(-)-Epigallocatechin-3-gallate (EGCG) maintains k-casein in its pre-fibrillar state without redirecting its aggregation pathway

The polyphenol (-)-epigallocatechin-3-gallate (EGCG) has recently attracted much research interest in the field of protein-misfolding diseases because of its potent anti-amyloid activity against amyloid-beta, alpha-synuclein and huntingtin, the amyloid-fibril-forming proteins involved in Alzheimer\u27s, Parkinson\u27s and Huntington\u27s diseases, respectively. EGCG redirects the aggregation of these polypeptides to a disordered off-folding pathway that results in the formation of non-toxic amorphous aggregates. whether this anti-fibril activity is specific to these disease-related target proteins or ismore generic remains to be established. In addition, the mechanism by which EGCG exerts its effects, as with all anti-amyloidogenic polyphenols, remains unclear. To address these aspects, we have investigated the ability of EGCG to inhibit amyloidogenesis of the generic model fibril-forming protein RCMkappa-CN (reduced and carboxymethylated kappa-casein) and thereby protect pheochromocytoma-12 cells from RCMkappa-CN amyloid-induced toxicity. We found that EGCG potently inhibits in vitro fibril formation byRCMkappa-CN [the IC50 for 50 uM RCMkappa-CN is 1 uM]. Biophysical studies reveal that EGCG prevents RCMkappa-CN fibril formation by stabilising RCMkappa-CN in its nativelike state rather than by redirecting its aggregation to the disordered, amorphous aggregation pathway. Thus, while it appears that EGCG is a generic inhibitor of amyloid-fibril formation, the mechanism by which it achieves this inhibition is specific to the target fibril-forming polypeptide. It is proposed that EGCG is directed to the amyloidogenic sheet-turn-sheet motif of monomeric RCMkappa-CN with high affinity by strong non-specific hydrophobic associations. Additional non-covalent pi-pi stacking interactions between the polyphenolic and aromatic residues common to the amyloidogenic sequence are also implicated

(-)-Epigallocatechin-3-gallate (EGCG) maintains k-casein in its pre-fibrillar state without redirecting its aggregation pathway

The polyphenol (-)-epigallocatechin-3-gallate (EGCG) has recently attracted much research interest in the field of protein-misfolding diseases because of its potent anti-amyloid activity against amyloid-beta, alpha-synuclein and huntingtin, the amyloid-fibril-forming proteins involved in Alzheimer\u27s, Parkinson\u27s and Huntington\u27s diseases, respectively. EGCG redirects the aggregation of these polypeptides to a disordered off-folding pathway that results in the formation of non-toxic amorphous aggregates. whether this anti-fibril activity is specific to these disease-related target proteins or ismore generic remains to be established. In addition, the mechanism by which EGCG exerts its effects, as with all anti-amyloidogenic polyphenols, remains unclear. To address these aspects, we have investigated the ability of EGCG to inhibit amyloidogenesis of the generic model fibril-forming protein RCMkappa-CN (reduced and carboxymethylated kappa-casein) and thereby protect pheochromocytoma-12 cells from RCMkappa-CN amyloid-induced toxicity. We found that EGCG potently inhibits in vitro fibril formation byRCMkappa-CN [the IC50 for 50 uM RCMkappa-CN is 1 uM]. Biophysical studies reveal that EGCG prevents RCMkappa-CN fibril formation by stabilising RCMkappa-CN in its nativelike state rather than by redirecting its aggregation to the disordered, amorphous aggregation pathway. Thus, while it appears that EGCG is a generic inhibitor of amyloid-fibril formation, the mechanism by which it achieves this inhibition is specific to the target fibril-forming polypeptide. It is proposed that EGCG is directed to the amyloidogenic sheet-turn-sheet motif of monomeric RCMkappa-CN with high affinity by strong non-specific hydrophobic associations. Additional non-covalent pi-pi stacking interactions between the polyphenolic and aromatic residues common to the amyloidogenic sequence are also implicated

Defective lung macrophage function in lung cancer +/- chronic obstructive pulmonary disease (COPD/emphysema)-mediated by cancer cell production of PGE2?

