Degree Of Protein Aggregation Research Articles

Protein Corona Formation and Aggregation of Amyloid β 1-40-Coated Gold Nanocolloids.

Amyloid fibrillogenesis is a pathogenic protein aggregation process that occurs through a highly ordered process of protein-protein interactions. To better understand the protein-protein interactions involved in amyloid fibril formation, we formed nanogold colloid aggregates by stepwise additions of ∼2 nmol of amyloid β 1-40 peptide (Aβ1-40) at pH ∼3.7 and ∼25 °C. The processes of protein corona formation and building of gold colloid [diameters (d) of 20 and 80 nm] aggregates were confirmed by a red-shift of the surface plasmon resonance (SPR) band, λpeak, as the number of Aβ1-40 peptides [N(Aβ1-40)] increased. The normalized red-shift of λpeak, Δλ, was correlated with the degree of protein aggregation, and this process was approximated as the adsorption isotherm explained by the Langmuir-Freundlich model. As the coverage fraction (θ) was analyzed as a function of ϕ, which is the N(Aβ1-40) per total surface area of nanogold colloids available for adsorption, the parameters for explaining the Langmuir-Freundlich model were in good agreement for both 20 and 80 nm gold, indicating that ϕ could define the stage of the aggregation process. Surface-enhanced Raman scattering (SERS) imaging was conducted at designated values of ϕ and suggested that a protein-gold surface interaction during the initial adsorption stage may be dependent on the nanosize. The 20 nm gold case seems to prefer a relatively smaller contacting section, such as a -C-N or C═C bond, but a plane of the benzene ring may play a significant role for 80 nm gold. Regardless of the size of the particles, the β-sheet and random coil conformations were considered to be used to form gold colloid aggregates. The methodology developed in this study allows for new insights into protein-protein interactions at distinct stages of aggregation.

Effect of high-temperature drying at different moisture levels on texture of dried noodles: Insights into gluten aggregation and pore distribution

In this study, the impacts of high-temperature drying (80 °C) at varying moisture levels HT1 (30%) and HT2 (12%) on texture of dried noodles were investigated by considering the protein aggregation and pore distribution in both dried and cooked noodles. In comparison to medium-temperature drying (45 °C), an increase in protein aggregation was observed in dried noodles obtained under HT1. Additionally, HT1 conditions led to uneven gluten network and significant variations in pore size, which ultimately reduced the bending resistance of dried noodles from 9.45% to 31.82%. Upon cooking, the protein in noodles from HT1 underwent slight depolymerization, resulting in lower protein aggregation as compared to noodles from HT2, which negatively affected the edible quality of products from HT1. Our findings demonstrated that dried noodle qualities under HT2 increased up to 32.73% compared to medium-temperature drying and HT1, due to suitable protein aggregation and smaller pore distribution associated with HT2. Finally, the degree of protein aggregation in cooked noodles is a more accurate quality indicator than analyzing dried noodles.

A dual-functional probe that allows cascade response to hydrogen peroxide oxidative stress-induced protein aggregation in live cells

The misfolding and aggregation of specific proteins induced by endogenous oxidation are closely related to some incurable diseases including cancers, diabetes and neurodegenerative diseases. Reactive oxygen species (ROS), as a common oxidizing substance in vivo would induce the oxidation of endogenous biomacromolecules like proteins. However, it remains a challenge to detect protein aggregation induced by specific ROS. Herein, we demonstrate that the developed probe (P2) with a dual function that can simultaneously detect hydrogen peroxide (H2O2) levels and protein aggregates formed by oxidative stress. In this probe, the boronic acid pinacol ester group was conjugated to fluorescent molecular rotor 4-hydroxybenzylidene-imidazolinone (HBI) and served as the H2O2 detection group. Experimental results show that the fluorescence of P2 can be activated by H2O2 and exhibited a positive correlation with the degree of protein aggregation. Moreover, combined with AggTag method P2 was successfully applied to dual-channel imaging of protein aggregation that is stressed by H2O2 in cellular. In general, this work not only provides a potential method to study protein aggregation that associates with oxidation in live cells but also can be generally applied to study other biological processes.

