Maturation of dendritic cells leads to up-regulation of cellular FLICE-inhibitory protein and concomitant down-regulation of death ligand-mediated apoptosis.

Dendritic cells (DCs) disappear from lymph nodes 1 to 2 days after antigen presentation, presumably by apoptosis. To evaluate the role of death ligands in elimination of DCs, we analyzed the sensitivity of human DCs to CD95 ligand (CD95L) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We found mature DCs to be resistant to killing via CD95L or TRAIL, whereas only immature DCs were partially sensitive. However, all DC populations expressed CD95, TRAIL-R2, and TRAIL-R3 at comparable levels, suggesting that sensitivity to death ligand-induced DC apoptosis is not regulated at the receptor level. Interestingly, mature DCs highly expressed the caspase 8 inhibitory protein cFLIP, whereas only low levels were detected in immature DCs. Thus, death ligand sensitivity proved to be dependent on DC maturation and inversely correlated with expression levels of cFLIP. Induction of apoptosis by TRAIL or CD95L does not seem to play a role in the elimination of mature DCs, but instead might serve to regulate immature DC populations.

Listeria monocytogenes-infected human dendritic cells: uptake and host cell response.

Dendritic cells (DCs) are potent antigen-presenting cells and play a crucial role in initiation and modulation of specific immune responses. Various pathogens are able to persist inside DCs. However, internalization of the gram-positive bacterium Listeria monocytogenes into human DCs has not yet been shown. In the present study, we demonstrate that human monocyte-derived immature DCs can efficiently phagocytose L. monocytogenes. This uptake is independent of listerial adhesion factors internalin A and internalin B but requires cytoskeletal motion and factors present in human plasma. A major portion of internalized bacteria is found in membrane-bound phagosomes and is rarely free in the cytosol, as shown by transmission electron microscopy and by using an L. monocytogenes strain expressing green fluorescent protein when in the host cell cytosol. The infection caused maturation of the immature DCs into mature DCs displaying high levels of CD83, CD25, major histocompatibility complex class II, and the CD86 costimulator molecule. This effect appeared to be largely mediated by listerial lipoteichoic acid. Although L. monocytogenes infection is known to induce death in other cell types, infection of human DCs was found to induce necrotic but not apoptotic death in fewer than 20% of DCs. Therefore, the ability of DCs to act as effective antigen-presenting cells for listerial immunity is probably enhanced by their resistance to cell death, as well as their ability to rapidly differentiate into mature, immunostimulatory DCs upon encountering bacteria.

The ATPase activity of purified CDC48p from Saccharomyces cerevisiae shows complex dependence on ATP-, ADP-, and NADH-concentrations and is completely inhibited by NEM.

The cell cycle protein CDC48p from Saccharomyces cerevisiae is a member of a protein superfamily (AAA superfamily) characterized by a common region of approximately 200 amino-acid residues including an ATP binding consensus. CDC48p purified to homogeneity showed considerable ATPase activity which could be completely abolished by preincubation with NEM in the absence of ATP. ATP protects the protein from NEM and stabilizes the otherwise labile enzyme. The ATPase activity is reversibly inhibited by NADH and shows cooperativity with its substrate ATP. The application of the in vitro ATPase activity to the identification of physiologically interacting molecules is discussed. By electron microscopy, the enzyme was shown to consist of hexameric ring structures similar to its vertebrate homologue.

The ins and outs of Raf kinases.

Raf kinases are signal-integrating enzymes that have the ability to switch tyrosine kinase signalling to serine/threonine phosphorylation and connect growth factor receptors with transcription factors. The connection involves a cascade of protein kinases that is essential for cellular proliferation and differentiation of species ranging from worms to humans. This cascade also mediates transformation by most oncogenes.

Yeast cell cycle protein CDC48p shows full-length homology to the mammalian protein VCP and is a member of a protein family involved in secretion, peroxisome formation, and gene expression.

Yeast mutants of cell cycle gene cdc48-1 arrest as large budded cells with microtubules spreading aberrantly throughout the cytoplasm from a single spindle plaque. The gene was cloned and disruption proved it to be essential. The CDC48 sequence encodes a protein of 92 kD that has an internal duplication of 200 amino acids and includes a nucleotide binding consensus sequence. Vertebrate VCP has a 70% identity over the entire length of the protein. Yeast Sec18p and mammalian N-ethylmaleimide-sensitive fusion protein, which are involved in intracellular transport, yeast Pas1p, which is essential for peroxisome assembly, and mammalian TBP-1, which influences HIV gene expression, are 40% identical in the duplicated region. Antibodies against CDC48 recognize a yeast protein of apparently 115 kD and a mammalian protein of 100 kD. Both proteins are bound loosely to components of the microsomal fraction as described for Sec18p and N-ethylmaleimide-sensitive fusion protein. This similarity suggests that CDC48p participates in a cell cycle function related to that of N-ethylmaleimide-sensitive fusion protein/Sec18p in Golgi transport.