17 research outputs found
Crystal structure of the GTPase domain and the bundle signalling element of dynamin in the GDP state
Dynamin is the prototype of a family of large multi-domain GTPases. The 100 kDa protein is a key playerin clathrin-mediated endocytosis, where it cleaves off vesicles from membranes using the energy fromGTP hydrolysis. We have solved the high resolution crystal structure of a fusion protein of the GTPasedomain and the bundle signalling element (BSE) of dynamin 1 liganded with GDP. The structure providesa hitherto missing snapshot of the GDP state of the hydrolytic cycle of dynamin and reveals how theswitch I region moves away from the active site after GTP hydrolysis and release of inorganic phosphate.Comparing our structure of the GDP state with the known structures of the GTP state, the transition stateand the nucleotide-free state of dynamin 1 we describe the structural changes through the hydrolyticcycle
A New Model for the Transition of APAF-1 from Inactive Monomer to Caspase-activating Apoptosome*
The cytosolic adaptor protein Apaf-1 is a key player in the intrinsic pathway of apoptosis. Binding of mitochondrially released cytochrome c and of dATP or ATP to Apaf-1 induces the formation of the heptameric apoptosome complex, which in turn activates procaspase-9. We have re-investigated the chain of events leading from monomeric autoinhibited Apaf-1 to the functional apoptosome in vitro. We demonstrate that Apaf-1 does not require energy from nucleotide hydrolysis to eventually form the apoptosome. Despite a low intrinsic hydrolytic activity of the autoinhibited Apaf-1 monomer, nucleotide hydrolysis does not occur at any stage of the process. Rather, mere binding of ATP in concert with the binding of cytochrome c primes Apaf-1 for assembly. Contradicting the current view, there is no strict requirement for an adenine base in the nucleotide. On the basis of our results, we present a new model for the mechanism of apoptosome assembly
Toxoplasma gondii inhibits cytochrome c-induced caspase activation in its host cell by interference with holo-apoptosome assembly
Inhibition of programmed cell death pathways of mammalian cells often facilitates the sustained survival of intracellular microorganisms. The apicomplexan parasite Toxoplasma gondii is a master regulator of host cell apoptotic pathways. Here, we have characterized a novel anti-apoptotic activity of T. gondii. Using a cell-free cytosolic extract model, we show that T. gondii interferes with the activities of caspase 9 and caspase 3/7 which have been induced by exogenous cytochrome c and dATP. Proteolytic cleavage of caspases 9 and 3 is also diminished suggesting inhibition of holo-apoptosome function. Parasite infection of Jurkat T cells and subsequent triggering of apoptosome formation by exogenous cytochrome c in vitro and in vivo indicated that T. gondii also interferes with caspase activation in infected cells. Importantly, parasite inhibition of cytochrome c-induced caspase activation considerably contributes to the overall anti-apoptotic activity of T. gondii as observed in staurosporine-treated cells. Co-immunoprecipitation showed that T. gondii abolishes binding of caspase 9 to Apaf-1 whereas the interaction of cytochrome c with Apaf-1 remains unchanged. Finally, T. gondii lysate mimics the effect of viable parasites and prevents holo-apoptosome functionality in a reconstituted in vitro system comprising recombinant Apaf-1 and caspase 9. Beside inhibition of cytochrome c release from host cell mitochondria, T. gondii thus also targets the holo-apoptosome assembly as a second mean to efficiently inhibit the caspase-dependent intrinsic cell death pathway
Expression of viral CD45 ligand E3/49K on porcine cells reduces human anti-pig immune responses
Abstract Transgenic expression of protective molecules in porcine cells and tissues is a promising approach to prevent xenograft rejection. Viruses have developed various strategies to escape the host’s immune system. We generated porcine B cells (B cell line L23) expressing the human adenovirus protein E3/49K or the human cytomegalovirus protein pUL11 and investigated how human T, NK and B cell responses are affected by the expression of the viral proteins. Binding studies revealed that E3/49K and pUL11 interact with CD45 on human but not porcine peripheral blood mononuclear cells. T cell proliferation in response to L23-E3/49K cells was significantly reduced and accompanied by development of an anti-inflammatory cytokine milieu (low: TNF-alpha, IFN-gamma, IL-6; high: IL-4, IL-10). Human peripheral blood mononuclear cells which had been primed for four weeks by L23-E3/49K cells included an extended population of regulatory T cells. Cytotoxicity of effector T and natural killer cells against L23 cells was significantly reduced (40 to 50%) by E3/49K expression. B cell activation and antibody production to E3/49K expressing cells was also diminished. Surprisingly, pUL11 expression showed no effects. Reduction of human anti-pig immune responses by transgenic expression of selected viral genes may be a novel approach for protection of porcine xenografts
Crystal structure of the leucine-rich repeat domain of the NOD-like receptor NLRP1: Implications for binding of muramyl dipeptide
Structural Biology of NOD-Like Receptors
The nucleotide-binding domain (NBD) and leucine-rich repeat (LRR) containing (NLR) proteins are a large family of intracellular immune receptors conserved in both animals and plants. Mammalian NLRs function as pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) or host-derived danger associated molecular patterns (DAMPs). PAMP or DAMP perception activates NLRs which consequently recruit pro-caspase-1 directly or indirectly. These sequential events result in formation of large multimeric protein complexes termed inflammasomes that mediate caspase-1 activation for pyroptosis and cytokine secretion. Recent structural and biochemical studies provide significant insights into the acting mechanisms of NLR proteins. In this chapter, we review and discuss these studies concerning autoinhibition, ligand recognition, activation of NLRs, and assembly of NLR inflammasomes