High Molecular Weight Aggregates Research Articles

In vitro gastroduodenal and jejunal brush border membrane digestion of raw and roasted tree nuts

Heat treatments induce chemical/physical modifications, which may affect the stability to enzymatic digestion and consequently the allergenicity of food proteins to a varying extent, depending on the time/temperature regimen. Herein, we evaluated the stability to digestion of whole tree nut (walnuts, hazelnuts and almonds) allergens in a food digestion model reflecting the real one by, taking into consideration the allergen-containing processed (roasted) food. To this aim, whole raw and roasted tree nuts were subjected to in vitro digestion combining the harmonized oral-gastric-duodenal digestion models with brush border membrane enzymes (BBM) to simulate the jejunal degradation of peptides. The degradation of allergens was monitored by integrated proteomic/peptidomic and bio-informatic tools. Roasting increased digestibility of tree nuts, since very few peptides were detected in digested samples (<6.5 kDa fraction). After BBM digestion step, the degradation of peptides was enhanced in roasted walnuts and hazelnuts compared to the raw counterpart. Conversely, almond allergens showed a different behaviour, since the presence of resistant peptides was more evident for roasted almonds, probably because of the hydrolysis of high molecular weight aggregates generated during roasting. Our results provide new insight into the relationship between thermal processing and metabolic fate of tree nut allergens, highlighting the importance of investigating the digestion stability of whole allergenic food, rather than purified proteins.

Rubisco: A promising plant protein to enrich wheat-based food without impairing dough viscoelasticity and protein polymerisation

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), a leaf protein, has an interesting amino acid profile and promising functional properties. Incorporated in wheat-based products, it would increase their protein content and improve their essential amino acid profile, particularly lysine. The impact of rubisco enrichment on wheat dough mechanical properties and protein-protein interactions was investigated using Dynamic Mechanical Thermal Analysis and Size-Exclusion chromatography, respectively. Experiments were also performed on gluten and pea protein enriched doughs as a comparison. Wheat doughs with increasing concentrations of rubisco, gluten or pea proteins (from 0 to 33% of total proteins) were prepared using a 2 g-mixograph at constant hydration. In contrast to pea proteins and gluten, rubisco does not reduce dough stiffening during heating, probably due to its own reactivity to temperature and to low competition with starch for water. Detailed analysis of protein interactions showed that rubisco is part of the gluten network formed during dough mixing through the establishment of weak and disulphide bonds. In addition, rubisco subunits form new covalent bonds during the heat treatment thereby increasing the concentration of SDS insoluble high molecular weight aggregates. These results suggest that rubisco actively participates in the formation of the dough protein network. The colocation of gluten and rubisco proteins on micrographs supports the hypothesis that they form a co-protein network.

Aggregation pathways of human γ D crystallin induced by metal ions revealed by time dependent methods.

Cataract formation is a slow accumulative process due to protein aggregates promoted by different factors over time. Zinc and copper ions have been reported to induce the formation of aggregates opaque to light in the human gamma D crystallin (HγD) in a concentration and temperature dependent manner. In order to gain insight into the mechanism of metal-induced aggregation of HγD under conditions that mimic more closely the slow, accumulative process of the disease, we have studied the non-equilibrium process with the minimal metal dose that triggers HγD aggregation. Using a wide variety of biophysics techniques such as turbidimetry, dynamic light scattering, fluorescence, nuclear magnetic resonance and computational methods, we obtained information on the molecular mechanisms for the formation of aggregates. Zn(II) ions bind to different regions at the protein, probably with similar affinities. This binding induces a small conformational rearrangement within and between domains and aggregates via the formation of metal bridges without any detectable unfolded intermediates. In contrast, Cu(II)-induced aggregation includes a lag time, in which the N-terminal domain partially unfolds while the C-terminal domain and parts of the N-terminal domain remain in a native-like conformation. This partially unfolded intermediate is prone to form the high-molecular weight aggregates. Our results clearly show that different external factors can promote protein aggregation following different pathways.

