Peptidic inhibitors of alpha-Synuclein preventing Parkinson's disease-associated fibrilization and cytotoxicity

Blagojce Jovcevski and Giovanni Abbenant

C-Phycocyanin from Spirulina Inhibits alpha-synuclein and amyloid-beta fibril formation but not amorphous aggregation

Proteinopathies including cataracts and neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, are characterized by a series of aberrant protein folding events, resulting in amorphous aggregate or amyloid fibril formation. In the latter case, research has heavily focused on the development of small-molecule inhibitors with limited success during clinical trials. However, very few studies have focused on utilizing exogenous proteins as potential aggregation inhibitors. C-Phycocyanin, derived from Spirulina sp., has been known to exert anti-inflammatory properties; however, the ability of C-phycocyanin to inhibit protein aggregation has yet to be investigated. We have demonstrated that C-phycocyanin is an effective inhibitor of A53Tα-synuclein at extremely low substoichiometric ratios (200-fold excess of α-synuclein) and Aβ40/42 fibril formation. However, C-phycocyanin is relatively ineffective in inhibiting the reduction-induced amorphous aggregation of ADH and heat-induced aggregation of catalase. In addition, 2D NMR, ion mobility-mass spectrometry, and analytical-SEC demonstrate that the interaction between C-phycocyanin and α-synuclein is through nonstable interactions, indicating that transient interactions are likely to be responsible for preventing fibril formation. Overall, this work highlights how biomolecules from natural sources could be used to aid in the development of therapeutics to combat protein misfolding diseases.Yanqin Liu, Blagojce Jovcevski and Tara L. Pukal

Designer D-peptides targeting the N-terminal region of α-synuclein to prevent parkinsonian-associated fibrilization and cytotoxicity

The deposition of α-synuclein (αS) aggregates in the gut and the brain is ever present in cases of Parkinson’s disease. While the central non-amyloidogenic-component (NAC) region of αS plays a critical role in fibrilization, recent studies have identified a specific sequence from within the N-terminal region (NTR, residues 36–42) as a key modulator of αS fibrilization. Due to the lack of effective therapeutics which specifically target αS aggregates, we have developed a strategy to prevent the aggregation and subsequent toxicity attributed to αS fibrilization utilizing NTR targeting peptides. In this study, L- and D-isoforms of a hexa- (VAQKTV-Aib, 77–82 NAC) and heptapeptide (GVLYVGS-Aib, 36–42 NTR) containing a self-recognition component unique to αS, as well as a Cterminal disruption element, were synthesized to target primary sequence regions of αS that modulate fibrilization. The D-peptide that targets the NTR (NTR-TP-D) was shown by ThT fluorescence assays and TEM to be the most effective at preventing fibril formation and elongation, as well as increasing the abundance of soluble monomeric αS. In addition, NTR-TP-D alters the conformation of destabilised monomers into a less aggregationprone state and reduces the hydrophobicity of αS fibrils via fibril remodelling. Furthermore, both NTR-TP isoforms alleviate the cytotoxic effects of αS aggregates in both Neuro-2a and Caco-2 cells. Together, this study highlights how targeting the NTR of αS using D-isoform peptide inhibitors may effectively combat the deleterious effects of αS fibrilization and paves the way for future drug design to utilise such an approach to treat Parkinson’s disease.John R. Horsley, Blagojce Jovcevski, Tara L. Pukala, Andrew D. Abel

The molecular chaperone beta-casein prevents amorphous and fibrillar aggregation of alpha-lactalbumin by stabilisation of dynamic disorder

