The role of pH on instability and aggregation of sickle hemoglobin solutions

Understanding the physical basis of protein aggregation covers strong physical and biomedical interests. Sickle hemoglobin (HbS) is a point-mutant form of normal human adult hemoglobin (HbA). It is responsible for the first identified "molecular disease," as its propensity to aggregation is responsible for sickle cell disease. At moderately higher than physiological pH value, this propensity is inhibited: The rate of aggregate nucleation becomes exceedingly small and solubility after polymerization increases. These order-of-magnitude effects on polymer nucleation rates and concurrent relatively modest changes of solubility after polymerization are here shown to be related to both pH-induced changes of location and shape of the liquid-liquid demixing (LLD) region. This allows establishment of a self-consistent contact between the thermodynamics of the solution as such (i.e., the LLD region), the kinetics of fiber nucleation, the theory of percolation, and the thermodynamics of gelation. The observed pH-induced changes are largely attributable to strong perturbations of hydrophobic hydration configurations and related free energy by electric charges. Similar mechanisms of effective control of aggregate nucleation rates by means of agents such as cosolutes, pH, salts, and additives, shifting the LLD and associated regions of anomalous fluctuations, promise to be relevant to the whole field of protein aggregation pathologies.     

Irreversible formation of intermediate BSA oligomers requires and induces conformational changes

Understanding the relation between protein conformational changes and aggregation, and the physical mechanisms leading to such processes, is of primary importance, due to its direct relation to a vast class of severe pathologies. Growing evidence also suggests that oligomeric intermediates, which may occur early in the aggregation pathway, can be themselves pathogenic. The possible cytotoxicity of oligomers of non-disease-associated proteins adds generality to such suggestion and to the interest of studies of oligomer formation. Here we study the early stages of aggregation of Bovine Serum Albumin (BSA), a non pathogenic protein which has proved to be a useful model system. Dynamic light scattering and circular dichroism measurements in kinetic experiments following step-wise temperature rises, show that the "intermediate" form, which initiates large-scale aggregation, is the result of structural and conformational changes and concurrent formation of oligomers, of average size in the range of 100-200 A. Two distinct thresholds are observed. Beyond the first one oligomerization starts and causes partial irreversibility of conformational changes. Beyond the second threshold, additional secondary structural changes occurring in proteins being recruited progress on the same time scale of oligomerization. The concurrent behavior causes a mutual stabilization of oligomerization, and of structural and conformational changes, evidenced by a progressive increase of their irreversibility. This process interaction appears to be pivotal in producing irreversible oligomers.     

Effect of T-R conformational change on sickle-cell hemoglobin interactions and aggregation

We compare the role of a conformational switch and that of a point mutation in the thermodynamic stability of a protein solution and in the consequent propensity toward aggregation. We study sickle-cell hemoglobin (HbS), the beta6 Glu-Val point mutant of adult human hemoglobin (HbA), in its R (CO-liganded) conformation, and compare its aggregation properties to those of both HbS and HbA in their T (unliganded) conformation. Static and dynamic light scattering measurements performed for various hemoglobin concentrations showed critical divergences with mean field exponents as temperature was increased. This allowed determining spinodal data points T(S)(c) by extrapolation. These points were fitted to theoretical expressions of the T(S)(c) spinodal line, which delimits the region where the homogeneous solution becomes thermodynamically unstable against demixing in two sets of denser and dilute mesoscopic domains, while remaining still liquid. Fitting provided model-free numerical values of enthalpy and entropy parameters measuring the stability of solutions against demixing, namely, 93.2 kJ/mol and 314 J/ degrees K-mol, respectively. Aggregation was observed also for R-HbS, but in amorphous form and above physiological temperatures close to the spinodal, consistent with the role played in nucleation by anomalous fluctuations governed by the parameter epsilon = (T - T(S))/T(S). Fourier transform infrared (FTIR) and optical spectroscopy showed that aggregation is neither preceded nor followed by denaturation. Transient multiple interprotein contacts occur in the denser liquid domains for R-HbS, T-HbS, and T-HbA. The distinct effects of their specific nature and configurations, and those of desolvation on the demixing and aggregation thermodynamics, and on the aggregate structure are highlighted.     