Asp isomerization increases aggregation of α-crystallin and decreases its chaperone activity in human lens of various ages

α-Crystallin, comprising 40–50 subunits of αA- and αB-subunits, is a long-lived major soluble chaperone protein in lens. During aging, α-crystallin forms aggregates of high molecular weight (HMW) protein and eventually becomes water-insoluble (WI). Isomerization of Asp in α-crystallin has been proposed as a trigger of protein aggregation, ultimately leading to cataract formation. Here, we have investigated the relationship between protein aggregation and Asp isomerization of αA-crystallin by a series of analyses of the soluble α-crystallin, HMW and WI fractions from human lens samples of different ages (10–76 years). Analytical ultracentrifugation showed that the HMW fraction had a peak sedimentation coefficient of 40 S and a wide distribution of values (10–450 S) for lens of all ages, whereas the α-crystallin had a much smaller peak sedimentation coefficient (10–20 S) and was less heterogeneous, regardless of lens age. Measurement of the ratio of isomers (Lα-, Lβ-, Dα-, Dβ-) at Asp58, Asp91/92 and Asp151 in αA-crystallin by liquid chromatography–mass spectrometry showed that the proportion of isomers at all three sites increased in order of aggregation level (α-crystallin < HMW < WI fractions). Among the abnormal isomers of Asp58 and Asp151, Dβ-isomers were predominant with a very few exceptions. Notably, the chaperone activity of HMW protein was minimal for lens of all ages, whereas that of α-crystallin decreased with increasing lens age. Thus, abnormal aggregation caused by Asp isomerization might contribute to the loss of chaperone activity of α-crystallin in aged human lens.

Gut Microbiome-Modified Polyphenolic Compounds Inhibit α-Synuclein Seeding and Spreading in α-Synucleinopathies

Misfolding, aggregation and deposition of α-synuclein (α-syn) are major pathologic characteristics of Parkinson’s disease (PD) and the related synucleinopathy, multiple system atrophy (MSA). The spread of α-syn pathology across brain regions is thought to play a key role in the onset and progression of clinical phenotypes. Thus, there is increasing interest in developing strategies that target and attenuate α-syn aggregation and spread. Recent studies of brain-penetrating polyphenolic acids, namely, 3-hydroxybenzoic acid (3-HBA), 3,4-dihydroxybenzoic acid (3,4-diHBA), and 3-(3-hydroxyphenyl)propionic acid (3-HPPA) that are derived from gut microbiota metabolism of dietary polyphenols, show in vitro ability to effectively modulate α-syn misfolding, oligomerization, and mediate aggregated α-syn neurotoxicity. Here we investigate whether 3-HBA, 4-hydroxybenzoic acid (4-HBA), 3,4-diHBA, or 3-HPPA interfere with α-syn spreading in a cell-based system. Using HEK293 cells overexpressing α-syn-A53T-CFP/YFP, we assessed α-syn seeding activity using Fluorescence Resonance Energy Transfer (FRET) to detect and quantify α-syn aggregation. We demonstrated that 3-HPPA, 3,4-diHBA, 3-HBA, and 4-HBA significantly attenuated intracellular α-syn seeding aggregation. To determine whether our compounds could inhibit brain-derived seeding activity, we utilized insoluble α-syn extracted from post-mortem MSA or PD brain specimens. We found that 3-HPPA effectively attenuated MSA-induced aggregation of monomer into high molecular weight aggregates capable of inducing intracellular aggregation. Outcomes from our studies suggest interactions between gut microbiome and certain dietary factors may form the basis for effective therapies that modulate pathologic α-syn propagation. Collectively, our findings provide the basis for future developments of probiotic, prebiotic, or synbiotic approaches for modulating the onset and/or progression of α-synucleinopathies.

