SpectraSage unveils specific proteolytic patterns of 20S on mono-ubiquitylated Tau proteoforms involved in neurodegeneration.

The ubiquitin proteasome system is a critical regulator of proteostasis and shows altered activity and composition in neurodegenerative diseases affecting both the brain (, Alzheimer's disease) and the retina/optic nerve (, age-related macular degeneration, glaucoma). A common feature of neurodegeneration is the progressive accumulation of amyloidogenic proteins such as beta-amyloid and tau protein (MAPT gene). There is compelling evidence that the aggregation propensity of tau protein is regulated by post-synthetic modifications including phosphorylation and ubiquitylation. These alterations are gaining increasing pathological relevance not only for brain tauopathies but also for the retinal/optic nerve degenerative diseases. In this regard, site-specific mono-ubiquitylated (Ub) tau proteoforms, have been recently identified in neurodegenerative brains. In this work, the cleavage patterns of the uncapped 20S proteasome acting on mono-Ub regio-isomers of tauK18, which covers the 4RD domain, have been unveiled by using SpectraSage, a novel proteomics software conceived for the MS1 identification of complex branched peptide and here introduced for the first time. Ub position was found to affect regio-isomers susceptibility to proteolysis and unexpectedly long Ub-tauK18 branched peptides have been identified, proving distinct catalytic preferences. These findings show that the 20S digests mono-Ub proteins through specific enzymatic mechanisms and the implications of the latter on neurodegeneration are discussed.