Tertiary motifs as building blocks for the design of protein‐binding peptides

Despite advances in protein engineering, the de novo design of small proteins or peptides that bind to a desired target remains a difficult task. Most computational methods search for binder structures in a library of candidate scaffolds, which can lead to designs with poor target complementarity and low success rates. Instead of choosing from pre‐defined scaffolds, we propose that custom peptide structures can be constructed to complement a target surface. Our method mines tertiary motifs (TERMs) from known structures to identify surface‐complementing fragments or “seeds.” We combine seeds that satisfy geometric overlap criteria to generate peptide backbones and score the backbones to identify the most likely binding structures. We found that TERM‐based seeds can describe known binding structures with high resolution: the vast majority of peptide binders from 486 peptide‐protein complexes can be covered by seeds generated from single‐chain structures. Furthermore, we demonstrate that known peptide structures can be reconstructed with high accuracy from peptide‐covering seeds. As a proof of concept, we used our method to design 100 peptide binders of TRAF6, seven of which were predicted by Rosetta to form higher‐quality interfaces than a native binder. The designed peptides interact with distinct sites on TRAF6, including the native peptide‐binding site. These results demonstrate that known peptide‐binding structures can be constructed from TERMs in single‐chain structures and suggest that TERM information can be applied to efficiently design novel target‐complementing binders.     

Oligodendrocytes regulate formation of nodes of Ranvier via the recognition molecule OMgp

The molecular mechanisms underlying the involvement of oligodendrocytes in formation of the nodes of Ranvier (NORs) remain poorly understood. Here we show that oligodendrocyte-myelin glycoprotein (OMgp) aggregates specifically at NORs. Nodal location of OMgp does not occur along demyelinated axons of either Shiverer or proteolipid protein (PLP) transgenic mice. Over-expression of OMgp in OLN-93 cells facilitates process outgrowth. In transgenic mice in which expression of OMgp is down-regulated, myelin thickness declines, and lateral oligodendrocyte loops at the node-paranode junction are less compacted and even join together with the opposite loops, which leads to shortened nodal gaps. Notably, each of these structural abnormalities plus modest down-regulation of expression of Na(+) channel alpha subunit result in reduced conduction velocity in the spinal cords of the mutant mice. Thus, OMgp that is derived from glia has distinct roles in regulating nodal formation and function during CNS myelination.     

Phenotypic and genotypic characterization of Klebsiella pneumoniae isolates recovered from nonhuman primates

Klebsiella pneumoniae is a zoonotic, Gram-negative member of the family Enterobacteriaceae and is the causative agent of nosocomial septicemic, pneumonic, and urinary tract infections. Recently, pathogenic strains of K. pneumoniae sharing a hypermucoviscosity (HMV) phenotype have been attributed to multisystemic abscessation in both human and nonhuman primates. Although K. pneumoniae is a well-recognized zoonotic agent, there is a lack of general information including adequate diagnostic methods or treatments for nonhuman primates. In an effort to increase the body of knowledge of this enigmatic pathogen, K. pneumoniae isolates from African green monkeys (Chlorocebus aethiops sabaeus) on the island of St. Kitts, West Indies were genotypically and phenotypically characterized. Genetic fingerprints generated by PCR-mediated genomic fingerprinting, phenotypic characterization, and antimicrobial susceptibility all identified a high degree of similarity between the HMV and non-HMV K. pneumoniae isolates. The results obtained from this work will help establish a baseline for the development of efficacious diagnostic methods and treatment strategies for both human and nonhuman primates.     

