Modification Of Keratins Research Articles

Relationships between polymorphisms in keratin genes and Alzheimer’s disease phenotypes.

AbstractBackgroundKeratins are genetically polymorphic and are the largest subset of intermediate filaments. Keratins are also implicated in signaling pathways, inflammation, and disease states. Keratin 9 (KRT9) protein has been identified to have high diagnostic accuracy for Alzheimer’s disease (AD) (PMID:22045497); it was detected in cerebrospinal fluid (CSF) of AD patients but not healthy controls (PMID:24959311). Plasma keratin 9 concentrations also positively correlated with AD‐associated proteins (e.g., apolipoprotein E and tau) in AD patients (PMID:26973255). Post‐translational modification of keratin 83 (KRT83) protein has been associated with serum asymmetric dimethylarginine levels (PMID:34181092), a vascular risk factor implicated in AD. However, our understanding of the role of genetic variation in AD risk is incomplete. Here, we investigate associations between single nucleotide polymorphisms (SNPs) in KRT9, KRT83, and KRT2 (Keratin 2, couples with keratin 9) and AD biomarker phenotypes.MethodIndividuals with mild cognitive impairment (MCI) or AD, and clinically normal controls were selected among ADNI participants. Participants underwent genome‐wide genotyping. Keratin gene variants were identified from the genome‐wide data using dbSNP variant IDs and genotypes were coded additively. Linkage‐disequilibrium‐based clumping on minor allele frequency (MAF) was applied to prioritize more common SNPs. CSF‐Aβ42, ‐tau, and phosphorylated‐tau (p‐tau) levels were quantified via Roche Elecsys immunoassays. Brain‐Aβ deposition was evaluated via normalized PET‐18F‐AV‐45 cortical summary measures (SUVR). White matter hyperintensities (WMH) were obtained from MRI 3D‐T1 and FLAIR sequences via an automated atlas‐based segmentation pipeline. Linear regression models controlling for age, sex, Mini‐Mental State Exam, APOE‐ε4 status, and head size were used to assess association and interaction effects between SNPs and the AD phenotypes in R.ResultAmong included participants (n = 907, 73±7.2 years, 44% female), SNPs in KRT2 (e.g. 3’UTR‐variant rs117041267‐G/A; MAF = 1.4%) and KRT83 (e.g. upstream‐variant KRT83‐rs17119838‐C/T; MAF = 15.6%) were associated with higher CSF‐Aβ42 concentrations (F(2,905) = 4.26,p = 0.014; and F(2,905) = 6.61,p = 0.0014, respectively). Upstream‐variant KRT83‐rs182354391‐C/G (MAF = 1%) showed an association with lower CSF‐tau (F(1,904) = 5.44,p = 0.020). Multiple KRT9 and KRT2 SNPs also showed association with WMH, while KRT2 and KRT83 intron variants showed associations with PET‐Aβ.ConclusionThese candidate gene analyses suggest involvement of multiple keratin family genes in AD, as indicated by differences in amyloid and tau biomarkers, and white matter disease.

Chemical modification of keratin using Schiff bases to prepare cation exchangers and study their adsorption activity

In this research, keratin was chemically modified by Grafting keratin with 4-nitro-aniline, and then a reducing reaction was carried out to reduce the nitro group in order to generate an aromatic amino group on keratin structure to be used in the preparation of Schiff bases. Four exchangers of Schiff bases then were synthesized by reacting five derivatives of benzaldehyde with the crafted keratin. The FTIR, DSC spectra of the prepared exchanges were recorded. The effectiveness of the compounds was tested in the adsorption of some heavy metal ions (copper and lead), as the compounds gave promising results by removing ions from their aqueous solutions in pH (6.5–7), the percentage of removal of copper and lead ions reached about 40%.

Therapeutic Textiles Functionalized with Keratin-Based Particles Encapsulating Terbinafine for the Treatment of Onychomycosis.

