Dramatic Structural Changes Resulting from the Loss of a Crucial Hydrogen Bond in the Hinge Region Involved in C-Terminal Helix Swapping in SurE: A Survival Protein from Salmonella typhimurium

Domain swapping is an interesting feature of some oligomeric proteins in which each protomer of the oligomer provides an identical surface for exclusive interaction with a segment or domain belonging to another protomer. Here we report results of mutagenesis experiments on the structure of C-terminal helix swapped dimer of a stationary phase survival protein from Salmonella typhimurium (StSurE). Wild type StSurE is a dimer in which a large helical segment at the C-terminus and a tetramerization loop comprising two beta strands are swapped between the protomers. Key residues in StSurE that might promote C-terminal helix swapping were identified by sequence and structural comparisons. Three mutants in which the helix swapping is likely to be avoided were constructed and expressed in E. coli. Three-dimensional X-ray crystal structures of the mutants H234A and D230A/H234A could be determined at 2.1 angstrom and 2.35 angstrom resolutions, respectively. Contrary to expectations, helix swapping was mostly retained in both the mutants. The loss of the crucial D230 OD2- H234 NE2 hydrogen bond (2.89 angstrom in the wild type structure) in the hinge region was compensated by new inter and intra-chain interactions. However, the two fold molecular symmetry was lost and there were large conformational changes throughout the polypeptide. In spite of these changes, the dimeric structure and an approximate tetrameric organization were retained, probably due to the interactions involving the tetramerization loop. Mutants were mostly functionally inactive, highlighting the importance of precise inter-subunit interactions for the symmetry and function of StSurE.

Regional variations in the nonlinearity and anisotropy of bovine aortic elastin

Arterial walls have a regular and lamellar organization of elastin present as concentric fenestrated networks in the media. In contrast, elastin networks are longitudinally oriented in layers adjacent to the media. In a previous model exploring the biomechanics of arterial elastin, we had proposed a microstructurally motivated strain energy function modeled using orthotropic material symmetry. Using mechanical experiments, we showed that the neo-Hookean term had a dominant contribution to the overall form of the strain energy function. In contrast, invariants corresponding to the two fiber families had smaller contributions. To extend these investigations, we use biaxial force-controlled experiments to quantify regional variations in the anisotropy and nonlinearity of elastin isolated from bovine aortic tissues proximal and distal to the heart. Results from this study show that tissue nonlinearity significantly increases distal to the heart as compared to proximally located regions (). Distally located samples also have a trend for increased anisotropy (), with the circumferential direction stiffer than the longitudinal, as compared to an isotropic and relatively linear response for proximally located elastin samples. These results are consistent with the underlying tissue histology from proximally located samples that had higher optical density (), fiber thickness (), and trend for lower tortuosity () in elastin fibers as compared to the thinner and highly undulating elastin fibers isolated from distally located samples. Our studies suggest that it is important to consider elastin fiber orientations in investigations that use microstructure-based models to describe the contributions of elastin and collagen to arterial mechanics.

STAT-1 expression is regulated by IGFBP-3 in malignant glioma cells and is a strong predictor of poor survival in patients with glioblastoma Laboratory investigation

Object. Insulin-like growth factor binding proteins (IGEBPs) have been implicated in the pathogenesis of glioma. In a previous study the authors demonstrated that IGFBP-3 is a novel glioblastoma biomarker associated with poor survival. Since signal transducer and activator of transcription 1 (STAT-1) has been shown to be regulated by IGFBP-3 during chondrogenesis and is a prosurvival and radioresistant molecule in different tumors, the aim in the present study was to explore the functional significance of IGFBP-3 in malignant glioma cells, to determine if STAT-1 is indeed regulated by IGFBP-3, and to study the potential of STAT-1 as a biomarker in glioblastoma. Methods. The functional significance of IGFBP-3 was investigated using the short hairpin (sh)RNA gene knockdown approach on U251MG cells. STAT-1 regulation by IGFBP-3 was tested on U251MG and U87MG cells by shRNA gene knockdown and exogenous treatment with recombinant IGFBP-3 protein. Subsequently, the expression of STAT-1 was analyzed with real-time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) in glioblastoma and control brain tissues. Survival analyses were done on a uniformly treated prospective cohort of adults with newly diagnosed glioblastoma (136 patients) using Kaplan-Meier and Cox regression models. Results. IGFBP-3 knockdown significantly impaired proliferation, motility, migration, and invasive capacity of U251MG cells in vitro (p < 0.005). Exogenous overexpression of IGFBP-3 in U251MG and U87MG cells demonstrated STAT-1 regulation. The mean transcript levels (by real-time RT-PCR) and the mean labeling index of STAT-1 (by IHC) were significantly higher in glioblastoma than in control brain tissues (p = 0.0239 and p < 0.001, respectively). Multivariate survival analysis revealed that STAT-1 protein expression (HR 1.015, p = 0.033, 95% CI 1.001-1.029) along with patient age (HR 1.025, p = 0.005, 95% CI 1.008-1.042) were significant predictors of shorter survival in patients with glioblastoma. Conclusions. IGFBP-3 influences tumor cell proliferation, migration, and invasion and regulates STAT-1 expression in malignant glioma cells. STAT-1 is overexpressed in human glioblastoma tissues and emerges as a novel prognostic biomarker.

