RESUMO
Human epithelial keratin is an intermediate filament protein that serves as a backbone to maintain the stability of the cell nucleus and mechanical stability of the whole cells. The present study focused on two point mutations, F231L and S233L, of the 1B domain of keratin K 1/10 related to the rare genetic skin disease palmoplantar keratoderma (PPK). We used molecular dynamics simulation to study the effects of the mutations on various hierarchical structures, including heterodimers, tetramers, and octamers of the K1/10 1B domain at the atomic scale. The initial results demonstrated that the wild type and mutant proteins were highly similar at the dimer level but had different microstructures and mechanics at a higher-level assembly. A decrease in the hydrophobic interactions and hydrogen bonds at the terminus resulted in weakened mechanical properties of the tetramer and octamer of the F231L mutant. The asymmetrical structure of the S233L tetramer with an uneven distribution of the hydrogen bonds decreased its mechanical properties. However, the S233L mutation provided extra hydrophobic interactions between these mutated amino acid residues in the octamer, leading to improved mechanical properties. The results of the present study provided a deeper understanding of how the differences in point mutations induced the changes in the configuration and mechanical properties at the molecular scale. The differences in these properties may influence keratin assembly at the microscopic scale and ultimately cause diseases at the macroscopic scale.
Assuntos
Queratina-10 , Queratina-1 , Mutação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Queratina-1/química , Queratina-1/genética , Queratina-1/metabolismo , Queratina-10/química , Queratina-10/genética , Queratina-10/metabolismo , Estrutura MolecularRESUMO
Palmoplantar keratodermas (PPKs) are characterized by thickness of stratum corneum and epidermal hyperkeratosis localized in palms and soles. PPKs can be epidermolytic (EPPK) or non epidermolytic (NEPPK). Specific mutations of keratin 16 (K16) and keratin 1 (K1) have been associated to EPPK, and NEPPK. Cases of mosaicism in PPKs due to somatic keratin mutations have also been described in scientific literature. We evaluated a patient presenting hyperkeratosis localized monolaterally in the right palmar area, characterized by linear yellowish hyperkeratotic lesions following the Blaschko lines. No other relatives of the patient showed any dermatological disease. Light and confocal histological analysis confirmed the presence of epidermolityic hyperkeratosis. Genetic analysis performed demonstrates the heterozygous deletion NM_006121.4:r.274_472del for a total of 198 nucleotides, in KRT1 cDNA obtained by a palmar lesional skin biopsy, corresponding to the protein mutation NP_006112.3:p.Gly71_Gly137del. DNA extracted from peripheral blood lymphocytes did not display the presence of the mutation. These results suggest a somatic mutation causing an alteration in K1 N-terminal variable domain (V1). The deleted sequence involves the ISIS subdomain, containing a lysine residue already described as fundamental for epidermal transglutaminases in the crosslinking of IF cytoskeleton. Moreover, a computational analysis of the wild-type and V1-mutated K1/K10 keratin dimers, suggests an unusual interaction between these keratin filaments. The mutation taster in silico analysis also returned a high probability for a deleterious mutation. These data demonstrate once again the importance of the head domain (V1) of K1 in the formation of a functional keratinocyte cytoskeleton. Moreover, this is a further demonstration of the presence of somatic mutations arising in later stages of the embryogenesis, generating a mosaic phenotype.
Assuntos
Queratina-10/química , Queratina-1/química , Queratina-1/genética , Nevo/etiologia , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência , Neoplasias Cutâneas/etiologia , Sequência de Aminoácidos , Sequência de Bases , Biópsia , Análise Mutacional de DNA , Imunofluorescência , Humanos , Imuno-Histoquímica , Queratina-1/metabolismo , Queratina-10/metabolismo , Modelos Moleculares , Nevo/metabolismo , Nevo/patologia , Conformação Proteica , Multimerização Proteica , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Relação Estrutura-AtividadeRESUMO
Ichthyosis with confetti (IWC) is a genodermatosis associated with dominant-negative variants in keratin 10 (KRT10) or keratin 1 (KRT1). These frameshift variants result in extended aberrant proteins, localized to the nucleus rather than the cytoplasm. This mislocalization is thought to occur as a result of the altered carboxy (C)-terminus, from poly-glycine to either a poly-arginine or -alanine tail. Previous studies on the type of C-terminus and subcellular localization of the respective mutant protein are divergent. In order to fully elucidate the pathomechanism of IWC, a greater understanding is critical. This study aimed to establish the consequences for localization and intermediate filament formation of altered keratin 10 (K10) C-termini. To achieve this, plasmids expressing distinct KRT10 variants were generated. Sequences encoded all possible reading frames of the K10 C-terminus as well as a nonsense variant. A keratinocyte line was transfected with these plasmids. Additionally, gene editing was utilized to introduce frameshift variants in exon 6 and exon 7 at the endogenous KRT10 locus. Cellular localization of aberrant K10 was observed via immunofluorescence using various antibodies. In each setting, immunofluorescence analysis demonstrated aberrant nuclear localization of K10 featuring an arginine-rich C-terminus. However, this was not observed with K10 featuring an alanine-rich C-terminus. Instead, the protein displayed cytoplasmic localization, consistent with wild-type and truncated forms of K10. This study demonstrates that, of the various 3' frameshift variants of KRT10, exclusively arginine-rich C-termini lead to nuclear localization of K10.