Onychomycosis is the most common nail fungal infection worldwide. There are several therapy options available for onychomycosis, such as oral antifungals, topicals, and physical treatments. Terbinafine is in the frontline for the treatment of onychomycosis; however, several adverse effects are associated to its oral administration. In this work, innovative keratin-based carriers encapsulating terbinafine were designed to overcome the drawbacks related to the use this drug. Therapeutic textiles functionalized with keratin-based particles (100% keratin; 80% keratin/20% keratin-PEG) encapsulating terbinafine were developed. The controlled release of terbinafine from the functionalized textiles was evaluated against different mimetic biologic solutions (PBS buffer-pH = 7.4, micellar solution and acidic sweat solution-pH = 4.3). The modification of keratin with polyethylene glycol (PEG) moieties favored the release of terbinafine at the end of 48 h for all the solution conditions. When the activity of functionalized textiles was tested against Trichophyton rubrum, a differentiated inhibition was observed. Textiles functionalized with 80% keratin/20% keratin-PEG encapsulating terbinafine showed a 2-fold inhibition halo compared with the textiles containing 100% keratin-encapsulating terbinafine. No activity was observed for the textiles functionalized with keratin-based particles without terbinafine. The systems herein developed revealed therapeutic potential towards nail fungal infections, taking advantage of keratin-based particles affinity to keratin structures and of the keratinase activity of T. rubrum.

Antibacterial and biodegradable keratin-based quaternary ammonium salt surfactant potential as hair care additive

Biomass-based surfactants have excellent surface activities, showing the common characteristics of natural biomass including biocompatibility, biodegradability and non-toxicity, which are environmental-friendly and accord with the green development direction. In this study, a keratin based quaternary ammonium salt surfactant (denoted as E-K) was prepared by cationic modification of keratin with epoxypropyldodecyldimethylammonium chloride (EPDDMAC). The characterization of E-K by FTIR and the conversion rate of -NH2 in keratin confirmed the successful preparation of E-K. Subsequently, surface activity such as surface tension (γcmc) and critical micelle concentration (cmc), physicochemical properties such as HLB value, isoelectric point, foaming property and emulsifying property, as well as antibacterial activity and biodegradability were investigated. The results show that E-K possesses good surface activity, acceptable emulsifying and foaming ability. The HLB of E-K is 14-15, and it has excellent compatibility with commercial surfactants commonly used in hair care products. Particularly, E-K is a betaine zwitterionic surfactant, and its isoelectric point increases from 3.8 to 9.2 due to cationization. This means that the quaternized keratin hydrolysate is easily adsorbed on the hair surface, and makes the hair surface smoother and denser, which was proved by the adsorption test and SEM observation. Furthermore, E-K has outstanding antibacterial activity against E. coli and S. aureus, and the BOD5/CODCr value indicated that it is easily biodegradable. Therefore, the prepared biomass-based surfactant (E-K) has great potential application in hair care products and other products related to antibacterial and biodegradable surfactants.

Chemically modified wool waste keratin for flame retardant cotton finishing

Environmentally friendly flame retardant chemicals are being developed in recent studies as legislation for eco-sustainable textile production becomes more stringent around the world. Utilisation of waste generated by the wool spinning industry is a prime concern with present work. The extraction and modification of keratin from waste wool for flame retardant cotton has not been attempted previously. The research describes a unique method for using chemically modified keratin derived from wool waste as an environmentally acceptable nitrogen/phosphorus containing flame retardant for cotton fabrics. Melamine and sodium pyrophosphate were used as nitrogen and phosphorus sources, respectively, along with glyoxal as a cross-linking agent. The improved thermal stability of modified wool waste keratin and treated cotton fabrics was confirmed by TGA, while structural changes were established by FTIR and XRD analyses. Due to the synergistic impact of nitrogen and phosphorous, the modified wool waste keratin resulted in the formation of a continuous and intact layer of char that confers flame retardancy on treated cotton. The limiting oxygen index of cotton after application of the modified keratin showed a nearly 66.7% increase in flame retardancy compared to untreated cotton. Overall, the findings in the paper show a sustainable approach for the textile industry that combines waste utilisation from one textile sector (minimizing solid waste from the wool industry) with waste management elements to add value to another (multifunctional textile finishing agent for the cotton industry).

