Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.

Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions of Leishmania HSPs. Here we used a gel-based quantitative approach to assess the Leishmania donovani phosphoproteome and revealed that 38% of the proteins showed significant stage-specific differences, with a strong focus of amastigote-specific phosphoproteins on chaperone function. We identified STI1/HOP-containing chaperone complexes that interact with ribosomal client proteins in an amastigote-specific manner. Genetic analysis of STI1/HOP phosphorylation sites in conditional sti1

In the human gastric bacterium Helicobacter pylori, two metalloenzymes, hydrogenase and urease, are essential for in vivo colonization, the latter being a major virulence factor. The UreA and UreB structural subunits of urease and UreG, one of the accessory proteins for Ni 2؉ incorporation into apourease, were taken as baits for tandem affinity purification. The method allows the purification of protein complexes under native conditions and physiological expression levels of the bait protein. Protein-protein interactions are operative at the majority of cellular processes; thus, their study gives valuable insight into proteomic and functional associations. Helicobacter pylori is a Gram-negative bacterium that is infecting the stomach of about half of the human population. It is responsible for the development of gastric pathologies such as gastritis, gastroduodenal ulcer, and adenocarcinoma (1). One remarkable feature of H. pylori is its capacity to persist (often during decades) and multiply in the hostile environment of the stomach. In H. pylori, urease is a major virulence factor and is essential for the resistance to acidity because of its capacity to hydrolyze urea into bicarbonate and ammonia (2), which results in pH homeostasis of the bacterium. The protein is an extremely active and highly regulated member of the nickel metalloenzyme family. Activation of urease, i.e. incorporation of nickel into the active site, requires urease-specific accessory proteins whose homologues have been extensively studied genetically and biochemically in Klebsiella pneumoniae (3)(4)(5)(6)(7)(8). It is still enigmatic why vast amounts of urease, up to 10% of the total protein, are produced, whereas only a minor active portion is sufficient for acid resistance (9). Furthermore the observation that urease is even indispensable for the colonization under neutral conditions (10) suggested additional essential functions for this central enzyme that remained unidentified.H. pylori possesses another nickel-containing enzyme, a [NiFe] hydrogenase, allowing this organism to utilize hydrogen as an energy source. Accordingly hydrogenase has been shown to be essential for H. pylori colonization in the mouse model (11). The assembly of the [NiFe] hydrogenase metal center located in the large hydrogenase subunit is partially understood and requires several proteins involved in the sequential delivery of iron before nickel (for a review, see Ref. 12). In addition, the [NiFe] hydrogenase possesses a small subunit containing [Fe-S] clusters. For the assembly of the latter no distinct biosynthetic pathway has been attributed so far, suggesting that the housekeeping [Fe-S] cluster assembly system might be involved (12). H. pylori is unique in that the maturation events of these two nickel-containing essential proteins are interconnected as H. pylori mutants deficient in one of the accessory proteins, HypA or HypB, involved in nickel insertion into hydrogenase, lack significant activation of urease (13).

Detection of intracerebral targets with imaging probes is challenging due to the non-permissive nature of blood-brain barrier (BBB). The present work describes two novel single-domain antibodies (VHHs or nanobodies) that specifically recognize extracellular amyloid deposits and intracellular tau neurofibrillary tangles, the two core lesions of Alzheimer's disease (AD). Following intravenous administration in transgenic mouse models of AD, in vivo real-time two-photon microscopy showed gradual extravasation of the VHHs across the BBB, diffusion in the parenchyma and labeling of amyloid deposits and neurofibrillary tangles. Our results demonstrate that VHHs can be used as specific BBB-permeable probes for both extracellular and intracellular brain targets and suggest new avenues for therapeutic and diagnostic applications in neurology.