In chronic obstructive pulmonary disease (COPD/emphysema) we have shown a reduced ability of lung and alveolar (AM) macrophages to phagocytose apoptotic cells (defective ‘efferocytosis’), associated with evidence of secondary cellular necrosis and a resultant inflammatory response in the airway. It is unknown whether this defect is present in cancer (no COPD) and if so, whether this results from soluble mediators produced by cancer cells. We investigated efferocytosis in AM (26 controls, 15 healthy smokers, 37 COPD, 20 COPD+ non small cell lung cancer (NSCLC) and 8 patients with NSCLC without COPD) and tumor and tumor-free lung tissue macrophages (21 NSCLC with/13 without COPD). To investigate the effects of soluble mediators produced by lung cancer cells we then treated AM or U937 macrophages with cancer cell line supernatant and assessed their efferocytosis ability. We qualitatively identified Arachidonic Acid (AA) metabolites in cancer cells by LC-ESI-MSMS, and assessed the effects of COX inhibition (using indomethacin) on efferocytosis. Decreased efferocytosis was noted in all cancer/COPD groups in all compartments. Conditioned media from cancer cell cultures decreased the efferocytosis ability of both AM and U937 macrophages with the most pronounced effects occurring with supernatant from SCLC (an aggressive lung cancer type). AA metabolites identified in cancer cells included PGE2. The inhibitory effect of PGE2 on efferocytosis, and the involvement of the COX-2 pathway were shown. Efferocytosis is decreased in COPD/emphysema and lung cancer; the latter at least partially a result of inhibition by soluble mediators produced by cancer cells that include PGE2.Francis C. Dehle, Violet R. Mukaro, Craig Jurisevic, David Moffat, Jessica Ahern, Greg Hodge, Hubertus Jersmann, Paul N. Reynolds, Sandra Hodg

(-)-Epigallocatechin-3-gallate (EGCG) maintains κ-casein in its pre-fibrillar state without redirecting its aggregation pathway

The polyphenol (-)-epigallocatechin-3-gallate (EGCG) has recently attracted much research interest in the field of protein-misfolding diseases because of its potent anti-amyloid activity against amyloid-β, α-synuclein and huntingtin, the amyloid-fibril

Gallic acid is the major component of grape seed extract that inhibits amyloid fibril formation

Many protein misfolding diseases, for example, Alzheimer's, Parkinson's and Huntington's, are characterised by the accumulation of protein aggregates in an amyloid fibrillar form. Natural products which inhibit fibril formation are a promising avenue to

Methionine Oxidation Enhances κ-Casein Amyloid Fibril Formation

The effects of protein oxidation, for example of methionine residues, are linked to many diseases, including those of protein misfolding, such as Alzheimer’s disease. Protein misfolding diseases are characterized by the accumulation of insoluble proteinaceous aggregates comprised mainly of amyloid fibrils. Amyloid-containing bodies known as corpora amylacea (CA) are also found in mammary secretory tissue, where their presence slows milk flow. The major milk protein κ-casein readily forms amyloid fibrils under physiological conditions. Milk exists in an extracellular oxidizing environment. Accordingly, the two methionine residues in κ-casein (Met₉₅ and Met₁₀₆) were selectively oxidized and the effects on the fibril-forming propensity, cellular toxicity, chaperone ability, and structure of κ-casein were determined. Oxidation resulted in an increase in the rate of fibril formation and a greater level of cellular toxicity. β-Casein, which inhibits κ-casein fibril formation in vitro, was less effective at suppressing fibril formation of oxidized κ-casein. The ability of κ-casein to prevent the amorphous aggregation of target proteins was slightly enhanced upon methionine oxidation, which may arise from the protein’s greater exposed surface hydrophobicity. No significant changes to κ-casein’s intrinsically disordered structure occurred upon oxidation. The enhanced rate of fibril formation of oxidized κ-casein, coupled with the reduced chaperone ability of β-casein to prevent this aggregation, may affect casein–casein interaction within the casein micelle and thereby promote κ-casein aggregation and contribute to the formation of CA

