Flavones Suppress Aggregation and Amyloid Fibril Formation of Human Lysozyme under Macromolecular Crowding Conditions.

The crowded milieu of a biological cell significantly impacts protein aggregation and interactions. Understanding the effects of macromolecular crowding on the aggregation and fibrillation of amyloidogenic proteins is crucial for the treatment of many amyloid-related disorders. Most studies of protein amyloid formation and its inhibition by small molecules are conducted in dilute buffers, which do not mimic the complexity of the cellular environment.

Cyclic-NDGA Effectively Inhibits Human γ-Synuclein Fibrillation, Forms Nontoxic Off-Pathway Species, and Disintegrates Preformed Mature Fibrils.

Parkinson's disease arises from protein misfolding, aggregation, and fibrillation and is characterized by LB (Lewy body) deposits, which contain the protein α-synuclein (α-syn) as their major component. Another synuclein, γ-synuclein (γ-syn), coexists with α-syn in Lewy bodies and is also implicated in various types of cancers, especially breast cancer. It is known to seed α-syn fibrillation after its oxidation at methionine residue, thereby contributing in synucleinopathy.

Modulation of the conformation, fibrillation, and fibril morphologies of human brain α-, β-, and γ-synuclein proteins by the disaccharide chemical chaperone trehalose.

Human α-, β-, and γ-synuclein (syn) are natively unfolded proteins present in the brain. Deposition of aggregated α-syn in Lewy bodies is associated with Parkinson's disease (PD) and γ-syn is known to be involved in both neurodegeneration and breast cancer. At physiological pH, while α-syn has the highest propensity for fibrillation followed by γ-syn, β-syn does not form any fibrils.

Kinetic control in amyloid polymorphism: Different agitation and solution conditions promote distinct amyloid polymorphs of alpha-synuclein.

Aggregation of neuronal protein α-synuclein is implicated in synucleinopathies, including Parkinson's disease. Despite abundant in vitro studies, the mechanism of α-synuclein assembly process remains ambiguous. In this work, α-synuclein aggregation was induced by its constant mixing in two separate modes, either by agitation in a 96-well microplate reader (MP) or in microcentrifuge tubes using a shaker incubator (SI).

The Anthocyanidin Peonidin Interferes with an Early Step in the Fibrillation Pathway of α-Synuclein and Modulates It toward Amorphous Aggregates.

Parkinson's disease (PD) is characterized by progressive degeneration of the dopaminergic neurons in the brain, accompanied by the accumulation of proteinaceous inclusions, Lewy bodies (LB), mainly comprised of alpha synuclein (α-syn) aggregates. The heterogeneity and the transient nature of the intermediate species formed in the α-syn fibrillation pathway have made it difficult to develop an effective therapeutic intervention. Therefore, any therapeutic molecule that could prevent as well as treat PD would be of great interest.

Modulation of assembly of TDP-43 low-complexity domain by heparin: From droplets to amyloid fibrils.

TAR DNA-binding protein 43 (TDP-43) is an RNA-regulating protein that carries out many cellular functions through liquid-liquid phase separation (LLPS). The LLPS of TDP-43 is mediated by its C-terminal low-complexity domain (TDP43-LCD) corresponding to the region 267-414. In neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia, pathological inclusions of the TDP-43 are found that are rich in the C-terminal fragments of ∼25 and ∼35 kDa, of which TDP43-LCD is a part.

A tribute to Dr. Serge N. Timasheff, our mentor.

Dr. Serge N. Timasheff, our mentor and friend, passed away in 2019.

Two polyphenols with diverse mechanisms towards amyloidosis: differential modulation of the fibrillation pathway of human lysozyme by curcumin and EGCG.

The effect of two widely used polyphenols, curcumin and EGCG was investigated on the amyloid fibrillogenesis of the well-characterized model protein human lysozyme (HuL), associated with non-neuropathic systemic amyloidosis, towards exploring their efficacy as modulators of HuL amyloid aggregation and toxicity and unravelling their mechanism of action. Curcumin exerts its inhibitory influence towards HuL fibrillation by interacting with the prefibrillar and fibrillar intermediates resulting in complete suppression of fibrillation at ∼200 µM and effectively disaggregates preformed fibrils of HuL. EGCG on the other hand suppresses fibrillation only upto 70% at ∼400 µM, modulates the pathway towards large, β-sheet rich amyloid fibril-like aggregates and modifies the preformed fibrils into similar type of large, clustered aggregate assemblies.

