Asparagine deamidation and the role of higher order protein structure

The 'protein world' exhibits additional complexity caused by post-translational modifications. One such process is nonenzymic deamidation of asparagine which is controlled partly by primary sequence, but also higher order protein structure. We have studied the deamidation of an N-terminal peptide in muscle glyceraldehyde 3-phosphate dehydrogenase to relate three-dimensional structure, proteolysis, and deamidation. This work has significant consequences for identification of proteins using peptide mass fingerprinting.     

Clonal characterization of mouse mammary luminal epithelial and myoepithelial cells separated by fluorescence-activated cell sorting

Summary Lineage analysis in vitro of heterogeneous tissues such as mammary epithelium requires the separation of constituent cell types and their growth as clones. The separation of virgin mouse mammary luminal epithelial and myoepithelial cells by fluorescence-activated cell-sorting, their growth at clonal density, and the phenotyping of the clones obtained with cell-type specific markers are described in this paper. Epithelial cells were isolated by collagenase digestion followed by trypsinization, and the luminal and myoepithelial cells were flow-sorted with the rat monoclonal antibodies 33A10 and JB6, respectively. Sorted cells were cloned under, using low oxygen conditions (<5% vol/vol), in medium containing cholera toxin and insulin, with an irradiated feeder layer of 3T3-L1 cells. Clones were characterized morphologically, and antigenically by multiple immunofluorescence with a panel of antibodies to cytoskeletal antigens specific to either luminal epithelial or myoepithelial cells in situ. Whereas sorted myoepithelial cells gave a single clone type, sorted luminal cells gave three morphological clone types, two of which grew rapidly. All myoepithelially derived clones showed a limited proliferative capacity in vitro, in contrast to their rat and human counterparts, as shown in previous studies. The present results with sorted mouse cells have also allowed the stability of the differentiated phenotype in mouse, rat, and human mammary luminal epithelial and myoepithelial cells in primary clonal culture to be compared. They show that the mouse mammary cells are the least stable in terms of expression of differentiation-specific cytoskeletal markers in vitro.     

Creating Novel Activated Factor XI Inhibitors through Fragment Based Lead Generation and Structure Aided Drug Design

Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrinolytic effects with a low bleeding risk. This motivated a structure aided fragment based lead generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on docking experiments, was performed to generate a FXIa targeted fragment library for an NMR screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket. The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine structures are novel FXIa P1 fragments. The expansion of these fragments towards the FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa inhibitors that we found to bind in the S1-S1’-S2’ FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1’-S2’ binding reference compounds enabled structure guided linking and expansion work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC₅₀ of 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1’-S2’ binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds.     

The chaperone domain BRICHOS prevents CNS toxicity of amyloid-β peptide in Drosophila melanogaster

Aggregation of the amyloid-β peptide (Aβ) into toxic oligomers and amyloid fibrils is linked to the development of Alzheimer’s disease (AD). Mutations of the BRICHOS chaperone domain are associated with amyloid disease and recent in vitro data show that BRICHOS efficiently delays Aβ42 oligomerization and fibril formation. We have generated transgenic Drosophila melanogaster flies that express the Aβ42 peptide and the BRICHOS domain in the central nervous system (CNS). Co-expression of Aβ42 and BRICHOS resulted in delayed Aβ42 aggregation and dramatic improvements of both lifespan and locomotor function compared with flies expressing Aβ42 alone. Moreover, BRICHOS increased the ratio of soluble:insoluble Aβ42 and bound to deposits of Aβ42 in the fly brain. Our results show that the BRICHOS domain efficiently reduces the neurotoxic effects of Aβ42, although significant Aβ42 aggregation is taking place. We propose that BRICHOS-based approaches should be explored with an aim towards the future prevention and treatment of AD.KEY WORDS: Amyloid, Alzheimer’s disease, Protein misfolding, Chaperone     

Porphyromonas gingivalis and Treponema denticola Exhibit Metabolic Symbioses

Porphyromonas gingivalis and Treponema denticola are strongly associated with chronic periodontitis. These bacteria have been co-localized in subgingival plaque and demonstrated to exhibit symbiosis in growth in vitro and synergistic virulence upon co-infection in animal models of disease. Here we show that during continuous co-culture a P. gingivalis:T. denticola cell ratio of 6∶1 was maintained with a respective increase of 54% and 30% in cell numbers when compared with mono-culture. Co-culture caused significant changes in global gene expression in both species with altered expression of 184 T. denticola and 134 P. gingivalis genes. P. gingivalis genes encoding a predicted thiamine biosynthesis pathway were up-regulated whilst genes involved in fatty acid biosynthesis were down-regulated. T. denticola genes encoding virulence factors including dentilisin and glycine catabolic pathways were significantly up-regulated during co-culture. Metabolic labeling using ¹³C-glycine showed that T. denticola rapidly metabolized this amino acid resulting in the production of acetate and lactate. P. gingivalis may be an important source of free glycine for T. denticola as mono-cultures of P. gingivalis and T. denticola were found to produce and consume free glycine, respectively; free glycine production by P. gingivalis was stimulated by T. denticola conditioned medium and glycine supplementation of T. denticola medium increased final cell density 1.7-fold. Collectively these data show P. gingivalis and T. denticola respond metabolically to the presence of each other with T. denticola displaying responses that help explain enhanced virulence of co-infections.     