Deficits in protein homeostasis (proteostasis) are typified by the partial unfolding or misfolding of native proteins leading to amorphous or fibrillar aggregation, events that have been closely associated with diseases including Alzheimer's and Parkinson's Disease. Molecular chaperones are intimately involved in maintaining proteostasis, and their mechanisms of action are in part dependent on the morphology of aggregation-prone proteins. This study utilised native ion-mobility mass spectrometry to provide molecular insights into the conformational properties and dynamics of a model protein, α-lactalbumin (α-LA), which aggregates in an amorphous or amyloid fibrillar manner controlled by appropriate selection of experimental conditions.  The molecular chaperone β-casein (b-CN) is effective at inhibiting amorphous and fibrillar aggregation of α-LA at sub-stoichiometric ratios, with greater efficiency against fibril formation. Analytical size-exclusion chromatography demonstrates the interaction between β-CN and amorphously aggregating α-LA is stable, forming a soluble high molecular weight complex, whilst with fibril-forming α-LA the interaction is transient. Moreover, ion-mobility-mass spectrometry (IM-MS) coupled with collision-induced unfolding (CIU) revealed that α-LA monomers undergo distinct conformational transitions during the initial stages of amorphous (order to disorder) and fibrillar (disorder to order) aggregation. The structural heterogeneity of monomeric α-LA during fibrillation is reduced in the presence of β-CN along with an enhancement in stability, which provides a potential means for preventing fibril formation. Together, this study demonstrates how IM-MS and CIU can investigate the unfolding of proteins as well as examine transient and dynamic protein-chaperone interactions, and thereby provides detailed insight into the mechanism of chaperone action and proteostasis mechanisms.Henry M. Sanders, Blagojce Jovcevski, John A. Carver and Tara L. Pukal

Polyphenol honokiol and flavone 2′,3′,4′-trihydroxyflavone differentially interact with α-synuclein at distinct phases of aggregation

The association between protein aggregation and neurodegenerative diseases such as Parkinson’s disease continues to be well interrogated but poorly elucidated at a mechanistic level. Nevertheless, the formation of amyloid fibrils from the destabilization and misfolding of native proteins is a molecular hallmark of disease. Consequently, there is ongoing demand for the identification and development of small molecules which prevent fibril formation. This study comprehensively assesses the inhibitory properties of two small molecules, the lignan polyphenol honokiol and the flavonoid 2′,3′,4′-trihydroxyflavone, in preventing α-synuclein fibrilization. The data shows that honokiol does not prevent α-synuclein fibril elongation, while 2′,3′,4′-trihydroxyflavone is effective at inhibiting fibril elongation and induces oligomer formation (for both wild-type α-synuclein and the disease-associated A53T mutation). Moreover, the exposed hydrophobicity of α-synuclein fibrils is reduced in the presence of 2′,3′,4′-trihydroxyflavone, whereas the addition of honokiol did not reduce the hydrophobicity of fibrils. In addition, ion mobility–mass spectrometry revealed that the conformation of α-synuclein wild-type and A53T monomers after disassembly is restored to a nonaggregation-prone state upon 2′,3′,4′-trihydroxyflavone treatment. Collectively, this study shows that the mechanisms by which these polyphenols and flavonoids prevent fibril formation are distinct by their interactions at various phases of the fibril-forming pathway. Furthermore, this study highlights how thorough biophysical interrogation of the interaction is required for understanding the ability of inhibitors to prevent protein aggregation associated with disease.Blagojce Jovcevski, Sukanya Das, Scott Smid, and Tara Louise Pukal

Evaluating the effect of phosphorylation on the structure and dynamics of Hsp27 dimers by means of ion mobility mass spectrometry

The quaternary structure and dynamics of the human small heat-shock protein Hsp27 are linked to its molecular chaperone function and influenced by post-translational modifications, including phosphorylation. Phosphorylation of Hsp27 promotes oligomer dissociation and can enhance chaperone activity. This study explored the impact of phosphorylation on the quaternary structure and dynamics of Hsp27. Using mutations that mimic phosphorylation, and ion mobility mass spectrometry, we show that successive substitutions result in an increase in the conformational heterogeneity of Hsp27 dimers. In contrast, we did not detect any changes in the structure of an Hsp27 12-mer, representative of larger Hsp27 oligomers. Our data suggest that oligomer dissociation and increased flexibility of the dimer contribute to the enhanced chaperone activity of phosphorylated Hsp27. Thus, post-translational modifications such as phosphorylation play a crucial role in modulating both the tertiary and quaternary structure of Hsp27, which is pivotal to its function as a key component of the proteostasis network in cells. Our data demonstrate the utility of ion mobility mass spectrometry for probing the structure and dynamics of heterogeneous proteins

Evaluating the effect of phosphorylation on the structure and dynamics of Hsp27 dimers by means of ion mobility mass spectrometry

