Biology subjects

Sheta, R.

Publications and source records attributed to Sheta, R..

4 recordsLinked to original sources

bioRxiv2026

WDR44 drives de novo α-synuclein aggregation at the lysosomal membrane and promotes neuronal dysfunction in Parkinson's Disease

The aggregation of -synuclein (-SYN) into Lewy bodies (LBs) is a central event in the pathogenesis of Parkinsons disease (PD) and related synucleinopathies1,2. Despite significant advances in understanding -SYN self-assembly, the precise sequence of early aggregation steps has not been directly visualized in living neurons. Here, we use an optogenetic-induced protein aggregation system with a high temporal resolution to monitor the onset of -SYN assembly in neurons. We found that the initiation and accumulation of -SYN aggregates occur predominantly at the lysosomal membrane, an event driven by the -SYN N-terminus and modulated by the membrane-associated adaptor protein WD repeat-containing protein 44 (WDR44). Remarkably, we demonstrate that WDR44 knockdown markedly reduced de novo -SYN aggregation in both neuronal cultures and in vivo, whereas WDR44 overexpression enhances -SYN aggregation in PD patient-derived iPSC neurons. Consistent with its potential pathogenic involvement, WDR44 aberrantly accumulates in vivo and in the brains of PD patients, where it colocalizes with LB inclusions. Finally, we show that lysosome-associated -SYN aggregates compromised lysosomal structure and function, leading to neuronal impairment, a phenotype worsened by WDR44 overexpression, linking early aggregation events to downstream toxicity. Together, these findings reveal the earliest dynamic stages of -SYN oligomerization in living neurons and identify the WDR44--SYN interaction as a promising therapeutic target for reducing -SYN pathology and enabling early intervention in PD.

neuroscience↗

bioRxiv2026

Modulating Innate Immune Responses to Curli Fibers Through Protein Engineering

Curli fibers produced by Escherichia coli are functional amyloids that activate Toll-like receptor 2 (TLR2), initiating innate immune responses at mucosal surfaces. While microbiome-derived curli contribute to host-microbe interactions, their intrinsic immunostimulatory activity limits their utility as programmable scaffolds for engineered probiotic systems, and dysregulated TLR2 activation has been linked to inflammatory bowel disease, systemic lupus erythematosus, neurodegeneration, and sepsis. Here, we engineered E. coli Nissle 1917 to produce modified curli fibers designed to inhibit TLR2 through two mechanistically distinct strategies: steric shielding via silk-elastin-like protein sequences, and direct receptor antagonism via a known TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3). Both engineered variants assembled into structurally intact amyloid fibers and exhibited significantly reduced intrinsic TLR2-dependent NF-{kappa}B activation in reporter cells. In competitive inhibition assays against structurally diverse TLR2 agonists, the SSL3 fusion achieved near-complete inhibition maintained under rising agonist load, while steric shielding provided moderate, agonist-class-dependent inhibition. In primary human monocyte-derived dendritic cells, the SSL3 fusion robustly attenuated IL-8 secretion and transcriptional induction of IL-8, IL-6, and IL-1{beta}, whereas steric shielding produced only partial attenuation that did not translate to broad inflammatory suppression. These results establish engineered curli as a tunable platform for receptor-specific modulation of innate immune signaling and highlight the broader potential of modular microbial amyloids as programmable interfaces for engineering host-microbe interactions at mucosal surfaces. IMPORTANCEBacteria residing in the gut produce protein fibers called curli that potently activate the immune system through a receptor called Toll-like receptor 2 (TLR2). While TLR2 plays a beneficial role in maintaining gut health, its overactivation drives chronic inflammation in conditions including inflammatory bowel disease, autoimmune diseases, neurodegenerative diseases, and sepsis, and curli fibers have been directly implicated in several of these conditions. Here, we engineered curli fibers produced by the probiotic E. coli Nissle 1917 to inhibit TLR2 activation, transforming a naturally inflammatory bacterial fiber into a programmable immune modulator. We demonstrated that direct receptor antagonism, rather than steric shielding, is required for effective immune modulation in primary human immune cells, establishing a design principle for engineering bacteria-derived fibers as programmable interfaces with host immunity. The modularity of the curli scaffold positions this platform as a broader tool for programming interactions between probiotic bacteria and the mucosal immune system.

synthetic biology↗

bioRxiv2025

Recapitulating Parkinson's pathology in human iPSC dopaminergic neurons reveals new mechanistic insights into Lewy body formation and heterogeneity.

The accumulation of alpha-synuclein (aSyn) into intraneuronal inclusions of heterogeneous morphology, known as Lewy bodies (LB), is one of the defining diagnostic features of Parkinsons disease (PD); yet, our understanding of the mechanisms underpinning their formation and heterogeneity remains incomplete. Here, we present a human isogenic iPSC-derived dopaminergic neuron (iDA) model that faithfully recapitulates the diverse biochemical, morphological, and ultrastructural features of LB neuropathology in PD. Unlike other iDA seeding models, our model does not rely on aSyn overexpression, mutations, or genetic engineering, making it a more physiologically relevant system for studying PD. We demonstrate that the iDA model accurately reproduces the temporal relationships between neuritic and cell-body aSyn pathology, recapitulating the full biochemical spectrum, post-translational modifications (PTM), and morphological diversity of aSyn aggregates found in human PD tissue. Moreover, our work provides critical insight into how different pathways to aSyn fibrillization and the complex interaction between aSyn fibrils and membranous organelles influence the morphological diversity of LB-like inclusions. This model represents a versatile platform for investigating the mechanisms of pathology formation, maturation, and neuronal dysfunction, as well as supporting the development of diagnostics that capture the diversity of aSyn pathology in PD and related synucleinopathies.

neuroscience↗

bioRxiv2024

The combination of optogenetic-induced protein aggregation and proximity biotinylation assays strongly implicates endolysosomal proteins in the early stages of α-synuclein aggregation

Alpha-synuclein (-syn) aggregation is a defining feature of Parkinsons disease (PD) and related synucleinopathies. Despite significant research efforts focused on understanding -syn aggregation mechanisms, the early stages of this process remain elusive, largely due to limitations in experimental tools that lack the temporal resolution to capture these dynamic events. Here, we introduce UltraID-LIPA, an innovative platform that combines the Light-Inducible Protein Aggregation (LIPA) system with the UltraID proximity-dependent biotinylation assay to identify -syn-interacting proteins and uncover key mechanisms driving its oligomerization. UltraID-LIPA successfully identified 38 -syn-interacting proteins, including both established and novel candidates, highlighting the accuracy and robustness of the approach. Notably, a strong interaction with endolysosomal and membrane-associated proteins was observed, supporting the hypothesis that interactions with membrane-bound organelles are pivotal in the early stages of -syn aggregation. This powerful platform provides new insights into dynamic protein aggregation events, enhancing our understanding of synucleinopathies and other proteinopathies.

neuroscience↗