Apoptosis is a genetically regulated cell suicide programme mediated by activation of the effector caspases 3, 6 and 7. If apoptotic cells are not scavenged, they progress to a lytic and inflammatory phase called secondary necrosis. The mechanism by which this occurs is unknown. Here we show that caspase-3 cleaves the GSDMD-related protein DFNA5 after Asp270 to generate a necrotic DFNA5-N fragment that targets the plasma membrane to induce secondary necrosis/pyroptosis. Cells that express DFNA5 progress to secondary necrosis, when stimulated with apoptotic triggers such as etoposide or vesicular stomatitis virus infection, but disassemble into small apoptotic bodies when DFNA5 is deleted. Our findings identify DFNA5 as a central molecule that regulates apoptotic cell disassembly and progression to secondary necrosis, and provide a molecular mechanism for secondary necrosis. Because DFNA5-induced secondary necrosis and GSDMD-induced pyroptosis are dependent on caspase activation, we propose that they are forms of programmed necrosis.

Cytosolic proteins bearing a classical nuclear localization signal enter the nucleus bound to a heterodimer of importin-alpha and importin-beta (also called karyopherin-alpha and -beta). The formation of this heterodimer involves the importin-beta-binding (IBB) domain of importin-alpha, a highly basic amino-terminal region of roughly 40 amino-acid residues. Here we report the crystal structure of human importin-beta bound to the IBB domain of importin-alpha, determined at 2.5 A and 2.3 A resolution in two crystal forms. Importin-beta consists of 19 tandemly repeated HEAT motifs and wraps intimately around the IBB domain. The association involves two separate regions of importin-beta, recognizing structurally distinct parts of the IBB domain: an amino-terminal extended moiety and a carboxy-terminal helix. The structure indicates that significant conformational changes occur when importin-beta binds or releases the IBB domain domain and suggests how dissociation of the importin-alpha/beta heterodimer may be achieved upon nuclear entry.

ATP synthase is a membrane-bound, rotary motor enzyme that is critical for cellular energy metabolism in all kingdoms of life. Despite conservation of its basic structure and function, auto-inhibition by one of its rotary stalk subunits occurs in bacteria and chloroplasts but not in mitochondria. The crystal structure of the ATP synthase catalytic complex (F1) from Escherichia coli described here reveals the structural basis for this inhibition. The C-terminal domain of subunit ε adopts a novel, highly extended conformation that inserts deeply into the central cavity of the enzyme and engages both rotor and stator subunits in extensive contacts that are incompatible with functional rotation. As a result, the three catalytic subunits are stabilized in a set of conformations and rotational positions distinct from previous F1 structures.

DNA viruses such as bacteriophages and herpesviruses deliver their genome into and out of the capsid through large proteinaceous assemblies, known as portal proteins. Here we report two snapshots of the dodecameric portal protein of bacteriophage P22. The 3.25 Å resolution structure of the portal protein core bound to twelve copies of gp4 reveals a ~1.1 MDa assembly formed by 24 proteins. Unexpectedly, a lower resolution structure of the full length portal protein unveils the unique topology of the C-terminal domain, which forms a ~200 Å long, α-helical barrel. This domain inserts deeply into the virion and is highly conserved in the Podoviridae family. We propose that the barrel domain facilitates genome spooling onto the interior surface of the capsid during genome packaging and, in analogy to a rifle barrel, increases the accuracy of genome ejection into the host cell.

The human genome encodes seven isoforms of importin α which are grouped into three subfamilies known as α1, α2 and α3. All isoforms share a fundamentally conserved architecture that consists of an N-terminal, autoinhibitory, importin-β-binding (IBB) domain and a C-terminal Arm (Armadillo)-core that associates with nuclear localization signal (NLS) cargoes. Despite striking similarity in amino acid sequence and 3D structure, importin-α isoforms display remarkable substrate specificity in vivo. In the present review, we look at key differences among importin-α isoforms and provide a comprehensive inventory of known viral and cellular cargoes that have been shown to associate preferentially with specific isoforms. We illustrate how the diversification of the adaptor importin α into seven isoforms expands the dynamic range and regulatory control of nucleocytoplasmic transport, offering unexpected opportunities for pharmacological intervention. The emerging view of importin α is that of a key signalling molecule, with isoforms that confer preferential nuclear entry and spatiotemporal specificity on viral and cellular cargoes directly linked to human diseases.

Phosphorylation is the most common and pleiotropic modification in biology, which plays a vital role in regulating and finely tuning a multitude of biological pathways. Transport across the nuclear envelope is also an essential cellular function and is intimately linked to many degeneration processes that lead to disease. It is therefore not surprising that phosphorylation of cargos trafficking between the cytoplasm and nucleus is emerging as an important step to regulate nuclear availability, which directly affects gene expression, cell growth and proliferation. However, the literature on phosphorylation of nucleocytoplasmic trafficking cargos is often confusing. Phosphorylation, and its mirror process dephosphorylation, has been shown to have opposite and often contradictory effects on the ability of cargos to be transported across the nuclear envelope. Without a clear connection between attachment of a phosphate moiety and biological response, it is difficult to fully understand and predict how phosphorylation regulates nucleocytoplasmic trafficking. In this review, we will recapitulate clue findings in the field and provide some general rules on how reversible phosphorylation can affect the nuclear-cytoplasmic localization of substrates. This is only now beginning to emerge as a key regulatory step in biology.

Nuclear import of proteins containing a classical nuclear localization signal (NLS) involves NLS recognition by importin alpha, which associates with importin beta via the IBB domain. Other proteins, including parathyroid hormone-related protein (PTHrP), are imported into the nucleus by direct interaction with importin beta. We solved the crystal structure of a fragment of importin beta-1 (1-485) bound to the nonclassical NLS of PTHrP. The structure reveals a second extended cargo binding site on importin beta distinct from the IBB domain binding site. Using a permeabilized cell import assay we demonstrate that importin beta (1-485) can import PTHrP-coupled cargo in a Ran-dependent manner. We propose that this region contains a prototypical nuclear import receptor domain, which could have evolved into the modern importin beta superfamily.

Mycobacterium tuberculosis (Mtb) induces necrosis of infected cells to evade immune responses. Recently, we found that Mtb utilizes the protein CpnT to kill human macrophages by secreting its C-terminal domain, named tuberculosis necrotizing toxin (TNT) that induces necrosis by an unknown mechanism. Here we show that TNT gains access to the cytosol of Mtb-infected macrophages, where it hydrolyzes the essential co-enzyme nicotinamide adenine dinucleotide (NAD+). Expression or injection of a non-catalytic TNT mutant showed no cytotoxicity in macrophages or zebrafish zygotes, respectively, demonstrating that the NAD+-glycohydrolase activity is required for TNT-induced cell death. To prevent self-poisoning, Mtb produces an immunity factor for TNT (IFT) that binds TNT and inhibits its activity. The crystal structure of the TNT-IFT complex revealed a novel NAD+-glycohydrolase fold of TNT, which constitutes the founding member of a toxin family wide-spread in pathogenic microorganisms.