Kríz Z

Analysis of the Nse3/MAGE-Binding Domain of the Nse4/EID Family Proteins

Background The Nse1, Nse3 and Nse4 proteins form a tight sub-complex of the large SMC5-6 protein complex. hNSE3/MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and the Nse4 kleisin subunit is related to the EID (E1A-like inhibitor of differentiation) family of proteins. We have recently shown that human MAGE proteins can interact with NSE4/EID proteins through their characteristic conserved hydrophobic pocket. Methodology/Principal Findings Using mutagenesis and protein-protein interaction analyses, we have identified a new Nse3/MAGE-binding domain (NMBD) of the Nse4/EID proteins. This short domain is located next to the Nse4 N-terminal kleisin motif and is conserved in all NSE4/EID proteins. The central amino acid residues of the human NSE4b/EID3 domain were essential for its binding to hNSE3/MAGEG1 in yeast two-hybrid assays suggesting they form the core of the binding domain. PEPSCAN ELISA measurements of the MAGEC2 binding affinity to EID2 mutant peptides showed that similar core residues contribute to the EID2-MAGEC2 interaction. In addition, the N-terminal extension of the EID2 binding domain took part in the EID2-MAGEC2 interaction. Finally, docking and molecular dynamic simulations enabled us to generate a structure model for EID2-MAGEC2. Combination of our experimental data and the structure modeling showed how the core helical region of the NSE4/EID domain binds into the conserved pocket characteristic of the MAGE protein family. Conclusions/Significance We have identified a new Nse4/EID conserved domain and characterized its binding to Nse3/MAGE proteins. The conservation and binding of the interacting surfaces suggest tight co-evolution of both Nse4/EID and Nse3/MAGE protein families.

In silico mutagenesis and docking studies of Pseudomonas aeruginosa PA-IIL lectin predicting binding modes and energies.

This article is focused on the application of two types of docking software, AutoDock and DOCK. It is aimed at studying the interactions of a calcium-dependent bacterial lectin PA-IIL (from Pseudomonas aeruginosa) and its in silico mutants with saccharide ligands. The effect of different partial charges assigned to the calcium ions was tested and evaluated in terms of the best agreement with the crystal structure. The results of DOCK were further optimized by molecular dynamics and rescored using AMBER. For both software, the agreement of the docked structures and the provided binding energies were evaluated in terms of prediction accuracy. This was carried out by comparing the computed results to the crystal structures and experimentally determined binding energies, respectively. The performance of both docking software applied on a studied problem was evaluated as well. The molecular docking methods proved efficient in identifying the correct binding modes in terms of geometry and partially also in predicting the preference changes caused by mutation. Obtaining a reasonable in silico method for the prediction of lectin-saccharide interactions may be possible in the future.

Pedal bypass for limb salvage

Abstract Background: Pedal bypass grafting is often the only method of limb salvage in patients with chronic critical lower limb ischemia due to atherosclerotic obliteration of the crural arteries, including patients with diabetic foot gangrene. It involves arterial reconstruction with distal anastomosis to one of the pedal arteries. Material and Methods: Between January 2000 and June 2004, 54 pedal bypasses were performed in 53 patients with chronic critical lower limb ischemia. Forty-seven (87%) patients had gangrene or ischemic ulcer, 36 (68%) had diabetes. In some of the patients (16.7%), previous percutaneous transluminal angioplasty (PTA) of the crural arteries had failed. Preoperative angiographic findings were unsatisfactory in the majority of the patients; the plantar arch was not visualized in 36 (66.7%) limbs. Results: In the period investigated (54 months) 11 grafts (20.4%) failed. Early thrombectomy resulting in long-term graft patency salvaged five limbs. One limb with graft occlusion occurring after foot ulcer healing was also salvaged. However, one amputation had to be performed despite a patent graft. The perioperative mortality rate was 3.8%. Cumulative primary and secondary graft patency rates and limb-salvage rates at 54 months were 76%, 78% and 81%, respectively. Conclusion: Pedal bypass grafting is a safe method with very good long-term outcomes. The absence of the pedal arteries or plantar arch on preoperative angiograms need not be taken as a contraindication to pedal vascular reconstruction. In discussions on the plantar arch it is recommended to discriminate between its actual absence and a mere “angiographic” absence.