Kinetic models reveal the interplay of protein production and aggregation.

Protein aggregation is a key process in the development of many neurodegenerative disorders, including dementias such as Alzheimer's disease. Significant progress has been made in understanding the molecular mechanisms of aggregate formation in pure buffer systems, much of which was enabled by the development of integrated rate laws that allowed for mechanistic analysis of aggregation kinetics. However, in order to translate these findings into disease-relevant conclusions and to make predictions about the effect of potential alterations to the aggregation reactions by the addition of putative inhibitors, the current models need to be extended to account for the altered situation encountered in living systems. In particular, in vivo, the total protein concentrations typically do not remain constant and aggregation-prone monomers are constantly being produced but also degraded by cells. Here, we build a theoretical model that explicitly takes into account monomer production, derive integrated rate laws and discuss the resulting scaling laws and limiting behaviours. We demonstrate that our models are suited for the aggregation-prone Huntington's disease-associated peptide HttQ45 utilizing a system for continuous in situ monomer production and the aggregation of the tumour suppressor protein P53. The aggregation-prone HttQ45 monomer was produced through enzymatic cleavage of a larger construct in which a fused protein domain served as an internal inhibitor. For P53, only the unfolded monomers form aggregates, making the unfolding a rate-limiting step which constitutes a source of aggregation-prone monomers. The new model opens up possibilities for a quantitative description of aggregation in living systems, allowing for example the modelling of inhibitors of aggregation in a dynamic environment of continuous protein synthesis.

Molecular mechanism of α-synuclein aggregation on lipid membranes revealed.

The central hallmark of Parkinson's disease pathology is the aggregation of the α-synuclein protein, which, in its healthy form, is associated with lipid membranes. Purified monomeric α-synuclein is relatively stable in vitro, but its aggregation can be triggered by the presence of lipid vesicles. Despite this central importance of lipids in the context of α-synuclein aggregation, their detailed mechanistic role in this process has not been established to date. Here, we use chemical kinetics to develop a mechanistic model that is able to globally describe the aggregation behaviour of α-synuclein in the presence of DMPS lipid vesicles, across a range of lipid and protein concentrations. Through the application of our kinetic model to experimental data, we find that the reaction is a co-aggregation process involving both protein and lipids and that lipids promote aggregation as much by enabling fibril elongation as by enabling their initial formation. Moreover, we find that the primary nucleation of lipid-protein co-aggregates takes place not on the surface of lipid vesicles in bulk solution but at the air-water and/or plate interfaces, where lipids and proteins are likely adsorbed. Our model forms the basis for mechanistic insights, also in other lipid-protein co-aggregation systems, which will be crucial in the rational design of drugs that inhibit aggregate formation and act at the key points in the α-synuclein aggregation cascade.

Aß Oligomer Dissociation Is Catalyzed by Fibril Surfaces.

Oligomeric assemblies consisting of only a few protein subunits are key species in the cytotoxicity of neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Their lifetime in solution and abundance, governed by the balance of their sources and sinks, are thus important determinants of disease. While significant advances have been made in elucidating the processes that govern oligomer production, the mechanisms behind their dissociation are still poorly understood. Here, we use chemical kinetic modeling to determine the fate of oligomers formed in vitro and discuss the implications for their abundance in vivo. We discover that oligomeric species formed predominantly on fibril surfaces, a broad class which includes the bulk of oligomers formed by the key Alzheimer's disease-associated Aß peptides, also dissociate overwhelmingly on fibril surfaces, not in solution as had previously been assumed. We monitor this "secondary nucleation in reverse" by measuring the dissociation of Aß42 oligomers in the presence and absence of fibrils via two distinct experimental methods. Our findings imply that drugs that bind fibril surfaces to inhibit oligomer formation may also inhibit their dissociation, with important implications for rational design of therapeutic strategies for Alzheimer's and other amyloid diseases.

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.

