Spatiotemporal modulations in heterotypic condensates of prion and α-synuclein control phase transitions and amyloid conversion.

Biomolecular condensation via liquid-liquid phase separation of proteins and nucleic acids is associated with a range of critical cellular functions and neurodegenerative diseases. Here, we demonstrate that complex coacervation of the prion protein and α-synuclein within narrow stoichiometry results in the formation of highly dynamic, reversible, thermo-responsive liquid droplets via domain-specific electrostatic interactions between the positively-charged intrinsically disordered N-terminal segment of prion and the acidic C-terminal tail of α-synuclein. The addition of RNA to these coacervates yields multiphasic, vesicle-like, hollow condensates. Picosecond time-resolved measurements revealed the presence of transient electrostatic nanoclusters that are stable on the nanosecond timescale and can undergo breaking-and-making of interactions on slower timescales giving rise to a liquid-like behavior in the mesoscopic regime. The liquid-to-solid transition drives a rapid conversion of complex coacervates into heterotypic amyloids. Our results suggest that synergistic prion-α-synuclein interactions within condensates provide mechanistic underpinnings of their physiological role and overlapping neuropathological features.

Prion Protein Biology Through the Lens of Liquid-Liquid Phase Separation.

Conformational conversion of the α-helix-rich cellular prion protein into the misfolded, ß-rich, aggregated, scrapie form underlies the molecular basis of prion diseases that represent a class of invariably fatal, untreatable, and transmissible neurodegenerative diseases. However, despite the extensive and rigorous research, there is a significant gap in the understanding of molecular mechanisms that contribute to prion pathogenesis. In this review, we describe the historical perspective of the development of the prion concept and the current state of knowledge of prion biology including structural, molecular, and cellular aspects of the prion protein. We then summarize the putative functional role of the N-terminal intrinsically disordered segment of the prion protein. We next describe the ongoing efforts in elucidating the prion phase behavior and the emerging role of liquid-liquid phase separation that can have potential functional relevance and can offer an alternate non-canonical pathway involving conformational conversion into a disease-associated form. We also attempt to shed light on the evolutionary perspective of the prion protein highlighting the potential role of intrinsic disorder in prion protein biology and summarize a few important questions associated with the phase transitions of the prion protein. Delving deeper into these key aspects can pave the way for a detailed understanding of the critical molecular determinants of the prion phase transition and its relevance to physiology and neurodegenerative diseases.

An intrinsically disordered pathological prion variant Y145Stop converts into self-seeding amyloids via liquid-liquid phase separation.

Biomolecular condensation via liquid-liquid phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) along with other biomolecules is proposed to control critical cellular functions, whereas aberrant phase transitions are associated with a range of neurodegenerative diseases. Here, we show that a disease-associated stop codon mutation of the prion protein (PrP) at tyrosine 145 (Y145Stop), resulting in a truncated, highly disordered, N-terminal IDR, spontaneously phase-separates into dynamic liquid-like droplets. Phase separation of this highly positively charged N-terminal segment is promoted by the electrostatic screening and a multitude of weak, transient, multivalent, intermolecular interactions. Single-droplet Raman measurements, in conjunction with an array of bioinformatic, spectroscopic, microscopic, and mutagenesis studies, revealed a highly mobile internal organization within the liquid-like condensates. The phase behavior of Y145Stop is modulated by RNA. Lower RNA:protein ratios promote condensation at a low micromolar protein concentration under physiological conditions. At higher concentrations of RNA, phase separation is abolished. Upon aging, these highly dynamic liquid-like droplets gradually transform into ordered, ß-rich, amyloid-like aggregates. These aggregates formed via phase transitions display an autocatalytic self-templating characteristic involving the recruitment and binding-induced conformational conversion of monomeric Y145Stop into amyloid fibrils. In contrast to this intrinsically disordered truncated variant, the wild-type full-length PrP exhibits a much lower propensity for both condensation and maturation into amyloids, hinting at a possible protective role of the C-terminal domain. Such an interplay of molecular factors in modulating the protein phase behavior might have much broader implications in cell physiology and disease.

