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Dev Cell. Author manuscript; available in PMC 2016 July 22. Published in final edited form as: Dev Cell. 2016 April 4; 37(1): 1–2. doi:10.1016/j.devcel.2016.03.016.

Krebs Cycle Moonlights in Caspase Regulation Adi Minis1 and Hermann Steller1,* 1Strang

Laboratory of Apoptosis and Cancer Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065, USA

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In this issue of Developmental Cell, Aram et al. (2016) identify a mechanism that uses a Krebs cycle protein to control local activation of a ubiquitin ligase complex at the mitochondrial outer membrane for temporally and spatially restricted caspase activation during Drosophila sperm differentiation.

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Caspases, a conserved family of cysteine proteases, have been intensely studied as key executioners of apoptosis, the most common form of natural cell death. In addition, caspases also play important non-apoptotic roles in development and differentiation (Yi and Yuan, 2009). A striking example is the non-lethal use of apoptotic effector caspases for cellular remodeling (Fuchs and Steller, 2011; Schuldiner and Yaron, 2015). Apoptotic proteins are required for the regulated destruction of axons and dendrites to sculpt the nervous system, the degrading of the nucleus and membrane-bound organelles during lens fiber differentiation, and terminal differentiation of spermatids. This raises the intriguing question of how the activation of apoptotic caspases can be restricted in time and space to avoid death of the entire cell. In this issue of Developmental Cell, Aram et al. (2016) report a major advance that sheds new light onto this question. Using Drosophila melanogaster spermatogenesis as a model system, the authors uncover an unexpected role for a Krebs cycle protein, Succinyl-CoA synthetase β (A-Sβ), in promoting temporally and spatially restricted caspase activation at the mitochondrial outer membrane (MOM) (Figure 1).

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During terminal sperm differentiation in Drosophila, the vast majority of proteins are degraded to generate highly specialized and minimalistic cells devoid of major organelles. This process occurs in cysts containing 64 haploid cells that remain interconnected due to incomplete cytokinesis (Fuller, 1993). The terminal differentiation of spermatids involves dramatic cell elongation and separation into individual spermatids; hence, this process is termed “individualization.” Importantly, this process involves the degradation of the majority of proteins and organelles and depends on apoptotic proteins, including caspase-3type effector caspases (Arama et al., 2003). Earlier studies had already revealed the critical importance of the caspase inhibitor dBruce, the apoptosome activator Cytochrome C, and a Cullin-3-based ubiquitin ligase complex (CRL3) for regulating caspase activity in a nonlethal manner (Arama et al., 2007). Furthermore, the Arama lab previously identified a pseudosubstrate inhibitor for CRL3, termed Soti, which inhibits the activity of this complex

*

Correspondence: [email protected].

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in a spatially graded manner and thereby restricts caspase activity in time and space (Kaplan et al., 2010). This fine-tuning of caspase levels is crucial for male fertility, as either too low or too high caspase activity has disastrous consequences.

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In the current study, Aram et al. (2016) used a yeast two-hybrid approach followed by coimmunoprecipitation experiments to identify an ATP-specific form of the Succinyl-CoA synthetase β subunit (A-Sβ) as another key regulator of CRL3 activity. Typically, A-Sβ is found in the mitochondrial matrix and functions within the Krebs cycle. However, in Drosophila spermatids, a significant portion of A-Sβ is localized on the MOM where it can bind to CRL3. In a series of elegant experiments that combine cell fractionation, immunoEM, biochemical analyses, domain swapping experiments, and genetic and structurefunction studies, Aram et al. (2016) now demonstrate that A-Sβ promotes caspase activation by antagonizing Soti to activate the CRL3 complex at the MOM. They also show that the localization and function of A-Sβ in developing sperm depends on its N-terminal domain. This is interesting because although the CRL3-binding domain of A-Sβ, located at its C terminus, is sufficient for caspase activation in S2 culture cells, it is not sufficient for caspase activation in spermatids. Furthermore, although the authors suggest that the binding of A-Sβ to the CRL3 complex is important for its activation by neddylation, their RNAi experiments show a more prominent effect on the localization to the mitochondria than on the levels of neddylation. Collectively, these results imply that Cul3-neddylation and caspase activation are uncoupled in spermatids and that another layer of regulation operates at the MOM. This could be mediated through an interaction with other mitochondrial proteins or lipids, posttranslational modification, or the proximity to other apoptosis-related proteins that are necessary for caspase activation, such as Cytochrome C.

