Presumed regulation of cell death by caspase degradation

Three of the seven Drosophila caspases (Dronc, Dredd, and Strica) appear to be similar to mammalian “initiator” caspases that cleave and activate “effector” caspases causing apoptosis [7], whereas the remaining four Drosophila caspases resemble effector caspases. The main initiator caspase Dronc (homologous to mammalian caspases-2 and -9) plays a major role in activating cell death during fly development and following injury by activating the effector caspases Drice and Dcp-1 (homologous to mammalian caspases-3 and -7) [8]. Dronc activation is dependent on direct binding of its N-terminal caspase recruitment domain (CARD) to the CARD domain in the adapter protein Dark (homologous to mammalian Apaf1), together forming the death-inducing apoptosome [9].

Caspases are tightly controlled by several negative regulatory mechanisms. Active Dronc, presumably derived from the apoptosome, is inhibited by ubiquitylation, a process largely dependent on the E3 ubiquitin ligase Diap1 (Drosophila inhibitor of apoptosis; homologous to human XIAP, cIAP1/2). The proposed mechanism is the poly-ubiquitylation of Dronc by Diap1 to trigger either Dronc degradation via the proteasome or its inhibition by other means [10, 11]. However, there is a lack of clarity in the mechanisms of Dronc regulation, as both the proteasome and autophagy pathways must be ablated to stabilize Dronc levels [12].

Non-degradative inhibition of Dronc by mono-ubiquitylation

Prior to apoptosome formation, unprocessed Dronc is negatively regulated by ubiquitylation; surprisingly, this does not require Dronc degradation [11]. Research from the Bergmann lab [6] sheds light on the regulation of Dronc activity by mono-ubiquitylation, rather than poly-ubiquitylation, in living cells. Mono-ubiquitylation of mammalian caspases has been described [13], and is also thought to alter function rather than promote degradation [14].

Kamber Kaya et al. [6] report that overexpressed Dronc in living tissues is mono-ubiquitylated on lysine 78 (K78) in the CARD domain. Using mass spectrometry and immunoblot analysis, they detected mono- but not poly-ubiquitylated Dronc in developing larval and pupal extracts, a finding further supported by in vitro ubiquitylation on K78 in extracts. Based on a recent crystal structure analysis, K78 forms an intermolecular hydrogen bond with the Dronc residue that directly contacts Dark at the center of the CARD–CARD interface between Dronc and Dark [9]. Therefore, mono-ubiquitylation at lysine 78 is likely to alter or inhibit apoptosome formation and subsequent cell death. Consistent with mono-ubiquitylation being exclusive to living cells, Kamber Kaya et al. [6] failed to find any lysine 78–ubiquitylated Dronc in larval and pupal extracts after hs-hid induced apoptosis. Although mutation of the lysine 78 site resulted in a strong inhibition of mono-ubiquitylation, residual mono-ubiquitylation suggested that other sites might be modified when the major site is mutated.

This work [6] provides considerable additional genetic and biochemical data indicating that Dronc K78 mono-ubiquitylation inhibits apoptosis. Overexpression of Dronc and Dark in the eye-antenna imaginal disc epithelial tissues triggers massive cell death in the developing eye, providing a model system to dissect function [10]. Ubiquitylation-resistant Dronc (K78R) was demonstrated to enhance cell death in the developing eye and pupal lethality more than wild-type (WT) Dronc expressed at similar levels. The enhanced apoptotic function of Dronc K78R correlates with higher proteolytic function in vivo, but this is not due to higher inherent catalytic activity, as recombinant proteins cleave Drice similarly in vitro [6]. The possibility that mono-ubiquitylation inhibits Dronc—Dark interaction is suggested by increased binding of K78R to Dark in co-immunoprecipitations [6], consistent with structural data [9].

The E3 ubiquitin ligase responsible for mono-ubiquitylation of Dronc K78 may be Diap1. This is supported by the finding that heterozygosity for diap1 reduced Dronc mono-ubiquitylation [6]. Additionally, Diap1 is sufficient to mono-ubiquitylate Dronc in vitro. Thus, it appears that in living cells, Diap1 can inhibit apoptosome formation through mono-ubiquitylation of Dronc. However, Dronc inhibition by mono-ubiquitylation has additional functions besides inhibiting apoptosis (Fig 1).

