Biology subjects

Rangachari, V.

Publications and source records attributed to Rangachari, V..

16 recordsLinked to original sources

bioRxiv2026

Leveraging Supramolecular Polymers to Induce the Targeted Protein Degradation of α-Synuclein

Halting the progression of neurodegenerative diseases remains one of the foremost challenges in medicinal chemistry due to the complex biology that drives disease progression. For example, a hallmark of synucleinopathies, such as Parkinsons disease, is the misfolding and aggregation of the protein -Synuclein (-Syn), driving the formation of toxic oligomers and fibrils that avoid natural intracellular clearance mechanisms, participate in unusual protein-protein interactions, and ultimately contribute to the death of dopaminergic neurons. The field of targeted protein degradation (TPD) has emerged as an innovative therapeutic route to selectively degrade proteins of interest that leverage natural intracellular protein degradation machinery. First generation TPD therapeutics have traditionally been designed as bifunctional, chimeric compounds in which a short covalent linker tethers a ligand designed to bind target proteins to a ligand that initiates an either proteosome- or lysosome-dependent protein degradation cascade. While initial studies have indicated the promise of these approaches, translation to the clinical setting has been challenging due to difficulties in achieving cellular internalization, long-term stability, and establishment of a generalizable strategy. To overcome these obstacles, this work has focused on adding modularity and dynamic capability to this classical model by leveraging a multivalent macromolecular approach to TPD. Specifically, peptide amphiphiles (PAs) were designed to self-assemble into high-aspect-ratio supramolecular nanofibers and present peptide epitopes on the surface of the fibers to target simultaneous binding of -Syn and recruitment of enzymes that facilitate entry into the lysosome-dependent chaperone-mediated autophagy protein degradation pathway. In vitro application of these bioactive PA nanofibers has demonstrated the ability to independently internalize in cells and reduce -Syn protein levels selectively and effectively. While further optimization of this model has the potential to be a viable therapeutic against -Syn aggregation, the modularity of these supramolecular nanofibers through facile monomer design and incorporation illustrates the potential of establishing a platform technology for targeting a diverse range of pathologic proteins. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/739556v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@d031adorg.highwire.dtl.DTLVardef@6d792dorg.highwire.dtl.DTLVardef@12e80c1org.highwire.dtl.DTLVardef@71a2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Disulfide cross-linked redox-sensitive peptide condensates are efficient cell delivery vehicles of molecular cargo.

Biomolecular condensates (BCs) are phase-separated viscoelastic hubs within demixed solutions enriched in proteins and nucleic acids. Such condensates, also called membraneless organelles, are increasingly observed in cells and serve as transient hubs for spatial organization and compartmentalization of biomolecules. Along with the transiency of formation and dissolution, their ability to sequester molecules has inspired us to develop BCs as potential vehicles to transport and deliver molecular cargo. We recently reported the design of disulfide bond cross-linked phase-separating peptide (PSP) condensates that spontaneously dissolve in reducing conditions (JACS, 2024, 146, 255299). Based on the premise that the highly reducing cytoplasm could dissolve PSP condensates and release partitioned cargo, here, we demonstrate the ability of PSP condensates to deliver molecular cargo to the cytoplasm of HeLa cells efficiently. We show that PSP condensates deliver a variety of cargos that differ in their sizes and chemistries, including small molecules, peptides, GFP protein (31 kDa), DNA (1.7 kbp), and mRNA. The transfection efficiencies of PSP condensates for delivering DNA and mRNA were also significantly greater than those of a commercial transfection agent. With room to tailor the condensate properties based on cargo and cell types, these results showcase the potential of disulfide-cross-linked PSPs as effective and customizable cellular delivery vehicles, filling a critical demand gap for such delivery systems.

biophysics↗

bioRxiv2024

Amyloid peptide - synthetic polymer blends with enhanced mechanical and biological properties