Assuntos
Arginina/genética , Núcleo Celular/genética , Eritrodermia Ictiosiforme Congênita/genética , Queratina-10/genética , Mutação , Transporte Ativo do Núcleo Celular/genética , Alanina/genética , Alanina/metabolismo , Arginina/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Éxons/genética , Mutação da Fase de Leitura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Eritrodermia Ictiosiforme Congênita/metabolismo , Eritrodermia Ictiosiforme Congênita/patologia , Queratina-10/química , Queratina-10/metabolismo , Queratinócitos/metabolismo , Microscopia ConfocalRESUMO
To characterize keratin intermediate filament assembly mechanisms at atomic resolution, we determined the crystal structure of wild-type human keratin-1/keratin-10 helix 1B heterotetramer at 3.0 Å resolution. It revealed biochemical determinants for the A11 mode of axial alignment in keratin filaments. Four regions on a hydrophobic face of the K1/K10-1B heterodimer dictated tetramer assembly: the N-terminal hydrophobic pocket (defined by L227K1, Y230K1, F231K1, and F234K1), the K10 hydrophobic stripe, K1 interaction residues, and the C-terminal anchoring knob (formed by F314K1 and L318K1). Mutation of both knob residues to alanine disrupted keratin 1B tetramer and full-length filament assembly. Individual knob residue mutant F314AK1, but not L318AK1, abolished 1B tetramer formation. The K1-1B knob/pocket mechanism is conserved across keratins and many non-keratin intermediate filaments. To demonstrate how pathogenic mutations cause skin disease by altering filament assembly, we additionally determined the 2.39 Å structure of K1/10-1B containing a S233LK1 mutation linked to epidermolytic palmoplantar keratoderma. Light scattering and circular dichroism measurements demonstrated enhanced aggregation of K1S233L/K10-1B in solution without affecting secondary structure. The K1S233L/K10-1B octamer structure revealed S233LK1 causes aberrant hydrophobic interactions between 1B tetramers.
Assuntos
Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/metabolismo , Queratina-10 , Queratina-1 , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica/fisiologia , Substituição de Aminoácidos , Dicroísmo Circular , Cristalografia por Raios X , Citoesqueleto/química , Citoesqueleto/metabolismo , Difusão Dinâmica da Luz , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Filamentos Intermediários/genética , Queratina-1/química , Queratina-1/genética , Queratina-1/metabolismo , Queratina-10/química , Queratina-10/genética , Queratina-10/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas/genética , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Dermatopatias/genética , Dermatopatias/metabolismo , Dermatopatias/patologiaRESUMO
Ichthyosis Hystrix of Curth-Macklin (IH-CM) is a rare manifestation of epidermolytic ichthyosis (EI) that is characterised by generalised spiky or verrucous hyperkeratosis. The disorder is further distinguished by the presence of binucleated cells in the affected skin, whereas epidermolysis and clumping of tonofilaments, as seen in EI, are absent. While IH-CM is associated with mutations in the keratin 1 (KRT1) gene, reports to date have indicated that mutations in the KRT1 gene result in an aberrant and truncated protein tail, essentially affecting the function of the V2 domain. Here, we studied a female sporadic patient who was born with diffused erythrodermic hyperkeratosis and who presented at the age of 13 months with an intense and widespread hyperkeratosis with a papillomatous appearance and typical palmoplantar keratoderma. Genetic analysis demonstrated a "de novo" mutation in the keratin 10 gene (KRT10) consisting of a three-base-pair deletion, resulting in the substitution of amino acids p.Glu445 and p.Ile446 by Asp at the end of the 2B domain of the protein. We performed structural and functional studies showing that this mutation modifies the structure of the paired 2B and V2 K1/10 domains, leading to the disease phenotype. Our results highlight the importance and complexity of the KRT1/10 V2 domain in keratin dimer formation and the potential consequences of its alteration.