Abstract P2073: Hyperbaric Oxygen Therapy Protects The Myocardium From Reductive Stress-induced Proteotoxic Remodeling

Background: Oxygen is the prime source of reactive oxygen species (ROS) in cells. We hypothesize that hyperbaric oxygen therapy (HBOT) can be used as an effective intervention to maintain redox homeostasis in the myocardium. Here, we assessed whether HBOT could prevent RS through enhanced ROS signaling, thereby protecting the hearts from proteotoxic stress. Methods: Cardiac-specific human Nrf2 transgenic (hNrf2-TG) and their non-transgenic littermates (NTG) were used in this study. Mice at the age of 8 weeks were exposed to hyperbaric oxygen (2.5 Bars, 2 hr/day) for 8 weeks. The TG and NTG (n=6) mice without HBOT served as controls. Pre- and post- echocardiography (Vevo 2100) analysis were carried out to assess the structural and functional changes over time in response to HBOT exposure. The influence of HBOT on matrixins (MMP2/9), redox (GSH & NQO1), and cardiac hypertrophy markers (ANF, BNF) were examined using zymography, immunoblotting, and qPCR analysis. The aggregation of proteins in myocardial tissues was detected by Proteostat staining. Results: While the expression of hNrf2 induces RS in the mouse heart, we noticed that HBOT ameliorates the RS through enhanced ROS signaling. A decrease in systemic GSH levels along with the downregulation of ANF and BNF mRNA levels observed in the HBOT group indicates the restoration of the redox homeostasis as well as reduced remodeling of the myocardium. Interestingly, hNRF2-induced RS was suppressed by HBOT as supported by changes in the protein levels for NQO1, GCLC, and GCLM. The degree of protein aggregation in RS hearts was significantly reduced by HBOT. Vevo strain analysis reveals that 8 weeks of HBOT restored the diastolic stretch seen in the RS hearts to a normal diastole-systole velocity equal to NTG mice. A significant decrease in the ejection fraction (80% vs. 55% in TG vs. TG-HBOT), was evident in the TG mice that received HBOT. Repression of matrixins, observed in the hearts of HBOT-TG mice signifies the benefits of HBOT in RS hearts. Conclusion: HBOT directly improves myocardial structure at the tisuue level; maintain the redox status at systemic level, and improves the myocardial performance at functional level. Our study established that HBOT can rescue the myocardium from RS-induced proteotoxic insults.

Preparation, Characterization and Antioxidant Activity of Glycosylated Whey Protein Isolate/Proanthocyanidin Compounds.

A glycosylated protein/procyanidin complex was prepared by self-assembly of glycosylated whey protein isolate and proanthocyanidins (PCs). The complex was characterized through endogenous fluorescence spectroscopy, polyacrylamide gel electrophoresis, Fourier infrared spectroscopy, oil-water interfacial tension, and transmission electron microscopy. The results showed that the degree of protein aggregation could be regulated by controlling the added amount of procyanidin, and the main interaction force between glycosylated protein and PCs was hydrogen bonding or hydrophobic interaction. The optimal binding ratio of protein:PCs was 1:1 (w/w), and the solution pH was 6.0. The resulting glycosylated protein/PC compounds had a particle size of about 119 nm. They exhibited excellent antioxidant and free radical-scavenging abilities. Moreover, the thermal denaturation temperature rose to 113.33 °C. Confocal laser scanning microscopy (CLSM) images show that the emulsion maintains a thick interface layer and improves oxidation resistance with the addition of PCs, increasing the application potential in the functional food industry.

Understanding the influence of curdlan on the quality of frozen cooked noodles during the cooking process

The aim of this study was to clarify the influence of curdlan on the quality of frozen cooked noodles (FCN) by exploring the changes in textural properties, color profile, thermal stability, and micromorphology of FCN during cooking. Herein, an improvement in hardness, springiness, and tensile fracture distance was observed after adding 0.5% of curdlan, from 2876.82 ± 14.59 to 3371.08 ± 64.75 g, 0.85 ± 0.01 to 0.91 ± 0.02, and 90.60 ± 0.79 to 94.29 ± 1.37 mm, respectively. Combined with the decrease in weight loss and total color difference, from 9.01 ± 0.25 to 7.03 ± 0.07 and 76.00 ± 0.16 to 75.71 ± 0.24%, respectively, it can be inferred that the improved quality of FCN was mainly attributed to the stable gluten network mediated by curdlan. Moreover, results of micromorphology demonstrated that FCN with 0.5% curdlan had higher protein percentage area (35.20 ± 0.32%), junction density (5.97 ± 0.09 × 10−4), total protein length (35733.97 ± 456.84 μm), and degree of protein aggregation than those of FCN without curdlan, confirming that curdlan helped in maintaining the stability of gluten network and inhibited the depolymerization of gluten proteins induced by excessive cooking.

