Novel peptide antagonists of adrenomedullin and calcitonin gene-related peptide receptors:identification, pharmacological characterization, and interactions with position 74 in receptor activity-modifying protein 1/3

Human adrenomedullin (AM) is a 52-amino acid peptide belonging to the calcitonin peptide family, which also includes calcitonin gene-related peptide (CGRP) and AM2. The two AM receptors, AM(1) and AM(2), are calcitonin receptor-like receptor (CL)/receptor activity-modifying protein (RAMP) (RAMP2 and RAMP3, respectively) heterodimers. CGRP receptors comprise CL/RAMP1. The only human AM receptor antagonist (AM(22-52)) is a truncated form of AM; it has low affinity and is only weakly selective for AM(1) over AM(2) receptors. To develop novel AM receptor antagonists, we explored the importance of different regions of AM in interactions with AM(1), AM(2), and CGRP receptors. AM(22-52) was the framework for generating further AM fragments (AM(26-52) and AM(30-52)), novel AM/alphaCGRP chimeras (C1-C5 and C9), and AM/AM(2) chimeras (C6-C8). cAMP assays were used to screen the antagonists at all receptors to determine their affinity and selectivity. Circular dichroism spectroscopy was used to investigate the secondary structures of AM and its related peptides. The data indicate that the structures of AM, AM2, and alphaCGRP differ from one another. Our chimeric approach enabled the identification of two nonselective high-affinity antagonists of AM(1), AM(2), and CGRP receptors (C2 and C6), one high-affinity antagonist of AM(2) receptors (C7), and a weak antagonist selective for the CGRP receptor (C5). By use of receptor mutagenesis, we also determined that the C-terminal nine amino acids of AM seem to be responsible for its interaction with Glu74 of RAMP3. We provide new information on the structure-activity relationship of AM, alphaCGRP, and AM2 and how AM interacts with CGRP and AM(2) receptors.

Identification of nesfatin-1 in human and murine adipose tissue:a novel depot-specific adipokine with increased levels in obesity

Nesfatin-1 is a recently identified anorexigenic peptide derived from its precursor protein, nonesterified fatty acid/nucleobindin 2 (NUCB2). Although the hypothalamus is pivotal for the maintenance of energy homeostasis, adipose tissue plays an important role in the integration of metabolic activity and energy balance by communicating with peripheral organs and the brain via adipokines. Currently no data exist on nesfatin-1 expression, regulation, and secretion in adipose tissue. We therefore investigated NUCB2/nesfatin-1 gene and protein expression in human and murine adipose tissue depots. Additionally, the effects of insulin, dexamethasone, and inflammatory cytokines and the impact of food deprivation and obesity on nesfatin-1 expression were studied by quantitative RT-PCR and Western blotting. We present data showing NUCB2 mRNA (P < 0.001), nesfatin-1 intracellular protein (P < 0.001), and secretion (P < 0.01) were significantly higher in sc adipose tissue compared with other depots. Also, nesfatin-1 protein expression was significantly increased in high-fat-fed mice (P < 0.01) and reduced under food deprivation (P < 0.01) compared with controls. Stimulation of sc adipose tissue explants with inflammatory cytokines (TNFa and IL-6), insulin, and dexamethasone resulted in a marked increase in intracellular nesfatin-1 levels. Furthermore, we present evidence that the secretion of nesfatin-1 into the culture media was dramatically increased during the differentiation of 3T3-L1 preadipocytes into adipocytes (P < 0.001) and after treatments with TNF-a, IL-6, insulin, and dexamethasone (P < 0.01). In addition, circulating nesfatin-1 levels were higher in high-fat-fed mice (P < 0.05) and showed positive correlation with body mass index in human. We report that nesfatin-1 is a novel depot specific adipokine preferentially produced by sc tissue, with obesity- and food deprivation-regulated expression.

The V-ATPase in Paramecium:functional specialization by multiple gene isoforms

The vacuolar H(+)-ATPase (V-ATPase), a multisubunit, adenosine triphosphate (ATP)-driven proton pump, is essential for numerous cellular processes in all eukaryotes investigated so far. While structure and catalytic mechanism are similar to the evolutionarily related F-type ATPases, the V-ATPase's main function is to establish an electrochemical proton potential across membranes using ATP hydrolysis. The holoenzyme is formed by two subcomplexes, the transmembraneous V(0) and the cytoplasmic V(1) complexes. Sequencing of the whole genome of the ciliate Paramecium tetraurelia enabled the identification of virtually all the genes encoding V-ATPase subunits in this organism and the studying of the localization of the enzyme and roles in membrane trafficking and osmoregulation. Surprisingly, the number of V-ATPase genes in this free-living protozoan is strikingly higher than in any other species previously studied. Especially abundant are V(0)-a-subunits with as many as 17 encoding genes. This abundance creates the possibility of forming a large number of different V-ATPase holoenzymes by combination and has functional consequences by differential targeting to various organelles.

