Multiple substitutions of methionine 129 in human prion protein reveal its importance in the amyloid fibrillation pathway

The role of the polymorphism Met or Val in position 129 in the human prion protein is well documented regarding disease susceptibility and clinical manifestations. However, little is known about the molecular background to this phenomenon. We investigated herein the conformational stability, amyloid fibrillation kinetics, and seeding propensity of different 129 mutants, located in β-strand 1 of PrP (Met(129) (WT), M129A, M129V, M129L, M129W, M129P, M129E, M129K, and M129C) in HuPrP(90-231). The mutations M129V, M129L, M129K, and M129C did not affect stability (midpoints of thermal denaturation, T(m) = 65-66 °C), whereas the mutants M129A and M129E and the largest side chain M129W were destabilized by 3-4 °C. The most destabilizing substitution was M129P, which lowered the T(m) by 7.2 °C. All mutants, except for M129C, formed amyloid-like fibrils within hours during fibril formation under near physiological conditions. Fibril-forming mutants showed a sigmoidal kinetic profile and showed shorter lag times during seeding with preformed amyloid fibrils implicating a nucleated polymerization reaction. In the spontaneous reactions, the lag time of fibril formation was rather uniform for the mutants M129A, M129V, and M129L resembling the wild type. When the substituted amino acid had a distinct feature discriminating it from the wild type, such as size (M129W), charge (M129E, M129K), or rotational constraint (M129P), the fibrillation was impeded. M129C did not form ThT/Congo red-positive fibrils, and non-reducing SDS-PAGE of M129C during fibrillation conditions at different time points revealed covalent dimer formation already 15 min after fibrillation reaction initiation. Position 129 appears to be a key site for dictating PrP receptiveness toward recruitment into the amyloid state.     

Cytokine and CXC chemokine expression patterns in aqueous humor of patients with presumed tuberculous uveitis

Aqueous humor (AH) samples from 14 patients with presumed tuberculous uveitis (PTU), and 30 control patients were assayed for the proinflammatory cytokines interleukin IL-4, IL-12, IL-15, IL-17, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, the immunosuppressive cytokine IL-10, and the chemokines GRO-α/CXCL1, IL-8/CXCL8, MIG/CXCL9, IP-10/CXCL10 and SDF-1/CXCL12 with the use of a multiplex assay. Among cytokines, IL-4 and IL-12 were not detected. IL-15, IL-17, IFN-γ, TNF-α and IL-10 levels in AH were significantly higher in patients than in controls (p<0.001; p=0.004; p<0.001; p<0.001; p<0.001, respectively). Among chemokines, SDF-1 levels did not differ significantly between patients and controls, whereas GRO-α, IL-8, MIG and IP-10 levels were significantly higher in patients than in controls (p=0.001; p<0.001; p<0.001; p<0.001, respectively). Mean GRO-α levels in AH of PTU patients were 6-fold higher than IL-8 levels and mean IP-10 levels were 15-fold higher than MIG levels. Clinical disease activity correlated significantly with the levels of IL-15, IFN-γ, TNF-α and IP-10. Logistic regression analysis demonstrated a significant positive association between PTU and high levels of IFN-γ, IL-8, MIG and IP-10. These data suggest that both T helper (Th) Th(1) and Th(17) cells are involved in PTU and that the cytokine profile is polarized toward a Th(1) response. GRO-α and IP-10 might be involved in neutrophil and activated T lymphocyte chemoattraction in PTU, respectively.     

14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding experiments reveal weak affinity of the more selective isoform 14-3-3σ but both 14-3-3 isoforms η and σ significantly stimulate hEXO1 activity, indicating that these regulatory proteins exert a common regulation mode on hEXO1. Results demonstrate that binding involves the phosphorable amino acid S746 in hEXO1 and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate with PCNA in distinct replication foci and respond to DNA double strand breaks (DSBs), indicating that 14-3-3 binding does not involve regulating the subcellular distribution of hEXO1. Altogether, these results suggest that association may be related to regulation of hEXO1 availability during the DNA damage response to plausibly prevent extensive DNA resection at the damage site, as supported by recent studies.     

Selection of DNA nanoparticles with preferential binding to aggregated protein target

High affinity and specificity are considered essential for affinity reagents and molecularly-targeted therapeutics, such as monoclonal antibodies. However, life''s own molecular and cellular machinery consists of lower affinity, highly multivalent interactions that are metastable, but easily reversible or displaceable. With this inspiration, we have developed a DNA-based reagent platform that uses massive avidity to achieve stable, but reversible specific recognition of polyvalent targets. We have previously selected these DNA reagents, termed DeNAno, against various cells and now we demonstrate that DeNAno specific for protein targets can also be selected. DeNAno were selected against streptavidin-, rituximab- and bevacizumab-coated beads. Binding was stable for weeks and unaffected by the presence of soluble target proteins, yet readily competed by natural or synthetic ligands of the target proteins. Thus DeNAno particles are a novel biomolecular recognition agent whose orthogonal use of avidity over affinity results in uniquely stable yet reversible binding interactions.     