The thermodynamics of neurodegenerative disease.

The formation of protein aggregates in the brain is a central aspect of the pathology of many neurodegenerative diseases. This self-assembly of specific proteins into filamentous aggregates, or fibrils, is a fundamental biophysical process that can easily be reproduced in the test tube. However, it has been difficult to obtain a clear picture of how the biophysical insights thus obtained can be applied to the complex, multi-factorial diseases and what this means for therapeutic strategies. While new, disease-modifying therapies are now emerging, for the most devastating disorders, such as Alzheimer's and Parkinson's disease, they still fall well short of offering a cure, and few drug design approaches fully exploit the wealth of mechanistic insights that has been obtained in biophysical studies. Here, I attempt to provide a new perspective on the role of protein aggregation in disease, by phrasing the problem in terms of a system that, under constant energy consumption, attempts to maintain a healthy, aggregate-free state against the thermodynamic driving forces that inexorably push it toward pathological aggregation.

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.

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.

Phase Separation and Fibrillization of Human Annexin A7 Are Mediated by Its Proline-Rich Domain.

Human annexin A7, a calcium- and phospholipid-binding protein, governs calcium homeostasis, plasma membrane repair, apoptosis, and tumor progression. A7 contains an N-terminal proline-rich domain (PRD; 180 residues, ∼24% prolines) that determines its functional specificity. Using microscopy and dye-binding assays, we show that recombinant A7 and its isolated PRD spontaneously phase separate into spherical condensates, which subsequently transform into ß-sheet-rich fibrils. We demonstrate that fibrillization of A7-PRD proceeds via primary nucleation and fibril-catalyzed secondary nucleation processes, as determined by chemical kinetics, providing a mechanistic basis for its amyloid assembly. This study confirms and highlights a subclass of eukaryotic PRDs prone to forming aggregates with important physiological and pathological implications.

Secondary Processes Dominate the Quiescent, Spontaneous Aggregation of α-Synuclein at Physiological pH with Sodium Salts.

The accurate recapitulation in an in vitro assay of the aggregation process of α-synuclein in Parkinson's disease has been a significant challenge. As α-synuclein does not aggregate spontaneously in most currently used in vitro assays, primary nucleation is triggered by the presence of surfaces such as lipid membranes or interfaces created by shaking, to achieve aggregation on accessible time scales. In addition, secondary nucleation is typically only observed by lowering the pH below 5.8. Here we investigated assay conditions that enables spontaneous primary nucleation and secondary nucleation at pH 7.4. Using 400 mM sodium phosphate, we observed quiescent spontaneous aggregation of α-synuclein and established that this aggregation is dominated by secondary processes. Furthermore, the presence of potassium ions enhanced the reproducibility of quiescent α-synuclein aggregation. This work provides a framework for the study of spontaneous α-synuclein aggregation at physiological pH.

Super-resolution imaging unveils the self-replication of tau aggregates upon seeding.

Tau is a soluble protein interacting with tubulin to stabilize microtubules. However, under pathological conditions, it becomes hyperphosphorylated and aggregates, a process that can be induced by treating cells with exogenously added tau fibrils. Here, we employ single-molecule localization microscopy to resolve the aggregate species formed in early stages of seeded tau aggregation. We report that entry of sufficient tau assemblies into the cytosol induces the self-replication of small tau aggregates, with a doubling time of 5 h inside HEK cells and 1 day in murine primary neurons, which then grow into fibrils. Seeding occurs in the vicinity of the microtubule cytoskeleton, is accelerated by the proteasome, and results in release of small assemblies into the media. In the absence of seeding, cells still spontaneously form small aggregates at lower levels. Overall, our work provides a quantitative picture of the early stages of templated seeded tau aggregation in cells.

Microfluidic antibody affinity profiling of alloantibody-HLA interactions in human serum.

