The Alliance for Cellular Signaling Plasmid Collection: A Flexible Resource for Protein Localization Studies and Signaling Pathway Analysis

Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway® entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines

Transgenic Expression of RecA of the Spirochetes Borrelia burgdorferi and Borrelia hermsii in Escherichia coli Revealed Differences in DNA Repair and Recombination Phenotypes

After unsuccessful attempts to recover a viable RecA-deficient mutant of the Lyme borreliosis agent Borrelia burgdorferi, we characterized the functional activities of RecA of B. burgdorferi, as well as RecA of the relapsing fever spirochete Borrelia hermsii and the free-living spirochete Leptospira biflexa, in a recA mutant of Escherichia coli. As a control, E. coli RecA was expressed from the same plasmid vector. DNA damage repair activity was assessed after exposure of the transgenic cells to UV light or the radiomimetic chemicals methyl methanesulfonate and mitomycin C. Recombination activity in the cells was assessed by using an assay for homologous recombination between repeats in the chromosome and by measuring the ability of the cells to foster lytic growth by red gam mutant bacteriophage λ. Overall, we found that transgenic cells with recA genes of B. burgdorferi, B. hermsii, and L. biflexa had approximately equivalent activities in promoting homologous recombination in the lacZ duplication assay, but cells with B. burgdorferi recA and, most notably, B. hermsii recA were significantly less capable than cells with L. biflexa recA or E. coli recA in responding to DNA damage or in facilitating plaque formation in the phage assay. The comparatively poor function of Borrelia recA in the latter set of assays may be the consequence of impaired coordination in the loading of the transgenic RecA by RecBCD and/or RecFOR in E. coli

Horizontally Acquired Genes for Purine Salvage in Borrelia spp. Causing Relapsing Fever

Unlike Borrelia burgdorferi, the relapsing fever agent Borrelia hermsii and the related Borrelia miyamotoi had purA and purB genes of the purine salvage pathway. These were located among the rRNA genes. Phylogenetic analysis indicated that these genes had a different evolutionary history than those of orthologs in other spirochetes

Differential Telomere Processing by Borrelia Telomere Resolvases In Vitro but Not In Vivo

Causative agents of Lyme disease and relapsing fever, including Borrelia burgdorferi and Borrelia hermsii, respectively, are unusual among bacteria in that they possess a segmented genome with linear DNA molecules terminated by hairpin ends, known as telomeres. During replication, these telomeres are processed by the essential telomere resolvase, ResT, in a unique biochemical reaction known as telomere resolution. In this study, we report the identification of the B. hermsii resT gene through cross-species hybridization. Sequence comparison of the B. hermsii protein with the B. burgdorferi orthologue revealed 67% identity, including all the regions currently known to be crucial for telomere resolution. In vitro studies, however, indicated that B. hermsii ResT was unable to process a replicated B. burgdorferi type 2 telomere substrate. In contrast, in vivo cross-species complementation in which the native resT gene of B. burgdorferi was replaced with B. hermsii resT had no discernible effect, even though B. burgdorferi strain B31 carries at least two type 2 telomere ends. The B. burgdorferi ResT protein was also able to process two telomere spacing mutants in vivo that were unresolvable in vitro. The unexpected differential telomere processing in vivo versus in vitro by the two telomere resolvases suggests the presence of one or more accessory factors in vivo that are normally involved in the reaction. Our current results are also expected to facilitate further studies into ResT structure and function, including possible interaction with other Borrelia proteins

Suppression of LPS-Induced TNF-α Production in Macrophages by cAMP Is Mediated by PKA-AKAP95-p105

The activation of macrophages through Toll-like receptor (TLR) pathways leads to the production of a broad array of cytokines and mediators that coordinate the immune response. The inflammatory potential of this response can be reduced by compounds, such as prostaglandin E_2, that induce the production of cyclic adenosine monophosphate (cAMP). Through experiments with cAMP analogs and multigene RNA interference (RNAi), we showed that key anti-inflammatory effects of cAMP were mediated specifically by cAMP-dependent protein kinase (PKA). Selective inhibitors of PKA anchoring, time-lapse microscopy, and RNAi screening suggested that differential mechanisms of PKA action existed. We showed a specific role for A kinase–anchoring protein 95 in suppressing the expression of the gene encoding tumor necrosis factor–, which involved phosphorylation of p105 (also known as Nfkb1) by PKA at a site adjacent to the region targeted by inhibitor of nuclear factor B kinases. These data suggest that crosstalk between the TLR4 and cAMP pathways in macrophages can be coordinated through PKA-dependent scaffolds that localize specific pools of the kinase to distinct substrates

SMG1 Identified as a Regulator of Parkinson’s Disease-Associated alpha-Synuclein through siRNA Screening

<div><p>Synucleinopathies are a broad class of neurodegenerative disorders characterized by the presence of intracellular protein aggregates containing α-synuclein protein. The aggregated α-synuclein protein is hyperphosphorylated on serine 129 (S129) compared to the unaggregated form of the protein. While the precise functional consequences of S129 hyperphosphorylation are still being clarified, numerous <i>in vitro</i> and <i>in vivo</i> studies suggest that S129 phosphorylation is an early event in α-synuclein dysfunction and aggregation. Identifying the kinases and phosphatases that regulate this critical phosphorylation event may ultimately prove beneficial by allowing pharmacological mitigation of synuclein dysfunction and toxicity in Parkinson’s disease and other synucleinopathies. We report here the development of a high-content, fluorescence-based assay to quantitate levels of total and S129 phosphorylated α-synuclein protein. We have applied this assay to conduct high-throughput loss-of-function screens with siRNA libraries targeting 711 known and predicted human kinases and 206 phosphatases. Specifically, knockdown of the phosphatidylinositol 3-kinase related kinase SMG1 resulted in significant increases in the expression of pS129 phosphorylated α-synuclein (p-syn). Moreover, SMG1 protein levels were significantly reduced in brain regions with high p-syn levels in both dementia with Lewy bodies (DLB) and Parkinson’s disease with dementia (PDD). These findings suggest that SMG1 may play an important role in increased α-synuclein pathology during the course of PDD, DLB, and possibly other synucleinopathies.</p></div

Kinases and Phosphatases that increase the ratio of p-syn/t-syn.

<p>Kinases and Phosphatases that increase the ratio of p-syn/t-syn.</p

Silencing of the stress granule protein TIA1 increases p-syn.

<p>Shown are results wherein SMG1 and TIA1 were knocked down either individually or in combination to assess effects on α-synuclein. Expression levels of p-syn and t-syn for each siRNA condition normalized to the non-silencing (NS) siRNA control are shown beneath each lane on the westerns. Results are representative of at least three independent replicates. Antibodies used were as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077711#pone-0077711-g003" target="_blank">Figure 3</a>, with anti-TIA1 antibody (α-TIA1) included. siRNAs used are indicated across the top of the westerns.</p