A. Anoop

The Parkinson’s Disease-Associated H50Q Mutation Accelerates α-Synuclein Aggregation in Vitro

α-Synuclein (α-Syn) aggregation is directly linked with Parkinsons disease (PD) pathogenesis. Here, we analyzed the aggregation of newly discovered α-Syn missense mutant H50Q in vitro and found that this mutation significantly accelerates the aggregation and amyloid formation of α-Syn. This mutation, however, did not alter the overall secondary structure as suggested by two-dimensional nuclear magnetic resonance and circular dichroism spectroscopy. The initial oligomerization study by cross-linking and chromatographic techniques suggested that this mutant oligomerizes to an extent similar to that of the wild-type α-Syn protein. Understanding the aggregation mechanism of this H50Q mutant may help to establish the aggregation and phenotypic relationship of this novel mutant in PD.

Nanomaterials: amyloids reflect their brighter side

Amyloid fibrils belong to the group of ordered nanostructures that are self-assembled from a wide range of polypeptides/proteins. Amyloids are highly rigid structures possessing a high mechanical strength. Although amyloids have been implicated in the pathogenesis of several human diseases, growing evidence indicates that amyloids may also perform native functions in host organisms. Discovery of such amyloids, referred to as functional amyloids, highlight their possible use in designing novel nanostructure materials. This review summarizes recent advances in the application of amyloids for the development of nanomaterials and prospective applications of such materials in nanotechnology and biomedicine.

CSF Biomarkers for Alzheimer's Disease Diagnosis

Alzheimers disease (AD) is the most common form of dementia that affects several million people worldwide. The major neuropathological hallmarks of AD are the presence of extracellular amyloid plaques that are composed of Aβ40 and Aβ42 and intracellular neurofibrillary tangles (NFT), which is composed of hyperphosphorylated protein Tau. While the amyloid plaques and NFT could define the disease progression involving neuronal loss and dysfunction, significant cognitive decline occurs before their appearance. Although significant advances in neuroimaging techniques provide the structure and physiology of brain of AD cases, the biomarker studies based on cerebrospinal fluid (CSF) and plasma represent the most direct and convenient means to study the disease progression. Biomarkers are useful in detecting the preclinical as well as symptomatic stages of AD. In this paper, we discuss the recent advancements of various biomarkers with particular emphasis on CSF biomarkers for monitoring the early development of AD before significant cognitive dysfunction.

Elucidating the role of disulfide bond on amyloid formation and fibril reversibility of somatostatin-14: Relevance to its storage and secretion

Background: Peptide/protein hormones are stored as amyloids within endocrine secretory granules. Results: Disulfide bond cleavage enhances conformational dynamics and aggregation kinetics in somatostatin-14, resulting in amyloid fibrils with increased resistance to denaturing conditions and decreased reversibility. Conclusion: Disulfide bond could be a key modulating factor in somatostatin-14 amyloid formation associated with secretory granule biogenesis. Significance: Defective disulfide bonding might cause dysregulation of hormone storage/secretion. The storage of protein/peptide hormones within subcellular compartments and subsequent release are crucial for their native function, and hence these processes are intricately regulated in mammalian systems. Several peptide hormones were recently suggested to be stored as amyloids within endocrine secretory granules. This leads to an apparent paradox where storage requires formation of aggregates, and their function requires a supply of non-aggregated peptides on demand. The precise mechanism behind amyloid formation by these hormones and their subsequent release remain an open question. To address this, we examined aggregation and fibril reversibility of a cyclic peptide hormone somatostatin (SST)-14 using various techniques. After proving that SST gets stored as amyloid in vivo, we investigated the role of native structure in modulating its conformational dynamics and self-association by disrupting the disulfide bridge (Cys3–Cys14) in SST. Using two-dimensional NMR, we resolved the initial structure of somatostatin-14 leading to aggregation and further probed its conformational dynamics in silico. The perturbation in native structure (S-S cleavage) led to a significant increase in conformational flexibility and resulted in rapid amyloid formation. The fibrils formed by disulfide-reduced noncyclic SST possess greater resistance to denaturing conditions with decreased monomer releasing potency. MD simulations reveal marked differences in the intermolecular interactions in SST and noncyclic SST providing plausible explanation for differential aggregation and fibril reversibility observed experimentally in these structural variants. Our findings thus emphasize that subtle changes in the native structure of peptide hormone(s) could alter its conformational dynamics and amyloid formation, which might have significant implications on their reversible storage and secretion.

