Anti-apoptotic MCL-1 promotes long-chain fatty acid oxidation through interaction with ACSL1.

MCL-1 is essential for promoting the survival of many normal cell lineages and confers survival and chemoresistance in cancer. Beyond apoptosis regulation, MCL-1 has been linked to modulating mitochondrial metabolism, but the mechanism(s) by which it does so are unclear. Here, we show in tissues and cells that MCL-1 supports essential steps in long-chain (but not short-chain) fatty acid ß-oxidation (FAO) through its binding to specific long-chain acyl-coenzyme A (CoA) synthetases of the ACSL family. ACSL1 binds to the BH3-binding hydrophobic groove of MCL-1 through a non-conventional BH3-domain. Perturbation of this interaction, via genetic loss of Mcl1, mutagenesis, or use of selective BH3-mimetic MCL-1 inhibitors, represses long-chain FAO in cells and in mouse livers and hearts. Our findings reveal how anti-apoptotic MCL-1 facilitates mitochondrial metabolism and indicate that disruption of this function may be associated with unanticipated cardiac toxicities of MCL-1 inhibitors in clinical trials.

BOK Is a Non-canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation.

The mitochondrial pathway of apoptosis is initiated by mitochondrial outer membrane permeabilization (MOMP). The BCL-2 family effectors BAX and BAK are thought to be absolutely required for this process. Here, we report that BCL-2 ovarian killer (BOK) is a bona fide yet unconventional effector of MOMP that can trigger apoptosis in the absence of both BAX and BAK. However, unlike the canonical effectors, BOK appears to be constitutively active and unresponsive to antagonistic effects of the antiapoptotic BCL-2 proteins. Rather, BOK is controlled at the level of protein stability by components of the endoplasmic reticulum (ER)-associated degradation pathway. BOK is ubiquitylated by the AMFR/gp78 E3 ubiquitin ligase complex and targeted for proteasomal degradation in a VCP/p97-dependent manner, which allows survival of the cell. When proteasome function, VCP, or gp78 activity is compromised, BOK is stabilized to induce MOMP and apoptosis independently of other BCL-2 proteins.

MLKL Requires the Inositol Phosphate Code to Execute Necroptosis.

Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.

A killer metamorphosis: catching BAK in action at the membrane.

Permeabilization of the outer mitochondrial membrane initiates apoptotic cell death. B-cell lymphoma 2 (BCL-2) antagonist killer (BAK) and BCL-2-associated X (BAX) mediate mitochondrial poration, but how this process unfolds remains poorly defined. Two studies in this issue investigate the transition of dormant, inactive BAK monomer to a highly dynamic membrane-associated, pore-forming oligomer.

Apoptotic mitochondrial poration by a growing list of pore-forming BCL-2 family proteins.

The pore-forming BCL-2 family proteins are effectors of mitochondrial poration in apoptosis initiation. Two atypical effectors-BOK and truncated BID (tBID)-join the canonical effectors BAK and BAX. Gene knockout revealed developmental phenotypes in the absence the effectors, supporting their roles in vivo. During apoptosis effectors are activated and change shape from dormant monomers to dynamic oligomers that associate with and permeabilize mitochondria. BID is activated by proteolysis, BOK accumulates on inhibition of its degradation by the E3 ligase gp78, while BAK and BAX undergo direct activation by BH3-only initiators, autoactivation, and crossactivation. Except tBID, effector oligomers on the mitochondria appear as arcs and rings in super-resolution microscopy images. The BH3-in-groove dimers of BAK and BAX, the tBID monomers, and uncharacterized BOK species are the putative building blocks of apoptotic pores. Effectors interact with lipids and bilayers but the mechanism of membrane poration remains elusive. I discuss effector-mediated mitochondrial poration.

Sequential Engagement of Distinct MLKL Phosphatidylinositol-Binding Sites Executes Necroptosis.

Necroptosis is a cell death pathway regulated by the receptor interacting protein kinase 3 (RIPK3) and the mixed lineage kinase domain-like (MLKL) pseudokinase. How MLKL executes plasma membrane rupture upon phosphorylation by RIPK3 remains controversial. Here, we characterize the hierarchical transduction of structural changes in MLKL that culminate in necroptosis. The MLKL brace, proximal to the N-terminal helix bundle (NB), is involved in oligomerization to facilitate plasma membrane targeting through the low-affinity binding of NB to phosphorylated inositol polar head groups of phosphatidylinositol phosphate (PIP) phospholipids. At the membrane, the NB undergoes a "rolling over" mechanism to expose additional higher-affinity PIP-binding sites responsible for robust association to the membrane and displacement of the brace from the NB. PI(4,5)P2 is the preferred PIP-binding partner. We investigate the specific association of MLKL with PIPs and subsequent structural changes during necroptosis.