Ice crystal recrystallization inhibition of type I antifreeze protein, type III antifreeze protein, and antifreeze glycoprotein: effects of AF(G)Ps concentration and heat treatment.

This study compared ice recrystallization behaviors of frozen dessert model systems containing type I antifreeze protein (AFP I), type III antifreeze protein (AFP III), and antifreeze glycoprotein (AFGP) at -10 °C. Specifically, effects of AF(G)P concentration and heat treatment (95 °C for 10 min) were examined. The concentration dependence of the ice recrystallization rate constant reasonably well fit a sigmoidal function: the fitting procedure was proposed, along with cooperative coefficient α,and a new index of AF(G)P ice recrystallization inhibition (IRI) activity (C50). After 95 °C heat treatment for 10 min, AFP III lost its ice crystal recrystallization inhibitory activity the most: AFP I was less affected; AFGP was almost entirely unaffected. These different thermal treatment effects might reflect a lower degree of protein aggregation because of hydrophobic interaction after heat treatment or might reflect the simplicity and flexibility of the higher order structures of AFP I and AFGP.

The inhibitory effects of natural antioxidants on protein glycation as well as aggregation induced by methylglyoxal and underlying mechanisms

The non-enzymatic glycation of protein can result in the formation of advanced glycation end-products (AGEs), leading to the deposition of amyloid proteins, and it’s essential for the pathogenesis of diabetes complications and amyloid diseases. Reactive dicarbonyl compounds, such as methylglyoxal (MGO), are one of the most reactive glycating agents. Therefore, it’s crucially necessary to inhibit protein glycation and aggregation induced by MGO. In the present study, we aimed to systemically investigate the anti-glycation and anti-fibrillization activities of eight natural antioxidants, including apigenin, quercetin (Que), catechin, resveratrol (Res), and gallic acid (GA), L-ascorbic acid (L-AA), limonene, and β-carotene, during MGO-induced protein glycation and aggregation. Furthermore, the underlying mechanisms were clarified. The formation of AGEs and the degree of protein aggregation were characterized by optical detection, flow cytometry, and so on. The results demonstrated that eight selected natural antioxidants could inhibit glycation and protein aggregation induced by MGO via the synergy of scavenging free radicals, capturing MGO, and interacting with proteins, among which GA (300 μM) and Res (15 μM) had higher inhibition rates on both argpyrimidine (specific fluorescent AGEs, 17% and 22%, respectively) and protein amyloid aggregation (42% and 29%, respectively). These findings suggested that antioxidants could act as potential inhibitors of AGEs and glycation-induced protein aggregation, which were expected to become a new strategy for the prevention and treatment of diabetes and amyloid diseases. Besides, these inhibition mechanisms provided valuable insights into the design and development of candidate drugs for the prevention and treatment of AGEs and protein aggregation-related diseases.

Insights on lysozyme aggregation in protic ionic liquid solvents by using small angle X-ray scattering and high throughput screening

Solvents play a crucial role in protein instability and aggregation, which are undesirable in biomedical applications. The development of quantitative, high throughput methods of evaluating protein aggregation is important in the search for novel solvent mixtures/systems, such as ionic liquids (ILs). In this study, three different small-angle X-ray scattering setups were employed to determine lysozyme size, shape, and aggregation in IL-water mixtures, including a high throughput plate approach, capillary autoloader, and size-exclusion setup, in order of increasing measurement time and scattering data quality. Protein size and shape were determined in low concentrations of ethylammonium nitrate, and the degree of protein aggregation was evaluated by a pseudo-radius of gyration (pseudo-Rg). The short collection time, and wide viscosity tolerance render the plate setup ideal for high throughput screening of protein aggregation in complex IL systems. We then investigated the effect of five additives, known to stabilize proteins, on lysozyme in 144 different IL-water mixtures. We showed that lysozyme lost its shape and had increased aggregation with over 17 mol% IL present, though lysozyme was less aggregated in ethylammonium nitrate (EAN) and ethanolammonium formate (EtAF) solutions. Further, lysozyme aggregation was slightly suppressed by glycerol in EAN-water mixtures, and by trimethylamine N-oxide in EtAF and ethylammonium formate aqueous solutions. This work develops a new quantitative and high throughput approach for evaluating protein aggregation in solvent systems, including viscous solvents.

