Significant Expansion of Exon-bordering Protein Domains During Animal Proteome Evolution

Nucleic Acids Research. 2005  |  Pubmed ID: 15640447

We present evidence of remarkable genome-wide mobility and evolutionary expansion for a class of protein domains whose borders locate close to the borders of their encoding exons. These exon-bordering domains are more numerous and widely distributed in the human genome than other domains. They also co-occur with more diverse domains to form a larger variety of domain architectures in human proteins. A systematic comparison of nine animal genomes from nematodes to mammals revealed that exon-bordering domains expanded faster than other protein domains in both abundance and distribution, as well as the diversity of co-occurring domains and the domain architectures of harboring proteins. Furthermore, exon-bordering domains exhibited a particularly strong preference for class 1-1 intron phase. Our findings suggest that exon-bordering domains were amplified and interchanged within a genome more often and/or more successfully than other domains during evolution, probably the result of extensive exon shuffling and gene duplication events. The diverse biological functions of these domains underscore the important role they play in the expansion and diversification of animal proteomes.

Exon-domain Correlation and Its Corollaries

Bioinformatics (Oxford, England). Aug, 2005  |  Pubmed ID: 15905272

A Recombinant Cell-permeable P53 Fusion Protein is Selectively Stabilized Under Hypoxia and Inhibits Tumor Cell Growth

Cancer Letters. Jun, 2009  |  Pubmed ID: 19232460

More than 50% of human tumors contain a mutation in p53. Over 90% of tumors are solid tumors. Solid tumors have low oxygenated regions, called hypoxic regions where the tumor cells are more resistant to radio- and chemo-therapy than their well-oxygenated counterparts. In this study, we constructed a cell-permeable p53 fusion protein with selective stability in the hypoxic region. The fusion protein contained the TAT peptide for transduction across membranes, the oxygen-dependent degradation domain of hypoxia-inducible factor-1alpha and wild-type p53. This protein was effectively delivered into tumor cells where it exerted anticancer activity leading to the inhibition of cancer cell growth in vitro and the reduction of tumor weight in vivo. Hence, the fusion protein can be a novel protein drug for antitumor therapies, especially for hypoxic tumor cells.

Vitamin D Receptor Deletion Leads to Reduced Level of IkappaBalpha Protein Through Protein Translation, Protein-protein Interaction, and Post-translational Modification

The International Journal of Biochemistry & Cell Biology. Feb, 2010  |  Pubmed ID: 19931640

Vitamin D receptor plays an essential role in the regulation of inflammation. Previous studies demonstrate that vitamin D receptor negatively modulates the proinflammatory NF-kappaB pathway. However, it is unknown how vitamin D receptor regulates IkappaBalpha, the endogenous inhibitor of NF-kappaB. Here we investigated the molecular mechanism of vitamin D receptor deletion and IkappaBalpha expression. We found that cells lacking vitamin D receptor had significantly increased levels of IkappaBalpha mRNA and simultaneously decreased levels of IkappaBalpha protein. Lacking vitamin D receptor abolished its binding to the IkappaBalpha promoter. Moreover, the levels of protein translation regulators and the rate of protein synthesis were both decreased in cells lacking vitamin D receptor. At the post-translational level, IkappaBalpha ubiquitination was enhanced, indicating increased degradation of IkappaBalpha in the absence of vitamin D receptor. We further transfected cells with a plasmid carrying either wild-type or mutant IkappaBalpha. The expression of wild-type IkappaBalpha was much higher in the cells with vitamin D receptor than in the cells without vitamin D receptor, whereas the expression of exogenous IkappaBalpha was equally high in both cell lines. In summary, vitamin D receptor deletion affects IkappaBalpha through mRNA transcription, protein translation, protein-protein interaction, post-translational modification, and protein degradation, thus reducing the level of IkappaBalpha protein. Cells lacking vitamin D receptor are known in a proinflammatory state with activation of NF-kappaB. Our study provides new insight into vitamin D receptor regulation of an inhibitor of NF-kappaB in inflammation. Deletion of vitamin D receptor contributes to the activation of NF-kappaB on multiple levels.

