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Intronic polymorphic TOMM40 variants increasing TOMM40 mRNA expression are strongly correlated to late onset Alzheimer’s Disease. The gene product, hTomm40, encoded in the APOE gene cluster, is a core component of TOM, the translocase that imports nascent proteins across the mitochondrial outer membrane. We used Drosophila melanogaster eyes as an in vivo model to investigate the relationship between elevated Tom40 (the Drosophila homologue of hTomm40) expression and neurodegeneration. Here we provide evidence that an overabundance of Tom40 in mitochondria invokes caspase-dependent cell death in a dose-dependent manner, leading to degeneration of the primarily neuronal eye tissue. Degeneration is contingent on the availability of co-assembling TOM components, indicating that an increase in assembled TOM is the factor that triggers apoptosis and degeneration in a neural setting. Eye death is not contingent on inner membrane translocase components, suggesting it is unlikely to be a direct consequence of impaired import. Another effect of heightened Tom40 expression is upregulation and co-association of a mitochondrial oxidative stress biomarker, DmHsp22, implicated in extension of lifespan, providing new insight into the balance between cell survival and death. Activation of regulated death pathways, culminating in eye degeneration, suggests a possible causal route from TOMM40 polymorphisms to neurodegenerative disease.

Activating the intrinsic apoptosis pathway with small molecules is now a clinically validated approach to cancer therapy. In contrast, blocking apoptosis to prevent the death of healthy cells in disease settings has not been achieved. Caspases have been favored, but they act too late in apoptosis to provide long-term protection. The critical step in committing a cell to death is activation of BAK or BAX, pro-death BCL-2 proteins mediating mitochondrial damage. Apoptosis cannot proceed in their absence. Here we show that WEHI-9625, a novel tricyclic sulfone small molecule, binds to VDAC2 and promotes its ability to inhibit apoptosis driven by mouse BAK. In contrast to caspase inhibitors, WEHI-9625 blocks apoptosis before mitochondrial damage, preserving cellular function and long-term clonogenic potential. Our findings expand on the key role of VDAC2 in regulating apoptosis and demonstrate that blocking apoptosis at an early stage is both advantageous and pharmacologically tractable.

Overexpression of members of the BCL-2 family of pro-survival proteins is commonly associated with unfavourable pathogenesis in cancer. The convergence of cytotoxic stress signals on the extended BCL-2 protein family provides the biological rationale for directly targeting this family to induce apoptotic cell death. Recently, several compounds have been described that inhibit the interaction between BCL-2 family members and their natural ligand, a helical peptide sequence known as the BH3 domain. Here, we review preclinical and clinical data on these compounds, and recommend four criteria that define antagonists of the BCL-2 protein family.

Apoptosis is an important part of the host's defense mechanism for eliminating invading pathogens. Some viruses express proteins homologous in sequence and function to mammalian pro-survival Bcl-2 proteins. Anti-apoptotic F1L expressed by vaccinia virus is essential for survival of infected cells, but it bears no discernable sequence homology to proteins other than its immediate orthologues in related pox viruses. Here we report that the crystal structure of F1L reveals a Bcl-2-like fold with an unusual N-terminal extension. The protein forms a novel domain-swapped dimer in which the alpha1 helix is the exchanged domain. Binding studies reveal an atypical BH3-binding profile, with sub-micromolar affinity only for the BH3 peptide of pro-apoptotic Bim and low micromolar affinity for the BH3 peptides of Bak and Bax. This binding interaction is sensitive to F1L mutations within the predicted canonical BH3-binding groove, suggesting parallels between how vaccinia virus F1L and myxoma virus M11L bind BH3 domains. Structural comparison of F1L with other Bcl-2 family members reveals a novel sequence signature that redefines the BH4 domain as a structural motif present in both pro- and anti-apoptotic Bcl-2 members, including viral Bcl-2-like proteins.

The pathway to cell death in Caenorhabditis elegans is well established. In cells undergoing apoptosis, the Bcl-2 homology domain 3 (BH3)-only protein EGL-1 binds to CED-9 at the mitochondrial membrane to cause the release of CED-4, which oligomerises and facilitates the activation of the caspase CED-3. However, despite many studies, the biophysical features of the CED-4/CED-9 complex have not been fully characterised. Here, we report the purification of a soluble and stable 2 : 2 heterotetrameric complex formed by recombinant CED-4 and CED-9 coexpressed in bacteria. Consistent with previous studies, synthetic peptides corresponding to the BH3 domains of worm BH3-only proteins (EGL-1, CED-13) dissociate CED-4 from CED-9, but not from the gain-of-function CED-9 (G169E) mutant. Surprisingly, the ability of worm BH3 domains to dissociate CED-4 was specific since mammalian BH3-only proteins could not do so.

The fusion of viral membranes with target-cell membranes is an essential step in the entry of enveloped viruses into cells, and recent X-ray structures of paramyxoviral envelope proteins have provided new insights into protein-mediated plasma-membrane fusion. Here, we review our understanding of the structural transitions that are involved in this fusion pathway, compare it to our understanding of influenza virus membrane fusion, and discuss the implications for retroviral membrane fusion.

The BH3-only protein Bim is a potent direct activator of the proapoptotic effector protein Bax, but the structural basis for its activity has remained poorly defined. Here we describe the crystal structure of the BimBH3 peptide bound to BaxΔC26 and structure-based mutagenesis studies. Similar to BidBH3, the BimBH3 peptide binds into the cognate surface groove of Bax using the conserved hydrophobic BH3 residues h1–h4. However, the structure and mutagenesis data show that Bim is less reliant compared with Bid on its ‘h0’ residues for activating Bax and that a single amino-acid difference between Bim and Bid encodes a fivefold difference in Bax-binding potency. Similar to the structures of BidBH3 and BaxBH3 bound to BaxΔC21, the structure of the BimBH3 complex with BaxΔC displays a cavity surrounded by Bax α1, α2, α5 and α8. Our results are consistent with a model in which binding of an activator BH3 domain to the Bax groove initiates separation of its core (α2–α5) and latch (α6–α8) domains, enabling its subsequent dimerisation and the permeabilisation of the mitochondrial outer membrane.

The three-dimensional structure of plastocyanin, a ‘blue’ or ‘Type 1’ copper-protein, has been determined at a resolution of 2.7 A. The copper atom has a highly distorted tetrahedral coordination geometry. It is coordinated by a cysteine thiol group, a methionine thioether group, and two histidine imidazole groups.

The structure of a χ-type Bence-Jones protein variable fragment Au has been determined by molecular replacement methods using the known structure of an other Bence-Jones variable fragment Rei (Epp et al., Eur. J. Biochem. 45, 513 (1974)). The crystallographic R factor is 0.31 for about 4000 significantly measured reflections between 6.8 to 2.5 a. The Au protein forms a dimer across a crystallographic two fold axis. The spatial relationship of the two monomers, the conformation of the backbones and of the internal residues is extremely similar to that found in Rei.

Phaseolin, the main reserve globulin in seeds of the French bean (Phaseolus vulgaris L.), has been purified for sedimentation equilibrium analysis. The major protomer at neutral pH has a molecular weight of 150 000±5 000 which associates to a tetrameric form of molecular weight 596 000±20 000 at pH 4.5. The trimeric nature of the protomer is apparent from protein crosslinking experiments. Initial structural studies were made using optical rotatory dispersion and circular dichroism measurements. Two different crystal forms of the protein have been grown and characterised by X-ray diffraction; one of these forms appears suitable for high resolution X-ray analysis.