A conserved interdomain interaction is a determinant of folding cooperativity in the GST fold

The canonical glutathione transferase (GST) fold found in many monomeric and dimeric proteins consists of two domains that differ in structure and conformational dynamics. However, no evidence exists that the two domains unfold/fold independently at equilibrium, indicating the significance of interdomain interactions in governing cooperativity between domains. Bioinformatics analyses indicate the interdomain interface of the GST fold is large, predominantly hydrophobic with a high packing density explaining cooperative interdomain behavior. Structural alignments reveal a topologically conserved lock-and-key interaction across the domain interface in which a bulky hydrophobic residue ("key") protrudes from the surface of the N-domain and inserts into a pocket ("lock") in the C-domain. To better understand the molecular basis for the contribution of interdomain interactions toward cooperativity within the GST fold in the absence of any influence from quaternary interactions, studies were done with two monomeric GST proteins: Escherichia coli Grx2 (EcGrx2) and human CLIC1 (hCLIC1). Replacing the methionine "key" residue with alanine is structurally nondisruptive, whereas it significantly diminishes the folding cooperativity of both proteins. The loss in cooperativity between domains in the mutants is reflected by a change in the equilibrium folding mechanism from a wild-type two-state process to a three-state process, populating a stable folding intermediate.     

Cytoplasmic granule formation in mouse pancreatic acinar cells. Evidence for formation of immature granules (condensing vacuoles) by aggregation and fusion of progranules of unit size,and for reductions in membrane surface area and immature granule volume during granule maturation

We used a computer-assisted morphometry approach to analyze quantitatively the process of cytoplasmic granule formation in mouse pancreatic acinar cells stimulated with pilocarpine to induce secretion. Our findings suggest that each condensing vacuole/immature granule of pancreatic acinar cells is formed by the progressive aggregation of 106 to 128 unit progranules of narrowly fixed volume, define a range of 7.7 to 9.2 for the factor of volume condensation between the largest immature granules and the mature unit granule, and predict that the formation of a single mature unit granule by the aggregation and fusion of unit progranules involves a net reduction of at least 95% in the amount of membrane surface area associated with these structures.     

Calreticulin Induces Dilated Cardiomyopathy

Background

Calreticulin, a Ca²⁺-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart.

Methodology/Principal Findings

We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca²⁺ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca²⁺-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca²⁺-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin.

Conclusions/Significance

We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy in vivo leading to heart failure. This is due to an alternation in changes in a subset of Ca²⁺ handling genes, gap junction components and left ventricle remodeling.     

The gut anaerobe Faecalibacterium prausnitzii uses an extracellular electron shuttle to grow at oxic–anoxic interphases

Faecalibacterium prausnitzii is one of the most abundant bacteria in the human gut ecosystem and it is an important supplier of butyrate to the colonic epithelium. Low numbers of faecalibacteria have been associated with inflammatory bowel disease. Despite being extremely oxygen sensitive, F. prausnitzii is found adherent to the gut mucosa where oxygen diffuses from epithelial cells. This paradox is now explained on the basis of gas tube experiments, flavin-dependent reduction of 5,5′-dithiobis-2-nitrobenzoate and microbial fuel cell experiments. The results show that F. prausnitzii employs an extracellular electron shuttle of flavins and thiols to transfer electrons to oxygen. Both compounds are present in the healthy human gut. Our observations may have important implications for the treatment of patients with Crohn''s disease, for example, with flavin- or antioxidant rich diets, and they provide a novel key insight in host–microbe interactions at the gut barrier.     

BaySTDetect: detecting unusual temporal patterns in small area data via Bayesian model choice

Space-time modeling of small area data is often used in epidemiology for mapping chronic disease rates and by government statistical agencies for producing local estimates of, for example, unemployment or crime rates. Although there is typically a general temporal trend, which affects all areas similarly, abrupt changes may occur in a particular area, e.g. due to emergence of localized predictors/risk factor(s) or impact of a new policy. Detection of areas with "unusual" temporal patterns is therefore important as a screening tool for further investigations. In this paper, we propose BaySTDetect, a novel detection method for short-time series of small area data using Bayesian model choice between two competing space-time models. The first model is a multiplicative decomposition of the area effect and the temporal effect, assuming one common temporal pattern across the whole study region. The second model estimates the time trends independently for each area. For each area, the posterior probability of belonging to the common trend model is calculated, which is then used to classify the local time trend as unusual or not. Crucial to any detection method, we provide a Bayesian estimate of the false discovery rate (FDR). A comprehensive simulation study has demonstrated the consistent good performance of BaySTDetect in detecting various realistic departure patterns in addition to estimating well the FDR. The proposed method is applied retrospectively to mortality data on chronic obstructive pulmonary disease (COPD) in England and Wales between 1990 and 1997 (a) to test a hypothesis that a government policy increased the diagnosis of COPD and (b) to perform surveillance. While results showed no evidence supporting the hypothesis regarding the policy, an identified unusual district (Tower Hamlets in inner London) was later recognized to have higher than national rates of hospital readmission and mortality due to COPD by the National Health Service, which initiated various local enhanced services to tackle the problem. Our method would have led to an early detection of this local health issue.     

