A lipid signature of BAK-driven apoptotic pore formation.

Apoptotic cell death is regulated by the BCL-2 protein family, with clusters of BAK or BAX homodimers driving pore formation in the mitochondrial outer membrane via a poorly understood process. There is growing evidence that, in addition to BAK and BAX, lipids play an important role in pore formation. Towards a better understanding of the lipidic drivers of apoptotic pore formation in isolated mitochondria, two complementary approaches were taken.

Sequence differences between BAX and BAK core domains manifest as differences in their interactions with lipids.

The B-cell lymphoma 2 (BCL2) family members, BCL2-associated protein X (BAX) and BCL2 homologous antagonist killer (BAK), are required for programmed cell death via the mitochondrial pathway. When cells are stressed, damaged or redundant, the balance of power between the BCL2 family of proteins shifts towards BAX and BAK, allowing their transition from an inactive, monomeric state to a membrane-active oligomeric form that releases cytochrome c from the mitochondrial intermembrane space. That oligomeric state has an essential intermediate, a symmetric homodimer of BAX or BAK.

NAb-seq: an accurate, rapid, and cost-effective method for antibody long-read sequencing in hybridoma cell lines and single B cells.

Despite their common use in research, monoclonal antibodies are currently not systematically sequenced. This can lead to issues with reproducibility and the occasional loss of antibodies with loss of cell lines. Hybridoma cell lines have been the primary means of generating monoclonal antibodies from immunized animals, including mice, rats, rabbits, and alpacas.

The BCL-2 family member BID plays a role during embryonic development in addition to its BH3-only protein function by acting in parallel to BAX, BAK and BOK.

The intrinsic apoptosis pathway, regulated by the BCL-2 protein family, is essential for embryonic development. Using mice lacking all known apoptosis effectors, BAX, BAK and BOK, we have previously defined the processes during development that require apoptosis. Rare Bok Bax Bak triple knockout (TKO) mice developed to adulthood and several tissues that were thought to require apoptosis during development appeared normal.

Structure of detergent-activated BAK dimers derived from the inert monomer.

A body of data supports the existence of core (α2-α5) dimers of BAK and BAX in the oligomeric, membrane-perturbing conformation of these essential apoptotic effector molecules. Molecular structures for these dimers have only been captured for truncated constructs encompassing the core domain alone. Here, we report a crystal structure of BAK α2-α8 dimers (i.

Robust autoactivation for apoptosis by BAK but not BAX highlights BAK as an important therapeutic target.

BAK and BAX, which drive commitment to apoptosis, are activated principally by certain BH3-only proteins that bind them and trigger major rearrangements. One crucial conformation change is exposure of their BH3 domain which allows BAK or BAX to form homodimers, and potentially to autoactivate other BAK and BAX molecules to ensure robust pore formation and cell death. Here, we test whether full-length BAK or mitochondrial BAX that are specifically activated by antibodies can then activate other BAK or BAX molecules.

Probing BAK and BAX Activation and Pore Assembly with Cytochrome c Release, Limited Proteolysis, and Oxidant-Induced Linkage.

Mitochondrial permeabilization is a key event in the intrinsic pathway of apoptosis, and is mediated by either of the BCL-2 family members BAK or BAX. These two proteins generate pores in the mitochondrial outer membrane that release factors such as cytochrome c into the cytosol to trigger caspase activation and apoptotic cell death. To generate pores, BAK and BAX undergo major changes including BAX translocation to the outer membrane, and partial unfolding, dimerization, and oligomerization.

Pore formation by dimeric Bak and Bax: an unusual pore?

Apoptotic cell death via the mitochondrial pathway occurs in all vertebrate cells and requires the formation of pores in the mitochondrial outer membrane. Two Bcl-2 protein family members, Bak and Bax, form these pores during apoptosis, and how they do so has been investigated for the last two decades. Many of the conformation changes that occur during their transition to pore-forming proteins have now been delineated.

Bak core and latch domains separate during activation, and freed core domains form symmetric homodimers.

Apoptotic stimuli activate and oligomerize the proapoptotic proteins Bak and Bax, resulting in mitochondrial outer-membrane permeabilization and subsequent cell death. This activation can occur when certain BH3-only proteins interact directly with Bak and Bax. Recently published crystal structures reveal that Bax separates into core and latch domains in response to BH3 peptides.

