The Bcl-2 relative Bak is thought to drive apoptosis by forming homo-oligomers that permeabilize mitochondria, but how it is activated and oligomerizes is unclear. To clarify these pivotal steps toward apoptosis, we have characterized multiple random loss-of-function Bak mutants and explored the mechanism of Bak conformation change during apoptosis. Single missense mutations located to the alpha helix 2-5 region of Bak, with most altering the BH3 domain or hydrophobic groove (BH1 domain). Loss of function invariably corresponded to impaired ability to oligomerize. An essential early step in Bak activation was shown to be exposure of the BH3 domain, which became reburied in dimers. We demonstrate that oligomerization involves insertion of the BH3 domain of one Bak molecule into the groove of another and may produce symmetric Bak dimers. We conclude that this BH3:groove interaction is essential to nucleate Bak oligomerization, which in turn is required for its proapoptotic function.

Receptor Interacting Protein Kinase 1 (RIPK1) is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is post-translationally regulated by well characterised ubiquitylation and phosphorylation events, as well as caspase-8 mediated cleavage 1-7. The physiological relevance of this cleavage remains unclear, though it is believed to inhibit activation of RIPK3 and necroptosis 8. Here we show that heterozygous missense mutations p.D324N, p.D324H and p.D324Y prevent caspase cleavage of RIPK1 in humans and result in early-onset periodic fever episodes and severe intermittent lymphadenopathy, a condition we designate 'Cleavage-resistant RIPK1-Induced Autoinflammatory' (CRIA) syndrome. To define the mechanism for this disease we generated a cleavage-resistant Ripk1 D325A mutant mouse strain. While Ripk1-/mice die postnatally from systemic inflammation, Ripk1 D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by combined loss of Casp8 and Ripk3 but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1 D325A/D325A embryonic lethality, however the mice died before weaning from multi organ inflammation in a RIPK3 dependent manner. Consistently, Ripk1 D325A/D325A and Ripk1 D325A/+ cells were hypersensitive to RIPK3 dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1 D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but maintains inflammatory homeostasis throughout life. Members of three families presented with a previously undescribed autoinflammatory disorder characterised by fevers and pronounced lymphadenopathy beginning in early childhood and continuing throughout adulthood (Fig. 1a). From birth or shortly thereafter, all affected individuals experienced fevers usually occurring approximately every 2-4 weeks, Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

A pivotal step toward apoptosis is oligomerization of the Bcl-2 relative Bak. We recently reported that its oligomerization initiates by insertion of an exposed BH3 domain into the groove of another Bak monomer. We now report that the resulting BH3:groove dimers can be converted to the larger oligomers that permeabilize mitochondria by an interface between alpha6 helices. Cysteine residues placed in alpha6 could be crosslinked only after apoptotic signaling. Cysteines placed at both interfaces established that the BH3:groove dimer is symmetric and that the alpha6:alpha6 interface can link these dimers into homo-oligomers containing at least 18 Bak molecules. A putative zinc-binding site in alpha6 was not required to form the alpha6:alpha6 interface, and its mutation in full-length Bak did not affect Bak conformation, oligomerization, or function. We conclude that alpha6:alpha6 interaction occurs during Bak oligomerization and proapoptotic function, but we find no evidence that zinc binding to that interface regulates apoptosis.

During apoptotic cell death, Bax and Bak change conformation and homo-oligomerize to permeabilize mitochondria. We recently reported that Bak homodimerizes via an interaction between the BH3 domain and hydrophobic surface groove, that this BH3:groove interaction is symmetric, and that symmetric dimers can be linked via the a6-helices to form the high order oligomers thought responsible for pore formation. We now show that Bax also dimerizes via a BH3:groove interaction after apoptotic signaling in cells and in mitochondrial fractions. BH3:groove dimers of Bax were symmetric as dimers but not higher order oligomers could be linked by cysteine residues placed in both the BH3 and groove. The BH3:groove interaction was evident in the majority of mitochondrial Bax after apoptotic signaling, and correlated strongly with cytochrome c release, supporting its central role in Bax function. A second interface between the Bax a6-helices was implicated by cysteine linkage studies, and could link dimers to higher order oligomers. We also found that a population of Bax:Bak heterodimers generated during apoptosis formed via a BH3:groove interaction, further demonstrating that Bax and Bak oligomerize via similar mechanisms. These findings highlight the importance of BH3:groove interactions in apoptosis regulation by the Bcl-2 protein family.

Inhibitor of apoptosis (IAP) proteins, which bind to caspases via their baculoviral IAP repeat domains, also bear RING domains that enable them to promote ubiquitylation of themselves and other interacting proteins. Here we show that the RING domain of cIAP1 allows it to bind directly to the RING of X-linked IAP, causing its ubiquitylation and degradation by the proteasome, thus revealing a mechanism by which IAPs can regulate their abundance. Expression of a construct containing the RING of cellular IAP1 was able to deplete melanoma cells of endogenous X-linked IAP, promoted apoptosis, and also markedly reduced their clonogenicity when treated with cisplatin. Cross control of protein levels by RING domains may therefore enable their levels to be manipulated therapeutically.apoptosis ͉ ubiquitin ͉ homeostasis ͉ E3 ligase I nhibitor of apoptosis (IAP) proteins were initially identified in baculoviruses, where they prevent defensive apoptosis of the host cell (1), thereby increasing the time available for viral replication. Cellular IAP (cIAP) homologues, which all bear one to three baculoviral IAP repeat (BIR) domains, have been identified in yeasts and metazoans. Those that bear a RING domain in addition to BIR domains [X-linked IAP (XIAP), cIAP1, cIAP2, and ML-IAP͞Livin] appear to function as cell death inhibitors (reviewed in ref.2).The RING domains of IAPs can act as E3 ubiquitin ligases to promote the ubiquitylation of associated proteins such as TNF receptor-associated factors (TRAFs), Smac͞Diablo, and caspases (3-6). However, the importance of the RING domain for the antiapoptotic activity of the IAPs is unclear; on the one hand, a RING-less DIAP1 protein overexpressed in Drosophila had increased antiapoptotic activity (7); on the other hand, alleles of DIAP1 with mutations in the RING finger are null for Reaperinduced cell death, although more potent at blocking Hid-induced cell death (5,8,9).Our initial experiments showed that cIAP1 and XIAP can heterodimerize via a RING-RING interaction, but we also observed that expression of a stably integrated cIAP1 gene caused a specific reduction in the abundance of endogenous XIAP. Deletion studies revealed that the RING finger of cIAP1 was necessary and sufficient to cause loss of XIAP in a proteasome-dependent manner. cIAP1 RING-stimulated depletion of XIAP was seen in several cell types and associated with greatly increased sensitivity of several melanoma cells to cisplatin. Because several other E3 ligases such as BRCA1, BARD1, and RAG1 can interact via their RING fingers, it is possible that other RING-containing E3 ligases act to regulate the abundance of each other following heterodimerization. In this regard, it is striking that the E3 ligase activity of BRCA1 is greatly enhanced by heterodimerization with BARD1 (10, 11), suggesting a possible mechanism for homeostatic control of protein levels by RING domains. Materials and MethodsTransfections and Constructs. The complete sequence of all constructs used can be obtained upon request. Full length IAPs, RIN...