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Protein aggregation is a significant challenge in the development of monoclonal antibodies (mAbs), which can be exacerbated by stress conditions encountered along its production pipeline. In this study, we examine how thermal and pH stress conditions influence mAb aggregation mechanisms. We observe a complex interplay between these factors that significantly affects mAb stability, particularly under combined stress conditions. The mAb aggregates formed also varied distinctly in size and properties depending on the pH and thermal conditions, suggesting differences in their underlying mechanisms. Using a combination of experimental methods and kinetic modelling, we found that acidic pH conditions primarily promoted aggregation via the mAb unfolding step, while higher temperature conditions facilitated the formation of larger aggregates via monomer-independent cluster-cluster aggregation steps. These insights underscore the importance of extrinsic stress conditions in determining mAb aggregation propensity, and potentially provides a quantitative framework to holistically assess this across various accelerated stress conditions for the development of stable biologics. [ABSTRACT FROM AUTHOR]

Fibrinogen (Fg), an essential plasma glycoprotein involved in the coagulation cascade, undergoes structural alterations upon exposure to various chemicals, impacting its functionality and contributing to pathological conditions. This research article explored the effects of 4-Chloro- o -phenylenediamine (4-Cl-o-PD), a common hair dye component (IUPAC = 1-Chloro-3,4-diaminobenzene), on human fibrinogen through comprehensive computational, biophysical, and biochemical approaches. The formation of a stable ligand-protein complex is confirmed through molecular docking and molecular dynamics simulations, revealing possible interaction having a favorable −4.8 kcal/mol binding energy. Biophysical results, including UV–vis and fluorescence spectroscopies, corroborated with the computational findings, whereas Fourier transform infrared spectroscopy (FT-IR) and circular dichroism spectroscopy (CD) provide insights into the alterations of secondary structures upon interaction with 4-Cl-o-PD. Anilinonaphthalene-sulfonic acid (ANS) fluorescence showed a partially unfolded protein, with enhanced α to β-sheet transition as evidenced by thioflavin T (ThT) spectroscopy and microscopy. Moreover, biochemical assays confirmed the formation of carbonyl compounds that may be responsible for the oxidation of methionine residues in fibrinogen. Electrophoresis and electron microscopy confirmed the formation of aggregates. Our findings elucidate the interaction pattern of 4-Cl-o-PD with Fg, leading to structural perturbation, which may have potential implications for fibrinogen misfolding or its aggregation. Protein aggregation or its misfolded products affect peripheral tissues and the central nervous system. Many chronic progressive diseases, like type II diabetes mellitus, Alzheimer's disease, Parkison's disease, and Creutzfeldt-Jakob disease are associated with intrinsically aberrant disordered proteins. Understanding these interactions may offer new perspectives on the safety and biocompatibility of dye compounds, which may contribute to developing improved strategies for acquired amyloidogenesis. [ABSTRACT FROM AUTHOR]

Lignin-carbohydrate complexes (LCCs) represent a group of macromolecules with diverse biological functions such as antioxidative properties. Polyglutamine (polyQ) diseases such as spinocerebellar ataxia type 3 (SCA3) comprise a set of neurodegenerative disorders characterized by the formation of polyQ protein aggregates in patient neurons. LCCs have been reported to prevent such protein aggregation. In this study, we identified a potential mechanism underlying the above anti-protein aggregation activity. We isolated and characterized multiple LCC fractions from bamboo and poplar and found that lignin-rich LCCs (BM-LCC-AcOH and PR-LCC-AcOH) effectively eliminated both monomeric and aggregated mutant ataxin-3 (ATXN3polyQ) proteins in neuronal cells and a Drosophila melanogaster SCA3 disease model. In addition, treatment with BM-LCC-AcOH or PR-LCC-AcOH rescued photoreceptor degeneration in vivo. At the mechanistic level, we demonstrated that BM-LCC-AcOH and PR-LCC-AcOH upregulated proteasomal activity. When proteasomal function was impaired, the ability of the LCCs to suppress ATXN3polyQ aggregation was abolished. Thus, we identified a previously undescribed proteasome-inducing function of LCCs and showed that such activity is indispensable for the beneficial effects of LCCs on SCA3 neurotoxicity. [ABSTRACT FROM AUTHOR]

Double PHD fingers 3 (DPF3) is a zinc finger protein, found in the BAF chromatin remodelling complex, and is involved in the regulation of gene expression. Two DPF3 isoforms have been identified, respectively named DPF3b and DPF3a. Very limited structural information is available for these isoforms, and their specific functionality still remains poorly studied. In a previous work, we have demonstrated the first evidence of DPF3a being a disordered protein sensitive to amyloid fibrillation. Intrinsically disordered proteins (IDPs) lack a defined tertiary structure, existing as a dynamic conformational ensemble, allowing them to act as hubs in protein-protein interaction networks. In the present study, we have more thoroughly characterised DPF3a in vitro behaviour, as well as unravelled and compared the structural properties of the DPF3b isoform, using an array of predictors and biophysical techniques. Predictions, spectroscopy, and dynamic light scattering have revealed a high content in disorder: prevalence of random coil, aromatic residues partially to fully exposed to the solvent, and large hydrodynamic diameters. DPF3a appears to be more disordered than DPF3b, and exhibits more expanded conformations. Furthermore, we have shown that they both time-dependently aggregate into amyloid fibrils, as revealed by typical circular dichroism, deep-blue autofluorescence, and amyloid-dye binding assay fingerprints. Although spectroscopic and microscopic analyses have unveiled that they share a similar aggregation pathway, DPF3a fibrillates at a faster rate, likely through reordering of its C-terminal domain. [ABSTRACT FROM AUTHOR]

Previously, N -acetyl- l -arginine (NALA) suppressed the aggregation of intravenous immunoglobulins (IVIG) more effectively and with a minimum decrease in transition temperature (T m) than arginine monohydrochloride. In this study, we performed a comparative study with etanercept (commercial product: Enbrel®), where 25 mM arginine monohydrochloride (arginine) was added to the prefilled syringe. The biophysical properties were investigated using differential scanning calorimetry (DSC), dynamic light scattering (DLS), size-exclusion chromatography (SEC), and flow-imaging microscopy (FI). NALA retained the transition temperature of etanercept better than arginine, where arginine significantly reduced the T m by increasing its concentration. End-over-end rotation was applied to each formulation for 5 days to accelerate protein aggregation and subvisible particle formation. Higher monomeric content was retained with NALA with a decrease in particle level. Higher aggregation onset temperature (T agg) was detected for etanercept with NALA than arginine. The results of this comparative study were consistent with previous study, suggesting that NALA could be a better excipient for liquid protein formulations. Agitated IVIG and etanercept were injected into C57BL/6J female mice to observe immunogenic response after 24 h. In the presence of silicone oil, NALA dramatically reduced IL-1 expression, implying that decreased aggregation was related to reduced immunogenicity of both etanercept and IVIG. [Display omitted] [ABSTRACT FROM AUTHOR]

The interaction of human proteins and metal species, both ions and nanoparticles, is poorly understood despite their growing importance. These materials are the by-products of corrosion processes and are of relevance for food and drug manufacturing, nanomedicine, and biomedical implant corrosion. Here, we study the interaction of Cr(III) ions and chromium oxide nanoparticles with bovine serum albumin in physiological conditions. This study combined electrophoretic mobility measurements, spectroscopy, and time-of-flight secondary ion mass spectrometry with principal component analysis. It was determined that neither metal species resulted in global albumin unfolding. The Cr(III) ions interacted strongly with amino acids found in previously discovered metal binding sites, but also were most strongly implicated in the interaction with negatively charged acid residues, suggesting an electrostatic interaction. Bovine serum albumin (BSA) was found to bind to the Cr 2 O 3 nanoparticles in a preferential orientation, due to electrostatic interactions between the positive amino acid residues and the negative chromium oxide nanoparticle surface. These findings ameliorate our understanding of the interaction between trivalent chromium ions and nanoparticles, and biological macromolecules. [Display omitted] • Corrosion of implants can release both chromium ions and nanoparticles. • Chromium ions and chromium oxide nanoparticles interact with albumin differently. • Trivalent chromium ions induce albumin aggregation through salt-bridging. • Chromium oxide nanoparticles also cause serum albumin aggregation. • Chromium oxide nanoparticles develop an albumin corona through electrostatic forces. [ABSTRACT FROM AUTHOR]