Antibody profiling is a fundamental component of understanding the humoral response in a wide range of disease areas. Most currently used approaches operate by capturing antibodies onto functionalised surfaces. Such measurements of surface binding are governed by an overall antibody titre, while the two fundamental molecular parameters, antibody affinity and antibody concentration, are challenging to determine individually from such approaches. Here, by applying microfluidic diffusional sizing (MDS), we show how we can overcome this challenge and demonstrate reliable quantification of alloantibody binding affinity and concentration of alloantibodies binding to Human Leukocyte Antigens (HLA), an extensively used clinical biomarker in organ transplantation, both in buffer and in crude human serum. Capitalising on the ability to vary both serum and HLA concentrations during MDS, we show that both affinity and concentration of HLA-specific antibodies can be determined directly in serum when neither of these parameters is known. Finally, we provide proof of principle in clinical transplant patient sera that our assay enables differentiation of alloantibody reactivity against HLA proteins of highly similar structure, providing information not attainable through currently available techniques. These results outline a path towards detection and in-depth profiling of humoral immunity and may enable further insights into the clinical relevance of antibody reactivity in clinical transplantation and beyond.

Continuous population-level monitoring of SARS-CoV-2 seroprevalence in a large European metropolitan region.

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.

Perphenazine-Macrocycle Conjugates Rapidly Sequester the Aß42 Monomer and Prevent Formation of Toxic Oligomers and Amyloid.

Alzheimer's disease is imposing a growing social and economic burden worldwide, and effective therapies are urgently required. One possible approach to modulation of the disease outcome is to use small molecules to limit the conversion of monomeric amyloid (Aß42) to cytotoxic amyloid oligomers and fibrils. We have synthesized modulators of amyloid assembly that are unlike others studied to date: these compounds act primarily by sequestering the Aß42 monomer. We provide kinetic and nuclear magnetic resonance data showing that these perphenazine conjugates divert the Aß42 monomer into amorphous aggregates that are not cytotoxic. Rapid monomer sequestration by the compounds reduces fibril assembly, even in the presence of pre-formed fibrillar seeds. The compounds are therefore also able to disrupt monomer-dependent secondary nucleation, the autocatalytic process that generates the majority of toxic oligomers. The inhibitors have a modular design that is easily varied, aiding future exploration and use of these tools to probe the impact of distinct Aß42 species populated during amyloid assembly.

Microfluidic antibody profiling after repeated SARS-CoV-2 vaccination links antibody affinity and concentration to impaired immunity and variant escape in patients on anti-CD20 therapy.

Background: Patients with autoimmune/inflammatory conditions on anti-CD20 therapies, such as rituximab, have suboptimal humoral responses to vaccination and are vulnerable to poorer clinical outcomes following SARS-CoV-2 infection. We aimed to examine how the fundamental parameters of antibody responses, namely, affinity and concentration, shape the quality of humoral immunity after vaccination in these patients. Methods: We performed in-depth antibody characterisation in sera collected 4 to 6 weeks after each of three vaccine doses to wild-type (WT) SARS-CoV-2 in rituximab-treated primary vasculitis patients (n = 14) using Luminex and pseudovirus neutralisation assays, whereas we used a novel microfluidic-based immunoassay to quantify polyclonal antibody affinity and concentration against both WT and Omicron (B.1.1.529) variants. We performed comparative antibody profiling at equivalent timepoints in healthy individuals after three antigenic exposures to WT SARS-CoV-2 (one infection and two vaccinations; n = 15) and in convalescent patients after WT SARS-CoV-2 infection (n = 30). Results: Rituximab-treated patients had lower antibody levels and neutralisation titres against both WT and Omicron SARS-CoV-2 variants compared to healthy individuals. Neutralisation capacity was weaker against Omicron versus WT both in rituximab-treated patients and in healthy individuals. In the rituximab cohort, this was driven by lower antibody affinity against Omicron versus WT [median (range) KD: 21.6 (9.7-38.8) nM vs. 4.6 (2.3-44.8) nM, p = 0.0004]. By contrast, healthy individuals with hybrid immunity produced a broader antibody response, a subset of which recognised Omicron with higher affinity than antibodies in rituximab-treated patients [median (range) KD: 1.05 (0.45-1.84) nM vs. 20.25 (13.2-38.8) nM, p = 0.0002], underpinning the stronger serum neutralisation capacity against Omicron in the former group. Rituximab-treated patients had similar anti-WT antibody levels and neutralisation titres to unvaccinated convalescent individuals, despite two more exposures to SARS-CoV-2 antigen. Temporal profiling of the antibody response showed evidence of affinity maturation in healthy convalescent patients after a single SARS-CoV-2 infection, which was not observed in rituximab-treated patients, despite repeated vaccination. Discussion: Our results enrich previous observations of impaired humoral immune responses to SARS-CoV-2 in rituximab-treated patients and highlight the significance of quantitative assessment of serum antibody affinity and concentration in monitoring anti-viral immunity, viral escape, and the evolution of the humoral response.

