Comparison of size distribution and (Pro249-Ser258) epitope exposure in in vitro and in vivo derived Tau fibrils.

BACKGROUND: Although several studies demonstrate prion-like properties of Tau fibrils, the effect of size in the seeding capacity of these aggregates is not fully understood. The aim of this study is to characterize Tau seeds by their size and seeding capacity. METHODS: Tau aggregates were isolated from postmortem AD brain tissue and separated from low molecular weight species by sucrose gradient ultracentrifugation. Biochemical characterization of the different fractions was done by non-reducing Western blotting and aggregate-specific immuno-assays using in house developed anti-Tau monoclonal antibodies, including PT76 which binds to an epitope close to the microtubule-binding domain and, hence, also to K18. Seeding efficiency was then assessed in HEK293 cells expressing K18 FRET sensors. RESULTS: We observed that upon sonication of Tau aggregates different size-distributed tau aggregates are obtained. In biochemical assays, these forms show higher signals than the non-sonicated material in some aggregation-specific Tau assays. This could be explained by an increased epitope exposure of the smaller aggregates created by the sonication. By analyzing human brain derived and recombinant (K18) Tau aggregates in a cellular FRET assay, it was observed that, in the absence of transfection reagent, sonicated aggregates showed higher aggregation induction. Preparations also showed altered profiles on native PAGE upon sonication and we could further separate different aggregate species based on their molecular weight via sucrose gradients. CONCLUSIONS: This study further elucidates the molecular properties regarding relative aggregate size and seeding efficiency of sonicated vs. non-sonicated high molecular weight Tau species. This information will provide a better knowledge on how sonication, a commonly used technique in the field of study of Tau aggregation, impacts the aggregates. In addition, the description of PT76-based aggregation specific assay is a valuable tool to quantify K18 and human AD Tau fibrils.

Epitope mapping of diverse influenza Hemagglutinin drug candidates using HDX-MS.

Epitope characterization is critical for elucidating the mechanism of action of drug candidates. However, traditional high-resolution epitope mapping techniques are not well suited for screening numerous drug candidates recognizing a similar target. Here, we use Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to explore the conformational impact of diverse drug molecules binding on Hemagglutinin (HA), the major surface antigen of influenza viruses. We optimized a semi-automated HDX-MS workflow to systematically probe distantly related HA subtypes in complex with 4 different drug candidates, ranging from a monoclonal antibody to a small synthetic peptide. This fast, cost-effective HDX-MS epitope mapping approach accurately determined the main antigenic site in all cases. Moreover, our studies reveal distinct changes in the local conformational dynamics of HA associated to the molecular mechanism of neutralization, establishing a marker for broad anti-HA activity. Taken together, these findings highlight the potential for HDX-MS epitope mapping-based screening to identify promising candidates against HA at early stages of drug discovery.

Naturally occurring antibodies isolated from PD patients inhibit synuclein seeding in vitro and recognize Lewy pathology.

Deposition of α-synuclein into Lewy bodies and Lewy neurites is the hallmark of Parkinson's disease (PD). It is hypothesized that α-synuclein pathology spreads by a "prion-like" mechanism (i.e., by seeded aggregation or templated misfolding). Therefore, various extracellular α-synuclein conformers and/or posttranslational modifications may serve as biomarkers of disease or potential targets for novel interventions. To explore whether the antibody repertoires of PD patients contain anti-α-synuclein antibodies that can potentially be used as markers or immunotherapy, we interrogated peripheral IgG+ memory B cells from PD patients for reactivity to α-synuclein. In total, ten somatically mutated antibodies were recovered, suggesting the presence of an ongoing antigen-driven immune response. The three antibodies that had the highest affinity to recombinant full-length α-synuclein, aSyn-323.1, aSyn-336.1 and aSyn-338.1, were characterized further and shown to recognize epitopes in the C terminus of α-synuclein with binding affinities between 0.3 and 2.8 µM. Furthermore, all three antibodies were able to neutralize the "seeding" of intracellular synuclein aggregates in an in vitro α-synuclein seeding assay. Finally, differential reactivities were observed for all three human anti-α-synuclein antibodies across tissue treatment conditions by immunohistochemistry. Our results suggest that the memory B-cell repertoire of PD patients might represent a potential source of biomarkers and therapies.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening.

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer's disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia.

