Stable Isotope Labeling Reveals Novel Insights Into Ubiquitin-Mediated Protein Aggregation With Age, Calorie Restriction, and Rapamycin Treatment

J Gerontol A Biol Sci Med Sci. 2018 Apr 17;73(5):561-570. doi: 10.1093/gerona/glx047.


Accumulation of protein aggregates with age was first described in aged human tissue over 150 years ago and has since been described in virtually every human tissue. Ubiquitin modifications are a canonical marker of insoluble protein aggregates; however, the composition of most age-related inclusions remains relatively unknown. To examine the landscape of age-related protein aggregation in vivo, we performed an antibody-based pulldown of ubiquitinated proteins coupled with metabolic labeling and mass spectrometry on young and old mice on calorie restriction (CR), rapamycin (RP)-supplemented, and control diets. We show increased abundance of many ubiquitinated proteins in old mice and greater retention of preexisting (unlabeled) ubiquitinated proteins relative to their unmodified counterparts-fitting the expected profile of age-increased accumulation of long-lived aggregating proteins. Both CR and RP profoundly affected ubiquitinome composition, half-live, and the insolubility of proteins, consistent with their ability to mobilize these age-associated accumulations. Finally, confocal microscopy confirmed the aggregation of two of the top predicted aggregating proteins, keratins 8/18 and catalase, as well as their attenuation by CR and RP. Stable-isotope labeling is a powerful tool to gain novel insights into proteostasis mechanisms, including protein aggregation, and could be used to identify novel therapeutic targets in aging and protein aggregation diseases.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aging / metabolism*
  • Animals
  • Caloric Restriction*
  • Female
  • Half-Life
  • Isotope Labeling*
  • Leucine / pharmacology
  • Mass Spectrometry
  • Mice
  • Mice, Inbred C57BL
  • Microscopy, Confocal
  • Protein Aggregates / drug effects*
  • Protein Biosynthesis / drug effects
  • Proteome / metabolism
  • Sirolimus / pharmacology*
  • TOR Serine-Threonine Kinases / metabolism
  • Ubiquitin / metabolism*


  • Protein Aggregates
  • Proteome
  • Ubiquitin
  • mTOR protein, mouse
  • TOR Serine-Threonine Kinases
  • Leucine
  • Sirolimus