UC Riverside

Proteins play major roles and participate in multiple pathways in the cell. Thus, cells have to rely on proper folding and localization of the proteome to avoid dysfunction. One of the key steps in maturation for secretory proteins is translocation into the endoplasmic reticulum (ER) rather than mistargeting to and accumulating in the cytosol. However, under ER stress, ER translocation becomes less efficient leading to higher accumulation of proteins in the cytosol. This potentially poses a threat to the cytosol. In our study, we identified an ER chaperone HSPA13 as a regulatory factor of protein translocation into the ER. Overexpression of HSPA13 in HEK293T cells impairs ER and cytosolic proteostasis by inhibiting ER translocation of substrates, disrupting their secretory pathway and promoting their intracellular aggregation. Additionally, we found that mistargeted protein in the cytosol is triaged on the basis of its stability. Highly destabilized mistargeted proteins are rapidly processed and degraded by the proteasome, while more stable proteins are degraded less efficiently. Furthermore, in the case of transthyretin, the processing is mediated by selective N-terminal proteolysis. Thus, we have found that mistargeted ER-directed proteins are subject to quality control in the cytosol.

Influenza infection requires viral escape from early endosomes into the cytosol, which is enabled by an acid-induced irreversible conformational transformation in the viral protein hemagglutinin. Despite the direct relationship between this conformational change and infectivity, label-free methods for characterizing this and other protein conformational changes in biological mixtures are limited. While the chemical reactivity of the protein backbone and side-chain residues is a proxy for protein conformation, coupling this reactivity to quantitative mass spectrometry is a challenge in complex environments. Herein, we developed electrophilic amidination coupled with reaction monitoring to detect the fusion-associated conformational transformation in recombinant hemagglutinin (rHA). We identified rHA peptides that are differentially amidinated between the pre- and post-fusion states, and validated that this difference relies upon the fusion-associated conformational switch. We further demonstrate that we can distinguish the fusion profile in a matrix of digested cellular lysate.