Retinol binding protein IV purified from Escherichia coli using intein-mediated cleavage as a suitable replacement for serum sources.

Retinol binding protein IV (RBP) functions as the principal carrier of retinol (Vitamin A) in the blood, where RBP circulates bound to another serum protein, transthyretin. Isolation of pure RBP from the transthyretin complex in human serum can be difficult, but expression of RBP in recombinant systems can circumvent these purification issues. Human recombinant RBP has previously been successfully expressed and purified from E. coli, but recovery of active protein typically requires extensive processing steps, such as denaturing and refolding, and complex purification steps, such as multi-modal chromatography. Furthermore, these methods produce recombinant proteins, often tagged, that display different functional and structural characteristics across systems. In this work, we optimized downstream processing by use of an intein-based expression system in E. coli to produce tag-free, human recombinant RBP (rRBP) with intact native amino termini at yields of up to ~15 mg/L off column. The novel method requires solubilization of inclusion bodies and subsequent oxidative refolding in the presence of retinol, but importantly allows for one-step chromatographic purification that yields high purity rRBP with no N-terminal Met or other tag. Previously reported purification methods typically require two or more chromatographic separation steps to recover tag-free rRBP. Given the interest in mechanistic understanding of RBP transport of retinol in health and disease, we characterized our purified product extensively to confirm rRBP is both structurally and functionally a suitable replacement for serum-derived RBP.

Retinol-Binding Protein Interferes with Transthyretin-Mediated ß-Amyloid Aggregation Inhibition.

ß-Amyloid (Aß) aggregation is causally linked to Alzheimer's disease. On the basis of in vitro and transgenic animal studies, transthyretin (TTR) is hypothesized to provide neuroprotection against Aß toxicity by binding to Aß and inhibiting its aggregation. TTR is a homotetrameric protein that circulates in blood and cerebrospinal fluid; its normal physiological role is as a carrier for thyroxine and retinol-binding protein (RBP). RBP forms a complex with retinol, and the holoprotein (hRBP) binds with high affinity to TTR. In this study, the role of TTR ligands in TTR-mediated inhibition of Aß aggregation was investigated. hRBP strongly reduced the ability of TTR to inhibit Aß aggregation. The effect was not due to competition between Aß and hRBP for binding to TTR, as Aß bound equally well to TTR-hRBP complexes and TTR. hRBP is known to stabilize the TTR tetrameric structure. We show that Aß partially destabilizes TTR and that hRBP counteracts this destabilization. Taken together, our results support a mechanism wherein TTR-mediated inhibition of Aß aggregation requires not only TTR-Aß binding but also destabilization of TTR quaternary structure.

A mechanistic model to predict effects of cathepsin B and cystatin C on ß-amyloid aggregation and degradation.

ß-Amyloid (Aß) aggregation is thought to initiate a cascade of neurodegenerative events in Alzheimer's disease (AD). Much effort is underway to develop strategies to reduce Aß concentration or inhibit aggregation. Cathepsin B (CatB) proteolytically degrades Aß into non-aggregating fragments but is potently inhibited by cystatin C (CysC). It has been suggested that decreasing CysC would facilitate Aß clearance by relieving CatB inhibition. However, CysC binds Aß and inhibits Aß aggregation, suggesting that an intervention that increases CysC would prevent Aß aggregation. Both approaches have been tested in animal models, yielding contradictory results, possibly because of the opposing influences of CysC on Aß degradation versus aggregation. Here, we sought to develop a model that quantitatively predicts the effects of CysC and CatB on Aß aggregation. Aß aggregation kinetics in the absence of CatB or CysC was measured. The rate constant for Aß degradation by CatB and the equilibrium constant for binding of CysC to Aß were determined. We derived a mathematical model that combines material balances and kinetic rate equations. The model accurately predicted Aß aggregation kinetics at various CatB and CysC concentrations. We derived approximate expressions for the half-times of degradation and aggregation and show that their ratio can be used to estimate, at any given Aß, CatB, or CysC concentration, whether Aß aggregation or degradation will result. Our results may be useful for designing experiments and interpreting results from investigations of manipulation of CysC concentration as an AD therapy.

