RNA Granules Hitchhike on Lysosomes for Long-Distance Transport, Using Annexin A11 as a Molecular Tether.

Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.

Mechanobiology of Protein Droplets: Force Arises from Disorder.

The use of optogenetic approaches has revealed new roles for intracellular protein condensates described in two papers in this issue of Cell (Bracha et. al., 2018; Shin et al., 2018). These results show that growing condensates are able to exert mechanical forces resulting in chromatin rearrangement, establishing a new role for liquid-liquid phase separation in the mechanobiology of the cell.

Direct observation of the interconversion of normal and toxic forms of α-synuclein.

Here, we use single-molecule techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.

Polysaccharide functionalization reduces lipid vesicle stiffness.

The biophysical properties of lipid vesicles are important for their stability and integrity, key parameters that control the performance when these vesicles are used for drug delivery. The vesicle properties are determined by the composition of lipids used to form the vesicle. However, for a given lipid composition, they can also be tailored by tethering polymers to the membrane. Typically, synthetic polymers like polyethyleneglycol are used to increase vesicle stability, but the use of polysaccharides in this context is much less explored. Here, we report a general method for functionalizing lipid vesicles with polysaccharides by binding them to cholesterol. We incorporate the polysaccharides on the outer membrane leaflet of giant unilamellar vesicles (GUVs) and investigate their effect on membrane mechanics using micropipette aspiration. We find that the presence of the glycolipid functionalization produces an unexpected softening of GUVs with fluid-like membranes. By contrast, the functionalization of GUVs with polyethylene glycol does not reduce their stretching modulus. This work provides the potential means to study membrane-bound meshworks of polysaccharides similar to the cellular glycocalyx; moreover, it can be used for tuning the mechanical properties of drug delivery vehicles.

Maximum entropy determination of mammalian proteome dynamics.

Full understanding of proteostasis and energy utilization in cells will require knowledge of the fraction of cell proteins being degraded with different half-lives and their rates of synthesis. We therefore developed a method to determine such information that combines mathematical analysis of protein degradation kinetics obtained in pulse-chase experiments with Bayesian data fitting using the maximum entropy principle. This approach will enable rapid analyses of whole-cell protein dynamics in different cell types, physiological states, and neurodegenerative disease. Using it, we obtained surprising insights about protein stabilities in cultured cells normally and upon activation of proteolysis by mTOR inhibition and increasing cAMP or cGMP. It revealed that >90% of protein content in dividing mammalian cell lines is long-lived, with half-lives of 24 to 200 h, and therefore comprises much of the proteins in daughter cells. The well-studied short-lived proteins (half-lives < 10 h) together comprise <2% of cell protein mass, but surprisingly account for 10 to 20% of measurable newly synthesized protein mass. Evolution thus appears to have minimized intracellular proteolysis except to rapidly eliminate misfolded and regulatory proteins.

Self-replication of Aß42 aggregates occurs on small and isolated fibril sites.

Self-replication of amyloid fibrils via secondary nucleation is an intriguing physicochemical phenomenon in which existing fibrils catalyze the formation of their own copies. The molecular events behind this fibril surface-mediated process remain largely inaccessible to current structural and imaging techniques. Using statistical mechanics, computer modeling, and chemical kinetics, we show that the catalytic structure of the fibril surface can be inferred from the aggregation behavior in the presence and absence of a fibril-binding inhibitor. We apply our approach to the case of Alzheimer's A[Formula: see text] amyloid fibrils formed in the presence of proSP-C Brichos inhibitors. We find that self-replication of A[Formula: see text] fibrils occurs on small catalytic sites on the fibril surface, which are far apart from each other, and each of which can be covered by a single Brichos inhibitor.

The role of shear forces in primary and secondary nucleation of amyloid fibrils.

Shear forces affect self-assembly processes ranging from crystallization to fiber formation. Here, the effect of mild agitation on amyloid fibril formation was explored for four peptides and investigated in detail for A[Formula: see text]42, which is associated with Alzheimer's disease. To gain mechanistic insights into the effect of mild agitation, nonseeded and seeded aggregation reactions were set up at various peptide concentrations with and without an inhibitor. First, an effect on fibril fragmentation was excluded by comparing the monomer-concentration dependence of aggregation kinetics under idle and agitated conditions. Second, using a secondary nucleation inhibitor, Brichos, the agitation effect on primary nucleation was decoupled from secondary nucleation. Third, an effect on secondary nucleation was established in the absence of inhibitor. Fourth, an effect on elongation was excluded by comparing the seeding potency of fibrils formed under idle or agitated conditions. We find that both primary and secondary nucleation steps are accelerated by gentle agitation. The increased shear forces facilitate both the detachment of newly formed aggregates from catalytic surfaces and the rate at which molecules are transported in the bulk solution to encounter nucleation sites on the fibril and other surfaces. Ultrastructural evidence obtained with cryogenic transmission electron microscopy and free-flow electrophoresis in microfluidics devices imply that agitation speeds up the detachment of nucleated species from the fibril surface. Our findings shed light on the aggregation mechanism and the role of detachment for efficient secondary nucleation. The results inform on how to modulate the relative importance of different microscopic steps in drug discovery and investigations.