The improvement of gel and physicochemical properties of porcine myosin under low salt concentrations by pulsed ultrasound treatment and its mechanism

The effects of pulsed ultrasound treatment (250 W, 0–12 min) on gel and physicochemical properties of porcine myosin at low-salt group (0.3 mol/L) and control groups (0.6 and 0.9 mol/L) were investigated. The texture and water holding capacity (WHC) of low-salt group gel were remarkably lower than in medium-salt (0.6 mol/L) and high-salt group (0.9 mol/L). However, 6-min ultrasound treatment could obviously improve the texture and WHC of low-salt group gel. After ultrasound treatment, the protein solubility was increased, as the degree of protein aggregation was reduced. Simultaneously, ultrasound treatment led to unfolding of protein structure and increasing surface hydrophobicity. The three-dimensional network of myosin gel gradually became uniform by 6-min ultrasound treatment. Under 12-min ultrasound treatment, the protein aggregated excessively during the gelation, which led to the deterioration of gel quality. These results suggested that moderate ultrasound treatment is promising to be used to enhance the quality of salt-reduced meat products.

The Role of Surface Properties on Protein Aggregation Behavior in Aqueous Solution of Different pH Values.

This study aimed to investigate the effect of pH-mediated surface properties of bovine serum albumin (BSA) on protein aggregation and the changes of protein structure and colloidal stability at different solution pH levels. The hydrophobicity of BSA surface was characterized by endogenous fluorescence spectroscopy, fluorescence quenching of acrylamide, and fluorescence probe. The results showed that the hydrophobicity of BSA surface was similar at pH5, 6, 7.4, followed by pH4, 8, 9, 10, and finally by pH3 and 11 with strong acidity and alkalinity. The positive charge on the BSA surface was increased gradually with the decrease of solution pH, while the negative charge on protein surface was increased gradually with the increase of solution pH. The degree of protein aggregation was examined by turbidimetry, flow cytometry, and SDS-PAGE. The results showed that the oscillating aggregation of BSA did not change with the solution pH, but was partially dependent on the relative contribution of electrostatic and hydrophobic interactions between the protein molecules. In addition, the secondary structure, conformational stability, unfolding degree, and colloidal stability of proteins were investigated by circular dichroism, fluorescence spectroscopy, protein pulse hydrolysis, and dynamic light scattering, respectively. The results suggested that the solution pH could change the structure and stability of the protein at different levels. Solution pH has distinct effects on the structural stability of protein at different levels. The change of protein surface properties mediated by solution pH is related to protein aggregation.

Influence of extreme alkaline pH induced unfolding and aggregation on PSE-like chicken protein edible film formation

The mechanisms of the unfolding and aggregation of PSE-like chicken meat protein at extreme alkaline pHs (pH 11.0, 11.5 and 12.0) were studied and related to the physical properties of edible films. Results of asymmetrical flow field-flow fractionation coupled with various detectors showed that pH 12.0 leads to a certain degree of protein aggregation, which could also be observed in atomic force microscopy (AFM) images. Higher levels of protein denaturation and unfolding, as proved by the circular dichroism spectrum, could result in aggregates formation, whereas all pH-adjusted groups exhibited similar flow behavior. The pH 11.0 sample exhibited films with higher water resistance compared to others. Comparatively, solubilizing PSE-like meat protein at pH 11.5 could result in better edible films with greater mechanical properties, water resistance and transparency. Our results elucidated the effects of the PSE-like chicken meat protein unfolding and aggregation on edible film properties under various extreme alkaline pH conditions.

A Kinase Anchor Protein 4 Is Vulnerable to Oxidative Adduction in Male Germ Cells.