Insights into the mechanism of cystatin C oligomer and amyloid formation and its interaction with ß-amyloid.

Cystatin C (CysC) is a versatile and ubiquitously-expressed member of the cysteine protease inhibitor family that is present at notably high concentrations in cerebrospinal fluid. Under mildly denaturing conditions, CysC forms inactive domain-swapped dimers. A destabilizing mutation, L68Q, increases the rate of domain-swapping and causes a fatal amyloid disease, hereditary cystatin C amyloid angiopathy. Wild-type (wt) CysC will also aggregate into amyloid fibrils under some conditions. Propagated domain-swapping has been proposed as the mechanism by which CysC fibrils grow. We present evidence that a CysC mutant, V57N, stabilized against domain-swapping, readily forms fibrils, contradicting the propagated domain-swapping hypothesis. Furthermore, in physiological buffer, wt CysC can form oligomers without undergoing domain-swapping. These non-swapped oligomers are identical in secondary structure to CysC monomers and completely retain protease inhibitory activity. However, unlike monomers or dimers, the oligomers bind fluorescent dyes that indicate they have characteristics of pre-amyloid aggregates. Although these oligomers appear to be a pre-amyloid assembly, they are slower than CysC monomers to form fibrils. Fibrillation of CysC therefore likely initiates from the monomer and does not require domain-swapping. The non-swapped oligomers likely represent a dead-end offshoot of the amyloid pathway and must dissociate to monomers prior to rearranging to amyloid fibrils. These prefibrillar CysC oligomers were potent inhibitors of aggregation of the Alzheimer's-related peptide, ß-amyloid. This result illustrates an example where heterotypic interactions between pre-amyloid oligomers prevent the homotypic interactions that would lead to mature amyloid fibrils.

Cerebrospinal Fluid Proteins as Regulators of Beta-amyloid Aggregation and Toxicity.

Amyloid disorders, such as Alzheimer's, are almost invariably late-onset diseases. One defining diagnostic feature of Alzheimer's disease is the deposition of beta-amyloid as extracellular plaques, primarily in the hippocampus. This raises the question: are there natural protective agents that prevent beta-amyloid from depositing, and is it loss of this protection that leads to onset of disease? Proteins in cerebrospinal fluid (CSF) have been suggested to act as just such natural protective agents. Here, we describe some of the early evidence that led to this suggestion, and we discuss, in greater detail, two CSF proteins that have garnered the bulk of the attention.

Expression, purification, and characterization of human cystatin C monomers and oligomers.

Human cystatin C (cysC) is a soluble basic protein belonging to the cysteine protease inhibitor family. CysC is a potent inhibitor of cathepsins--proteolytic enzymes that degrade intracellular and endocytosed proteins, remodel extracellular matrix, and trigger apoptosis. Inhibition is via tight reversible binding involving the N-terminus as well as two ß-hairpin loops of cysC. As a significant component of cerebrospinal fluid, cysC has numerous other functions, including support of neural stem cell growth and differentiation. Several studies suggest that cysC may bind to the Alzheimer-related protein beta-amyloid (Aß), and inhibit its aggregation and toxicity. Because of an increasing recognition of its important biological roles, there is considerable interest in methods to produce full-length recombinant human cysC. Several researchers have reported success, but with processes that require multiple purification steps. Here we report successful production of human cysC using an intein-based expression system and a simple one-column purification scheme. The recombinant protein so obtained was natively folded and active as an enzyme inhibitor. Unexpectedly, even mild concentration by ultrafiltration caused significant oligomerization. The oligomers are noncovalent and retain the native secondary structure and inhibitory activity of the monomer. The oligomers, but not the monomers, were highly effective at inhibiting aggregation of Aß. These results demonstrate the critical importance of careful physicochemical characterization of recombinant cysC protein prior to evaluation of its biological functions.

Asparagine Repeat Peptides: Aggregation Kinetics and Comparison with Glutamine Repeats.