Salmonella Regulation of Intestinal Stem Cells Through the Wnt/beta-catenin Pathway

FEBS Letters. Mar, 2010  |  Pubmed ID: 20083111

Recent studies have revealed that bacteria target stem cells for long-term survival in a Drosophila model. However, in mammalian models, little is known about bacterial infection and intestinal stem cells. Our study aims at understanding bacterial regulation of the intestinal stem cell in a Salmonella colitis mouse model. We found that Salmonella activates the Wnt/beta-catenin signaling pathway that is known to regulate stem cells. We identified Salmonella protein AvrA that modulates Wnt signaling including upregulating Wnt expression, modifying beta-catenin, increasing total beta-catenin expression, and activating Wnt/beta-catenin transcriptional activity in the intestinal epithelial cells. The numbers of stem cells and proliferative cells increased in the intestine infected with Salmonella expressing AvrA. Our study provides insights into bacterial infection and stem cell maintenance.

Chronic Effects of a Salmonella Type III Secretion Effector Protein AvrA in Vivo

PloS One. 2010  |  Pubmed ID: 20463922

Salmonella infection is a common public health problem that can become chronic and increase the risk of inflammatory bowel diseases and cancer. AvrA is a Salmonella bacterial type III secretion effector protein. Increasing evidence demonstrates that AvrA is a multi-functional enzyme with critical roles in inhibiting inflammation, regulating apoptosis, and enhancing proliferation. However, the chronic effects of Salmonella and effector AvrA in vivo are still unknown. Moreover, alive, mutated, non-invasive Salmonella is used as a vector to specifically target cancer cells. However, studies are lacking on chronic infection with non-pathogenic or mutated Salmonella in the host.

Salmonella Typhimurium Infection Increases P53 Acetylation in Intestinal Epithelial Cells

American Journal of Physiology. Gastrointestinal and Liver Physiology. May, 2010  |  Pubmed ID: 20224008

The ability of Salmonella typhimurium to enter intestinal epithelial cells constitutes a crucial step in pathogenesis. Salmonella invasion of the intestinal epithelium requires bacterial type three secretion system. Type three secretion system is a transport device that injects virulence proteins, called effectors, to paralyze or reprogram the eukaryotic cells. Avirulence factor for Salmonella (AvrA) is a Salmonella effector that inhibits the host's inflammatory responses. The mechanism by which AvrA modulates host cell signaling is not entirely clear. p53 is situated at the crossroads of a network of signaling pathways that are essential for genotoxic and nongenotoxic stress responses. We hypothesized that Salmonella infection activates the p53 pathway. We demonstrated that Salmonella infection increased p53 acetylation. Cells infected with AvrA-sufficient Salmonella have increased p53 acetylation, whereas cells infected with AvrA-deficient Salmonella have less p53 acetylation. In a cell-free system, AvrA possessed acetyltransferase activity and used p53 as a substrate. AvrA expression increased p53 transcriptional activity and induced cell cycle arrest. HCT116 p53-/- cells had less inflammatory responses. In a mouse model of Salmonella infection, intestinal epithelial p53 acetylation was increased by AvrA expression. Our studies provide novel mechanistic evidence that Salmonella modulates the p53 pathway during intestinal inflammation and infection.

Vitamin D Receptor Negatively Regulates Bacterial-stimulated NF-kappaB Activity in Intestine

The American Journal of Pathology. Aug, 2010  |  Pubmed ID: 20566739

Vitamin D receptor (VDR) plays an essential role in gastrointestinal inflammation. Most investigations have focused on the immune response; however, how bacteria regulate VDR and how VDR modulates the nuclear factor (NF)-kappaB pathway in intestinal epithelial cells remain unexplored. This study investigated the effects of VDR ablation on NF-kappaB activation in intestinal epithelia and the role of enteric bacteria on VDR expression. We found that VDR(-/-) mice exhibited a pro-inflammatory bias. After Salmonella infection, VDR(-/-) mice had increased bacterial burden and mortality. Serum interleukin-6 in noninfected VDR(+/+) mice was undetectable, but was easily detectable in VDR(-/-) mice. NF-kappaB p65 formed a complex with VDR in noninfected wild-type mouse intestine. In contrast, deletion of VDR abolished VDR/P65 binding. P65 nuclear translocation occurred in colonic epithelial cells of untreated VDR(-/-) mice. VDR deletion also elevated NF-kappaB activity in intestinal epithelia. VDR was localized to the surface epithelia of germ-free mice, but to crypt epithelial cells in conventionalized mice. VDR expression, distribution, transcriptional activity, and target genes were regulated by Salmonella stimulation, independent of 1,25-dihydroxyvitamin D3. Our study demonstrates that commensal and pathogenic bacteria directly regulate colonic epithelial VDR expression and location in vivo. VDR negatively regulates bacterial-induced intestinal NF-kappaB activation and attenuates response to infection. Therefore, VDR is an important contributor to intestinal homeostasis and host protection from bacterial invasion and infection.