Alzheimer's disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This "prion-like" spreading can be interrupted by antibodies capable of binding and "neutralizing" extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Structural Basis for Recognition of a Unique Epitope by a Human Anti-tau Antibody.

Aggregation of the hyperphosphorylated protein tau into neurofibrillary tangles and neuropil threads is a hallmark of Alzheimer disease (AD). Identification and characterization of the epitopes recognized by anti-tau antibodies might shed light on the molecular mechanisms of AD pathogenesis. Here we report on the biochemical and structural characterization of a tau-specific monoclonal antibody CBTAU-24.1, which was isolated from the human memory B cell repertoire. Immunohistochemical staining with CBTAU-24.1 specifically detects pathological tau structures in AD brain samples. The crystal structure of CBTAU-24.1 Fab with a phosphorylated tau peptide revealed recognition of a unique epitope (Ser235-Leu243) in the tau proline-rich domain. Interestingly, the antibody can bind tau regardless of phosphorylation state of its epitope region and also recognizes both monomeric and paired helical filament tau irrespective of phosphorylation status. This human anti-tau antibody and its unique epitope may aid in development of diagnostics and/or therapeutic AD strategies.

Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau.

Aggregation of tau protein and spreading of tau aggregates are pivotal pathological processes in a range of neurological disorders. Accumulating evidence suggests that immunotherapy targeting tau may be a viable therapeutic strategy. We have previously described the isolation of antibody CBTAU-22.1 from the memory B-cell repertoire of healthy human donors. CBTAU-22.1 was shown to specifically bind a disease-associated phosphorylated epitope in the C-terminus of tau (Ser422) and to be able to inhibit the spreading of pathological tau aggregates from P301S spinal cord lysates in vitro, albeit with limited potency. Using a combination of rational design and random mutagenesis we have derived a variant antibody with improved affinity while maintaining the specificity of the parental antibody. This affinity improved antibody showed greatly enhanced potency in a cell-based immunodepletion assay using paired helical filaments (PHFs) derived from human Alzheimer's disease (AD) brain tissue. Moreover, the affinity improved antibody limits the in vitro aggregation propensity of full length tau species specifically phosphorylated at position 422 produced by employing a native chemical ligation approach. Together, these results indicate that in addition to being able to inhibit the spreading of pathological tau aggregates, the matured antibody can potentially also interfere with the nucleation of tau which is believed to be the first step of the pathogenic process. Finally, the functionality in a P301L transgenic mice co-injection model highlights the therapeutic potential of human antibody dmCBTAU-22.1.

A common antigenic motif recognized by naturally occurring human VH5-51/VL4-1 anti-tau antibodies with distinct functionalities.

Misfolding and aggregation of tau protein are closely associated with the onset and progression of Alzheimer's Disease (AD). By interrogating IgG+ memory B cells from asymptomatic donors with tau peptides, we have identified two somatically mutated VH5-51/VL4-1 antibodies. One of these, CBTAU-27.1, binds to the aggregation motif in the R3 repeat domain and blocks the aggregation of tau into paired helical filaments (PHFs) by sequestering monomeric tau. The other, CBTAU-28.1, binds to the N-terminal insert region and inhibits the spreading of tau seeds and mediates the uptake of tau aggregates into microglia by binding PHFs. Crystal structures revealed that the combination of VH5-51 and VL4-1 recognizes a common Pro-Xn-Lys motif driven by germline-encoded hotspot interactions while the specificity and thereby functionality of the antibodies are defined by the CDR3 regions. Affinity improvement led to improvement in functionality, identifying their epitopes as new targets for therapy and prevention of AD.

Immunological memory to hyperphosphorylated tau in asymptomatic individuals.

Several reports have described the presence of antibodies against Alzheimer's disease-associated hyperphosphorylated forms of tau in serum of healthy individuals. To characterize the specificities that can be found, we interrogated peripheral IgG+ memory B cells from asymptomatic blood donors for reactivity to a panel of phosphorylated tau peptides using a single-cell screening assay. Antibody sequences were recovered, cloned, and expressed as full-length IgGs. In total, 52 somatically mutated tau-binding antibodies were identified, corresponding to 35 unique clonal families. Forty-one of these antibodies recognize epitopes in the proline-rich and C-terminal domains, and binding of 26 of these antibodies is strictly phosphorylation dependent. Thirteen antibodies showed inhibitory activity in a P301S lysate seeded in vitro tau aggregation assay. Two such antibodies, CBTAU-7.1 and CBTAU-22.1, which bind to the proline-rich and C-terminal regions of tau, respectively, were characterized in more detail. CBTAU-7.1 recognizes an epitope that is similar to that of murine anti-PHF antibody AT8, but has different phospho requirements. Both CBTAU-7.1 and CBTAU-22.1 detect pathological tau deposits in post-mortem brain tissue. CBTAU-7.1 reveals a similar IHC distribution pattern as AT8, immunostaining (pre)tangles, threads, and neuritic plaques. CBTAU-22.1 shows selective detection of neurofibrillary changes by IHC. Taken together, these results suggest the presence of an ongoing antigen-driven immune response against tau in healthy individuals. The wide range of specificities to tau suggests that the human immune repertoire may contain antibodies that can serve as biomarkers or be exploited for therapy.

