From Synucleinopathies to Cryptic Phosphosites: Experimental and Computational Approaches to Protein Structure Dependent Regulation

The level, fate, and regulation of a protein are decided not only by its mature, folded state but also by the transient window during and immediately after synthesis, when the nascent chain is still exposed to enzymes that the native fold would otherwise exclude. This thesis develops that idea along two complementary lines: a proteome-wide structural and evolutionary dissection of \emph{cryptic phosphorylation}, and the construction of a quantitative cellular assay to lower the pool of an aggregation-prone protein, $\alpha$-synuclein (ASYN). The first part concerns the therapeutic strategy of reducing monomeric ASYN before it can nucleate the amyloid pathology that defines the synucleinopathies. A live-cell, bimolecular fluorescence complementation (BiFC) assay, LIPS-ASYN, was built and validated by fusing GFP\textsubscript{11} to the C-terminus of ASYN and co-expressing it, under a doxycycline-inducible promoter, with constitutive cytosolic GFP\textsubscript{1--10} in HEK293 Flp-In T-REx cells; a reconstituted sfGFP signal reports directly on ASYN levels. Concordant flow cytometry, high-content imaging, and western blot read-outs, together with control clones lacking cytosolic complementation, established the specificity and screening quality of the system. The assay was deployed at high-content scale against the MicroSource Spectrum and NIIZ libraries ($\sim$2{,}166 compounds) in an automated 384-well format. Prioritised hits were characterised by eight-point dose--response curves and cross-validated against total-ASYN western blot, an orthogonal resazurin viability assay, and a LIPS-AKT\textsubscript{Cyt} counter-screen. Gedunin emerged as the most consistently validated ASYN reducer, producing a monotonic, dose-dependent decline across all orthogonal read-outs while remaining outside the cytotoxic range, with phenoxybenzamine hydrochloride providing further, partially toxicity-confounded support. The second part tests whether phosphosites buried in the native fold---accessible only during folding or translocation---are a marginal curiosity or a pervasive, functionally meaningful class of the human phosphoproteome. Per-residue relative solvent accessibility (RSA) was computed for every PhosphoSitePlus phosphosite mapped onto AlphaFold2 human models (pLDDT $>$ 65), defining a site as cryptic when RSA $<$ 0.15. Of 87{,}138 structured phosphosites, 22{,}982 (26.3\%) were cryptic; a SPECTRUS-based structure--dynamics filter retaining only quasi-rigid, robustly packed cores reduced this to a high-confidence set of roughly 5\% of all human phosphosites, yet still implied that about one-third of phosphorylated proteins harbour at least one cryptic site, arguing against a rare-exception interpretation. Evolutionary analysis across 137 eukaryotic proteomes (OrthoFinder2, Clustal Omega, Evolutionary Score) showed that cryptic phosphosites are more conserved than non-cryptic ones and at least as conserved as structurally matched buried residues, indicating they are as essential to the fold as the hydrophobic core itself. Consistent with a co-translational origin, cryptic-site proteins were significantly enriched in compartments reached through dedicated translocation machinery---mitochondrion, endoplasmic reticulum, Golgi, and the secretory pathway---and depleted in the nucleus and bulk cytoplasm. Together with cellular evidence from the cryptic sites of CHK1 (Thr382) and SMAD2 (Ser417), whose phosphomimetic substitution lowers steady-state protein levels, these findings support a model in which folding-coupled phosphorylation of buried residues destabilises the nascent protein and directs it towards degradation, constituting a general and previously overlooked layer of proteome regulation.