The quaternary structure and dynamics of the human small heat-shock protein Hsp27 are linked to its molecular chaperone function and influenced by post-translational modifications, including phosphorylation. Phosphorylation of Hsp27 promotes oligomer dissociation and can enhance chaperone activity. This study explored the impact of phosphorylation on the quaternary structure and dynamics of Hsp27. Using mutations that mimic phosphorylation, and ion mobility mass spectrometry, we show that successive substitutions result in an increase in the conformational heterogeneity of Hsp27 dimers. In contrast, we did not detect any changes in the structure of an Hsp27 12-mer, representative of larger Hsp27 oligomers. Our data suggest that oligomer dissociation and increased flexibility of the dimer contribute to the enhanced chaperone activity of phosphorylated Hsp27. Thus, post-translational modifications such as phosphorylation play a crucial role in modulating both the tertiary and quaternary structure of Hsp27, which is pivotal to its function as a key component of the proteostasis network in cells. Our data demonstrate the utility of ion mobility mass spectrometry for probing the structure and dynamics of heterogeneous proteins

The influence of the N-terminal region proximal to the core domain on the assembly and chaperone activity of αB-crystallin

αB-Crystallin (HSPB5) is a small heat-shock protein that is composed of dimers that then assemble into a polydisperse ensemble of oligomers. Oligomerisation is mediated by heterologous interactions between the C-terminal tail of one dimer and the core "α-crystallin" domain of another and stabilised by interactions made by the N-terminal region. Comparatively little is known about the latter contribution, but previous studies have suggested that residues in the region 54-60 form contacts that stabilise the assembly. We have generated mutations in this region (P58A, S59A, S59K, R56S/S59R and an inversion of residues 54-60) to examine their impact on oligomerisation and chaperone activity in vitro. By using native mass spectrometry, we found that all the αB-crystallin mutants were assembly competent, populating similar oligomeric distributions to wild-type, ranging from 16-mers to 30-mers. However, circular dichroism spectroscopy, intrinsic tryptophan and bis-ANS fluorescence studies demonstrated that the secondary structure differs to wild type, the 54-60 inversion mutation having the greatest impact. All the mutants exhibited a dramatic decrease in exposed hydrophobicity. We also found that the mutants in general were equally active as the wild-type protein in inhibiting the amorphous aggregation of insulin and seeded amyloid fibrillation of α-synuclein in vitro, except for the 54-60 inversion mutant, which was significantly less effective at inhibiting insulin aggregation. Our data indicate that alterations in the part of the N-terminal region proximal to the core domain do not drastically affect the oligomerisation of αB-crystallin, reinforcing the robustness of αB-crystallin in functioning as a molecular chaperone

Structural insights into the antifungal drug target guanosine monophosphate synthase from <i>Aspergillus fumigatus</i>

Purine biosynthesis is a fundamental cellular process that sustains life by maintaining the intracellular pool of purines for DNA/RNA synthesis and signal transduction. As an integral determinant of fungal survival and virulence, the enzymes in this metabolic pathway have been pursued as potential antifungal targets. Guanosine monophosphate (GMP) synthase has been identified as an attractive target as it is essential for virulence in the clinically prominent fungal pathogens Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. However, a lack of structural information on GMP synthase has hindered drug-design efforts. Here, the first structure of a GMP synthase of fungal origin, that from A. fumigatus (at 2.3 Å resolution), is presented. Structural analysis of GMP synthase shows a distinct absence of the D1 dimerization domain that is present in the human homologue. Interestingly, A. fumigatus GMP synthase adopts a dimeric state, as determined by native mass spectrometry and gel-filtration chromatography, in contrast to the monomeric human homologue. Analysis of the substrate-binding pockets of A. fumigatus GMP synthase reveals key differences in the ATP- and XMP-binding sites that can be exploited for species-specific inhibitor drug design. Furthermore, the inhibitory activities of the glutamine analogues acivicin (IC50 = 16.6 ± 2.4 µM) and 6-diazo-5-oxo-L-norleucine (IC50 = 29.6 ± 5.6 µM) against A. fumigatus GMP synthase are demonstrated. Together, these data provide crucial structural information required for specifically targeting A. fumigatus GMP synthase for future antifungal drug-discovery endeavours.</jats:p