A comparison of urinary mercury between children with autism spectrum disorders and control children

Background

Urinary mercury concentrations are used in research exploring mercury exposure. Some theorists have proposed that autism is caused by mercury toxicity. We set out to test whether mercury concentrations in the urine of children with autism were significantly increased or decreased compared to controls or siblings.

Methods

Blinded cohort analyses were carried out on the urine of 56 children with autism spectrum disorders (ASD) compared to their siblings (n = 42) and a control sample of children without ASD in mainstream (n = 121) and special schools (n = 34).

Results

There were no statistically significant differences in creatinine levels, in uncorrected urinary mercury levels or in levels of mercury corrected for creatinine, whether or not the analysis is controlled for age, gender and amalgam fillings.

Conclusions

This study lends no support for the hypothesis of differences in urinary mercury excretion in children with autism compared to other groups. Some of the results, however, do suggest further research in the area may be warranted to replicate this in a larger group and with clear measurement of potential confounding factors.     

Detection of PrPCWD in feces from naturally exposed Rocky Mountain elk (Cervus elaphus nelsoni) using protein misfolding cyclic amplification

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting captive and free-ranging cervids. Currently, tests for CWD in live animals involve relatively invasive procedures to collect lymphoid tissue biopsies and examine them for CWD-associated, protease-resistant cervid prion protein (PrP(CWD)) detected by immunohistochemistry (IHC). We adapted an ultrasensitive prion detection system, protein misfolding cyclic amplification (PMCA), to detect PrP(CWD) in Rocky Mountain elk (Cervus elaphus nelsoni) feces. Our PMCA reproducibly detected a 1.2 × 10(7) dilution of PrP(CWD) (a 10% infected brain homogenate diluted 1.2 × 10(6)-fold into 10% fecal homogenates), equivalent to approximately 100 pg of PrP(CWD)/g of feces. We developed a semiquantitative scoring system based on the first PMCA round at which PrP(CWD) was detected and fit a nonlinear regression curve to our serial dilutions to correlate PMCA scores with known PrP(CWD) concentrations. We used this PMCA scoring system to detect PrP(CWD) and estimate its concentration in feces from free-ranging elk from Rocky Mountain National Park, Colorado. We compared our results to PrP(CWD) IHC of rectoanal mucosa-associated lymphoid tissue and obex from the same animals. The PMCA successfully detected PrP(CWD) in feces from elk that were positive by IHC, with estimated prion loads from 100 to 5,000 pg PrP(CWD)/g of feces. These data show for the first time PrP(CWD) in feces from naturally exposed free-ranging elk and demonstrate the potential of PMCA as a new, noninvasive CWD diagnostic tool to complement IHC.     

Functional asymmetries of proteasome translocase pore

Degradation by proteasomes involves coupled translocation and unfolding of its protein substrates. Six distinct but paralogous proteasome ATPase proteins, Rpt1 to -6, form a heterohexameric ring that acts on substrates. An axially positioned loop (Ar-Φ loop) moves in concert with ATP hydrolysis, engages substrate, and propels it into a proteolytic chamber. The aromatic (Ar) residue of the Ar-Φ loop in all six Rpts of S. cerevisiae is tyrosine; this amino acid is thought to have important functional contacts with substrate. Six yeast strains were constructed and characterized in which Tyr was individually mutated to Ala. The mutant cells were viable and had distinct phenotypes. rpt3, rpt4, and rpt5 Tyr/Ala mutants, which cluster on one side of the ATPase hexamer, were substantially impaired in their capacity to degrade substrates. In contrast, rpt1, rpt2, and rpt6 mutants equaled or exceeded wild type in degradation activity. However, rpt1 and rpt6 mutants had defects that limited cell growth or viability under conditions that stressed the ubiquitin proteasome system. In contrast, the rpt3 mutant grew faster than wild type and to a smaller size, a defect that has previously been associated with misregulation of G1 cyclins. This rpt3 phenotype probably results from altered degradation of cell cycle regulatory proteins. Finally, mutation of five of the Rpt subunits increased proteasome ATPase activity, implying bidirectional coupling between the Ar-Φ loop and the ATP hydrolysis site. The present observations assign specific functions to individual Rpt proteins and provide insights into the diverse roles of the axial loops of individual proteasome ATPases.     

Anti-tumor effects of rigosertib in high-risk neuroblastoma

High-risk neuroblastoma has a poor prognosis despite intense treatment, demonstrating the need for new therapeutic strategies. Here we evaluated the effects of rigosertib (ON-01910.Na) in preclinical models of high-risk neuroblastoma. Among several hundred cancer cell lines representing 24 tumor types, neuroblastoma was the most sensitive to rigosertib. Treatment of MYCN-amplified neuroblastoma organoids resulted in organoid disintegration, decreased cell viability, and increased apoptotic cell death. Neuroblastoma response to rigosertib involved G2M cell cycle arrest and decreased phosphorylation of AKT (Ser473) and ERK1/2 (Thr202/Tyr204). Rigosertib delayed tumor growth and prolonged survival of mice carrying neuroblastoma MYCN-amplified PDX tumors (median survival: 31 days, treated; 22 days, vehicle) accompanied with increased apoptosis in treated tumors. We further identified vincristine and rigosertib as a potential promising drug combination treatment. Our results show that rigosertib might be a useful therapeutic agent for MYCN-amplified neuroblastomas, especially in combination with existing agents.