Congenital cataracts, a prevalent cause of blindness in children, are associated with protein aggregation. γD-crystallin, essential for sustaining lens transparency, exists as a monomer and exhibits excellent structural stability. In our cohort, we identified a nonsense mutation (c.451_452insGACT, p.Y151X) in the CRYGD gene. To explore the effect of truncation mutations on the structure of γD-crystallin, we examined the Y151X and T160RfsX8 mutations, both located in the Greek key motif 4 at the cellular and protein level in this study. Both truncation mutations induced protein misfolding and resulted in the formation of insoluble aggregates when overexpressed in HLE B3 and HEK 293T cells. Moreover, heat, UV irradiation, and oxidative stress increased the proportion of aggregates of mutants in the cells. We next purified γD-crystallin to estimate its structural changes. Truncation mutations led to conformational disruption and a concomitant decrease in protein solubility. Molecular dynamics simulations further demonstrated that partial deletion of the conserved domain within the Greek key motif 4 markedly compromised the overall stability of the protein structure. Finally, co-expression of α-crystallins facilitated the proper folding of truncated mutants and mitigated protein aggregation. In summary, the structural integrity of the Greek key motif 4 in γD-crystallin is crucial for overall structural stability. [Display omitted] • A nonsense mutation (c.451_452insGACT, p.Y151X) in CRYGD gene was identified in the pedigree of congenital nuclear cataracts • Both the truncation mutations Y151X and T160RfsX8 caused the exposure of the hydrophobic core within the Greek key motif 4 and disrupted the overall structural stability, which resulted in protein aggregation • Upregulating the expression of α-crystallin could mitigate the formation of intracellular aggregates induced by the truncated mutants Y151X and T160RfsX8 [ABSTRACT FROM AUTHOR]

The robust use of osmolytes (i.e., polyols and sugars) in the key therapeutic regimens/formulations has questioned their impact beyond the stability of therapeutic proteins as these osmolytes trigger structural alterations into proteins including misfolding and subsequent aggregation into amyloid fibrils. Therefore, the current study is the first to delineate the inhibitory effect of carvacrol (CRV) on the carbonyl osmolyte-induced aggregation as well as structural alterations to the bovine serum albumin (BSA) via a set of physicochemical as well as artificial intelligence (AI)-based molecular docking studies. Our initial findings from physicochemical investigations revealed that CRV exhibits substantial protection to BSA under carbonyl osmolyte stress as evident by the compromised hyperchromicity, Schiff's bases, carbonyl and hydroxymethyl furfural content, reduced fluorescent signals, low Rayleigh scattering and prevention of covalent modifications at Lys and Arg residues. The protection against aggregate formation by CRV was further confirmed through the reduced amyloid-specific congo red absorbance as well as fluorescent signals recorded after adding the fibril-specific extrinsic fluorophore probes (i.e., ThT and ANS). The AI-based molecular docking analysis further revealed that CRV (ΔG: −4.96 kcal/mol) competes with d -fructose (ΔG: −4.40 kcal/mol) to mask the Lys and Arg residues to restrict the osmolyte-mediated protein modifications. In conclusion, CRV exhibits substantial protective impact against carbonyl osmolyte-induced structural alterations and protein misfolding and aggregation. [ABSTRACT FROM AUTHOR]

Cataract is the most common pathogenic ophthalmic disease leading to blindness in children worldwide. Genetic disorder is the leading cause of congenital cataract, among which crystallin mutations have a high incidence. There are few reports on γA-crystallin, one critical member of crystallin superfamilies. In this study, we identified a novel pathogenic mutation (Ile82Met) in γA-crystallin from a three-generation Chinese family with cataract, and investigated the potential molecular mechanism in detail. To elucidate the pathogenic mechanism of I82M mutant, spectroscopic and solubility experiments were performed to determine the difference between the purified γA-crystallin wild type (WT) and I82M mutant under both physiological conditions and environmental stresses (UV irradiation, thermal denaturation or chemical denaturation). The I82M mutant did not affect the secondary/tertiary structure of monomeric γA-crystallin under physiological status, but decreased protein stability and increased aggregatory potency under the stressful treatment. Surprisingly, the chemical denaturation caused I82M to switch from the two-state unfolding of γA-crystallin to three-state unfolding involving an unfolding intermediate. This study expands the genetic variation map of cataract, and provides novel insights into the pathomechanism, in particular, filling in a gap in the understanding of γA-crystallin mutants causing cataract. [ABSTRACT FROM AUTHOR]

Intraneuronal neurofibrillary tangles composed of Tau aggregates have been widely accepted as an important pathological hallmark of Alzheimer's disease. Liquid-liquid phase separation (LLPS) of Tau can lead to its aggregation, and Tau aggregation can then be enhanced by zinc. However, it is unclear whether zinc modulates the formation of Tau stress granules in cells. We herein report that zinc promotes the formation of stress granules containing a pathological mutant ΔK280 of full-length human Tau. Furthermore, zinc promotes LLPS of ΔK280 of full-length Tau, shifting the equilibrium phase boundary to a lower protein concentration, and modulates the liquid nature of droplets formed by this pathological mutation. Zinc also promotes pathological phosphorylation of ΔK280 in neuronal cells, and aggravates mitochondrial damage and elevates reactive oxygen species production induced by Tau aggregation. Importantly, we show that treatment of cells with zinc increases the interaction between full-length Tau and G3BP1 inside stress granules to promote the formation of Tau filaments and increase Tau toxicity in neuronal cells. Collectively, these results demonstrate how Tau condensation and mitochondrial damages induced by Tau aggregation are enhanced by zinc to deteriorate the pathogenesis of Alzheimer's disease, bridging the gap between Tau LLPS and aggregation in neuronal cells. [ABSTRACT FROM AUTHOR]

As a major microtubule-associated protein, tau is involved in the assembly of microtubules in the central nervous system. However, under pathological conditions tau assembles into amyloid filaments. Liquid droplets formed by liquid−liquid phase separation (LLPS) are a recently identified assembly state of tau and may have a major effect on the physiological function of tau and the formation of tau aggregates. 14-3-3 proteins are ubiquitously expressed in various tissues and regulate a wide variety of biological processes. In this work, we demonstrate that 14-3-3ζ is recruited into tau droplets and regulates tau LLPS by in vitro assays. While the mobility of tau molecules inside the droplets is not affected in the presence of 14-3-3ζ, the amount and size of droplets can vary significantly. Mechanistic studies reveal that 14-3-3ζ regulates tau LLPS by electrostatic interactions and hydrophobic interactions with the proline-rich domain and the microtubule-binding domain of tau. Surprisingly, the disordered C-terminal tail rather than the amphipathic binding groove of 14-3-3ζ plays a key role. Our findings not only provide a novel dimension to understand the interactions between 14-3-3 proteins and tau, but also suggest that 14-3-3 proteins may play an important role in regulating the LLPS of their binding partners. [ABSTRACT FROM AUTHOR]

The formation of biomolecular condensates has emerged as a crucial player both in neuronal physiology and neurodegeneration. Phase separation of the Alzheimer's related protein tau into liquid condensates is facilitated by polyanions and is regulated by post-translational modifications. Given the central role of ubiquitination in proteostasis regulation and signaling, we investigated the behavior of monoubiquitinated tau during formation of condensates. We ubiquitinated the lysine-rich, four-repeat domain of tau either unspecifically via enzymatic conjugation or in a position-specific manner by semisynthesis. Ubiquitin conjugation at specific sites weakened multivalent tau/RNA interactions and disfavored tau/heparin condensation. Yet, heterogeneous ubiquitination was tolerated during phase separation and stabilized droplets against aggregation-linked dissolution. Thus, we demonstrated that cofactor chemistry and site of modification affect the mesoscopic and molecular signatures of ubiquitinated tau condensates. Our findings suggest that ubiquitination could influence the physiological states and pathological transformations of tau in cellular condensates. [Display omitted] • We ubiquitinated the tau repeat domain via enzymatic conjugation or semisynthesis. • Anionic cofactor-induced condensation of tau tolerates heterogeneous ubiquitination. • Enzymatic ubiquitination stabilizes droplets against aggregation-linked dissolution. • The site of ubiquitination affects the molecular signatures of tau condensates. [ABSTRACT FROM AUTHOR]