Amino acid repeat runs are common occurrences in eukaryotic proteins, with glutamine (Q) and asparagine (N) as particularly frequent repeats. Abnormal expansion of Q-repeat domains causes at least nine neurodegenerative disorders, most likely because expansion leads to protein misfolding, aggregation, and toxicity. The linkage between Q-repeats and disease has motivated several investigations into the mechanism of aggregation and the role of Q-repeat length in aggregation. Curiously, glutamine repeats are common in vertebrates, whereas N-repeats are virtually absent in vertebrates, but common in invertebrates. One hypothesis for the lack of N-repeats in vertebrates is biophysical; that is, there is strong selective pressure in higher organisms against aggregation-prone proteins. If true, then asparagine and glutamine repeats must differ substantially in their aggregation properties despite their chemical similarities. In this work, aggregation of peptides with asparagine repeats of variable length (12-24) were characterized and compared to that of similar peptides with glutamine repeats. As with glutamine, aggregation of N-repeat peptides was strongly length-dependent. Replacement of glutamine with asparagine caused a subtle shift in the conformation of the monomer, which strongly affected the rate of aggregation. Specifically, N-repeat peptides adopted ß-turn structural elements, leading to faster self-assembly into globular oligomers and much more rapid conversion into fibrillar aggregates, compared to Q-repeat peptides. These biophysical differences may account for the differing biological roles of N- versus Q-repeat domains.

Insights into the molecular mechanism of protein native-like aggregation upon glycation.

Several human neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Familial Amyloidotic Polyneuropathy, have long been associated with, structural and functional changes in disease related proteins leading to aggregation into amyloid fibrils. Such changes can be triggered by post-translational modifications. Methylglyoxal modifications have been shown to induce the formation of small and stable native-like aggregates in the case of the amyloidogenic proteins insulin and α-synuclein. However, the fundamental biophysical mechanism underlying such methylglyoxal-induced protein aggregation is not yet fully understood. In this work cytochrome c (Cyt c) was used as a model protein for the characterization of specific glycation targets and to study their impact on protein structure, stability, and ability to form native-like aggregates. Our results show that methylglyoxal covalently modifies Cyt c at a single residue and induces early conformational changes that lead to the formation of native-like aggregates. Furthermore, partially unfolded species are formed, but do not seem to be implicated in the aggregation process. This shows a clear difference from the amyloid fibril mechanisms which involve partially or totally unfolded intermediates. Equilibrium-unfolding experiments show that glycation strongly decreases Cyt c conformational stability, which is balanced with an increase of conformational stability upon aggregation. Data collected from analytical and spectroscopic techniques, along with kinetic analysis based on least-squares parameter fitting and statistical model discrimination are used to help to understand the driving force underlying glycation-induced native-like aggregation, and enable the proposal of a comprehensive thermodynamic and kinetic model for native-like aggregation of methylglyoxal glycated Cyt c.

Synthesis and disaggregation of asparagine repeat-containing peptides.

Of all amino acid repeats in eukaryotes, polyglutamine (polyQ) is the most frequent, followed by polyasparagine (polyN). Glutamine repeats are expanded in proteins associated with several neurodegenerative disorders. The expanded polyQ domain is known to induce aggregation, and it is hypothesized that aggregation is directly causative of pathology. Despite the widespread presence of asparagine repeats in invertebrate eukaryotes, polyN is curiously quite rare in vertebrates. Several investigators have characterized the conformational and aggregation properties of polyQ-containing peptides and proteins, and to a lesser extent, peptides containing mixed glutamine and asparagine, but to our knowledge, there is no detailed characterization of polyN-containing peptides. Such a comparison could elucidate reasons for the paucity of asparagine repeats in humans. In this study, we synthesized a peptide containing a 24-asparagine repeat (N24). For aggregation studies, it is critical to start with monomeric unaggregated peptide. A protocol involving dissolution in mixed trifluoroacetic acid and hexafluoroisopropanol (TFA + HFIP) solvents is widely used for disaggregation of polyQ peptides. We used the same protocol for N24 but discovered that there was both oxidative damage and insufficient disaggregation. Oxidation of tryptophan, used as a flanking residue, was common. Moreover, we found evidence of Förster resonance energy transfer between Trp and its oxidation product N-formylkynurenine, even in chemical denaturants. This suggested that N24 was insufficiently disaggregated, a conclusion that was further supported by gel electrophoresis analysis. Oxidation was reduced, but not eliminated, by addition of methionine to the buffer. Formic acid proved to be a better disaggregator and caused no oxidative damage. The glutamine repeat peptide Q24 also underwent some oxidation after extended incubation in TFA + HFIP, but there was no evidence of Förster resonance energy transfer, and samples appeared monomeric by gel electrophoresis. This result indicates that polyN-containing peptides self-associate more strongly than polyQ-containing peptides. Circular dichroism spectra reveal a greater propensity for ß-turn formation in polyN than polyQ, providing an explanation for the increased stability of polyN aggregates relative to polyQ.

