Co-aggregation and secondary nucleation in the life cycle of human prolactin/galanin functional amyloids.

Synergistic-aggregation and cross-seeding by two different proteins/peptides in the amyloid aggregation are well evident in various neurological disorders including Alzheimer's disease. Here, we show co-storage of human Prolactin (PRL), which is associated with lactation in mammals, and neuropeptide galanin (GAL) as functional amyloids in secretory granules (SGs) of the female rat. Using a wide variety of biophysical studies, we show that irrespective of the difference in sequence and structure, both hormones facilitate their synergic aggregation to amyloid fibrils.

Evolution of CPEB4 Dynamics Across its Liquid-Liquid Phase Separation Transition.

Knowledge about the structural and dynamic properties of proteins that form membrane-less organelles in cells via liquid-liquid phase separation (LLPS) is required for understanding the process at a molecular level. We used spin labeling and electron paramagnetic resonance (EPR) spectroscopy to investigate the dynamic properties (rotational diffusion) of the low complexity N-terminal domain of cytoplasmic polyadenylation element binding-4 protein (CPEB4) across its LLPS transition, which takes place with increasing temperature. We report the coexistence of three spin labeled CPEB4 (CPEB4*) populations with distinct dynamic properties representing different conformational spaces, both before and within the LLPS state.

Extraction and Dissection of the Domesticated Pig Brain.

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular systems, gastrointestinal systems, and nutrition, and the pig is increasingly being used as a large animal model in neuroscience. Furthermore, the pig is an accepted model to study neurodevelopment as it displays brain growth and development patterns similar to what occurs in humans. As a less common animal model in neuroscience, surgical and dissection procedures on pigs may not be as familiar or well-practiced among researchers.

α-Synuclein plasma membrane localization correlates with cellular phosphatidylinositol polyphosphate levels.

The Parkinson's disease protein α-synuclein (αSyn) promotes membrane fusion and fission by interacting with various negatively charged phospholipids. Despite postulated roles in endocytosis and exocytosis, plasma membrane (PM) interactions of αSyn are poorly understood. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP) and phosphatidylinositol 3,4,5-trisphosphate (PIP), two highly acidic components of inner PM leaflets, mediate PM localization of endogenous pools of αSyn in A2780, HeLa, SK-MEL-2, and differentiated and undifferentiated neuronal SH-SY5Y cells.

An NMR-Based Biosensor to Measure Stereospecific Methionine Sulfoxide Reductase Activities in Vitro and in Vivo*.

Oxidation of protein methionines to methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to methionine by two families of conserved methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the S- or R-diastereoisomers of methionine-sulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups.

Protein Nanofibrils as Storage Forms of Peptide Drugs and Hormones.

Amyloids are highly organized cross β-sheet protein nanofibrils that are associated with both diseases and functions. Thermodynamically amyloids are stable structures as they represent the lowest free energy state that proteins can attain. However, recent studies suggest that amyloid fibrils can be dissociated by a change in environmental parameters such as pH and ionic strength.

Amyloids Are Novel Cell-Adhesive Matrices.

Amyloids are highly ordered peptide/protein aggregates traditionally associated with multiple human diseases including neurodegenerative disorders. However, recent studies suggest that amyloids can also perform several biological functions in organisms varying from bacteria to mammals. In many lower organisms, amyloid fibrils function as adhesives due to their unique surface topography.

Time-Resolved NMR Analysis of Proteolytic α-Synuclein Processing in vitro and in cellulo.

Targeted proteolysis of the disordered Parkinson's disease protein alpha-synuclein (αSyn) constitutes an important event under physiological and pathological cell conditions. In this work, site-specific αSyn cleavage by different endopeptidases in vitro and by endogenous proteases in extracts of challenged and unchallenged cells was studied by time-resolved NMR spectroscopy. Specifically, proteolytic processing was monitored under neutral and low pH conditions and in response to Rotenone-induced oxidative stress.

Glycosaminoglycans have variable effects on α-synuclein aggregation and differentially affect the activities of the resulting amyloid fibrils.

Parkinson's disease is mainly a sporadic disorder in which both environmental and cellular factors play a major role in the initiation of this disease. Glycosaminoglycans (GAG) are integral components of the extracellular matrix and are known to influence amyloid aggregation of several proteins, including α-synuclein (α-Syn). However, the mechanism by which different GAGs and related biological polymers influence protein aggregation and the structure and intercellular spread of these aggregates remains elusive.

