Signal sequence insufficiency contributes to neurodegeneration caused by transmembrane prion protein

Protein translocation into the endoplasmic reticulum is mediated by signal sequences that vary widely in primary structure. In vitro studies suggest that such signal sequence variations may correspond to subtly different functional properties. Whether comparable functional differences exist in vivo and are of sufficient magnitude to impact organism physiology is unknown. Here, we investigate this issue by analyzing in transgenic mice the impact of signal sequence efficiency for mammalian prion protein (PrP). We find that replacement of the average efficiency signal sequence of PrP with more efficient signals rescues mice from neurodegeneration caused by otherwise pathogenic PrP mutants in a downstream hydrophobic domain (HD). This effect is explained by the demonstration that efficient signal sequence function precludes generation of a cytosolically exposed, disease-causing transmembrane form of PrP mediated by the HD mutants. Thus, signal sequences are functionally nonequivalent in vivo, with intrinsic inefficiency of the native PrP signal being required for pathogenesis of a subset of disease-causing PrP mutations.     

Conserved tertiary base pairing ensures proper RNA folding and efficient assembly of the signal recognition particle Alu domain

Proper folding of the RNA is an essential step in the assembly of functional ribonucleoprotein complexes. We examined the role of conserved base pairs formed between two distant loops in the Alu portion of the mammalian signal recognition particle RNA (SRP RNA) in SRP assembly and functions. Mutations disrupting base pairing interfere with folding of the Alu portion of the SRP RNA as monitored by probing the RNA structure and the binding of the protein SRP9/14. Complementary mutations rescue the defect establishing a role of the tertiary loop–loop interaction in RNA folding. The same mutations in the Alu domain have no major effect on binding of proteins to the S domain suggesting that the S domain can fold independently. Once assembled into a complete SRP, even particles that contain mutant RNA are active in arresting nascent chain elongation and translocation into microsomes, and, therefore, tertiary base pairing does not appear to be essential for these activities. Our results suggest a model in which the loop–loop interaction and binding of the protein SRP9/14 play an important role in the early steps of SRP RNA folding and assembly.     

Electrical detection of human immunoglobulins G from human serum using a microbiosensor

A biosensor for the electrical detection of human antibodies from serum has been fabricated and experimentally demonstrated. The device is based on the immobilization of proteins used as probes between a set of microelectrodes. Incubation with diluted human serum was followed by incubation with anti-human secondary antibodies labeled with gold nanoparticles (GNPs) and then precipitation of silver on the nanoparticles. The output of the device was defined as the percentage of short-circuited microelectrodes after silver deposition independently of the gap conductance. Two model probes were studied: protein A and goat antibodies. The effects of the microgap spacing (5, 10, 15 or 20 microm) and the duration of the silver treatment were examined. The data obtained showed that a large spacing (20 microm) led to poor sensitivity. Alternately, 5 microm gaps led to high sensitivity and saturation of the signal. Interestingly, 10-15 microm gaps enabled a non-saturated and distinct signal for both probes that was correlated with the GNP density between the microgaps as determined by atomic force microscopy. Different capture efficiencies could be easily distinguished. The biosensor described here is easy to use and thus can be applied to real detection experiments.     

Occurrence, biosynthesis and functions of N-acylphosphatidylethanolamines (NAPE): not just precursors of N-acylethanolamines (NAE)

N-acylphosphatidylethanolamine (NAPE) is a minor phospholipid resulting from the transfer of an acyl chain from an acyl donor to the primary amine of the ethanolamine moiety of phosphatidylethanolamine (PE). Occurring in plant and animal kingdoms as well as in prokaryotic cells, it is synthesized in higher amounts in membranes during cellular stresses and tissue damage, and it is widely thought to be the precursor of the lipid mediator, N-acylethanolamine (NAE), which modulates the endocannabinoid signaling pathway and therefore regulates various physiological processes. However, recent studies have shown that NAPE is also a bioactive molecule that is involved in several physiological functions. The present paper reviews the occurrence of NAPE in animals and plants and focuses on the various properties of NAPE observed in vitro and in vivo. The different metabolic pathways promoting the synthesis and degradation of NAPE are also discussed and the differences between animals and plants are underlined.     

Identification of germline genomic copy number variation in familial pancreatic cancer

Adenocarcinoma of the pancreas is a significant cause of cancer mortality, and up to 10?% of cases appear to be familial. Heritable genomic copy number variants (CNVs) can modulate gene expression and predispose to disease. Here, we identify candidate predisposition genes for familial pancreatic cancer (FPC) by analyzing germline losses or gains present in one or more high-risk patients and absent in a large control group. A total of 120 FPC cases and 1,194 controls were genotyped on the Affymetrix 500K array, and 36 cases and 2,357 controls were genotyped on the Affymetrix 6.0 array. Detection of CNVs was performed by multiple computational algorithms and partially validated by quantitative PCR. We found no significant difference in the germline CNV profiles of cases and controls. A total of 93 non-redundant FPC-specific CNVs (53 losses and 40 gains) were identified in 50 cases, each CNV present in a single individual. FPC-specific CNVs overlapped the coding region of 88 RefSeq genes. Several of these genes have been reported to be differentially expressed and/or affected by copy number alterations in pancreatic adenocarcinoma. Further investigation in high-risk subjects may elucidate the role of one or more of these genes in genetic predisposition to pancreatic cancer.     

