Sun2 is a novel mammalian inner nuclear membrane protein

Sun protein (Sun1 and Sun2) cDNAs were previously cloned based on the homology of their C-terminal regions (SUN (Sad1 and UNC) domain) with the Caenorhabditis elegans protein UNC-84 whose mutation disrupts nuclear migration/positioning. In this study, we raised an anti-Sun2 serum and identified Sun2 in mammalian cells. In HeLa cells, Sun2 displays a nuclear rim-like pattern typical for a nuclear envelope protein. The Sun2 antibody signal co-localizes with nuclear pore and INM markers signals. The rim-like pattern was also observed with the recombinant full-length Sun2 protein fused to either EGFP or V5 epitopes. In addition, we found that a recombinant truncated form of Sun2, extending from amino acids 26 to 339, is sufficient to specify the nuclear envelope localization. Biochemical analyses show that Sun2 is an 85-kDa protein that is partially insoluble in detergent with high salt concentration and in chaotropic agents. Furthermore, Sun2 is enriched in purified HeLa cell nuclei. Electron microscopy analysis shows that Sun2 localizes in the nuclear envelope with a sub-population present in small clusters. Additionally, we show that the SUN domain of Sun2 is localized to the periplasmic space between the inner and the outer nuclear membranes. From our data, we conclude that Sun2 is a new mammalian inner nuclear membrane protein. Because the SUN domain is conserved from fission yeast to mammals, we suggest that Sun2 belongs to a new class of nuclear envelope proteins with potential relevance to nuclear membrane function in the context of the involvement of its components in an increasing spectrum of human diseases.     

A stable aspirin-triggered lipoxin A4 analog blocks phosphorylation of leukocyte-specific protein 1 in human neutrophils

Lipoxins and their aspirin-triggered 15-epimers are endogenous anti-inflammatory agents that block neutrophil chemotaxis in vitro and inhibit neutrophil influx in several models of acute inflammation. In this study, we examined the effects of 15-epi-16-(p-fluoro)-phenoxy-lipoxin A(4) methyl ester, an aspirin-triggered lipoxin A(4)-stable analog (ATLa), on the protein phosphorylation pattern of human neutrophils. Neutrophils stimulated with the chemoattractant fMLP were found to exhibit intense phosphorylation of a 55-kDa protein that was blocked by ATLa (10-50 nM). This 55-kDa protein was identified as leukocyte-specific protein 1, a downstream component of the p38-MAPK cascade in neutrophils, by mass spectrometry, Western blotting, and immunoprecipitation experiments. ATLa (50 nM) also reduced phosphorylation/activation of several components of the p38-MAPK pathway in these cells (MAPK kinase 3/MAPK kinase 6, p38-MAPK, MAPK-activated protein kinase-2). These results indicate that ATLa exerts its anti-inflammatory effects, at least in part, by blocking activation of the p38-MAPK cascade in neutrophils, which is known to promote chemotaxis and other proinflammatory responses by these cells.     

High-throughput identification of refolding conditions for LXRbeta without a functional assay

The expression of human genes in bacteria is often one of the most efficient systems for generating proteins for drug discovery efforts. However, expression of mammalian cDNAs in Escherichia coli often results in the production of protein that is insoluble and misfolded and thus requires the development of a successful refolding procedure to generate active protein. To accelerate the process of developing protein refolding protocols, we have developed a semi-automated screening and assay system that utilizes an incomplete factorial approach to sample a large "space" of refolding conditions based on parameters known to influence protein stability and solubility. Testing of these conditions is performed readily in a 96-well plate format with minimal sample manipulation. The folded protein is resolved and detected using an HPLC equipped with a mini-column and a highly sensitive fluorescence detector. This simple method requires only a small amount of protein for the entire screen (<1 mg), and most importantly, a functional assay is not required to assess the refolding yields. Here, we validate the utility of this screening system using two model proteins, IL13 and MMP13, and demonstrate its successful application to the refolding of our target protein, the ligand-binding domain of rat liver X receptor beta.     

Gel-free and gel-based proteomics in Bacillus subtilis: a comparative study

The proteome of exponentially growing Bacillus subtilis cells was dissected by the implementation of shotgun proteomics and a semigel-based approach for a particular exploration of membrane proteins. The current number of 745 protein identifications that was gained by the use of two-dimensional gel electrophoresis could be increased by 473 additional proteins. Therefore, almost 50% of the 2500 genes expressed in growing B. subtilis cells have been demonstrated at the protein level. In terms of exploring cellular physiology and adaptation to environmental changes or stress, proteins showing an alteration in expression level are of primary interest. The large number of vegetative proteins identified by gel-based and gel-free approaches is a good starting point for comparative physiological investigations. For this reason a gel-free quantitation with the recently introduced iTRAQ (isobaric tagging for relative and absolute quantitation) reagent technique was performed to investigate the heat shock response in B. subtilis. A comparison with gel-based data showed that both techniques revealed a similar level of up-regulation for proteins belonging to well studied heat hock regulons (SigB, HrcA, and CtsR). However, additional datasets have been obtained by the gel-free approach indicating a strong heat sensitivity of specific enzymes involved in amino acid synthesis.     

