Large Scale Identification and Categorization of Protein Sequences Using Structured Logistic Regression

Background

Structured Logistic Regression (SLR) is a newly developed machine learning tool first proposed in the context of text categorization. Current availability of extensive protein sequence databases calls for an automated method to reliably classify sequences and SLR seems well-suited for this task. The classification of P-type ATPases, a large family of ATP-driven membrane pumps transporting essential cations, was selected as a test-case that would generate important biological information as well as provide a proof-of-concept for the application of SLR to a large scale bioinformatics problem.

Results

Using SLR, we have built classifiers to identify and automatically categorize P-type ATPases into one of 11 pre-defined classes. The SLR-classifiers are compared to a Hidden Markov Model approach and shown to be highly accurate and scalable. Representing the bulk of currently known sequences, we analysed 9.3 million sequences in the UniProtKB and attempted to classify a large number of P-type ATPases. To examine the distribution of pumps on organisms, we also applied SLR to 1,123 complete genomes from the Entrez genome database. Finally, we analysed the predicted membrane topology of the identified P-type ATPases.

Conclusions

Using the SLR-based classification tool we are able to run a large scale study of P-type ATPases. This study provides proof-of-concept for the application of SLR to a bioinformatics problem and the analysis of P-type ATPases pinpoints new and interesting targets for further biochemical characterization and structural analysis.     

A Coreceptor-Independent Transgenic Human TCR Mediates Anti-Tumor and Anti-Self Immunity in Mice

Recent advancements in T cell immunotherapy suggest that T cells engineered with high-affinity TCR can offer better tumor regression. However, whether a high-affinity TCR alone is sufficient to control tumor growth, or the T cell subset bearing the TCR is also important remains unclear. Using the human tyrosinase epitope-reactive, CD8-independent, high-affinity TCR isolated from MHC class I-restricted CD4(+) T cells obtained from tumor-infiltrating lymphocytes (TIL) of a metastatic melanoma patient, we developed a novel TCR transgenic mouse with a C57BL/6 background. This HLA-A2-restricted TCR was positively selected on both CD4(+) and CD8(+) single-positive cells. However, when the TCR transgenic mouse was developed with a HLA-A2 background, the transgenic TCR was primarily expressed by CD3(+)CD4(-)CD8(-) double-negative T cells. TIL 1383I TCR transgenic CD4(+), CD8(+), and CD4(-)CD8(-) T cells were functional and retained the ability to control tumor growth without the need for vaccination or cytokine support in vivo. Furthermore, the HLA-A2(+)/human tyrosinase TCR double-transgenic mice developed spontaneous hair depigmentation and had visual defects that progressed with age. Our data show that the expression of the high-affinity TIL 1383I TCR alone in CD3(+) T cells is sufficient to control the growth of murine and human melanoma, and the presence or absence of CD4 and CD8 coreceptors had little effect on its functional capacity.     

Lysophosphatidic acid stimulates thrombomodulin lectin-like domain shedding in human endothelial cells

Thrombomodulin (TM) is an anticoagulant glycoprotein highly expressed on endothelial cell surfaces. Increased levels of soluble TM in circulation have been widely accepted as an indicator of endothelial damage or dysfunction. Previous studies indicated that various proinflammatory factors stimulate TM shedding in various cell types such as smooth muscle cells and epithelial cells. Lysophosphatidic acid (LPA) is a bioactive lipid mediator present in biological fluids during endothelial damage or injury. In the present study, we first observed that LPA triggered TM shedding in human umbilical vein endothelial cells (HUVECs). By Cyflow analysis, we showed that the LPA-induced accessibility of antibodies to the endothelial growth factor (EGF)-like domain of TM is independent of matrix metalloproteinases (MMPs), while LPA-induced TM lectin-like domain shedding is MMP-dependent. Furthermore, a stable cell line expressing TM without its lectin-like domain exhibited a higher cell proliferation rate than a stable cell line expressing full-length TM. These results imply that LPA induces TM lectin-like domain shedding, which might contribute to the exposure of its EGF-like domain for EGF receptor (EGFR) binding, thereby stimulating subsequent cell proliferation. Based on our findings, we propose a novel mechanism for the exposure of TM EGF-like domain, which possibly mediates LPA-induced EGFR transactivation.     

IcsA surface presentation in Shigella flexneri requires the periplasmic chaperones DegP, Skp, and SurA

A Shigella flexneri degP mutant, which was defective for plaque formation in Henle cell monolayers, had a reduced amount of IcsA detectable on the bacterial surface with antibody. However, the mutant secreted IcsA to the outer membrane at wild-type levels. This suggests that IcsA adopts an altered conformation in the outer membrane of the degP mutant with reduced exposure on the cell surface. IcsA is, therefore, unlikely to be accessible to actin-nucleating proteins within the eukaryotic cell cytoplasm, which is required for bacterial movement within the host cell and cell-to-cell spread. The degP mutant was somewhat more sensitive to detergents, antibiotics, and the antimicrobial peptide magainin, indicating that the degP phenotype was not limited to IcsA surface presentation. The plaque defect of the degP mutant, which is independent of DegP protease activity, was suppressed by overexpression of the periplasmic chaperone Skp but not by SurA. S. flexneri skp and surA mutants failed to form plaques in Henle cell monolayers and were defective in cell surface presentation and polar localization of IcsA. Therefore, the three periplasmic folding factors DegP, Skp, and SurA were all required for IcsA localization and plaque formation by S. flexneri.     

