Aß induces its own prion protein N-terminal fragment (PrPN1)-mediated neutralization in amorphous aggregates.

Plasma membrane cellular prion protein (PrP(C)) is a high-affinity receptor for toxic soluble amyloid-ß (Aß) oligomers that mediates synaptic dysfunction. Secreted forms of PrP(C) resulting from PrP(C) α-cleavage (PrPN1) or shedding (shed PrP(C)) display neuroprotective activity in neuronal cultures and in mouse models of Aß-induced neuronal dysfunction. In vitro, recombinant PrPN1 and PrP inhibit Aß fibrillization. However, the mechanism by which PrPN1 and shed PrP(C) neutralize Aß oligomers is unclear, and evidence of such neuroprotective activity in Alzheimer's disease (AD) patients is lacking. Here, we show that PrPN1 association with Aß causes a conformational change resulting in the formation of amorphous and insoluble aggregates that are not compatible with the assembly of Aßs. Using postmortem brain tissues of AD patients, we were able to coimmunoprecipitate Aß with PrP(C) molecules and observed a coaggregation of Aß and PrPN1 in the guanidine-extractable fraction presumably representing insoluble amyloid plaques. Furthermore, PrP(C) α-cleavage is increased in AD brains, and we noticed a significant positive correlation between the levels of α-cleavage and of guanidine-extractable Aß. These data strongly support the hypothesis that PrP(C) α-cleavage is an endogenous neuroprotective mechanism in AD and support the development of PrP(C)-derived peptides as therapeutic molecules for AD.

Taking advantage of physiological proteolytic processing of the prion protein for a therapeutic perspective in prion and Alzheimer diseases.

Prion and Alzheimer diseases are fatal neurodegenerative diseases caused by misfolding and aggregation of the cellular prion protein (PrP(C)) and the ß-amyloid peptide, respectively. Soluble oligomeric species rather than large aggregates are now believed to be neurotoxic. PrP(C) undergoes three proteolytic cleavages as part of its natural life cycle, α-cleavage, ß-cleavage, and ectodomain shedding. Recent evidences demonstrate that the resulting secreted PrP(C) molecules might represent natural inhibitors against soluble toxic species. In this mini-review, we summarize recent observations suggesting the potential benefit of using PrP(C)-derived molecules as therapeutic agents in prion and Alzheimer diseases.

Homodimerization as a molecular switch between low and high efficiency PrP C cell surface delivery and neuroprotective activity.

PrP (C) is associated with a variety of functions, and its ability to interact with a multitude of partners, including itself, may largely explain PrP multifunctionality and the lack of consensus on the genuine physiological function of the protein in vivo. In contrast, there is a consensus in the literature that alterations in PrP (C) trafficking and intracellular retention result in neuronal degeneration. In addition, a proteolytic modification in the late secretory pathway termed the α-cleavage induces the secretion of PrPN1, a PrP (C) -derived metabolite with fascinating neuroprotective activity against toxic oligomeric Aß molecules implicated in Alzheimer disease. Thus, studies focusing on understanding the regulation of PrP (C) trafficking to the cell surface and the modulation of α-cleavage are essential. The objective of this commentary is to highlight recent evidences that PrP (C) homodimerization stimulates trafficking of the protein to the cell surface and results in high levels of PrPN1 secretion. We also discuss a hypothetical model for these results and comment on future challenges and opportunities.

PrP(C) homodimerization stimulates the production of PrPC cleaved fragments PrPN1 and PrPC1.