Protein-protein and protein-lipid interactions of pore-forming BCL-2 family proteins in apoptosis initiation.

Apoptosis is a common cell death program that is important in human health and disease. Signaling in apoptosis is largely driven through protein-protein interactions. The BCL-2 family proteins function in protein-protein interactions as key regulators of mitochondrial poration, the process that initiates apoptosis through the release of cytochrome c, which activates the apoptotic caspase cascade leading to cellular demolition. The BCL-2 pore-forming proteins BAK and BAX are the key executors of mitochondrial poration. We review the state of knowledge of protein-protein and protein-lipid interactions governing the apoptotic function of BAK and BAX, as determined through X-ray crystallography and NMR spectroscopy studies. BAK and BAX are dormant, globular α-helical proteins that participate in protein-protein interactions with other pro-death BCL-2 family proteins, transforming them into active, partially unfolded proteins that dimerize and associate with and permeabilize mitochondrial membranes. We compare the protein-protein interactions observed in high-resolution structures with those derived in silico by AlphaFold, making predictions based on combining experimental and in silico approaches to delineate the structural basis for novel protein-protein interaction complexes of BCL-2 family proteins.

Metabolic activation of CaMKII by coenzyme A.

Active metabolism regulates oocyte cell death via calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated phosphorylation of caspase-2, but the link between metabolic activity and CaMKII is poorly understood. Here we identify coenzyme A (CoA) as the key metabolic signal that inhibits Xenopus laevis oocyte apoptosis by directly activating CaMKII. We found that CoA directly binds to the CaMKII regulatory domain in the absence of Ca(2+) to activate CaMKII in a calmodulin-dependent manner. Furthermore, we show that CoA inhibits apoptosis not only in X. laevis oocytes but also in Murine oocytes. These findings uncover a direct mechanism of CaMKII regulation by metabolism and further highlight the importance of metabolism in preserving oocyte viability.

Linker Histone H1.2 Directly Activates BAK through the K/RVVKP Motif on the C-Terminal Domain.

H1.2 is a key mediator of apoptosis following DNA double-strand breaks. The link between H1.2 and canonical apoptotic pathways is unclear. One study found that H1.2 stimulates cytochrome c (Cyt c) release; in contrast, apoptosis-inducing factor was found to be released in another study. The C-terminal domain (CTD) of H1.2 has been implicated in the latter pathway, but activation of the proapoptotic protein BCL-2 homologous antagonist/killer (BAK) is a common denominator in both pathways. This study aimed to determine whether the CTD of H1.2 is also responsible for mitochondrial Cyt c release and whether a previously identified K/RVVKP motif in the CTD mediates the response. This study investigated if H1.2 mediates apoptosis induction through direct interaction with BAK. We established that the CTD of H1.2 stimulates mitochondrial Cyt c release in vitro in a mitochondrial permeability transition-independent manner and that the substitution of a single valine with threonine in the K/RVVKP motif abolishes Cyt c release. Additionally, we showed that H1.2 directly interacts with BAK with weak affinity and that the CTD of H1.2 mediates this binding. Using two 20-amino acid peptides derived from the CTD of H1.2 and H1.1 (K/RVVKP motif inclusive), we determined the main residues involved in the direct interaction with BAK. We propose that H1.2 operates through the K/RVVKP motif by directly activating BAK through inter- and intramolecular interactions. These findings expand the view of H1.2 as a signal-transducing molecule that can activate apoptosis in a BAK-dependent manner.

A unified model of mammalian BCL-2 protein family interactions at the mitochondria.

During apoptosis, the BCL-2 protein family controls mitochondrial outer membrane permeabilization (MOMP), but the dynamics of this regulation remain controversial. We employed chimeric proteins composed of exogenous BH3 domains inserted into a tBID backbone that can activate the proapoptotic effectors BAX and BAK to permeabilize membranes without being universally sequestered by all antiapoptotic BCL-2 proteins. We thus identified two "modes" whereby prosurvival BCL-2 proteins can block MOMP, by sequestering direct-activator BH3-only proteins ("MODE 1") or by binding active BAX and BAK ("MODE 2"). Notably, we found that MODE 1 sequestration is less efficient and more easily derepressed to promote MOMP than MODE 2. Further, MODE 2 sequestration prevents mitochondrial fusion. We provide a unified model of BCL-2 family function that helps to explain otherwise paradoxical observations relating to MOMP, apoptosis, and mitochondrial dynamics.

Many players in BCL-2 family affairs.

During apoptotic cell death, cellular stress signals converge at the mitochondria to induce mitochondrial outer-membrane permeabilization (MOMP) through B cell lymphoma-2 (BCL-2) family proteins and their effectors. BCL-2 proteins function through protein-protein interactions, the mechanisms and structural aspects of which are only now being uncovered. Recently, the elucidation of the dynamic features underlying their function has highlighted their structural plasticity and the consequent complex thermodynamic landscape governing their protein-protein interactions. These studies show that canonical interactions involve a conserved, hydrophobic groove, whereas non-canonical interactions function allosterically outside the groove. We review the latest structural advances in understanding the interactions and functions of mammalian BCL-2 family members, and discuss new opportunities to modulate these proteins in health and disease.

