Mitochondrial stress activates a concerted mitonuclear response to safeguard and repair mitochondrial function. Quirós et al. assessed the transcriptome, proteome, and metabolome of mammalian cells under four types of stressors and combine population genetic analyses and in vivo studies to show that ATF4 coordinates the mitochondrial stress response.

USP14 is a major regulator of the proteasome and one of three proteasome-associated deubiquitinating enzymes1–9. Its effects on protein turnover are substrate specific, for unknown reasons. We report that USP14 shows a dramatic preference for ubiquitin-cyclin B conjugates that carry more than one ubiquitin modification or chain. This specificity is conserved from yeast to humans and is independent of chain linkage type. USP14 has been thought to cleave single ubiquitin groups from the distal tip of a chain but we find that it removes chains from cyclin B en bloc, proceeding until a single chain remains. The suppression of degradation by USP14’s catalytic activity reflects its capacity to act on a millisecond time scale, before the proteasome can initiate degradation of the substrate. In addition, single-molecule studies showed that the dwell time of ubiquitin conjugates at the proteasome was reduced by USP14-dependent deubiquitination. In summary, the specificity of the proteasome can be regulated by rapid ubiquitin chain removal, which resolves substrates based on a novel aspect of ubiquitin chain architecture.

SUMMARY System-wide quantitative analysis of ubiquitylomes has proven to be a valuable tool for elucidating targets and mechanisms of the ubiquitin-driven signaling systems, as well as gaining insights into neurodegenerative diseases and cancer. Current mass spectrometry methods for ubiquitylome detection require large amounts of starting material and rely on stochastic data collection to increase replicate analyses. We describe a method compatible with cell line and tissue samples for large-scale quantification of 5,000–9,000 ubiquitylation forms across ten samples simultaneously. Using this method, we reveal site-specific ubiquitylation in mammalian brain and liver tissues, as well as in cancer cells undergoing proteasome inhibition. To demonstrate the power of the approach for signal-dependent ubiquitylation, we examined protein and ubiquitylation dynamics for mitochondria undergoing PARKIN- and PINK1-dependent mitophagy. This analysis revealed the largest collection of PARKIN- and PINK1-dependent ubiquitylation targets to date in a single experiment, and it also revealed a subset of proteins recruited to the mitochondria during mitophagy.

During terminal differentiation, the global protein complement is remodeled, as epitomized by erythrocytes, whose cytosol is ~98% globin. The erythroid proteome undergoes a rapid transition at the reticulocyte stage; however, the mechanisms driving programmed elimination of preexisting cytosolic proteins are unclear. We found that a mutation in the murine Ube2o gene, which encodes a ubiquitin-conjugating enzyme induced during erythropoiesis, results in anemia. Proteomic analysis suggested that UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome. In particular, ribosome elimination, a hallmark of reticulocyte differentiation, was defective in Ube2o−/− mutants. UBE2O recognized ribosomal proteins and other substrates directly, targeting them to proteasomes for degradation. Thus, in reticulocytes, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome.

The ubiquitin-proteasome system (UPS) is responsible for most selective protein degradation in eukaryotes and regulates numerous cellular processes, including cell cycle control and protein quality control. A component of this system, the deubiquitinating enzyme USP14, associates with the proteasome where it can rescue substrates from degradation by removal of the ubiquitin tag. We previously found that a small-molecule inhibitor of USP14, known as IU1, can increase the rate of degradation of a subset of proteasome substrates. We report here the synthesis and characterization of 87 variants of IU1, which resulted in the identification of a 10-fold more potent USP14 inhibitor that retains specificity for USP14. The capacity of this compound, IU1-47, to enhance protein degradation in cells was tested using as a reporter the microtubule-associated protein tau, which has been implicated in many neurodegenerative diseases. Using primary neuronal cultures, IU1-47 was found to accelerate the rate of degradation of wild-type tau, the pathological tau mutants P301L and P301S, and the A152T tau variant. We also report that a specific residue in tau, lysine 174, is critical for the IU1-47-mediated tau degradation by the proteasome. Finally, we show that IU1-47 stimulates autophagic flux in primary neurons. In summary, these findings provide a powerful research tool for investigating the complex biology of USP14.