De novo design of protein binders that target DELE1 to inhibit the mitochondrial stress response
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eLife Assessment
This potentially valuable study describes the development of protein binders targeting DELE1, a protein involved in activating the integrated stress response when mitochondria are perturbed (the mitoISR pathway. The strategy appears to be successful, as several designed proteins were shown to bind DELE1, disrupt DELE1 oligomerization, and attenuate ISR activation. However, the demonstration of the utility of these inhibitory binders is incomplete, particularly given the limited biological outcomes examined in the current study, thus limiting the significance of the paper in its current form.
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Abstract
Mitochondrial stress activates the integrated stress response (ISR) through the mitochondrial protein DELE1, which relays stress signals to the cytosolic kinase HRI to induce ATF4. Dysregulation of DELE1-mediated signaling has been implicated in pathological conditions, yet molecular strategies to modulate DELE1 remain unavailable. Here, we report de novo designed proteins that bind DELE1, block its oligomerization, and inhibit DELE1-mediated ISR activation. Several designs form stable complexes with DELE1 and disrupt its oligomerization in vitro while preserving DELE1’s ability to bind HRI. In cells, these designs suppress ATF4 induction during mitochondrial stress and impair the recovery of elongated mitochondrial morphology following transient insult. Crystal structure of a representative binder, together with structural modeling and targeted mutagenesis, confirm that the designed proteins engage a critical interface required for DELE1 oligomerization. These findings establish DELE1 as a druggable target and demonstrate that de novo designed proteins offer precise tools to modulate this pathway, providing a foundation for future therapeutic exploration.
SIGNIFICANCE
Mitochondrial stress activates the integrated stress response through the signaling protein DELE1, but no molecular tools have been available to directly target DELE1 and selectively modulate this pathway. We developed de novo designed protein binders that recognize a critical oligomerization interface in DELE1, disrupt its assembly, and suppress mitochondrial stress-induced ISR activation in cells. These binders also impair recovery of mitochondrial network morphology following transient stress, linking DELE1 assembly to adaptive remodeling. Our study establishes DELE1 as a tractable and druggable target in mitochondrial stress signaling and demonstrates that de novo protein design can generate precise modulators of intracellular stress-response pathways.
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eLife Assessment
This potentially valuable study describes the development of protein binders targeting DELE1, a protein involved in activating the integrated stress response when mitochondria are perturbed (the mitoISR pathway. The strategy appears to be successful, as several designed proteins were shown to bind DELE1, disrupt DELE1 oligomerization, and attenuate ISR activation. However, the demonstration of the utility of these inhibitory binders is incomplete, particularly given the limited biological outcomes examined in the current study, thus limiting the significance of the paper in its current form.
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Reviewer #1 (Public review):
Summary:
The protein DELE1 is a critical component to signal mitochondrial stress to the cytosol: under stress conditions, a truncated form of DELE1, termed DELE1(CTD) accumulates in the cytosol as an oligomer, binds the HRI kinase, which triggers the integrated stress response.
Leveraging the structural knowledge of the DELE1(CTD) oligomer, this study attempts to interfere with the oligomerization process, using an AI-designed protein that binds to the DELE1(CTD) oligomerization interface. The starting hypothesis is that such a binder shall selectively inhibit the DELE1-signalled mitochondrial stress response. The authors use established AI pipelines (RFdiffusion) to make a series of such binders, characterize them with biochemical methods and a crystal structure of the binder in its free state. When …
Reviewer #1 (Public review):
Summary:
The protein DELE1 is a critical component to signal mitochondrial stress to the cytosol: under stress conditions, a truncated form of DELE1, termed DELE1(CTD) accumulates in the cytosol as an oligomer, binds the HRI kinase, which triggers the integrated stress response.
