A fast growing spectrum of biological functions of γ-secretase in development and disease

γ-secretase, which assembles as a tetrameric complex, is an aspartyl protease that proteolytically cleaves substrate proteins within their membrane-spanning domain; a process also known as regulated intramembrane proteolysis (RIP). RIP regulates signaling pathways by abrogating or releasing signaling molecules. Since the discovery, already > 15 years ago, of its catalytic component, presenilin, and even much earlier with the identification of amyloid precursor protein as its first substrate, γ-secretase has been commonly associated with Alzheimer's disease. However, starting with Notch and thereafter a continuously increasing number of novel substrates, γ-secretase is becoming linked to an equally broader range of biological processes. This review presents an updated overview of the current knowledge on the diverse molecular mechanisms and signaling pathways controlled by γ-secretase, with a focus on organ development, homeostasis and dysfunction. This article is part of a Special Issue entitled: Intramembrane Proteases.     

An immunoaffinity purification method for the proteomic analysis of ubiquitinated protein complexes

Protein ubiquitination plays an important role in the regulation of many cellular processes, including protein degradation, cell cycle regulation, apoptosis, and DNA repair. To study the ubiquitin proteome we have established an immunoaffinity purification method for the proteomic analysis of endogenously ubiquitinated protein complexes. A strong, specific enrichment of ubiquitinated factors was achieved using the FK2 antibody bound to protein G-beaded agarose, which recognizes monoubiquitinated and polyubiquitinated conjugates. Mass spectrometric analysis of two FK2 immunoprecipitations (IPs) resulted in the identification of 296 FK2-specific proteins in both experiments. The isolation of ubiquitinated and ubiquitination-related proteins was confirmed by pathway analyses (using Ingenuity Pathway Analysis and Gene Ontology-annotation enrichment). Additionally, comparing the proteins that specifically came down in the FK2 IP with databases of ubiquitinated proteins showed that a high percentage of proteins in our enriched fraction was indeed ubiquitinated. Finally, assessment of protein–protein interactions revealed that significantly more FK2-specific proteins were residing in protein complexes than in random protein sets. This method, which is capable of isolating both endogenously ubiquitinated proteins and their interacting proteins, can be widely used for unraveling ubiquitin-mediated protein regulation in various cell systems and tissues when comparing different cellular states.     

Notes on the genus Amorphophallus (Araceae) – 13. Evolution of pollen ornamentation and ultrastructure in Amorphophallus and Pseudodracontium

A strict consensus tree based on chloroplast and nuclear sequences (rbcL, matK, trnL, FLint2) from 46 Amorphophallus species, two Pseudodracontium species and six outgroups is used to develop a hypothesis for the evolution of ornamentation and ectexine ultrastructure in the pollen of Amorphophallus. There are four main clades: an exclusively African, largely psilate clade (‘African clade’), an Asian, largely psilate clade (‘Asian psilate clade’) and an Asian, largely striate clade consisting of a mainly continental SE Asian clade (‘continental SE Asian striate clade’) and one centred in Malesia (‘Malesian striate clade’). Ultrastructure provides a valuable contribution towards understanding pollen ornamentation in Amorphophallus. Pollen with a thin psilate ectexine without dark granules might be plesiomorphic in Amorphophallus. Then the diverse striate type would be derived. Within both striate clades, reversals to the psilate type occur. Striate pollen with psilate caps, which is nested in the continental SE Asian striate clade, is a synapomorphy of Pseudodracontium. The fossulate type is also diverse, and its distribution in the tree indicates a polyphyletic origin. Areolate, echinate and verrucate ornamentation, occur in single species in the tree, but are found also in species not included in the molecular analysis. All three are heterogeneous and probably polyphyletic too. Reticulate, scabrate and striate/scabrate ornamentation are autapomorphies, of which the reticulate type and the striate/scabrate type may derive from psilate and striate ornamentation, respectively. Of the four main clades, the Asian psilate and African clade seem to be basal, while both striate clades might have evolved from the Asian psilate clade via a species like A. rhizomatosus. Dark granules evolved more than once, which might explain their diverse size, shape and distribution.     

Structure of an early native‐like intermediate of β2‐microglobulin amyloidogenesis

To investigate early intermediates of β2‐microglobulin (β2m) amyloidogenesis, we solved the structure of β2m containing the amyloidogenic Pro32Gly mutation by X‐ray crystallography. One nanobody (Nb24) that efficiently blocks fibril elongation was used as a chaperone to co‐crystallize the Pro32Gly β2m monomer under physiological conditions. The complex of P32G β2m with Nb24 reveals a trans peptide bond at position 32 of this amyloidogenic variant, whereas Pro32 adopts the cis conformation in the wild‐type monomer, indicating that the cis to trans isomerization at Pro32 plays a critical role in the early onset of β2m amyloid formation.     

Improving the iterative Linear Interaction Energy approach using automated recognition of configurational transitions

Recently an iterative method was proposed to enhance the accuracy and efficiency of ligand-protein binding affinity prediction through linear interaction energy (LIE) theory. For ligand binding to flexible Cytochrome P450s (CYPs), this method was shown to decrease the root-mean-square error and standard deviation of error prediction by combining interaction energies of simulations starting from different conformations. Thereby, different parts of protein-ligand conformational space are sampled in parallel simulations. The iterative LIE framework relies on the assumption that separate simulations explore different local parts of phase space, and do not show transitions to other parts of configurational space that are already covered in parallel simulations. In this work, a method is proposed to (automatically) detect such transitions during the simulations that are performed to construct LIE models and to predict binding affinities. Using noise-canceling techniques and splines to fit time series of the raw data for the interaction energies, transitions during simulation between different parts of phase space are identified. Boolean selection criteria are then applied to determine which parts of the interaction energy trajectories are to be used as input for the LIE calculations. Here we show that this filtering approach benefits the predictive quality of our previous CYP 2D6-aryloxypropanolamine LIE model. In addition, an analysis is performed of the gain in computational efficiency that can be obtained from monitoring simulations using the proposed filtering method and by prematurely terminating simulations accordingly.     