Transthyretin as both a sensor and a scavenger of ß-amyloid oligomers.

Transthyretin (TTR) is a homotetrameric transport protein, assembled from monomers that each contain two four-stranded ß-sheets and a short α-helix and loop. In the tetramer, the "inner" ß-sheet forms a hydrophobic pocket while the helix and loop are solvent-exposed. ß-Amyloid (Aß) aggregates bind to TTR, and the level of binding is significantly reduced in mutants L82A (on the loop) and L110A (on the inner ß-sheet). Protection against Aß toxicity was demonstrated for wild-type TTR but not L82A or L110A, providing a direct link between TTR-Aß binding and TTR-mediated cytoprotection. Protection is afforded at substoichiometric (1:100) TTR:Aß molar ratios, and the level of binding of Aß to TTR is highest for partially aggregated materials and decreased for freshly prepared or heavily aggregated Aß, suggesting that TTR binds selectively to soluble toxic Aß aggregates. A novel technique, nanoparticle tracking, is used to show that TTR arrests Aß aggregation by both preventing formation of new aggregates and inhibiting growth of existing aggregates. TTR tetramers are normally quite stable; tetrameric structure is necessary for the protein's transport functions, and mutations that decrease tetramer stability have been linked to TTR amyloid diseases. However, TTR monomers bind more Aß than do tetramers, presumably because the hydrophobic inner sheet is solvent-exposed upon tetramer disassembly. Wild-type and L110A tetramers, but not L82A, were destabilized upon being co-incubated with Aß, suggesting that binding of Aß to L82 triggers tetramer dissociation. Taken together, these results suggest a novel mechanism of action for TTR: the EF helix/loop "senses" the presence of soluble toxic Aß oligomers, triggering destabilization of TTR tetramers and exposure of the hydrophobic inner sheet, which then "scavenges" these toxic oligomers and prevents them from causing cell death.

An in vitro model for vitamin A transport across the human blood-brain barrier.

Vitamin A, supplied by the diet, is critical for brain health, but little is known about its delivery across the blood-brain barrier (BBB). Brain microvascular endothelial-like cells (BMECs) differentiated from human-derived induced pluripotent stem cells (iPSC) form a tight barrier that recapitulates many of the properties of the human BBB. We paired iPSC-derived BMECs with recombinant vitamin A serum transport proteins, retinol binding protein (RBP) and transthyretin (TTR), to create an in vitro model for the study of vitamin A (retinol) delivery across the human BBB. iPSC-derived BMECs display a strong barrier phenotype, express key vitamin A metabolism markers and can be used for quantitative modeling of retinol accumulation and permeation. Manipulation of retinol, RBP and TTR concentrations, and the use of mutant RBP and TTR, yielded novel insights into the patterns of retinol accumulation in, and permeation across, the BBB. The results described herein provide a platform for deeper exploration of the regulatory mechanisms of retinol trafficking to the human brain.

An in vitro model for vitamin A transport across the human blood-brain barrier.