An endoproteolytic cleavage termed α-cleavage between residues 111/112 is a characteristic feature of the cellular prion protein (PrP(C)). This cleavage generates a soluble N-terminal fragment (PrPN1) and a glycosylphosphatidylinositol-anchored C-terminal fragment (PrPC1). Independent studies demonstrate that modulating PrP(C) α-cleavage represents a potential therapeutic strategy in prion diseases. The regulation of PrP(C) α-cleavage is unclear. The only known domain that is essential for the α-cleavage to occur is a hydrophobic domain (HD). Importantly, the HD is also essential for the formation of PrP(C) homodimers. To explore the role of PrP(C) homodimerization on the α-cleavage, we used a well described inducible dimerization strategy whereby a chimeric PrP(C) composed of a modified FK506-binding protein (Fv) fused with PrP(C) and termed Fv-PrP is incubated in the presence of a dimerizer AP20187 ligand. We show that homodimerization leads to a considerable increase of PrP(C) α-cleavage in cultured cells and release of PrPN1 and PrPC1. Interestingly, enforced homodimerization increased PrP(C) levels at the plasma membrane, and preventing PrP(C) trafficking to the cell surface inhibited dimerization-induced α-cleavage. These observations were confirmed in primary hippocampal neurons from transgenic mice expressing Fv-PrP. The proteases responsible for the α-cleavage are still elusive, and in contrast to initial studies we confirm more recent investigations that neither ADAM10 nor ADAM17 are involved. Importantly, PrPN1 produced after PrP(C) homodimerization protects against toxic amyloid-ß (Aß) oligomers. Thus, our results show that PrP(C) homodimerization is an important regulator of PrP(C) α-cleavage and may represent a potential therapeutic avenue against Aß toxicity in Alzheimer's disease.

The prion protein unstructured N-terminal region is a broad-spectrum molecular sensor with diverse and contrasting potential functions.

The physiological function of the prion protein (PrP(C) ) and its conversion into its infectious form (PrP(Sc) ) are central issues to understanding the pathogenesis of prion diseases. The N-terminal moiety of PrP(C) (NH(2) -PrP(C) ) is an unstructured region with the characteristic of interacting with a broad range of partners. These interactions endow PrP(C) with multifunctional and sometimes contrasting capabilities, including neuroprotection and neurotoxicity. Recently, binding of ß-sheet rich conformers to NH(2) -PrP(C) demonstrated a probable neurotoxic function for PrP(C) in Alzheimer's disease. NH(2) -PrP(C) also enhances the propagation of prions in vivo and is the target of the most potent antiprion compounds. Another level of complexity is provided by endoproteolysis and release of most of NH(2) -PrP(C) into the extracellular space. Further studies will be necessary to understand how NH(2) -PrP(C) regulates the physiological function of PrP(C) and how it is involved in the corruption of its normal function in diseases.

Proteomics-based confirmation of protein expression and correction of annotation errors in the Brucella abortus genome.

BACKGROUND: Brucellosis is a major bacterial zoonosis affecting domestic livestock and wild mammals, as well as humans around the globe. While conducting proteomics studies to better understand Brucella abortus virulence, we consolidated the proteomic data collected and compared it to publically available genomic data. RESULTS: The proteomic data was compiled from several independent comparative studies of Brucella abortus that used either outer membrane blebs, cytosols, or whole bacteria grown in media, as well as intracellular bacteria recovered at different times following macrophage infection. We identified a total of 621 bacterial proteins that were differentially expressed in a condition-specific manner. For 305 of these proteins we provide the first experimental evidence of their expression. Using a custom-built protein sequence database, we uncovered 7 annotation errors. We provide experimental evidence of expression of 5 genes that were originally annotated as non-expressed pseudogenes, as well as start site annotation errors for 2 other genes. CONCLUSIONS: An essential element for ensuring correct functional studies is the correspondence between reported genome sequences and subsequent proteomics studies. In this study, we have used proteomics evidence to confirm expression of multiple proteins previously considered to be putative, as well as correct annotation errors in the genome of Brucella abortus strain 2308.

A novel PhoP-regulated locus encoding the cytolysin ClyA and the secreted invasin TaiA of Salmonella enterica serovar Typhi is involved in virulence.

