Periodic Reporting for period 4 - SOLID (Suppression of Organelle Defects in Human Disease)
Período documentado: 2024-01-01 hasta 2025-04-30
Failure or dysfunction of cellular machines is a common cause of severe human disease, with unmet clinical need in many cases. This is particularly evident for mitochondria, cellular ‘organelles’ which are best known for their role in cellular respiration but also play critical roles in processes ranging from metabolism to signaling and programmed cell death. Consequently, mitochondrial dysfunction is a hallmark of aging and major age-related diseases, including neurodegeneration, heart failure, and cancer. Because mitochondria originated from once-free-living bacteria, they still rely on elaborate communication with the rest of the cell to sense damage and mount repairs—a process termed ‘mito-nuclear communication’, which falters under heavy stress and/or in advanced age. Given its biomedical importance, this biology has been studied extensively and breakthrough discoveries have been made using invertebrate model organisms like baker’s yeast and worms. However, surprisingly little is known about how mitochondrial homeostasis and thus cellular health are maintained in the human system. This knowledge gap represents significant ‘biological dark matter’ that hinders therapeutic progress for prevalent and severe diseases.
Our objective was to systematically identify the genetic inputs on healthy and perturbed organelles like the endoplasmic reticulum and mitochondria through genome-scale experimental genetics in human cells. We set out to mechanistically clarify the pathways that maintain organelle homeostasis and cellular health to advance efforts to combat malfunction of this critical cellular machine in human disease settings. In particular, we pursued three aims: (1) map the genetic repertoire governing organelle function in the steady state and under stress; (2) identify, through a unique screening strategy, genetic interactions that can be exploited to restore homeostasis in a disease state; and (3) generate reagents and workflows that can be transferred to other cellular machines and processes involved in human disease.
Our objective was to systematically identify the genetic inputs on healthy and perturbed organelles like the endoplasmic reticulum and mitochondria through genome-scale experimental genetics in human cells. We set out to mechanistically clarify the pathways that maintain organelle homeostasis and cellular health to advance efforts to combat malfunction of this critical cellular machine in human disease settings. In particular, we pursued three aims: (1) map the genetic repertoire governing organelle function in the steady state and under stress; (2) identify, through a unique screening strategy, genetic interactions that can be exploited to restore homeostasis in a disease state; and (3) generate reagents and workflows that can be transferred to other cellular machines and processes involved in human disease.
1. Genome-wide screens identify a central mitochondrial stress pathway
We conducted several genome-scale genetic screens in human cells to catalog genes involved in organelle homeostasis and stress signaling. This unmasked the long-sought relay that signals a broad range of mitochondrial defects to the cytosol: the OMA1-DELE1-HRI axis (Fessler et al., Nature 2020). We discovered that activation of this pathway—the mitochondrial integrated stress response (mitoISR)—can be either cytoprotective or cytotoxic, depending on the nature of the insult (also reviewed in Fieler & Jae, BIOspektrum 2024; Eckl et al., CMLS 2021).
2. Mechanistic dissection of the OMA1-DELE1-HRI axis
Using synthetic biology, cell biology and biochemistry, we uncovered that DELE1 is a sentinel protein monitoring mitochondrial fidelity through its perpetual de novo synthesis and destruction in the mitochondrial interior. Dysfunctional protein import into the organelle allows the protease OMA1 to cleave DELE1 precursors during transit into the mitochondrial matrix. This trimming removes N-terminal targeting signals and liberates the structured C-terminal fragment, which exits the organelle to bind and activate the eIF2α kinase HRI, triggering the integrated stress response—a cellular program that remodels gene expression in the context of stress (Fessler et al., Nat Commun 2022).
3. Alternative modes of activity
Surprisingly, iron deficiency—one of the most prevalent nutritional deficits worldwide—also engages DELE1 signaling, but by a distinct mechanism that is independent of OMA1 protease activity and results in stalling of full-length DELE1 precursors at the mitochondrial surface (Sekine et al., Mol Cell 2023), hinting at a deeper entanglement of this axis with mitochondrial metabolism and other quality control processes of the organelle.