The BCL-2 family reunion.

B cell CLL/lymphoma-2 (BCL-2) and its relatives comprise the BCL-2 family of proteins, which were originally characterized with respect to their roles in controlling outer mitochondrial membrane integrity and apoptosis. Current observations expand BCL-2 family function to include numerous cellular pathways. Here we will discuss the mechanisms and functions of the BCL-2 family in the context of these pathways, highlighting the complex integration and regulation of the BCL-2 family in cell fate decisions.

Concerted multi-pronged attack by calpastatin to occlude the catalytic cleft of heterodimeric calpains.

The Ca(2+)-dependent cysteine proteases, calpains, regulate cell migration, cell death, insulin secretion, synaptic function and muscle homeostasis. Their endogenous inhibitor, calpastatin, consists of four inhibitory repeats, each of which neutralizes an activated calpain with exquisite specificity and potency. Despite the physiological importance of this interaction, the structural basis of calpain inhibition by calpastatin is unknown. Here we report the 3.0 A structure of Ca(2+)-bound m-calpain in complex with the first calpastatin repeat, both from rat, revealing the mechanism of exclusive specificity. The structure highlights the complexity of calpain activation by Ca(2+), illustrating key residues in a peripheral domain that serve to stabilize the protease core on Ca(2+) binding. Fully activated calpain binds ten Ca(2+) atoms, resulting in several conformational changes allowing recognition by calpastatin. Calpain inhibition is mediated by the intimate contact with three critical regions of calpastatin. Two regions target the penta-EF-hand domains of calpain and the third occupies the substrate-binding cleft, projecting a loop around the active site thiol to evade proteolysis.

Inhibition of BAK-mediated apoptosis by the BH3-only protein BNIP5.

BCL-2 family proteins regulate apoptosis by initiating mitochondrial outer membrane permeabilization (MOMP). Activation of the MOMP effectors BAX and BAK is controlled by the interplay of anti-apoptotic BCL-2 proteins (e.g., MCL-1) and pro-apoptotic BH3-only proteins (e.g., BIM). Using a genome-wide CRISPR-dCas9 transactivation screen we identified BNIP5 as an inhibitor of BAK-, but not BAX-induced apoptosis. BNIP5 blocked BAK activation in different cell types and in response to various cytotoxic therapies. The BH3 domain of BNIP5 was both necessary and sufficient to block BAK activation. Mechanistically, the BH3 domain of BNIP5 acts as a selective BAK activator, but a poor de-repressor of complexes between BAK and pro-survival BCL-2 family proteins. By promoting the binding of activated BAK to MCL-1 or BCL-xL, BNIP5 inhibits apoptosis when BAX is absent. Based on our observations, BNIP5 can act functionally as an anti-apoptotic BH3-only protein.

BH3 domains other than Bim and Bid can directly activate Bax/Bak.

Bcl-2 family proteins regulate a critical step in apoptosis referred to as mitochondrial outer membrane permeabilization (MOMP). Members of a subgroup of the Bcl-2 family, known as the BH3-only proteins, activate pro-apoptotic effectors (Bax and Bak) to initiate MOMP. They do so by neutralizing pro-survival Bcl-2 proteins and/or directly activating Bax/Bak. Bim and Bid are reported to be direct activators; however, here we show that BH3 peptides other than Bim and Bid exhibited various degrees of direct activation of the effector Bax or Bak, including Bmf and Noxa BH3s. In the absence of potent direct activators, such as Bim and Bid, we unmasked novel direct activator BH3 ligands capable of inducing effector-mediated cytochrome c release and liposome permeabilization, even when both Bcl-xL- and Mcl-1-type anti-apoptotic proteins were inhibited. The ability of these weaker direct activator BH3 peptides to cause MOMP correlated with that of the corresponding full-length proteins to induce apoptosis in the absence of Bim and Bid. We propose that, in certain contexts, direct activation by BH3-only proteins other than Bim and Bid may significantly contribute to MOMP and apoptosis.

Structural basis of BAK activation in mitochondrial apoptosis initiation.

BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric "BH3-in-groove" triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.

Small molecule SJ572946 activates BAK to initiate apoptosis.