Discovery of potent inhibitors of α-synuclein aggregation using structure-based iterative learning.

Machine learning methods hold the promise to reduce the costs and the failure rates of conventional drug discovery pipelines. This issue is especially pressing for neurodegenerative diseases, where the development of disease-modifying drugs has been particularly challenging. To address this problem, we describe here a machine learning approach to identify small molecule inhibitors of α-synuclein aggregation, a process implicated in Parkinson's disease and other synucleinopathies. Because the proliferation of α-synuclein aggregates takes place through autocatalytic secondary nucleation, we aim to identify compounds that bind the catalytic sites on the surface of the aggregates. To achieve this goal, we use structure-based machine learning in an iterative manner to first identify and then progressively optimize secondary nucleation inhibitors. Our results demonstrate that this approach leads to the facile identification of compounds two orders of magnitude more potent than previously reported ones.

Design of amyloidogenic peptide traps.

Segments of proteins with high ß-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in ß-strand and ß-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein-peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid ß1-42 (Aß42). The Aß binders block the assembly of Aß fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aß42 species.

The amyloid state and its association with protein misfolding diseases.

The phenomenon of protein aggregation and amyloid formation has become the subject of rapidly increasing research activities across a wide range of scientific disciplines. Such activities have been stimulated by the association of amyloid deposition with a range of debilitating medical disorders, from Alzheimer's disease to type II diabetes, many of which are major threats to human health and welfare in the modern world. It has become clear, however, that the ability to form the amyloid state is more general than previously imagined, and that its study can provide unique insights into the nature of the functional forms of peptides and proteins, as well as understanding the means by which protein homeostasis can be maintained and protein metastasis avoided.

Formation of amyloid loops in brain tissues is controlled by the flexibility of protofibril chains.

Neurodegenerative diseases, such as Alzheimer's disease (AD), are associated with protein misfolding and aggregation into amyloid fibrils. Increasing evidence suggests that soluble, low-molecular-weight aggregates play a key role in disease-associated toxicity. Within this population of aggregates, closed-loop pore-like structures have been observed for a variety of amyloid systems, and their presence in brain tissues is associated with high levels of neuropathology. However, their mechanism of formation and relationship with mature fibrils have largely remained challenging to elucidate. Here, we use atomic force microscopy and statistical theory of biopolymers to characterize amyloid ring structures derived from the brains of AD patients. We analyze the bending fluctuations of protofibrils and show that the process of loop formation is governed by the mechanical properties of their chains. We conclude that ex vivo protofibril chains possess greater flexibility than that imparted by hydrogen-bonded networks characteristic of mature amyloid fibrils, such that they are able to form end-to-end connections. These results explain the diversity in the structures formed from protein aggregation and shed light on the links between early forms of flexible ring-forming aggregates and their role in disease.

Nonspecificity fingerprints for clinical-stage antibodies in solution.

Monoclonal antibodies (mAbs) have successfully been developed for the treatment of a wide range of diseases. The clinical success of mAbs does not solely rely on optimal potency and safety but also require good biophysical properties to ensure a high developability potential. In particular, nonspecific interactions are a key developability parameter to monitor during discovery and development. Despite an increased focus on the detection of nonspecific interactions, their underlying physicochemical origins remain poorly understood. Here, we employ solution-based microfluidic technologies to characterize a set of clinical-stage mAbs and their interactions with commonly used nonspecificity ligands to generate nonspecificity fingerprints, providing quantitative data on the underlying physical chemistry. Furthermore, the solution-based analysis enables us to measure binding affinities directly, and we evaluate the contribution of avidity in nonspecific binding by mAbs. We find that avidity can increase the apparent affinity by two orders of magnitude. Notably, we find that a subset of these highly developed mAbs show nonspecific electrostatic interactions, even at physiological pH and ionic strength, and that they can form microscale particles with charge-complementary polymers. The group of mAb constructs flagged here for nonspecificity are among the worst performers in independent reports of surface and column-based screens. The solution measurements improve on the state-of-the-art by providing a stand-alone result for individual mAbs without the need to benchmark against cohort data. Based on our findings, we propose a quantitative solution-based nonspecificity score, which can be integrated in the development workflow for biological therapeutics and more widely in protein engineering.

Spontaneous nucleation and fast aggregate-dependent proliferation of α-synuclein aggregates within liquid condensates at neutral pH.