Fitness components of Drosophila melanogaster developed on a standard laboratory diet or a typical natural food source

Drosophila melanogaster is often used as a model organism in evolutionary biology and ecophysiology to study evolutionary processes and their physiological mechanisms. Diets used to feed Drosophila cultures differ between laboratories and are often nutritious and distinct from food sources in the natural habitat. Here we rear D. melanogaster on a standard diet used in our laboratory and a field diet composed of decomposing apples collected in the field. Flies developed on these two diet compositions are tested for heat, cold, desiccation, and starvation resistance as well as developmental time, dry body mass and fat percentage. The nutritional compositions of the standard and field diets were analyzed, and discussed in relation to the phenotypic observations. Results showed marked differences in phenotype of flies from the two types of diets. Flies reared on the field diet are more starvation resistant and they are smaller, leaner, and have lower heat resistance compared to flies reared on the standard diet. Sex specific effects of diet type are observed for several of the investigated traits and the strong sexual dimorphism usually observed in desiccation resistance in D. melanogaster disappeared when rearing the flies on the field diet. Based on our results we conclude that care should be taken in extrapolating results from one type of diet to another and especially from laboratory to field diets.     

Telomere length and cardiovascular risk factors in a middle-aged population free of overt cardiovascular disease

Evidence assembled over the last decade shows that average telomere length (TL) acts as a biomarker for biological aging and cardiovascular disease (CVD) in particular. Although essential for a more profound understanding of the underlying mechanisms, little reference information is available on TL. We therefore sought to provide baseline TL information and assess the association of prevalent CVD risk factors with TL in subjects free of overt CVD within a small age range. We measured mean telomere restriction fragment length of peripheral blood leukocytes in a large, representative Asklepios study cohort of 2509 community-dwelling, Caucasian female and male volunteers aged approximately 35-55 years and free of overt CVD. We found a manifest age-dependent telomere attrition, at a significantly faster rate in men as compared to women. No significant associations were established with classical CVD risk factors such as cholesterol status and blood pressure, yet shorter TL was associated with increased levels of several inflammation and oxidative stress markers. Importantly, shorter telomere length was associated with an increasingly unhealthy lifestyle, particularly in men. All findings were age and gender adjusted where appropriate. With these cross-sectional results we show that TL of peripheral blood leukocytes primarily reflects the burden of increased oxidative stress and inflammation, whether or not determined by an increasingly unhealthy lifestyle, while the association with classical CVD risk factors is limited. This further clarifies the added value of TL as a biomarker for biological aging and might improve our understanding of how TL is associated with CVD.     

Citrullinated vimentin and biglycan protein fingerprints as candidate serological biomarkers for disease activity in systemic sclerosis: a pilot study

Purpose: Extracellular matrix (ECM) deposition and remodelling in skin and lungs of systemic sclerosis (SSc) subjects lead to release of metabolites/biomarkers into circulation. We investigated if biomarkers of ECM degradation (biglycan and elastin) and macrophage activation (citrullinated vimentin) could identify diffuse SSc (dSSc) subjects from controls and the biomarkers discriminative power.

Methods: DSSc subjects (n?=?40) fulfilling the 2013 EULAR/ACR classification criteria were divided in early (<2years of symptoms) and late (≥10 years of symptoms). Early were subdivided into intermediate and rapid skin thickness progression rate (STPR). Twenty controls were included. Citrullinated and matrix metalloproteinase (MMP)-2/8-degraded vimentin (VICM), MMP-9/12-degraded biglycan (BGM) and MMP-7-degraded elastin (ELM-7) were assessed in serum. Analysis between groups was by Kruskal–Wallis and ROC AUC for discriminative power.

Results: VICM and BGM levels were increased in early compared with late dSSc (p<?=0.023). VICM was increased in rapid and intermediate STPR compared with controls (p<?=0.025). No differences in ELM-7 levels were observed. AUC of VICM was 0.71 for early versus late dSSc and BGM had an AUC of 0.79 for dSSc versus controls.

Conclusion: This pilot study found differences in biomarker levels between early and late dSSc. This study offers new perspectives of ECM metabolites as potential biomarkers of dSSc.