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Utilizing Mass Spectrometry to Reveal the Behavior of Lysosomal Enzymes and the Consequences of Protein Structure


The biochemical processes responsible for proper cellular operation are wholly dependent on interactions between biomolecules constructed with canonical yet alterable structures. Proteins represent a unique class of biomolecules with multiple levels of structure that can be spontaneously or enzymatically modified, resulting in unintended consequences. Spontaneous deamidation of asparagine and isomerization of aspartic acid into four products (L-Asp, D-Asp, L-isoAsp, D-isoAsp), and epimerization of L-serine into D-serine, are prevalent modifications that alter primary structure. Additionally, modifications can occur in higher order structure as observed in the infamous fibrilization of amyloid beta, resulting in beta sheet structures which further associate into fibrils. One cellular aspect of particular interest is the lysosome-dependent autophagy system. As the protein degradation process that occurs in the lysosome requires binding between cathepsin proteases and protein substrates, this system is intimately affected by the underlying structures To understand the behavior of these enzymes and their relation to substrate structure, we utilized mass spectrometry to identify products and characterize the effect of modifications on normal protease operation. Initial experiments revealed the inhibition of digestion by both isomerization and higher order modifications in amyloid beta fibrilization. Subsequent studies employing fluorescence microscopy affirmed the consequence of structural alterations on the lysosome in a quantitative manner when amino acid isomers were introduced into live cells. Importantly, digestion was found to be inhibited by a sequence containing D-isoAsp residues which prevented cathepsin binding. During these experiments, another enzymatic behavior was revealed in cathepsin B, where a previously unknown structural ligation reaction occurred between peptides. We performed further mass spectrometry experiments to characterize this reaction and determined that the ligation reaction occurs as a reversal of the proteolytic action by attaching a dipeptide to the C-terminus. Additionally, it was found that ligation could operate via an L-isoAsp interaction that could attach peptides of any length together. Finally, to expand our toolkit for examination of protein structure with mass spectrometry, we characterized the use of a commercially available iodophenyl molecule for facile attachment of a radical precursor using isothiocyanate chemistry. Photodissociation of the iodine bond generated a radical for structurally sensitive radical-directed dissociation experiments.

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