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α-Synuclein (α-Syn) aggregation and amyloid formation is directly linked with Parkinson’s disease pathogenesis. However, the early events involved in this process remain unclear. Here, using the in vitro reconstitution and cellular model, we show that liquid–liquid phase separation of α-Syn precedes its aggregation. In particular, in vitro generated α-Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form an amyloid hydrogel that contains oligomers and fibrillar species. Factors known to aggravate α-Syn aggregation, such as low pH, phosphomimetic substitution and familial Parkinson’s disease mutations, also promote α-Syn liquid–liquid phase separation and its subsequent maturation. We further demonstrate α-Syn liquid-droplet formation in cells. These cellular α-Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. This work provides detailed insights into the phase-separation behaviour of natively unstructured α-Syn and its conversion to a disease-associated aggregated state, which is highly relevant in Parkinson’s disease pathogenesis.The mechanism of nucleation for α-synuclein (α-Syn) aggregation and amyloid formation in Parkinson’s disease is unclear. Now, α-Syn has been shown to undergo liquid–liquid phase separation and a liquid-to-solid-like transition leading to amyloid fibril formation. This raises the possibility that liquid–liquid phase separation is a key pathogenic mechanism behind α-Syn aggregation in Parkinson’s disease.

Liquid–liquid phase separation has emerged as an important means of intracellular RNA compartmentalization. Some membraneless organelles host two or more compartments serving different putative biochemical roles. The mechanisms for, and functional consequences of, this subcompartmentalization are not yet well understood. Here we show that adjacent phases of decapeptide-based multiphase model membraneless organelles differ markedly in their interactions with RNA. Single- and double-stranded RNAs preferentially accumulate in different phases within the same droplet, and one phase is more destabilizing for RNA duplexes than the other. Single-phase peptide droplets did not capture this behaviour. Phase coexistence introduces new thermodynamic equilibria that alter RNA duplex stability and RNA sorting by hybridization state. These effects require neither biospecific RNA-binding sites nor full-length proteins. As such, they are more general and point to primitive versions of mechanisms operating in extant biology that could aid understanding and enable the design of functional artificial membraneless organelles. The biochemical roles and mechanisms of multiphase membraneless organelles are not yet well understood. Now, multiphase peptide droplets have been shown to sort RNA based on whether it is single- or double-stranded, as well as impact RNA duplexation through in-droplet thermodynamic equilibria. This work provides insight into possible primitive mechanisms for multicompartment intracellular condensates and can aid in the design of functional artificial membraneless organelles.

Biological cells are highly organized, with numerous subcellular compartments. Phosphorylation has been hypothesized as a means to control the assembly/disassembly of liquid-like RNA- and protein-rich intracellular bodies, or liquid organelles, that lack delimiting membranes. Here, we demonstrate that charge-mediated phase separation, or complex coacervation, of RNAs with cationic peptides can generate simple model liquid organelles capable of reversibly compartmentalizing biomolecules. Formation and dissolution of these liquid bodies was controlled by changes in peptide phosphorylation state using a kinase/phosphatase enzyme pair. The droplet-generating phase transition responded to modification of even a single serine residue. Electrostatic interactions between the short cationic peptides and the much longer polyanionic RNAs drove phase separation. Coacervates were also formed on silica beads, a primitive model for localization at specific intracellular sites. This work supports phosphoregulation of complex coacervation as a viable mechanism for dynamic intracellular compartmentalization in membraneless organelles. Intracellular bodies called liquid organelles are rich in nucleic acids and proteins, and are thought to occur by liquid–liquid phase coexistence. Now, enzymatic control over the phosphorylation state of a simple cationic peptide, thereby altering its electrostatic interaction with RNA, has been shown to drive formation and dissolution of droplets that mimic these intracellular liquid bodies.

Membraneless organelles within cells have unique microenvironments that play a critical role in their functions. However, how microenvironments of biomolecular condensates affect their structure and function remains unknown. In this study, we investigated the micropolarity and microviscosity of model biomolecular condensates by fluorescence lifetime imaging coupling with environmentally sensitive fluorophores. Using both in vitro and in cellulo systems, we demonstrated that sufficient micropolarity difference is key to forming multilayered condensates, where the shells present more polar microenvironments than the cores. Furthermore, micropolarity changes were shown to be accompanied by conversions of the layered structures. Decreased micropolarities of the granular components, accompanied by the increased micropolarities of the dense fibrillar components, result in the relocation of different nucleolus subcompartments in transcription-stalled conditions. Our results demonstrate the central role of the previously overlooked micropolarity in the regulation of structures and functions of membraneless organelles. Ye et al. reveal the critical role of micropolarity in controlling the structure and miscibility of subcompartments in multiphasic biomolecular condensates, thereby providing new insights into multiphasic condensation regulation.

Phase separation of intrinsically disordered proteins (IDPs) is a remarkable feature of living cells to dynamically control intracellular partitioning. Despite the numerous new IDPs that have been identified, progress towards rational engineering in cells has been limited. To address this limitation, we systematically scanned the sequence space of native IDPs and designed artificial IDPs (A-IDPs) with different molecular weights and aromatic content, which exhibit variable condensate saturation concentrations and temperature cloud points in vitro and in cells. We created A-IDP puncta using these simple principles, which are capable of sequestering an enzyme and whose catalytic efficiency can be manipulated by the molecular weight of the A-IDP. These results provide a robust engineered platform for creating puncta with new, phase-separation-mediated control of biological function in living cells.Artificial intrinsically disordered proteins (A-IDPs) have now been shown to form exclusionary, intracellular droplets that can be designed using simple principles that are based on the aromatic/aliphatic ratio and molecular weight. Droplets that sequester an enzyme and modulate enzyme efficiency on the basis of the molecular weight of the A-IDPs were also engineered using A-IDPs as a minimal condensate scaffold.

