Protein feature analysis of heat shock induced ubiquitination sites reveals preferential modification site localization

Protein aggregation is indicative of failing protein quality control systems. These systems are responsible for the refolding or degradation of aberrant and misfolded proteins. Heat stress can cause proteins to misfold, triggering cellular responses including a marked increase in the ubiquitination of proteins. This response has been characterized in yeast, however more studies are needed within mammalian cells. Herein, we examine proteins that become ubiquitinated during heat shock in human tissue culture cells using diGly enrichment coupled with mass spectrometry. A majority of these proteins are localized in the nucleus or cytosol. Proteins which are conjugated under stress display longer sequence lengths, more interaction partners, and more hydrophobic patches than controls but do not show lower melting temperatures. Furthermore, heat-induced conjugation sites occur less frequently in disordered regions and are closer to hydrophobic patches than other ubiquitination sites; perhaps providing novel insight into the molecular mechanism mediating this response. Nuclear and cytosolic pools of modified proteins appear to have different protein features. Using a pulse-SILAC approach, we found that both long-lived and newly-synthesized proteins are conjugated under stress. Modified long-lived proteins are predominately nuclear and were distinct from newly-synthesized proteins, indicating that different pathways may mediate the heat-induced increase of polyubiquitination.