Slow Evolution toward “Super-Aggregation” of the Oligomers Formed through the Swapping of RNase A N-Termini: A Wish for Amyloidosis?

Abstract

Natively monomeric RNase A can oligomerize upon lyophilization from 40% acetic acid solutions or when it is heated at high concentrations in various solvents. In this way, it produces many dimeric or oligomeric conformers through the three-dimensional domain swapping (3D-DS) mechanism involving both RNase A N- or/and C-termini. Here, we found many of these oligomers evolving toward not negligible amounts of large derivatives after being stored for up to 15 months at 4 °C in phosphate buffer. We call these species super-aggregates (SAs). Notably, SAs do not originate from native RNase A monomer or from oligomers characterized by the exclusive presence of the C-terminus swapping of the enzyme subunits as well. Instead, the swapping of at least two subunits’ N-termini is mandatory to produce them. Through immunoblotting, SAs are confirmed to derive from RNase A even if they retain only low ribonucleolytic activity. Then, their interaction registered with Thioflavin-T (ThT), in addition to TEM analyses, indicate SAs are large and circular but not “amyloid-like” derivatives. This confirms that RNase A acts as an “auto-chaperone”, although it displays many amyloid-prone short segments, including the 16–22 loop included in its N-terminus. Therefore, we hypothesize the opening of RNase A N-terminus, and hence its oligomerization through 3D-DS, may represent a preliminary step favoring massive RNase A aggregation. Interestingly, this process is slow and requires low temperatures to limit the concomitant oligomers’ dissociation to the native monomer. These data and the hypothesis proposed are discussed in the light of protein aggregation in general, and of possible future applications to contrast amyloidosis.

Effects of Pathogenic Mutants of the Neuroprotective RNase 5-Angiogenin in Amyotrophic Lateral Sclerosis (ALS)

“…However, and notably, fibrils have also recently been detected from wt-RNase A when the enzyme overpasses the supersaturation barrier [ 182 ], or when it acts in macromolecular crowding environments [ 183 ], that can in turn also affect the extent of RNase A-controlled oligomerization [ 184 ]. Again, the RNase A N-term-swapped oligomers, whilst neither the C-term-swapped ones, nor the monomer, can promote the slow formation of “super-aggregates” [ 185 ] that resemble the protofibrils detected in crowding contexts [ 183 ].…”

Protein Oligomerization

“…Finally, the article of Gotte et al [ 18 ] reports the peculiar behavior displayed by some of the oligomers that RNase A forms through the mentioned 3D-DS mechanism [ 13 ]. After storage at 4 °C for about one year, certain oligomers, separated from the others, formed detectable amounts of very large aggregates, referred to as “super-aggregates” (SAs), along with low-soluble derivatives.…”

“…Additionally, one review provides detailed insights into the structural and functional features of the oligomeric family of N-Ribohydrolases [ 11 ], while another review lists and exhaustively describes various approaches and methods useful for modeling and determining the structure of homo-oligomeric proteins in general [ 10 ]. Finally, another paper reveals the peculiar long-term evolution behavior of RNase A 3D-DS oligomers [ 18 ], highlighting again how protein 3D-DS oligomerization could represent a preliminary, sometimes necessary, step favoring the uncontrolled aggregation of proteins.…”