Leveraging the structural knowledge of the DELE1(CTD) oligomer, this study attempts to interfere with the oligomerization process, using an AI-designed protein that binds to the DELE1(CTD) oligomerization interface. The starting hypothesis is that such a binder shall selectively inhibit the DELE1-signalled mitochondrial stress response. The authors use established AI pipelines (RFdiffusion) to make a series of such binders, characterize them with biochemical methods and a crystal structure of the binder in its free state. When over-expressing the binders in HEK293T cells, the authors report that mitochondrial stress - induced with a drug - does indeed not lead to triggering the stress response, confirming their starting hypothesis.
The work is an elegant demonstration of how AI-designed proteins can specifically interfere with cellular mechanisms.
The conclusions of the work are mostly well supported by data; there are some mechanistic gaps, however, about the interaction mechanisms.
Strengths:
The study is a nice combination of (i) a clear structure-derived hypothesis on how to interfere with a signalling mechanism, (ii) state-of-the-art protein design tools, (iii) a mostly robust biochemical characterization, and (iv) cellular experiments to demonstrate the effects of the binders.
Weaknesses:
The crystal structure of the binder5, while confirming its AlphaFold model, does not provide direct evidence of the binding mode to DELE1. Direct structure determination, using crystallography (which may require cleaving the MBP domain) would make their mechanistic arguments stronger.
The demonstration that the binders do not inhibit the DELE1-HRI interaction is interesting; however, the underlying mechanism, in particular where the DELE1-HRI binding occurs, is not explored.
While this study opens perspectives on how to interfere with DELE1-signalling, it is unlikely that these binders are actually useful for medical applications (compared to small-molecule drugs), as acknowledged in the manuscript.
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Reviewer #2 (Public review):
Summary:
Previous structural analyses of DELE1 by the authors revealed that the first α-helix within the TPR repeat domain provides the oligomeric interface of DELE1, and that DELE1 octamer formation is required for maximal ISR activation. Based on these findings, the authors designed peptides intended to bind this oligomeric interface and showed that these peptides interfere with DELE1 oligomerization in vitro and attenuate ISR activation in cultured cells.
Strengths:
The series of in-vitro data sets showing direct binding of the designed peptides to DELE1 and inhibitory effects on its oligomerization are convincing.
Weaknesses:
The physiological (or experimental) significance of inhibiting the DELE1-HRI-ISR pathway using these peptides has not been clearly demonstrated, particularly given that the very …
Reviewer #2 (Public review):
Summary:
Previous structural analyses of DELE1 by the authors revealed that the first α-helix within the TPR repeat domain provides the oligomeric interface of DELE1, and that DELE1 octamer formation is required for maximal ISR activation. Based on these findings, the authors designed peptides intended to bind this oligomeric interface and showed that these peptides interfere with DELE1 oligomerization in vitro and attenuate ISR activation in cultured cells.
Strengths:
The series of in-vitro data sets showing direct binding of the designed peptides to DELE1 and inhibitory effects on its oligomerization are convincing.
Weaknesses:
The physiological (or experimental) significance of inhibiting the DELE1-HRI-ISR pathway using these peptides has not been clearly demonstrated, particularly given that the very limited cell biological outcomes are tested in the current manuscript.