A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen.

The identification of human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem revitalized hopes of developing a universal influenza vaccine. Using a rational design and library approach, we engineered stable HA stem antigens ("mini-HAs") based on an H1 subtype sequence. Our most advanced candidate exhibits structural and bnAb binding properties comparable to those of full-length HA, completely protects mice in lethal heterologous and heterosubtypic challenge models, and reduces fever after sublethal challenge in cynomolgus monkeys. Antibodies elicited by this mini-HA in mice and nonhuman primates bound a wide range of HAs, competed with human bnAbs for HA stem binding, neutralized H5N1 viruses, and mediated antibody-dependent effector activity. These results represent a proof of concept for the design of HA stem mimics that elicit bnAbs against influenza A group 1 viruses.

Characterization and immunogenicity of a novel mosaic M HIV-1 gp140 trimer.

UNLABELLED: The extraordinary diversity of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein poses a major challenge for the development of an HIV-1 vaccine. One strategy to circumvent this problem utilizes bioinformatically optimized mosaic antigens. However, mosaic Env proteins expressed as trimers have not been previously evaluated for their stability, antigenicity, and immunogenicity. Here, we report the production and characterization of a stable HIV-1 mosaic M gp140 Env trimer. The mosaic M trimer bound CD4 as well as multiple broadly neutralizing monoclonal antibodies, and biophysical characterization suggested substantial stability. The mosaic M trimer elicited higher neutralizing antibody (nAb) titers against clade B viruses than a previously described clade C (C97ZA.012) gp140 trimer in guinea pigs, whereas the clade C trimer elicited higher nAb titers than the mosaic M trimer against clade A and C viruses. A mixture of the clade C and mosaic M trimers elicited nAb responses that were comparable to the better component of the mixture for each virus tested. These data suggest that combinations of relatively small numbers of immunologically complementary Env trimers may improve nAb responses. IMPORTANCE: The development of an HIV-1 vaccine remains a formidable challenge due to multiple circulating strains of HIV-1 worldwide. This study describes a candidate HIV-1 Env protein vaccine whose sequence has been designed by computational methods to address HIV-1 diversity. The characteristics and immunogenicity of this Env protein, both alone and mixed together with a clade C Env protein vaccine, are described.

The GroEL/GroES cis cavity as a passive anti-aggregation device.

The GroEL/GroES chaperonin folding chamber is an encapsulated space of approximately 65 A diameter with a hydrophilic wall, inside of which many cellular proteins reach the native state. The question of whether the cavity wall actively directs folding reactions or is playing a passive role has been open. We review past and recent observations and conclude that the chamber functions as a passive "Anfinsen cage" that prevents folding monomers from multimolecular aggregation.

Chaperonin chamber accelerates protein folding through passive action of preventing aggregation.

The original experiments reconstituting GroEL-GroES-mediated protein folding were carried out under "nonpermissive" conditions, where the chaperonin system was absolutely required and substrate proteins could not achieve the native state if diluted directly from denaturant into solution. Under "permissive" conditions, however, employing lower substrate concentration and lower temperature, some substrate proteins can be refolded both by the chaperonin system and while free in solution. For several of these, the protein refolds more rapidly inside the GroEL-GroES cis chamber than free in solution, suggesting that the chamber may have an active role in assisting protein folding. Here, we observe that the difference is caused by reversible multimolecular association while folding in solution, an avenue of kinetic partitioning that slows the overall rate of renaturation relative to the chaperonin chamber, where such associations cannot occur. For Rubisco, reversible aggregation during folding in solution was observed by gel filtration. For a mutant of maltose-binding protein (DM-MBP), the rate of folding in solution declined with increasing concentration, and the folding reaction produced light scattering. Under solution conditions where chloride was absent, however, light scattering no longer occurred, and DM-MBP folded at the same rate as in the cis cavity. In a further test, dihydrofolate reductase, thermally inactivated in the cis cavity or in solution, was substantially reactivated upon temperature downshift in the cis cavity but not in solution, where aggregation occurred. We conclude that the GroEL-GroES chamber behaves as a passive "Anfinsen cage" whose primary role is to prevent multimolecular association during folding.