Interest in utilizing amyloids to develop biomaterials is increasing due to their potential for biocompatibility, unique assembling morphology, mechanical stability, and biophysical properties. However, challenges include the complexity of peptide chemistry and the practical techniques required for processing amyloids into bulk materials. In this work, two decapeptides with fibrillar and globular morphologies were selected, blended with poly(ethylene oxide), and fabricated into composite mats via electrospinning. Notable enhancements in mechanical properties were observed, attributed to the uniform distribution of the decapeptide assemblies within the PEO matrix. Morphological differences, such as the production of thinner nanofibers, are attributed to the increased conductivity from the zwitterionic nature of the decapeptides. Blend rheology and post-processing analysis revealed how processing might affect the amyloid aggregation and secondary structure of the peptides. Both decapeptides demonstrated good biocompatibility and strong antioxidant activity, indicating their potential for safe and effective use as biomaterials. By evaluating these interdependencies, this research lays the foundation for understanding the structure-property-processing relationships of peptide-polymer blends and highlights the strong potential for developing applications in biotechnology.

biophysics↗

bioRxiv2024

Reversible disulfide bond crosslinks as tunable levers of phase separation in designer biomolecular condensates.

Biomolecular condensates (BCs) are membraneless hubs enriched in proteins and nucleic acids that have become important players in many cellular functions. Uncovering the sequence determinants of proteins for phase separation is important in understanding the biophysical and biochemical properties of BCs. Despite significant discoveries in the last decade, the role of cysteine residues in BC formation and dissolution has remained unknown. Here, to determine the involvement of disulfide crosslinks and their redox sensitivity in BCs, we designed a stickers and spacers model of phase-separating peptides interspersed with cysteines. Through biophysical investigations, we learned that cysteines promote liquid-liquid phase separation in oxidizing conditions and perpetuate liquid condensates through disulfide crosslinks, which can be reversibly tuned with redox chemistry. By varying the composition of cysteines, subtle but distinct changes in the viscoelastic behavior of the condensates were observed. Empirically, we conclude that cysteines are neither stickers nor spacers but function as covalent nodes to lower the effective concentrations for sticker interactions and inhibit system-spanning percolation networks. Together, we unmask the role of cysteines in protein phase behavior and the potential to develop tunable, redox-sensitive viscoelastic materials.

biophysics↗

bioRxiv2023

α-Synuclein emulsifies TDP-43 prion-like domain - RNA liquid droplets to promote heterotypic amyloid fibrils

Many neurodegenerative diseases including frontotemporal lobar degeneration (FTLD), Lewy body disease (LBD), multiple system atrophy (MSA), etc., show colocalized deposits of TDP-43 and -synuclein (S) aggregates. To understand whether these colocalizations are driven by specific molecular interactions between the two proteins, we previously showed that the prion-like C-terminal domain of TDP-43 (TDP-43PrLD) and S synergistically interact to form neurotoxic heterotypic amyloids in homogeneous buffer conditions. However, it remains unclear whether and how S modulates TDP-43 present within liquid droplets and biomolecular condensates called stress granules (SGs). Here, using cell culture and in vitro TDP-43PrLD - RNA liquid droplets as models along with microscopy, nanoscale spatially-resolved spectroscopy, and other biophysical analyses, we uncover the interactions of S with phase-separated droplets. We learn that S acts as a Pickering agent by forming clusters on the surface of TDP-43PrLD - RNA droplets and emulsifying them. The hardening of the droplets that follow by S aggregates on the periphery, nucleates the formation of heterotypic TDP-43PrLD amyloid fibrils with structures distinct from those derived from homogenous solutions. Together, these results reveal an intriguing property of S as a Pickering agent in interacting with SGs and unmask the hitherto unknown role of S in modulating TDP-43 proteinopathies.

biophysics↗

bioRxiv2023

Sugar distributions on gangliosides guide the formation and stability of amyloid-β oligomers

Aggregation of A{beta} peptides has been known as a key contributor to the etiology of Alzheimers disease. Being intrinsically disordered, the monomeric A{beta} is susceptible to conformational excursions, especially in the presence of key interacting partners such as membrane lipids, to adopt specific aggregation pathways. Furthermore, key components such as gangliosides in membranes and lipid rafts are known to play important roles in the adoption of pathways and the generation of discrete neurotoxic oligomers. Yet, what roles the carbohydrates on gangliosides play in this process remains unknown. Here, using GM1, GM3, and GD3 ganglioside micelles as models, we show that the sugar distributions and cationic amino acids within A{beta} N-terminal region modulate oligomerization of A{beta} temporally, and dictate the stability and maturation of oligomers.