The aggregation of α-synuclein into amyloid fibrils has been under scrutiny in recent years because of its association with Parkinson's disease. This process can be triggered by a lipid-dependent nucleation process, and the resulting aggregates can proliferate through secondary nucleation under acidic pH conditions. It has also been recently reported that the aggregation of α-synuclein may follow an alternative pathway, which takes place within dense liquid condensates formed through phase separation. The microscopic mechanism of this process, however, remains to be clarified. Here, we used fluorescence-based assays to enable a kinetic analysis of the microscopic steps underlying the aggregation process of α-synuclein within liquid condensates. Our analysis shows that at pH 7.4, this process starts with spontaneous primary nucleation followed by rapid aggregate-dependent proliferation. Our results thus reveal the microscopic mechanism of α-synuclein aggregation within condensates through the accurate quantification of the kinetic rate constants for the appearance and proliferation of α-synuclein aggregates at physiological pH.

The liquid-to-solid transition of FUS is promoted by the condensate surface.

A wide range of macromolecules can undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed reversibly, and carry out essential functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics, and mechanics in time and space, that single-component FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases coexist simultaneously within the same condensate, resulting in highly inhomogeneous structures. Furthermore, our results show that this transition originates at the interface between the condensate and the dilute continuous phase, and once initiated, the gelation process propagates toward the center of the condensate. To probe such spatially inhomogeneous rheology during condensate aging, we use a combination of established micropipette aspiration experiments together with two optical techniques, spatial dynamic mapping and reflective confocal dynamic speckle microscopy. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a critical element in driving pathological protein aggregation.

Surface patches induce nonspecific binding and phase separation of antibodies.

Nonspecific interactions are a key challenge in the successful development of therapeutic antibodies. The tendency for nonspecific binding of antibodies is often difficult to reduce by rational design, and instead, it is necessary to rely on comprehensive screening campaigns. To address this issue, we performed a systematic analysis of the impact of surface patch properties on antibody nonspecificity using a designer antibody library as a model system and single-stranded DNA as a nonspecificity ligand. Using an in-solution microfluidic approach, we find that the antibodies tested bind to single-stranded DNA with affinities as high as KD = 1 µM. We show that DNA binding is driven primarily by a hydrophobic patch in the complementarity-determining regions. By quantifying the surface patches across the library, the nonspecific binding affinity is shown to correlate with a trade-off between the hydrophobic and total charged patch areas. Moreover, we show that a change in formulation conditions at low ionic strengths leads to DNA-induced antibody phase separation as a manifestation of nonspecific binding at low micromolar antibody concentrations. We highlight that phase separation is driven by a cooperative electrostatic network assembly mechanism of antibodies with DNA, which correlates with a balance between positive and negative charged patches. Importantly, our study demonstrates that both nonspecific binding and phase separation are controlled by the size of the surface patches. Taken together, these findings highlight the importance of surface patches and their role in conferring antibody nonspecificity and its macroscopic manifestation in phase separation.

Theoretical and Data-Driven Approaches for Biomolecular Condensates.

Biomolecular condensation processes are increasingly recognized as a fundamental mechanism that living cells use to organize biomolecules in time and space. These processes can lead to the formation of membraneless organelles that enable cells to perform distinct biochemical processes in controlled local environments, thereby supplying them with an additional degree of spatial control relative to that achieved by membrane-bound organelles. This fundamental importance of biomolecular condensation has motivated a quest to discover and understand the molecular mechanisms and determinants that drive and control this process. Within this molecular viewpoint, computational methods can provide a unique angle to studying biomolecular condensation processes by contributing the resolution and scale that are challenging to reach with experimental techniques alone. In this Review, we focus on three types of dry-lab approaches: theoretical methods, physics-driven simulations and data-driven machine learning methods. We review recent progress in using these tools for probing biomolecular condensation across all three fields and outline the key advantages and limitations of each of the approaches. We further discuss some of the key outstanding challenges that we foresee the community addressing next in order to develop a more complete picture of the molecular driving forces behind biomolecular condensation processes and their biological roles in health and disease.

Aging can transform single-component protein condensates into multiphase architectures.