Screening of Chemical Modifications in Human Skin Keratins by Mass Spectrometry-Based Proteomic Analysis via Noninvasive Sampling and On-Tape Digestion.

Proteins are continuously exposed to diverse chemical stresses, and the resulting chemical modifications can provide significant information on biological events. Keratins are the main constituent of human skin and are the major target proteins of various chemical modifications. We have previously developed a mass spectrometry-based noninvasive proteomic methodology to screen oxidative modifications in human skin keratins. We have improved this methodology in terms of sample preparation time and amino acid sequence coverage using an on-tape digestion method. After sampling by tape stripping, skin proteins on the tape were subjected to reduction/alkylation, followed by trypsin digestion without a presolubilization step using detergents. To screen chemical modifications in keratins, target modifications and tryptic target peptides carrying the modification sites were determined from in vitro experiments with major reactive chemical species (4-hydroxy-2(E)-nonenal (HNE), 4-oxo-2(E)-nonenal, glucose, methylglyoxal, peroxynitrite, and hydrogen peroxide). The developed method was used to screen target modifications in controls and patients with a swollen red rash. Basal levels of lipid-derived modification, oxidation, nitration, and glycation in keratins were detected in controls. Principal component analysis based on the relative chemical modification resulted in a clear classification of both groups within a 95% confidence interval. Lipid-derived HNE modification increased most significantly in the patient group. This methodology can be easily applied to patients with other diseases, and the target modifications can be used as biomarkers of certain physiological conditions.

Liver disease–associated keratin 8 and 18 mutations modulate keratin acetylation and methylation

Keratin 8 (K8) and keratin 18 (K18) are the intermediate filament proteins whose phosphorylation/transamidation associate with their aggregation in Mallory-Denk bodies found in patients with various liver diseases. However, the functions of other post-translational modifications in keratins related to liver diseases have not been fully elucidated. Here, using a site-specific mutation assay combined with nano-liquid chromatography-tandem mass spectrometry, we identified K8-Lys108 and K18-Lys187/426 as acetylation sites, and K8-Arg47 and K18-Arg55 as methylation sites. Keratin mutation (Arg-to-Lys/Ala) at the methylation sites, but not the acetylation sites, led to decreased stability of the keratin protein. We compared keratin acetylation/methylation in liver disease-associated keratin variants. The acetylation of K8 variants increased or decreased to various extents, whereas the methylation of K18-del65-72 and K18-I150V variants increased. Notably, the highly acetylated/methylated K18-I150V variant was less soluble and exhibited unusually prolonged protein stability, which suggests that additional acetylation of highly methylated keratins has a synergistic effect on prolonged stability. Therefore, the different levels of acetylation/methylation of the liver disease-associated variants regulate keratin protein stability. These findings extend our understanding of how disease-associated mutations in keratins modulate keratin acetylation and methylation, which may contribute to disease pathogenesis.-Jang, K.-H., Yoon, H.-N., Lee, J., Yi, H., Park, S.-Y., Lee, S.-Y., Lim, Y., Lee, H.-J., Cho, J.-W., Paik, Y.-K., Hancock, W. S., Ku, N.-O. Liver disease-associated keratin 8 and 18 mutations modulate keratin acetylation and methylation.

Mass spectrometry data from proteomic analysis of human skin keratins after exposure to UV radiation.