• Effect of SY over HSA in an in vitro manner was investigated. • HSA (15 μM) was incubated for 6 days at 65°C. 30-180 μM SY was used in the study. • SY showed decrease in fluorescence intensity with the increasing dye concentration. • Molecular docking was performed to understand the interacting residues. • Maximum inhibition was found to be in presence of 180 μM of sunset yellow. Intensive research in the field of protein aggregation confirmed that the deposition of amyloid fibrils of proteins are the major cause for the development of various neurotoxic and neurodegenerative diseases, which could be controlled by ensuring the efficient inhibition of aggregation using anti aggregation strategies. Herein, we elaborated the anti amyloidogenic potential of Sunset Yellow (SY) dye against Human Serum Albumin (HSA) fibrillogenesis utilising different biophysical, computational and microscopic techniques. The inhibitory effect of sunset yellow was confirmed by Rayleigh Light Scattering (RLS) measurements along with different dye binding assays (ANS, ThT and CR) by showing concentration dependent reduction in scattering intensity and fluorescence intensity respectively. Further, destabilization and anti fibrillation activity of HSA aggregates were characterized through spectroscopic techniques like Circular Dichroism (CD) and other microscopic techniques like Transmission Electron Microscopy (TEM) for elucidating the structural properties. The SDS-PAGE was also carried out that render the disaggregation effect of the dye on the protein. Moreover, Molecular Docking studies revealed the binding parameters justifying the stable protein-dye complex. Simulation studies were also performed accordingly. Thus, this dye which is used as food additive can serve as a potential aggregation inhibiting agent that can aid in the prevention of amyloidogenic diseases. [ABSTRACT FROM AUTHOR]

Peptidyl prolyl isomerases (PPIases) accelerate the rate limiting step of protein folding by catalyzing cis/trans isomerization of peptidyl prolyl bonds. The larger PPIases have been shown to be multi-domain proteins, with functions other than isomerization of the proline-containing peptide bond. Recently, a few smaller PPIases have also been described for their ability to stabilize folding intermediates. The yeast Fpr1 (FK506-sensitive proline rotamase) is a homologue of the mammalian prolyl isomerase FKBP12 (FK506-binding protein of 12 kDa). Its ability to stabilize stressed cellular proteins has not been reported yet. We had earlier reported upregulation of Fpr1 in yeast cells exposed to proteotoxic stress conditions. In this work, we show that yeast Fpr1 exhibits characteristics typical of a general chaperone of the proteostasis network. Aggregation of mutant huntingtin fragment was higher in Fpr1 -deleted as compared to parental yeast cells. Overexpression of Fpr1 led to reduced protein aggregation by decreasing the amount of oligomers and diverting the aggregation pathway towards the formation of detergent-soluble species. This correlated well with higher survival of these cells. Purified and enzymatically active yeast Fpr1 was able to inhibit aggregation of mutant huntingtin fragment and luciferase in vitro in a concentration-dependent manner; suggesting a direct action for aggregation inhibitory action of Fpr1. Overexpression of yeast Fpr1 was able to protect E. coli cells against thermal shock. This work establishes the role of Fpr1 in the protein folding network and will be used for the identification of novel pharmacological leads in disease conditions. [ABSTRACT FROM AUTHOR]

The loss of crystallins solubility with aging and the formation of amyloid-like aggregates is considered the hallmark characteristic of cataract pathology. The present study was carried out to assess the effect of temperature on the soluble lens protein and the formation of protein aggregates with typical amyloid characteristics. The soluble fraction of lens proteins was subjected for heat treatment in the range of 40–60 °C, and the nature of protein aggregates was assessed by using Congo red (CR), thioflavin T (ThT), and 8-anilinonaphthalene-1-sulfonic acid (ANS) binding assays, circular dichroism (CD), Fourier-transform infrared (FT-IR) spectroscopy, and transmission electron microscopy (TEM). The heat-treated protein samples displayed a substantial bathochromic shift (≈15 nm) in the CR's absorption maximum (λ max) and increased ThT and ANS binding. The heat treatment of lens soluble proteins results in the formation of nontoxic, β-sheet rich, non-fibrillar, protein aggregates similar to the structures evident in the insoluble fraction of proteins isolated from the cataractous lens. The data obtained from the present study suggest that the exposure of soluble lens proteins to elevated temperature leads to the formation of non-fibrillar aggregates, establishing the role of amyloid in the heat-induced augmentation of cataracts pathology. Schematic model of heat-induced aggregation of the soluble fraction of lens proteins and their amyloid-like characteristics. The total proteins in an age-related cataract lens are partitioned into a soluble and insoluble fraction of proteins. The insoluble fraction contains large aggregates that might have formed through amyloid-like aggregation or sequestration of β-sheet rich crystallins, displaying typical amyloid characteristics. On the contrary, the soluble fraction of proteins might consist of soluble amyloid oligomers along with the native crystallins and other lens proteins, which upon heat treatment, sequestered as amyloids. Although both the aggregates display typical amyloid characteristics and identical morphology, they significantly differ in their stability in the presence of SDS. The heat-induced aggregated soluble fraction of protein gets disaggregated in the presence of SDS, whereas the insoluble fraction of protein remains stable. [Display omitted] [ABSTRACT FROM AUTHOR]

Hen egg white lysozyme (HEWL) is a structural homolog of human lysozyme and is widely used as a model protein to investigate protein aggregation. The effect of N-benzylisatin on stress-induced aggregation of HEWL has been investigated in the present study. Interaction of the isatin derivative with HEWL induced changes in protein secondary and tertiary structural conformation as evident from different biophysical and spectroscopic studies. In addition, modification with N-benzylisatin was found to increase the conformational stability of HEWL and afford considerable resistance to the protein to stress-induced aggregation as indicated from subsequent experimental studies, including thioflavin T fluorescence, microscopic imaging and dynamic light scattering analysis. Protein modification was analysed and confirmed by MALDI-TOF and ESI-MS studies. The results highlight possible clinical implications of isatin derivative in the treatment of protein misfolding and conformational disorders. • Interaction with N-benzylisatin causes structural changes of hen egg white lysozyme. • N-benzylisatin induces changes in surface hydrophobicity of hen egg white lysozyme. • N-benzylisatin induced modification of hen egg white lysozyme was analysed by mass spectrometric studies. • N-benzylisatin modification increases conformational stability of hen egg white lysozyme. • N-benzylisatin modification restricts stress-induced aggregation of hen egg white lysozyme. [ABSTRACT FROM AUTHOR]

Intrinsically disordered proteins (IDPs), involved in the regulation and function of various cellular processes like transcription, translation, cell cycle etc., exist as ensembles of rapidly interconverting structures with functional plasticity. Among numerous cellular regulatory mechanisms involved in structural and functional regulation of IDPs, osmolytes are emerging as promising regulatory agents due to their ability to affect the structure-function integrity of IDPs. The present study investigated the effect of methylamine osmolytes on β-casein, an IDP essential for maintaining the overall stability of casein complex in milk. It was observed that trimethylamine N-oxide induces a compact structural state in β-casein with slightly decreased chaperone activity and insignificant aggregation propensity. However, the other two osmolytes from this group, i.e., sarcosine and betaine, had no significant effect on the overall structure and chaperone activity of the IDP. The present study hints towards the possible evolutionary selection of higher structural disorder in β-casein, compared to α-casein, for stability of the casein complex and prevention of amyloidosis in the mammary gland. [ABSTRACT FROM AUTHOR]