Reduced BMP Signaling Results in Hindlimb Fusion with Lethal Pelvic/Urogenital Organ Aplasia: A New Mouse Model of Sirenomelia

Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of phenotypes observed. Here, we provide new evidences that reduced BMP (Bone Morphogenetic Protein) signaling disrupts caudal body formation in mice and phenocopies sirenomelia. Bmp4 is strongly expressed in the developing caudal body structures including the peri-cloacal region and hindlimb field. In order to address the function of Bmp4 in caudal body formation, we utilized a conditional Bmp4 mouse allele (Bmp4^flox/flox) and the Isl1 (Islet1)-Cre mouse line. Isl1-Cre is expressed in the peri-cloacal region and the developing hindimb field. Isl1Cre;Bmp4^flox/flox conditional mutant mice displayed sirenomelia phenotypes including hindlimb fusion and pelvic/urogenital organ dysgenesis. Genetic lineage analyses indicate that Isl1-expressing cells contribute to both the aPCM (anterior Peri-Cloacal Mesenchyme) and the hindlimb bud. We show Bmp4 is essential for the aPCM formation independently with Shh signaling. Furthermore, we show Bmp4 is a major BMP ligand for caudal body formation as shown by compound genetic analyses of Bmp4 and Bmp7. Taken together, this study reveals coordinated development of caudal body structures including pelvic/urogenital organs and hindlimb orchestrated by BMP signaling in Isl1-expressing cells. Our study offers new insights into the pathogenesis of sirenomelia.     

Role of aromatic interactions in amyloid formation by peptides derived from human Amylin

Numerous polypeptides and proteins form amyloid deposits in vivo or in vitro. The mechanism of amyloid formation is not well-understood particularly in the case where unstructured polypeptides assemble to form amyloid. Aromatic-aromatic interactions are known to be important in globular proteins, and the possibility that they might play a key role in amyloid formation has been raised. The results of Ala-scanning experiments on short polypeptides derived from Amylin have suggested that aromatic interactions could be particularly important for this system. Here, we examine a set of Amylin-derived polypeptides in which the single aromatic residue has been substituted with a Leu and Ala. A peptide corresponding to residues 21-29 with a Phe-23 to Leu substitution, a free N terminus, and amidated C terminus readily forms amyloid. Shorter peptides derived from the putative minimal amyloid-forming segment of Amylin, residues 22-27, also form amyloid when Phe-23 is replaced by Leu. Amyloid formation is more facile when the N terminus is deprotonated and the peptide is uncharged. Substitution of the Phe with Ala results in a peptide that is noticeably less prone to form amyloid. A peptide corresponding to residues 10-19 of human Amylin with blocked termini and the sole aromatic residue, Phe-15, substituted by Leu readily forms amyloid. A Phe-15 to Ala substitution reduces significantly the ability to form amyloid. These results indicate that an aromatic residue is not required for amyloid formation in these systems and indicates that other factors such as size, beta-sheet propensity, and hydrophobicity of the side chain in question are also important.     

MicroCore: mapping genome expression to cell pathways and networks

The MicroCore toolkit is a suite of analysis programs for microarray and proteomics data that is open source and programmed exclusively in Java. MicroCore provides a flexible and extensible environment for the interpretation of functional genomics data through visualization. The first version of the application (downloadable from the MicroCore website: http://www.ucl.ac.uk/oncology/MicroCore/microcore.htm), implements two programs-PIMs (protein interaction maps) and MicroExpress-and is soon to be followed by an extended version which will also feature a fuzzy k-means clustering application and a Java-based R plug-in for microarray analysis. PIMs and MicroExpress provide a simple yet powerful way of graphically relating large quantities of expression data from multiple experiments to cellular pathways and biological processes in a statistically meaningful way.