Prion protein amyloid formation under native-like conditions involves refolding of the C-terminal alpha-helical domain.

Transmissible spongiform encephalopathies are associated with conformational conversion of the cellular prion protein, PrP(C), into a proteinase K-resistant, amyloid-like aggregate, PrP(Sc). Although the structure of PrP(Sc) remains enigmatic, recent studies have afforded increasingly detailed characterization of recombinant PrP amyloid. However, all previous studies were performed using amyloid fibrils formed in the presence of denaturing agents that significantly alter the folding state(s) of the precursor monomer. Here we report that PrP amyloid can also be generated under physiologically relevant conditions, where the monomeric protein is natively folded. Remarkably, site-directed spin labeling studies reveal that these fibrils possess a beta-core structure nearly indistinguishable from that of amyloid grown under denaturing conditions, where the C-terminal alpha-helical domain of the PrP monomer undergoes major refolding to a parallel and in-register beta-structure upon conversion. The structural similarity of fibrils formed under drastically different conditions strongly suggests that the common beta-sheet architecture within the approximately 160-220 core region represents a distinct global minimum in the PrP conversion free energy landscape. We also show that the N-terminal region of fibrillar PrP displays conformational plasticity, undergoing a reversible structural transition with an apparent pK(a) of approximately 5.3. The C-terminal region, on the other hand, retains its beta-structure over the pH range 1-11, whereas more alkaline buffer conditions denature the fibrils into constituent PrP monomers. This profile of pH-dependent stability is reminiscent of the behavior of brain-derived PrP(Sc), suggesting a substantial degree of structural similarity within the beta-core region of these PrP aggregates.

The emerging principles of mammalian prion propagation and transmissibility barriers: Insight from studies in vitro.

Self-perpetuating conformational conversion of the cellular prion protein PrP(C) into the beta-sheet-rich "scrapie" conformer (PrP(Sc)) is believed to be the central molecular event in pathogenesis of a group of diseases known as transmissible spongiform encephalopathies. Recent advances provide growing support for the notion that a misfolded protein alone might act as an infectious agent. Furthermore, findings regarding the mechanism of prion protein structural rearrangement, the role of folding intermediates in conformational conversion, and "conformational adaptability" in the propagation of prion amyloids in vitro yield molecular-level insight into such phenomena as inherited prion diseases, prion transmission barriers, and prion strains.

Early intermediate in human prion protein folding as evidenced by ultrarapid mixing experiments.

An important step toward understanding the mechanism of the PrP(C)-to-PrP(Sc) conversion is to elucidate the folding pathway(s) of the prion protein. On the basis of stopped-flow measurements, we recently proposed that the prion protein folds via a transient intermediate formed on the submillisecond time scale, and mutations linked to familial diseases result in a pronounced increase in the population of this intermediate. Here, we have extended these studies to continuous-flow measurements using a capillary mixing system with a time resolution of approximately 100 micros. This allowed us to directly observe two distinct phases in folding of the recombinant human prion protein 90-231, providing unambiguous evidence for rapid accumulation of an early intermediate (with a time constant of approximately 50 micros), followed by a rate-limiting folding step (with a time constant of approximately 700 micros). The present study also clearly demonstrates that the population of the intermediate is significantly increased at mildly acidic pH and in the presence of urea. A similar three-state folding behavior was observed for the Gerstmann-Straussler-Scheinker disease-associated F198S mutant, in which case the population of an intermediate was greatly increased as compared to that of the wild-type protein. Overall, the present data strongly suggest that this partially structured intermediate may be a direct monomeric precursor of the misfolded PrP(Sc) oligomer.

Polymorphism at residue 129 modulates the conformational conversion of the D178N variant of human prion protein 90-231.