Physico-chemical, structural and techno-functional properties of gelatin from saithe (Pollachius virens) skin

Gelatin from saithe (Pollachius virens) skin, with high mineral content, was characterized and compared to marine gelatin from the market. As expected, gelatin extracted from the saithe skin had a high mineral content (>20%), compared to commercial gelatin (<0.2%). SDS-PAGE analysis revealed the presence of α-chains, β-chains and other high molecular weight aggregates. The absorption bands of gelatin in FTIR spectra were mainly situated in the amide band region (amide A, amide B, amide I, amide II and amide III), whereas DSC thermograms showed typical gelatin behavior. Interesting viscoelastic and higher foaming properties were observed for the saithe gelatins, compared to the market gelatins, probably due to the presence of high sea mineral content. The potential applications in food and cosmetic area of gelatin with high mineral content are presented and discussed in the conclusions.

Effect of hydrocolloids on structural and functional properties of wheat/potato (50/50) flour dough

Abstract Physicochemical analyses were performed to study the effect of hydrocolloids on wheat/potato (50/50) flour doughs. Pectin, hydroxypropilmethylcellulose, Arabic gum and konjac glucomannan were tested at 1.5 g/100 g (flour basis). Textural and rheological assays indicated great differences between the control dough (100 % wheat flour) and doughs containing wheat/potato flour (50/50). These differences were reduced when hydrocolloids were added with Arabic gum and hydroxypropilmethylcellulose being the gums that better mimic the control dough properties. The analysed dough showed a bimodal distribution of relaxation times indicating that more than one molecular mechanism was responsible for relaxation. The presence of potato flour led to a marked decrease in relaxation times indicating a faster decay of stress as a function of time. The presence of gums probably increased relaxation times by establishing stronger non-covalent interaction with the dough components, rendering high molecular weight aggregates, which slowed down relaxation behaviour. Differential scanning calorimetry results showed that potato flour did not significantly changed the gelatinization temperature, but the addition of hydrocolloids shifted this parameter to higher values. Samples containing gums broadened the endotherm making the process slower and more energy demanding. The microstructure was studied at different levels (optical, confocal and scanning electron microscopy) and it was observed that potato flour made the microstructure smoother and more uniform. On the other hand, the addition of hydrocolloids led to rougher structures. These observations were confirmed by modelling image texture with different algorithms such as grey level co-occurrence matrix, fractal dimension and lacunarity.

Revisiting CB1 cannabinoid receptor detection and the exploration of its interacting partners

BackgroundCannabinoid receptor 1 (CB1) identification by western blot (WB) has generated a great deal of controversial data making the interpretation of the results difficult. Our purpose is to find the most adequate experimental conditions to detect CB1 by WB and immunoprecipitation (IP) as a first step towards the study of CB1 interactome. New methodWe use CB1 knockout mice tissue as negative controls and describe appropriate sample handling conditions for CB1 detection by WB and IP from brain and cortical neuron cultures. ResultsSample heating above 65 °C greatly impaired CB1 detection by WB, since it favored the formation of high molecular weight aggregates. We also show the convenience of using n-dodecyl-β-d-maltoside (DDM) as a detergent for the detection of CB1 by WB and, mostly, for IP. Comparison with existing method(s)We obtain consistent and specific CB1 detection by WB and IP using four different commercial antibodies and KO tissue for an accurate CB1 identification. We clarify the identification of the receptor in complex samples compared with the diverse and unclear results obtained using standard WB methods. ConclusionsWe establish experimental guidelines for the detection of CB1 by WB and the study of CB1 interacting proteins by IP. We propose a new interpretation of CB1 WB and IP data based on the folding and packing state of the protein and the detergent used. The standardization of the most advantageous conditions for coimmunoprecipitation (CoIP) would be a useful tool for the future study of the interactome of CB1.

