The heat shock protein Gp96 has been shown to induce specific immune responses. On one hand, this phenomenon is based on the specific interaction with CD91 that mediates endocytosis and results in major histocompatibility complex class I-restricted representation of the Gp96-associated peptides. On the other hand, Gp96 induces activation of professional antigen-presenting cells, resulting in the production of pro-inflammatory cytokines and up-regulation of costimulatory molecules by unknown mechanisms. In this study, we have analyzed the consequences of Gp96 interaction with cells expressing different Toll-like receptors (TLRs) and with bone marrow-derived dendritic cells from mice lacking functional TLR2 and/or TLR4 molecules. We find that the Gp96-TLR2/4 interaction results in activation of nuclear factor B-driven reporter genes and mitogen-and stress-activated protein kinases and induces IB␣ degradation. Bone marrow-derived dendritic cells of C3H/HeJ and more pronounced C3H/HeJ/ TLR2 ؊/؊ mice fail to respond to Gp96. Interestingly, activation of bone marrow-derived dendritic cells depends on endocytosis of Gp96 molecules. Our results provide, for the first time, the molecular basis for understanding the Gp96-mediated activation of antigen-presenting cells by describing the simultaneous stimulation of the innate and adaptive immune system. This feature explains the remarkable ability of Gp96 to induce specific immune responses against tumors and pathogens.

Hepsin is a membrane-anchored, trypsin-like serine protease with prominent expression in the human liver and tumours of the prostate and ovaries. To better understand the biological functions of hepsin, we identified macromolecular substrates employing a tetrapeptide PS-SCL (positional scanning-synthetic combinatorial library) screen that rapidly determines the P1-P4 substrate specificity. Hepsin exhibited strong preference at the P1 position for arginine over lysine, and favoured threonine, leucine or asparagine at the P2, glutamine or lysine at the P3, and proline or lysine at the P4 position. The relative activity of hepsin toward individual AMC (7-amino-4-methylcoumarin)-tetrapeptides was generally consistent with the overall peptide profiling results derived from the PC-SCL screen. The most active tetrapeptide substrate Ac (acetyl)-KQLR-AMC matched with the activation cleavage site of the hepatocyte growth factor precursor sc-HGF (single-chain HGF), KQLR downward arrowVVNG (where downward arrow denotes the cleavage site), as identified by a database analysis of trypsin-like precursors. X-ray crystallographic studies with KQLR chloromethylketone showed that the KQLR peptide fits well into the substrate-binding cleft of hepsin. This hepsin-processed HGF induced c-Met receptor tyrosine phosphorylation in SKOV-3 ovarian cancer cells, indicating that the hepsin-cleaved HGF is biologically active. Activation cleavage site mutants of sc-HGF with predicted non-preferred sequences, DPGR downward arrowVVNG or KQLQ downward arrowVVNG, were not processed, illustrating that the P4-P1 residues can be important determinants for substrate specificity. In addition to finding macromolecular hepsin substrates, the extracellular inhibitors of the HGF activator, HAI-1 and HAI-2, were potent inhibitors of hepsin activity (IC50 4+/-0.2 nM and 12+/-0.5 nM respectively). Together, our findings suggest that the HGF precursor is a potential in vivo substrate for hepsin in tumours, where hepsin expression is dysregulated and may influence tumorigenesis through inappropriate activation and/or regulation of HGF receptor (c-Met) functions.

