1H, 13C, and 15N NMR assignments of the Pyrococcus abyssi DNA polymerase II intein

Protein splicing is a precise post-translational process mediated by inteins. Inteins are intervening proteins that cleave themselves from a precursor protein while joining the flanking sequences. Here we report the ¹⁵N, ¹³C, and ¹H chemical shift assignments of the intein from DNA polymerase II of Pyrococcus abyssi (Pab PolII intein), which has been recombinantly overexpressed and isotopically labeled. The NMR assignments of Pab PolII intein are essential for solution structure determination and protein dynamics study.     

The TNF-alpha-238 polymorphism and cancer risk: a meta-analysis

Background and Objectives

Tumor necrosis factor-α (TNF-α) plays a very important role in the development and progress of cancer. Some TNF-α polymorphisms have been confirmed to increase cancer risks; however, the association between TNF-α-238 polymorphism and cancers remains controversial and ambiguous. The aim of this study is to explore a more precise estimation of its relationship with cancer using meta-analysis.

Methods

Electronic searches of several databases were conducted for all publications on the association between this variant and cancer through March 2011. Odds ratios (OR) with 95% confidence intervals (95% CI) were used to access the strength of this association in the random-effect model.

Results

Thirty four studies with 34,679 cancer patients and 41,186 healthy controls were included. This meta-analysis showed no significant association between TNF-α-238 polymorphism and cancers (AA+GA vs GG: OR = 1.09, 95%CI = 0.88–1.34). In Caucasian and Asian subgroups, OR values (95% CI) were 1.14 (0.91–1.43) and 0.97 (0.58–1.61), respectively. In the subgroups of cancer type, no significant association was detected. The sensitivity analysis further strengthened the validity of these negative associations. No publication bias was observed in this study.

Conclusions

No significant association was found between the TNF-α-238 polymorphism and the risk for cancer.     

Impaired thymic export and apoptosis contribute to regulatory T-cell defects in patients with chronic heart failure

Objective

Animal studies suggest that regulatory T (T_reg) cells play a beneficial role in ventricular remodeling and our previous data have demonstrated defects of T_reg cells in patients with chronic heart failure (CHF). However, the mechanisms behind T_reg-cell defects remained unknown. We here sought to elucidate the mechanism of T_reg-cell defects in CHF patients.

Methods and Results

We performed flow cytometry analysis and demonstrated reduced numbers of peripheral blood CD4⁺CD25⁺FOXP3⁺CD45RO⁻CD45RA⁺ naïve T_reg (nT_reg) cells and CD4⁺CD25⁺FOXP3⁺CD45RO⁺CD45RA⁻ memory T_reg (mT_reg) cells in CHF patients as compared with non-CHF controls. Moreover, the nT_reg/mT_reg ratio (p<0.01), CD4⁺CD25⁺FOXP3⁺CD45RO⁻ CD45RA⁺CD31⁺ recent thymic emigrant T_reg cell (RTE-T_reg) frequency (p<0.01), and T-cell receptor excision circle levels in T_reg cells (p<0.01) were lower in CHF patients than in non-CHF controls. Combined annexin-V and 7-AAD staining showed that peripheral T_reg cells from CHF patients exhibited increased spontaneous apoptosis and were more prone to interleukin (IL)-2 deprivation- and CD95 ligand-mediated apoptosis than those from non-CHF individuals. Furthermore, analyses by both flow cytometry and real-time polymerase chain reaction showed that T_reg-cell frequency in the mediastinal lymph nodes or Foxp3 expression in hearts of CHF patients was no higher than that of the non-CHF controls.

Conclusion

Our data suggested that the T_reg-cell defects of CHF patients were likely caused by decreased thymic output of nascent T_reg cells and increased susceptibility to apoptosis in the periphery.     

DomPep--a general method for predicting modular domain-mediated protein-protein interactions

Protein-protein interactions (PPIs) are frequently mediated by the binding of a modular domain in one protein to a short, linear peptide motif in its partner. The advent of proteomic methods such as peptide and protein arrays has led to the accumulation of a wealth of interaction data for modular interaction domains. Although several computational programs have been developed to predict modular domain-mediated PPI events, they are often restricted to a given domain type. We describe DomPep, a method that can potentially be used to predict PPIs mediated by any modular domains. DomPep combines proteomic data with sequence information to achieve high accuracy and high coverage in PPI prediction. Proteomic binding data were employed to determine a simple yet novel parameter Ligand-Binding Similarity which, in turn, is used to calibrate Domain Sequence Identity and Position-Weighted-Matrix distance, two parameters that are used in constructing prediction models. Moreover, DomPep can be used to predict PPIs for both domains with experimental binding data and those without. Using the PDZ and SH2 domain families as test cases, we show that DomPep can predict PPIs with accuracies superior to existing methods. To evaluate DomPep as a discovery tool, we deployed DomPep to identify interactions mediated by three human PDZ domains. Subsequent in-solution binding assays validated the high accuracy of DomPep in predicting authentic PPIs at the proteome scale. Because DomPep makes use of only interaction data and the primary sequence of a domain, it can be readily expanded to include other types of modular domains.     

Novel structural components of the ventral disc and lateral crest in Giardia intestinalis

Giardia intestinalis is a ubiquitous parasitic protist that is the causative agent of giardiasis, one of the most common protozoan diarrheal diseases in the world. Giardia trophozoites attach to the intestinal epithelium using a specialized and elaborate microtubule structure, the ventral disc. Surrounding the ventral disc is a less characterized putatively contractile structure, the lateral crest, which forms a continuous perimeter seal with the substrate. A better understanding of ventral disc and lateral crest structure, conformational dynamics, and biogenesis is critical for understanding the mechanism of giardial attachment to the host. To determine the components comprising the ventral disc and lateral crest, we used shotgun proteomics to identify proteins in a preparation of isolated ventral discs. Candidate disc-associated proteins, or DAPs, were GFP-tagged using a ligation-independent high-throughput cloning method. Based on disc localization, we identified eighteen novel DAPs, which more than doubles the number of known disc-associated proteins. Ten of the novel DAPs are associated with the lateral crest or outer edge of the disc, and are the first confirmed components of this structure. Using Fluorescence Recovery After Photobleaching (FRAP) with representative novel DAP::GFP strains we found that the newly identified DAPs tested did not recover after photobleaching and are therefore structural components of the ventral disc or lateral crest. Functional analyses of the novel DAPs will be central toward understanding the mechanism of ventral disc-mediated attachment and the mechanism of disc biogenesis during cell division. Since attachment of Giardia to the intestine via the ventral disc is essential for pathogenesis, it is possible that some proteins comprising the disc could be potential drug targets if their loss or disruption interfered with disc biogenesis or function, preventing attachment.