A mass spectrometry (MS)-based proteomic methodology was employed to monitor oxidative modifications in keratins, the main constituents of human skin (“Non-invasive proteomic analysis of human skin keratins: screening of methionine oxidation in keratins by mass spectrometry” [1], “UV irradiation-induced methionine oxidation in human skin keratins: mass spectrometry-based non-invasive proteomic analysis” [2]). Human skin proteins were obtained non-invasively by tape stripping and solubilized in sodium dodecyl sulfate (SDS) buffer, followed by purification and digestion using the filter-aided sample preparation method. The tryptic peptides were then analyzed by liquid chromatography (LC)/electrospray ionization (ESI)-MS, tandem MS (MS/MS), and LC/ESI-selected reaction monitoring (SRM)/MS. The MS/MS data were generated to confirm amino acid sequences and oxidation sites of tryptic peptides D290VDGAYMTK298 (P1) and N258MQDMVEDYR267 (P2), which contain the most susceptible oxidation sites (Met259, Met262, and Met296 in K1 keratin) upon UVA irradiation [2]. Subsequently, quantitative determination of the relative oxidation levels of P1 and P1 [2] was achieved by LC/ESI-SRM/MS analyses of P1 and P2 together with their oxidized forms after exposure to UVA radiation or treatment with hydrogen peroxide (H2O2).

A Novel High Throughput High Content Analysis Assay for Intermediate Filament Perturbing Drugs

Keratins form the intermediate filament (IF) component of the epithelial cytoskeleton. Depolymerisation of these filaments causes the cell to collapse and become more plastic. We have previously shown that short chain fatty acids may trigger depolymerisation of keratins through altered protein acetylation. Currently, there is no single functional assay for screening of the cytoskeleton. The aim of this study is to develop a high-throughput assay to quantify IF depolymerisation and to apply as a screen for IF-perturbing nutrients and drugs. Three treatments were used in a proof-of-principle study: the anti-fungal drugs griseofulvin and cordycepin (the former a c-mitotic drug known to suppress microtubule growth, the latter inducing abnormal mitosis by suppressing microtubule dynamics) and sodium butyrate, a histone deacetylase inhibitor which disrupts IF formation in cancer cells via post-translational modification of keratins. Methods were optimised for cell fixation using methanol or formalin, permeabilising agents for Keratin 8 (krt8) antibody (triton-x100, digitonin and saponin) and blocking of non-specific binding prior to cell staining using BSA. High Content Analysis (HCA) was employed to quantify cell staining intensities by comparing co-occurrence of adjacent pixel intensities. Immunocytochemistry was used to identify Krt 8 intermediate filaments. Indicators of depolymerisation include Krt 8 fluorescence intensity, filamentousness or texture intensity, fibre spot count and fibre spot total area. All treatments resulted in significant decreases for texture intensity. Assessment of this assay using a Z prime calculation recorded gace a statistic of 0.95, indicating suitability for high-throughput applications. In conclusion, a HCA assay for intermediate filament integrity has been demonstrated, establishing proof of principle with griseofulvin, and cross-validating with two further treatments assayable using this method. Normal 0 false false false ES X-NONE X-NONE

Non-invasive proteomic analysis of human skin keratins: Screening of methionine oxidation in keratins by mass spectrometry

Keratins are the main constituent of human skin and have been identified as major oxidative target proteins. However, there has been a lack of studies aimed at identifying the oxidation sites of keratins because of the difficulties associated with their insolubility and handling. Here, we introduce a mass spectrometry (MS)-based proteomic methodology to screen oxidative modifications in human skin keratins. Human skin proteins were obtained non-invasively by tape stripping and solubilized in SDS buffer, followed by purification and digestion using the modified filter-aided sample preparation method. The tryptic peptides were then analyzed by MALDI-TOF/MS, LC-ESI/MS, and MS/MS. PMF analyses have identified keratins K1 and K10 as the major proteins of human skin. Met(259), Met(262), Met(296), and Met(469), located in the α-helical rod domain of K1, were the most susceptible sites to oxidation induced by hydrogen peroxide in vitro and in vivo. Our results indicate a potential use of the identified methionine residues as biomarkers of oxidative skin damage. The present methodology is the first MS-based approach to detecting oxidative modifications in keratins obtained directly from human skin and can be easily applied to the monitoring of other keratin modifications in various skin conditions.

