TDP-43 dysregulation of polyadenylation site selection is a defining feature of RNA misprocessing in ALS/FTD and related disorders.

Nuclear clearance and cytoplasmic aggregation of the RNA-binding protein TDP-43 are observed in many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and fronto- temporal dementia (FTD). Although TDP-43 dysregulation of splicing has emerged as a key event in these diseases, TDP-43 can also regulate polyadenylation; yet, this has not been adequately studied. Here, we applied the dynamic analysis of polyadenylation from RNA-seq (DaPars) tool to ALS/FTD transcriptome datasets, and report extensive alternative polyadenylation (APA) upon TDP-43 alteration in ALS/FTD cell models and postmortem ALS/FTD neuronal nuclei. Importantly, many identified APA genes highlight pathways implicated in ALS/FTD pathogenesis. To determine the functional significance of APA elicited by TDP-43 nuclear depletion, we examined microtubule affinity regulating kinase 3 (MARK3). Nuclear loss of TDP-43 yielded increased expression of MARK3 transcripts with longer 3'UTRs, resulting in greater transcript stability and elevated MARK3 protein levels, which promotes increased neuronal tau S262 phosphorylation. Our findings define changes in polyadenylation site selection as a previously unrecognized feature of TDP-43-driven disease pathology in ALS/FTD and highlight a potentially novel mechanistic link between TDP-43 dysfunction and tau regulation.

Emergence of Catalytic Triad by Short Peptide Based Nanofibrillar Assemblies.

Through millions of years of the evolutionary journey, contemporary enzymes observed in extant metabolic pathways have evolved to become specialized, in contrast to their ancestors, which displayed promiscuous activities with wider substrate specificities. However, there remain critical gaps in our understanding of how these early enzymes could show such catalytic versatility despite lacking the complex three-dimensional folds of the existing modern-day enzymes. Herein, we report the emergence of a promiscuous catalytic triad by short amyloid peptide based nanofibers that access paracrystalline folds of ß-sheets to expose three residues (lysine, imidazole, and tyrosine) toward solvent. The ordered folded nanostructures could simultaneously catalyze two metabolically relevant chemical transformations via C-O and C-C bond manipulations, displaying both hydrolase and retro-aldolase-like activities. Further, the latent catalytic capabilities of the short peptide based promiscuous folds also helped in processing a cascade transformation, suggesting the important role they might have played in protometabolism and early evolutionary processes.

Conformational Plasticity in α-Synuclein and How Crowded Environment Modulates It.

A 140-residue intrinsically disordered protein (IDP), α-synuclein (αS), is known to adopt conformations that are vastly plastic and susceptible to environmental cues and crowders. However, the inherently heterogeneous nature of αS has precluded a clear demarcation of its monomeric precursor between aggregation-prone and functionally relevant aggregation-resistant states and how a crowded environment could modulate their mutual dynamic equilibrium. Here, we identify an optimal set of distinct metastable states of αS in aqueous media by dissecting a 73 µs-long molecular dynamics ensemble via building a comprehensive Markov state model (MSM). Notably, the most populated metastable state corroborates with the dimension obtained from PRE-NMR studies of αS monomer, and it undergoes kinetic transition at diverse time scales with a weakly populated random-coil-like ensemble and a globular protein-like state. However, subjecting αS to a crowded environment results in a nonmonotonic compaction of these metastable conformations, thereby skewing the ensemble by either introducing new tertiary contacts or by reinforcing the innate contacts. The early stage of dimerization process is found to be considerably expedited in the presence of crowders, albeit promoting nonspecific interactions. Together with this, using an extensively sampled ensemble of αS, this exposition demonstrates that crowded environments can potentially modulate the conformational preferences of IDP that can either promote or inhibit aggregation events.

Short peptide-based cross-ß amyloids exploit dual residues for phosphoesterase like activity.

Herein, we report that short peptides are capable of exploiting their anti-parallel registry to access cross-ß stacks to expose more than one catalytic residue, exhibiting the traits of advanced binding pockets of enzymes. Binding pockets decorated with more than one catalytic residue facilitate substrate binding and process kinetically unfavourable chemical transformations. The solvent-exposed guanidinium and imidazole moieties on the cross-ß microphases synergistically bind to polarise and hydrolyse diverse kinetically stable model substrates of nucleases and phosphatase. Mutation of either histidine or arginine results in a drastic decline in the rate of hydrolysis. These results not only support the argument of short amyloid peptides as the earliest protein folds but also suggest their interactions with nucleic acid congeners, foreshadowing the mutualistic biopolymer relationships that fueled the chemical emergence of life.