Comprehensive biophysical and functional study of ziv-aflibercept: characterization and forced degradation

Aflibercept (AFL) is an Fc fusion protein used in the treatment of colorectal cancers and different ophthalmological diseases. There are two medicines in which AFL is the active substance: Zaltrap and Eylea, referred as ziv-AFL and AFL respectively. No proper accelerated degradation studies were published on either AFL or ziv-AFL. These studies are essential during research, development and manufacturing stages. Here, we characterized ziv-AFL and submitted it to different stress conditions: light, 60 °C, freeze-thaw cycles, changes in pH, high hypertonic solution and strong denaturing conditions. We used an array of techniques to detect aggregation (SE-HPLC/DAD and DLS), changes in secondary structure (Far-UV circular dichroism), changes in conformation or tertiary structure (Intrinsic tryptophan fluorescence) and alterations in functionality (ELISA). Results indicate that aggregation is common degradation pathway. Two different types of aggregates were detected: dimers and high molecular weight aggregates attributed to β-amyloid-like structures. Secondary structure was maintained in most of the stress tests, while conformation was altered by almost all the tests except for the freeze-thaw cycles. Functionality, evaluated by its immunochemical reaction with VEGF, was found to be stable but with decrease when exposed to light and with likely partial inactivation of the drug when pH was altered.

Impact of industrial cream heat treatments on the protein composition of the milk fat globule membrane.

The impact of cream processing on milk fat globule membrane (MFGM) was assessed in an industrial setting for the first time. Three creams and their derived MFGM fractions from different stages of the pasteurization procedure at a butter dairy were investigated and compared to a native control as well as a commercial MFGM fraction. The extent of cross-linking of serum proteins to MFGM proteins increased progressively with each consecutive pasteurization step. Unresolved high molecular weight aggregates were found to consist of both indigenous MFGM proteins and β-lactoglobulin as well as αs1- and β-casein. With regards to fat globule stability and in terms of resistance towards coalescence and flocculation after cream washing, single-pasteurized cream exhibited reduced sensitivity to cream washing compared to non- and double-pasteurized creams. Inactivation of the agglutination mechanism and the increased presence of non-MFGM proteins may determine this balance between stable and non-stable fat globules.

Chlorpyrifos Oxon-Induced Isopeptide Bond Formation in Human Butyrylcholinesterase.

A newly recognized action of organophosphates (OP) is the ability to crosslink proteins through an isopeptide bond. The first step in the mechanism is covalent addition of the OP to the side chain of lysine. This activates OP-lysine for reaction with a nearby glutamic or aspartic acid to make a gamma glutamyl epsilon lysine bond. Crosslinked proteins are high molecular weight aggregates. Our goal was to identify the residues in the human butyrylcholinesterase (HuBChE) tetramer that were crosslinked following treatment with 1.5 mM chlorpyrifos oxon. High molecular weight bands were visualized on an SDS gel. Proteins in the gel bands were digested with trypsin, separated by liquid chromatography and analyzed in an Orbitrap mass spectrometer. MSMS files were searched for crosslinked peptides using the Batch-Tag program in Protein Prospector. MSMS spectra were manually evaluated for the presence of ions that supported the crosslinks. The crosslink between Lys544 in VLEMTGNIDEAEWEWK544AGFHR and Glu542 in VLEMTGNIDEAEWE542WK satisfied our criteria including that of spatial proximity. Distances between Lys544 and Glu542 were 7.4 and 9.5 Å, calculated from the cryo-EM (electron microscopy) structure of the HuBChE tetramer. Paraoxon ethyl, diazoxon, and dichlorvos had less pronounced effects as visualized on SDS gels. Our proof-of-principle study provides evidence that OP have the ability to crosslink proteins. If OP-induced protein crosslinking occurs in the brain, OP exposure could be responsible for some cases of neurodegenerative disease.