Human Alzheimer’s disease synaptic <Emphasis Type=Italic>O</Emphasis>-GlcNAc site mapping and iTRAQ expression proteomics with ion trap mass spectrometry

Neuronal synaptic functional deficits are linked to impaired learning and memory in Alzheimer’s disease (AD). We recently demonstrated that O-GlcNAc, a novel cytosolic and nuclear carbohydrate post-translational modification, is enriched at neuronal synapses and positively regulates synaptic plasticity linked to learning and memory in mice. Reduced levels of O-GlcNAc have been observed in AD, suggesting a possible link to deficits in synaptic plasticity. Using lectin enrichment and mass spectrometry, we mapped several human cortical synaptic O-GlcNAc modification sites. Overlap in patterns of O-GlcNAcation between mouse and human appears to be high, as previously mapped mouse synaptic O-GlcNAc sites in Bassoon, Piccolo, and tubulin polymerization promoting protein p25 were identified in human. Novel O-GlcNAc modification sites were identified on Mek2 and RPN13/ADRM1. Mek2 is a signaling component of the Erk 1/2 pathway involved in synaptic plasticity. RPN13 is a component of the proteasomal degradation pathway. The potential interplay of phosphorylation with mapped O-GlcNAc sites, and possible implication of those sites in synaptic plasticity in normal versus AD states is discussed. iTRAQ is a powerful differential isotopic quantitative approach in proteomics. Pulsed Q dissociation (PQD) is a recently introduced fragmentation strategy that enables detection of low mass iTRAQ reporter ions in ion trap mass spectrometry. We optimized LTQ ion trap settings for PQD-based iTRAQ quantitation and demonstrated its utility in O-GlcNAc site mapping. Using iTRAQ, abnormal synaptic expression levels of several proteins previously implicated in AD pathology were observed in addition to novel changes in synaptic specific protein expression including Synapsin II.     

A comparison of two techniques for measuring and crossdating tree rings

Crossdating is the core principle of dendrochronology. Our study compared two techniques for measuring and crossdating tree rings using Juniperus virginiana L. (eastern redcedar) as a case study. We used a pseudo 2 × 2 study design comparing the traditional skeleton plot/sliding measuring stage technique to a semi-automatic image analysis program across two technicians. Crossdating was evaluated in COFECHA. Raw measurements of total, earlywood, and latewood widths from the two methods were analyzed using the Verify for Windows program, ANOVA, and correlation matrices. Total ring width and earlywood width were well correlated between techniques and technicians but questionable ring boundaries from image analysis program should be checked under a stereoscope. Juniperus virginiana latewood widths were significantly different between techniques and technicians; therefore, we do not recommend combining latewood measurements from species with limited latewood variability for dendrochronological analysis. A standard definition of the earlywood-latewood boundary that can be replicated across technicians is needed to combine latewood measurements from the sliding measuring stage and image analysis systems.     

Nucleic acid binding activity of pns6 encoded by genome segment 6 of rice ragged stunt oryzavirus

Rice ragged stunt disease, caused by rice ragged stuntoryzavirus (RRSV), was first discovered in 1976–1977 inIndonesia and Philippines [1]. Subsequently the diseasewas found in most rice-growing countries in south-easternand far-eastern Asia [2] and may inflict heavy loss on thecrop. RRSV is the type species of the genus Oryzavirus in thefamily Reoviridae. The virus particle is icosahedral witha diameter of about 65–70 nm and the genome consistsof 10 double stranded RNA (dsRNA) segm…     

RNA-seq analysis reveals a key role of brassinolide-regulated pathways in NaCl-stressed cotton

Brassinolide (BL) alleviates salt injury in cotton seedlings; however, little is known about the molecular mechanisms of this response. In this study, digital gene expression analysis was performed to better understand the regulatory pathways of BL in NaCl-stressed cotton (Gossypium hirsutum L.). Compared with control plants (CK), a total of 1 162 and 7 659 differentially expressed genes (DEGs) were detected in the leaves and roots of NaCl-treated plants, respectively. Most of the DEGs in NaCl-treated plants, compared to CK, were regulated by BL. Moreover, expression patterns of DEGs in BL+NaCl treated plants were similar to those in CK plants; however, the responses of DEGs in the leaves and roots of NaCl-treated plants to BL differed. In the roots, BL-regulated DEGs were involved in protein biosynthesis, whereas in the leaves, BL promoted photosynthesis in NaCl-stressed cotton. BL treatment also significantly increased the overall biomass, chlorophyll a + b content in leaves, and the protein content in roots in NaCl-stressed cotton. The downregulation of stress-responsive genes in BL+NaCl-stressed leaves was also found. These results suggest that BL can alleviate NaCl injury in cotton plants.