Exploring protein aggregation and self-propagation using lattice models: phase diagram and kinetics

Many seemingly unrelated neurodegenerative disorders, such as amyloid and prion diseases, are associated with propagating fibrils whose structures are dramatically different from the native states of the corresponding monomers. This observation, along with the experimental demonstration that any protein can aggregate to form either fibrils or amorphous structures (inclusion bodies) under appropriate external conditions, suggest that there must be general principles that govern aggregation mechanisms. To probe generic aspects of prion-like behavior we use the model of Harrison, Chan, Prusiner, and Cohen. In this model, aggregation of a structure, that is conformationally distinct from the native state of the monomer, occurs by three parallel routes. Kinetic partitioning, which leads to parallel assembly pathways, occurs early in the aggregation process. In all pathways transient unfolding precedes polymerization and self-propagation. Chain polymerization is consistent with templated assembly, with the dimer being the minimal nucleus. The kinetic effciency of R(n-1) + G --> R(n) (R is the aggregation prone state and G is either U, the unfolded state, or N, the native state of the monomer) is increased when polymerization occurs in the presence of a "seed" (a dimer). These results support the seeded nucleated-polymerization model of fibril formation in amyloid peptides. To probe generic aspects of aggregation in two-state proteins, we use lattice models with side chains. The phase diagram in the (T,C) plane (T is the temperature and C is the polypeptide concentration) reveals a bewildering array of "phases" or structures. Explicit computations for dimers show that there are at least six phases including ordered structures and amorphous aggregates. In the ordered region of the phase diagram there are three distinct structures. We find ordered dimers (OD) in which each monomer is in the folded state and the interaction between the monomers occurs via a well-defined interface. In the domain-swapped structures a certain fraction of intrachain contacts are replaced by interchain contacts. In the parallel dimers the interface is stabilized by favorable intermolecular hydrophobic interactions. The kinetics of folding to OD shows that aggregation proceeds directly from U in a dynamically cooperative manner without populating partially structured intermediates. These results support the experimental observation that ordered aggregation in the two-state folders U1A and CI2 takes place from U. The contrasting aggregation processes in the two models suggest that there are several distinct mechanisms for polymerization that depend not only on the polypeptide sequence but also on external conditions (such as C, T, pH, and salt concentration).     

Insight into the correlation between lag time and aggregation rate in the kinetics of protein aggregation

Under favorable conditions, many proteins can assemble into macroscopically large aggregates such as the amyloid fibrils that are associated with Alzheimer's, Parkinson's, and other neurological and systemic diseases. The overall process of protein aggregation is characterized by initial lag time during which no detectable aggregation occurs in the solution and by maximal aggregation rate at which the dissolved protein converts into aggregates. In this study, the correlation between the lag time and the maximal rate of protein aggregation is analyzed. It is found that the product of these two quantities depends on a single numerical parameter, the kinetic index of the curve quantifying the time evolution of the fraction of protein aggregated. As this index depends relatively little on the conditions and/or system studied, our finding provides insight into why for many experiments the values of the product of the lag time and the maximal aggregation rate are often equal or quite close to each other. It is shown how the kinetic index is related to a basic kinetic parameter of a recently proposed theory of protein aggregation. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Prion‐like protein aggregates exploit the RHO GTPase to cofilin‐1 signaling pathway to enter cells

Protein aggregation is a hallmark of diverse neurodegenerative diseases. Multiple lines of evidence have revealed that protein aggregates can penetrate inside cells and spread like prions. How such aggregates enter cells remains elusive. Through a focused siRNA screen targeting genes involved in membrane trafficking, we discovered that mutant SOD1 aggregates, like viruses, exploit cofilin‐1 to remodel cortical actin and enter cells. Upstream of cofilin‐1, signalling from the RHO GTPase and the ROCK1 and LIMK1 kinases controls cofilin‐1 activity to remodel actin and modulate aggregate entry. In the spinal cord of symptomatic SOD1^G93A transgenic mice, cofilin‐1 phosphorylation is increased and actin dynamics altered. Importantly, the RHO to cofilin‐1 signalling pathway also modulates entry of tau and α‐synuclein aggregates. Our results identify a common host cell signalling pathway that diverse protein aggregates exploit to remodel actin and enter cells.     

