Role of STRAP in regulating GSK3β function and Notch3 stabilization

Glycogen synthase kinase 3β (GSK3β) can regulate a broad range of cellular processes in a variety of cell types and tissues through its ability to phosphorylate its substrates in a cell- and time-specific manner. Although it is known that Axin and presenilin help to recruit β-catenin/Smad3 and tau protein to GSK3β, respectively, it is not clear how many of the other GSK3β substrates are recruited to it. Here, we have established the binding of GSK3β with a novel scaffold protein, STRAP, through its WD40 domains. In a new finding, we have observed that STRAP, GSK3β and Axin form a ternary complex together. We show for the first time that intracellular fragment of Notch3 (ICN3) binds with GSK3β through the ankyrin repeat domain. This binding between STRAP and GSK3β is reduced by small-molecule inhibitors of GSK3β. Further studies revealed that STRAP also binds ICN3 through the ankyrin repeat region, and this binding is enhanced in a proteasomal inhibition-dependent manner. In vivo ubiquitination studies indicate that STRAP reduces ubiquitination of ICN3, suggesting a role of STRAP in stabilizing ICN3. This is supported by the fact that STRAP and Notch3 are co-upregulated and co-localized in 59% of non-small cell lung cancers, as observed in an immunohistochemical staining of tissue microarrays. These results provide a potential mechanism by which STRAP regulates GSK3β function and Notch3 stabilization and further support the oncogenic functions of STRAP.Key words: STRAP, GSK3β, Notch3, axin, lung cancer, ubiquitination     

Heterologous expression, purification and characterization of heterodimeric monellin

Monellin is an intensely sweet-tasting protein present in the berry of Dioscoreophyllum cumminsii. Naturally occurring monellin (double chain monellin) is a heterodimer of two subunits commonly referred to as chain A and chain B. Monellin is a good model system for structural and dynamic studies of proteins. Single chain monellin has been generated by covalently linking the two subunits of naturally occurring double chain monellin, and has been used extensively for folding and unfolding studies, as well as for protein aggregation studies. There are, however, relatively few reports on such studies with double chain monellin. The primary difficulty associated with studies using double chain monellin appears to be the lack of a standard purification method. Here, a simple method for the purification of double chain monellin is presented. The genes encoding the two chains of monellin were cloned into a modified pETDUET vector under separate T7 promoters. The expression vector containing the genes of the two chains was expressed in E. coli BL21 Star (DE3). The expressed protein was purified using two steps of chromatography, ion exchange chromatography and gel filtration chromatography. This expression system consistently produced 40 mg of pure double chain monellin per litre of E. coli culture, in the correctly folded native state. The purity of the protein was confirmed by mass spectrometry and SDS-PAGE analysis. The purified protein was characterized using different spectroscopic methods, and the spectra obtained were in good agreement with the published spectra of naturally occurring double chain monellin.     

RNA approaches the B‐form in stacked single strand dinucleotide contexts

Duplex RNA adopts an A‐form structure, while duplex DNA interconverts between the A‐ and B‐forms depending on the environment. The C2′‐endo sugar pucker seen in B‐form DNA can occur infrequently in ribose sugars as well, but RNA is not understood to assume B‐form conformations. Through analysis of over 45,000 stacked single strand dinucleotide (SSD) crystal structure conformations, this study demonstrates that RNA is capable of adopting a wide conformational range between the canonical A‐ and B‐forms at the localized SSD level, including many B‐form‐like conformations. It does so through C2′‐endo ribose conformations in one or both nucleotides, and B‐form‐like neighboring base stacking patterns. As chemical reactions on nucleic acids involve localized changes in chemical bonds, the understanding of how enzymes distinguish between DNA and RNA nucleotides is altered by the energetic accessibility of these rare B‐form‐like RNA SSD conformations. The existence of these conformations also has direct implications in parametrization of molecular mechanics energy functions used extensively to model nucleic acid behavior., 2016. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 65–82, 2016     

Proteome-Wide Profiling of the MCF10AT Breast Cancer Progression Model

Background

Mapping the expression changes during breast cancer development should facilitate basic and translational research that will eventually improve our understanding and clinical management of cancer. However, most studies in this area are challenged by genetic and environmental heterogeneities associated with cancer.

