Biology subjects

Madrazo, N.

Publications and source records attributed to Madrazo, N..

4 recordsLinked to original sources

bioRxiv2025

Drug Repurposing Screen Identifies an HRI Activating Compound that Promotes Adaptive Mitochondrial Remodeling in MFN2-deficient Cells

Pathogenic variants in the mitochondrial outer membrane GTPase MFN2 cause the peripheral neuropathy Charcot-Marie-Tooth Type 2A (CMT2A). These mutations disrupt MFN2-dependent regulation of diverse aspects of mitochondrial biology including organelle morphology, motility, mitochondrial-endoplasmic reticulum (ER) contacts (MERCs), and respiratory chain activity. However, no therapies currently exist to mitigate the mitochondrial dysfunction linked to genetic deficiencies in MFN2. Herein, we performed a drug repurposing screen to identify compounds that selectively activate the integrated stress response (ISR) - the predominant stress-responsive signaling pathway responsible for regulating mitochondrial morphology and function. This screen identified the compounds parogrelil and MBX-2982 as potent and selective activators of the ISR through the OMA1-DELE1-HRI signaling axis. We show that treatment with these compounds promotes adaptive, ISR-dependent remodeling of mitochondrial morphology and protects mitochondria against genetic and chemical insults. Moreover, we show that pharmacologic ISR activation afforded by parogrelil restores mitochondrial tubular morphology, promotes mitochondrial motility, rescues MERCs, and enhances mitochondrial respiration in MFN2-deficient cells. These results demonstrate the potential for pharmacologic HRI activation as a viable strategy to mitigate mitochondrial dysfunction in CMT2A and other pathologies associated with MFN2 deficiency.

cell biology↗

bioRxiv2024

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.

cell biology↗

bioRxiv2023

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.

biochemistry↗

bioRxiv2021

Comprehensive analysis of meiosis-derived cDNA libraries reveals gene isoforms and mitochondrial proteins important for competitive fitness

Gametogenesis is a highly regulated and dynamic developmental program where a diploid progenitor cell differentiates into haploid gametes, the precursors for sexual reproduction. During meiosis, several pathways converge to initiate ploidy reduction and organelle remodelling to render gametes competent for zygote formation and subsequent organismal development. Additionally, meiosis inherently rejuvenates the newly formed gametes resulting in lifespan resetting. Here, we construct five stage-specific, inducible meiotic cDNA libraries that represent over 84% of the yeast genome. We employ computational strategies to detect stage-specific meiotic transcript isoforms in each library and develop a robust screening pipeline to test the effect of each cDNA on competitive fitness. Our multi-day proof-of-principle time course reveals gene isoforms that are important for competitive fitness as well as mitochondrial proteins that cause dose-dependent disruption of respiration. Together, these novel meiotic cDNA libraries provide an important resource for systematically studying meiotic genes and gene isoforms in future studies. HIGHLIGHTSO_LIConstruction of five stage-specific, inducible meiotic cDNA libraries in budding yeast that collectively represent 5563 genes, which is over 84% of the genome C_LIO_LIAnalysis of the cDNA libraries reveal the presence of meiosis-specific transcript isoforms that are largely uncharacterized C_LIO_LIDevelopment of a robust gain-of-function screening pipeline identifies previously characterized genes and novel gene isoforms important for competitive fitness C_LIO_LIMulti-day proof-of-principle screen reveals mitochondrial proteins that cause dosage-specific respiration defects C_LI

genetics↗