Lysosomal ubiquitin and the demise of Mycobacterium tuberculosis

The antimicrobial activity of macrophages is mediated by both oxidative and non-oxidative mechanisms. Oxidative mechanisms include the action of reactive oxygen and nitrogen intermediates on bacteria. Non-oxidative mechanisms include the maturation of the phagosome into an acidified, hydrolytically active compartment as well as the action of antimicrobial peptides. Mycobacterium tuberculosis parasitizes the host macrophage by arresting the normal maturation of its phagosome and resides in a compartment that fails to fuse with lysosomes. When bacteria are unable to regulate phagosome maturation, such as in activated macrophages, they are delivered to lysosomal compartments, where they are killed. Recent data indicate that the antimycobacterial mechanism of the lysosome is due in part to the action of ubiquitin-derived peptides.     

Laser ablation inductively coupled plasma mass spectrometry imaging of metals in experimental and clinical Wilson's disease

Wilson's disease is an autosomal recessive disorder in which the liver does not properly release copper into bile, resulting in prominent copper accumulation in various tissues. Affected patients suffer from hepatic disorders and severe neurological defects. Experimental studies in mutant mice in which the copper‐transporting ATPase gene (Atp7b) is disrupted revealed a drastic, time‐dependent accumulation of hepatic copper that is accompanied by formation of regenerative nodes resembling cirrhosis. Therefore, these mice represent an excellent exploratory model for Wilson's disease. However, the precise time course in hepatic copper accumulation and its impact on other trace metals within the liver is yet poorly understood. We have recently established novel laser ablation inductively coupled plasma mass spectrometry protocols allowing quantitative metal imaging in human and murine liver tissue with high sensitivity, spatial resolution, specificity and quantification ability. By use of these techniques, we here aimed to comparatively analyse hepatic metal content in wild‐type and Atp7b deficient mice during ageing. We demonstrate that the age‐dependent accumulation of hepatic copper is strictly associated with a simultaneous increase in iron and zinc, while the intrahepatic concentration and distribution of other metals or metalloids is not affected. The same findings were obtained in well‐defined human liver samples that were obtained from patients suffering from Wilson's disease. We conclude that in Wilson's disease the imbalances of hepatic copper during ageing are closely correlated with alterations in intrahepatic iron and zinc content.     

Metabolic changes during carbon monoxide poisoning: An experimental study

Carbon monoxide (CO) is the leading cause of death by poisoning worldwide. The aim was to explore the effects of mild and severe poisoning on blood gas parameters and metabolites. Eleven pigs were exposed to CO intoxication and had blood collected before and during poisoning. Mild CO poisoning (carboxyhaemoglobin, COHb 35.2 ± 7.9%) was achieved at 32 ± 13 minutes, and severe poisoning (69.3 ± 10.2% COHb) at 64 ± 23 minutes from baseline (2.9 ± 0.5% COHb). Blood gas parameters and metabolites were measured on a blood gas analyser and nuclear magnetic resonance spectrometer, respectively. Unsupervised principal component, analysis of variance and Pearson's correlation tests were applied. A P-value ≤ .05 was considered statistically significant. Mild poisoning resulted in a 28.4% drop in oxyhaemoglobin (OHb) and 12-fold increase in COHb, while severe poisoning in a 65% drop in OHb and 24-fold increase in COHb. Among others, metabolites implicated in regulation of metabolic acidosis (lactate, P < .0001), energy balance (pyruvate, P < .0001; 3-hydroxybutyrc acid, P = .01), respiration (citrate, P = .007; succinate, P = .0003; fumarate, P < .0001), lipid metabolism (glycerol, P = .002; choline, P = .0002) and antioxidant-oxidant balance (glutathione, P = .03; hypoxanthine, P < .0001) were altered, especially during severe poisoning. Our study adds new insights into the deranged metabolism of CO poisoning and leads the way for further investigation.     

Synthesis and pharmacological characterization of novel fluorescent histamine H2-receptor ligands derived from aminopotentidine

In an effort to develop a non-radioactive alternative to the [3H]tiotidine and [125I]iodoaminopotentidine binding assays for the histamine H2-receptor (H2R), primary amines related to aminopotentidine were prepared and coupled with the succinimidyl esters of the bulky fluorescent dyes S0536 and BODIPY 650/665-X. The primary amines exhibited different degrees of antagonistic potency at the human and guinea pig H2R. Surprisingly, one compound (5) coupled to the cyanine dye S0536 acted as potent partial agonist/antagonist at the H2R (KB approximately 50 nM; EC50 approximately 100-150 nM). Compounds coupled to the BODIPY dye exhibited moderately high H2R-affinity, too. Thus, the H2R accommodates bulky fluorophores, probably through interaction with extracellular receptor domains. The compounds presented herein provide a starting point for the optimization of fluorescent H2R ligands with respect to affinity and fluorescence as valuable tools to analyze the molecular mechanisms of H2R activation.     

Effects of host plant environment and Ustilago maydis infection on the fungal endophyte community of maize (Zea mays)

The focus of many fungal endophyte studies has been how plants benefit from endophyte infection. Few studies have investigated the role of the host plant as an environment in shaping endophyte community diversity and composition. The effects that different attributes of the host plant, that is, host genetic variation, host variation in resistance to the fungal pathogen Ustilago maydis and U. maydis infection, have on the fungal endophyte communities in maize (Zea mays) was examined. The internal transcribed spacer (ITS) region of the rDNA was sequenced to identify fungi and the endophyte communities were compared in six maize lines that varied in their resistance to U. maydis. It was found that host genetic variation, as determined by maize line, had significant effects on species richness, while the interactions between line and U. maydis infection and line and field plot had significant effects on endophyte community composition. However, the effects of maize line were not dependent on whether lines were resistant or susceptible to U. maydis. Almost 3000 clones obtained from 58 plants were sequenced to characterize the maize endophyte community. These results suggest that the endophyte community is shaped by complex interactions and factors, such as inoculum pool and microclimate, may be important.