Oxidative stress is a leading causative agent in the defective sperm function associated with male infertility. Such stress commonly manifests via the accumulation of pathological levels of the electrophilic aldehyde, 4-hydroxynonenal (4HNE), generated as a result of lipid peroxidation. This highly reactive lipid aldehyde elicits a spectrum of cytotoxic lesions owing to its propensity to form stable adducts with biomolecules. Notably however, not all elements of the sperm proteome appear to display an equivalent vulnerability to 4HNE modification, with only a small number of putative targets having been identified to date. Here, we validate one such target of 4HNE adduction, A-Kinase Anchor Protein 4 (AKAP4); a major component of the sperm fibrous sheath responsible for regulating the signal transduction and metabolic pathways that support sperm motility and capacitation. Our data confirm that both the precursor (proAKAP4), and mature form of AKAP4, are conserved targets of 4HNE adduction in primary cultures of post-meiotic male germ cells (round spermatids) and in mature mouse and human spermatozoa. We further demonstrate that 4HNE treatment of round spermatids and mature spermatozoa results in a substantial reduction in the levels of both proAKAP4 and AKAP4 proteins. This response proved refractory to pharmacological inhibition of proteolysis, but coincided with an apparent increase in the degree of protein aggregation. Further, we demonstrate that 4HNE-mediated protein degradation and/or aggregation culminates in reduced levels of capacitation-associated phosphorylation in mature human spermatozoa, possibly due to dysregulation of the signaling framework assembled around the AKAP4 scaffold. Together, these findings suggest that AKAP4 plays an important role in the pathophysiological responses to 4HNE, thus strengthening the importance of AKAP4 as a biomarker of sperm quality, and providing the impetus for the design of an efficacious antioxidant-based intervention strategy to alleviate sperm dysfunction.

Application of disaccharides alone and in combination, for the improvement of stability and particle properties of spray-freeze dried IgG

Purpose: Spray-freeze drying (SFD) is a recently applied method to develop pharmaceutical powders. This study aimed to analyze the competence of Trehalose, Mannitol, Lactose, and Sorbitol instability and aerosolization of Immunoglobulin G (IgG) via SFD.Methods: Induced soluble aggregates were quantified at 0 and 3 months, and 45 °C using size-exclusion chromatography. Conformation and thermogravimetric assessments were done by Fourier transform infrared spectroscopy and differential scanning calorimetry. Laser light scattering was performed to determine the particle sizes. Aerodynamic features were characterized by twin stage impinger and scanning electron microscopy.Results: Although sugars/polyols preferably stabilized IgG following the process, storage stabilization was achieved in Trehalose, Trehalose-Lactose, Lactose, and Trehalose-Mannitol-based powders with soluble aggregates <5%. The conformation of antibody was preserved with β sheet content from 66.28% to 76.37%. Particle sizes ranged from 5.23 to 8.12 µm. Mannitol exhibited the best aerodynamic behavior, fine particle fraction (FPF: 70%) but high degree of protein aggregation during storage.Conclusions: SFD could favorably stabilize antibody using Trehalose and its combination with Lactose and Mannitol, and also, Lactose alone. Sorbitol disturbed IgG powder recovery. Incorporation of other types of excipient is required for efficient respiratory delivery of IgG molecules.

Proteolysis process in fermented sausage model systems as studied by NMR relaxometry.

Proton NMR relaxation analyses were performed in sausage model systems (SMS) at different manufacturing times (0, 1, 3, 5, 7, and 9 days) to evaluate changes in water distribution and mobility. Three different water populations were identified, T2b (5-10 ms), T21 (30-70 ms), and T22 (100-300 ms), and the progress of ripening could be followed as a shift toward shorter relaxation times. In addition, the combined effect of adding commercial proteases (Pronase E and aspartyl proteinase) on protein breakdown and structural integrity of sausage models (SMS+P) was investigated, resulting in the formation of a more fluid and less organized meat matrix that led to changes in water populations T2b2 and T22 compared with SMS. A very different protein degradation pattern between SMS and SMS+P was observed by means of SDS-PAGE and fluorescamine assay, supporting that some degree of protein aggregation is needed for the presence of the T22 population in fermented sausages.

The effects of salt concentration on conformational changes in cod ( Gadus morhua) proteins during brine salting

The effects of different salt concentrations (6%, 15%, 18% and 24% NaCl (w/w)) on the conformational changes of cod muscle proteins during brine salting were examined in this study. Proteins were extracted from the brine salted samples with solutions of 1 M (5.9%) and 4 M (23.4%) NaCl and the quantity of salt soluble proteins (SSP), disulfide bond (S–S), total sulfhydryl (SH) and available SH content in the soluble fraction were determined. Increased salt concentration in cod muscle promoted protein denaturation and aggregation. The SSP and total SH content decreased, whereas the S–S bond and available SH content increased with increased salt concentration in cod muscle. Disulfide bond formation correlated ( r = −0.6) with a decrease in total SH groups. Higher SSP and available SH groups of the samples at lower brine concentrations was explained by smaller concentration gradients and salt diffusion rates, resulting in stronger salting-in at early stages of the brining process. There was a significant difference in conformational changes in proteins extracted with a salt solution of 1 and 4 M, mainly due to a different degree of protein aggregation.