Understanding the removal of V, Ni and S in crude oil atmospheric residue hydrodemetallization and hydrodesulfurization

This study describes the use of gel permeation chromatography inductively coupled plasma high-resolution mass spectrometry (GPC ICP HRMS) to examine and explain two important petroleum industry catalytic processes: hydrodemetallization (HDM) and hydrodesulfurization (HDS).The sulfur, nickel and vanadium species size distributions in atmospheric residue fractions were studied to track their evolution during both catalytic processes and examine their mechanisms, especially those mechanisms linked to changes in temperature and initial deactivation via coke laydown. Chromatogram shapes as well as peak areas were used to study the V, Ni and S aggregate types and concentrations in the feedstock as well as in the product after varying the operating temperature and residence time at a constant temperature. For the V and Ni compounds, the low and medium molecular weight (LMW and MMW, respectively) aggregates are easily hydrotreated under all conditions, while the high molecular weight (HMW) compounds are more refractory. For the S compounds, a different reactivity pattern was observed whereby the LMW, MMW and HMW aggregates were more similar in their reactivity, showing that the catalyst is less selective towards a certain aggregate size for the S compounds compared to the V or Ni compounds.

Thermal Inactivation of a Cold-Active Esterase PMGL3 Isolated from the Permafrost Metagenomic Library.

PMGL3 is a cold-adapted esterase which was recently isolated from the permafrost metagenomic library. It exhibits maximum activity at 30 °C and low stability at elevated temperatures (40 °C and higher). Sequence alignment has revealed that PMGL3 is a member of the hormone-sensitive lipase (HSL) family. In this work, we demonstrated that incubation at 40 °C led to the inactivation of the enzyme (t1/2 = 36 min), which was accompanied by the formation of tetramers and higher molecular weight aggregates. In order to increase the thermal stability of PMGL3, its two cysteines Cys49 and Cys207 were substituted by the hydrophobic residues, which are found at the corresponding positions of thermostable esterases from the HSL family. One of the obtained mutants, C207F, possessed improved stability at 40 °C (t1/2 = 169 min) and increased surface hydrophobicity, whereas C49V was less stable in comparison with the wild type PMGL3. Both mutants exhibited reduced values of Vmax and kcat, while C207F demonstrated increased affinity to the substrate, and improved catalytic efficiency.

Modification of Aβ Peptide Aggregation via Covalent Binding of a Series of Ru(III) Complexes.

Alzheimer's disease (AD) is the most common form of dementia, leading to loss of cognition, and eventually death. The disease is characterized by the formation of extracellular aggregates of the amyloid-beta (Aβ) peptide and neurofibrillary tangles of tau protein inside cells, and oxidative stress. In this study, we investigate a series of Ru(III) complexes (Ru-N) derived from NAMI-A in which the imidazole ligand has been substituted for pyridine derivatives, as potential therapeutics for AD. The ability of the Ru-N series to bind to Aβ was evaluated by NMR and ESI-MS, and their influence on the Aβ peptide aggregation process was investigated via electrophoresis gel/western blot, TEM, turbidity, and Bradford assays. The complexes were shown to bind covalently to the Aβ peptide, likely via a His residue. Upon binding, the complexes promote the formation of soluble high molecular weight aggregates, in comparison to peptide precipitation for peptide alone. In addition, TEM analysis supports both amorphous and fibrillar aggregate morphology for Ru-N treatments, while only large amorphous aggregates are observed for peptide alone. Overall, our results show that the Ru-N complexes modulate Aβ peptide aggregation, however, the change in the size of the pyridine ligand does not substantially alter the Aβ aggregation process.