Microcarrier-Based Culture of Human Pluripotent Stem-Cell-Derived Retinal Pigmented Epithelium.

Dry age-related macular degeneration (AMD) is estimated to impact nearly 300 million individuals globally by 2040. While no treatment options are currently available, multiple clinical trials investigating retinal pigmented epithelial cells derived from human pluripotent stem cells (hPSC-RPE) as a cellular replacement therapeutic are currently underway. It has been estimated that a production capacity of >109 RPE cells annually would be required to treat the afflicted population, but current manufacturing protocols are limited, being labor-intensive and time-consuming. Microcarrier technology has enabled high-density propagation of many adherent mammalian cell types via monolayer culture on surfaces of uM-diameter matrix spheres; however, few studies have explored microcarrier-based culture of RPE cells. Here, we provide an approach to the growth, maturation, and differentiation of hPSC-RPE cells on Cytodex 1 (C1) and Cytodex 3 (C3) microcarriers. We demonstrate that hPSC-RPE cells adhere to microcarriers coated with Matrigel, vitronectin or collagen, and mature in vitro to exhibit characteristic epithelial cell morphology and pigmentation. Microcarrier-grown hPSC-RPE cells (mcRPE) are viable; metabolically active; express RPE signature genes including BEST1, RPE65, TYRP1, and PMEL17; secrete the trophic factors PEDF and VEGF; and demonstrate phagocytosis of photoreceptor outer segments. Furthermore, we show that undifferentiated hESCs also adhere to Matrigel-coated microcarriers and are amenable to directed RPE differentiation. The capacity to support hPSC-RPE cell cultures using microcarriers enables efficient large-scale production of therapeutic RPE cells sufficient to meet the treatment demands of a large AMD patient population.

Dynamical Manifestations of Supercooling in Amyloid Hydration.

The effect of extreme temperature on amyloidogenic species remains sparsely explored. In a recent study (J. Phys. Chem. Lett., 2019, 10, (10)), we employed exhaustive molecular dynamics simulations to explore the cold thermal response of a putative small amyloid oligomer and to elicit the role of solvent modulation. Herein, we investigate the dynamical response of the hydration waters of the oligomer within the supercooled states. Using NMR-based formalism, we delineate the entropic response in terms of the side-chain conformational entropy that corroborates the weakening of the hydrophobic core with lowering of temperature. The translational dynamics of the protein and hydration waters reveal the coupling of protein dynamical fluctuations with solvent dynamics under supercooled conditions. Probing the translational motion as a space-time correlation indicates glassy dynamics exhibited by hydration waters in the supercooled regime. Caging of the water molecules with lowering of temperature and the resultant hopping dynamics are reflected in the longer ß-relaxation timescales of translational motion. Furthermore, we utilized mode-coupling theory (MCT) and derived the ideal glass transition temperature from translational and rotational dynamics, around ∼196 and 209 K, respectively. Interestingly, rotational motion in the supercooled regime deviates from the MCT law, exhibits Arrhenius motion, and marks a fragile-to-strong crossover at 227 K. The low-frequency vibrational modes also coincide with the dynamical transition. This exposition lends dynamical insights into the hydration coupling of an amyloid aggregate under cryogenic conditions.

Nanoscale Interplay of Membrane Composition and Amyloid Self-Assembly.

Cell membranes are complex assemblies of lipids and proteins exhibiting lipid compositional heterogeneity between the inner and outer leaflets of the bilayer. Aberrant protein aggregation, implicated in a number of neurodegenerative diseases including Alzheimer's, is known to result in both extracellular and intracellular deposits with divergent pathophysiological effects. Mounting evidence substantiates membrane-mediated amyloid effects and indicates membrane composition, particularly gangliosides, as a plausible factor influencing the fibrillation process. By employing exhaustive molecular dynamics simulations using a coarse-grained model, we probed the assembly behavior of amyloidogenic Aß(12-28) peptides on the chemically heterogeneous extracellular (outer) and cytosolic (inner) leaflets of a mammalian plasma membrane. Our results indicate that the compositional nature of the membrane has a crucial impact on the peptide self-assembly. Peptide oligomerization is hindered on the outer leaflet relative to the inner leaflet due to a competition between interpeptide and peptide-membrane interactions, resulting in higher population of smaller oligomers. The weaker associations among peptides on the outer membrane can be attributed to the favorable interactions of the peptides with gangliosides (GM) that characterize the extracellular membrane. At a higher peptide:GM ratio, we observe enhanced nanoclustering of GM lipids mediated by preferential GM-Aß binding. Interaction between peptide and GM further impacts local membrane curvature; there is a concomitant loss in membrane concavity due to looser GM packing. Our simulations provide molecular insights into the role of membrane composition on Aß aggregation and lend credence to earlier reports of ganglioside-mediated Aß aggregation in the outer membrane. We also demonstrate the effects of local peptide assemblies on the membrane structure and dynamics.