Methodology/Principal Findings

We conducted proteomics of the MCF10AT breast cancer model, which comprises of 4 isogenic xenograft-derived human cell lines that mimic different stages of breast cancer progression, using iTRAQ-based tandem mass spectrometry. Of more than 1200 proteins detected, 98 proteins representing at least 20 molecular function groups including kinases, proteases, adhesion, calcium binding and cytoskeletal proteins were found to display significant expression changes across the MCF10AT model. The number of proteins that showed different expression levels increased as disease progressed from AT1k pre-neoplastic cells to low grade CA1h cancer cells and high grade cancer cells. Bioinformatics revealed that MCF10AT model of breast cancer progression is associated with a major re-programming in metabolism, one of the first identified biochemical hallmarks of tumor cells (the “Warburg effect”). Aberrant expression of 3 novel breast cancer-associated proteins namely AK1, ATOX1 and HIST1H2BM were subsequently validated via immunoblotting of the MCF10AT model and immunohistochemistry of progressive clinical breast cancer lesions.

Conclusion/Significance

The information generated by this study should serve as a useful reference for future basic and translational cancer research. Dysregulation of ATOX1, AK1 and HIST1HB2M could be detected as early as the pre-neoplastic stage. The findings have implications on early detection and stratification of patients for adjuvant therapy.     

PSGL-1 derived from human neutrophils is a high-efficiency ligand for endothelium-expressed E-selectin under flow

P-selectin glycoprotein ligand-1 (PSGL-1) has been proposed as an important tethering ligand for E-selectin and is expressed at a modest level on human leukocytes. Sialyl Lewis x (sLe^x)-like glycans bind to E-selectin and are expressed at a relatively high level on circulating leukocytes. It is unclear whether PSGL-1 has unique biochemical attributes that contribute to its role as an E-selectin ligand. To probe this issue, we conjugated microspheres with either sLe^x or PSGL-1 purified from myeloid cells (neutrophils and HL-60) and compared their adhesion to endothelial expressed E-selectin under defined shear conditions. We found that both sLe^x and PSGL-1 microspheres adhere to 4 h of IL-1-activated human umbilical vein endothelial cells predominantly through E-selectin. Analysis of the adhesion revealed that the rate of initial tethering of the PSGL-1 microspheres to E-selectin was significantly greater than the rate of initial tethering of the sLe^x microspheres despite the fact that the sLe^x microspheres tested had higher ligand densities than the PSGL-1 microspheres. We also found that pretreatment of the PSGL-1 or sLe^x microspheres with HECA-452 had no significant effect on initial tethering to E-selectin. These results support the hypotheses that 1) PSGL-1 is a high-efficiency tethering ligand for E-selectin, 2) ligand biochemistry can significantly influence initial tethering to E-selectin, and 3) PSGL-1 tethering to E-selectin can occur via non-HECA-452 reactive epitopes. adhesion; leukocyte; inflammation     

A genomewide linkage study of 1,933 families affected by premature coronary artery disease: The British Heart Foundation (BHF) Family Heart Study