Eukaryotic Signaling Pathways Targeted by Salmonella Effector Protein AvrA in Intestinal Infection in Vivo

BMC Microbiology. 2010  |  Pubmed ID: 21182782

The Salmonella AvrA gene is present in 80% of Salmonella enterica serovar strains. AvrA protein mimics the activities of some eukaryotic proteins and uses these activities to the pathogen's advantage by debilitating the target cells, such as intestinal epithelial cells. Therefore, it is important to understand how AvrA works in targeting eukaryotic signaling pathways in intestinal infection in vivo. In this study, we hypothesized that AvrA interacts with multiple stress pathways in eukaryotic cells to manipulate the host defense system. A whole genome approach combined with bioinformatics assays was used to investigate the in vivo genetic responses of the mouse colon to Salmonella with or without AvrA protein expression in the early stage (8 hours) and late stage (4 days). Specifically, we examined the gene expression profiles in mouse colon as it responded to pathogenic Salmonella stain SL1344 (with AvrA expression) or SB1117 (without AvrA expression).

Introduction of Hypoxia-targeting P53 Fusion Protein for the Selective Therapy of Non-small Cell Lung Cancer

Cancer Biology & Therapy. Jan, 2011  |  Pubmed ID: 21248471

Non-small cell lung cancer (NSCLC), which accounts for ~85% of lung cancer, is the major cause of malignancy mortality around the world. TP53 dysfunction and hypoxia are the typical biological features of the diverse solid tumors, including NSCLC. To develop an effective and low cytotoxic biological agent for targeted therapy, a p53 fusion protein, which was conjugated with the minimum motif of oxygen-dependent degradation domain (ODD) and the basic domain of TAT of HIV-1 named as TAT-ODD-p53, was evaluated for the treatment of NSCLC established by grafting H1299 cell line in which TP53 is homozygously deleted. We provide the evidence that this p53 fusion protein could significantly induce the cell-cycle arrest and/or apoptosis to inhibit H1299 cells' growth via p53-dependent pathways, including up-regulation of p21 expression and activation of pro-caspase-3, especially under hypoxia in vitro. The results in vivo indicated that this protein could selectively accumulate in the low oxygen tension areas of solid tumor tissues, inhibiting tumor growth via a similar mechanism to that in vitro. No obvious side effects were observed. Therefore, this recombinant p53 protein is likely to become a good candidate for targeted therapy of NSCLC.

Deletion of Vitamin D Receptor Leads to Premature Emphysema/COPD by Increased Matrix Metalloproteinases and Lymphoid Aggregates Formation

Biochemical and Biophysical Research Communications. Mar, 2011  |  Pubmed ID: 21300024

Deficiency of vitamin D is associated with accelerated decline in lung function. Vitamin D is a ligand for nuclear hormone vitamin D receptor (VDR), and upon binding it modulates various cellular functions. The level of VDR is reduced in lungs of patients with chronic obstructive pulmonary disease (COPD) which led us to hypothesize that deficiency of VDR leads to significant alterations in lung phenotype that are characteristics of COPD/emphysema associated with increased inflammatory response. We found that VDR knock-out (VDR(-/-)) mice had increased influx of inflammatory cells, phospho-acetylation of nuclear factor-kappaB (NF-κB) associated with increased proinflammatory mediators, and up-regulation of matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MMP-12 in the lung. This was associated with emphysema and decline in lung function associated with lymphoid aggregates formation compared to WT mice. These findings suggest that deficiency of VDR in mouse lung can lead to an early onset of emphysema/COPD because of chronic inflammation, immune dysregulation, and lung destruction.