Influence of crowding and surfaces on protein amyloidogenesis: A thermo-kinetic perspective.

The last few decades have irreversibly implicated protein self-assembly and aggregation leading to amyloid fibril formation in proteopathies that include several neurodegenerative diseases. Emerging studies recognize the importance of eliciting the pathways leading to protein aggregation in the context of the crowded intracellular environment rather than in conventional in vitro conditions. It is found that crowded environments can have acceleratory as well as inhibitory effects on protein aggregation, depending on the interplay of underlying factors on the crucial rate limiting steps. The aggregation mechanism and transient species formed along the pathway are further altered when they interface with natural and artificial surfaces in the cellular milieu. An increasing number of studies probe the autocatalytic nature of amyloid surfaces as well as membrane bilayer effects on amyloidogenesis. Moreover, exposure to modern nanosurfaces via nanomedicines and other sources potentially invokes beneficial or deleterious biological response that needs rigorous investigation. Mounting evidences indicate that nanoparticles can either promote or impede amyloid aggregation, spurring efforts to tune their interactions for developing effective anti-amyloid strategies. Mechanistic insights into nanoparticle mediated aggregation pathways are therefore crucial for engineering anti-amyloid nanoparticle strategies that are biocompatible and sustainable. This review is a compilation of studies that contribute to the current understanding of the altering effects of molecular crowding as well as natural and artificial surfaces on protein amyloidogenesis.

The Cold Thermal Response of an Amyloid Oligomer Differs from Typical Globular Protein Cold Denaturation.

In contrast with the general behavior of folded proteins, the cold thermal response of amyloid assemblies is difficult to elicit with simple models. We exploit exhaustive simulations to evaluate the thermal response of a barrel-shaped model amyloid oligomer, with a distinct hydrophobic core akin to that of folded proteins. Cumulative thermal data over the range of 210-483 K indicate a sharp inflection and rise in structural stability as the temperature is decreased below the melting temperature of the water model. This is not commensurate with the equilibrium free energy profile obtained with core packing as the order parameter. However, energetic analyses and the size of their fluctuations indicate the crucial role of hydration in mediating structural transitions, beyond the expected temperature-dependent hydrophobic effect. Structural ordering of the hydration layer over bulk water is maximized at the transition and vanishes at high temperatures. This is a first direct demonstration of the microscopic influence of hydration water on the low-temperature response of an amyloid assembly close to the cryo-regime.

Influence of Hyperglycemic Conditions on Self-Association of the Alzheimer's Amyloid ß (Aß1-42) Peptide.

Clinical studies have identified a correlation between type-2 diabetes mellitus and cognitive decrements en route to the onset of Alzheimer's disease (AD). Recent studies have established that post-translational modifications of the amyloid ß (Aß) peptide occur under hyperglycemic conditions; particularly, the process of glycation exacerbates its neurotoxicity and accelerates AD progression. In view of the assertion that macromolecular crowding has an altering effect on protein self-assembly, it is crucial to characterize the effects of hyperglycemic conditions via crowding on Aß self-assembly. Toward this purpose, fully atomistic molecular dynamics simulations were performed to study the effects of glucose crowding on Aß dimerization, which is the smallest known neurotoxic species. The dimers formed in the glucose-crowded environment were found to have weaker associations as compared to that of those formed in water. Binding free energy calculations show that the reduced binding strength of the dimers can be mainly attributed to the overall weakening of the dispersion interactions correlated with substantial loss of interpeptide contacts in the hydrophobic patches of the Aß units. Analysis to discern the differential solvation pattern in the glucose-crowded and pure water systems revealed that glucose molecules cluster around the protein, at a distance of 5-7 Å, which traps the water molecules in close association with the protein surface. This preferential exclusion of glucose molecules and resulting hydration of the Aß peptides has a screening effect on the hydrophobic interactions, which in turn diminishes the binding strength of the resulting dimers. Our results imply that physical effects attributed to crowded hyperglycemic environments are incapable of solely promoting Aß self-assembly, indicating that further mechanistic studies are required to provide insights into the self-assembly of post-translationally modified Aß peptides, known to possess aggravated toxicity, under these conditions.