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Fig 1. The caspase Dronc functions in both living and dying cells.

The Drosophila caspase Dronc, which is required for fly development, is composed of an N-terminal CARD domain, a large subunit (L) containing the Cys active site required for proteolytic catalysis, and a small subunit (S) that is auto-catalytically cleaved from the precursor zymogen following recruitment to the Dark apoptosome. The ubiquitin ligase Diap1 mono-ubiquitylates lysine 78 in the CARD domain, which inhibits cell death by interfering with formation of the Dronc–Dark apoptosome. In contrast to non-apoptotic functions of Dronc in some model systems, Dronc’s proteolytic activity is unexpectedly not required for Dronc’s role in apoptosis-induced cell proliferation. Surprisingly, mono-ubiquitylation also suppresses this non-apoptotic function of Dronc, as Dark is still required. Thus, an alternative CARD-independent interaction with Dark or other factor could potentially mediate the observed non-death functions of Dronc.

https://doi.org/10.1371/journal.pgen.1006545.g001

Non-apoptotic, non-catalytic caspase activity

The authors of this study [6] used several strategies to investigate a physiological role for Dronc mono-ubiquitylation. Ubiquitous expressions of WT and mutant forms of Dronc were used to rescue organismal lethality of dronc null flies. Loss of zygotic dronc is known to cause a semi-lethal phenotype, with most animals dying during pupal stages, late in development [8]. Although these animals have extra cells, it is unknown whether the lack of cell death is the cause of organismal lethality. However, it was expected that organismal survival required the elimination of some cells by caspases because mutation of the catalytic cysteine (C318A), which abolishes enzymatic activity, also abolished rescue. Unexpectedly, Kamber Kaya et al. [6] found that catalytically inactive Dronc that cannot be mono-ubiquitylated (K78R/C318A) rescued the lethality of dronc nulls—a finding that challenges the general assumption that Dronc’s proteolytic activity is essential for normal organismal development. Rescue of lethality by Dronc K78R/C318A is not due to a restoration of catalytic function or of increased apoptosis-inducing activity as extra cells remain, at least in the developing eye and the developing wing [6]. This suggests that mono-ubiquitylation also inhibits non-apoptotic functions of Dronc that are essential for the survival of the organism. Further experimentation using knock-in mutations will be required to confirm this hypothesis.

Future directions

Bergmann and colleagues’ study [6] opens up new directions for research in the regulation of non-apoptotic caspase functions. In addition to the model systems tested in their study, other non-death functions of caspases in flies and mammals will require further examination to distinguish between proteolytic and non-proteolytic caspase functions. For example, the notion that the Drosophila apoptotic system might be involved in determining the number of external sensory bristles (macrochaetae) stems from observations of extra macrochaetae in flies mutant for dark, dronc and cytochrome c, but growing evidence implicates non-apoptotic functions of these factors involving the regulation of cell signaling pathways (reviewed in [23]). Restricted activation of caspases is suggested to prune dendritic spines on neurons in the fly without killing the neuron [4]. However, caspases have also been implicated prior to the pruning steps [24], and the requirements for proteolytic activity can be difficult to evaluate, as demonstrated in the new study by Bergmann et al. [6]. Mammalian caspases and regulators of caspase activation have been demonstrated to regulate neuronal activity independent of apoptosis [25, 26], but whether these caspase functions are limited to their proteolytic activities has not been thoroughly investigated.

The most striking finding of the new work by Bergmann and colleagues [6] is that Dronc’s essential function during development may not be its proteolytic apoptosis function. However, this interpretation could be confounded by the presence of residual maternally-loaded Dronc protein in the zygotic dronc nulls [8]. Although zygotic dronc nulls are semilethal at pupal stages, mutants lacking both maternally loaded and zygotic Dronc are lethal earlier during embryonic stages, and lack almost all embryonic apoptosis. Kamber Kaya et al. do not report that the Dronc K78R/C318A mutant rescued this maternal/zygotic dronc null phenotype. In theory, overexpression of the Dronc K78R/C318A mutant could stabilize or activate maternally loaded WT Dronc protein, which could allow enough proteolytically competent Dronc to persist in carrying out functions required for organismal viability. Use of one of the new genetically encoded caspase sensors would allow testing of this possibility [27].