biophysics↗

bioRxiv2023

De novo amyloid peptides with subtle sequence variations differ in their self-assembly and nanomechanical properties

Proteinaceous amyloids are well known for their widespread pathological roles but lately have emerged also as key components in several biological functions. The remarkable ability of amyloid fibers to form tightly packed conformations in a cross {beta}-sheet arrangement manifests in their robust enzymatic and structural stabilities. These characteristics of amyloids make them attractive for designing proteinaceous biomaterials for various biomedical and pharmaceutical applications. In order to design customizable and tunable amyloid nanomaterials, it is imperative to understand the sensitivity of the peptide sequence for subtle changes based on amino acid position and chemistry. Here we report our results from four rationally-designed amyloidogenic decapeptides that subtly differ in hydrophobicity and polarity at positions 5 and 6. We show that making the two positions hydrophobic renders the peptide with enhanced aggregation and material properties while the introduction of polar residues in position 5 dramatically changes the structure and nanomechanical properties of the fibrils formed. A charged residue at position 6, however, completely abrogates amyloid formation. In sum, we show that subtle changes in the sequence do not make the peptide innocuous but rather sensitive to aggregation, reflected in the biophysical and nanomechanical properties of the fibrils. We conclude that tolerance of peptide amyloid for subtle changes in the sequence should not be neglected for the effective design of customizable amyloid nanomaterials.

biophysics↗

bioRxiv2022

Distinct neurotoxic TDP-43 fibril polymorphs can be generated by heterotypic interactions with α-synuclein

Amyloid aggregates of specific proteins form important pathological hallmarks in many neurodegenerative diseases, defining neuronal degeneration and disease onset. Recently, increasing numbers of patients show co-morbidities and overlaps between multiple neurodegenerative diseases, presenting distinct phenotypes. Such overlaps are often accompanied by co-localizations of more than one amyloid protein, prompting the question of whether direct interactions between different amyloid proteins could generate heterotypic amyloids. To answer this question, we investigated the effect of -synuclein (S) on TDP-43 aggregation inspired by their co-existence in pathologies such as Lewy body dementia and limbic predominant age-related TDP-43 encephalopathy. We previously showed that S and prion-like C-terminal domain (PrLD) of TDP-43 synergistically interact with one another to generate toxic heterotypic aggregates in vitro. Here, we extend these studies to investigate whether S induces structurally and functionally distinct polymorphs of PrLD aggregates. Using S -PrLD heterotypic aggregates generated in two different stoichiometric proportions, we show that S can effect PrLD fibril forms. The fibril samples have distinctive residue-level structural signatures in NMR spectra, dye-binding capability, proteinase K (PK) stability, and SDS-sensitive thermal stability. By gold nanoparticle labeling and TEM, we show the presence of both S and PrLD proteins within the same fibrils, and thus the existence of hetertypic hybrid fibrils. We also observe that S and PrLD co-localize in the cytosol of SH-SY5Y neuroblastoma cells, and show that the heterotypic PrLD fibrils selectively induce synaptic dysfunction in primary cortical neurons. These findings establish the existence of heterotypic amyloid polymorphs and provide a molecular basis for the observed overlap between synucleinopathies and TDP-43 proteinopathies.

biophysics↗

bioRxiv2022

Ganglioside enriched phospholipid vesicles induce cooperative Aβ oligomerization and membrane disruption