Non-native protein aggregation kinetics

Experimental kinetics of non-native protein aggregation are of practical importance in that they help dictate viable processing, formulation, and storage conditions for biotechnology products, and appear to play a role in determining the onset of a number of diseases. Fundamentally, aggregation kinetics provide insights into the identity of key intermediates in the process, and quantitative tests of available models of aggregation. Although aggregation kinetics often display seemingly disparate behaviors across different proteins and sample conditions, this review illustrates how many of these can be understood within a general framework that treats aggregation as a multi-stage process, and how most available kinetic models of aggregation can be grouped hierarchically in terms of which stage(s) they include. This provides an aid for workers seeking a mechanistic interpretation of in vitro aggregation kinetics, for discriminating among competing models, and in designing experiments to assess in vitro protein stability. Limitations and the utility of purely kinetic approaches to studying aggregation, clarifications of common misperceptions regarding experimental aggregation kinetics, and some outstanding challenges in the field are briefly discussed.     

Amyloids or prions? That is the question

ABSTRACT

Despite major efforts devoted to understanding the phenomenon of prion transmissibility, it is still poorly understood how this property is encoded in the amino acid sequence. In recent years, experimental data on yeast prion domains allow to start at least partially decrypting the sequence requirements of prion formation. These experiments illustrate the need for intrinsically disordered sequence regions enriched with a particularly high proportion of glutamine and asparagine. Bioinformatic analysis suggests that these regions strike a balance between sufficient amyloid nucleation propensity on the one hand and disorder on the other, which ensures availability of the amyloid prone regions but entropically prevents unwanted nucleation and facilitates brittleness required for propagation.     

Hierarchy and the mechanism of fibril formation in ADan peptides

Familial Danish dementia is a neurodegenerative disease which is a consequence of alterations in the BRI gene. The pathological signatures of the disease are cerebral amyloidolysis, parenchymal protein deposits and neuronal degeneration. Synthetic Danish dementia (ADan) peptides are capable of forming fibrillar assemblies in vitro at pH 4.8. However, the morphology of the aggregates formed depends greatly on the form of the peptides (oxidized or reduced). In addition to long slender assemblies (2-5 nm in diameter and several micrometers in length) we report ring-like or annular masses (8-9 nm in diameter and 1-2 mm in perimeter) in the case of the oxidized form of the peptides. The reduced forms mainly aggregate to produce granular heaps. The biophysical and kinetic characterization of the process of aggregation was carried out using different spectroscopic and imaging techniques. Neurotoxicity assays performed on both the forms reveal that the toxicity bears proportionality with the aggregate size.     

Genesis of tramsmissible protein states via deformed templating

Prion replication occurs via a template-assisted mechanism, which postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a template. The concept of prion-like template-assisted propagation of an abnormal protein conformation has been expanded to amyloidogenic proteins associated with Alzheimer, Parkinson, Huntington diseases, amyotrophic lateral sclerosis and others. Recent studies demonstrated that authentic PrP^Sc and transmissible prion disease could be generated in wild type animals by inoculation of recombinant prion protein amyloid fibrils, which are structurally different from PrP^Sc and lack any detectable PrP^Sc particles. Here we discuss a new replication mechanism designated as “deformed templating,” according to which fibrils with one cross-β folding pattern can seed formation of fibrils or particles with a fundamentally different cross-β folding pattern. Transformation of cross-β folding pattern via deformed templating provides a mechanistic explanation behind genesis of transmissible protein states induced by amyloid fibrils that are considered to be non-infectious. We postulate that deformed templating is responsible for generating conformationally diverse amyloid populations, from which conformers that are fit to replicate in a particular cellular environment are selected. We propose that deformed templating represents an essential step in the evolution of transmissible protein states.     

Genesis of tramsmissible protein states via deformed templating

Prion replication occurs via a template-assisted mechanism, which postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a template. The concept of prion-like template-assisted propagation of an abnormal protein conformation has been expanded to amyloidogenic proteins associated with Alzheimer, Parkinson, Huntington diseases, amyotrophic lateral sclerosis and others. Recent studies demonstrated that authentic PrP^Sc and transmissible prion disease could be generated in wild type animals by inoculation of recombinant prion protein amyloid fibrils, which are structurally different from PrP^Sc and lack any detectable PrP^Sc particles. Here we discuss a new replication mechanism designated as “deformed templating,” according to which fibrils with one cross-β folding pattern can seed formation of fibrils or particles with a fundamentally different cross-β folding pattern. Transformation of cross-β folding pattern via deformed templating provides a mechanistic explanation behind genesis of transmissible protein states induced by amyloid fibrils that are considered to be non-infectious. We postulate that deformed templating is responsible for generating conformationally diverse amyloid populations, from which conformers that are fit to replicate in a particular cellular environment are selected. We propose that deformed templating represents an essential step in the evolution of transmissible protein states.     