Staphylococcus aureus Sortase A-Mediated Incorporation of Peptides: Effect of Peptide Modification on Incorporation

The endogenous Staphylococcus aureus sortase A (SrtA) transpeptidase covalently anchors cell wall-anchored (CWA) proteins equipped with a specific recognition motif (LPXTG) into the peptidoglycan layer of the staphylococcal cell wall. Previous in situ experiments have shown that SrtA is also able to incorporate exogenous, fluorescently labelled, synthetic substrates equipped with the LPXTG motif (K(FITC)LPETG-amide) into the bacterial cell wall, albeit at high concentrations of 500 μM to 1 mM. In the present study, we have evaluated the effect of substrate modification on the incorporation efficiency. This revealed that (i) by elongation of LPETG-amide with a sequence of positively charged amino acids, derived from the C-terminal domain of physiological SrtA substrates, the incorporation efficiency was increased by 20-fold at 10 μM, 100 μM and 250 μM; (ii) Substituting aspartic acid (E) for methionine increased the incorporation of the resulting K(FITC)LPMTG-amide approximately three times at all concentrations tested; (iii) conjugation of the lipid II binding antibiotic vancomycin to K(FITC)LPMTG-amide resulted in the same incorporation levels as K(FITC)LPETG-amide, but much more efficient at an impressive 500-fold lower substrate concentration. These newly developed synthetic substrates can potentially find broad applications in for example the in situ imaging of bacteria; the incorporation of antibody recruiting moieties; the targeted delivery and covalent incorporation of antimicrobial compounds into the bacterial cell wall.     

Biomarkers in Exhaled Breath Condensate Are Not Predictive for Pulmonary Exacerbations in Children with Cystic Fibrosis: Results of a One-Year Observational Study

BackgroundCystic Fibrosis (CF) is characterized by chronically inflamed airways, and inflammation even increases during pulmonary exacerbations. These adverse events have an important influence on the well-being, quality of life, and lung function of patients with CF. Prediction of exacerbations by inflammatory markers in exhaled breath condensate (EBC) combined with early treatment may prevent these pulmonary exacerbations and may improve the prognosis.AimTo investigate the diagnostic accuracy of a set of inflammatory markers in EBC to predict pulmonary exacerbations in children with CF.MethodsIn this one-year prospective observational study, 49 children with CF were included. During study visits with an interval of 2 months, a symptom questionnaire was completed, EBC was collected, and lung function measurements were performed. The acidity of EBC was measured directly after collection. Inflammatory markers interleukin (IL)-6, IL-8, tumor necrosis factor α (TNF-α), and macrophage migration inhibitory factor (MIF) were measured using high sensitivity bead based flow immunoassays. Pulmonary exacerbations were recorded during the study and were defined in two ways. The predictive power of inflammatory markers and the other covariates was assessed using conditionally specified models and a receiver operating characteristic curve (SAS version 9.2). In addition, k-nearest neighbors (KNN) algorithm was applied (SAS version 9.2).ResultsSixty-five percent of the children had one or more exacerbations during the study. The conditionally specified models showed an overall correct prediction rate of 55%. The area under the curve (AUC) was equal to 0.62. The results obtained with the KNN algorithm were very similar.ConclusionAlthough there is some evidence indicating that the predictors outperform random guessing, the general diagnostic accuracy of EBC acidity and the EBC inflammatory markers IL-6, IL-8, TNF-α and MIF is low. At present it is not possible to predict pulmonary exacerbations in children with CF with the chosen biomarkers and the method of EBC analysis. The biochemical measurements of EBC markers should be improved and other techniques should be considered.     

Impact of Denture Cleaning Method and Overnight Storage Condition on Denture Biofilm Mass and Composition: A Cross-Over Randomized Clinical Trial

Background

Appropriate oral hygiene is required to maintain oral health in denture wearers. This study aims to compare the role of denture cleaning methods in combination with overnight storage conditions on biofilm mass and composition on acrylic removable dentures.

Methods

In a cross-over randomized controlled trial in 13 older people, 4 conditions with 2 different mechanical cleaning methods and 2 overnight storage conditions were considered: (i) brushing and immersion in water without a cleansing tablet, (ii) brushing and immersion in water with a cleansing tablet, (iii) ultrasonic cleaning and immersion in water without a cleansing tablet, and (iv) ultrasonic cleaning and immersion in water with a cleansing tablet. Each test condition was performed for 5 consecutive days, preceded by a 2-days wash-out period. Biofilm samples were taken at baseline (control) and at the end of each test period from a standardized region. Total and individual levels of selected oral bacteria (n = 20), and of Candida albicans were identified using the Polymerase Chain Reaction (PCR) technique. Denture biofilm coverage was scored using an analogue denture plaque score. Paired t-tests and Wilcoxon-signed rank tests were used to compare the test conditions. The level of significance was set at α< 5%.

Results

Overnight denture storage in water with a cleansing tablet significantly reduced the total bacterial count (p<0.01). The difference in total bacterial level between the two mechanical cleaning methods was not statistically significant. No significant effect was observed on the amount of Candida albicans nor on the analogue plaque scores.

Conclusions

The use of cleansing tablets during overnight denture storage in addition to mechanical denture cleaning did not affect Candida albicans count, but reduced the total bacterial count on acrylic removable dentures compared to overnight storage in water. This effect was more pronounced when combined with ultrasonic cleaning compared to brushing.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02454413","term_id":"NCT02454413"}}NCT02454413