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Effect of Multivalency on Phase-Separated Droplets Consisting of Poly(PR) Dipeptide Repeats and RNA at the Solid/Liquid Interface.

ABSTRACT: Dipeptide repeat proteins (DRPs) are considered a significant cause of amyotrophic lateral sclerosis (ALS), and their liquid-liquid phase separation (LLPS) formation with other biological molecules has been studied both in vitro and in vivo. The immobilization and wetting of the LLPS droplets on glass surfaces are technically crucial for the measurement with optical microscopy. In this work, we characterized the surface diffusion of LLPS droplets of the DRPs with different lengths to investigate the multivalent effect on the interactions of their LLPS droplets with the glass surface. Using fluorescence microscopy and the single-particle tracking method, we observed that the large multivalency drastically changed the surface behavior of the droplets. The coalescence and wetting of the droplets were accelerated by increasing the multivalency of peptides in the LLPS system. Our findings on the effect of multivalency on interactions between droplets and glass surfaces could provide a new insight to enhance the understanding of LLPS formation and biophysical properties related to the solid/liquid interface.

SUBMITTER: Chen C

PROVIDER: S-EPMC9201888 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Effect of Multivalency on Phase-Separated Droplets Consisting of Poly(PR) Dipeptide Repeats and RNA at the Solid/Liquid Interface.

Chen Chen C Jia Han H Nakamura Yoshiki Y Kanekura Kohsuke K Hayamizu Yuhei Y

ACS omega 20220603 23

Dipeptide repeat proteins (DRPs) are considered a significant cause of amyotrophic lateral sclerosis (ALS), and their liquid-liquid phase separation (LLPS) formation with other biological molecules has been studied both in vitro and in vivo. The immobilization and wetting of the LLPS droplets on glass surfaces are technically crucial for the measurement with optical microscopy. In this work, we characterized the surface diffusion of LLPS droplets of the DRPs with different lengths to investigate ...[more]

PMID: 35721931

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Project description:Liquid-Liquid Phase Separation (LLPS) plays a crucial role in cell biology and is closely associated with neurodegenerative diseases like Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). Recent studies connect mutations in the C9ORF72 gene to the production of arginine-rich dipeptide repeat proteins (R-DPRs), such as poly(PR) and poly(GR). These R-DPRs disrupt LLPS in membrane-less organelles (MLOs) and contribute to disease pathology. While traditional analysis techniques like nuclear magnetic resonance (NMR), fluorescence recovery after photobleaching (FRAP), and Förster resonance energy transfer (FRET) provide insights into LLPS's role in these diseases, their ability is limited in detecting weak intermolecular interactions within LLPS droplets. This study employs graphene field-effect transistors (GFETs) for their superior sensitivity in detecting these molecular interactions. We immobilized RNA (poly-A) on GFETs and measured the electrical conductivity of GFETs to characterize shifts in the voltage of the charge neutral point in GFETs, allowing for the detection of dipeptide repeat peptides, such as (PR)12, (GR)12, and R12. Our results show that interactions between peptides and RNA require a specific peptide concentration threshold and vary between peptide types. Notably, the minimal conductivity shift suggests that peptides containing proline residues exhibit a nonuniform spatial distribution during interactions with RNA on graphene surfaces. This finding indicates that peptide rigidity induced by prolines plays a vital role in these molecular interactions and their multivalent contacts with RNA, which agrees with findings reported in other recent works. The capability of GFETs to detect these interactions at nanomolar concentrations marks a significant advancement in sensitivity over existing methods. This research sheds light on the mechanisms of LLPS involving R-DPRs and opens avenues for further understanding of related neurodegenerative diseases.

Project description:ObjectiveTo appraise the utility as biomarkers of blood antibodies and immune complexes to neurofilaments and dipeptide repeat proteins, the products of translation of the most common genetic mutation in amyotrophic lateral sclerosis (ALS).MethodsAntibodies and immune complexes against neurofilament light, medium, heavy chains as well as poly-(GP)-(GR) dipeptide repeats were measured in blood samples from the ALS Biomarkers (n = 107) and the phenotype-genotype biomarker (n = 129) studies and in 140 healthy controls. Target analyte levels were studied longitudinally in 37 ALS cases. Participants were stratified according to the rate of disease progression estimated before and after baseline and C9orf72 genetic status. Survival and longitudinal analyses were undertaken with reference to matched neurofilament protein expression.ResultsCompared to healthy controls, total neurofilament proteins and antibodies, neurofilament light immune complexes (p < 0.0001), and neurofilament heavy antibodies (p = 0.0061) were significantly elevated in ALS, patients with faster progressing disease (p < 0.0001) and in ALS cases with a C9orf72 mutation (p < 0.0003). Blood neurofilament light protein discriminated better ALS from healthy controls (AUC: 0.92; p < 0.0001) and faster from slower progressing ALS (AUC: 0.86; p < 0.0001) compared to heavy-chain antibodies and light-chain immune complexes (AUC: 0.79; p < 0.0001 and AUC: 0.74; p < 0.0001). Lower neurofilament heavy antibodies were associated with longer survival (Log-rank Chi-square: 7.39; p = 0.0065). Increasing levels of antibodies and immune complexes between time points were observed in faster progressing ALS.ConclusionsWe report a distinctive humoral response characterized by raising antibodies against neurofilaments and dipeptide repeats in faster progressing and C9orf72 genetic mutation carriers ALS patients. We confirm the significance of plasma neurofilament proteins in the clinical stratification of ALS.

Dataset Information

Effect of Multivalency on Phase-Separated Droplets Consisting of Poly(PR) Dipeptide Repeats and RNA at the Solid/Liquid Interface.

Publications

Effect of Multivalency on Phase-Separated Droplets Consisting of Poly(PR) Dipeptide Repeats and RNA at the Solid/Liquid Interface.

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