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Reviewer #3 (Public review):
Significance of the findings and the strength of evidence:
The article presented by Yang et al. describes the development of protein binders targeting the C-terminal domain of the protein DELE1, which is involved in the mitochondrial integrated stress response (mitoISR) pathway. It was shown earlier that DELE1 is imported into the mitochondria and cleaved by the inner mitochondrial membrane protease OMA1, resulting in an N-terminal and C-terminal domain, the latter being transported back into the cytosol, where it interacts and activates the kinase HRI. HRI, in turn, phosphorylates eIF2α, resulting in selective translation of mRNAs encoding proteins involved in stress signalling, such as the transcription factor ATF4. ATF4 activates expression of genes involved in amino acid balance, redox homeostasis and …
Reviewer #3 (Public review):
Significance of the findings and the strength of evidence:
The article presented by Yang et al. describes the development of protein binders targeting the C-terminal domain of the protein DELE1, which is involved in the mitochondrial integrated stress response (mitoISR) pathway. It was shown earlier that DELE1 is imported into the mitochondria and cleaved by the inner mitochondrial membrane protease OMA1, resulting in an N-terminal and C-terminal domain, the latter being transported back into the cytosol, where it interacts and activates the kinase HRI. HRI, in turn, phosphorylates eIF2α, resulting in selective translation of mRNAs encoding proteins involved in stress signalling, such as the transcription factor ATF4. ATF4 activates expression of genes involved in amino acid balance, redox homeostasis and proteostasis. The C-terminal domain of DELE1 (DELE1CTD) was structurally and functionally characterized by earlier by cryo-EM by Jie Yang and co-workers. These studies suggest that it forms an octamer with D4 symmetry consisting of two tetramers arranged in a tail-to-tail arrangement. In this octamers two interfaces were identified, one between the monomers in the tetramers and one connecting the tetramers to form the octamer. In this earlier work, it was also shown by mutational studies that interrupting the first interface has an impact on the OMA1-DELE1-HRI-eIF2α-ATF4 pathway upon mitochondrial stress in human cells. To this end, the authors concluded in the current manuscript that it might be interesting and also of therapeutic interest to develop a protein binder that binds DELE1 and disrupts oligomer formation. The authors set up a de novo protein design approach using RFdiffusion to design a protein scaffold and ProteinMPNN to design the side chains to create protein binders targeting the α-helix α1 in DELE1CTD that is directly involved in the formation of the first interface forming the tetramer. As I am not an expert in protein design, I cannot judge the quality of this data. The candidates were evaluated by AlphaFold3 to confirm complexes formed between the designs and DELE1CTD. In the end, 12 designed protein binders were selected for further analyses. These proteins were recombinantly produced in E. coli and purified. The proteins DELE1 full-length (DELE1fl) and DELE1CTD were produced as MBP-fusion proteins to improve solubility and stability. Co-expression studies with mbp-delet1CTD revealed that 11 out of the 12 binders co-eluted with MBP-DELE1CTD from a size-exclusion chromatography column, indicating complex formation. Without the presence of the binders, MBP-DELE1CTD elutes as a higher oligomer, suggesting that the binders interfere with oligomerisation. Further analyses included the impact of the presence of selected binders on stress-induced ISR. The authors found that different binders had a slightly different impact on the outcome upon treatment with stressors, and also compared two different stressors. This was concluded by assessing the ATP4 protein level by immunoblotting. The interaction of selected binders with DELE1CTD was subsequently confirmed by co-immunoprecipitation experiments. To evaluate whether the impact of the binders is restricted to mitochondrial stress studies, eliciting endoplasmic reticulum stress showed no effect on ATF4 levels. The presence of the binders furthermore impaired recovery of tubulated mitochondria following mitochondrial stress induction, resulting in more fragmented mitochondria. The authors determined a crystal structure of one binder at a resolution of 2.6 Å and performed AlphaFold3 predictions to model the complex between binders and DELE1CTD. The interface is characterized by many hydrophobic residues. From this data, they concluded some interface mutants and tested those concerning their impact on the interaction. Indeed, mutation of these hydrophobic side chains to charged residues interfered with complex formation. Finally, the authors show that binder binding to DELE1CTD does not interfere with the binding of HRI kinase. Overall, the methodology applied is state-of-the-art, and the manuscript is well-written. The design of protein binders targeting DELE1 involved in mitochondrial stress signalling is interesting for basic science to study stress signalling, but also therapeutically. However, as ISR has a positive impact on disease development and ageing, but also a negative one, depending on the degree of activated ISR, a therapeutic use would need to be precisely applied. The study has some weaknesses, and particularly the structural data seems to have severe issues.
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