Interactome network analysis identifies multiple caspase-6 interactors involved in the pathogenesis of HD

Caspase-6 (CASP6) has emerged as an important player in Huntington disease (HD), Alzheimer disease (AD) and cerebral ischemia, where it is activated early in the disease process. CASP6 also plays a key role in axonal degeneration, further underscoring the importance of this protease in neurodegenerative pathways. As a protein's function is modulated by its protein-protein interactions we performed a high throughput yeast-2-hybrid (Y2H) screen against ∼17,000 human proteins to gain further insight into the function of CASP6. We identified a high confidence list of 87 potential CASP6 interactors. From this list, 61% are predicted to contain a CASP6 recognition site. Of nine candidate substrates assessed, six are cleaved by CASP6. Proteins that did not contain a predicted CASP6 recognition site were assessed using a LUMIER assay approach and 51% were further validated as interactors by this method. Of note, 54% of the high-confidence interactors identified show alterations in human HD brain at the mRNA level, and there is a significant enrichment for previously validated huntingtin (HTT) interactors. One protein of interest, STK3, a proapoptotic kinase, was validated biochemically to be a CASP6 substrate. Furthermore, our results demonstrate that in striatal cells expressing mutant huntingtin (mHTT) an increase in full length and fragment levels of STK3 are observed. We further show that caspase-3 is not essential for the endogenous cleavage of STK3. Characterization of the interaction network provides important new information regarding key pathways of interactors of CASP6 and highlights potential novel therapeutic targets for HD, AD and cerebral ischemia

Mutant Exon1 Huntingtin Aggregation is Regulated by T3 Phosphorylation-Induced Structural Changes and Crosstalk between T3 Phosphorylation and Acetylation at K6

Herein, we used protein semisynthesis to investigate, for the first time, the effect of lysine acetylation and phosphorylation, as well as the crosstalk between these modifications on the structure and aggregation of mutant huntingtin exon1 (Httex1). Our results demonstrate that phosphorylation at T3 stabilizes the α-helical conformation of the N-terminal 17 amino acids (Nt17) and significantly inhibits the aggregation of mutant Httex1. Acetylation of single lysine residues, K6, K9 or K15, had no effect on Httex1 aggregation. Interestingly, acetylation at K6, but not at K9 or K15, reversed the inhibitory effect of T3 phosphorylation. Together, our results provide novel insight into the role of Nt17 post-translational modifications in regulating the structure and aggregation of Httex1 and suggest that its aggregation and possibly its function(s) are controlled by regulatory mechanisms involving crosstalk between different PTMs

Identical oligomeric and fibrillar structures captured from the brains of R6/2 and knock-in mouse models of Huntington's disease

Huntington's disease (HD) is a late-onset neurodegenerative disorder that is characterized neuropathologically by the presence of neuropil aggregates and nuclear inclusions. However, the profile of aggregate structures that are present in the brains of HD patients or of HD mouse models and the relative contribution of specific aggregate structures to disease pathogenesis is unknown. We have used the Seprion ligand to develop a highly sensitive enzyme-linked immunosorbent assay (ELISA)-based method for quantifying aggregated polyglutamine in tissues from HD mouse models. We used a combination of electron microscopy, atomic force microscopy (AFM) and sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) to investigate the aggregate structures isolated by the ligand. We found that the oligomeric, proto-fibrillar and fibrillar aggregates extracted from the brains of R6/2 and HdhQ150 knock-in mice were remarkably similar. Using AFM, we determined that the nanometre globular oligomers isolated from the brains of both mouse models have dimensions identical to those generated from recombinant huntingtin exon 1 proteins. Finally, antibodies that detect exon 1 Htt epitopes differentially recognize the ligand-captured material on SDS–PAGE gels. The Seprion-ligand ELISA provides an assay with good statistical power for use in preclinical pharmacodynamic therapeutic trials or to assess the effects of the genetic manipulation of potential therapeutic targets on aggregate load. This, together with the ability to identify a spectrum of aggregate species in HD mouse tissues, will contribute to our understanding of how these structures relate to the pathogenesis of HD and whether their formation can be manipulated for therapeutic benefit

