Probing ion channel functional architecture and domain recombination compatibility by massively parallel domain insertion profiling

Protein domains are the basic units of protein structure and function. Comparative analysis of genomes and proteomes showed that domain recombination is a main driver of multidomain protein functional diversification and some of the constraining genomic mechanisms are known. Much less is known about biophysical mechanisms that determine whether protein domains can be combined into viable protein folds. Here, we use massively parallel insertional mutagenesis to determine compatibility of over 300,000 domain recombination variants of the Inward Rectifier K+ channel Kir2.1 with channel surface expression. Our data suggest that genomic and biophysical mechanisms acted in concert to favor gain of large, structured domain at protein termini during ion channel evolution. We use machine learning to build a quantitative biophysical model of domain compatibility in Kir2.1 that allows us to derive rudimentary rules for designing domain insertion variants that fold and traffic to the cell surface. Positional Kir2.1 responses to motif insertion clusters into distinct groups that correspond to contiguous structural regions of the channel with distinct biophysical properties tuned towards providing either folding stability or gating transitions. This suggests that insertional profiling is a high-throughput method to annotate function of ion channel structural regions

NF-kappaB Mediated Transcriptional Repression of Acid Modifying Hormone Gastrin

Helicobacter pylori is a major pathogen associated with the development of gastroduodenal diseases. It has been reported that H. pylori induced pro-inflammatory cytokine IL1B is one of the various modulators of acid secretion in the gut. Earlier we reported that IL1B-activated NFkB down-regulates gastrin, the major hormonal regulator of acid secretion. In this study, the probable pathway by which IL1B induces NFkB and affects gastrin expression has been elucidated. IL1B-treated AGS cells showed nine-fold activation of MyD88 followed by phosphorylation of TAK1 within 15 min of IL1B treatment. Furthermore, it was observed that activated TAK1 significantly up-regulates the NFkB subunits p50 and p65. Ectopic expression of NFkB p65 in AGS cells resulted in about nine-fold transcriptional repression of gastrin both in the presence and absence of IL1B. The S536A mutant of NFkB p65 is significantly less effective in repressing gastrin. These observations show that a functional NFkB p65 is important for IL1B-mediated repression of gastrin. ChIP assays revealed the presence of HDAC1 and NFkB p65 along with NCoR on the gastrin promoter. Thus, the study provides mechanistic insight into the IL1B-mediated gastrin repression via NFk

Effects of cell source, mouse strain, and immunosuppressive treatment on production of virulent and attenuated murine cytomegalovirus.

Murine cytomegalovirus pools from various in vitro and in vivo sources were compared for virulence in suckling mice in an effort to identify the conditions which were necessary for the production of virulent and attenuated viruses. Virus passaged in tracheal ring and salivary gland organ cultures, where virus is produced primarily by epithelial cells, was even more attenuated than virus passaged in mouse embryo fibroblasts. The attenuation observed after passage in all three of these in vitro systems did not appear to be due to defective interfering particles. We also found that virus produced in vivo in salivary glands became attenuated with time after infection. Virus harvested from salivary glands 5 to 6 weeks after infection was highly attenuated compared with both salivary gland-passaged virus harvested 2 to 3 weeks after infection and tissue culture-passaged virus. The attenuation of salivary gland-passaged virus with time was reversed when animals were treated with cyclophosphamide before the virus was harvested. A comparison of virus pools harvested from susceptible and resistant mouse strains indicated that the mouse strain had little effect on the virulence of the virus produced. When the various sources of virus tested in this study were ranked in terms of the virulence of the virus produced, salivary glands in intact mice either 2 to 3 weeks after infection or after cyclophosphamide treatment produced the most virulent virus, followed by mouse embryo fibroblast cultures, tracheal ring and salivary gland organ cultures, and, finally, salivary glands in intact mice 5 to 6 weeks after infection

Human cytomegalovirus uses two distinct pathways to enter retinal pigmented epithelial cells

Human cytomegalovirus infects multiple cell types, including fibroblasts and epithelial cells. It penetrates fibroblasts by fusion at the cell surface but is endocytosed into epithelial cells. In this report, we demonstrate by electron microscopy that the virus uses two different routes to enter retinal pigmented epithelial cells, depending on the cell type in which the infecting virus was produced. Virus produced in epithelial cells preferentially fuses with the plasma membrane, whereas fibroblast-derived virus mostly enters by receptor-mediated endocytosis. Treatment of epithelial cells with agents that block endosome acidification inhibited infection by virus produced in fibroblasts but had only a modest effect on infection by virus from epithelial cells. Epithelial cell-generated virions had higher intrinsic “fusion-from-without” activity than fibroblast-generated particles, and the two virus preparations triggered different cellular signaling responses, as evidenced by markedly different transcriptional profiles. We propose that the cell type in which a human cytomegalovirus particle is produced likely influences its subsequent spread and its contribution to pathogenesis

Oral Immunization with Recombinant Streptococcus gordonii Expressing Porphyromonas gingivalis FimA Domains

Porphyromonas gingivalis, a gram-negative anaerobe, is implicated in the etiology of adult periodontitis. P. gingivalis fimbriae are one of several critical surface virulence factors involved in both bacterial adherence and inflammation. P. gingivalis fimbrillin (FimA), the major subunit protein of fimbriae, is considered an important antigen for vaccine development against P. gingivalis-associated periodontitis. We have previously shown that biologically active domains of P. gingivalis fimbrillin can be expressed on the surface of the human commensal bacterium Streptococcus gordonii. In this study, we examined the effects of oral coimmunization of germfree rats with two S. gordonii recombinants expressing N (residues 55 to 145)- and C (residues 226 to 337)-terminal epitopes of P. gingivalis FimA to elicit FimA-specific immune responses. The effectiveness of immunization in protecting against alveolar bone loss following P. gingivalis infection was also evaluated. The results of this study show that the oral delivery of P. gingivalis FimA epitopes via S. gordonii vectors resulted in the induction of FimA-specific serum (immunoglobulin G [IgG] and IgA) and salivary (IgA) antibody responses and that the immune responses were protective against subsequent P. gingivalis-induced alveolar bone loss. These results support the potential usefulness of the S. gordonii vectors expressing P. gingivalis fimbrillin as a mucosal vaccine against adult periodontitis