Amyloid Fibrils: Versatile Biomaterials for Cell Adhesion and Tissue Engineering Applications.

Extracellular matrices (ECM) play an enormous role in any living system, controlling various factors and eventually fates of cells. ECM regulates cell fate by providing constant exogenous signals altering intracellular signal transduction for diverse pathways including proliferation, migration, differentiation, and apoptosis. Biomaterial scaffolds are designed to mimic the natural extracellular matrix such that the cells could recapitulate natural events alike their natural niche.

p53 amyloid formation leading to its loss of function: implications in cancer pathogenesis.

The transcriptional regulator p53 has an essential role in tumor suppression. Almost 50% of human cancers are associated with the loss of p53 functions, where p53 often accumulates in the nucleus as well as in cytoplasm. Although it has been previously suggested that amyloid formation could be a cause of p53 loss-of-function in subset of tumors, the characterization of these amyloids and its structure-function relationship is not yet established.

Comparison of Brain Development in Sow-Reared and Artificially Reared Piglets.

Introduction: Provision of adequate nutrients is critical for proper growth and development of the neonate, yet the impact of breastfeeding versus formula feeding on neural maturation has to be fully determined. Using the piglet as a model for the human infant, our objective was to compare neurodevelopment of piglets that were either sow-reared (SR) or artificially reared (AR) in an artificial setting.

Methods: Over a 25-day feeding study, piglets (1.

Effect of curcumin analogs onα-synuclein aggregation and cytotoxicity.

Alpha-synuclein (α-Syn) aggregation into oligomers and fibrils is associated with dopaminergic neuron loss occurring in Parkinson's disease (PD) pathogenesis. Compounds that modulate α-Syn aggregation and interact with preformed fibrils/oligomers and convert them to less toxic species could have promising applications in the drug development efforts against PD. Curcumin is one of the Asian food ingredient which showed promising role as therapeutic agent against many neurological disorders including PD.

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.

Atypical profiles and modulations of heme-enzymes catalyzed outcomes by low amounts of diverse additives suggest diffusible radicals' obligatory involvement in such redox reactions.

Background: Peroxidations mediated by heme-enzymes have been traditionally studied under a single-site (heme distal pocket), non-sequential (ping-pong), two-substrates binding scheme of Michaelis-Menten paradigm. We had reported unusual modulations of peroxidase and P450 reaction outcomes and explained it invoking diffusible reactive species [Manoj, 2006; Manoj et al., 2010; Andrew et al.

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.

Influence of retinoic acid on mesenchymal stem cell differentiation in amyloid hydrogels.

This paper presents data related to the research article "Self healing hydrogels composed of amyloid nano fibrils for cell culture and stem cell differentiation" [1]. Here we probed the collective influence of all-trans retinoic acid (RA) and substrate properties (amyloid hydrogel) on human mesenchymal stem cell (hMSC) differentiation. Stem cells were cultured on soft amyloid hydrogels [1], [2] in the presence and absence of matrix encapsulated RA.

Self healing hydrogels composed of amyloid nano fibrils for cell culture and stem cell differentiation.

Amyloids are highly ordered protein/peptide aggregates associated with human diseases as well as various native biological functions. Given the diverse range of physiochemical properties of amyloids, we hypothesized that higher order amyloid self-assembly could be used for fabricating novel hydrogels for biomaterial applications. For proof of concept, we designed a series of peptides based on the high aggregation prone C-terminus of Aβ42, which is associated with Alzheimer's disease.

Cytotoxic helix-rich oligomer formation by melittin and pancreatic polypeptide.

Conversion of amyloid fibrils by many peptides/proteins involves cytotoxic helix-rich oligomers. However, their toxicity and biophysical studies remain largely unknown due to their highly dynamic nature. To address this, we chose two helical peptides (melittin, Mel and pancreatic polypeptide, PP) and studied their aggregation and toxicity.

Structure based aggregation studies reveal the presence of helix-rich intermediate during α-Synuclein aggregation.

Mechanistic understanding of nucleation dependent polymerization by α-synuclein (α-Syn) into toxic oligomers and amyloids is important for the drug development against Parkinson's disease. However the structural and morphological characterization during nucleation and subsequent fibrillation process of α-Syn is not clearly understood. Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates.

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

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 Parkinson's disease associated α-synuclein (AS) amyloid assembly kinetics using conjugated polyfluorene polymers (PF, cationic; PFS, anionic).

CSF Biomarkers for Alzheimer's Disease Diagnosis.

Alzheimer's 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 Abeta40 and Abeta42 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.