Regulation of neurotrophin receptor (Trk) signaling: suppressor of cytokine signaling 2 (SOCS2) is a new player.

The classic neurotrophins Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF) and Neurotrophins NT-3 and NT-4 are well known to regulate various aspects of neuronal differentiation, survival and growth. They do this by binding to their cognate receptors, members of the Tropomyosin-related kinase (Trk) receptor tyrosine kinase family, namely TrkA, TrkB, and TrkC. These receptors are then internalized and localized to different cellular compartments, where signal transduction occurs.

A novel role of suppressor of cytokine signaling-2 in the regulation of TrkA neurotrophin receptor biology.

Suppressor of cytokine signaling-2 (SOCS2) is a regulator of intracellular responses to growth factors and cytokines. Cultured dorsal root ganglia neurons from neonatal mice with increased or decreased SOCS2 expression were examined for altered responsiveness to nerve growth factor (NGF). In the presence of NGF, SOCS2 over-expression increased neurite length and complexity, whereas loss of SOCS2 reduced neurite outgrowth.

Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins.

The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD-95 using gene targeting in mice.

Neurotransmitters drive combinatorial multistate postsynaptic density networks.

The mammalian postsynaptic density (PSD) comprises a complex collection of approximately 1100 proteins. Despite extensive knowledge of individual proteins, the overall organization of the PSD is poorly understood. Here, we define maps of molecular circuitry within the PSD based on phosphorylation of postsynaptic proteins.

Mitochondrial permeabilization relies on BH3 ligands engaging multiple prosurvival Bcl-2 relatives, not Bak.

The Bcl-2 family regulates apoptosis by controlling mitochondrial integrity. To clarify whether its prosurvival members function by sequestering their Bcl-2 homology 3 (BH3)-only ligands or their multidomain relatives Bak and Bax, we analyzed whether four prosurvival proteins differing in their ability to bind specific BH3 peptides or Bak could protect isolated mitochondria. Most BH3 peptides could induce temperature-dependent cytochrome c release, but permeabilization was prevented by Bcl-x(L), Bcl-w, Mcl-1, or BHRF1.

Mitochondrial release of pro-apoptotic proteins: electrostatic interactions can hold cytochrome c but not Smac/DIABLO to mitochondrial membranes.

A key step in the initiation of apoptosis is the release from the mitochondrial intermembrane space of cytochrome c and other pro-apoptotic proteins such as Smac/DIABLO, Omi/HtrA2, apoptosis-inducing factor (AIF), and endonuclease G (EndoG). Discrepancies have arisen, however, as to whether all these proteins are released in different systems. Our results suggest that failure to observe cytochrome c release may be due to the use of different buffers because after permeabilization by caspase-8 cleaved human Bid (tBid), cytochrome c dissociation from mitochondria was highly dependent on ionic strength and required 50-80 mm KCl, NaCl, or LiCl.

Biological evidence that SOCS-2 can act either as an enhancer or suppressor of growth hormone signaling.

Suppressor of cytokine signaling (SOCS)-2 is a member of a family of intracellular proteins implicated in the negative regulation of cytokine signaling. The generation of SOCS-2-deficient mice, which grow to one and a half times the size of their wild-type littermates, suggests that SOCS-2 may attenuate growth hormone (GH) signaling. In vitro studies indicate that, while SOCS-2 can inhibit GH action at low concentrations, at higher concentrations it may potentiate signaling.

SOCS-6 binds to insulin receptor substrate 4, and mice lacking the SOCS-6 gene exhibit mild growth retardation.

SOCS-6 is a member of the suppressor of cytokine signaling (SOCS) family of proteins (SOCS-1 to SOCS-7 and CIS) which each contain a central SH2 domain and a carboxyl-terminal SOCS box. SOCS-1, SOCS-2, SOCS-3, and CIS act to negatively regulate cytokine-induced signaling pathways; however, the actions of SOCS-4, SOCS-5, SOCS-6, and SOCS-7 remain less clear. Here we have used both biochemical and genetic approaches to examine the action of SOCS-6.