The kinetic behavior of insulin fibrillation is determined by heterogeneous nucleation pathways

When subjected to acidic conditions and high temperature, insulin is known to produce fibrils that display the common properties of disease amyloids. Thus, clarifying the mechanisms of insulin fibrillation can help the general understanding of amyloidal aggregation. Insulin fibrillation exhibits a very sharp time dependence, with a pronounced lag phase and subsequent explosive growth of amyloidal aggregates. Here we show that the initial stages of this process can be well described by exponential growth of the fibrillated proteins. This indicates that the process is mainly controlled by a secondary nucleation pathway.     

Huntington toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1

The cause of Huntington's disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone. Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity. Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1-151 mutations inhibited expansion of aggregates. The latter three mutants strongly suppressed the polyQ toxicity. Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved. Aggregation defects in these mutants and in sis1-85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance. Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity. We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ. Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ. We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity.     

Chemical probes for investigating protein liquid-liquid phase separation and aggregation

Protein liquid-liquid phase separation drives the dynamic assembly of membraneless organelles for fulfilling different physiological functions. Under diseased condition, protein may undergo liquid-to-solid condensation to form pathological amyloid aggregates closely associated with neurodegenerative diseases. Chemical probe serves as an important chemical tool not only for exploring the basic principle of the dynamic assembly of different protein condensates in vitro and in cell but also for clinical diagnosis and therapeutics of the related diseases. In this review, we first introduce chemical probes to image and regulate protein condensates. Then, we summarized three different categories of chemical probes including general amyloid dye, selective positron emission tomography tracer, and disaggregating binder, which feature distinct interaction pattern and activity upon binding to different pathological amyloid fibrillar aggregates. Next, we discuss the development of chemical probes for tracking protein amorphous aggregates in cells. Finally, we point out future direction in expanding the probes’ chemical space and applications.     

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

The Fc (fragment crystallizable) is a common structural region in immunoglobulin gamma (IgG) proteins, IgG-based multi-specific platforms, and Fc-fusion platform technologies. Changes in conformational stability, protein-protein interactions, and aggregation of NS0-produced human Fc1 were quantified experimentally as a function of pH (4 to 6) and temperature (30 to 77°C), using a combination of differential scanning calorimetry, laser light scattering, size-exclusion chromatography, and capillary electrophoresis. The Fc1 was O-glycosylated at position 3 (threonine), and confirmed to correspond to the intact IgG1 by comparison with Fc1 produced by cleavage of the parent IgG1. Changing the pH caused large effects for thermal unfolding transitions, but it caused surprisingly smaller effects for electrostatic protein-protein interactions. The aggregation behavior was qualitatively similar across different solution conditions, with soluble dimers and larger oligomers formed in most cases. Aggregation rates spanned approximately 5 orders of magnitude and could be divided into 2 regimes: (i) Arrhenius, unfolding-limited aggregation at temperatures near or above the midpoint-unfolding temperature of the C_H2 domain; (ii) a non-Arrhenius regime at lower temperatures, presumably as a result of the temperature dependence of the unfolding enthalpy for the C_H2 domain. The non-Arrhenius regime was most pronounced for lower temperatures. Together with the weak protein-protein repulsions, these highlight challenges that are expected for maintaining long-term stability of biotechnology products that are based on human Fc constructs.     

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

The Fc (fragment crystallizable) is a common structural region in immunoglobulin gamma (IgG) proteins, IgG-based multi-specific platforms, and Fc-fusion platform technologies. Changes in conformational stability, protein-protein interactions, and aggregation of NS0-produced human Fc1 were quantified experimentally as a function of pH (4 to 6) and temperature (30 to 77°C), using a combination of differential scanning calorimetry, laser light scattering, size-exclusion chromatography, and capillary electrophoresis. The Fc1 was O-glycosylated at position 3 (threonine), and confirmed to correspond to the intact IgG1 by comparison with Fc1 produced by cleavage of the parent IgG1. Changing the pH caused large effects for thermal unfolding transitions, but it caused surprisingly smaller effects for electrostatic protein-protein interactions. The aggregation behavior was qualitatively similar across different solution conditions, with soluble dimers and larger oligomers formed in most cases. Aggregation rates spanned approximately 5 orders of magnitude and could be divided into 2 regimes: (i) Arrhenius, unfolding-limited aggregation at temperatures near or above the midpoint-unfolding temperature of the C_H2 domain; (ii) a non-Arrhenius regime at lower temperatures, presumably as a result of the temperature dependence of the unfolding enthalpy for the C_H2 domain. The non-Arrhenius regime was most pronounced for lower temperatures. Together with the weak protein-protein repulsions, these highlight challenges that are expected for maintaining long-term stability of biotechnology products that are based on human Fc constructs.     