Alzheimer's disease is a neurodegenerative disease which severely impacts the health of the elderly. Current treatments are only able to alleviate symptoms, but not prevent or cure the disease. The neurofibrillary tangles formed by tau protein aggregation are one of the defining characteristics of Alzheimer's disease, so tau protein has become a key target for the drug design. In this study, we show that fisetin, a plant-derived polyphenol compound, can inhibit aggregation of the tau fragment, K18, and can disaggregate tau K18 filaments in vitro. Meanwhile it is able to prevent the formation of tau aggregates in cells. Both experimental and computational studies indicate that fisetin could directly interact with tau K18 protein. The binding is mainly created by hydrogen bond and van der Waal force, prevents the formation of β-strands at the two hexapeptide motifs, and does not perturb the secondary structure or the tubulin binding ability of tau protein. In summary, fisetin might be a candidate for further development as a potential preventive or therapeutic drug for Alzheimer's disease. • Fisetin inhibits tau aggregation in vitro and in cells. • Fisetin interacts with tau protein through favorable hydrogen bonds and van der Waals. • Fisetin prevents the formation of β-strands at the two critical hexapeptide motifs. [ABSTRACT FROM AUTHOR]

Heavy metal based toxicity has a direct relation with the perturbation of protein structure. We have investigated the progressive unfolding of ovalbumin, in the presence of increasing concentration mercury (0–6.25 μM) using different spectroscopic techniques. Formation of amorphous aggregate has been observed at the physiological pH. Initial addition of HgCl 2 resulted in the association of monomers to oligomers that proceeded to non-fibrillar aggregates on further addition. The sigmoidal curve obtained from the Stern-Volmer plot clearly divided into three stage transition. A strong lag phase is observed indicating the time dependence for the association of competent monomers. The second stage was resolved into non-cooperative binding. These results match very well with the data from atomic force microscopy and the free energy change observed in the regions. Raman spectroscopic studies indicated toxic antiparallel β-sheets structure. Time dependent atomic force microscopy study revealed the off-pathway nature of amorphous aggregates. At molten globular state, similar quenching behaviour is observed. The atomic force microscopy images clearly indicate at pH 2.2 the initiation of fibril formation occurs at lower concentration of HgCl 2 itself. Our results revealed the conformation switch of ovalbumin upon the contact of an environmental toxin and its possible way of toxicity. [ABSTRACT FROM AUTHOR]

Despite of increasingly accumulated genetic variations of autosomal dominant congenital cataracts (ADCC), the causative genes of many ADCC patients remains unknown. In this research, we identified a novel F30S mutation in γS-crystallin from a three-generation Chinese family with ADCC. The patients possessing the F30S mutation exhibited nuclear cataract phenotype. The potential molecular mechanism underlying ADCC by the F30S mutation was investigated by comparing the structural features, stability and aggregatory potency of the mutated protein with the wild type protein. Spectroscopic experiments indicated that the F30S mutation did not affect γS-crystallin secondary structure compositions, but modified the microenvironments around aromatic side-chains. Thermal and chemical denaturation studies indicated that the mutation destabilized the protein and increased its aggregatory potency. The mutation altered the two-state unfolding of γS-crystallin to a three-state unfolding with the accumulation of an unfolding intermediate. The almost identical values in the changes of Gibbs free energies for transitions from the native state to intermediate and from the intermediate to unfolded state suggested that the mutation probably disrupted the cooperativity between the two domains during unfolding. Our results expand the genetic variation map of ADCC and provide novel insights into the molecular mechanism underlying ADCC caused by mutations in β/γ-crystallins. [ABSTRACT FROM AUTHOR]

Misfolding and accumulation of the protein alpha synuclein in the brain cells characterize Parkinson's disease (PD). Electrochemical based aluminum interdigitated electrodes (ALIDEs) was fabricated by using conventional photolithography method and modified the surfaces with zinc oxide and gold nanorod by using spin coating method for the analysis of PD protein biomarker. The device surface modified with gold nanorod of 25 nm diameter was used. The bare devices and the surface modified devices were characterized by Scanning Electron Microscope, 3D-Profilometer, Atomic Force Microscope and high-power microscope. The above measurement was also performed to measure the interaction of antibody with aggregated alpha-synuclein for normal, aggregated and aggregated alpha synuclein in human serum and distinguished against 3 control proteins (PARK1, DJ-1 and Factor IX). The detection limit for normal alpha synuclein was 1 f. with the sensitivity of 1 f. on a linear regression (R2 = 0.9759). The detection limit for aggregated alpha synuclein was 10 aM with the sensitivity of 1 aM on a linear regression (R2 = 0.9797). Also, the detection limit of aggregated alpha synuclein in serum was 10 aM with the sensitivity of 1 aM on a linear regression (R2 = 0.9739). These results however indicate that, serum has only minimal amount of alpha synuclein. [ABSTRACT FROM AUTHOR]

A wide range of biophysical and theoretical analysis were employed to explore the formation of (α-syn) amyloid fibril formation as a model of Parkinson's disease in the presence of silica oxide nanoparticles (SiO 2 NPs). Also, different cellular and molecular assays such as MTT, LDH, caspase, ROS, and qPCR were performed to reveal the α-syn amyloid fibrils-associated cytotoxicity against SH-SY5Y cells. Fluorescence measurements showed that SiO 2 NPs accelerate the α-syn aggregation and exposure of hydrophobic moieties. Congo red absorbance, circular dichroism (CD), and transmission electron microscopy (TEM) analysis depicted the SiO 2 NPs accelerated the formation of α-syn amyloid fibrils. Molecular docking study showed that SiO 2 clusters preferably bind to the N-terminal of α-syn as the helix folding site. We also realized that SiO 2 NPs increase the cytotoxicity of α-syn amyloid fibrils through a significant decrease in cell viability, increase in membrane leakage, activation of caspase-9 and -3, elevation of ROS, and increase in the ratio of Bax/Bcl2 mRNA. The cellular assay indicated that α-syn amyloid fibrils formed in the presence of SiO 2 NPs induce their cytotoxic effects through the mitochondrial-mediated intrinsic apoptosis pathway. We concluded that these data may reveal some adverse effects of NPs on the progression of Parkinson's disease. [ABSTRACT FROM AUTHOR]

Even though arginine hydrochloride has been recognized as a protein aggregation suppressor in the biopharmaceutical industry, its use has been questioned due to decreasing transition unfolding temperatures (T m). Four compounds were designed to enhance the role of arginine by changing the length of the carbon chain with removal or N -acetylation of α-amino group. Biophysical properties were observed by differential scanning calorimetry (DSC), dynamic light scattering (DLS), size-exclusion chromatography (SEC), and flow imaging (FI). N -Acetyl-L-arginine (NALA) performed the best at minimizing decrease in T m with arginine at different pH. NALA also demonstrated relatively higher colloidal stability than arginine hydrochloride, especially in the acidic pH, thereby reducing agitation stress of IgG. Moreover, NALA exhibited a cooperative effect with commercially used glycine buffer for IVIG to maintain the monomer contents with almost no change and suppressed larger particle formation after agitation with heat. The study concludes that the decreasing T m of proteins by arginine hydrochloride is due to amide group in the α-carbon chain. Moreover, chemical modification on the group compared to removing it will be a breakthrough of arginine's limitations and optimize storage stability of protein therapeutics. Unlabelled Image [ABSTRACT FROM AUTHOR]