Assuntos
Substituição de Aminoácidos , Dermatite Seborreica/genética , Ictiose/genética , Queratina-10/genética , Queratina-1/genética , Feminino , Humanos , Lactente , Queratina-1/química , Queratina-10/química , Modelos Moleculares , Conformação Proteica , Domínios ProteicosRESUMO
Keratins 1 (K1) and 10 (K10) are the primary keratins expressed in differentiated epidermis. Mutations in K1/K10 are associated with human skin diseases. We determined the crystal structure of the complex between the distal (2B) helices of K1 and K10 to better understand how human keratin structure correlates with function. The 3.3 Å resolution structure confirms many features inferred by previous biochemical analyses, but adds unexpected insights. It demonstrates a parallel, coiled-coil heterodimer with a predominantly hydrophobic intermolecular interface; this heterodimer formed a higher order complex with a second K1-K10-2B heterodimer via a Cys401K10 disulfide link, although the bond angle is unanticipated. The molecular surface analysis of K1-K10-2B identified several pockets, one adjacent to the disulfide linkage and conserved in K5-K14. The solvent accessible surface area of the K1-K10 structure is 20-25% hydrophobic. The 2B region contains mixed acidic and basic patches proximally (N-terminal), whereas it is largely acidic distally (C-terminal). Mapping of conserved and nonconserved residues between K1-K10 and K5-K14 onto the structure demonstrated the majority of unique residues align along the outer helical ridge. Finally, the structure permitted a fresh analysis of the deleterious effects caused by K1/K10 missense mutations found in patients with phenotypic skin disease.
Assuntos
Queratina-10/genética , Queratina-1/genética , Estrutura Molecular , Dermatopatias/genética , Células Cultivadas , Células Epidérmicas , Epiderme/metabolismo , Humanos , Queratina-1/química , Queratina-10/química , Mutação de Sentido Incorreto , Sensibilidade e Especificidade , Dermatopatias/patologia , Propriedades de SuperfícieRESUMO
The K1 and K10 associated genodermatoses are characterized by clinical symptoms of mild to severe redness, blistering and hypertrophy of the skin. In this paper, we set out to computationally investigate the structural and functional effects of missense mutations on the 2B domain of K1/K10 heterodimer and its consequences in disease phenotype. We modeled the structure of the K1/K10 heterodimer based on crystal structures for the human homolog K5/K14 heterodimer, and identified that the missense mutations exert their effects on stability and assembly competence of the heterodimer by altering physico-chemical properties, interatomic interactions, and inter-residue atomic contacts. Comparative structural analysis between all the missense mutations and SNPs showed that the location and physico-chemical properties of the substituted amino acid are significantly correlated with phenotypic variations. In particular, we find evidence that a particular SNP (K10, p.E443K) is a pathogenic nsSNP which disrupts formation of the hydrophobic core and destabilizes the heterodimer through the loss of interatomic interactions. Our study is the first comprehensive report analyzing the mutations located on 2B domain of K1/K10 heterodimeric coiled-coil complex.
Assuntos
Simulação por Computador , Queratina-10/genética , Queratina-1/genética , Mutação de Sentido Incorreto , Dermatopatias Genéticas/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Humanos , Queratina-1/química , Queratina-1/metabolismo , Queratina-10/química , Queratina-10/metabolismo , Modelos Moleculares , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Pele/metabolismo , Pele/patologia , Dermatopatias Genéticas/metabolismoRESUMO
Keratins are cytoskeletal proteins that hierarchically arrange into filaments, starting with the dimer sub-unit. They are integral to the structural support of cells, in skin, hair and nails. In skin, keratin is thought to play a critical role in conferring the barrier properties and elasticity of skin. In general, the keratin dimer is broadly described by a tri-domain structure: a head, a central rod and a tail. As yet, no atomistic-scale picture of the entire dimer structure exists; this information is pivotal for establishing molecular-level connections between structure and function in intermediate filament proteins. The roles of the head and tail domains in facilitating keratin filament assembly and function remain as open questions. To address these, we report results of molecular dynamics simulations of the entire epithelial human K1/K10 keratin dimer. Our findings comprise: (1) the first three-dimensional structural models of the complete dimer unit, comprising of the head, rod and tail domains; (2) new insights into the chirality of the rod-domain twist gained from analysis of the full domain structure; (3) evidence for tri-subdomain partitioning in the head and tail domains; and, (4) identification of the residue characteristics that mediate non-covalent contact between the chains in the dimer. Our findings are immediately applicable to other epithelial keratins, such as K8/K18 and K5/K14, and to intermediate filament proteins in general.
