Oligomerization tendency and activity of RNase 1 in comparison with Onconase and RNase A

Many previous studies performed in the labs where I spent my PhD thesis revealed that monomeric RNase A can extensively oligomerize through the three dimensional domain swapping (3D-DS) mechanism when the protein is lyophilized from 40% acetic acid solutions, or when it is subjected to thermal incubation with EtOH, at very high protein concentrations. RNase A is the ribonuclease extract from the bovine pancreas, it has been extensively studied and has become the prototype of the ‘pancreatic-type’ RNases superfamily. In the recent years, some other members of the superfamily, like amphibian onconase and human angiogenin (RNase 5) showed the ability to dimerize following the same mechanism. Human pancreatic RNase 1 (or HP-RNases) is the human counterpart of RNase A and until recently only some RNase 1 mutants were found to self-associate through the 3D-DS mechanism and forming exclusively N-swapped dimers. During my PhD course, we found instead for the first time that RNase 1 can form dimers, trimers, and larger aggregates when it is subjected to lyophilization from 40% acetic acid solution. Specifically, RNase 1 self-associates through the 3D-DS of its N-terminus but also and at a greater extent, of its C-terminus. Notably, the amount of oligomers is even larger than the corresponding ones formed by RNase A. Since RNase 1 is four residues longer than RNase A, we further analysed the oligomerization tendency of a mutant lacking these four terminal residues. Importantly, RNase 1 reaches here the highest ability to extensively self-associate through 3D-DS among pt-RNases, paving the way for new investigations into the structural and biological properties of its oligomers. Considering that the toxicity of RNase A oligomeric species, and that RNase 1 cytotoxic activity is impaired by the ribonuclease inhibitor (RI) present in the cells, the ability of RNase 1 to extensively oligomerize opens the possibility to investigate if these oligomers can be active against malignant cell. Hence, these oligomers can be studied from a structural and biological perspective. When I reached the labs of the research group in which I spent my PhD thesis, a study focused on a peculiar behaviour of RNase A was already ongoing. In particular it a had been detected that some oligomers of RNase A collected and stored at 4°C for many months, surprisingly produce very large aggregates, that were called super-aggregates, (SAs). Although not abundant and accompanied by the concomitant dissociation of the oligomer toward the monomer, some studies focused on the analysis of these SAs species could be performed. In particular, we found that the oligomers containing subunits undergoing a N-terminal swapping event could form SAs, while the monomer and the exclusively C-swapped oligomers did not. This first evidence drove the studies performed with different techniques to suggest that SAs are not fibrillar but huge circular aggregates of the protein. All the results obtained have been discussed in the light of the known amyloidogenic potential of many short domains of RNase A, but taking into account that the protein itself is able to act as an “autochaperone” and protects it from a fibrillar destiny. In conclusion, the studies performed in this thesis confirm how the protein self-association through the 3D-DS mechanism can involve an increasing number of pancreatic type RNases, and how the 3D-DS event can be considered as an initial seed potentially driving toward massive and uncontrolled aggregation of a protein in general.