Phase-separated biomolecular condensates that contain multiple coexisting phases are widespread in vitro and in cells. Multiphase condensates emerge readily within multicomponent mixtures of biomolecules (e.g., proteins and nucleic acids) when the different components present sufficient physicochemical diversity (e.g., in intermolecular forces, structure, and chemical composition) to sustain separate coexisting phases. Because such diversity is highly coupled to the solution conditions (e.g., temperature, pH, salt, composition), it can manifest itself immediately from the nucleation and growth stages of condensate formation, develop spontaneously due to external stimuli or emerge progressively as the condensates age. Here, we investigate thermodynamic factors that can explain the progressive intrinsic transformation of single-component condensates into multiphase architectures during the nonequilibrium process of aging. We develop a multiscale model that integrates atomistic simulations of proteins, sequence-dependent coarse-grained simulations of condensates, and a minimal model of dynamically aging condensates with nonconservative intermolecular forces. Our nonequilibrium simulations of condensate aging predict that single-component condensates that are initially homogeneous and liquid like can transform into gel-core/liquid-shell or liquid-core/gel-shell multiphase condensates as they age due to gradual and irreversible enhancement of interprotein interactions. The type of multiphase architecture is determined by the aging mechanism, the molecular organization of the gel and liquid phases, and the chemical makeup of the protein. Notably, we predict that interprotein disorder to order transitions within the prion-like domains of intracellular proteins can lead to the required nonconservative enhancement of intermolecular interactions. Our study, therefore, predicts a potential mechanism by which the nonequilibrium process of aging results in single-component multiphase condensates.

Adsorption free energy predicts amyloid protein nucleation rates.

Primary nucleation is the fundamental event that initiates the conversion of proteins from their normal physiological forms into pathological amyloid aggregates associated with the onset and development of disorders including systemic amyloidosis, as well as the neurodegenerative conditions Alzheimer's and Parkinson's diseases. It has become apparent that the presence of surfaces can dramatically modulate nucleation. However, the underlying physicochemical parameters governing this process have been challenging to elucidate, with interfaces in some cases having been found to accelerate aggregation, while in others they can inhibit the kinetics of this process. Here we show through kinetic analysis that for three different fibril-forming proteins, interfaces affect the aggregation reaction mainly through modulating the primary nucleation step. Moreover, we show through direct measurements of the Gibbs free energy of adsorption, combined with theory and coarse-grained computer simulations, that overall nucleation rates are suppressed at high and at low surface interaction strengths but significantly enhanced at intermediate strengths, and we verify these regimes experimentally. Taken together, these results provide a quantitative description of the fundamental process which triggers amyloid formation and shed light on the key factors that control this process.

Phase-separating RNA-binding proteins form heterogeneous distributions of clusters in subsaturated solutions.

Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation are thought to follow the tenets of classical nucleation theory, and, therefore, subsaturated solutions should be devoid of clusters with more than a few molecules. We tested this prediction using in vitro biophysical studies to characterize subsaturated solutions of phase-separating RNA-binding proteins with intrinsically disordered prion-like domains and RNA-binding domains. Surprisingly, and in direct contradiction to expectations from classical nucleation theory, we find that subsaturated solutions are characterized by the presence of heterogeneous distributions of clusters. The distributions of cluster sizes, which are dominated by small species, shift continuously toward larger sizes as protein concentrations increase and approach the saturation concentration. As a result, many of the clusters encompass tens to hundreds of molecules, while less than 1% of the solutions are mesoscale species that are several hundred nanometers in diameter. We find that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. We also find that cluster formation and phase separation can be decoupled using solutes as well as specific sets of mutations. Our findings, which are concordant with predictions for associative polymers, implicate an interplay between networks of sequence-specific and solubility-determining interactions that, respectively, govern cluster formation in subsaturated solutions and the saturation concentrations above which phase separation occurs.

Cathepsin B Processing Is Required for the In Vivo Efficacy of Albumin-Drug Conjugates.

Targeted drug delivery approaches that selectively and preferentially deliver therapeutic agents to specific tissues are of great interest for safer and more effective pharmaceutical treatments. We investigated whether cathepsin B cleavage of a valine-citrulline [VC(S)]-containing linker is required for the release of monomethyl auristatin E (MMAE) from albumin-drug conjugates. In this study, we used an engineered version of human serum albumin, Veltis High Binder II (HBII), which has enhanced binding to the neonatal Fc (fragment crystallizable) receptor (FcRn) to improve drug release upon binding and FcRn-mediated recycling. The linker-payload was conjugated to cysteine 34 of albumin using a carbonylacrylic (caa) reagent which produced homogeneous and plasma stable conjugates that retained FcRn binding. Two caa-linker-MMAE reagents were synthesized─one with a cleavable [VC(S)] linker and one with a noncleavable [VC(R)] linker─to question whether protease-mediated cleavage is needed for MMAE release. Our findings demonstrate that cathepsin B is required to achieve efficient and selective antitumor activity. The conjugates equipped with the cleavable [VC(S)] linker had potent antitumor activity in vivo facilitated by the release of free MMAE upon FcRn binding and internalization. In addition to the pronounced antitumor activity of the albumin conjugates in vivo, we also demonstrated their preferable tumor biodistribution and biocompatibility with no associated toxicity or side effects. These results suggest that the use of engineered albumins with high FcRn binding combined with protease cleavable linkers is an efficient strategy to target delivery of drugs to solid tumors.