Vitamin A, supplied by the diet, is critical for brain health, but little is known about its delivery across the blood-brain barrier (BBB). Brain microvascular endothelial-like cells (BMECs) differentiated from human-derived induced pluripotent stem cells (iPSCs) form a tight barrier that recapitulates many of the properties of the human BBB. We paired iPSC-derived BMECs with recombinant vitamin A serum transport proteins, retinol-binding protein (RBP), and transthyretin (TTR), to create an in vitro model for the study of vitamin A (retinol) delivery across the human BBB. iPSC-derived BMECs display a strong barrier phenotype, express key vitamin A metabolism markers, and can be used for quantitative modeling of retinol accumulation and permeation. Manipulation of retinol, RBP, and TTR concentrations, and the use of mutant RBP and TTR, yielded novel insights into the patterns of retinol accumulation in, and permeation across, the BBB. The results described herein provide a platform for deeper exploration of the regulatory mechanisms of retinol trafficking to the human brain.

Nanoparticle tracking analysis and statistical mixture distribution analysis to quantify nanoparticle-vesicle binding.

Nanoparticle tracking analysis (NTA) is a single particle tracking technique that in principle provides a more direct measure of particle size distribution compared to dynamic light scattering (DLS). Here, we demonstrate how statistical mixture distribution analysis can be used in combination with NTA to quantitatively characterize the amount and extent of particle binding in a mixture of nanomaterials. The combined approach is used to study the binding of gold nanoparticles to two types of phospholipid vesicles, those containing and lacking the model ion channel peptide gramicidin A. This model system serves as both a proof of concept for the method and a demonstration of the utility of the approach in studying nano-bio interactions. Two diffusional models (Stokes-Einstein and Kirkwood-Riseman) were compared in the determination of particle size, extent of binding, and nanoparticle:vesicle binding ratios for each vesicle type. The combination of NTA and statistical mixture distributions is shown to be a useful method for quantitative assessment of the extent of binding between particles and determination of binding ratios.

Necroptosis is associated with Rab27-independent expulsion of extracellular vesicles containing RIPK3 and MLKL.

Extracellular vesicle (EV) secretion is an important mechanism used by cells to release biomolecules. A common necroptosis effector-mixed lineage kinase domain like (MLKL)-was recently found to participate in the biogenesis of small and large EVs independent of its function in necroptosis. The objective of the current study is to gain mechanistic insights into EV biogenesis during necroptosis. Assessing EV number by nanoparticle tracking analysis revealed an increased number of EVs released during necroptosis. To evaluate the nature of such vesicles, we performed a newly adapted, highly sensitive mass spectrometry-based proteomics on EVs released by healthy or necroptotic cells. Compared to EVs released by healthy cells, EVs released during necroptosis contained a markedly higher number of unique proteins. Receptor interacting protein kinase-3 (RIPK3) and MLKL were among the proteins enriched in EVs released during necroptosis. Further, mouse embryonic fibroblasts (MEFs) derived from mice deficient of Rab27a and Rab27b showed diminished basal EV release but responded to necroptosis with enhanced EV biogenesis as the wildtype MEFs. In contrast, necroptosis-associated EVs were sensitive to Ca2+ depletion or lysosomal disruption. Neither treatment affected the RIPK3-mediated MLKL phosphorylation. An unbiased screen using RIPK3 immunoprecipitation-mass spectrometry on necroptotic EVs led to the identification of Rab11b in RIPK3 immune-complexes. Our data suggests that necroptosis switches EV biogenesis from a Rab27a/b dependent mechanism to a lysosomal mediated mechanism.

Location trumps length: polyglutamine-mediated changes in folding and aggregation of a host protein.