Das @Rathaus : Zeitschr. für Kommunalpolitik ; das Fachorgan d. Bundesvereinigung Liberaler Kommunalpolitiker (VLK)

When not incorporated into the casein micelle, κ-casein, a major milk protein, rapidly forms amyloid fibrils at physiological pH and temperature. In this study, the effects of milk components (calcium, lactose, lipids, and heparan sulfate) and crowding agents on reduced and carboxymethylated (RCM) κ-casein fibril formation was investigated using far-UV circular dichroism spectroscopy, thioflavin T binding assays, and transmission electron microscopy. Longer-chain phosphatidylcholine lipids, which form the lining of milk ducts and milk fat globules, enhanced RCM κ-casein fibril formation irrespective of whether the lipids were in a monomeric or micellar state, whereas shorter-chain phospholipids and triglycerides had little effect. Heparan sulfate, a component of the milk fat globule membrane and catalyst of amyloid deposition in extracellular tissue, had little effect on the kinetics of RCM κ-casein fibril formation. Major nutritional components such as calcium and lactose also had no significant effect. Macromolecular crowding enhances protein–protein interactions, but in contrast to other fibril-forming species, the extent of RCM κ-casein fibril formation was reduced by the presence of a variety of crowding agents. These data are consistent with a mechanism of κ-casein fibril formation in which the rate-determining step is dissociation from the oligomer to give the highly amyloidogenic monomer. We conclude that the interaction of κ-casein with membrane-associated phospholipids along its secretory pathway may contribute to the development of amyloid deposits in mammary tissue. However, the formation of spherical oligomers such as casein micelles is favored over amyloid fibrils in the crowded environment of milk, within which the occurrence of amyloid fibrils is low

Prostaglandin inhibition of efferocytosis involves COX-2.

<p>U937 cells were incubated in normal RPMI media or SBC-1 supernatant that had been treated with indomethacin. Following 24 hrs incubation, an efferocytosis assay was performed. (n = 5 experiments performed in triplicate). Data presented as box plots as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061573#pone-0061573-g001" target="_blank">Figure 1</a>. Values are presented as percentage of macrophages ingesting apoptotic cells *, p<0.05 compared with RPMI media treatment (one-way ANOVA, Dunnett’s test).</p

Efferocytosis ability of alveolar and lung tissue macrophages.

<p><b>A.</b> Efferocytosis of BAL-derived alveolar macrophages was assessed for controls (‘C’), smokers, current- and ex- smokers with COPD (‘COPD Cur’ and ‘COPD Ex’), COPD subjects with lung cancer (‘COPD Cancer’) and patients with lung cancer and no COPD (‘Cancer’); <b>B.</b> Tissue from Controls (‘C Non-Tumor’) (non-cancer area from patients with cancer/no COPD), ‘COPD Non-Tumor’ (non-cancer area from patients with cancer+COPD), ‘COPD Tumor’ (cancer site from patients with cancer+COPD) and ‘Control Tumor’ (cancer site from patients with cancer/no COPD). *significantly (p<0.05) lower expression vs. controls (non-parametric Kruskal-Wallis test)Box plots present median±25th and 75th percentiles (solid box) with the 10th and 90th percentiles shown by whiskers outside the box.</p

Effect of cancer cell line supernatants on efferocytosis.

<p><b>A.</b> Effect of cancer cell line supernatants on the phagocytosis of apoptotic bronchial epithelial cells by alveolar macrophages. Macrophages from (A) control subjects or (B) subjects with lung cancer were incubated in normal RPMI media or cancer cell line supernatants (H2009, H1466, SBC1) for 24 hrs prior to phagocytosis assay. Values are presented as percentage of macrophages ingesting apoptotic cells *, p< 0.05 compared with RPMI media treatment (n = 5 experiments performed in triplicate; one-way ANOVA, Dunnett’s test). Data presented as box plots as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061573#pone-0061573-g001" target="_blank">Figure 1</a>.</p