Disorder under stress: Role of polyol osmolytes in modulating fibrillation and aggregation of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) comprise ~30-40% of the proteome, have key roles in cellular processes, and have been reported to be involved in stress regulation working in synergy with osmolytes. Osmolytes are known to accumulate against various stresses in living systems and are known to stabilize the native conformation of globular proteins. However, little is known of their effect on IDPs and their mechanism of action is unclear.

Binding of Noradrenaline to Native and Intermediate States during the Fibrillation of α-Synuclein Leads to the Formation of Stable and Structured Cytotoxic Species.

Parkinson's disease is characterized by the deterioration of dopaminergic neurons of substantia nigra pars compacta along with a substantial loss of noradrenergic neurons of the locus coeruleus, which is the major source of noradrenaline (NA) in the brain. We have investigated the interaction of NA with α-synuclein (α-syn), the major protein constituent of Lewy bodies that are the pathological hallmark of Parkinson's disease (PD). It is expected that NA, like dopamine, could bind to α-syn and modulate its aggregation propensity and kinetics, which could also contribute to the onset of PD.

Suppression, disaggregation, and modulation of γ-Synuclein fibrillation pathway by green tea polyphenol EGCG.

Oligomerization of γ-Synuclein is known to have implications for both neurodegeneration and cancer. Although it is known to co-exist with the fibrillar deposits of α-Synuclein (Lewy bodies), a hallmark in Parkinson's disease (PD), the effect of potential therapeutic modulators on the fibrillation pathway of γ-Syn remains unexplored. By a combined use of various biophysical tools and cytotoxicity assays we demonstrate that the flavonoid epigallocatechin-3-gallate (EGCG) significantly suppresses γ-Syn fibrillation by affecting its nucleation and binds with the unstructured, nucleus forming oligomers of γ-Syn to modulate the pathway to form α-helical containing higher-order oligomers (~158 kDa and ~ 670 kDa) that are SDS-resistant and conformationally restrained in nature.

Effect of polyols on the structure and aggregation of recombinant human γ-Synuclein, an intrinsically disordered protein.

Polyol osmolytes accumulated in cells under stress are known to promote stability in globular proteins with respect to their increasing hydroxyl groups but their effect on the structure, stability and aggregation of intrinsically disordered proteins (IDPs) is still elusive. The lack of a natively folded structure in intrinsically disordered proteins under physiological conditions results in their aggregation and fibrillation that gives rise to a number of diseases. We have investigated the effect of a series of polyols, ethylene glycol (EG), glycerol, erythritol, xylitol and sorbitol on the fibrillation pathway of recombinant human γ-Synuclein, used as a model, for an IDP known to form fibrils that play a role in neurodegeneration and cancer.

Comparative Analysis of the Conformation, Aggregation, Interaction, and Fibril Morphologies of Human α-, β-, and γ-Synuclein Proteins.

The human synuclein (syn) family is comprised of α-, β-, and γ-syn proteins. α-syn has the highest propensity for aggregation, and its aggregated forms accumulate in Lewy bodies (LB) and Lewy neurites, which are involved in Parkinson's disease (PD). β- and γ-syn are absent in LB, and their exact role is still enigmatic.

Resveratrol Interferes with an Early Step in the Fibrillization Pathway of Human Lysozyme and Modulates it towards Less-Toxic, Off-Pathway Aggregates.

The effect of resveratrol, a polyphenol in red wine, on the amyloid fibril formation of human lysozyme (HuL) was investigated, towards elucidating the mechanism of resveratrol action and probing its role as a possible modulator of lysozyme aggregation and toxicity. By using a number of biophysical tools, resveratrol was observed to alter the fibrillization kinetics of HuL and inhibit its fibrillization by binding with weak to moderate affinity to the conformations populated at the early stages of the pathway with concomitant stabilization of these initial conformations. The marginal decrease in the lifetime of HuL in the presence of resveratrol by time-resolved fluorescence measurements indicated the involvement of a static quenching mechanism in the interaction between HuL and resveratrol.

Importance of the α10 helix for DevR activation: A road map for screening inhibitors against DevR-mediated dormancy of Mycobacterium tuberculosis.

Objective/background: Bacterial persistence is the hallmark of tuberculosis (TB) and poses the biggest threat to the success of any antitubercular drug regimen. The DevR/DosR dormancy regulator of Mycobacterium tuberculosis belongs to the NarL subfamily of response regulators and is essential for M. tuberculosis persistence in macaque models of TB.

The α10 helix of DevR, the Mycobacterium tuberculosis dormancy response regulator, regulates its DNA binding and activity.

