FE65 interaction with the ApoE receptor ApoEr2

The adaptor protein FE65 interacts with the beta-amyloid precursor protein (APP) via its C-terminal phosphotyrosine binding (PTB) domain and affects APP processing and Abeta production. Our previous data demonstrate that the apoE receptor ApoEr2 co-precipitated with APP and suggest that there are extracellular and intracellular interactions between these two transmembrane proteins. We hypothesized that FE65 acts as an intracellular link between ApoEr2 and APP. Co-immunoprecipitation experiments in COS7 cells demonstrated an interaction between ApoEr2 and FE65 that depended on the N-terminal PTB domain of FE65. Full-length FE65 increased co-immunoprecipitation of ApoEr2 and APP. Full-length FE65 also increased surface expression of ApoEr2, as determined by surface protein biotinylation and live cell surface staining. Constructs containing both the C- and N-terminal PTB domains of FE65 increased secreted APP, secreted ApoEr2, APP C-terminal fragment, and ApoEr2 C-terminal fragment, but constructs containing only single PTB domains did not affect APP or ApoEr2 processing. In addition, full-length FE65 decreased Abeta to a significantly greater extent than individual FE65 domains. These data suggest that FE65 can bind APP and ApoEr2 at the same time and affect the processing of each.     

The ColRS two-component system regulates membrane functions and protects Pseudomonas putida against phenol

As reported, the two-component system ColRS is involved in two completely different processes. It facilitates the root colonization ability of Pseudomonas fluorescens and is necessary for the Tn4652 transposition-dependent accumulation of phenol-utilizing mutants in Pseudomonas putida. To determine the role of the ColRS system in P. putida, we searched for target genes of response regulator ColR by use of a promoter library. Promoter screening was performed on phenol plates to mimic the conditions under which the effect of ColR on transposition was detected. The library screen revealed the porin-encoding gene oprQ and the alginate biosynthesis gene algD occurring under negative control of ColR. Binding of ColR to the promoter regions of oprQ and algD in vitro confirmed its direct involvement in regulation of these genes. Additionally, the porin-encoding gene ompA(PP0773) and the type I pilus gene csuB were also identified in the promoter screen. However, it turned out that ompA(PP0773) and csuB were actually affected by phenol and that the influence of ColR on these promoters was indirect. Namely, our results show that ColR is involved in phenol tolerance of P. putida. Phenol MIC measurement demonstrated that a colR mutant strain did not tolerate elevated phenol concentrations. Our data suggest that increased phenol susceptibility is also the reason for inhibition of transposition of Tn4652 in phenol-starving colR mutant bacteria. Thus, the current study revealed the role of the ColRS two-component system in regulation of membrane functionality, particularly in phenol tolerance of P. putida.     

The structure of the Lingo-1 ectodomain, a module implicated in central nervous system repair inhibition

Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200 A2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane.     

Regulation of growth differentiation factor 9 expression in oocytes in vivo: a key role of the E-box

Growth differentiation factor 9 (GDF9) is preferentially expressed in oocytes and is essential for female fertility. To identify regulatory elements that confer high-level expression of GDF9 in the ovary but repression in other tissues, we generated transgenic mice in which regions of the Gdf9 locus were fused to reporter genes. Two transgenes (-10.7/+5.6mGdf9-GFP) and (-3.3/+5.6mGdf9-GFP) that contained sequences either 10.7 or 3.3 kb upstream and 5.6 kb downstream of the Gdf9 initiation codon demonstrated expression specifically in oocytes, thereby mimicking endogenous Gdf9 expression. In contrast, transgenes -10.7mGdf9-Luc and -3.3mGdf9-Luc, which lacked the downstream 5.6-kb region, demonstrated reporter expression not only in oocytes but also high expression in male germ cells. This suggests that the downstream 5.6-kb sequence contains a testis-specific repressor element and that 3.3 kb of 5'-flanking sequence contains all the cis-acting elements for directing high expression of Gdf9 to female (and male) germ cells. To define sequences responsible for oocyte expression of Gdf9, we analyzed sequences of Gdf9 genes from 16 mammalian species. The approximately 400 proximal base pairs upstream of these Gdf9 genes are highly conserved and contain a perfectly conserved E-box (CAGCTG) sequence. When this 400-bp region was placed upstream of a luciferase reporter (-0.4mGdf9-Luc), oocyte-specific expression was observed. However, a similar transgene construct (-0.4MUT-mGdf9-Luc) with a mutation in the E-box abolished oocyte expression. Likewise, the presence of an E-box mutation in a longer construct (-3.3MUT-mGdf9-Luc) abolished expression in the ovary but not in the testis. These observations indicate that the E-box is a key regulatory sequence for Gdf9 expression in the ovary.     

