Bruce suppresses autophagy-regulated caspase activity and wing tissue growth in Drosophila

Reviewer #1 (Public review):

Summary:

The authors clearly demonstrate that overexpressed Dcp-1, but not Drice, is activated without canonical apoptosome components. Using TurboID-based proximity labeling, they revealed distinct proximal proteomes, among which Sirtuin 1, an Atg8a deacetylase, which promotes autophagy, was specifically required for Dcp-1 activation. Additionally, the show that autophagy-related genes, including Bcl-2 family members Debcl and Buffy, are required for Dcp-1 activation.

Using structure-based prediction using AlphaFold3, they identified that Bruce, an autophagy-regulated inhibitor of apoptosis, acts as a Dcp-1-specific regulator acting outside the apoptosome-mediated pathway. Finally, they show that Bruce suppresses wing tissue growth. These findings indicate that non-lethal Dcp-1 activity is governed by the autophagy-Bruce axis, enabling distinct non-lethal functions independent of cell death.

Strengths:

This is an excellent paper with very good structure, excellent quality data and analysis.

Weaknesses:

This reviewer did not identify any weaknesses or recommendations for revision.

Reviewer #2 (Public review):

Summary:

The Drosophila executioner caspase Dcp-1 has established roles in cell death, autophagy, and imaginal disc growth. This study reports previously unrecognized factors that work together with Dcp-1. Specifically, the authors performed a turboID-based proximal ligation experiment to identify factors associated Dcp-1 and Drice. Dcp-1-specific interactors were further examined for their genetic interaction. The authors report autophagy-related genes, including Debcl and Buffy, to be required for Dcp-1 activation. In addition, the authors present evidence of an interaction between Bruce and Dcp-1. Bruce-expression blocks the Dcp-1 overexpression phenotype. Inhibition of effector caspases or overexpression of Bruce commonly reduced wing growth, suggesting a relationship between the two proteins.

Strengths:

On the positive side, the study identifies new Dcp-1-interacting proteins and provides a functional link between Dcp-1 and Sirt1, Fkbp59, Debcl, Buffy, Atg2, and Atg8a.

Weaknesses:

The data supporting the Dcp-1/Bruce interaction are not strong, even though the title of this manuscript highlights Bruce. For example, the authors' turboID data does not support Dcp-1/Bruce interaction. The case for the interaction is based on a single experiment that overexpresses a truncated Bruce transgene in S2 cells.

Reviewer #3 (Public review):

Summary:

The present paper by Shinoda et al. from the Miura group builds upon findings reported in an earlier study by the same team (Shinoda et al., PNAS, 2019), which identified a non-apoptotic role for the Drosophila executioner caspase Dcp-1 in promoting wing tissue growth. That earlier work attributed this function primarily to Dcp-1 and to Decay, a caspase structurally related to executioner caspases, but not to DrICE, the principal apoptotic executioner caspase. The authors further proposed that this non-apoptotic caspase activity operates independently of the initiator caspase Dronc.

In the current study, the authors both corroborate aspects of their previous findings and extend the investigation to mechanisms regulating Dcp-1 in this context. They identify roles for the giant IAP Bruce, two BCL-2 family members, and autophagy-related components in modulating non-apoptotic Dcp-1 activity. Moreover, they show that Bruce binds to a BIR-like peptide exposed upon Dcp-1 cleavage, but not to DrICE. The study further suggests that low levels of Dcp-1 activity promote wing tissue growth, whereas excessive activity induces cell death, as evidenced by impaired wing development following Dcp-1 overexpression. Overall, the manuscript provides several intriguing insights into the non-apoptotic regulation of the comparatively weak apoptotic executioner caspase Dcp-1 and complements the group's earlier work. However, several concerns remain regarding certain interpretations of the data and the experimental rigour of some of the results.