The crystal structures of several bacterial response regulators provide insight into the various interdomain molecular interactions potentially involved in maintaining their 'active' or 'inactive' states. However, the requirement of high concentrations of protein, an optimal pH and ionic strength buffers during crystallization may result in a structure somewhat different from that observed in solution. Therefore, functional assessment of the physiological relevance of the crystal structure data is imperative.

Significantly enhanced heme retention ability of myoglobin engineered to mimic the third covalent linkage by nonaxial histidine to heme (vinyl) in synechocystis hemoglobin.

Heme proteins, which reversibly bind oxygen and display a particular fold originally identified in myoglobin (Mb), characterize the "hemoglobin (Hb) superfamily." The long known and widely investigated Hb superfamily, however, has been enriched by the discovery and investigation of new classes and members. Truncated Hbs typify such novel classes and exhibit a distinct two-on-two α-helical fold.

Modulation of human α-synuclein aggregation by a combined effect of calcium and dopamine.

Parkinson's disease is characterized by the deposition of aggregated α-syn and its familial mutants into Lewy bodies leading to death of dopaminergic neurons. α-syn is involved in Ca(II) and dopamine (DA) signaling and their adequate balance inside neuronal cytoplasm is essential for maintaining healthy dopaminergic neurons. We have probed the binding energetics of Ca(II) and DA to human α-syn and its familial mutants A30P, A53T and E46K using isothermal titration calorimetry and have investigated the conformational and aggregation aspects using circular dichroism and fluorescence spectroscopy.

Polyol osmolytes stabilize native-like cooperative intermediate state of yeast hexokinase A at low pH.

Osmolytes produced under stress in animal and plant systems have been shown to increase thermal stability of the native state of a number of proteins as well as induce the formation of molten globule (MG) in acid denatured states and compact conformations in natively unfolded proteins. However, it is not clear whether these solutes stabilize native state relative to the MG state under partially denaturing conditions. Yeast hexokinase A exists as a MG state at pH 2.

Stereo-selectivity of human serum albumin to enantiomeric and isoelectronic pollutants dissected by spectroscopy, calorimetry and bioinformatics.

1-naphthol (1N), 2-naphthol (2N) and 8-quinolinol (8H) are general water pollutants. 1N and 2N are the configurational enantiomers and 8H is isoelectronic to 1N and 2N. These pollutants when ingested are transported in the blood by proteins like human serum albumin (HSA).

Directed evolution of an anti-human red blood cell antibody.

We have earlier described a haemagglutination-based assay for on-site detection of antibodies to HIV using whole blood. The reagent in this assay comprises of monovalent Fab fragment of an anti-human RBC antibody fused to immunodominant antigens of HIV-1 and HIV-2. In the present work, we describe a rational and systematic method for directed evolution of scFv and Fab antihuman RBC antibody fragments.

Dissecting the role of critical residues and substrate preference of a Fatty Acyl-CoA Synthetase (FadD13) of Mycobacterium tuberculosis.

Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb) severely limit the treatment options for tuberculosis (TB); hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.

Differential behavior of missense mutations in the intersubunit contact domain of the human pyruvate kinase M2 isozyme.

In this study, we attempted to understand the mechanism of regulation of the activity and allosteric behavior of the pyruvate kinase M(2) enzyme and two of its missense mutations, H391Y and K422R, found in cells from Bloom syndrome patients, prone to develop cancer. Results show that despite the presence of mutations in the intersubunit contact domain, the K422R and H391Y mutant proteins maintained their homotetrameric structure, similar to the wild-type protein, but showed a loss of activity of 75 and 20%, respectively. Interestingly, H391Y showed a 6-fold increase in affinity for its substrate phosphoenolpyruvate and behaved like a non-allosteric protein with compromised cooperative binding.

Inhibition of protein aggregation: supramolecular assemblies of arginine hold the key.

Background: Aggregation of unfolded proteins occurs mainly through the exposed hydrophobic surfaces. Any mechanism of inhibition of this aggregation should explain the prevention of these hydrophobic interactions. Though arginine is prevalently used as an aggregation suppressor, its mechanism of action is not clearly understood.

CHIP chaperones wild type p53 tumor suppressor protein.

Wild type p53 exists in a constant state of equilibrium between wild type and mutant conformation and undergoes conformational changes at elevated temperature. We have demonstrated that the co-chaperone CHIP (carboxyl terminus of Hsp70-interacting protein), which suppressed aggregation of several misfolded substrates and induced the proteasomal degradation of both wild type and mutant p53, physically interacts with the amino terminus of WT53 and prevented it from irreversible thermal inactivation. CHIP preferentially binds to the p53 mutant phenotype and restored the DNA binding activity of heat-denatured p53 in an ATP-independent manner.