Characterization of Amyloid Formation by Glucagon-Like Peptides: Role of Basic Residues in Heparin-Mediated Aggregation

Glycosaminoglycans (GAGs) have been reported to play a significant role in amyloid formation of a wide range of proteins/peptides either associated with diseases or native biological functions. The exact mechanism by which GAGs influence amyloid formation is not clearly understood. Here, we studied two closely related peptides, glucagon-like peptide 1 (GLP1) and glucagon-like peptide 2 (GLP2), for their amyloid formation in the presence and absence of the representative GAG heparin using various biophysical and computational approaches. We show that the aggregation and amyloid formation by these peptides follow distinct mechanisms: GLP1 follows nucleation-dependent aggregation, whereas GLP2 forms amyloids without any significant lag time. Investigating the role of heparin, we also found that heparin interacts with GLP1, accelerates its aggregation, and gets incorporated within its amyloid fibrils. In contrast, heparin neither affects the aggregation kinetics of GLP2 nor gets embedded within its fibrils. Furthermore, we found that heparin preferentially influences the stability of the GLP1 fibrils over GLP2 fibrils. To understand the specific nature of the interaction of heparin with GLP1 and GLP2, we performed all-atom MD simulations. Our in silico results show that the basic-nonbasic-basic (B-X-B) motif of GLP1 (K28-G29-R30) facilitates the interaction between heparin and peptide monomers. However, the absence of such a motif in GLP2 could be the reason for a significantly lower strength of interaction between GLP2 and heparin. Our study not only helps to understand the role of heparin in inducing protein aggregation but also provides insight into the nature of heparin-protein interaction.

Familial Parkinson Disease-associated Mutations Alter the Site-specific Microenvironment and Dynamics of α-Synuclein

Background: Aggregation of α-Syn is associated with PD pathogenesis. Results: Despite being natively unfolded, a site-specific structure exists in α-Syn that is significantly altered by familial PD-associated E46K, A53T, and A30P mutations. Conclusion: Altered site-specific structure of the PD-associated mutants may attribute to their different aggregation propensity. Significance: This study contributes to understanding the relationship between structure and aggregation of α-Syn. Human α-synuclein (α-Syn) is a natively unstructured protein whose aggregation into amyloid fibrils is associated with Parkinson disease (PD) pathogenesis. Mutations of α-Syn, E46K, A53T, and A30P, have been linked to the familial form of PD. In vitro aggregation studies suggest that increased propensity to form non-fibrillar oligomers is the shared property of these familial PD-associated mutants. However, the structural basis of the altered aggregation propensities of these PD-associated mutants is not yet clear. To understand this, we studied the site-specific structural dynamics of wild type (WT) α-Syn and its three PD mutants (A53T, E46K, and A30P). Tryptophan (Trp) was substituted at the N terminus, central hydrophobic region, and C terminus of all α-Syns. Using various biophysical techniques including time-resolved fluorescence studies, we show that irrespective of similar secondary structure and early oligomerization propensities, familial PD-associated mutations alter the site-specific microenvironment, solvent exposure, and conformational flexibility of the protein. Our results further show that the common structural feature of the three PD-associated mutants is more compact and rigid sites at their N and C termini compared with WT α-Syn that may facilitate the formation of a partially folded intermediate that eventually leads to their increased oligomerization propensities.

Complexation of amyloid fibrils with charged conjugated polymers.