Influence of denaturants on amyloid ß42 aggregation kinetics.

Amyloid formation is linked to devastating neurodegenerative diseases, motivating detailed studies of the mechanisms of amyloid formation. For Aß, the peptide associated with Alzheimer's disease, the mechanism and rate of aggregation have been established for a range of variants and conditions in vitro and in bodily fluids. A key outstanding question is how the relative stabilities of monomers, fibrils and intermediates affect each step in the fibril formation process. By monitoring the kinetics of aggregation of Aß42, in the presence of urea or guanidinium hydrochloride (GuHCl), we here determine the rates of the underlying microscopic steps and establish the importance of changes in relative stability induced by the presence of denaturant for each individual step. Denaturants shift the equilibrium towards the unfolded state of each species. We find that a non-ionic denaturant, urea, reduces the overall aggregation rate, and that the effect on nucleation is stronger than the effect on elongation. Urea reduces the rate of secondary nucleation by decreasing the coverage of fibril surfaces and the rate of nucleus formation. It also reduces the rate of primary nucleation, increasing its reaction order. The ionic denaturant, GuHCl, accelerates the aggregation at low denaturant concentrations and decelerates the aggregation at high denaturant concentrations. Below approximately 0.25 M GuHCl, the screening of repulsive electrostatic interactions between peptides by the charged denaturant dominates, leading to an increased aggregation rate. At higher GuHCl concentrations, the electrostatic repulsion is completely screened, and the denaturing effect dominates. The results illustrate how the differential effects of denaturants on stability of monomer, oligomer and fibril translate to differential effects on microscopic steps, with the rate of nucleation being most strongly reduced.

Characterization of full-length p53 aggregates and their kinetics of formation.

Mutations in the TP53 gene are common in cancer with the R248Q missense mutation conferring an increased propensity to aggregate. Previous p53 aggregation studies showed that, at micromolar concentrations, protein unfolding to produce aggregation-prone species is the rate-determining step. Here we show that, at physiological concentrations, aggregation kinetics of insect cell-derived full-length wild-type p53 and p53R248Q are determined by a nucleation-growth model, rather than formation of aggregation-prone monomeric species. Self-seeding, but not cross-seeding, increases aggregation rate, confirming the aggregation process as rate determining. p53R248Q displays enhanced aggregation propensity due to decreased solubility and increased aggregation rate, forming greater numbers of larger amorphous aggregates that disrupt lipid bilayers and invokes an inflammatory response. These results suggest that p53 aggregation can occur under physiological conditions, a rate enhanced by R248Q mutation, and that aggregates formed can cause membrane damage and inflammation that may influence tumorigenesis.

A conformational switch controlling the toxicity of the prion protein.

Prion infections cause conformational changes of the cellular prion protein (PrPC) and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ('H-latch'), altering the flexibility of the α2-α3 and ß2-α2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is an important reporter of prion neurotoxicity.

