysine residue (typically Lys48) of the previously conjugated molecule. This generated polyubiquitin chain is regarded as the targeting signal for the downstream 26 S proteasome. However, in view of recent findings, several alternative mechanisms have been proposed (for a recent review, see Ref. 2). Li (3) demonstrated in a reconstituted cell-free system that a preformed polyubiquitin chain can be initially assembled on the active-site cysteine of E2 (UBE2G2), presumably by the action of an exogenous E2 acting in and cleaving after basic residues), the 5-subunit has a chymotryptic activity (cleaving after hydrophobic residues), and SR 3677 dihydrochloride the 1-subunit has a caspase-like or post-acidic activity (supplemental Fig. S1). The proteolytic active sites of SR 3677 dihydrochloride the proteasome are facing the lumen of the barrel and are sequestered from the bulk solution. In addition, the gated channel in the -ring, through which substrates enter the 20 S particle, is narrow. Thus, only unfolded polypeptides can enter the 20 S proteasome. Consequently, a globular substrate must be unfolded (probably by the ATPases of the 19 S complex) to be translocated and digested within the 20 S particle. The 19 S regulatory particle is a large complex of 1 1 MDa and consists of at least 19 different subunits (supplemental Table S1). Nine of these subunits form a lid, whereas the other 10 subunits, including the six ATPases, compose the base of the 19 S particle (Fig. 1 em B /em ). Electron micrographs (21,C23) and cross-linking experiments (24, 25) demonstrated that the six homologous ATPases are associated with the -rings of the 20 S particle. In addition to these six ATPases, which are termed (in yeast) Rpt1C6, the base contains four non-ATPase subunits (Rpn1, Rpn2, Rpn10, and SR 3677 dihydrochloride Rpn13). Despite the high sequence homology between the six ATPases, some of their amino acid sequences SR 3677 dihydrochloride are significantly divergent (mainly at the N-terminal domains), and the different subunits may have distinct functions as indicated previously (26). One ATPase, Rpt2, was shown to play a role in opening the gated -ring to facilitate substrate entry (27), whereas Rpt5 was.