Metabolically activated proteostasis regulators that protect against erastin-induced ferroptosis.

We previously showed that the proteostasis regulator compound AA147 (N-(2-hydroxy-5-methylphenyl)benzenepropanamide) potently protects against neurotoxic insults, such as glutamate-induced oxytosis. Though AA147 is a selective activator of the ATF6 arm of the unfolded protein response in non-neuronal cells, AA147-dependent protection against glutamate toxicity in cells of neuronal origin is primarily mediated through activation of the NRF2 oxidative stress response. AA147 activates NRF2 through a mechanism involving metabolic activation of AA147 by endoplasmic reticulum (ER) oxidases, affording an AA147-based quinone methide that covalently targets the NRF2 repressor protein KEAP1. Previous results show that the 2-amino-p-cresol A-ring of AA147 is required for NRF2 activation, while the phenyl B-ring of AA147 is amenable to modification. Here we explore whether the protease-sensitive amide linker between the A- and B-rings of this molecule can be modified to retain NRF2 activation. We show that replacement of the amide linker of AA147 with a carbamate linker retains NRF2 activation in neuronal cells and improves protection against neurotoxic insults, including glutamate-induced oxytosis and erastin-induced ferroptosis. Moreover, we demonstrate that inclusion of this carbamate linker facilitates identification of next-generation AA147 analogs with improved cellular tolerance and activity in disease-relevant assays.

Mapping stress-responsive signaling pathways induced by mitochondrial proteostasis perturbations.

Imbalances in mitochondrial proteostasis are associated with pathologic mitochondrial dysfunction implicated in etiologically diverse diseases. This has led to considerable interest in defining the mechanisms responsible for regulating mitochondria in response to mitochondrial stress. Numerous stress-responsive signaling pathways have been suggested to regulate mitochondria in response to proteotoxic stress. These include the integrated stress response (ISR), the heat shock response (HSR), and the oxidative stress response (OSR). Here, we define the stress signaling pathways activated in response to chronic mitochondrial proteostasis perturbations by monitoring the expression of sets of genes regulated downstream of each of these signaling pathways in published Perturb-seq datasets from K562 cells CRISPRi-depleted of mitochondrial proteostasis factors. Interestingly, we find that the ISR is preferentially activated in response to chronic, genetically-induced mitochondrial proteostasis stress, with no other pathway showing significant activation. Further, we demonstrate that CRISPRi depletion of other mitochondria-localized proteins similarly shows preferential activation of the ISR relative to other stress-responsive signaling pathways. These results both establish our gene set profiling approach as a viable strategy to probe stress responsive signaling pathways induced by perturbations to specific organelles and identify the ISR as the predominant stress-responsive signaling pathway activated in response to chronic disruption of mitochondrial proteostasis.

Mapping Stress-Responsive Signaling Pathways Induced by Mitochondrial Proteostasis Perturbations.

Imbalances in mitochondrial proteostasis are associated with pathologic mitochondrial dysfunction implicated in etiologically-diverse diseases. This has led to considerable interest in defining the biological mechanisms responsible for regulating mitochondria in response to mitochondrial stress. Numerous stress responsive signaling pathways have been suggested to regulate mitochondria in response to proteotoxic stress, including the integrated stress response (ISR), the heat shock response (HSR), and the oxidative stress response (OSR). Here, we define the specific stress signaling pathways activated in response to mitochondrial proteostasis stress by monitoring the expression of sets of genes regulated downstream of each of these signaling pathways in published Perturb-seq datasets from K562 cells CRISPRi-depleted of individual mitochondrial proteostasis factors. Interestingly, we find that the ISR is preferentially activated in response to mitochondrial proteostasis stress, with no other pathway showing significant activation. Further expanding this study, we show that broad depletion of mitochondria-localized proteins similarly shows preferential activation of the ISR relative to other stress-responsive signaling pathways. These results both establish our gene set profiling approach as a viable strategy to probe stress responsive signaling pathways induced by perturbations to specific organelles and identify the ISR as the predominant stress-responsive signaling pathway activated in response to mitochondrial proteostasis disruption.

Divergent Proteome Reactivity Influences Arm-Selective Activation of the Unfolded Protein Response by Pharmacological Endoplasmic Reticulum Proteostasis Regulators.