Ultrasensitive Characterization of the Prion Protein by Surface-Enhanced Raman Scattering: Selective Enhancement via Electrostatic Tethering of the Intrinsically Disordered Domain with Functionalized Silver Nanoparticles.

Surface-enhanced Raman scattering (SERS) circumvents the inherent insensitivity of Raman spectroscopy and offers a powerful tool for the ultrasensitive detection and characterization of biomolecules at low concentrations. Here we show that SERS via electrostatic tethering between surface-modified negatively charged silver nanoparticles and highly positively charged intrinsically disordered N-terminal domain of the prion protein allows highly sensitive and reproducible protein detection and characterization at as low as hundreds of nanomolar protein concentrations. These measurements preferentially illuminate a selective part of the protein due to a sharp dependence of the near-field intensity on the distance between the nanoparticle surface and the protein. We also demonstrate that by shortening the length of the disordered tail it is possible to achieve a domain-selective Raman enhancement to study the C-terminal globular domain. Our tether-length-dependent SERS methodology will serve as a potent, noninvasive, and label-free strategy to detect and characterize a wide range of proteins possessing disordered segments.

National Health Burden of Aspiration Pneumonia on Clinical and Operational Outcomes in Patients With Small Bowel Obstruction Requiring Nasogastric Decompression.

BACKGROUND: Nasogastric tubes (NGTs) are used for decompression in patients with acute small bowel obstruction (SBO); however, their role remains controversial. There is evidence that NGT use is still associated with high incidence of aspiration pneumonia. The aims of this study were to define the prevalence of aspiration pneumonia in patients with SBO managed with an NGT and estimate the association of aspiration pneumonia with 30-day mortality rates, length of stay (LOS), and hospital costs. MATERIALS AND METHODS: A retrospective cohort study was done using Medicare Inpatient Standard Analytic Files from 2016 to 2018. Patients hospitalized with SBO and managed with NGT were identified using an algorithm of ICD-10-CM codes. The key exposure was aspiration pneumonia. Outcome measures included 30-day mortality rates, LOS, and hospital costs. RESULTS: 53 715 patients hospitalized with SBO and managed with an NGT were identified and included in the analysis. We observed a prevalence of aspiration pneumonia of 7.3%. The 30-day mortality rate was 31% for those who developed aspiration pneumonia vs. 10% for those without pneumonia (P < .001). Those with aspiration pneumonia, on average, were hospitalized 7.0 days longer (P < .001) and accrued $20,543 greater hospitalization costs (P < .001) than those without pneumonia. Controlling for hospital size and hospital teaching status, we noted a significant association between aspiration pneumonia and increased mortality (P < .001), longer length of stay (P < .001), and higher hospital costs (P < .001). DISCUSSION: Among patients hospitalized for SBO who required an NGT, aspiration pneumonia was associated with a higher mortality rate, longer hospital LOS, and higher total hospital costs. vv.

Energy migration captures membrane-induced oligomerization of the prion protein.

Excitation energy migration via homo-Förster resonance energy transfer (homo-FRET) can serve as an intermolecular proximity ruler within complex biomolecular assemblies. Here we present a unique case to demonstrate that energy migration can be a novel and sensitive readout to capture the membrane-mediated misfolding and oligomerization of the human prion protein (PrP), which is known to undergo an aberrant conformational conversion from an α-helical form into a self-propagating aggregated ß-rich state causing deadly transmissible neurodegenerative diseases. Using site-specific energy migration studies by monitoring steady-state and time-resolved fluorescence anisotropy of fluorescently-tagged PrP, we elucidate the molecular details of lipid membrane-induced oligomers. We show that the intrinsically disordered N-terminal segment is critical for lipid-induced conformational sequestration of PrP into higher-order, ß-rich oligomeric species that exhibit membrane permeabilization. Our results revealed that the N-terminal regions constitute the central core of the oligomeric architecture, whereas the distal C-terminal ends participate in peripheral association with the lipid membrane. Our study will find applications in the sensitive detection and in the structural characterization of membrane-induced protein misfolding and aggregation in a variety of deadly amyloid diseases.