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Irrespective of the exact underlying biochemical mechanism, the current work by Aram et al. (2016) reveals an unexpected “moonlighting” function of A-Sβ for the spatio-temporal regulation of a Cullin-3-based ubiquitin ligase complex and caspase activity. At the same time, these findings raise many new and intriguing questions. Foremost, why would a Krebs cycle protein involve a function at the surface of mitochondria to regulate protein degradation? The important role of mitochondria in the regulation of apoptosis has long been recognized, as a source of regulatory proteins, but also as a platform for the sequestration of apoptotic proteins (Fuchs and Steller, 2011). Furthermore, sperm differentiation in insects involves very dramatic changes in the structure of mitochondria. Therefore, it is possible that A-Sβ helps to sense the appropriate metabolic state for the initiation of caspase activity. Intriguingly, two additional non-canonical functions of A-Sβ outside mitochondria have also been suggested, namely in modulating the activity of a voltage-gated potassium channel and Skp-1-mediated centrosome regulation (Gao et al., 2008; Hughes et al., 2008). Therefore, it is possible that A-Sβ has multiple moonlight jobs, or perhaps all these observations reflect a more general role of this protein in Cullin-based ubiquitin ligase complexes. In addition, although the CRL3 complex is clearly important for caspase activation during sperm differentiation, there may be other substrates with relevant biological functions. Another important unresolved question is how exactly A-Sβ overcomes the inhibition of CRL3 by Soti. Furthermore, it will be interesting to investigate the precise mechanism that determines when and how A-Sβ becomes located to the MOM, since this appears to be the rate-limiting step in activating CRL3 in this system. Finally, it will be Dev Cell. Author manuscript; available in PMC 2016 July 22.

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important to explore whether these findings are restricted to Drosophila sperm differentiation or represent a more general regulatory mechanism. Given the tremendous importance of understanding both mechanisms that restrict caspase activity and the regulation of CRL3 complexes, it is likely that the answers to many of these questions will emerge soon.

REFERENCES

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Aram L, Braun T, Braverman C, Kaplan Y, Ravid L, Levin-Zaidman S, Arama E. Dev. Cell. 2016; 37(this issue):15–33. [PubMed: 27052834] Arama E, Agapite J, Steller H. Dev. Cell. 2003; 4:687–697. [PubMed: 12737804] Arama E, Bader M, Rieckhof GE, Steller H. PLoS Biol. 2007; 5:e251. [PubMed: 17880263] Fuchs Y, Steller H. Cell. 2011; 147:742–758. [PubMed: 22078876] Fuller, MT. The Development of Drosophila melanogaster. Bate, M.; Arias, AM., editors. Cold Spring Harbor Laboratory Press; 1993. p. 71-147. Gao L, Fei H, Connors NC, Zhang J, Levitan IB. J. Neurophysiol. 2008; 99:2736–2740. [PubMed: 18385479] Hughes JR, Meireles AM, Fisher KH, Garcia A, Antrobus PR, Wainman A, Zitzmann N, Deane C, Ohkura H, Wakefield JG. PLoS Biol. 2008; 6:e98. [PubMed: 18433294] Kaplan Y, Gibbs-Bar L, Kalifa Y, Feinstein-Rotkopf Y, Arama E. Dev. Cell. 2010; 19:160–173. [PubMed: 20643358] Schuldiner O, Yaron A. Cell. Mol. Life Sci. 2015; 72:101–119. [PubMed: 25213356] Yi CH, Yuan J. Dev. Cell. 2009; 16:21–34. [PubMed: 19154716]

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Figure 1. Spatially Restricted Caspase Activation at the Mitochondrial Membrane during Sperm Differentiation

(A) The non-lethal activity apoptotic effector caspases is regulated by multiple pathways at the mitochondrial surface. First, Cytochrome C is necessary to promote caspase activation. Second, the pseudosubstrate Soti and A-Sβt, a testis-specific isoform of the Krebs cycle enzyme Succinyl-CoA synthase β, have antagonistic effects on the activity of a Culin-3based ubiquitin ligase (CRL3) complex. Activation of CRL3 promotes degradation of the caspase-inhibitor dBruce, which in turn restricts the potentially lethal activity of caspases in time and space. The interplay between Soti and A-Sβt achieves spatial and temporal Dev Cell. Author manuscript; available in PMC 2016 July 22.

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restriction of caspase activity that is necessary for the selective destruction of subcellular structures during sperm terminal differentiation. (B) Soti and A-Sβt regulate CRL3 to promote caspase activation. Soti inhibits CRL3 by blocking substrate binding. Upon the onset of sperm differentiation, A-Sβt levels increase on the mitochondrial outer membrane (MOM) and compete with Soti for binding to CRL3. This allows for spatially restricted activation of CRL3 at the MOM, which in turn promotes degradation of dBruce and caspase activation.

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Krebs Cycle Moonlights in Caspase Regulation.

In this issue of Developmental Cell, Aram et al. (2016) identify a mechanism that uses a Krebs cycle protein to control local activation of a ubiquiti...
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