Effects of different pre-salting methods on protein aggregation during heavy salting of cod fillets

The use of injection and brining as the first step in heavy salting of cod increases weight yields of the products through both salting and rehydration, compared to other pre-salting methods, like brining only and pickling. This is interesting since salt content of the muscle exceeds 20% NaCl, in all procedures. Therefore, the dissimilarities in yield were presumed to depend on the degree of protein denaturation and aggregation as influenced by the different salting procedures. This hypothesis was studied and confirmed with the aid of SDS–PAGE and DSC-analysis. Higher water retention of injected products was explained by stronger salting-in effects on proteins during pre-salting, reducing aggregation of muscle proteins during the dry salting step. The degree of protein aggregation during salting increased in the following order with regard to the different pre-salting methods: injection and brining < brining < pickling. These effects were still observed after rehydration. Furthermore, differences in denaturation/aggregation were assigned to both myosin and collagen.

Effect of hydroxycinnamic acids on lipid oxidation and protein changes as well as water holding capacity in frozen minced horse mackerel white muscle

The antioxidant effectiveness of different hydroxycinnamic acids for inhibiting the formation of off-flavours associated to rancidity in minced frozen horse mackerel white muscle stored at −10 and −18 °C was studied. The influence of lipid oxidation on protein aggregation, protein denaturation and water holding capacity was also determined. Caffeic acid, o-coumaric acid and ferulic acid were the antioxidants tested. The order of antioxidant effectiveness was caffeic acid > ferulic acid > o-coumaric acid accordingly with previous results obtained in chilled horse mackerel. A strong dependence of antioxidant effectiveness to the ratio lipid/antioxidant concentration was observed. There was no evidence to suggest that antioxidants reduce protein aggregation or water holding capacity changes. Protein aggregation was not accompanied by gross protein denaturation, as monitored by differential scanning calorimetry. No evidence was found for a link between extent of lipid oxidation and degree of protein aggregation or water holding capacity changes.

Immunoreactivity and digestibility of high-pressure-treated whey proteins

The effect of high-pressure (HP) treatments on the IgG- and immunoglobulin E (IgE)-binding properties and digestibility of whey proteins was investigated. The formation of protein aggregates was important in whey protein isolate (WPI) and β-lactoglobulin (β-Lg) treated at 200 and 400 MPa at pH 6.8, but was not detected at pH 2.5. Treatment of β-Lg and WPI at 200 and 400 MPa increased binding to β-Lg-specific rabbit IgG and did not affect binding to IgE from allergic patients, but there was no apparent relationship between these responses and the degree of protein aggregation. HP treatment at 400 MPa promoted the hydrolysis of β-Lg by pepsin, but this increased susceptibility of β-Lg to proteolysis was progressively lost during the refrigerated storage of the HP-treated WPI. The higher digestibility of the HP-treated WPI with pepsin did not have an impact on the IgE-binding of the proteolysis products, as compared to non-HP-treated WPI, probably because of the release, in both cases, of specific IgE-binding peptides. Some of these immunoreactive fragments remained after hydrolysis with Corolase PP and probably accounted for a low, but detectable, IgE-binding response.

Polyhistidine tract expansions in HOXA1 result in intranuclear aggregation and increased cell death

HOXA1 gene is part of a cluster of homeotic selector genes that regulates the anteroposterior patterning of mammals during embryonic development. HOXA1 encodes two alternatively spliced mRNAs with two isoforms, A and B, the former contains the homeodomain and expressed in early embryonic development. HOXA1 contains a string of 10 histidine repeats. However, individuals heterozygous for 7, 9, 11, and 12 histidine repeat variants were present among the Japanese population, notably in some autism cases. To determine the biological implications of the different polyhistidine repeat lengths, we expressed these variants in COS-7 and a human neuroblastoma cell line (SK-N-SH). Expression of expanded variants of HOXA1 isoform A, containing 11 and 12 polyhistidine, resulted in early and great degree of protein aggregation in the nucleus. This aggregation resulted in accelerated cell death in cells expressing 11 and 12 expanded variants compared to those transfected with 7 and 10 polyhistidine variants. Furthermore, we showed that these aggregates were ubiquitinated and were inhibited by a histidine-modifying compound, DEPC. These data suggest that HOXA1 protein with polyhistidine tract expansions misfold, aggregate, and have a toxic effect on cell.