A major hallmark of Alzheimer disease (AD) is the accumulation of extracellular aggregates of amyloid-{beta} (A{beta}). Structural polymorphism observed among A{beta} fibrils in AD brains seem to correlate with the clinical sub-types suggesting a link between fibril polymorphism and pathology. Since fibrils emerge from a templated growth of low-molecular weight oligomers, understanding the factors affecting oligomer generation is important. The membrane lipids are key factors that influence early stages of A{beta} aggregation and oligomer generation, and cause membrane disruption. We have previously demonstrated that conformationally discrete A{beta} oligomers can be generated by modulating the charge, composition, chain length of lipids and surfactants. Here, we extend our studies into liposomal models by investigating A{beta} oligomerization on large unilamellar vesicles (LUVs) of total brain extracts (TBE), reconstituted lipid rafts (LRs) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Specifically, we varied the vesicle composition by varying the amount of GM1 gangliosides added as a constituent. We found that liposomes enriched in GM1 induce the formation of toxic, low-molecular weight oligomers that are isolable in a lipid-complexed form. Importantly, the data indicate that oligomer formation and membrane disruption are highly cooperative processes. Numerical simulations on the experimental data confirm cooperativity and reveal that GM1-enriched liposomes form twice as many numbers of pores as those without GM1. Overall, this study uncovers mechanisms of cooperativity between oligomerization and membrane disruption under controlled lipid compositional bias, and refocuses the significance of the early stages of A{beta} aggregation in polymorphism, propagation, and toxicity in AD. TOC figure O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biophysics↗

bioRxiv2021

αS oligomers generated from polyunsaturated fatty acid and dopamine metabolite differentially interact with Aβ to enhance neurotoxicity

It is increasingly becoming clear that neurodegenerative diseases are not as discrete as originally thought to be but rather display significant overlap in histopathological and clinical presentations. For example, nearly half of the patients with Alzheimer disease (AD) and synucleinopathies such as Parkinson disease (PD) show symptoms and pathological features of one another. Yet, the molecular events and features that underlie such comorbidities in neurodegenerative diseases remain poorly understood. Here, inspired to uncover the molecular underpinnings of the overlap between AD and PD, we investigated the interactions between amyloid-{beta} (A{beta}) and -synuclein (S), aggregates of which form the major components of amyloid plaques and Lewy bodies, respectively. Specifically, we focused on S oligomers generated from the dopamine metabolite called dihydroxyphenylacetaldehyde (DOPAL), and a polyunsaturated fatty acid docosahexaenoic acid (DHA). Both S oligomers showed structural and conformational differences confirmed by their disparity in size, secondary structure, susceptibility to proteinase K digestion and cytotoxicity. More importantly, the two oligomers differentially modulated A{beta} aggregation. While both oligomers inhibited A{beta} aggregation to varying extents, they induced structurally different A{beta} assemblies. Furthermore, A{beta} seeded with DHA-derived S oligomers showed greater toxicity than DOPAL-derived S oligomers in SH-SY5Y neuroblastoma cells. These results provide insights into the interactions between two amyloid proteins with empirically distinctive biophysical and cellular manifestations, enunciating a basis for potentially ubiquitous cross-amyloid interactions across many neurodegenerative diseases.

biophysics↗

bioRxiv2021

Cytoplasmic Colocalization of Granulins and TDP-43 Prion-like Domain Involves Electrostatically Driven Complex Coacervation Tuned by the Redox State of Cysteines.

Cytoplasmic inclusions containing aberrant proteolytic fragments of TDP-43 are associated with frontotemporal lobar degeneration (FTLD) and other related pathologies. In FTLD, TDP-43 is translocated into the cytoplasm and proteolytically cleaved to generate a prion-like domain (PrLD) containing C-terminal fragments (C25 and C35) that form toxic inclusions. Under stress, TDP-43 partitions into membraneless organelles called stress granules (SGs) by coacervating with RNA and other proteins. We were interested in understanding if and how cysteine-rich granulins (GRNs 1-7), which are the proteolytic products of a genetic risk factor in FTLD called progranulin, interact with TDP-43. We show that extracellular GRNs internalize and colocalize with PrLD as puncta in the cytoplasm of neuroblastoma cells but show no presence in SGs. In addition, we show GRNs and PrLD coacervate to undergo liquid-liquid phase separation (LLPS) or form gel- or solid-like aggregates. Identification of the sequence determinants within GRNs for the observed phase transitions reveal the negative charges to be the drivers of LLPS modulated by the positive charges and the redox state of cysteines. Furthermore, RNA and GRNs compete and expunge one another from PrLD condensates, providing a basis for GRNs absence in SGs. Together, the results illustrate the potential mechanisms by which extracellular GRNs, formed during chronic inflammatory conditions, could internalize, and modulate cytoplasmic TDP-43 inclusions in proteinopathies.