Perturbations in inter-domain associations may trigger the onset of pathogenic transformations in PrP(C): insights from atomistic simulations.

Conversion of the predominantly α-helical cellular prion protein (PrP(C)) to the misfolded ß-sheet enriched Scrapie form (PrP(Sc)) is a critical event in prion pathogenesis. However, the conformational triggers that lead to the isoform conversion (PrP(C) to PrP(Sc)) remain obscure, and conjectures about the role of unusually hydrophilic, short helix H1 of the C-terminal globular domain in the transition are varied. Helix H1 is anchored to helix H3 via a few stabilizing polar interactions. We have employed fully atomistic molecular dynamics simulations to study the effects triggered by a minor perturbation in the network of these non-bonded interactions in PrP(C). The elimination of just one of the key H1-H3 hydrogen bonds led to a cascade of conformational changes that are consistent with those observed in partially unfolded intermediates of PrP(C), with pathogenic mutations and in low pH environments. Our analyses reveal that the perturbation results in the enhanced conformational flexibility of the protein. The resultant enhancement in the dynamics leads to overall increased solvent exposure of the hydrophobic core residues and concomitant disruption of the H1-H3 inter-domain salt bridge network. This study lends credence to the hypothesis that perturbing the cooperativity of the stabilizing interactions in the PrP(C) globular domain can critically affect its dynamics and may lead to structural transitions of pathological relevance.

In silico profiling and structural insights of missense mutations in RET protein kinase domain by molecular dynamics and docking approach.

A major challenge remaining in drug design efforts towards protein kinase is due to the development of drug resistance initiated by the missense mutations in the kinase catalytic domain. Gain or loss of function mutations in the REarranged during Transfection (RET) tyrosine kinase gene have been associated with the development of a wide range of human associated cancers and Hirschsprung's disease. However, to what extent these mutations might affect bio-molecular functions remains unclear. In this article, the functionally significant mutations in RET were screened with the aid of various sequence and structure based in silico prediction methods. We mapped the deleterious mutants, modelled mutant proteins and deciphered the impact of mutations on drug binding mechanisms in the RET crystal structure of PDB ID: with the potential inhibitor vandetanib by docking analysis. Furthermore, molecular dynamics simulations were undertaken to understand the mechanistic action of cancer associated mutations in altering the protein kinase structure, dynamics, and stability. According to our results, the overall effect of V804M, M918T and S922Y were destabilizing and mostly alter the electrostatic component of the binding energy. Specifically, the mutation of gatekeeper residue valine 804 present in the ATP binding pocket affects the protein stability and confers resistance to the drug vandetanib, which was consistent with previously published experimental results. Overall, our findings may provide useful structural insights for in-depth understanding of the molecular mechanism underlying RET mutation and developing effective drugs.

Influence of stress on gonadotrophin induced testicular recrudescence in the lizard Mabuya Carinata.

Administration (ip) of FSH (10 IU/0.1 ml distilled water (dw)/lizard/alternate days/30 days) to adult male lizards, Mabuya carinata, during the early recrudescence phase of the reproductive cycle caused activation of spermatogenic and steroidogenic activity of the testis, as shown by a significant increase in mean number of spermatogonia, primary spermatocytes and spermatids, and serum levels of testosterone, as compared to initial controls. In addition, there were abundant spermatozoa in the lumen of the seminiferous tubules. Interestingly, administration of a similar dosage of FSH to lizards exposed to stressors (handling, chasing, and noise randomly applied, five times a day for 30 days) resulted in a significant increase in mean number of spermatogonia and primary spermatocytes over initial control values, whereas the number of secondary spermatocytes and spermatids and serum levels of testosterone did not significantly differ from those of initial controls, and were significantly lower than FSH treated normal lizards. Further, spermatozoa were infrequently found in the seminiferous tubules of these lizards. Treatment controls (receiving 0.1 ml dw/lizard/alternate days for 30 days) did not show significant variation in mean number of spermatogonia, spermatocytes and spermatids, and serum levels of testosterone from initial controls. Another group of lizards was exposed to stressors and did not receive FSH. These lizards showed a significant decrease in mean number of secondary spermatocytes compared to treatment controls and all other parameters did not significantly differ from those of both control groups. The results reveal that gonadotrophin-induced spermatogonial proliferation occurs under stressful conditions, whereas progress of spermatogenesis beyond primary spermatocyte stage is impaired due to inhibition (under stress) of gonadotrophin induced steroidogenic activity in M. carinata.