Salmonella enterica serovar Typhi causes a human-restricted systemic infection called typhoid fever. We have identified a Typhi genomic region encoding two ORFs, STY1498 and STY1499, that are expressed during infection of human macrophages and organized in an operon. STY1498 corresponds to clyA, which encodes a pore-forming cytolysin, and STY1499 encodes a 27 kDa protein, without any attributed function, which we have named TaiA (Typhi-associated invasin A). In order to evaluate the roles of these genes in Typhi pathogenesis, isogenic Typhi strains harbouring a non-polar mutation of either clyA or taiA were constructed. In macrophages, taiA was involved in increasing phagocytosis, as taiA deletion reduced bacterial uptake, whereas clyA reduced or controlled bacterial growth, as clyA deletion enhanced Typhi survival within macrophages without affecting cytotoxicity. In epithelial cells, deletion of taiA had no effect on invasion, whereas deletion of clyA enhanced the Typhi invasion rate, and reduced cytotoxicity. Overexpression of taiA in Typhi or in Escherichia coli resulted in a higher invasion rate of epithelial cells. We have demonstrated that TaiA is secreted independently of both the Salmonella pathogenicity island (SPI)-1 and the SPI-2 type three secretion systems. We have shown that this operon is regulated by the virulence-associated regulator PhoP. Moreover, our results revealed that products of this operon might be involved in promoting the use of macrophages as a sheltered reservoir for Typhi and allowing long-term persistence inside the host.

Escherichia coli O157:H7 survives within human macrophages: global gene expression profile and involvement of the Shiga toxins.

Escherichia coli O157:H7 is an important food-borne pathogen that specifically binds to the follicle-associated epithelium in the intestine, which rapidly brings this bacterial pathogen in contact with underlying human macrophages. Very little information is available about the interaction between E. coli O157:H7 and human macrophages. We evaluated the uptake and survival of strain EDL933 during infection of human macrophages. Surprisingly, EDL933 survived and multiplied in human macrophages at 24 h postinfection. The global gene expression profile of this pathogen during macrophage infection was determined. Inside human macrophages, upregulation of E. coli O157:H7 genes carried on O islands (such as pagC, the genes for both of the Shiga toxins, and the two iron transport system operons fit and chu) was observed. Genes involved in acid resistance and in the SOS response were upregulated. However, genes of the locus of enterocyte effacement or genes involved in peroxide resistance were not differentially expressed. Many genes with putative or unknown functions were upregulated inside human macrophages and may be newly discovered virulence factors. As the Shiga toxin genes were upregulated in macrophages, survival and cytotoxicity assays were performed with isogenic Shiga toxin mutants. The initial uptake of Shiga toxins mutants was higher than that of the wild type; however, the survival rates were significantly lower at 24 h postinfection. Thus, Shiga toxins are implicated in the interaction between E. coli O157:H7 and human macrophages. Understanding the molecular mechanisms used by E. coli to survive within macrophages may help in the identification of targets for new therapeutic agents.

The presence of the tet gene from cloning vectors impairs Salmonella survival in macrophages.

Cloning, mutagenesis and complementation of virulence factors are key steps to understand the mechanisms of bacterial pathogenesis and cloning vectors are routinely utilized for these processes. We have investigated the effect of the presence of commonly used cloning vectors on the survival of the intracellular bacterial pathogen Salmonella during macrophage infection. We demonstrate that the presence of the pSC101 derived tetracycline resistance gene on plasmids causes a lower survival rate of Salmonella in macrophages. The decrease in survival caused by the presence of the tet gene was not due to a higher susceptibility to gentamicin, a growth defect, or to increased sensitivity to acid. Higher susceptibility to hydrogen peroxide was observed in vitro for strain containing plasmid with the tet gene when the strains were grown at high densities but not when they were grown at low densities. Our findings demonstrate that the use of the tet gene for mutation or complementation can have deleterious effects and should thus be carefully considered.