Protein aggregation is the major cause of several acute amyloid diseases such as Parkinson's, Huntington's, Alzheimer's, Lysozyme Systemic amyloidosis, Diabetes-II etc. While these diseases have attracted much attention but the cure is still unavailable. In the present study, Human Serum Albumin (HSA) and Human Lysozyme (HL) were used as the model proteins to investigate their aggregations. Nanoclays are hydrous silicates found in clay fraction of soil and known as natural nanomaterials. They have long been used in several applications in health-related products. In the present paper, the different types of nanoclays (MMT K-10, MMT K-30, Halloysite, Bentonite) were used to inhibit the process of HSA and HL aggregation. Aggregation experiments were evaluated using several biophysical tools such as Turbidity measurements, Intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate (ANS), Thioflavin T (Th T), congo red (CR) binding assays and Circular dichroism. Results demonstrated that all the nanoclays inhibit the DTT-induced aggregation. However, bentonite and MMT K-10 were progressively intense and potent as they slowed down nucleation stage which can be perceived using several biophysical techniques. Hence, nanoclays can be used as an artificial chaperone and might provide effective treatment against several protein aggregation related disorders. Unlabelled Image • HSA and HL were used as convenient model to investigate the protein aggregation. • Nanoclays are potent therapeutic molecule that inhibited the HSA and HL aggregation. • MMT K-10 and BN were the most potent nanoclays that prevented the aggregation. • Nanoclays stabilize the exposed hydrophobic patches of protein in aggregation inducing condition. [ABSTRACT FROM AUTHOR]

The aggregation of β-crystallins in the human eye lens constitutes a critical step during the development of cataract. We anticipated that the presence of Aggregation-Prone Regions (APRs) in their primary structure, which might be responsible for conformational change required for the self-assembly. To examine the presence of APRs, we systematically analyzed the primary structures of β-crystallins. Out of seven subtypes, the βB1-crystallin found to possess the highest aggregation score with 9 APRs in its primary structure. To confirm the amyloidogenic nature of these newly identified APRs, we further studied the aggregation behavior of one of the APRs spanning from 174 to 180 residues (174LWVYGFS180) of βB1-crystallin, which is referred as βB1 (174-180). Under in vitro conditions, the synthetic analogue of βB1 (174-180) peptide formed visible aggregates and displayed high Congo red (CR) bathochromic shift, Thioflavin T (ThT) binding and fibrilar morphology under transmission electron microscopy, which are the typical characteristics of amyloids. Further, the aggregated βB1 (174-180) was found to induce aggregation of the soluble fraction of proteins isolated from the human cataractous lens. This observation suggests that the presence of APRs in βB1-crystallin might be serving as one of the intrinsic supplementary factors responsible for constitutive aggregation behavior of βB1-crystallin and development of cataract. [ABSTRACT FROM AUTHOR]

The drugs-protein binding study is of growing importance for drug-repurposing against amyloidosis. In this work, we study the binding of teicoplanin (TPN), a glycopeptide antibiotic, with bovine serum albumin (BSA) in its neutral (N), physiological (P) and basic (B) forms, which exist at pH 6, pH 7.4 and pH 9, respectively. The binding and thermodynamic parameters of TPN binding were determined by isothermal titration calorimetry (ITC) and fluorescence quench titration methods. Two binding sites were observed for N and P forms, whereas B form showed only one binding site. ITC and molecular docking results indicated that TPN-BSA complex formation is stabilized by hydrogen bonds, salt bridges and hydrophobic interaction. The red-edge excitation shift (REES) study indicated an ordered compact and spatial arrangement of the TPN bound protein molecule. TPN was found to affect the secondary and tertiary structures of B form only. The TPN binding was observed to marginally stabilize BSA isomers. TPN was also found to inhibit BSA aggregation as monitored by Rayleigh light scattering and thioflavin T binding assay. The current in vitro study will open a new path to explore the possible use of TPN as potential drugs to treat amyloidosis. [ABSTRACT FROM AUTHOR]

The misfolding of soluble protein to amyloid fibers or oligomers leads to cell membrane rupture, cell death, and a variety of amyloid-related diseases. Hence, inhibition of protein fibrillation is an important and promising method to prevent and treat these diseases. In this study, we have investigated the inhibitory effect of entacapone (Ent) on human lysozyme (HL) amyloid fibrillation using a combination of biophysical techniques; Rayleigh scattering (RLS) data indicated that Ent can reduce the aggregation of HL amyloid fibrillation with the inhibition constant (Λ) of (3.0 ± 0.5) × 103 M−1. This finding was further confirmed by thioflavin-T (ThT), 8-Anilino-1-naphthalenesulfonic acid (ANS) fluorescence assays and congo red (CR) binding absorption assays with an IC 50 value of 125.89 ± 1.25 μM. Meanwhile, dynamic light scattering (DLS) showed that the size of HL amyloids decreases sharply after Ent treatment. This effect was positively correlated with Ent concentration. Atomic force microscopy (AFM) techniques confirmed that the formation of the fibril decreased significantly when HL was co-incubated with Ent. In addition, steady-state fluorescence spectra and synchronous fluorescence analysis suggested that the formation of stable complexes between Ent and HL contributes to maintain the alpha-helical structure of HL. The molecular docking study revealed that the Ent binds at the active pocket of HL with Glu35, Asp53, Gln58, Trp 64, Ala108 and Trp109 residues via hydrogen bonds, van-der-Waals forces and hydrophobic interactions. The epitope mapping of HL for its interaction with Ent was further elucidated using two-dimensional solution-state nuclear magnetic resonance (NMR) experiments. NMR results showed that the Trp64 and Trp109 of HL plays an important role for binding to Ent, correlating well with our docking result. Thus our study showed the potential of Ent to serve as an effective therapeutic agent for the therapy of amyloid-related diseases. Unlabelled Image [ABSTRACT FROM AUTHOR]

Protein aggregation and glycation are directly associated with many pathological conditions including several neurodegenerative disorders. This study investigates the potential of naturally occurring plant product, Rosmarinic acid (RA), to inhibit the glycation and aggregation process. In this study, we report that varying concentrations of methylglyoxal (MG) induce advanced glycation end products (AGEs) and aggregates formation in HSA in vitro on day 6 and day 8, respectively. AGEs specific fluorescence confirmed the formation of AGEs in HSA in the presence of MG and further characterized the inhibitory potential of RA. It was found that the presence of RA prevented AGEs formation in vitro. Further, aggregates of HSA were characterized employing multi spectroscopic and microscopic techniques and RA was found to inhibit this process. This study proposes that RA could be a potential natural molecule to treat disorders where AGEs and aggregates of proteins play a pivotal role. • MG induces AGEs and aggregates formation in HSA. • Rosmarinic acid restrains glycation. • Rosmarinic acid inhibits fibrillation. • Rosmarinic acid can be used as a therapeutic molecule. [ABSTRACT FROM AUTHOR]

A bioinformatics analysis of the currently predicted GroEL-like proteins encoded by bacteriophage genomes was carried out in comparison with the phage double-ring EL and single-ring OBP chaperonins, previously described by us, as well as with the known chaperonins of group I and group II. A novel GroEL-like protein predicted in the genome of phage AR9 Bacillus subtilis was expressed in E. coli cells, purified and characterised by various physicochemical methods. As shown by native electrophoresis, analytical ultracentrifugation and single-particle electron microscopy analysis, the putative AR9 chaperonin is a single-ring heptamer. Like the EL and OBP chaperonins, the new AR9 chaperonin possesses chaperone activity and does not require co-chaperonin to function. It was shown to prevent aggregation and provide refolding of the denatured substrate protein, endolysin, in an ATP-dependent manner. A comparison of its structural and biochemical properties with those of the EL and OBP chaperonins suggests outstanding diversity in this group of phage chaperonins. [ABSTRACT FROM AUTHOR]

The notion that nanoscale surfaces influence protein conformational transitions stimulates the investigation of fibrillogenic polypeptides adsorbing to nanomaterials. Alpha-synuclein (αS) is a prototypical amyloidogenic protein whose aggregation is associated with severe neurodegenerative disorders. We explored the interaction of αS with silica nanoparticles (SNPs) in diverse solution conditions, ranging from protein-free to protein-rich media. We found that the SNP-binding region of αS, determined by site-resolved NMR spectroscopy, was similar in simple buffer and blood serum. Competition binding experiments with isotopic homologues and different proteins showed that cosolutes elicited molecular exchange in a protein-specific manner. The interaction of an oxidized, fibrillation-resistant protein form with SNPs was similar to that of unmodified αS. SNPs, however, did not stimulate fibrillation of the oxidized protein, which remained fibrillation incompetent. CD experiments revealed SNP-induced perturbations of the structural properties of oxidized and non-oxidized αS. Thus, while αS binding to SNPs is essentially orthogonal to fibril formation, the interaction perturbs the distribution of conformational states populated by the protein in the colloidal suspension. This study sheds light on the dynamic nature of αS interactions with NPs, an aspect that crucially impacts on our ability to control aggregation of αS. Unlabelled Image [ABSTRACT FROM AUTHOR]