Resonant waveguide grating biosensor for living cell sensing

This article presents theoretical analysis and experimental data for the use of resonant waveguide grating (RWG) biosensors to characterize stimulation-mediated cell responses including signaling. The biosensor is capable of detecting redistribution of cellular contents in both directions that are perpendicular and parallel to the sensor surface. This capability relies on online monitoring cell responses with multiple optical output parameters, including the changes in incident angle and the shape of the resonant peaks. Although the changes in peak shape are mainly contributed to stimulation-modulated inhomogeneous redistribution of cellular contents parallel to the sensor surface, the shift in incident angle primarily reflects the stimulation-triggered dynamic mass redistribution (DMR) perpendicular to the sensor surface. The optical signatures are obtained and used to characterize several cellular processes including cell adhesion and spreading, detachment and signaling by trypsinization, and signaling through either epidermal growth factor receptor or bradykinin B2 receptor. A mathematical model is developed to link the bradykinin-mediated DMR signals to the dynamic relocation of intracellular proteins and the receptor internalization during B2 receptor signaling cycle. This model takes the form of a set of nonlinear, ordinary differential equations that describe the changes in four different states of B2 receptors, diffusion of proteins and receptor-protein complexes, and the DMR responses. Classical analysis shows that the system converges to a unique optical signature, whose dynamics (amplitudes, transition time, and kinetics) is dependent on the bradykinin signal input, and consistent with those observed using the RWG biosensors. This study provides fundamentals for probing living cells with the RWG biosensors, in general, optical biosensors.     

Ubiquinone accumulates in the mitochondria of yeast mutated in the ubiquinone binding protein, Qcr8p

In Saccharomyces cerevisiae, the trans-membrane helix of Qcr8p, the ubiquinone binding protein of complex III, contributes to the Q binding site. In wild-type cells, residue 62 of the helix is non-polar (proline). Substitution of proline 62 with a polar, uncharged residue does not impair the ability of the cells to respire, complex III assembly is unaffected, ubiquinone occupancy of the Q binding site is unchanged, and mitochondrial ubiquinone levels are in the wild-type range. Substitution with a +1 charged residue is associated with partial respiratory competence, impaired complex III assembly, and loss of cytochrome b. Although ubiquinone occupancy of the Q binding site is similar to wild-type, total mitochondrial ubiquinone doubled in these mutants. Mutants with a +2 charged substitution at position 62 are unable to respire. These results suggest that the accumulation of ubiquinone in the mitochondria may be a compensatory mechanism for impaired electron transport at cytochrome b.     

The proteome of dissimilatory metal-reducing microorganism Geobacter sulfurreducens under various growth conditions

The proteome of Geobacter sulfurreducens, a model for the Geobacter species that predominate in many Fe(III)-reducing subsurface environments, was characterized with ultra high-pressure liquid chromatography and mass spectrometry using accurate mass and time (AMT) tags as well as with more traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Cells were grown under six different growth conditions in order to enhance the potential that a wide range of genes would be expressed. The AMT tag approach was able to identify a much greater number of proteins than could be detected with the 2-D PAGE approach. With the AMT approach over 3,000 gene products were identified, representing about 90% of the total predicted gene products in the genome. A high proportion of predicted proteins in most protein role categories were detected; the highest number of proteins was identified in the hypothetical protein role category. Furthermore, 91 c-type cytochromes of 111 predicted genes in the G. sulfurreducens genome were identified. Differences in the abundance of cytochromes and other proteins under different growth conditions provided information for future functional analysis of these proteins. These results demonstrate that a high percentage of the predicted proteins in the G. sulfurreducens genome are produced and that the AMT tag approach provides a rapid method for comparing differential expression of proteins under different growth conditions in this organism.     

Invariant chain transmembrane domain trimerization: a step in MHC class II assembly

The transmembrane (TM) domain of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) has long been implicated in both correct folding and function of the MHC class II complex. To function correctly, Ii must form a trimer, and the TM domain is one of the domains thought to stabilize the trimeric state. Specific mutations in the TM domain have been shown previously to disrupt MHC class II functions such as mature complex formation and antigen presentation, possibly due to disruption of Ii TM helix-helix interactions. Although this hypothesis has been reported several times in the literature, thus far no experimental measurements have been made to explore the relationship between TM domain structure and TM mutations that affect Ii function. We have applied biophysical and computational methods to study the folding and assembly of the Ii TM domain in isolation and find that the TM domain strongly self-associates. According to analytical ultracentrifugation analyses, the primary oligomeric state for this TM domain is a strongly associated trimer with a dissociation constant of approximately 120 nM in DPC micelles. We have also examined the effect of functionally important mutations of glutamine and threonine residues in the TM domain on its structure, providing results that now link the disruption of TM helix interactions to previously reported losses of Ii function.     

Detection and quantification of Legionella pneumophila in water samples using competitive PCR

The presence of high levels of Legionella pneumophila in man-made aquatic systems correlates with the incidence of nosocomial Legionnaires' disease. This requires a rapid, reliable, and sensitive quantification of L. pneumophila concentrations in suspected water systems. In this research, a homologous competitor was developed and evaluated in a L. pneumophila competitive polymerase chain reaction (cPCR) to quantify this human pathogen in a quick, cost-effective, and reliable way. Accuracy of cPCR was evaluated by analyzing cooling tower and tap water samples spiked with known concentrations of L. pneumophila bacteria, in parallel with the standard culture method. Legionella pneumophila amounts detected and calculated from cPCR and culture correlated very well: r = 0.998, P = 0.002 for tap water and r = 0.990, P = 0.009 for cooling tower water. Nevertheless, for both kinds of water samples, mean numbers of L. pneumophila calculated from cPCR results were always higher than those obtained by culture. This study makes it clear that the rapid, sensitive, and cost-effective L. pneumophila cPCR is a promising alternative to the standard time-consuming culture method and expensive real-time PCR to enumerate L. pneumophila bacteria in environmental water samples.