Influence of low-molecular-weight aggregates on aggregate growth kinetics and physical properties of solid-state proteins during storage

Low-molecular-weight (LMW) aggregate is a critical determinant of subsequent protein aggregation, but the aggregate growth kinetics of solid proteins have not been fully characterized. In this study, the high-molecular weight (HMW) aggregate formation process for solid-state proteins and the relationship between aggregation and physical properties of tablets were evaluated using proteins with various initial aggregate ratios. Quantitative changes in monomers, LMW aggregates, and HMW aggregates during storage were measured. The monomer amount decreased uniformly for all proteins. Proteins with low initial LMW aggregates showed remarkable increases in LMW aggregates but little increases in HMW aggregates during storage. Proteins with high initial LMW aggregates showed decreases in LMW aggregates but remarkable increases in HMW aggregates. A correlation analysis and logistic regression indicated that HMW aggregate formation depended on the initial quantity of LMW aggregates. Furthermore, the initial LMW aggregate ratio was related to the disintegratability of protein-containing tablets after storage. These results provide novel insight into solid-state protein aggregation and may guide the prediction of the long-term quality of solid protein-containing pharmaceuticals and foods without storage.

Biology of Inherited Cataracts and Opportunities for Treatment.

Cataract, the clinical correlate of opacity or light scattering in the eye lens, is usually caused by the presence of high-molecular-weight (HMW) protein aggregates or disruption of the lens microarchitecture. In general, genes involved in inherited cataracts reflect important processes and pathways in the lens including lens crystallins, connexins, growth factors, membrane proteins, intermediate filament proteins, and chaperones. Usually, mutations causing severe damage to proteins cause congenital cataracts, while milder variants increasing susceptibility to environmental insults are associated with age-related cataracts. These may have different pathogenic mechanisms: Congenital cataracts induce the unfolded protein response and apoptosis. By contrast, denatured crystallins in age-related cataracts are bound by α-crystallin and form light-scattering HMW aggregates. New therapeutic approaches to age-related cataracts use chemical chaperones to solubilize HMW aggregates, while attempts are being made to regenerate lenses using endogenous stem cells to treat congenital cataracts.

The l-isoaspartate modification within protein fragments in the aging lens can promote protein aggregation

Transparency in the lens is accomplished by the dense packing and short-range order interactions of the crystallin proteins in fiber cells lacking organelles. These features are accompanied by a lack of protein turnover, leaving lens proteins susceptible to a number of damaging modifications and aggregation. The loss of lens transparency is attributed in part to such aggregation during aging. Among the damaging post-translational modifications that accumulate in long-lived proteins, isomerization at aspartate residues has been shown to be extensive throughout the crystallins. In this study of the human lens, we localize the accumulation of l-isoaspartate within water-soluble protein extracts primarily to crystallin peptides in high-molecular weight aggregates and show with MS that these peptides are from a variety of crystallins. To investigate the consequences of aspartate isomerization, we investigated two αA crystallin peptides 52LFRTVLDSGISEVR65 and 89VQDDFVEIH98, identified within this study, with the l-isoaspartate modification introduced at Asp58 and Asp91, respectively. Importantly, whereas both peptides modestly increase protein precipitation, the native 52LFRTVLDSGISEVR65 peptide shows higher aggregation propensity. In contrast, the introduction of l-isoaspartate within a previously identified anti-chaperone peptide from water-insoluble aggregates, αA crystallin 66SDRDKFVIFL(isoAsp)VKHF80, results in enhanced amyloid formation in vitro The modification of this peptide also increases aggregation of the lens chaperone αB crystallin. These findings may represent multiple pathways within the lens wherein the isomerization of aspartate residues in crystallin peptides differentially results in peptides associating with water-soluble or water-insoluble aggregates. Here the eye lens serves as a model for the cleavage and modification of long-lived proteins within other aging tissues.

Cation exchange frontal chromatography for the removal of monoclonal antibody aggregates

A low ligand density cation exchange (CEX) chromatography resin, Eshmuno® CP-FT resin, was investigated for the removal of aggregates from monoclonal antibody (mAb) feeds using a continuous loading process. Removing mAb aggregates with a CEX resin using continuous loading is advantageous relative to a bind/elute loading process, because the resin can use nearly its full capacity to bind the aggregates enabling much higher loadings. The removal of mAb aggregates with Eshmuno® CP-FT resin using a continuous loading process was found to be consistent with a frontal chromatography mechanism where the mAb monomer initially binds to the column and is subsequently displaced by dimers and higher molecular weight aggregates. The removal of mAb aggregates with Eshmuno® CP-FT resin using a continuous loading process was compared with six other commercially available strong CEX chromatography resins and found to correlate with their ionic densities, but not their mAb static binding capacities. The influence of pH, conductivity, residence time, and mAb concentration on the removal of aggregates with Eshmuno® CP-FT resin using a continuous loading process was also investigated. Finally, the percentage of aggregates in a mAb feed was varied to examine the effect on the removal of aggregates with Eshmuno® CP-FT resin using a continuous loading process.