Metal ions like Cu2+ and Zn2+ have been shown to impact protein misfolding pathways in neurodegenerative proteinopathies like Alzheimer's and Parkinson's. Also, due to their strong interaction with Ubiquitin, they interfere in degradation of misfolded proteins by impairing the ubiquitin-proteasome system (UPS). In this work, we have studied the interaction of these metal ions with a small Ubiquitin like post-translation modifier SUMO1, which is known to work co-operatively with Ubiquitin to regulate UPS system. Between Cu2+ and Zn2+, the former binds more strongly with SUMO1 as determined using fluorescence spectroscopy. SUMO1 aggregates, forming trimer and higher oligomers in presence of Cu2+ ions which were characterized using gel electrophoresis, Bradford assay, and transmission electron microscopy. Chemical shift analysis using 15N/1H based NMR spectroscopy revealed that SUMO1 retains its structural fold in its trimeric state. Cu2+ induced paramagnetic quenching and Zn2+ induced chemical shift perturbation of 15N-1H cross-peaks were used to identify their respective binding sites in SUMO1. Binding sites so obtained were further validated with molecular dynamics studies. Our findings provide structural insights into the SUMO1-Cu2+/Zn2+ interaction, and its impact on aggregation of SUMO1 which might affect its ability to modify functions of target proteins. [ABSTRACT FROM AUTHOR]

Accurate pairing of amino acids and tRNAs is a prerequisite for faithful translation of genetic information during protein biosynthesis. Here we present the effects of proteome-wide mistranslation of isoleucine (Ile) by canonical valine (Val) or non-proteinogenic norvaline (Nva) in a genetically engineered Escherichia coli strain with an editing-defective isoleucyl-tRNA synthetase (IleRS). Editing-defective IleRS efficiently mischarges both Val and Nva to tRNAIle and impairs the translational accuracy of Ile decoding. When mistranslation was induced by the addition of Val or Nva to the growth medium, an Ile-to-Val or Ile-to-Nva substitution of up to 20 % was measured by high-resolution mass spectrometry. This mistranslation level impaired bacterial growth, promoted the SOS response and filamentation during stationary phase, caused global proteome dysregulation and upregulation of the cellular apparatus for maintaining proteostasis, including the major chaperones (GroES/EL, DnaK/DnaJ/GrpE and HtpG), the disaggregase ClpB and the proteases (Lon, HslV/HslU, ClpA, ClpS). The most important consequence of mistranslation appears to be non-specific protein aggregation, which is effectively counteracted by the disaggregase ClpB. Our data show that E. coli can sustain high isoleucine mistranslation levels and remain viable despite excessive protein aggregation and severely impaired translational fidelity. However, we show that inaccurate translation lowers bacterial resilience to heat stress and decreases bacterial survival at elevated temperatures. [Display omitted] • Escherichia coli is resilient to unexpectedly high levels of isoleucine mistranslation. • Translational errors increase protein aggregation, and demand for chaperone-assisted protein folding and degradation. • Excessive protein aggregation is efficiently counteracted by cellular disaggregase. • Biological process most compromised by mistranslation is protein translation. • Isoleucine mistranslation inhibits the division of E. coli cells in the stationary phase. [ABSTRACT FROM AUTHOR]

The small heat-shock protein (sHSP) from the archaea Methanocaldococcus jannaschii , MjsHSP16.5, functions as a broad substrate ATP-independent holding chaperone protecting misfolded proteins from aggregation under stress conditions. This protein is the first sHSP characterized by X-ray crystallography, thereby contributing significantly to our understanding of sHSPs. However, despite numerous studies assessing its functions and structures, the precise arrangement of the N-terminal domains (NTDs) within this sHSP cage remains elusive. Here we present the cryo-electron microscopy (cryo-EM) structure of MjsHSP16.5 at 2.49-Å resolution. The subunits of MjsHSP16.5 in the cryo-EM structure exhibit lesser compaction compared to their counterparts in the crystal structure. This structural feature holds particular significance in relation to the biophysical properties of MjsHSP16.5, suggesting a close resemblance to this sHSP native state. Additionally, our cryo-EM structure unveils the density of residues 24–33 within the NTD of MjsHSP16.5, a feature that typically remains invisible in the majority of its crystal structures. Notably, these residues show a propensity to adopt a β-strand conformation and engage in antiparallel interactions with strand β1, both intra- and inter-subunit modes. These structural insights are corroborated by structural predictions, disulfide bond cross-linking studies of Cys-substitution mutants, and protein disaggregation assays. A comprehensive understanding of the structural features of MjsHSP16.5 expectedly holds the potential to inspire a wide range of interdisciplinary applications, owing to the renowned versatility of this sHSP as a nanoscale protein platform. [ABSTRACT FROM AUTHOR]

Arginine shows Jekyll and Hyde behavior in several respects. It participates in protein folding via ionic and H-bonds and cation-pi interactions; the charge and hydrophobicity of its side chain make it a disorder-promoting amino acid. Its methylation in histones; RNA binding proteins; chaperones regulates several cellular processes. The arginine-centric modifications are important in oncogenesis and as biomarkers in several cardiovascular diseases. The cross-links involving arginine in collagen and cornea are involved in pathogenesis of tissues but have also been useful in tissue engineering and wound-dressing materials. Arginine is a part of active site of several enzymes such as GTPases, peroxidases, and sulfotransferases. Its metabolic importance is obvious as it is involved in production of urea, NO, ornithine and citrulline. It can form unusual functional structures such as molecular tweezers in vitro and sprockets which engage DNA chains as part of histones in vivo. It has been used in design of cell-penetrating peptides as drugs. Arginine has been used as an excipient in both solid and injectable drug formulations; its role in suppressing opalescence due to liquid-liquid phase separation is particularly very promising. It has been known as a suppressor of protein aggregation during protein refolding. It has proved its usefulness in protein bioseparation processes like ion-exchange, hydrophobic and affinity chromatographies. Arginine is an amino acid, whose importance in biological sciences and biotechnology continues to grow in diverse ways. [ABSTRACT FROM AUTHOR]

The aggregation of recombinant proteins in the different stages of purification leads to the loss of a considerable portion of target protein and reduction in the process efficiency. As the active HBsAg used in Hepatitis B vaccine production is in the form of virus-like particle (VLP), therefore the time and stages at which the VLP assembling happened through the process would be important. The aim of this study was to explore the product aggregation during different stages of large scale production of rHBsAg in Pichia pastoris at production unit of the Pasteur Institute of Iran. Dynamic light scattering (DLS) and transmission electron microscopy (TEM), and also size exclusion-high-performance liquid chromatography (SE-HPLC) were carried out on samples taken from each downstream processes steps to determine the rate of VLPs formation as the desired product and the aggregated form at each stage of the purification. Based on the results, it was found that VLPs formation started at the acid precipitation stage and reached up to 80% at the thermal treatment stage. The ultrafiltration, ion exchange chromatography and immunoaffinity chromatography stages were disclosed to have the highest contribution in the formation of VLP (virus like particle) 22 nm. Unlabelled Image [ABSTRACT FROM AUTHOR]

