Tailoring stress responses
When faced with environmental stress, cells respond by shutting down cellular processes such as translation and nucleocytoplasmic transport. At the same time, cells preserve cytoplasmic messenger RNAs in structures known as stress granules, and many cellular proteins are modified by the covalent addition of ubiquitin, which has long been presumed to reflect degradation of stress-damaged proteins (see the Perspective by Dormann). Maxwell et al. show that cells generate distinct patterns of ubiquitination in response to different stressors. Rather than reflecting the degradation of stress-damaged proteins, this ubiquitination primes cells to dismantle stress granules and reinitiate normal cellular activities once the stress is removed. Gwon et al. show that persistent stress granules are degraded by autophagy, whereas short-lived granules undergo a process of disassembly that is autophagy independent. The mechanism of this disassembly depends on the initiating stress.
In response to many types of stress, eukaryotic cells initiate an adaptive and reversible response that includes down-regulation of key cellular activities along with sequestration of cytoplasmic mRNAs into structures called stress granules. Accompanying these stress responses is a global increase in ubiquitination that has been conventionally ascribed to the need for degradation of misfolded or damaged proteins. However, detailed characterization of how the ubiquitinome is reshaped in response to stress is lacking. Furthermore, it is unclear whether stress-dependent ubiquitination plays a more complex role in the larger stress response beyond its known protective function in targeting hazardous proteins for proteasomal degradation.
To explore the role of ubiquitination in the stress response, we used tandem ubiquitin binding entity (TUBE) proteomics to investigate changes to the ubiquitination landscape in response to five different types of stress in cultured mammalian cells, including human induced pluripotent stem cell (iPSC)–derived neurons. The discovery of unanticipated patterns of ubiquitination prompted a detailed analysis of the ubiquitination pattern specifically induced by heat shock by using diGly ubiquitin remnant profiling along with tandem mass tag quantitative proteomics in combination with additional total proteome and transcriptome analyses. Insights from this newly defined “heat shock ubiquitinome” guided subsequent investigation of the functional importance of this posttranslational modification in the cellular response to heat shock.
Each of the five different types of stress induced a distinctive pattern of ubiquitination. The heat shock ubiquitinome in human embryonic kidney 293T cells was defined by ubiquitination of specific proteins that function within cellular activities that are down-regulated during stress (e.g., translation and nucleocytoplasmic transport), and this pattern was similar in U2OS cells, primary mouse neurons, and human iPSC-derived neurons. The heat shock ubiquitinome was also enriched in protein constituents of stress granules. Suprisingly, this stress-induced ubiquitination was dispensable for the formation of stress granules and shutdown of cellular pathways; rather, heat shock–induced ubiquitination was a prerequisite for p97/valosin-containing protein (VCP)–mediated stress granule disassembly and for resumption of normal cellular activities, including nucleocytoplasmic transport and translation, upon recovery from stress. Many ubiquitination events were specific to one or another stress. For example, ubiquitination was required for disassembly of stress granules induced by heat stress but dispensable for disassembly for stress granules induced by oxidative (arsenite) stress.
Ubiquitination patterns are specific to different types of stress and indicate additional regulatory functions for stress-induced ubiquitination beyond the removal of misfolded or damaged proteins. Specifically, heat shock–induced ubiquitination primes the cell for recovery from stress by targeting specific proteins involved several pathways down-regulated during stress. Furthermore, some key stress granule constituents are ubiquitinated in response to heat stress but not arsenite stress, thus engaging a mechanism of VCP mediated–disassembly of heat shock–induced granules that is not shared by arsenite stress–induced granules. Finally, our deep proteomics datasets provide a rich community resource illuminating additional aspects of the roles of ubiquitination in response to stress.
Eukaryotic cells respond to stress through adaptive programs that include reversible shutdown of key cellular processes, the formation of stress granules, and a global increase in ubiquitination. The primary function of this ubiquitination is thought to be for tagging damaged or misfolded proteins for degradation. Here, working in mammalian cultured cells, we found that different stresses elicited distinct ubiquitination patterns. For heat stress, ubiquitination targeted specific proteins associated with cellular activities that are down-regulated during stress, including nucleocytoplasmic transport and translation, as well as stress granule constituents. Ubiquitination was not required for the shutdown of these processes or for stress granule formation but was essential for the resumption of cellular activities and for stress granule disassembly. Thus, stress-induced ubiquitination primes the cell for recovery after heat stress.