Identification of candidate genes for dyslexia susceptibility on chromosome 18

Six independent studies have identified linkage to chromosome 18 for developmental dyslexia or general reading ability. Until now, no candidate genes have been identified to explain this linkage. Here, we set out to identify the gene(s) conferring susceptibility by a two stage strategy of linkage and association analysis. Methodology/Principal Findings: Linkage analysis: 264 UK families and 155 US families each containing at least one child diagnosed with dyslexia were genotyped with a dense set of microsatellite markers on chromosome 18. Association analysis: Using a discovery sample of 187 UK families, nearly 3000 SNPs were genotyped across the chromosome 18 dyslexia susceptibility candidate region. Following association analysis, the top ranking SNPs were then genotyped in the remaining samples. The linkage analysis revealed a broad signal that spans approximately 40 Mb from 18p11.2 to 18q12.2. Following the association analysis and subsequent replication attempts, we observed consistent association with the same SNPs in three genes; melanocortin 5 receptor (MC5R), dymeclin (DYM) and neural precursor cell expressed, developmentally down-regulated 4-like (NEDD4L). Conclusions: Along with already published biological evidence, MC5R, DYM and NEDD4L make attractive candidates for dyslexia susceptibility genes. However, further replication and functional studies are still required.

Identification, mutagenesis, and transcriptional analysis of the methanesulfonate transport operon of methylosulfonomonas methylovora

Recently identified genes located downstream (3') of the msmEF (transport encoding) gene cluster, msmGH, and located 5' of the structural genes for methanesulfonate monooxygenase (MSAMO) are described from Methylosulfonomonas methylovora. Sequence analysis of the derived polypeptide sequences encoded by these genes revealed a high degree of identity to ABC-type transporters. MsmE showed similarity to a putative periplasmic substrate binding protein, MsmF resembled an integral membraneassociated protein, and MsmG was a putative ATP-binding enzyme. MsmH was thought to be the cognate permease component of the sulfonate transport system. The close association of these putative transport genes to the MSAMO structural genes msmABCD suggested a role for these genes in transport of methanesulfonic acid (MSA) into M. methylovora. msmEFGH and msmABCD constituted two operons for the coordinated expression of MSAMO and the MSA transporter systems. Reverse-transcription-PCR analysis of msmABCD and msmEFGH revealed differential expression of these genes during growth on MSA and methanol. The msmEFGH operon was constitutively expressed, whereas MSA induced expression of msmABCD. A mutant defective in msmE had considerably slower growth rates than the wild type, thus supporting the proposed role of MsmE in the transport of MSA into M. methylovora.

Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing:identification of novel zinc finger proteins.

We have successfully linked protein library screening directly with the identification of active proteins, without the need for individual purification, display technologies or physical linkage between the protein and its encoding sequence. By using 'MAX' randomization we have rapidly constructed 60 overlapping gene libraries that encode zinc finger proteins, randomized variously at the three principal DNA-contacting residues. Expression and screening of the libraries against five possible target DNA sequences generated data points covering a potential 40,000 individual interactions. Comparative analysis of the resulting data enabled direct identification of active proteins. Accuracy of this library analysis methodology was confirmed by both in vitro and in vivo analyses of identified proteins to yield novel zinc finger proteins that bind to their target sequences with high affinity, as indicated by low nanomolar apparent dissociation constants.

EnDNA-Prot:identification of DNA-binding proteins by applying ensemble learning

DNA-binding proteins are crucial for various cellular processes, such as recognition of specific nucleotide, regulation of transcription, and regulation of gene expression. Developing an effective model for identifying DNA-binding proteins is an urgent research problem. Up to now, many methods have been proposed, but most of them focus on only one classifier and cannot make full use of the large number of negative samples to improve predicting performance. This study proposed a predictor called enDNA-Prot for DNA-binding protein identification by employing the ensemble learning technique. Experiential results showed that enDNA-Prot was comparable with DNA-Prot and outperformed DNAbinder and iDNA-Prot with performance improvement in the range of 3.97-9.52% in ACC and 0.08-0.19 in MCC. Furthermore, when the benchmark dataset was expanded with negative samples, the performance of enDNA-Prot outperformed the three existing methods by 2.83-16.63% in terms of ACC and 0.02-0.16 in terms of MCC. It indicated that enDNA-Prot is an effective method for DNA-binding protein identification and expanding training dataset with negative samples can improve its performance. For the convenience of the vast majority of experimental scientists, we developed a user-friendly web-server for enDNA-Prot which is freely accessible to the public. © 2014 Ruifeng Xu et al.