biophysics↗

bioRxiv2020

Prion-like C-terminal domain of TDP-43 and α-Synuclein interact synergistically to generate neurotoxic hybrid fibrils

Aberrant aggregation and amyloid formation of tar DNA binding protein (TDP-43) and -synuclein (S) underlie frontotemporal dementia (FTD) and Parkinsons disease (PD), respectively. Amyloid inclusions of TDP-43 and S are also commonly co-observed in amyotrophic lateral sclerosis (ALS), dementia with Lewy bodies (DLB) and Alzheimer disease (AD). Emerging evidence from cellular and animal models show colocalization of the TDP-43 and S aggregates, raising the possibility of direct interactions and co-aggregation between the two proteins. In this report, we set out to answer this question by investigating the interactions between S and prion-like pathogenic C-terminal domain of TDP-43 (TDP-43 PrLD). PrLD is an aggregation-prone fragment generated both by alternative splicing as well as aberrant proteolytic cleavage of full length TDP-43. Our results indicate that two proteins interact in a synergistic manner to augment each others aggregation towards hybrid fibrils. While monomers, oligomers and sonicated fibrils of S seed TDP-43 PrLD monomer aggregation, TDP-43 PrLD fibrils failed to seed S monomers indicating selective interactions. Furthermore, S modulates liquid droplets formed by TDP-43 PrLD and RNA to promote insoluble amyloid aggregates. Importantly, the cross-seeded hybrid aggregates show greater cytotoxicity as compared to the individual homotypic aggregates suggesting that the interactions between the two proteins have a discernable impact on cellular functions. Together, these results bring forth insights into TDP-43 PrLD - S interactions that could help explain clinical and pathological presentations in patients with co-morbidities involving the two proteins.

biophysics↗

bioRxiv2020

Are Granulins Copper Sequestering Proteins?

Granulins (GRN 1-7) are short ([~]6 kDa), cysteine-rich proteins that are generated upon the proteolytic processing of progranulin (PGRN). These modules, along with their precursor, have been implicated in multiple pathophysiological roles, especially in neurodegenerative diseases. Our previous investigations into GRN-3 and GRN-5 reveal them to be fully disordered in the reduced form and implicate redox sensitive attributes to the proteins. Such redox-dependent modulation has become associated with proteins involved in oxidative stress regulation and maintaining metal-homeostasis within cells. To probe whether GRNs play a contributory role in such functions, we tested the metal binding potential of the reduced form of GRNs -3 and -5 under neutral and acidic pH mimicking cytosolic and lysosomal conditions, respectively. We found, at neutral pH, both GRNs selectively bind Cu(II) and no other divalent cations. Binding of Cu(II) also partly triggered the oxidative multimerization of GRNs via uncoordinated cystines at both pH conditions. Furthermore, binding did not induce gain in secondary structure and the protein remained disordered. Overall, the results indicate that GRN-3 and -5 have a surprisingly strong affinity for Cu(II) in the pM range, comparable to known copper sequestering proteins. This data also hints at a potential of GRNs to reduce Cu(II) to Cu(I), a process that has significance in mitigating Cu-induced ROS cytotoxicity in cells. Together, this report uncovers a metal-coordinating capability of GRNs for the first time, which could have profound significance in their structure and function.

biophysics↗

bioRxiv2019

Granulins modulate liquid-liquid phase separation and aggregation of TDP-43 C-terminal domain