Targeted protein degradation via “hijacking” of the ubiquitin-proteasome system using proteolysis targeting chimeras (PROTACs) has evolved into a novel therapeutic modality. The design of PROTACs is challenging; multiple steps involved in PROTAC-induced degradation make it difficult to establish coherent structure-activity relationships. Herein, we characterize PROTAC-mediated ternary complex formation and degradation by employing von Hippel–Lindau protein (VHL) recruiting PROTACs for two different target proteins, SMARCA2 and BRD4. Ternary-complex attributes and degradation activity parameters are evaluated by varying components of the PROTAC’s architecture. Ternary complex binding affinity and cooperativity correlates well with degradation potency and initial rates of degradation. Additionally, we develop a ternary-complex structure modeling workflow to calculate the total buried surface area at the interface, which is in agreement with the measured ternary complex binding affinity. Our findings establish a predictive framework to guide the design of potent degraders. Targeted protein degradation using proteolysis targeting chimeras (PROTACs) represents an emergent therapeutic modality, however, the design of PROTACs is challenging due to multiple steps involved in PROTAC-induced degradation. Here, the authors establish a predictive framework to guide the design of potent degraders.

Amyloid-β peptide (Aβ) forms metastable oligomers >50 kDa, termed AβOs, that are more effective than Aβ amyloid fibrils at triggering Alzheimer’s disease-related processes such as synaptic dysfunction and Tau pathology, including Tau mislocalization. In neurons, Aβ accumulates in endo-lysosomal vesicles at low pH. Here, we show that the rate of AβO assembly is accelerated 8,000-fold upon pH reduction from extracellular to endo-lysosomal pH, at the expense of amyloid fibril formation. The pH-induced promotion of AβO formation and the high endo-lysosomal Aβ concentration together enable extensive AβO formation of Aβ42 under physiological conditions. Exploiting the enhanced AβO formation of the dimeric Aβ variant dimAβ we furthermore demonstrate targeting of AβOs to dendritic spines, potent induction of Tau missorting, a key factor in tauopathies, and impaired neuronal activity. The results suggest that the endosomal/lysosomal system is a major site for the assembly of pathomechanistically relevant AβOs. Aβ oligomers (AβO) are thought to represent the main toxic species in Alzheimer’s disease but very high Aβ concentrations are required to study them in vitro and it remains unknown what role these off-pathway oligomers play in vivo. Here, the authors use a dimeric variant of Aβ termed dimAβ, where two Aβ40 units are linked, which facilitates to study AβO formation kinetics and they observe that Aβ off-pathway oligomer formation is strongly accelerated at endo-lysosomal pH, while amyloid fibril formation is delayed. Furthermore, the authors demonstrate that dimAβ is a disease-relevant model construct for pathogenic AβO formation by showing that dimAβ AβOs target dendritic spines and induce AD-like somatodendritic Tau missorting.

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) is involved in both intracellular membraneless organelles and extracellular tissues. Despite growing understanding of LLPS, molecular-level mechanisms behind this process are still not fully established. Here, we use histidine-rich squid beak proteins (HBPs) as model IDPs to shed light on molecular interactions governing LLPS. We show that LLPS of HBPs is mediated though specific modular repeats. The morphology of separated phases (liquid-like versus hydrogels) correlates with the repeats’ hydrophobicity. Solution-state NMR indicates that LLPS is a multistep process initiated by deprotonation of histidine residues, followed by transient hydrogen bonding with tyrosine, and eventually by hydrophobic interactions. The microdroplets are stabilized by aromatic clustering of tyrosine residues exhibiting restricted molecular mobility in the nano-to-microsecond timescale according to solid-state NMR experiments. Our findings provide guidelines to rationally design pH-responsive peptides with LLPS ability for various applications, including bioinspired protocells and smart drug-delivery systems. Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins plays an important part in the formation of extracellular biological materials. Here, the authors show that repeats of the peptide motif GHGLY are necessary for the LLPS of pH-responsive histidine-rich squid beak proteins.

Long-term stability of monoclonal antibodies to be used as biologics is a key aspect in their development. Therefore, its possible early prediction from accelerated stability studies is of major interest, despite currently being regarded as not sufficiently robust. In this work, using a combination of accelerated stability studies (up to 6 months) and first order degradation kinetic model, we are able to predict the long-term stability (up to 3 years) of multiple monoclonal antibody formulations. More specifically, we can robustly predict the long-term stability behaviour of a protein at the intended storage condition (5 °C), based on up to six months of data obtained for multiple quality attributes from different temperatures, usually from intended (5 °C), accelerated (25 °C) and stress conditions (40 °C). We have performed stability studies and evaluated the stability data of several mAbs including IgG1, IgG2, and fusion proteins, and validated our model by overlaying the 95% prediction interval and experimental stability data from up to 36 months. We demonstrated improved robustness, speed and accuracy of kinetic long-term stability prediction as compared to classical linear extrapolation used today, which justifies long-term stability prediction and shelf-life extrapolation for some biologics such as monoclonal antibodies. This work aims to contribute towards further development and refinement of the regulatory landscape that could steer toward allowing extrapolation for biologics during the developmental phase, clinical phase, and also in marketing authorisation applications, as already established today for small molecules.