Treatment of keratin intermediate filaments with sulfur mustard analogs

Sulfur mustard (SM) is an alkylating agent with a history of use as a chemical weapon. The chemical reactivity of sulfur mustard toward both proteins and nucleic acids coupled with the hours long delay between exposure and appearance of blisters has prevented the determination of the mechanism of blister formation. We have treated assembled keratin intermediate filaments with analogs of sulfur mustard to simulate exposure to SM. We find that treatment of intact filaments with chloroethyl ethyl sulfide (CEES) or mechlorethamine (MEC) produces aggregates of keratin filaments with little native appearing structure. Treatment of a mix of epidermal keratins 1/10 (keratin pair 1 and 10) and keratins 5/14 with a sulfhydryl-specific modification reagent also results in filament abnormalities. Our results are consistent with the hypothesis that modification of keratins by SM would result in keratin filament destruction, leading to lysis of epidermal basal cells and skin blistering.

Requirement for ERK activation in acetone extract identified from Bupleurrum scorzonerifolium induced A549 tumor cell apoptosis and keratin 8 phosphorylation

We previously demonstrated that the crude acetone extract of Bupleurum scorzonerifolium (AE-BS) 60 μg/ml has anti-proliferation activity and apoptosis effects to A549 human lung cancer cells. They can also cause tumor cell arrest in G2/M phase. To better understand its target protein in A549 cell, two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry were applied. The modification of keratin 8 was identified. By immunoblot, the expression of phosphorylated keratin 8 at Ser-73 was increased from 2.0 to 3.0-fold after AE-BS treatment 24 to 48 hr respectively as compared with untreated A549 control cells. Furthermore, the A549 cells were pretreated with 50 μM PD98059, a specific inhibitor of the upstream regulator of ERK1/2, or with the p38 kinase inhibitor 20 μM SB203580 or JNK inhibitor 20 μM SP600125 for 30 min, followed by 24 h of incubation with AE-BS, PD98059 can inhibit K8-Ser-73 hyperphosphorylation and prevented cell apoptosis which was induced by AE-BS significantly. By immunoblot, AE-BS also can induce ERK 1/2 phosphorylation. In conclusion, our data indicate that the AE-BS induced tumor apoptosis in A549 cells was related to ERK 1/2 activation. The molecular mechanism of hyperphosphorylation of K8 on Ser-73 was associated with ERK 1/2 activation rather than JNK and p38 kinase. The apoptosis induced by AE-BS may be related to K8 phosphorylation.

Mitotic arrest-associated enhancement of O-linked glycosylation and phosphorylation of human keratins 8 and 18.

Arrest of the human colonic cell line HT29 at the G2/M phase of the cell cycle resulted in changes in keratin assembly that were coupled with a significant increase in the O-linked glycosylation and serine phosphorylation of keratin polypeptides 8 and 18 (K8/18). With mitotic arrest, enhanced keratin phosphorylation occurred preferentially on K8, whereas K18 showed a higher glycosylation level than K8. Removal of the arresting agent allowed cells to proceed through the cell cycle with a concomitant decrease in K8/18 glycosylation. In contrast, keratins isolated from S phase-enriched cells, obtained after synchronization with aphidicolin, did not show enhanced glycosylation. Tryptic peptide analysis of keratins in G2/M-arrested cells showed changes in the glycopeptide pattern of K8 and in the phosphopeptide patterns of K8 and K18. Labeling of K8/18 immunoprecipitates, isolated from G2/M-arrested cells, with [3H]galactose followed by beta-elimination showed that K8/18 glycosylation consisted of single N-acetylglucosamine residues. Threonine was identified as the site of glycosylation after comparing acid hydrolysis products of beta-eliminated and non-beta-eliminated K8 and K18. Specific cleavage at tryptophan residues indicated that K18 glycosylation and phosphorylation were restricted to the head and proximal rod domains, whereas K8 did not show the same restriction. Our results show a unique association of the single O-linked N-acetylglucosamine type of modification of keratins with mitotic arrest in HT29 cells. There was no reciprocal relationship between K8/18 glycosylation and phosphorylation, and each keratin showed a preferential G2/M cell cycle-associated increase in either serine phosphorylation or threonine glycosylation.

Modification of keratin by the chemotherapeutic drug mitoxantrone

Modification of keratin by the chemotherapeutic drug mitoxantrone