Purification and characterization of an endogenous inhibitor specific to the Z‐Leu‐Leu‐Leu‐MCA degrading activity in proteasome and its identification as heat‐shock protein 90

Abstract

We previously identified a benzyloxycarbonyl(Z)-Leu-Leu-Leu-4-methylcoumaryl-7-amide (ZLLL-MCA) degrading activity in proteasome as a candidate for the regulator of neurite outgrowth. As its counterpart, we purified a protein from bovine brain that specifically inhibits the ZLLL-MCA degrading activity in proteasome. This protein is heat stable and has no effect on the other catalytic activities in proteasome, or on the activities of trypsin, chymotrypsin, or m-and p-calpains either. The molar ratio ofinhibitor-to-proteasome that inhibits 50% of the ZLLL-MCA degrading activity of proteasome is 1:1. The inhibitory mechanism of the inhibitor against proteasome is non-competitive. Finally, the inhibitor was identified as heat-shock protein 90 (HSP90) by partial amino acid sequencing and immunodetection. The results suggest that HSP90 initiates neurite outgrowth through the inhibition of the ZLLL-MCA degrading activity in proteasome.

Proteasomal Inhibition by α-Synuclein Filaments and Oligomers

“…However, it does not require the aggregates to be in the insoluble filamentous state but merely requires a structural transition to a form present on both filaments and soluble oligomers, a transition that correlates to emergence of the epitopes recognized by the FILA-1 antibody. The inhibitory mode is noncompetitive and thus resembles the inhibitory effect by HSP-90 (49). It likely represents a structure change by the binding of the aggregated AS to the exterior of the ␤5 subunit, responsible for the chymotrypsin-like activity, and relayed to its catalytic site on its interior surface of the proteasomal barrel.…”

Lactacystin, Proteasome Function, and Cell Fate

“…It is possible that neurite outgrowth through lactacystin-mediated proteasome inhibition mimics a normal mechanism involved in cellular differentiation. A number of endogenous inhibitors of the proteasome have been discovered, some of which affect multiple activities, whereas others are more specific (32)(33)(34). Endogenous regulators of proteasome activities might influence cell physiology and fate.…”

Proteasomes and Molecular Chaperones : Cellular Machinery Responsible for Folding and Destruction of Unfolded Proteins

“…Hsp90 also shows direct interaction with the proteasome and might possess regulatory roles, other than determination of the fate of unfolded proteins that cooperate with co-chaperones, PA28 and CHIP. 31,40 Initially, Hsp90 was considered to inhibit the 20S proteasome 51,52 and also to protect it from oxidative stress. 53 Proteomics analysis of proteasome-interacting proteins revealed physical interactions between Hsp70 and Hsp90 with the 26S proteasome.…”