From Single Molecules to Single Cells: Biophysics of Interactions between Small Regulators and Proteasome

Proteasome is the essential multicatalytic protease from the ubiquitin-proteasome pathway, responsible for the regulated degradation of the bulk of intracellular proteins. Cancer cells are more susceptible than healthy cells to blocking of the proteasome catalytic activity. Consequently, proteasome inhibitors are used as successful drugs against blood cancers. Still, many patients do not respond to the treatment or develop resistance. Since all the currently used proteasome-targeting drugs are competitive inhibitors blocking the active sites, we turned our attention to novel noncompetitive regulators affecting protein-protein interactions (PPI), especially attractive for the multisubunit and modular enzyme like the proteasome. Testing effects of competitive inhibitors requires only monitoring of peptidase activity of the proteasome catalytic core. The PPI-targeting compounds often perform disappointingly weak in such tests. Here we show that biophysical methods provide straightforward tools to follow the impact of PPI disruption on proteasome. We assessed small-molecule compounds that distinctly affect assembly and activity of the proteasome: (i) analogues of the rapamycin binding domain, (ii) Tat peptide mimetics modeled after the proteasome-binding domain of HIV-1Tat protein, (iii) derivatives of Pro and Arg-rich fragment of cathelicidin PR39, and (iv) TCH imidazoline derivatives. Probing topography of the catalytic core with AFM identified the gate status and alpha face dynamics as an excellent sensor of a type of the compounds proteasome interactions. Intrinsic fluorescence of tryptophans revealed divergent communication pathways affected by the compounds. AFM based analysis of proteasomal super-assemblies demonstrated distinct modes of compounds-induced disruption of PPIs. Finally, Peak Force QNM AFM demonstrated profound changes in cell elasticity, adhesiveness and roughness of cultured cells treated with the compounds. Based on these findings we attempted to build a model that can help predict the effects of small molecules on proteasome and cell physiology.