Strengths:

A major strength of the work is its systematic genetic and biochemical approaches, which combine tissue-specific manipulation with protein interaction mapping to explore how Dcp-1 is regulated. The identification of several regulatory factors, including an inhibitor of cell death protein and components linked to autophagy, provides a coherent framework for understanding how Dcp-1 activity might be tuned.

Weaknesses:

The evidence supporting some key claims remains incomplete. In particular, the type of cell death form induced when Dcp-1 is overexpressed is not clearly established, and additional tests would be needed to distinguish between the different cell death types.

Likely impact:

The study contributes to a growing body of work showing that proteins traditionally associated with cell death can have broader roles in tissue development. This conceptual advance is likely to be of interest to researchers studying growth control and tissue maintenance.

Specific points:

(1) Nature of the wing ablation phenotype

A central concern is whether the wing ablation phenotype observed upon Dcp-1 overexpression truly reflects apoptotic cell death. The authors show in Figure 1c that nuclei in cells overexpressing Dcp-1, but not DrICE, zymogens are highly condensed, which is suggestive of apoptosis. However, it is equally plausible that this phenotype reflects a form of non-apoptotic, Dcp-1-dependent cell death (e.g. autophagy-dependent cell death). This distinction could be readily addressed using TUNEL labelling and direct caspase activity assays. The latter would be particularly informative, as it remains unclear whether zymogen Dcp-1 is capable of cleaving standard effector caspase reporters in vivo. Does the anti-cleaved Dcp-1 antibody detect Dcp-1 activation following overexpression of the Dcp-1 zymogen?

(2) Role of Decay

In their earlier study, the authors identified Decay as another caspase influencing wing growth, albeit more modestly than Dcp-1. It is therefore unclear why this line of investigation was not pursued further in the current work. This omission is notable, as Decay is not implicated in apoptosis and, to date, no substantial physiological function has been assigned to this caspase in any system. At a minimum, this point should be discussed explicitly.

(3) Figure 2: Proximity labelling analysis

The authors use TurboID-mediated proximity labelling to reveal distinct Dcp-1- and DrICE-associated proteomes across tissues, with a particular focus on the wing disc. They further demonstrate that RNAi-mediated knockdown of the Dcp-1-associated proteins Sirt1 and Fkbp59 suppresses the wing ablation phenotype induced by Dcp-1 overexpression, suggesting that these factors are required for Dcp-1 activity. However, it should be clarified whether Bruce was identified as a Dcp-1 interactor in the proximity labelling dataset, given its proposed central regulatory role. In addition, further discussion of Fkbp59, its known functions and how it might mechanistically influence Dcp-1 activity would be valuable.

(4) Figure 3: Autophagy-related factors

Given that Sirt1 is known to promote autophagy, the authors next examine autophagy-related proteins and identify roles for Atg2, Atg8a, Debcl, and Buffy in Dcp-1 activation. Notably, these proteins do not promote cell death in the Hid-induced canonical apoptotic pathway. However, it is important to determine whether knockdown of Debcl, Buffy, Atg2, or Atg8a alone affects wing development in the absence of Dcp-1 overexpression, to exclude the possibility that these perturbations independently impair wing formation.

(5) Evidence for canonical autophagy

The involvement of autophagy would be more convincingly demonstrated by testing additional core autophagy genes, such as Atg7, Atg5, and Atg12, as well as performing a combined knockdown of Atg8a and Atg8b. Moreover, direct assessment of autophagy at the cellular level using established genetic reporters would substantially strengthen the conclusions.

(6) Figures 4-5: Functional consequences

It would be informative to determine whether Synr, Debcl, or Buffy influence wing size on their own and whether their overexpression enhances wing growth.

(7) Terminology and interpretation of cell death

Taken together, the results suggest that Dcp-1 zymogen overexpression induces a form of non-apoptotic cell death, potentially autophagy-dependent or related. The reviewer does not understand the authors' insistence on referring to this process as apoptosis. The authors should be more cautious in their terminology: there is no canonical versus non-canonical apoptosis; there is simply apoptosis. Without stronger evidence, these effects should not be described as apoptotic cell death.