It has been suggested that conjugated charged polymers are amyloid imaging agents and promising therapeutic candidates for neurological disorders. However, very less is known about their efficacy in modulating the amyloid aggregation pathway. Here, we studied the modulation of Parkinsons disease associated α-synuclein (AS) amyloid assembly kinetics using conjugated polyfluorene polymers (PF, cationic; PFS, anionic). We also explored the complexation of these charged polymers with the various AS aggregated species including amyloid fibrils and oligomers using multidisciplinary biophysical techniques. Our data suggests that both polymers irrespective of their different charges in the side chains increase the fibrilization kinetics of AS and also remarkably change the morphology of the resultant amyloid fibrils. Both polymers were incorporated/aligned onto the AS amyloid fibrils as evident from electron microscopy (EM) and atomic force microscopy (AFM), and the resultant complexes were structurally distinct from their pristine form of both polymers and AS supported by FTIR study. Additionally, we observed that the mechanism of interactions between the polymers with different species of AS aggregates were markedly different.

Amyloid formation of growth hormone in presence of zinc: Relevance to its storage in secretory granules.

Amyloids are cross-β-sheet fibrillar aggregates, associated with various human diseases and native functions such as protein/peptide hormone storage inside secretory granules of neuroendocrine cells. In the current study, using amyloid detecting agents, we show that growth hormone (GH) could be stored as amyloid in the pituitary of rat. Moreover, to demonstrate the formation of GH amyloid in vitro, we studied various conditions (solvents, glycosaminoglycans, salts and metal ions) and found that in presence of zinc metal ions (Zn(II)), GH formed short curvy fibrils. The amyloidogenic nature of these fibrils was examined by Thioflavin T binding, Congo Red binding, transmission electron microscopy and X-ray diffraction. Our biophysical studies also suggest that Zn(II) initiates the early oligomerization of GH that eventually facilitates the fibrillation process. Furthermore, using immunofluorescence study of pituitary tissue, we show that GH in pituitary significantly co-localizes with Zn(II), suggesting the probable role of zinc in GH aggregation within secretory granules. We also found that GH amyloid formed in vitro is capable of releasing monomers. The study will help to understand the possible mechanism of GH storage, its regulation and monomer release from the somatotrophs of anterior pituitary.

Cell adhesion on amyloid fibrils lacking integrin recognition motif

Amyloids are highly ordered, cross-β-sheet-rich protein/peptide aggregates associated with both human diseases and native functions. Given the well established ability of amyloids in interacting with cell membranes, we hypothesize that amyloids can serve as universal cell-adhesive substrates. Here, we show that, similar to the extracellular matrix protein collagen, amyloids of various proteins/peptides support attachment and spreading of cells via robust stimulation of integrin expression and formation of integrin-based focal adhesions. Additionally, amyloid fibrils are also capable of immobilizing non-adherent red blood cells through charge-based interactions. Together, our results indicate that both active and passive mechanisms contribute to adhesion on amyloid fibrils. The present data may delineate the functional aspect of cell adhesion on amyloids by various organisms and its involvement in human diseases. Our results also raise the exciting possibility that cell adhesivity might be a generic property of amyloids.

Site-specific fluorescence dynamics of α-synuclein fibrils using time-resolved fluorescence studies: effect of familial Parkinson's disease-associated mutations.

α-Synuclein (α-Syn) aggregation is directly implicated in both the initiation and spreading of Parkinsons Diseases (PD) pathogenesis. Although the familial PD-associated mutations (A53T, E46K, and A30P) are known to affect the aggregation kinetics of α-Syn in vitro, their structural differences in resultant fibrils are largely unknown. In this report we studied the site-specific dynamics of wild type (wt) α-Syn and its three PD mutant fibrils using time-resolved fluorescence intensity, anisotropy decay kinetics, and fluorescence quenching. Our data suggest that the N- and C-terminus are more flexible and exposed compared to the middle non-amyloid-β component (NAC) region of wt and PD mutant α-Syn fibrils. Yet the N-terminus showed great conformational heterogeneity compared to the C-terminus for all these proteins. 71 position of E46K showed more flexibility and solvent exposure compared to other α-Syns, whereas both E46K and A53T fibrils possess a more rigid C-terminus compared to wt and A30P. The present data suggest that wt and PD mutant fibrils possess large differences in flexibility and solvent exposure at different positions, which may contribute to their different pathogenicity in PD.