Coronary artery disease (CAD) and its most important complication, myocardial infarction (MI), are the leading cause of premature death in the Western world. CAD has a substantial genetic basis, especially when it occurs early. We investigated the genetic determinants of premature CAD by performing a genomewide linkage analysis of 4,175 affected subjects from 1,933 families recruited throughout the United Kingdom. Each family had at least two available siblings with CAD, with validated onset before age 66 years. Linkage analysis was performed using 416 microsatellite markers. We observed suggestive linkage, for both CAD and MI, to a region on chromosome 2. For CAD, a LOD score of 1.86 was observed at marker D2S2271, which, in an ordered subset analysis, increased to 2.70 in families (n=1,698) with a minimum age at diagnosis of 56 years or younger. For MI, an overlapping peak with a LOD score of 1.15 was observed at marker D2S2216, which increased to 2.1 in families (n=801) with a minimum age at diagnosis of 59 years or younger. Exclusion mapping showed that 100% of the autosomal genome could be excluded for locus-specific sibling relative risks of 1.5 and 1.6 for CAD and MI, respectively. The region identified on chromosome 2 overlaps linked regions observed in two other smaller genome scans for CAD. Together, these findings strongly suggest that there is a locus on chromosome 2 that influences coronary atherosclerosis risk. The exclusion of a common locus that increases risk of CAD to siblings by >50% has important implications for strategies for further defining the genetic basis of CAD.     

Linkage analysis using co-phenotypes in the BRIGHT study reveals novel potential susceptibility loci for hypertension

Identification of the genetic influences on human essential hypertension and other complex diseases has proved difficult, partly because of genetic heterogeneity. In many complex-trait resources, additional phenotypic data have been collected, allowing comorbid intermediary phenotypes to be used to characterize more genetically homogeneous subsets. The traditional approach to analyzing covariate-defined subsets has typically depended on researchers' previous expectations for definition of a comorbid subset and leads to smaller data sets, with a concomitant attrition in power. An alternative is to test for dependence between genetic sharing and covariates across the entire data set. This approach offers the advantage of exploiting the full data set and could be widely applied to complex-trait genome scans. However, existing maximum-likelihood methods can be prohibitively computationally expensive, especially since permutation is often required to determine significance. We developed a less computationally intensive score test and applied it to biometric and biochemical covariate data, from 2,044 sibling pairs with severe hypertension, collected by the British Genetics of Hypertension (BRIGHT) study. We found genomewide-significant evidence for linkage with hypertension and several related covariates. The strongest signals were with leaner-body-mass measures on chromosome 20q (maximum LOD = 4.24) and with parameters of renal function on chromosome 5p (maximum LOD = 3.71). After correction for the multiple traits and genetic locations studied, our global genomewide P value was .046. This is the first identity-by-descent regression analysis of hypertension to our knowledge, and it demonstrates the value of this approach for the incorporation of additional phenotypic information in genetic studies of complex traits.     

RETRACTED ARTICLE: Monoaminergic and Nitriergic Modulation for Antidepressant-like Activity of Naringin in Unstressed and Acute Immobilization-Induced Stressed Mice

Neurochemical Research -     

Kinetic studies of the folding of heterodimeric monellin: evidence for switching between alternative parallel pathways

Determining whether or not a protein uses multiple pathways to fold is an important goal in protein folding studies. When multiple pathways are present, defined by transition states that differ in their compactness and structure but not significantly in energy, they may manifest themselves by causing the dependence on denaturant concentration of the logarithm of the observed rate constant of folding to have an upward curvature. In this study, the folding mechanism of heterodimeric monellin [double-chain monellin (dcMN)] has been studied over a range of protein and guanidine hydrochloride (GdnHCl) concentrations, using the intrinsic tryptophan fluorescence of the protein as the probe for the folding reaction. Refolding is shown to occur in multiple kinetic phases. In the first stage of refolding, which is silent to any change in intrinsic fluorescence, the two chains of monellin bind to one another to form an encounter complex. Interrupted folding experiments show that the initial encounter complex folds to native dcMN via two folding routes. A productive folding intermediate population is identified on one route but not on both of these routes. Two intermediate subpopulations appear to form in a fast kinetic phase, and native dcMN forms in a slow kinetic phase. The chevron arms for both the fast and slow phases of refolding are shown to have upward curvatures, suggesting that at least two pathways each defined by a different intermediate are operational during these kinetic phases of structure formation. Refolding switches from one pathway to the other as the GdnHCl concentration is increased.