Protein modification and alteration are important factors in many age-related diseases such as diabetes and Alzheimer's disease. Modifications like the formation of advanced glycation end-products (AGE) and advanced oxidation protein products (AOPP) can cause harm to the organism and may contribute to protein aggregation and amyloid fibrils formation. Carnosine was used as a potential solution for protein modification complications. Furthermore, some organs like the brain are difficult to reach due to the blood-brain barrier. As such, new nano-engineered formulations were sought to bypass unwanted interactions and degradation. Thus, we propose the encapsulation of carnosine in niosomes as a potential solution. Initially, carnosine niosomes were synthesized and characterized. Then, modifications of bovine serum albumin (glycation, oxidation, and aggregation) were induced in vitro where carnosine and carnosine niosomes were added at different concentrations (2.5, 5, and 10 mM) to the reactions. In addition, biocomputational and docking studies were performed to elucidate the potential interactions. Data showed a dose-dependent inhibition of AGE, AOPP, and aggregation for both carnosine and niosome carnosine. Furthermore, the results suggest that carnosine interacts with specific amino acids implicated in the protein modification process. Carnosine nano-formulation shows promising potential in age-related protein modification and needs further exploration of its mechanisms. Unlabelled Image • Altered proteins are important targets in age-related diseases. • Bovine serum albumin is a good asset to assess such protein modification. • L-carnosine is a natural antioxidant known for its high presence in the body. • Niosomes are a good drug delivery tool able to reach organs like the brain. • Carnosine encapsulated niosomes shows a good anti-aggregative effect on BSA. [ABSTRACT FROM AUTHOR]

Apart from its relevance to pathology, protein misfolding disease like Type-II Diabetes Mellitus, caused by amyloids of amylin protein has attracted more attention due to structural changes occurring during the aggregation process. We report extensive spectroscopy data of amylin during fibril formation through Raman, FTIR, CD, UV–vis absorption and photoluminescence (PL) spectroscopy. UV–vis and PL spectrum showed the sigmoidal growth of fibril with a lag time of ~2 days, which is consistent with earlier reported work using dynamic light scattering (DLS). Raman spectra revealed the formation of parallel and anti-parallel β-sheet from 0% to 20% with ageing (1st day to 21st day) at pH 6.5 ± 0.1. The results are corroborated by CD and FTIR data. These show the change in β-sheet by 23% at pH 6.5 ± 0.1, 26% at pH = 1.0 ± 0.1 and 30% at pH = 12 ± 0.1. It is also shown that the formation and conversion of other secondary structures into β-sheet is very sensitive towards the pH and ageing. The study may be used for the development of therapeutic strategies that could inhibit or even reverse the process of aggregation. [ABSTRACT FROM AUTHOR]

Structural roles of loop regions are frequently overlooked in proteins. Nevertheless, they may be key players in the definition of protein topology and in the self-assembly processes occurring through domain swapping. We here investigate the effects on structure and stability of replacing the loop connecting the last two β-strands of RNase A with the corresponding region of the more thermostable Onconase. The crystal structure of this chimeric variant (RNaseA-ONC) shows that its terminal loop size better adheres to the topological rules for the design of stabilized proteins, proposed by Baker and coworkers [43]. Indeed, RNaseA-ONC displays a thermal stability close to that of RNase A, despite the lack of Pro at position 114, which, due to its propensity to favor a cis peptide bond, has been identified as an important stabilizing factor of the native protein. Accordingly, RNaseA-ONC is significantly more stable than RNase A variants lacking Pro114; RNaseA-ONC also displays a higher propensity to form oligomers in native conditions when compared to either RNase A or Onconase. This finding demonstrates that modifications of terminal loops should to be carefully controlled in terms of size and sequence to avoid unwanted and/or potentially harmful aggregation processes. [ABSTRACT FROM AUTHOR]

Many neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, Prion's disease, polyQ and Huntington's disease share abnormal folding of potentially cytotoxic protein species associated with degeneration and death of specific neuronal populations. In order to maintain cellular protein homeostasis, neurons have developed an intrinsic protein quality control system as a strategy to counteract protein aggregation and their toxicity. Heat shock proteins are an essential component for regulating protein quality control and contribute potentially in the process of protein folding, prevent protein aggregation and in disaggregation in several neurodegenerative diseases. Therefore, molecular chaperones are considered an exciting therapeutic target. In this book chapter, we will focus on the potential importance of different heat shock proteins in neurodegenerative diseases and understand their mechanisms to protect neurons form aggregates and their toxicity. [ABSTRACT FROM AUTHOR]

Abstract Protein aggregation and amyloid fibrillation are associated with many serious human pathophysiologies like Alzheimer's, Parkinson's diseases, type II diabetes etc. A powerful strategy for controlling and understanding amyloid protein aggregation is the modulation of protein self-assembly. In this study, anti-fibrillation activity of vitamin A (VA) and its effect on the kinetics of amyloid formation of Aβ-42 peptide was investigated by employing various spectroscopic, imaging and computational approaches. The present data of Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), dynamic light scattering assay, transmission electron microscopy and cell cytotoxicity assay demonstrated that vitamin A significantly inhibits fibril formation. Our experimental studies inferred that Vitamin A protects human neuroblastoma cell line (SH-SY5Y) and the neuroprotective effect against amyloid induced cytotoxicity is through modification of the amyloid formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin A interacts with Aβ-42 through hydrophobic interactions as well as hydrogen bonding. Therefore, the study signifies the role of vitamin A as a potential molecule in preventing Aβ-42 aggregation and associated pathophysiology. Hence, Vitamin A and related compounds can thus act as effective inhibitors in the therapeutic development to combat systemic amyloidosis. [ABSTRACT FROM AUTHOR]

Abstract Extracellular fungal cellobiases develop large stable aggregates by reversible concentration driven interaction. In-vitro addition of trehalose resulted in bigger cellobiase assemblies with increased stability against heat and dilution induced dissociation. In presence of 0.1 M trehalose, the size of aggregates increased from 344 nm to 494 nm. The increase in size was also observed in zymography of cellobiase. Activation energy of the trehalose stabilised enzyme (Ea = 220.9 kJ/mol) as compared to control (Ea = 257.734 kJ/mol), suggested enhanced thermostability and also showed increased resistance to chaotropes. Purified cellobiase was found to contain 196.27 μg of sugar/μg of protein. It was proposed that presence of glycan on protein's surface impedes and delays trehalose docking. Consequently, self-association of cellobiase preceded coating by trehalose leading to stabilisation of bigger cellobiase aggregates. In unison with the hypothesis, ribosylated BSA failed to get compacted by trehalose and developed into bigger aggregates with average size increasing from 210 nm to 328 nm. Wheat Germ Lectin, in presence of trehalose, showed higher molecular weight assemblies in DLS, native-PAGE and fluorescence anisotropy. This is the first report of cross-linking independent stabilisation of purified fungal glycosidases providing important insights towards understanding the aggregation and stability of glycated proteins. Graphical abstract Unlabelled Image Highlights • A novel method of stabilizing extracellular fungal cellobiase aggregate is presented. • Trehalose, a well known protein stabilizer, binds to the bigger cellobiase aggregates and protects them against heat and chaotrope induced denaturation. • High glycan content on cellobiase impedes fast docking of trehalose thus giving ample time for intermolecular association of the enzyme. • Eventually trehalose binds to and stabilizes the larger assemblies of cellobiase. • The studies are believed to report for the first time the effect of trehalose on aggregation and stability of glycoproteins. [ABSTRACT FROM AUTHOR]

Abstract Anti-amyloidogenic activity of safranal towards induced HSA amyloids has been observed using a variety of techniques including fluorescence, UV–visible, CD, DLS and microscopies. The HSA solution was pre-incubated at 65 °C for 120 h and, in between, the growth of amyloid fibrils, using ThT aggregation kinetics, was monitored at different time intervals. It was found that the amyloid fibril formation of HSA diminishes in presence of safranal and the inhibition was concentration dependent. The surface hydrophobicity of HSA amyloid fibrils also decreased in presence of safranal. The increased CR binding of HSA fibrils also decreased and high concentration of safranal causes the CR binding to resemble like that of native HSA. Both RLS and turbidity intensities were also in inverse relation to the safranal concentration. Safranal also has a good impact to protect the secondary structure of incubated HSA. From the electron microscopy it was seen that the fibrillar network of HSA amyloids gradually vanishes as the concentration of safranal increased. The largely decreased population of HSA aggregates in safranal containing solution as compared to the one without it also suggests the inhibition of formation of large fibrillar aggregates. Highlights • Safranal significantly inhibited the formation of amyloid fibrils of HSA. • The inhibition was dependent on the concentration of safranal. • Safranal stabilizes the HSA against thermal denaturation and also protects the secondary structure of incubated HSA. [ABSTRACT FROM AUTHOR]