The amyloid fibril-forming properties of the amphibian antimicrobial peptide uperin 3.5

The amphibian skin is a vast resource for bioactive peptides, which form the basis of the animals' innate immune system. Key components of the secretions of the cutaneous glands are antimicrobial peptides (AMPs), which exert their cytotoxic effects often as a result of membrane disruption. It is becoming increasingly evident that there is a link between the mechanism of action of AMPs and amyloidogenic peptides and proteins. In this work, we demonstrate that the broad-spectrum amphibian AMP uperin 3.5, which has a random-coil structure in solution but adopts an α-helical structure in membrane-like environments, forms amyloid fibrils rapidly in solution at neutral pH. These fibrils are cytotoxic to model neuronal cells in a similar fashion to those formed by the proteins implicated in neurodegenerative diseases. The addition of small quantities of 2,2,2-trifluoroethanol accelerates fibril formation by uperin 3.5, and is correlated with a structural stabilisation induced by this co-solvent. Uperin 3.5 fibril formation and the associated cellular toxicity are inhibited by the polyphenol (-)-epigallocatechin-3-gallate (EGCG). Furthermore, EGCG rapidly dissociates fully formed uperin 3.5 fibrils. Ion mobility-mass spectrometry reveals that uperin 3.5 adopts various oligomeric states in solution. Combined, these observations imply that the mechanism of membrane permeability by uperin 3.5 is related to its fibril-forming properties.Antonio N. Calabrese, Yanqin Liu, Tianfang Wang, Ian F. Musgrave, Tara L. Pukala, Rico F. Tabor, Lisandra L. Martin, John A. Carver, John H. Bowi

Supplemental Information 2: Kinetics of alpha-synuclein fibril formation

A range of diseases is associated with amyloid fibril formation. Despite different proteins being responsible for each disease, all of them share similar features including beta-sheet-rich secondary structure and fibril-like protein aggregates. A number of proteins can form amyloid-like fibrils in vitro, resembling structural features of disease-related amyloids. Given these generic structural properties of amyloid and amyloid-like fibrils, generic inhibitors of fibril formation would be of interest for treatment of amyloid diseases. Recently, we identified five outstanding inhibitors of insulin amyloid-like fibril formation among the pool of 265 commercially available flavone derivatives. Here we report testing of these five compounds and of epi-gallocatechine-3-gallate (EGCG) on aggregation of alpha-synuclein and beta-amyloid. We used a Thioflavin T (ThT) fluorescence assay, relying on halftimes of aggregation as the measure of inhibition. This method avoids large numbers of false positive results. Our data indicate that four of the five flavones and EGCG inhibit alpha-synuclein aggregation in a concentration-dependent manner. However none of these derivatives were able to increase halftimes of aggregation of beta-amyloid

Epigallocatechin-3-gallate (EGCG) for Clinical Trials: More Pitfalls than Promises?

Epigallocatechin-3-gallate (EGCG), the main and most significant polyphenol in green tea, has shown numerous health promoting effects acting through different pathways, as antioxidant, anti-inflammatory and anti-atherogenic agent, showing gene expression activity, functioning through growth factor-mediated pathways, the mitogen-activated protein kinase-dependent pathway, the ubiquitin/proteasome degradation pathway, as well as eliciting an amyloid protein remodeling activity. However, epidemiological inferences are sometimes conflicting and in vitro and in vivo studies may seem discrepant. Current knowledge on how to enhance bioavailability could be the answer to some of these issues. Furthermore, dose levels, administration frequency and potential side effects remain to be examined

The green tea polyphenol (-)-epigallocatechin gallate prevents the aggregation of tau protein into toxic oligomers at substoichiometric ratios