One of the arguments in favor of the protein-only hypothesis of transmissible spongiform encephalopathies is the link between inherited prion diseases and specific mutations in the PRNP gene. One such mutation (Asp178 --> Asn) is associated with two distinct disorders: fatal familial insomnia or familial Creutzfeldt-Jakob disease, depending upon the presence of Met or Val at position 129, respectively. In this study, we have characterized the biophysical properties of recombinant human prion proteins (huPrP90-231) corresponding to the polymorphic variants D178N/M129 and D178N/V129. In comparison to the wild-type protein, both polymorphic forms of D178N huPrP show a greatly increased propensity for a conversion to beta-sheet-rich oligomers (at acidic pH) and thioflavine T-positive amyloid fibrils (at neutral pH). Importantly, the conversion propensity for the D178N variant is strongly dependent upon the M/V polymorphism at position 129, whereas under identical experimental conditions, no such dependence is observed for the wild-type protein. Amyloid fibrils formed by wild-type huPrP90-231 and the D178N variant are characterized by different secondary structures, and these structures are further modulated by residue 129 polymorphism. Although on the basis of only in vitro data, this study strongly suggests that polymorphism-dependent phenotypic variability of familial prion diseases may be linked to differences in biophysical properties of prion protein variants.

The effect of disease-associated mutations on the folding pathway of human prion protein.

Propagation of transmissible spongiform encephalopathies is believed to involve the conversion of cellular prion protein, PrP(C), into a misfolded oligomeric form, PrP(Sc). An important step toward understanding the mechanism of this conversion is to elucidate the folding pathway(s) of the prion protein. We reported recently (Apetri, A. C., and Surewicz, W. K. (2002) J. Biol. Chem. 277, 44589-44592) that the folding of wild-type prion protein can best be described by a three-state sequential model involving a partially folded intermediate. Here we have performed kinetic stopped-flow studies for a number of recombinant prion protein variants carrying mutations associated with familial forms of prion disease. Analysis of kinetic data clearly demonstrates the presence of partially structured intermediates on the refolding pathway of each PrP variant studied. In each case, the partially folded state is at least one order of magnitude more populated than the fully unfolded state. The present study also reveals that, for the majority of PrP variants tested, mutations linked to familial prion diseases result in a pronounced increase in the thermodynamic stability, and thus the population, of the folding intermediate. These data strongly suggest that partially structured intermediates of PrP may play a crucial role in prion protein conversion, serving as direct precursors of the pathogenic PrP(Sc) isoform.

Atypical effect of salts on the thermodynamic stability of human prion protein.

Prion diseases are associated with the conversion of cellular prion protein, PrPC, into a misfolded oligomeric form, PrPSc. Previous studies indicate that salts promote conformational conversion of the recombinant prion protein into a PrPSc-like form. To gain insight into the mechanism of this effect, here we have studied the influence of a number of salts (sodium sulfate, sodium fluoride, sodium acetate, and sodium chloride) on the thermodynamic stability of the recombinant human prion protein. Chemical unfolding studies in urea show that at low concentrations (below approximately 50 mm), all salts tested significantly reduced the thermodynamic stability of the protein. This highly unusual response to salts was observed for both the full-length prion protein as well as the N-truncated fragments huPrP90-231 and huPrP122-231. At higher salt concentrations, the destabilizing effect was gradually reversed, and salts behaved according to their ranking in the Hofmeister series. The present data indicate that electrostatic interactions play an unusually important role in the stability of the prion protein. The abnormal effect of salts is likely because of the ion-induced destabilization of salt bridges (Asp144-Arg148 and/or Asp147-Arg151) in the extremely hydrophilic helix 1. Contrary to previous suggestions, this effect is not due to the interaction of ions with the glycine-rich flexible N-terminal region of the prion protein. The results of this study suggest that ionic species present in the cellular environment may control the PrPC to PrPSc conversion by modulating the thermodynamic stability of the native PrPC isoform.

Kinetic intermediate in the folding of human prion protein.

Transmissible spongiform encephalopathies are associated with the conversion of cellular prion protein, PrP(C), into a misfolded oligomeric form, PrP(Sc). Here we have examined the kinetics of folding and unfolding reactions for the recombinant human prion protein C-terminal fragment 90-231 at pH 4.8 and 7.0. The stopped-flow data provide clear evidence for the population of an intermediate on the refolding pathway of the prion protein as indicated by a pronounced curvature in chevron plots and the presence of significant burst phase amplitude in the refolding kinetics. In addition to its role in the normal prion protein folding, this intermediate likely represents a crucial monomeric precursor of the pathogenic PrP(Sc) isoform.