4. Disease-related genetic interactions
Functional studies linked the OMA1-DELE1-HRI pathway to mitochondrial myopathy driven by mutant CHCHD10, a gene also implicated in Parkinson’s disease, ALS and FTD (Shammas et al., J Clin Invest 2022). We also identified genetic interactions between DELE1/HRI and the machinery that removes and degrades the sorting signals of mitochondrial precursor proteins, mutations in which underlie neurological defects (Fessler et al., Nat Commun 2022). Similarly, we discovered a new pathway controlling mitochondrial dynamics and identified actionable genetic suppressors (Schuler et al., in preparation).
5. Methodological advances enabling broader applications
To support these studies, we developed and refined high-throughput screening, fitness profiling, and genome-editing methods. These tools facilitated the engineering of patient variants in an essential DNA-repair gene (Reinking et al., Mol Cell 2020), profiling of protein AMPylation (Hoffmann et al., bioRxiv 2025), discovery of histone-remodeling regulators (Mandemaker et al., Cell Rep 2023) and analysis of NOD2-mediated innate immunity (Stafford et al., Nature 2022).
We conducted several genome-scale genetic screens in human cells to catalog genes involved in organelle homeostasis and stress signaling. This unmasked the long-sought relay that signals a broad range of mitochondrial defects to the cytosol: the OMA1-DELE1-HRI axis (Fessler et al., Nature 2020). We discovered that activation of this pathway—the mitochondrial integrated stress response (mitoISR)—can be either cytoprotective or cytotoxic, depending on the nature of the insult (also reviewed in Fieler & Jae, BIOspektrum 2024; Eckl et al., CMLS 2021).
2. Mechanistic dissection of the OMA1-DELE1-HRI axis
Using synthetic biology, cell biology and biochemistry, we uncovered that DELE1 is a sentinel protein monitoring mitochondrial fidelity through its perpetual de novo synthesis and destruction in the mitochondrial interior. Dysfunctional protein import into the organelle allows the protease OMA1 to cleave DELE1 precursors during transit into the mitochondrial matrix. This trimming removes N-terminal targeting signals and liberates the structured C-terminal fragment, which exits the organelle to bind and activate the eIF2α kinase HRI, triggering the integrated stress response—a cellular program that remodels gene expression in the context of stress (Fessler et al., Nat Commun 2022).
3. Alternative modes of activity
Surprisingly, iron deficiency—one of the most prevalent nutritional deficits worldwide—also engages DELE1 signaling, but by a distinct mechanism that is independent of OMA1 protease activity and results in stalling of full-length DELE1 precursors at the mitochondrial surface (Sekine et al., Mol Cell 2023), hinting at a deeper entanglement of this axis with mitochondrial metabolism and other quality control processes of the organelle.
4. Disease-related genetic interactions
Functional studies linked the OMA1-DELE1-HRI pathway to mitochondrial myopathy driven by mutant CHCHD10, a gene also implicated in Parkinson’s disease, ALS and FTD (Shammas et al., J Clin Invest 2022). We also identified genetic interactions between DELE1/HRI and the machinery that removes and degrades the sorting signals of mitochondrial precursor proteins, mutations in which underlie neurological defects (Fessler et al., Nat Commun 2022). Similarly, we discovered a new pathway controlling mitochondrial dynamics and identified actionable genetic suppressors (Schuler et al., in preparation).
5. Methodological advances enabling broader applications
To support these studies, we developed and refined high-throughput screening, fitness profiling, and genome-editing methods. These tools facilitated the engineering of patient variants in an essential DNA-repair gene (Reinking et al., Mol Cell 2020), profiling of protein AMPylation (Hoffmann et al., bioRxiv 2025), discovery of histone-remodeling regulators (Mandemaker et al., Cell Rep 2023) and analysis of NOD2-mediated innate immunity (Stafford et al., Nature 2022).
The discoveries made as part of the project have sparked a new area of cellular stress response research that has attracted strong interest from adjacent disciplines. Likewise, the reagents and methodologies we developed in the project are showing broad utility across diverse aspects of biology, including DNA-damage repair, epigenetics, post-translational modifications, and innate immunity. Going forward, it will be critical to unravel how the identified biology is embedded into the wider landscape of cellular organization and intercellular stress communication. It will be of particular interest to deeply explore the biomedical potential of the identified signaling nodes and genetic suppressor constellations.