Tar DNA binding protein (TDP-43) has emerged as a key player in many neurodegenerative pathologies including frontotemporal lobar degeneration (FTLD) and amyotropic lateral sclerosis (ALS). Important hallmarks of FTLD and ALS are the toxic cytoplasmic inclusions of C-terminal fragments of TDP-43 (TDP-43CTD), which are formed upon proteolytic cleavage of full-length TDP-43 in the nucleus and subsequent transport to the cytoplasm. TDP-43CTD is also known to form stress granules (SGs) by coacervating with RNA in cytoplasm under stress conditions and are believed to be involved in modulating the pathologies. Among other factors affecting these pathologies, the pleiotropic protein called progranulin (PGRN) has gained significant attention lately. The haploinsufficiency of PGRN, caused by autosomal dominant mutations in GRN gene, results in its loss-of-function linked to FTLD and ALS. But precisely how the protein contributes to the pathology remains unknown. Recently, cleavage to GRNs were observed to be a significant part of FTLD and ALS progression with specific GRNs exacerbating TDP-43-induced toxicity in C.elegans. In this report, we show that GRNs -3 and -5 directly interact with TDP-43CTD to modulate latters aggregation or stress granule formation in disparate ways in vitro. These results constitute the first observation of direct interaction between GRNs and TDP-43 and suggest a mechanism by which the loss of PGRN function could lead to FTLD and ALS.

biophysics↗

bioRxiv2019

A game theoretic approach to deciphering the dynamics of amyloid-β aggregation along competing pathways

Aggregation of amyloid {beta} (A{beta}) peptides is a significant event that underpins Alzheimer disease (AD). A{beta} aggregates, especially the low-molecular weight oligomers, are the primary toxic agents in AD pathogenesis. Therefore, there is increasing interest in understanding their formation and behavior. In this paper, we use our previously established investigations on heterotypic interactions between A{beta} and fatty acids (FAs) that adopt off-fibril formation pathway under the control of FA concentrations, to develop a mathematical framework in defining this complex mechanism. We bring forth the use of novel game theoretic framework based on the principles of Nash equilibria to define and simulate the competing on- and off-pathways of A{beta} aggregation. Together with detailed simulations and biophysical experiments, our mathematical models define the dynamics involved in the mechanisms of A{beta} aggregation in the presence of FAs to adopt multiple pathways. Specifically, our game theoretic model indicates that the emergence of off- or on-pathway aggregates are tightly controlled by a narrow set of rate constant parameters, and one could alter such parameters to populate a particular oligomeric species. These models agree with the detailed simulations and experimental data on using FA as a heterotypic partner to modulate temporal parameters. Predicting spatiotemporal landscape along competing pathways for a given heterotypic partner such as biological lipids is a first step towards simulating physiological scenarios in which the generation of specific conformeric strains of A{beta} could be predicted. Such an approach could be profoundly significant in deciphering the biophysics of amyloid aggregation and oligomer generation, which is ubiquitously observed in many neurodegenerative diseases.

biophysics↗

bioRxiv2018

Large Fatty Acid-derived Aβ42 oligomers Form Ring-like Assemblies

As the primary toxic species in the etiology of Alzheimer disease (AD) are low molecular weight oligomers of A{beta}, it is crucial to understand the structure of A{beta} oligomers for gaining molecular insights into AD pathology. We have earlier demonstrated that in the presence of fatty acids A{beta}42 peptides assemble as 12-24mer oligomers. These Large Fatty Acid-derived Oligomers (LFAOs) exist predominantly as 12mers at low, and as 24mers at high concentrations. The 12mers are more neurotoxic than the 24mers and undergo self-replication, while the latter propagate to morphologically distinct fibrils with succinct pathological consequences. In order to glean into their functional differences and similarities, we have determined their structures in greater detail by combining molecular dynamic simulations with biophysical measurements. We conjecture that the LFAO are made of A{beta} units in an S-shaped conformation, with the 12mers forming a double-layered hexamer ring (6 x 2) while the structure of 24mers is a double-layered dodecamer ring (12 x 2). A closer inspection of the (6 x 2) and (12 x 2) structures reveals a concentration and pH dependent molecular reorganization in the assembly of 12 to 24mers, that seems to be the underlying mechanism for the observed biophysical and cellular properties of LFAOs.

biophysics↗