Procaspase 9 is the initiator caspase for apoptosis, but how its levels and activities are maintained remains unclear. The gigantic Inhibitor-of-Apoptosis Protein BIRC6/BRUCE/Apollon inhibits both apoptosis and autophagy by promoting ubiquitylation of proapoptotic factors and the key autophagic protein LC3, respectively. Here we show that BIRC6 forms an anti-parallel U-shaped dimer with multiple previously unannotated domains, including a ubiquitin-like domain, and the proapoptotic factor Smac/DIABLO binds BIRC6 in the central cavity. Notably, Smac outcompetes the effector caspase 3 and the pro-apoptotic protease HtrA2, but not procaspase 9, for binding BIRC6 in cells. BIRC6 also binds LC3 through its LC3-interacting region, probably following dimer disruption of this BIRC6 region. Mutation at LC3 ubiquitylation site promotes autophagy and autophagic degradation of BIRC6. Moreover, induction of autophagy promotes autophagic degradation of BIRC6 and caspase 9, but not of other effector caspases. These results are important to understand how the balance between apoptosis and autophagy is regulated under pathophysiological conditions.
© 2024. The Author(s).

ABSTRACT Caspase-9 is the initiator caspase for the intrinsic apoptotic cell death pathway, and is critical to the activation of effector caspases during apoptosis, but how its levels and activities are maintained remains unclear. The gigantic Inhibitor of Apoptosis Protein (IAP) BIRC6/BRUCE/Apollon not only inhibits apoptosis, but also promotes ubiquitination of the key autophagic protein LC3 and inhibits autophagy. Here we show that BIRC6 forms an anti-parallel U-shaped dimer in a 3.6-Å cryo-EM structure with multiple previously unannotated domains, including a ubiquitin-like domain, and discover that the mitochondria-derived pro-apoptotic factor Smac/DIABLO binds BIRC6 by interacting with one BIR domain, two carbohydrate-binding modules and two helices in the central cavity. Notably, Smac outcompetes the effector caspase 3 and the pro-apoptotic protease HtrA2, but not caspase 9, for binding BIRC6. BIRC6 strongly inhibits cellular activity of caspase 9, but weakly suppresses that of caspase 3. Meanwhile, BIRC6 binds LC3 through an LC3-interacting region, probably following dimer disruption of this BIRC6 region. Deficiency in LC3 ubiquitination promotes autophagy and autophagic degradation of BIRC6, and inhibits apoptosis. Moreover, induction of autophagy promotes autophagic degradation of both procaspase-9 and active caspase-9, but not of effector caspases. These results are important to understand how the balance between apoptosis and autophagy is regulated under pathophysiological conditions.

Autophagy has an important role in cellular homeostasis by degrading and recycling cytotoxic components. Ubiquitination is known to target cargoes for autophagy; however, key components of this pathway remain elusive. Here we performed an RNAi screen to uncover ubiquitin modifiers that are required for starvation-induced macroautophagy in mammalian cells. Our screen uncovered BRUCE/Apollon/Birc6, an IAP protein, as a new autophagy regulator. Depletion of BRUCE leads to defective fusion of autophagosomes and lysosomes. Mechanistically, BRUCE selectively interacts with two ATG8 members GABARAP and GABARAPL1, as well as with Syntaxin 17, which are all critical regulators of autophagosome-lysosome fusion. In addition, BRUCE colocalizes with LAMP2. Interestingly, a non-catalytic N-terminal BRUCE fragment that is sufficient to bind GABARAP/GABARAPL1 and Syntaxin 17, and to colocalize with LAMP2, rescues autolysosome formation in Bruce -/- cells. Thus, BRUCE promotes autolysosome formation independently of its ubiquitin-conjugating activity and is a regulator of both macroautophagy and apoptosis.

Neuroblastoma are pediatric tumors of the sympathetic nervous system with a poor prognosis. Apoptosis is often deregulated in cancer cells, but only a few defects in apoptotic routes have been identified in neuroblastoma.
Here we investigated genomic aberrations affecting genes of the intrinsic apoptotic pathway in neuroblastoma. We analyzed DNA profiling data (CGH and SNP arrays) and mRNA expression data of 31 genes of the intrinsic apoptotic pathway in a dataset of 88 neuroblastoma tumors using the R2 bioinformatic platform ( http://r2.amc.nl). BIRC6 was selected for further analysis as a tumor driving gene. Knockdown experiments were performed using BIRC6 lentiviral shRNA and phenotype responses were analyzed by Western blot and MTT-assays. In addition, DIABLO levels and interactions were investigated with immunofluorescence and co-immunoprecipitation.
We observed frequent gain of the BIRC6 gene on chromosome 2, which resulted in increased mRNA expression. BIRC6 is an inhibitor of apoptosis protein (IAP), that can bind and degrade the cytoplasmic fraction of the pro-apoptotic protein DIABLO. DIABLO mRNA expression was exceptionally high in neuroblastoma but the protein was only detected in the mitochondria. Upon silencing of BIRC6 by shRNA, DIABLO protein levels increased and cells went into apoptosis. Co-immunoprecipitation confirmed direct interaction between DIABLO and BIRC6 in neuroblastoma cell lines.
Our findings indicate that BIRC6 may have a potential oncogenic role in neuroblastoma by inactivating cytoplasmic DIABLO. BIRC6 inhibition may therefore provide a means for therapeutic intervention in neuroblastoma.

Caspase activation is a central event in numerous forms of apoptosis and results in the proteolytic degradation of multiple substrate proteins that contribute to the apoptotic phenotype. An important route to caspase activation proceeds via assembly of the "apoptosome" as a result of the cell stress-associated release of mitochondrial cytochrome c. Previous studies have shown that primary neutrophils are largely incapable of mitochondrial respiration, suggesting that these cells either lack functional mitochondria or possess a defective respiratory chain. This prompted us to examine whether neutrophils retain an intact cytochrome c/apoptotic protease-activating factor 1 (Apaf-1) pathway to caspase activation and apoptosis. We show that primary human neutrophils contain barely detectable levels of cytochrome c as well as other mitochondrial proteins. Surprisingly, neutrophil cell-free extracts readily supported Apaf-1-dependent caspase activation, suggesting that these cells may assemble cytochrome c-independent apoptosomes. However, further analysis revealed that the trace amount of cytochrome c present in neutrophils is both necessary and sufficient for Apaf-1-dependent caspase activation in these cells. Thus, neutrophils have a lowered threshold requirement for cytochrome c in the Apaf-1-dependent cell death pathway. These observations suggest that neutrophils retain cytochrome c for the purpose of assembling functional apoptosomes rather than for oxidative phosphorylation.