Aggregation Pathways of Native‐Like Ubiquitin Promoted by Single‐Point Mutation, Metal Ion Concentration, and Dielectric Constant of the Medium

Abstract

Ubiquitin-positive protein aggregates are biomarkers of neurodegeneration, but the molecular mechanism responsible for their formation and accumulation is still unclear. Possible aggregation pathways of human ubiquitin (hUb) promoted by both intrinsic and extrinsic factors, are here investigated. By a computational analysis, two different hUb dimers are indicated as possible precursors of amyloid-like structures, but their formation is disfavored by an electrostatic repulsion involving Glu16 and other carboxylate residues present at the dimer interface. Experimental data on the E16V mutant of hUb shows that this single-point mutation, although not affecting the overall protein conformation, promotes protein aggregation. It is sufficient to shift the same mutation by only two residues (E18V) to regain the behavior of wild-type hUb. The neutralization of Glu16 negative charge by a metal ion and a decrease of the dielectric constant of the medium by addition of trifluoroethanol (TFE), also promote hUb aggregation. The outcomes of this research have important implications for the prediction of physiological parameters that favor aggregate formation.

The <italic>pros</italic> and <italic>cons</italic> of ubiquitination on the formation of protein condensates

“…Ub also exhibits a high tendency to assemble in the presence of metal ions with high concentrations. Biophysical experiments revealed that Hg 2+ , Cd 2+ , Pt 2+ , or Zn 2+ could bridge residues from two or more Ub molecules, leading to the formation of protein clusters [ 188 – 191 ] . Intriguingly, the relevance of Zn 2+ -mediated Ub aggregation is supported based on the detection of high zinc ions concentrations in neurons, which is related to the onset of Alzheimer’s, Parkinson’s diseases, and other neurodegenerations [ 192 – 194 ] .…”