The accumulation of amyloid-beta (Abeta) and tau aggregates is a pathological hallmark of Alzheimer's disease. Both polypeptides form fibrillar deposits, but several lines of evidence indicate that Abeta and tau form toxic oligomeric aggregation intermediates. Depleting such structures could thus be a powerful therapeutic strategy. We generated a fragment of tau (His-K18DeltaK280) that forms stable, toxic, oligomeric tau aggregates in vitro. We show that (-)-epigallocatechin gallate (EGCG), a green tea polyphenol that was previously found to reduce Abeta aggregation, inhibits the aggregation of tau K18DeltaK280 into toxic oligomers at ten- to hundred-fold substoichiometric concentrations, thereby rescuing toxicity in neuronal model cells

Morphology-Specific Inhibition of β-Amyloid Aggregates by 17β-Hydroxysteroid Dehydrogenase Type 10

A major hallmark of Alzheimer's disease (AD) is the formation of toxic aggregates of the β-amyloid peptide (Aβ). Given that Aβ peptides are known to localise within mitochondria and interact with 17β-HSD10, a mitochondrial protein expressed at high levels in AD brains, we investigated the inhibitory potential of 17β-HSD10 against Aβ aggregation under a range of physiological conditions. Fluorescence self-quenching (FSQ) of Aβ(1-42) labelled with HiLyte Fluor 555 was used to evaluate the inhibitory effect under conditions established to grow distinct Aβ morphologies. 17β-HSD10 preferentially inhibits the formation of globular and fibrillar-like structures but has no effect on the growth of amorphous plaque-like aggregates at endosomal pH 6. This work provides insights into the dependence of the Aβ-17β-HSD10 interaction with the morphology of Aβ aggregates and how this impacts enzymatic function. 17 β-HSD10 interaction with Aβ amyloid: what type of amyloid? 17β-hydroxysteroid dehydrogenase type 10 interacts with β-amyloid (Aβ) aggregates and suppresses Aβ-induced apoptosis in neurons, but the aggregate morphology inhibited by 17β-HSD10 remains unknown. Fluorescence self-quenching demonstrated that fibrils and globular aggregates, but not plaques, are targeted by 17β-HSD10

Effects of hydroxyl group variations on a flavonoid backbone toward modulation of metal-free and metal-induced amyloid-?? aggregation

Amyloid-?? (A??) and metal ions are suggested to be involved in the pathogenesis of Alzheimer???s disease(AD). Cu(II) and Zn(II) can interact with A?? and facilitate peptide aggregation producing toxic oligomeric peptide species. Additionally, redox-active metal-bound A?? is shown to generate reactive oxygen species(ROS). Although the interaction of metal ions with A?? and the reactivity of metal-associated A?? (metal-A??) are indicated, the relationship between metal-A?? and AD etiology is still unclear. Some naturally occurring flavonoids capable of redirecting metal-A?? peptides into nontoxic, off-pathway A?? aggregates have been presented as valuable tools for elucidating the role of metal-A?? in AD. The structural moieties of the flavonoids responsible for their reactivity toward metal-A?? are not identified, however. To determine a structure-interaction-reactivity relationship between flavonoids and metal-free A?? or metal-A??, four flavonoids (morin, quercetin, galangin, and luteolin) were rationally selected based on structural variations(i.e., number and position of hydroxyl groups). These four flavonoids could noticeably modulate metal-A?? aggregation over metal-free analogue to different extents. Moreover, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) studies reveal that the direct interactions of the flavonoids with metal-free and/or metal-bound A?? are distinct. Overall, our studies demonstrate that alternation of the hydroxyl groups on the B and C rings of flavonoids (structure) could differentiate their metal/metal-free A??/metal-A?? interactions (interaction) and subsequently direct their effects on metal-free A?? and metal-A?? aggregation in vitro and A??-/metal-A??-triggered toxicity in living cells (reactivity), suggesting a structure-interaction-reactivity relationship.open