Pharmacological activation of the activating transcription factor 6 (ATF6) arm of the unfolded protein response (UPR) has proven useful for ameliorating proteostasis deficiencies in cellular and mouse models of numerous etiologically diverse diseases. Previous high-throughput screening efforts identified the small molecule AA147 as a potent and selective ATF6 activating compound that operates through a mechanism involving metabolic activation of its 2-amino-p-cresol substructure affording a quinone methide, which then covalently modifies a subset of endoplasmic reticulum (ER) protein disulfide isomerases (PDIs). Another compound identified in this screen, AA132, also contains a 2-amino-p-cresol moiety; however, this compound showed less transcriptional selectivity, instead globally activating all three arms of the UPR. Here, we show that AA132 activates global UPR signaling through a mechanism analogous to that of AA147, involving metabolic activation and covalent modification of proteins including multiple PDIs. Chemoproteomic-enabled analyses show that AA132 covalently modifies PDIs to a greater extent than AA147. However, the extent of PDI labeling by AA147 approaches a plateau more rapidly than PDI labeling by AA132. These observations together suggest that AA132 can access a larger pool of proteins for covalent modification, possibly because its activated form is less susceptible to quenching than activated AA147. In other words, the lower reactivity of activated AA132 allows it to persist longer and modify more PDIs in the cellular environment. Collectively, these results suggest that AA132 globally activates the UPR through increased engagement of ER PDIs. Consistent with this, reducing the cellular concentration of AA132 decreases PDI modifications and enables selective ATF6 activation. Our results highlight the relationship between metabolically activatable-electrophile stability, ER proteome reactivity, and the transcriptional response observed with the enaminone chemotype of ER proteostasis regulators, enabling continued development of next-generation ATF6 activating compounds.

Divergent Proteome Reactivity Influences Arm-Selective Activation of Pharmacological Endoplasmic Reticulum Proteostasis Regulators.

Pharmacological activation of the activating transcription factor 6 (ATF6) arm of the Unfolded Protein Response (UPR) has proven useful for ameliorating proteostasis deficiencies in a variety of etiologically diverse diseases. Previous high-throughput screening efforts identified the small molecule AA147 as a potent and selective ATF6 activating compound that operates through a mechanism involving metabolic activation of its 2-amino- p -cresol substructure affording a quinone methide, which then covalently modifies a subset of ER protein disulfide isomerases (PDIs). Intriguingly, another compound identified in this screen, AA132, also contains a 2-amino- p -cresol moiety; however, this compound showed less transcriptional selectivity, instead globally activating all three arms of the UPR. Here, we show that AA132 activates global UPR signaling through a mechanism analogous to that of AA147, involving metabolic activation and covalent PDI modification. Chemoproteomic-enabled analyses show that AA132 covalently modifies PDIs to a greater extent than AA147. Paradoxically, activated AA132 reacts slower with PDIs, indicating it is less reactive than activated AA147. This suggests that the higher labeling of PDIs observed with activated AA132 can be attributed to its lower reactivity, which allows this activated compound to persist longer in the cellular environment prior to quenching by endogenous nucleophiles. Collectively, these results suggest that AA132 globally activates the UPR through increased engagement of ER PDIs. Consistent with this, reducing the cellular concentration of AA132 decreases PDI modifications and allows for selective ATF6 activation. Our results highlight the relationship between metabolically activatable-electrophile stability, ER proteome reactivity, and the transcriptional response observed with the enaminone chemotype of ER proteostasis regulators, enabling continued development of next-generation ATF6 activating compounds.

Meiotic cDNA libraries reveal gene truncations and mitochondrial proteins important for competitive fitness in Saccharomyces cerevisiae.

Gametogenesis is an evolutionarily conserved developmental program whereby a diploid progenitor cell undergoes meiosis and cellular remodeling to differentiate into haploid gametes, the precursors for sexual reproduction. Even in the simple eukaryotic organism Saccharomyces cerevisiae, the meiotic transcriptome is very rich and complex, thereby necessitating new tools for functional studies. Here, we report the construction of 5 stage-specific, inducible complementary DNA libraries from meiotic cells that represent over 84% of the genes found in the budding yeast genome. We employed computational strategies to detect endogenous meiotic transcript isoforms as well as library-specific gene truncations. Furthermore, we developed a robust screening pipeline to test the effect of each complementary DNA on competitive fitness. Our multiday proof-of-principle time course revealed 877 complementary DNAs that were detrimental for competitive fitness when overexpressed. The list included mitochondrial proteins that cause dose-dependent disruption of cellular respiration as well as library-specific gene truncations that expose a dominant negative effect on competitive growth. Together, these high-quality complementary DNA libraries provide an important tool for systematically identifying meiotic genes, transcript isoforms, and protein domains that are important for a specific biological function.

The PERK Arm of the Unfolded Protein Response Regulates Mitochondrial Morphology during Acute Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) stress is transmitted to mitochondria and is associated with pathologic mitochondrial dysfunction in diverse diseases. The PERK arm of the unfolded protein response (UPR) protects mitochondria during ER stress through the transcriptional and translational remodeling of mitochondrial molecular quality control pathways. Here, we show that ER stress also induces dynamic remodeling of mitochondrial morphology by promoting protective stress-induced mitochondrial hyperfusion (SIMH). ER-stress-associated SIMH is regulated by the PERK arm of the UPR and activated by eIF2α phosphorylation-dependent translation attenuation. We show that PERK-regulated SIMH is a protective mechanism to prevent pathologic mitochondrial fragmentation and promote mitochondrial metabolism in response to ER stress. These results identify PERK-dependent SIMH as a protective stress-responsive mechanism that regulates mitochondrial morphology during ER stress. Furthermore, our results show that PERK integrates transcriptional and translational signaling to coordinate mitochondrial molecular and organellar quality control in response to pathologic ER insults.