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. 2023 May 30;14:3126. doi: 10.1038/s41467-023-38404-w

Fig. 10. C-PLoop function similar to other CCRs.

Fig. 10

a 20S proteasomes binding region derived from peptide array is highlighted on the C-PLoop structure (PDB-6C2U). β-strands and α-helices are shown in cyan and red, respectively. b Peptide array-based C-PLoop binding peptides are mapped on the structure of the archaeal 20S proteasome β-subunit. An enlargement of the β-subunit binding site is shown in the inset. The catalytic activities of the human c 20S and d 26S proteasomes were measured in the presence of increasing concentrations of the C-PLoop. A concentration-dependent increase in inhibition of the 20S proteasome is detected, while the 26S proteasome was not inhibited. Bars represent mean values from three independent experiments; error bars represent SD. e Overexpression of C-PLoop-FLAG leads to increase in p53 and α-synuclein (α-syn) levels in T47D-760S cells. Cerulean was used as an overexpression control, and GAPDH as a loading control. f Quantification demonstrating the average of at least three independent experiments. Measurements were subjected to a one-tailed Student t-test analysis. Error bars represent SEM. * for p53 and α-syn represent p-values = 0.0318 and 0.0213, respectively. g CCRs act as allosteric regulators of the 20S proteasome. CCR (cyan) functions by binding to the PSMB4 subunit of the 20S proteasome (magenta). This interaction induces an allosteric structural transition within the 20S proteasome that perturbs the three enzymatic activities of the complex. We detected one CCR bound to the 20S proteasome; however, given the symmetrical architecture of the 20S proteasome, we cannot exclude the possibility that two CCRs bind, occupying the two PSMB4 subunits. A balance between structural rigidity and flexibility is required for CCR function. Increased structural flexibility will lead to CCR degradation by the 20S proteasome (bright cyan), like any other intrinsically unstructured protein, whereas a rigid CCR structure (dark cyan) will prevent the CCR/20S interaction. Source data are provided with this paper.