Pathological Tau From Alzheimer’s Brain Induces Site-Specific Hyperphosphorylation and SDS- and Reducing Agent-Resistant Aggregation of Tau in vivo

Neurofibrillary tangles (NFTs) made up of hyperphosphorylated tau are a histopathological hallmark of Alzheimer’s disease (AD) and related tauopathies. Hyperphosphorylation of tau is responsible for its loss of normal physiological function, gain of toxicity and its aggregation to form NFTs. Injection of misfolded tau seeds into mouse brain induces tau aggregation, but the nature of tau phosphorylation in pathologic tau seeded pathology is unclear. In the present study, we injected hyperphosphorylated and oligomeric tau isolated from AD brain (AD P-tau) into hippocampus of human tau transgenic mice and found that in addition to tau aggregation/pathology, tau was hyperphosphorylated at Ser202/Thr205, Thr212, Ser214, Thr217, Ser262, and Ser422 in AD P-tau injected hippocampus and at Ser422 in the contralateral hippocampus and in the ipsilateral cortex. AD P-tau-induced AD-like high molecular weight aggregation of tau that was SDS- and reducing agent-resistant and site-specifically hyperphosphorylated in the ipsilateral hippocampus. There were no detectable alterations in levels of tau phosphatases or tau kinases in AD P-tau-injected brains. Furthermore, we found that hyperphosphorylated tau was easier to be captured by AD P-tau and that aggregated tau was more difficult to be dephosphorylated than the non-aggregated tau by protein phosphatase 2A (PP2A). Based on these findings, we speculate that AD P-tau seeds hyperphosphorylated tau to form aggregates, which resist to the dephosphorylation by PP2A, resulting in hyperphosphorylation and pathology of tau.

Kinetic Stability of Long-Lived Human γ-D and γS Lens Crystallins, Derived in Part from their Domain Interfaces, May Protect against Cataract

The γ-crystallins of the eye lens, are among the longest-lived proteins in the human body. Synthesized in utero, they must remain folded and soluble throughout adulthood to maintain lens transparency and avoid cataracts. All vertebrate crystallin structures exhibit homologous duplicated Greek Key β-sheet domains, presumably representing gene duplication earlier in their evolution. γD- and γS-crystallin are two major monomeric crystallins of the human lens. Oxidative damage to the crystallins leads to partial unfolding followed by polymerization to high molecular weight aggregates which scatter light causing cataract disease. The kinetic stabilities of the complete γD- and γS-crystallins and their respective isolated N-terminal and C-terminal domains were determined and compared. Kinetic unfolding experiments were performed in different concentrations of GdnHCl and monitored by fluorescence spectroscopy. Kinetic rate constants and half-lives of the crystallins were calculated using linear extrapolation to define the unfolding parameters in the absence of denaturant. The extrapolated t1/2 for the initial unfolding step of γD-crystallin was ∼19 years. The extrapolated t1/2 for γS-crystallin was not as long, ∼1.6 years, though still very long. The unfolding kinetic half-lives of each of the four isolated domains were much shorter than their respective full-length duplicated parent proteins. The simplest interpretation is that the domain interface is the barrier to initiation of unfolding and source of the high kinetic stability. Given the propensity of partially folded crystallins to aggregate at the high protein concentrations present in the lens, the high kinetic stability of the crystallins would protect the lens from the initiation of aggregation reactions.