Oncogenic activation of the BRAF serine/threonine kinase has been associated with initiation and maintenance of melanoma tumors. As such, development of pharmacologic agents to target RAF proteins or their effector kinases is an area of intense investigation. Here we report the biological properties of GDC-0879, a highly selective, potent, and orally bioavailable RAF small-molecule inhibitor. We used extracellular signal-regulated kinase (ERK)-1/2 and mitogen-activated protein kinase/ERK kinase (MEK)-1/2 phosphorylation as biomarkers to explore the relationship between tumor outcome and pharmacodynamic inhibition of the RAF-MEK-ERK pathway. In GDC-0879-treated mice, both cell line-and patient-derived BRAF V600E tumors exhibited stronger and more sustained pharmacodynamic inhibition (>90% for 8 hours) and improved survival compared with mutant KRAS-expressing tumors. Despite the involvement of activated RAF signaling in RAS-induced tumorigenesis, decreased time to progression was observed for some KRAS-mutant tumors following GDC-0879 administration. Moreover, striking differences were noted for RAF and MEK inhibition across a panel of 130 tumor cell lines. Whereas GDC-0879-mediated efficacy was associated strictly with BRAF V600E status, MEK inhibition also attenuated proliferation and tumor growth of cell lines expressing wild-type BRAF (81% KRAS mutant, 38% KRAS wild type). The responsiveness of BRAF V600E melanoma cells to GDC-0879 could be dramatically altered by pharmacologic and genetic modulation of phosphatidylinositol 3-kinase pathway activity. These data suggest that GDC-0879-induced signaling changes are dependent on the point of oncogenic activation within the RAS network. Taken together, these studies increase our understanding of the molecular determinants for antitumor efficacy resulting from RAF pathway inhibition and have implications for therapeutic intervention in the clinic.

Murine cytomegalovirus encodes three regulators of antigen presentation to antiviral CD8 T cells. According to current paradigms, all three regulators are committed to the inhibition of the presentation of antigenic peptides. Whereas m152/gp40 catalyzes the retention of peptide-loaded major histocompatibility complex (MHC) class I molecules in a cis-Golgi compartment, m06/gp48 binds stably to class I molecules and directs them into the cellular cargo-sorting pathway of lysosomal degradation. Regulator m04/gp34 also binds stably to class I molecules, but unlike m152 and m06, it does not downmodulate MHC class I cell surface expression. It has entered the literature as a direct inhibitor of T-cell recognition of the MHC-peptide complex at the cell surface. In this work, we have studied the presentation of antigenic viral peptides in cells infected with a comprehensive set of mutant viruses expressing the three regulators separately as well as in all possible combinations. The results redefine m04 as a positive regulator dedicated to the facilitation of antigen presentation. When expressed alone, it did not inhibit T-cell recognition, and when expressed in the presence of m152, it restored antigen presentation by antagonizing the inhibitory function of m152. Its intrinsic positive function, however, was antagonized and even slightly overcompensated for by the negative regulator m06. In an adoptive cell transfer model, the opposing forces of the three regulators were found to govern immune surveillance in the infected host. While negative regulators, also known as immunoevasins, are common, the existence of a positive regulator is without precedent and indicates an intriguing genetic potential of this virus to influence antigen presentation.

In the facultative autotrophic organism Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium, the Calvin cycle does not appear to be operative in autotrophic carbon assimilation. An alternative cyclic pathway, the 3-hydroxypropionate cycle, has been proposed. In this pathway, acetyl coenzyme A (acetyl-CoA) is assumed to be converted to malate, and two CO 2 molecules are thereby fixed. Malyl-CoA is supposed to be cleaved to acetyl-CoA, the starting molecule, and glyoxylate, the carbon fixation product. Malyl-CoA cleavage is shown here to be catalyzed by malyl-CoA lyase; this enzyme activity is induced severalfold in autotrophically grown cells. Malate is converted to malyl-CoA via an inducible CoA transferase with succinyl-CoA as a CoA donor. Some enzyme activities involved in the conversion of malonyl-CoA via 3-hydroxypropionate to propionyl-CoA are also induced under autotrophic growth conditions. So far, no clue as to the first step in glyoxylate assimilation has been obtained. One possibility for the assimilation of glyoxylate involves the conversion of glyoxylate to glycine and the subsequent assimilation of glycine. However, such a pathway does not occur, as shown by labeling of whole cells with [1,2-13 C 2 ]glycine. Glycine carbon was incorporated only into glycine, serine, and compounds that contained C 1 units derived therefrom and not into other cell compounds.