Protein fibrillation is commonly associated with pathologic amyloidosis. However, under appropriate conditions several proteins form fibrillar structures in vitro that can be used for biotechnological applications. MNEI and its variants, firstly designed as single chain derivatives of the sweet protein monellin, are also useful models for protein fibrillary aggregation studies. In this work, we have drawn attention to a protein dubbed Mut9, already characterized as a "super stable" MNEI variant. Comparative analysis of the respective X-ray structures revealed how the substitutions present in Mut9 eliminate several unfavorable interactions and stabilize the global structure. Molecular dynamic predictions confirmed the presence of a hydrogen-bonds network in Mut9 which increases its stability, especially at neutral pH. Thioflavin-T (ThT) binding assays and Fourier transform infrared (FTIR) spectroscopy indicated that the aggregation process occurs both at acidic and neutral pH, with and without addition of NaCl, even if with a different kinetics. Accordingly, Transmission Electron Microscopy (TEM) showed a fibrillar organization of the aggregates in all the tested conditions, albeit with some differences in the quantity and in the morphology of the fibrils. Our data underline the great potential of Mut9, which combines great stability in solution with the versatile conversion into nanostructured biomaterials. [Display omitted] • MNEI and its variants are useful models for protein fibrillary aggregation process studies. • Mut9 was designed as a super-stable and super-sweet MNEI mutant. • X-ray and MD studies highlighted the higher stability of Mut9 at neutral pH. • Differently from MNEI, Mut9 can form fibrils both at acidic and neutral pH. • Mut9 has a great potential as building-block to design nanostructured biomaterials. [ABSTRACT FROM AUTHOR]

Congenital cataract is the leading cause of childhood blindness, which primarily results from genetic factors. γD-crystallin is the most abundant γ-crystallin and is essential for maintaining lens transparency and refractivity. Numerous mutations in γD-crystallin have been reported with unclear pathogenic mechanism. Two different cataract-causing mutations Ser78Phe and Ser78Pro in γD-crystallin were previously identified at the same conserved Ser78 residue. In this work, firstly, we purified the mutants and characterized for the structural change using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and size-exclusion chromatography (SEC). Both mutants were prone to form insoluble precipitates when expressed in Escherichia coli strain BL21 (DE3) cells. Compared with wild-type (WT), both mutations caused structural disruption, increased hydrophobic exposure, decreased solubility, and reduced thermal stability. Next, we investigated the aggregation of the mutants at the cellular level. Overexpression the mutants in HLE-B3 and HEK 293T cells could induce aggresome formations. The environmental stresses (including heat, ultraviolet irradiation and oxidative stress) promoted the formation of aggregates. Moreover, the intracellular S78F and S78P aggregates could be reversed by lanosterol. Molecular dynamic simulation indicated that both mutations disrupted the structural integrity of Greek-key motif 2. Hence, our results reveal the vital role of conserved Ser78 in maintaining the structural stability, which can offer new insights into the mechanism of cataract formation. [ABSTRACT FROM AUTHOR]

Understanding the impact of pH and ionic strength on the physicochemical and structural properties of soy proteins at subunit level is essential for design and fabrication of many plant-based foods. In this study, soybean β-conglycinin and its subunit fractions αα′ and β were dispersed in solutions with different pH values (3.7, 7.6, and 9.0) at low (5 mM NaCl) and high (400 mM NaCl) ionic strengths, respectively. The solubility, rheology, particle size, zeta potential, microstructure, secondary structure, and tertiary structure of the different dispersions were analyzed using a range of analytical methods. The β-conglycinin, αα′- and β-subunits aggregated near the isoelectric point (pH 3.7). Increasing the ionic strength led to the assembly of more homogeneous units. An increase in ionic strength at pH 7.6 and pH 9.0 led to electrostatic screening, which promoted dissociation of the aggregates. The β-subunit showed a greater sensitivity to pH and ionic strength than the αα′-subunits. Based on the evidence from a range of analytical methods, the highly hydrophilic extension region of the αα′-subunits played an important role in determining the stability of the β-conglycinin dispersions under different environmental conditions. Moreover, the N-linked glycans appeared to impact the conformation and aggregation state of the β-conglycinin. [Display omitted] • The influences of pH and ionic strength on the properties of soybean β-conglycinin subunit fractions αα' and β were studied. • At pH 7.6 and 9.0, the increase in ionic strength resulted in the dissociation of the aggregates into trimers or monomers. • The β-subunit showed a greater sensitivity to pH and ionic strength than the αα'-subunits. [ABSTRACT FROM AUTHOR]

In this study, the properties of pigeon egg white (PEW) and chicken egg white (CEW) thermal gels were compared, with the aim of revealing the mechanisms involved in the high transparency of PEW thermal gels. Results demonstrated that PEW gels exhibited higher transparency than CEW gels. Scanning electron microscope (SEM) and transmission electron microscope (TEM) analysis revealed that PEW gels formed a fine chain gel network structure with an average diameter of thermal aggregates (89.84 ± 7.13 nm). The molecular properties of PEW proteins, such as higher content of β-sheet structures (32.73 %), reactive groups (free sulfhydryl groups, hydrophobic groups), and absolute zeta potential (−3.563 mV), were found to contribute to the formation of smaller thermal aggregates during thermal denaturation. The microrheology measurements showed that these features allowed PEW proteins to interact less with each other and form smaller thermal aggregates during thermal denaturation, which facilitated the formation of fine chain gel networks and thus improved the transparency of the gels. The present study initially reveals the molecular basis of the high transparency of PEW thermal gels and provides a theoretical reference for the development of new highly transparent protein materials. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract The study investigates the role of the electrostatic interactions in the fibrillation of the hen egg white lysozyme (HEWL). In order to achieve this aim the influence of the cations Na+, Mg2+ and Al3+ on the amyloid fibril formation and amorphous aggregation was tested. The amyloids are formed in the solution without added salt but the Thioflavin T fluorescence gives the false-negative result. In these conditions, the HEWL fibrils are long and curvy. If the ionic strength of the solution is sufficiently high, the formed amyloids are shorter and fragmented. Our study shows that the addition of the aluminium salt promotes protein fibrillation. The amorphous aggregation dominates in the high concentration of electrolyte. The in vitro amyloid fibril formation seems to be regulated by universal mechanisms. The theories implemented in the polymer science or for colloidal solutions give the qualitative description of the aggregation phenomena. However, the specific interactions and the additional effects (e.g. fibril fragmentation) modulate the amyloidogenesis. Highlights • Influence of cations on lysozyme fibrillation/amorphous aggregation was tested. • In low concentration of salt, ThT is negative but amyloids are present. • In high concentration of salt, amorphous aggregation dominates. • Aluminium ions promote amyloid fibril formation. [ABSTRACT FROM AUTHOR]

Abstract Stabilization of the enzymes under stress conditions is of special interest for modern biochemistry, bioengineering, as well as for formulation and target delivery of protein-based drugs. Aiming to achieve an efficient stabilization at elevated temperature with no influence on the enzyme under normal conditions, we studied chaperone-like activity of thermoresponsive polymers based on poly(dimethylaminoethyl methacrylate) (PDMAEMA) toward two different proteins, glyceraldehyde-3-phosphate dehydrogenase and chicken egg lysozyme. The polymers has been shown to do not interact with the folded protein at room temperature but form a complex upon heating to either protein unfolding or polymer phase transition temperature. A PDMAEMA-PEO block copolymer with a dodecyl end-group (d-PDMAEMA-PEO) as well as PDMAEMA-PEO without the dodecyl groups protected the denatured protein against aggregation in contrast to PDMAEMA homopolymer. No effect of the polymers on the enzymatic activity of the client protein was observed at room temperature. The polymers also partially protected the enzyme against inactivation at high temperature. The results provide a platform for creation of artificial chaperones with unfolded protein recognition which is a major feature of natural chaperones. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]