Vitamin D, Vitamin D Receptor, and Macroautophagy in Inflammation and Infection

Discovery Medicine. Apr, 2011  |  Pubmed ID: 21524386

Vitamin D is involved in mineral and bone homeostasis, immune responses, anti-inflammation, anti-infection, and cancer prevention. Vitamin D receptor (VDR) is a nuclear receptor that mediates most biological functions of 1,25(OH)(2)D(3) or vitamin D(3), the active form of vitamin D. Recently, vitamin D(3)-induced autophagy has been reported. Autophagy is a lysosome-mediated catabolic pathway classified into three different types: macroautophagy, microautophagy, and chaperone-mediated autophagy. Autophagy contributes to anti-aging, antimicrobial defense, and tumor suppression. The functions of autophagy overlap remarkably with those of vitamin D/VDR signaling. This review focuses on vitamin D(3), VDR, and macroautophagy in inflammation and infection. We place emphasis on the regulatory roles of vitamin D(3) on autophagy at different steps, including induction, nucleation, elongation to maturation, and degradation. We summarize the known molecular mechanisms of vitamin D/VDR signaling on autophagy homeostasis. The potential application of the insights gleaned from these research findings to anti-inflammation and anti-infection is also discussed.

A Novel Assay for Evaluating Fragile X Locus Repeats

The Journal of Molecular Diagnostics : JMD. Nov, 2011  |  Pubmed ID: 21798368

We have developed a novel fragile X locus repeat assay that is a simple and high-throughput method that, with clinical validation, may be suitable for screening. It uses amplification of the FMR1 trinucleotide repeat region, followed by a hybridization assay to quantify the number of repeats in the amplicons. To our knowledge, this is the first repeat-counting assay that uses fluorescent signals rather than electrophoresis or mass spectrometry as the signaling mechanism. We also report the development of a simple microfluidic electrophoresis reflex test that uses the same amplicons and reduces the need for Southern blots to differentiate homozygous female normal samples from full mutations. The new assay, which is based on a suspension-array hybridization method, was tested on a series of male and female reference samples spanning the range from normal to full mutations. It was also tested on DNA from 1008 dried blood spot samples from pregnant women in their first trimester. The hybridization assay identified 51 of those as potentially expanded alleles of ≥45 repeats or as intermediate or higher in FMR1 repeat classification. Of these screen-positive samples, eight were confirmed by microfluidic electrophoresis as premutations consisting of ≥55 repeats. The FMR1 repeat assay is straightforward to run in high throughput, and the results are in the form of numerical ratios for ease of initial interpretation.

Wingless Homolog Wnt11 Suppresses Bacterial Invasion and Inflammation in Intestinal Epithelial Cells

American Journal of Physiology. Gastrointestinal and Liver Physiology. Dec, 2011  |  Pubmed ID: 21903761

Wnt11 plays an essential role in gastrointestinal epithelial proliferation, and previous investigations have focused on development and immune responses. However, the roles of how enteric bacteria regulate Wnt11 and how Wnt11 modulates the host response to pathogenic bacteria remain unexplored. This study investigated the effects of Salmonella infection on Wnt activation in intestinal epithelial cells. We found that Wnt11 mRNA and protein expression were elevated after Salmonella colonization. Wnt11 protein secretion in epithelial cells was also elevated after bacterial infection. Furthermore, we demonstrated that pathogenic Salmonella regulated Wnt11 expression and localization in vivo. We found a decrease in Salmonella invasion in cells with Wnt11 overexpression compared with cells with normal Wnt11 level. IL-8 mRNA in Wnt11-transfected cells was low; however, it was enhanced in cells with a low level of Wnt11 expression. Functionally, Wnt11 overexpression inhibited Salmonella-induced apoptosis. AvrA is a known bacterial effector protein that stabilizes β-catenin, the downstream regulator of Wnt signaling, and inhibits bacterially induced intestinal inflammation. We observed that Wnt11 expression, secretion, and transcriptional activity were regulated by Salmonella AvrA. Overall, Wnt11 is involved in the protection of the host intestinal cells by blocking the invasion of pathogenic bacteria, suppressing inflammation, and inhibiting apoptosis. Wnt11 is a novel and important contributor to intestinal homeostasis and host defense.

Wnt2 Inhibits Enteric Bacterial-induced Inflammation in Intestinal Epithelial Cells

Inflammatory Bowel Diseases. Mar, 2012  |  Pubmed ID: 21674728

Wnt signaling plays an essential role in gastrointestinal epithelial proliferation. Most investigations have focused on developmental and immune responses. Bacterial infection can be chronic and increases the risk of inflammatory bowel disease and colitis-associated cancer. However, we lack studies on how bacteria regulate Wnt proteins and how Wnts modulate the host responses to enteric bacteria. This study investigated the effects of Salmonella and Escherichia coli on Wnt2, one of the Wnt family members, in intestinal epithelia cells.