Dissecting β-ring assembly pathway of the mammalian 20S proteasome

Abstract

The 20S proteasome is the catalytic core of the 26S proteasome. It comprises four stacked rings of seven subunits each, a 1-7 b 1-7 b 1-7 a 1-7 . Recent studies indicated that proteasome-specific chaperones and b-subunit appendages assist in the formation of a-rings and dimerization of halfproteasomes, but the process involved in the assembly of b-rings is poorly understood. Here, we clarify the mechanism of b-ring formation on a-rings by characterizing assembly intermediates accumulated in cells depleted of each b-subunit. Starting from b2, incorporation of b-subunits occurs in an orderly manner dependent on the propeptides of b2 and b5, and the C-terminal tail of b2. Unexpectedly, hUmp1, a chaperone functioning at the final assembly step, is incorporated as early as b2 and is required for the structural integrity of early assembly intermediates. We propose a model in which b-ring formation is assisted by both intramolecular and extrinsic chaperones, whose roles are partially different between yeast and mammals.

Involvement of Bag6 and the TRC pathway in proteasome assembly

“…Half-CPs then dimerize on the incorporation of b7 to form mature CPs, accompanied by cleavage of b-subunit propeptides and degradation of UMP1 and PAC1-PAC2 ( Supplementary Fig. S1) 14,26,[31][32][33][34][35][36] .…”

“…2f). PAC3 is associated with a-rings and detaches from them on b3 recruitment 36 . A small amount of PAC3 was already observed in the intermediates by b7 knockdown, which in principle halts the CP assembly just before b7 incorporation, suggesting that such intermediates are unstable and that some of the already incorporated b-subunits detach.…”

“…The following antibodies were purchased and used at the indicated dilutions: ubiquitin (1:1,000; Dako), actin (1:1,000; Chemicon), GAPDH (1:1,000; Serotec), Derlin1 (1:1,000; MBL), Flag (1:1,000; Sigma), and b3 and b7 (1:500; BioMol). Other antibodies were described previously 21,31,32,36 . Densitometry analysis of the bands was performed using ImageQuant LAS4000 (GE Healthcare).…”

“…siRNA-mediated knockdown was performed as described previously 21,31,32,36 . The targeted sequences are as follows: TRC40, 5 0 -CAGACAACUUUCAUCUGCGUAUGCA-3 0 ; Bag6, 5 0 -GCGCAUGUGCUGC AUUGCACAGAUA-3 0 ; b7, 5 0 -GCAUGCGAGUGCUGUACUACC-3 0 ; Rpt4, 5 0 -GAGAGAUUCAGAGAACGUUAAUGGA-3 0 .…”

Bufalin derivative BF211 inhibits proteasome activity in human lung cancer cells in vitro by inhibiting β1 subunit expression and disrupting proteasome assembly

“…The α subunits www.nature.com/aps Sun P et al Acta Pharmacologica Sinica npg maintain the structure, whereas the core β rings contains the three proteolytically active β subunits [32] . Notably, previous studies reported that the active proteasome β subunits exist in an immature precursor form before being assembled into the integral 26S proteasome [33][34][35][36][37] . The β subunits mature once the proteasome assembles [38,39] .…”