Expanded CAG diseases are progressive neurodegenerative disorders in which specific proteins have an unusually long polyglutamine stretch. Although these proteins share no other sequence or structural homologies, they all aggregate into intracellular inclusions that are believed to be pathological. We sought to determine what impact the position and number of glutamines have on the structure and aggregation of the host protein, apomyoglobin. Variable-length polyQ tracts were inserted either into the loop between the C- and D-helices (Q(n)CD) or at the N-terminus (Q(n)NT). The Q(n)CD mutants lost some α-helix and gained unordered and/or ß-sheet in a length-dependent manner. These mutants were partially unfolded and rapidly assembled into soluble chain-like oligomers. In sharp contrast, the Q(n)NT mutants largely retained wild-type tertiary structure but associated into long, fibrillar aggregates. Control proteins with glycine-serine repeats (GS(8)CD and GS(8)NT) were produced. GS(8)CD exhibited similar structural perturbations and aggregation characteristics to an analogously sized Q(16)CD, indicating that the observed effects are independent of amino acid composition. In contrast to Q(16)NT, GS(8)NT did not form fibrillar aggregates. Thus, soluble oligomers are produced through structural perturbation and do not require polyQ, whereas classic fibrils arise from specific polyQ intermolecular interactions in the absence of misfolding.

Length-dependent aggregation of uninterrupted polyalanine peptides.

Polyalanine (polyA) is the third-most prevalent homopeptide repeat in eukaryotes, behind polyglutamine and polyasparagine. Abnormal expansion of the polyA repeat is linked to at least nine human diseases, and the disease mechanism likely involves enhanced length-dependent aggregation. Because of the simplicity of its side chain, polyA has been a favorite target of computational studies, and because of their tendency to fold into α-helix, peptides containing polyA-rich domains have been a popular experimental subject. However, experimental studies on uninterrupted polyA are very limited. We synthesized polyA peptides containing uninterrupted sequences of 7 to 25 alanines (A7 to A25) and characterized their length-dependent conformation and aggregation properties. The peptides were primarily disordered, with a modest component of α-helix that increased with increasing length. From measurements of mean distance spanned by the polyA segment, we concluded that physiological buffers are neutral solvents for shorter polyA peptides and poor solvents for longer peptides. At moderate concentration and near-physiological temperature, polyA assembled into soluble oligomers, with a sharp transition in oligomer physical properties between A19 and A25. With A19, oligomers were large, contained only a small fraction of the total peptide mass, and slowly grew into loose clusters, while A25 rapidly and completely assembled into small stable oligomers of ~7 nm radius. At high temperatures, A19 assembled into fibrils, but A25 precipitated as dense, micrometer-sized particles. A comparison of these results to those obtained with polyglutamine peptides of similar design sheds light on the role of the side chain in regulating conformation and aggregation.

Membrane Remodeling and Stimulation of Aggregation Following α-Synuclein Adsorption to Phosphotidylserine Vesicles.

α-Synuclein is an intrinsically disordered protein abundant in presynaptic terminals in neurons and in synaptic vesicles. α-Synuclein's interaction with lipid bilayers is important not only for its normal physiological function but also in its pathological aggregation and deposition as Lewy bodies in Parkinson's disease. α-Synuclein binds preferentially to lipids with acidic head groups and to high-curvature vesicles and can modulate membrane curvature. The relationship between the protein's role as a membrane curvature sensor and generator and the role of membranes in facilitating its aggregation remains unknown. We investigated the interaction of α-synuclein with vesicles of 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) or 1,2-dilauroyl-sn-glycero-3-phospho-l-serine (DLPS). Using nanoparticle tracking along with electron microscopy, we demonstrate that α-synuclein induces extensive vesicle disruption and membrane remodeling into discoids, tubules, and ribbons with DLPS vesicles but not DOPS. Coarse-grained molecular dynamics simulations revealed that adsorption of α-synuclein to DLPS but not DOPS vesicles induced vesicle elongation and redistribution of protein to regions of higher curvature, a process that could drive protein aggregation. In agreement with this hypothesis, DLPS but not DOPS strongly stimulated α-synuclein aggregation. Our results provide new insights into the critical contribution of bilayer stability in the membrane response to α-synuclein adsorption and in stimulation of aggregation.

Characterization of the interaction of ß-amyloid with transthyretin monomers and tetramers.