Uncovering the universality of self-replication in protein aggregation and its link to disease.

Fibrillar protein aggregates are a hallmark of a range of human disorders, from prion diseases to dementias, but are also encountered in several functional contexts. Yet, the fundamental links between protein assembly mechanisms and their functional or pathological roles have remained elusive. Here, we analyze the aggregation kinetics of a large set of proteins that self-assemble by a nucleated-growth mechanism, from those associated with disease, over those whose aggregates fulfill functional roles in biology, to those that aggregate only under artificial conditions. We find that, essentially, all such systems, regardless of their biological role, are capable of self-replication. However, for aggregates that have evolved to fulfill a structural role, the rate of self-replication is too low to be significant on the biologically relevant time scale. By contrast, all disease-related proteins are able to self-replicate quickly compared to the time scale of the associated disease. Our findings establish the ubiquity of self-replication and point to its potential importance across aggregation-related disorders.

Both COVID-19 infection and vaccination induce high-affinity cross-clade responses to SARS-CoV-2 variants.

The B.1.1.529 (omicron) variant has rapidly supplanted most other SARS-CoV-2 variants. Using microfluidics-based antibody affinity profiling (MAAP), we have characterized affinity and IgG concentration in the plasma of 39 individuals with multiple trajectories of SARS-CoV-2 infection and/or vaccination. Antibody affinity was similar against the wild-type, delta, and omicron variants (K A ranges: 122 ± 155, 159 ± 148, 211 ± 307 µM-1, respectively), indicating a surprisingly broad and mature cross-clade immune response. Postinfectious and vaccinated subjects showed different IgG profiles, with IgG3 (p-value = 0.002) against spike being more prominent in the former group. Lastly, we found that the ELISA titers correlated linearly with measured concentrations (R = 0.72) but not with affinity (R = 0.29). These findings suggest that the wild-type and delta spike induce a polyclonal immune response capable of binding the omicron spike with similar affinity. Changes in titers were primarily driven by antibody concentration, suggesting that B-cell expansion, rather than affinity maturation, dominated the response after infection or vaccination.

Mechanistic Models of Protein Aggregation Across Length-Scales and Time-Scales: From the Test Tube to Neurodegenerative Disease.

Through advances in the past decades, the central role of aberrant protein aggregation has been established in many neurodegenerative diseases. Crucially, however, the molecular mechanisms that underlie aggregate proliferation in the brains of affected individuals are still only poorly understood. Under controlled in vitro conditions, significant progress has been made in elucidating the molecular mechanisms that take place during the assembly of purified protein molecules, through advances in both experimental methods and the theories used to analyse the resulting data. The determination of the aggregation mechanism for a variety of proteins revealed the importance of intermediate oligomeric species and of the interactions with promotors and inhibitors. Such mechanistic insights, if they can be achieved in a disease-relevant system, provide invaluable information to guide the design of potential cures to these devastating disorders. However, as experimental systems approach the situation present in real disease, their complexity increases substantially. Timescales increase from hours an aggregation reaction takes in vitro, to decades over which the process takes place in disease, and length-scales increase to the dimension of a human brain. Thus, molecular level mechanistic studies, like those that successfully determined mechanisms in vitro, have only been applied in a handful of living systems to date. If their application can be extended to further systems, including patient data, they promise powerful new insights. Here we present a review of the existing strategies to gain mechanistic insights into the molecular steps driving protein aggregation and discuss the obstacles and potential paths to achieving their application in disease. First, we review the experimental approaches and analysis techniques that are used to establish the aggregation mechanisms in vitro and the insights that have been gained from them. We then discuss how these approaches must be modified and adapted to be applicable in vivo and review the existing works that have successfully applied mechanistic analysis of protein aggregation in living systems. Finally, we present a broad mechanistic classification of in vivo systems and discuss what will be required to further our understanding of aggregate formation in living systems.