A Gene Transfer Agent and a Dynamic Repertoire of Secretion Systems Hold the Keys to the Explosive Radiation of the Emerging Pathogen Bartonella

Gene transfer agents (GTAs) randomly transfer short fragments of a bacterial genome. A novel putative GTA was recently discovered in the mouse-infecting bacterium Bartonella grahamii. Although GTAs are widespread in phylogenetically diverse bacteria, their role in evolution is largely unknown. Here, we present a comparative analysis of 16 Bartonella genomes ranging from 1.4 to 2.6 Mb in size, including six novel genomes from Bartonella isolated from a cow, two moose, two dogs, and a kangaroo. A phylogenetic tree inferred from 428 orthologous core genes indicates that the deadly human pathogen B. bacilliformis is related to the ruminant-adapted clade, rather than being the earliest diverging species in the genus as previously thought. A gene flux analysis identified 12 genes for a GTA and a phage-derived origin of replication as the most conserved innovations. These are located in a region of a few hundred kb that also contains 8 insertions of gene clusters for type III, IV, and V secretion systems, and genes for putatively secreted molecules such as cholera-like toxins. The phylogenies indicate a recent transfer of seven genes in the virB gene cluster for a type IV secretion system from a cat-adapted B. henselae to a dog-adapted B. vinsonii strain. We show that the B. henselae GTA is functional and can transfer genes in vitro. We suggest that the maintenance of the GTA is driven by selection to increase the likelihood of horizontal gene transfer and argue that this process is beneficial at the population level, by facilitating adaptive evolution of the host-adaptation systems and thereby expansion of the host range size. The process counters gene loss and forces all cells to contribute to the production of the GTA and the secreted molecules. The results advance our understanding of the role that GTAs play for the evolution of bacterial genomes.     

Infectious Chikungunya Virus in the Saliva of Mice,Monkeys and Humans

Chikungunya virus (CHIKV) is a reemerging, ordinarily mosquito-transmitted, alphavirus that occasionally produces hemorrhagic manifestations, such as nose bleed and bleeding gums, in human patients. Interferon response factor 3 and 7 deficient (IRF3/7^-/-) mice, which are deficient for interferon α/β responses, reliably develop hemorrhagic manifestations after CHIKV infection. Here we show that infectious virus was present in the oral cavity of CHIKV infected IRF3/7^-/- mice, likely due to hemorrhagic lesions in the olfactory epithelium that allow egress of infected blood into the nasal, and subsequently, oral cavities. In addition, IRF3/7^-/- mice were more susceptible to infection with CHIKV via intranasal and oral routes, with IRF3/7^-/- mice also able to transmit virus mouse-to-mouse without an arthropod vector. Cynomolgus macaques often show bleeding gums after CHIKV infection, and analysis of saliva from several infected monkeys also revealed the presence of viral RNA and infectious virus. Furthermore, saliva samples collected from several acute CHIKV patients with hemorrhagic manifestations were found to contain viral RNA and infectious virus. Oral fluids can therefore be infectious during acute CHIKV infections, likely due to hemorrhagic manifestations in the oral/nasal cavities.     

Natural Killer Cell Sensing of Infected Cells Compensates for MyD88 Deficiency but Not IFN-I Activity in Resistance to Mouse Cytomegalovirus

In mice, plasmacytoid dendritic cells (pDC) and natural killer (NK) cells both contribute to resistance to systemic infections with herpes viruses including mouse Cytomegalovirus (MCMV). pDCs are the major source of type I IFN (IFN-I) during MCMV infection. This response requires pDC-intrinsic MyD88-dependent signaling by Toll-Like Receptors 7 and 9. Provided that they express appropriate recognition receptors such as Ly49H, NK cells can directly sense and kill MCMV-infected cells. The loss of any one of these responses increases susceptibility to infection. However, the relative importance of these antiviral immune responses and how they are related remain unclear. In humans, while IFN-I responses are essential, MyD88 is dispensable for antiviral immunity. Hence, a higher redundancy has been proposed in the mechanisms promoting protective immune responses against systemic infections by herpes viruses during natural infections in humans. It has been assumed, but not proven, that mice fail to mount protective MyD88-independent IFN-I responses. In humans, the mechanism that compensates MyD88 deficiency has not been elucidated. To address these issues, we compared resistance to MCMV infection and immune responses between mouse strains deficient for MyD88, the IFN-I receptor and/or Ly49H. We show that selective depletion of pDC or genetic deficiencies for MyD88 or TLR9 drastically decreased production of IFN-I, but not the protective antiviral responses. Moreover, MyD88, but not IFN-I receptor, deficiency could largely be compensated by Ly49H-mediated antiviral NK cell responses. Thus, contrary to the current dogma but consistent with the situation in humans, we conclude that, in mice, in our experimental settings, MyD88 is redundant for IFN-I responses and overall defense against a systemic herpes virus infection. Moreover, we identified direct NK cell sensing of infected cells as one mechanism able to compensate for MyD88 deficiency in mice. Similar mechanisms likely contribute to protect MyD88- or IRAK4-deficient patients from viral infections.