ß-Amyloid (Aß) is the main protein component of the amyloid plaques associated with Alzheimer's disease. Transthyretin (TTR) is a homotetramer that circulates in both blood and cerebrospinal fluid. Wild-type (wt) TTR amyloid deposits are linked to senile systemic amyloidosis, a common disease of aging, while several TTR mutants are linked to familial amyloid polyneuropathy. Several recent studies provide support for the hypothesis that these two amyloidogenic proteins interact, and that this interaction is biologically relevant. For example, upregulation of TTR expression in Tg2576 mice was linked to protection from the toxic effects of Aß deposition [Stein, T. D., and Johnson, J. A. (2002) J. Neurosci. 22, 7380-7388]. We examined the interaction of Aß with wt TTR as well as two mutants: F87M/L110M, engineered to be a stable monomer, and T119M, a naturally occurring mutant with a tetrameric stability higher than that of the wild type. On the basis of enzyme-linked immunoassays as well as cross-linking experiments, we conclude that Aß monomers bind more to TTR monomers than to TTR tetramers. The data further suggest that TTR tetramers interact preferably with Aß aggregates rather than Aß monomers. Through tandem mass spectrometry analysis of cross-linked TTR-Aß fragments, we identified the A strand, in the inner ß-sheet of TTR, as well as the EF helix, as regions of TTR that are involved with Aß association. Light scattering and electron microscopy studies demonstrate that the outcome of the TTR-Aß interaction strongly depends on TTR quaternary structure. While TTR tetramers may modestly enhance aggregation, TTR monomers decidedly arrest Aß aggregate growth. These data provide important new insights into the nature of TTR-Aß interactions. Such interactions may regulate TTR-mediated protection against Aß toxicity.

Evaluation of Nanoparticle Tracking Analysis for the Detection of Rod-Shaped Particles and Protein Aggregates.

Nanoparticle tracking analysis (NTA) is an important technique for measuring hydrodynamic size of globular biological particles including liposomes and viruses. Less attention has been paid to NTA of rod-like particles, despite their considerable interest. For example, amyloid fibrils and protofibrils are protein aggregates with rod-like morphology, diameters of 2-15 nm, and lengths from 50 nm to 1 µm, and linked to diseases including Alzheimer's and Parkinson's. We used NTA to measure the concentration and hydrodynamic size of gold nanorods (10 nm diameter, 35-250 nm length) and myosin (2 nm diameter, 160 nm length), as models of rod-like particles. Measured hydrodynamic diameters of gold nanorods were consistent with theoretical calculations, as long as particle concentration and solution conditions were controlled. Myosin monomers were invisible by NTA, but a small population of aggregates was detected. We combined NTA results with other light scattering data to gain insight into number and size distribution of protein solutions containing both monomer and aggregates. Finally, we demonstrated the utility of NTA and its limitations by characterizing aggregates of alpha-synuclein. Of note is the use of NTA to detect a change in morphology from compact to elongated by analyzing the ratio of hydrodynamic size to intensity.

Model discrimination and mechanistic interpretation of kinetic data in protein aggregation studies.

Given the importance of protein aggregation in amyloid diseases and in the manufacture of protein pharmaceuticals, there has been increased interest in measuring and modeling the kinetics of protein aggregation. Several groups have analyzed aggregation data quantitatively, typically measuring aggregation kinetics by following the loss of protein monomer over time and invoking a nucleated growth mechanism. Such analysis has led to mechanistic conclusions about the size and nature of the nucleus, the aggregation pathway, and/or the physicochemical properties of aggregation-prone proteins. We have examined some of the difficulties that arise when extracting mechanistic meaning from monomer-loss kinetic data. Using literature data on the aggregation of polyglutamine, a mutant beta-clam protein, and protein L, we determined parameter values for 18 different kinetic models. We developed a statistical model discrimination method to analyze protein aggregation data in light of competing mechanisms; a key feature of the method is that it penalizes overparameterization. We show that, for typical monomer-loss kinetic data, multiple models provide equivalent fits, making mechanistic determination impossible. We also define the type and quality of experimental data needed to make more definitive conclusions about the mechanism of aggregation. Specifically, we demonstrate how direct measurement of fibril size provides robust discrimination.