Mycobacterium bovis BCG promotes tumor cell survival from tumor necrosis factor-alpha-induced apoptosis

Background: Increased incidence of lung cancer among pulmonary tuberculosis patients suggests mycobacteria-induced tumorigenic response in the host. The alveolar epithelial cells, candidate cells that form lung adenocarcinoma, constitute a niche for mycobacterial replication and infection. We thus explored the possible mechanism of M. bovis Bacillus Calmette-Guerin (BCG)-assisted tumorigenicity in type II epithelial cells, human lung adenocarcinoma A549 and other cancer cells. Methods: Cancer cell lines originating from lung, colon, bladder, liver, breast, skin and cervix were treated with tumor necrosis factor (TNF)-alpha in presence or absence of BCG infection. p53, COP1 and sonic hedgehog (SHH) signaling markers were determined by immunoblotting and luciferase assays, and quantitative real time PCR was done for p53-responsive pro-apoptotic genes and SHH signaling markers. MTT assays and Annexin V staining were utilized to study apoptosis. Gain-and loss-of-function approaches were used to investigate the role for SHH and COP1 signaling during apoptosis. A549 xenografted mice were used to validate the contribution of BCG during TNF-alpha treatment. Results: Here, we show that BCG inhibits TNF-alpha-mediated apoptosis in A549 cells via downregulation of p53 expression. Substantiating this observation, BCG rescued A549 xenografts from TNF-alpha-mediated tumor clearance in nude mice. Furthermore, activation of SHH signaling by BCG induced the expression of an E3 ubiquitin ligase, COP1. SHH-driven COP1 targeted p53, thereby facilitating downregulation of p53-responsive pro-apoptotic genes and inhibition of apoptosis. Similar effects of BCG could be shown for HCT116, T24, MNT-1, HepG2 and HELA cells but not for HCT116 p53(-/-) and MDA-MB-231 cells. Conclusion: Our results not only highlight possible explanations for the coexistence of pulmonary tuberculosis and lung cancer but also address probable reasons for failure of BCG immunotherapy of cancers.

Lactoylglutathione lyase, a critical enzyme in methylglyoxal detoxification, contributes to survival of Salmonella in the nutrient rich environment

Glyoxalase I which is synonymously known as lactoylglutathione lyase is a critical enzyme in methylglyoxal (MG) detoxification. We assessed the STM3117 encoded lactoylglutathione lyase (Lgl) of Salmonella Typhimurium, which is known to function as a virulence factor, due in part to its ability to detoxify methylglyoxal. We found that STM3117 encoded Lgl isomerises the hemithioacetal adduct of MG and glutathione (GSH) into S-lactoylglutathione. Lgl was observed to be an outer membrane bound protein with maximum expression at the exponential growth phase. The deletion mutant of S. Typhimurium (lgl) exhibited a notable growth inhibition coupled with oxidative DNA damage and membrane disruptions, in accordance with the growth arrest phenomenon associated with typical glyoxalase I deletion. However, growth in glucose minimal medium did not result in any inhibition. Endogenous expression of recombinant Lgl in serovar Typhi led to an increased resistance and growth in presence of external MG. Being a metalloprotein, Lgl was found to get activated maximally by Co2+ ion followed by Ni2+, while Zn2+ did not activate the enzyme and this could be attributed to the geometry of the particular protein-metal complex attained in the catalytically active state. Our results offer an insight on the pivotal role of the virulence associated and horizontally acquired STM3117 gene in non-typhoidal serovars with direct correlation of its activity in lending survival advantage to Salmonella spp.