Phototrophic CO 2 assimilation by the primitive, green eubacterium Chloroflexus aurantiacus has been shown earlier to proceed in a cyclic mode via 3-hydroxypropionate, propionyl-CoA, succinyl-CoA, and malyl-CoA. The metabolic cycle could be closed by cleavage of malylCoA affording glyoxylate (the primary CO 2 fixation product) with regeneration of acetyl-CoA serving as the starter unit of the cycle. Cell extracts of C. aurantiacus were also shown to catalyze the conversion of citramalate into pyruvate and acetyl-CoA in a succinyl-CoAdependent reaction. The data suggest that glyoxylate obtained by the cleavage of malyl-CoA can be utilized by condensation with propionyl-CoA affording erythro-␤-methylmalyl-CoA, which is converted to acetyl-CoA and pyruvate. This reaction sequence regenerates acetylCoA, which serves as the precursor of propionyl-CoA in the 3-hydroxypropionate cycle. Autotrophic CO 2 fixation proceeds by combination of the 3-hydroxypropionate cycle with the methylmalyl-CoA cycle. The net product of that bicyclic autotrophic CO 2 fixation pathway is pyruvate serving as an universal building block for anabolic reactions.Autotrophic CO 2 fixation in the phototrophic bacterium Chloroflexus aurantiacus has been proposed to proceed via a novel pathway, the 3-hydroxypropionate cycle ( Fig. 1) (1-7). Briefly, acetyl-CoA (1) serves as starting unit, and biotin-dependent carboxylation of acetyl-CoA and propionyl-CoA (4) are the main CO 2 fixation reactions. One turn of the proposed cycle results in conversion of acetyl-CoA into malyl-CoA (8) with consumption of 2 HCO 3 Ϫ and 3 NADPH. Malyl-CoA is cleaved by malyl-CoA lyase with regeneration of the starting molecule acetyl-CoA. Glyoxylate (9) is believed to be the initial CO 2 fixation product (7).The pathway of glyoxylate assimilation into cell material is incompletely understood (5-12). Glycine has been ruled out as an intermediate (7). So far, in vitro transformation of glyoxylate has not been observed, except for pyridine nucleotide-dependent reduction to glycolate (7). An acetyl-CoA-dependent conversion of glyoxylate to malyl-CoA and malate was ascribed to the reverse reaction of malyl-CoA lyase forming malyl-CoA, combined with a side reaction of citrate synthase or acyl-CoA thioesterase, which hydrolyzes malyl-CoA to malate and CoA (7,(13)(14)(15). Previous studies have shown that C. aurantiacus can use pyruvate for anaplerotic reactions (3,7,11,16). Pyruvate is converted to phosphoenolpyruvate (PEP) 1 by pyruvate phosphate dikinase, followed by PEP carboxylation to oxaloacetate by PEP carboxylase. However, pyruvate synthase activity was hardly detectable (12), and the origin of pyruvate in C. aurantiacus is still unknown. To serve as a central intermediate for anaplerotic reactions, it should be formed ultimately from one of the intermediates of the 3-hydroxypropionate cycle and/or from glyoxylate.The aim of this work was to elucidate reactions for glyoxylate assimilation. We show that a reaction sequence starting with glyoxylate and propionyl-CoA afford...

The 3-hydroxypropionate cycle is a bicyclic autotrophic CO 2 fixation pathway in the phototrophic Chloroflexus aurantiacus (Bacteria), and a similar pathway is operating in autotrophic members of the Sulfolobaceae

Influenza virus infection induces maturation of murine dendritic cells (DCs), which is most important for the initiation of an immune response. However, in contrast to EL-4 and MC57 cells, DCs present viral CTL epitopes with a delay of up to 10 h. This delay in Ag presentation coincides with the up-regulation of MHC class I molecules as well as costimulatory molecules on the cell surface and the accumulation of newly synthesized ubiquitinated proteins in large cytosolic structures, called DC aggresome-like-induced structures (DALIS). These structures were observed previously after LPS-induced maturation of DCs, and it was speculated that they play a role in the regulation of MHC class I Ag presentation. Our findings provide the first evidence for a connection between DC maturation, MHC class I-restricted Ag presentation, and DALIS formation, which is further supported by the observation that DALIS contain ubiquitinated influenza nucleoprotein.