Electrostatic modulation of hnRNPA1 low‐complexity domain liquid–liquid phase separation and aggregation

Membrane‐less organelles and RNP granules are enriched in RNA and RNA‐binding proteins containing disordered regions. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a key regulating protein in RNA metabolism, localizes to cytoplasmic RNP granules including stress granules. Dysfunctional nuclear‐cytoplasmic transport and dynamic phase separation of hnRNPA1 leads to abnormal amyloid aggregation and neurodegeneration. The intrinsically disordered C‐terminal domain (CTD) of hnRNPA1 mediates both dynamic liquid–liquid phase separation (LLPS) and aggregation. While cellular phase separation drives the formation of membrane‐less organelles, aggregation within phase‐separated compartments has been linked to neurodegenerative diseases. To understand some of the underlying mechanisms behind protein phase separation and LLPS‐mediated aggregation, we studied LLPS of hnRNPA1 CTD in conditions that probe protein electrostatics, modulated specifically by varying pH conditions, and protein, salt and RNA concentrations. In the conditions investigated, we observed LLPS to be favored in acidic conditions, and by high protein, salt and RNA concentrations. We also observed that conditions that favor LLPS also enhance protein aggregation and fibrillation, which suggests an aggregation pathway that is LLPS‐mediated. The results reported here also suggest that LLPS can play a direct role in facilitating protein aggregation, and that changes in cellular environment that affect protein electrostatics can contribute to the pathological aggregation exhibited in neurodegeneration.     

Different amyloid aggregation of smooth muscles titin in vitro

A comparative study of amyloid properties of the aggregates of smooth muscle titin (SMT) from chicken gizzard was carried out. These aggregates were formed in two solutions: 0.15 M glycine-KOH, pH 7.2–7.4 (SMT(Gly)) and 0.2 M KCl, 10 mM imidazole, pH 7.0 (SMT(KCl)). Electron microscopy data showed that SMT aggregates has an amorphous structure in both cases. The results of atomic-force microscopy demonstrated slight differences in morphology in two types of aggregates. The SMT(Gly) aggregates were represented as branching chains, composed of spherical aggregates approximately 300–500 nm in diameter and up to 35 nm in height. The SMT(KCl) aggregates formed sponge-like structures with strands of 8–10 nm in height. Structural analysis of SMT aggregates by X-ray diffraction revealed the presence of cross-β-sheet structure in the samples under study. In the presence of SMT(Gly) aggregates, thioflavine T fluorescence intensity was higher (~3-fold times) compared with that in the presence of SMT(KCl) aggregates. Congo red-stained SMT(Gly) aggregates had yellow to apple-green birefringence under polarized light, which was not observed for SMT(KCl) aggregates. Dynamic light scattering data showed the similar rate of aggregation for both types of aggregates, though SMT(KCl) aggregates were able to partially disaggregate under increased ionic strength of the solution. The ability of SMT to aggregation followed by disaggregation may be functionally significant in the cell.     

Kinetics of thermal aggregation of tobacco mosaic virus coat protein

The kinetics of thermal aggregation of coat protein (CP) of tobacco mosaic virus (TMV) have been studied at 42 and 52°C in a wide range of protein concentrations, [P]₀. The kinetics of aggregation were followed by monitoring the increase in the apparent absorbance (A) at 320 nm. At 52°C the kinetic curves may be approximated by the exponential law in the range of TMV CP concentrations from 0.02 to 0.30 mg/ml, the first order rate constant being linearly proportional to [P]₀ (50 mM phosphate buffer, pH 8.0). The analogous picture was observed at 42°C in the range of TMV CP concentrations from 0.01 to 0.04 mg/ml (100 mM phosphate buffer, pH 8.0). At higher TMV CP concentrations the time of half-conversion approaches a limiting value with increasing [P]₀ and at sufficiently high protein concentrations the kinetic curves fall on a common curve in the coordinates {A/A _lim; t} (t is time and A _lim is the limiting value of A at t ). According to a mechanism of aggregation of TMV CP proposed by the authors at rather low protein concentrations the rate of aggregation is limited by the stage of growth of aggregate, which proceeds as a reaction of the pseudo-first order, whereas at rather high protein concentrations the rate-limiting stage is the stage of protein molecule unfolding.