Poration of the outer mitochondrial membrane by the effector BCL-2 proteins BAK and BAX initiates apoptosis. BH3-only initiators BID and BIM trigger conformational changes in BAK and BAX transforming them from globular dormant proteins to oligomers of the apoptotic pores. Small molecules that can directly activate effectors are being sought for applications in cancer treatment. Here, we describe the small molecule SJ572946, discovered in a fragment-based screen that binds to the activation groove of BAK and selectively triggers BAK activation over that of BAX in liposome and mitochondrial permeabilization assays. SJ572946 independently kills BAK-expressing BCL2allKO HCT116 cells revealing on target cellular activity. In combination with apoptotic inducers and BH3 mimetics, SJ572946 kills experimental cancer cell lines. SJ572946 also cooperates with the endogenous BAK activator BID in activating a misfolded BAK mutant substantially impaired in activation. SJ572946 is a proof-of-concept tool for probing BAK-mediated apoptosis in preclinical cancer research.

Alzheimer's disease-associated U1 snRNP splicing dysfunction causes neuronal hyperexcitability and cognitive impairment.

Recent proteome and transcriptome profiling of Alzheimer's disease (AD) brains reveals RNA splicing dysfunction and U1 small nuclear ribonucleoprotein (snRNP) pathology containing U1-70K and its N-terminal 40-KDa fragment (N40K). Here we present a causative role of U1 snRNP dysfunction to neurodegeneration in primary neurons and transgenic mice (N40K-Tg), in which N40K expression exerts a dominant-negative effect to downregulate full-length U1-70K. N40K-Tg recapitulates N40K insolubility, erroneous splicing events, neuronal degeneration and cognitive impairment. Specifically, N40K-Tg shows the reduction of GABAergic synapse components (e.g., the GABA receptor subunit of GABRA2), and concomitant postsynaptic hyperexcitability that is rescued by a GABA receptor agonist. Crossing of N40K-Tg and the 5xFAD amyloidosis model indicates that the RNA splicing defect synergizes with the amyloid cascade to remodel the brain transcriptome and proteome, deregulate synaptic proteins, and accelerate cognitive decline. Thus, our results support the contribution of U1 snRNP-mediated splicing dysfunction to AD pathogenesis.

Apoptotic regulation by MCL-1 through heterodimerization.

Myeloid cell leukemia 1 (MCL-1), an anti-apoptotic BCL-2 family member active in the preservation of mitochondrial integrity during apoptosis, has fundamental roles in development and hematopoiesis and is dysregulated in human cancers. It bears a unique, intrinsically unstructured, N-terminal sequence, which leads to its instability in cells and hinders protein production and structural characterization. Here, we present collective data from NMR spectroscopy and titration calorimetry to reveal the selectivity of MCL-1 in binding BCL-2 homology 3 (BH3) ligands of interest for mammalian biology. The N-terminal sequence weakens the BH3 interactions but does not affect selectivity. Its removal by calpain-mediated limited proteolysis results in a stable BCL-2-like core domain of MCL-1 (cMCL-1). This core is necessary and sufficient for BH3 ligand binding. Significantly, we also characterized the in vitro protein-protein interaction between cMCL-1 and activated BID by size exclusion chromatography and NMR titrations. This interaction occurs in a very slow manner in solution but is otherwise similar to the interaction between cMCL-1 and BID-BH3 peptides. We also present the solution structure of complex cMCL-1xhBID-BH3, which completes the family portrait of MCL-1 complexes and may facilitate drug discovery against human tumors.

Diversifying selection and functional analysis of interleukin-4 suggests antagonism-driven evolution at receptor-binding interfaces.

BACKGROUND: Interleukin-4 (IL4) is a secreted immunoregulatory cytokine critically involved in host protection from parasitic helminths 1. Reasoning that helminths may have evolved mechanisms to antagonize IL4 to maximize their dispersal, we explored mammalian IL4 evolution. RESULTS: This analysis revealed evidence of diversifying selection at 15 residues, clustered in epitopes responsible for IL4 binding to its Type I and Type II receptors. Such a striking signature of selective pressure suggested either recurrent episodes of pathogen antagonism or ligand/receptor co-evolution. To test the latter possibility, we performed detailed functional analysis of IL4 allotypes expressed by Mus musculus musculus and Mus musculus castaneus, which happen to differ at 5 residues (including three at positively selected sites) in and adjacent to the site 1 epitope that binds the IL4Ralpha subunit shared by the Type I and Type II IL4 receptors. We show that this intra-species variation affects the ability of IL4 neither to bind IL4 receptor alpha (IL4Ralpha) nor to signal biological responses through its Type I receptor. CONCLUSIONS: Our results - reminiscent of clustered positively selected sites revealing functionally important residues at host-virus interaction interfaces - are consistent with IL4 having evolved to avoid recurrent pathogen antagonism, while maintaining the capacity to bind and signal through its cognate receptor. This work exposes what may be a general feature of evolutionary conflicts fought by pathogen antagonists at host protein-protein interaction interfaces involved in immune signaling: the emergence of receptor-binding ligand epitopes capable of buffering amino acid variation.