A Two-Pathway Analysis of Meiotic Crossing Over and Gene Conversion in Saccharomyces cerevisiae

Several apparently paradoxical observations regarding meiotic crossing over and gene conversion are readily resolved in a framework that recognizes the existence of two recombination pathways that differ in mismatch repair, structures of intermediates, crossover interference, and the generation of noncrossovers. One manifestation of these differences is that simultaneous gene conversion on both sides of a recombination-initiating DNA double-strand break (“two-sidedness”) characterizes only one of the two pathways and is promoted by mismatch repair. Data from previous work are analyzed quantitatively within this framework, and a molecular model for meiotic double-strand break repair based on the concept of sliding D-loops is offered as an efficient scheme for visualizing the salient results from studies of crossing over and gene conversion, the molecular structures of recombination intermediates, and the biochemical competencies of the proteins involved.EUKARYOTES transit from the diplophase to the haplophase via meiosis, which is associated with a number of interrelated processes, including crossing over and gene conversion. These processes involve meiosis-specific, programmed DNA double-strand breaks (DSBs) and their repair (DSBr). DSBr, in turn, is associated with mismatched base pairs and their rectification, referred to as “mismatch repair” or MMR (Bishop et al. 1987). Current efforts to accommodate both the genetic and molecular phenomena associated with meiotic DSBr in yeast (Saccharomyces cerevisiae) have been thoroughly reviewed (e.g., Hollingsworth and Brill 2004; Hoffmann and Borts 2004; Surtees et al. 2004; Hunter 2007; Berchowitz and Copenhaver 2010), but none of the reviews commits to an overall picture with quantitative predictions. This work aims to remedy that lack. Specifically, we have made use of salient published studies to develop, step-by-step, a comprehensive model of meiotic DSBr and MMR. The main features of this model are summarized in

Ddx42p--a human DEAD box protein with RNA chaperone activities

The human gene ddx42 encodes a human DEAD box protein highly homologous to the p68 subfamily of RNA helicases. In HeLa cells, two ddx42 poly(A)⁺ RNA species were detected both encoding the nuclear localized 938 amino acid Ddx42p polypeptide. Ddx42p has been heterologously expressed and its biochemical properties characterized. It is an RNA binding protein, and ATP and ADP modulate its RNA binding affinity. Ddx42p is an NTPase with a preference for ATP, the hydrolysis of which is enhanced by various RNA substrates. It acts as a non-processive RNA helicase. Interestingly, RNA unwinding by Ddx42p is promoted in the presence of a single-strand (ss) binding protein (T4gp32). Ddx42p, particularly in the ADP-bound form (the state after ATP hydrolysis), also mediates efficient annealing of complementary RNA strands thereby displacing the ss binding protein. Ddx42p therefore represents the first example of a human DEAD box protein possessing RNA helicase, protein displacement and RNA annealing activities. The adenosine nucleotide cofactor bound to Ddx42p apparently acts as a switch that controls the two opposing activities: ATP triggers RNA strand separation, whereas ADP triggers annealing of complementary RNA strands.     

The structure of the Lingo-1 ectodomain, a module implicated in central nervous system repair inhibition

Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200 A2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane.     

Food competition and social experience effects on V1a receptor binding in the forebrain of male Long-Evans hooded rats

The present study investigated the effect of social status in Long-Evans hooded rats established during food competition on V(1a) vasopressin receptor (V(1a)R) binding in the lateral septum (LS), medial preoptic area (MPOA), bed nucleus of the stria terminalis (BNST), anterior hypothalamus (AH), and central/basolateral amygdala (CeB). Serum concentration of testosterone (T) and corticosterone (CORT) was also measured. In Experiment 1, thirty-two lever-trained weight-matched rat pairs were placed in operant chambers where a single bar press provided access to milk reinforcement. A dominant-subordinate relationship, determined by the duration of drinking, was evident in 88% of the pairs. Sixteen rats were lever-trained but did not interact and served as no-treatment (NT) controls. In the LS, V(1a)R binding in the subordinate (SUB) group was significantly higher than in the dominant (DOM) group. V(1a)R binding was significantly higher in the LS, BNST, CeB, and AH in the NT group than in the other groups. The levels of CORT and T were not affected significantly by group membership. Experiment 2 investigated whether the binding effect in the LS was related to differences in fluid consumption. The results did not indicate a significant effect of fluid consumption. In the rat, V(1a)R binding in several forebrain areas seems to be affected by brief periods of social interactions, and, in the LS, it also appears to be related to dominance status.     

Impact of prehybridization PCR amplification on microarray detection of nitrifying bacteria in wastewater treatment plant samples

A gel-based microarray that included a set of 26 oligonucleotide probes targeting all nitrifying bacteria at varying levels of specificity suggested the presence of targeted microorganisms when hybridized to RNA isolated from a wastewater treatment plant, but could not discriminate between perfectly matched and mismatched sequences due in part to low signal intensity. To enhance sensitivity and improve discrimination, polymerase chain reaction was used to selectively amplify the 16S rRNA genes of specific nitrifier groups. RNA transcribed from these DNA templates was hybridized to the microarray and thermal dissociation analysis was used to characterize the specificity of hybridization. Amplification with Nitrospira-specific primers resulted in the selective amplification of this target group, confirmed by both a significant increase in signal intensity and a melting profile identical to the reference RNA. In contrast, Nitrobacter was not detected in the environmental samples with probe Nbac1000 despite pre-amplification with Nitrobacter-specific primers, indicating the absence of strains containing this Nitrobacter-specific sequence. Pre-amplification using primers specific for beta-Proteobacterial ammonia-oxidizing bacteria resulted in a significant increase in signal intensity for probe Nso190, but melting profiles for probe Nso190 showed a slight deviation between amplified RNA and the reference microorganism, suggesting that the amplification products contained some sequences that varied by a single nucleotide difference in the probe target region.