Assuntos
Dissulfetos/química , Filamentos Intermediários/química , Queratina-10/química , Queratina-1/química , Multimerização Proteica , Sequência de Aminoácidos , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Epidermolytic ichthyosis (EI) due to KRT10 mutations is a rare, typically autosomal dominant, disorder characterized by generalized erythema and cutaneous blistering at birth followed by hyperkeratosis and less frequent blistering later in life. We identified two KRT10 mutations p.Q434del and p.R441P in subjects presenting with a mild EI phenotype. Both occur within the mutational "hot spot" of the keratin 10 (K10) 2B rod domain, adjacent to severe EI-associated mutations. p.Q434del and p.R441P formed collapsed K10 fibers rather than aggregates characteristic of severe EI KRT10 mutations such as p.R156C. Upon differentiation, keratinocytes from p.Q434del showed significantly lower apoptosis (P-value<0.01) compared with p.R156C as assessed by the TUNEL assay. Conversely, the mitotic index of the p.Q434del epidermis was significantly higher compared with that of p.R156C (P-value<0.01) as estimated by the Ki67 assay. Structural basis of EI phenotype variation was investigated by homology-based modeling of wild-type and mutant K1-K10 dimers. Both mild EI mutations were found to affect the surface-exposed residues of the K10 alpha helix coiled-coil and caused localized disorganization of the K1-K10 heterodimer. In contrast, adjacent severe EI mutations disrupt key intermolecular dimer interactions. Our findings provide structural insights into phenotypic variation in EI due to KRT10 mutations.
Assuntos
Hiperceratose Epidermolítica/genética , Queratina-10/genética , Mutação , Adulto , Sequência de Aminoácidos , Criança , Feminino , Homeostase , Humanos , Queratina-10/química , Índice Mitótico , Modelos Moleculares , Dados de Sequência Molecular , Fenótipo , Multimerização Proteica , Estrutura Terciária de ProteínaRESUMO
Streptococcus pneumoniae is a major human pathogen, and a leading cause of disease and death worldwide. Pneumococcal invasive disease is triggered by initial asymptomatic colonization of the human upper respiratory tract. The pneumococcal serine-rich repeat protein (PsrP) is a lung-specific virulence factor whose functional binding region (BR) binds to keratin-10 (KRT10) and promotes pneumococcal biofilm formation through self-oligomerization. We present the crystal structure of the KRT10-binding domain of PsrP (BR187-385) determined to 2.0 Å resolution. BR187-385 adopts a novel variant of the DEv-IgG fold, typical for microbial surface components recognizing adhesive matrix molecules adhesins, despite very low sequence identity. An extended ß-sheet on one side of the compressed, two-sided barrel presents a basic groove that possibly binds to the acidic helical rod domain of KRT10. Our study also demonstrates the importance of the other side of the barrel, formed by extensive well-ordered loops and stabilized by short ß-strands, for interaction with KRT10.
Assuntos
Proteínas de Bactérias/metabolismo , Queratina-10/química , Modelos Moleculares , Fatores de Virulência/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Alanina/genética , Alanina/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Humanos , Queratina-10/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Eletricidade Estática , Streptococcus pneumoniae/metabolismo , Fatores de Virulência/química , Fatores de Virulência/genéticaRESUMO
Staphylococcus pseudintermedius, a commensal and pathogen of dogs and occasionally of humans, expresses surface proteins potentially involved in host colonization and pathogenesis. Here, we describe the cloning and characterization of SpsD, a surface protein of S. pseudintermedius reported as interacting with extracellular matrix proteins and corneocytes. A ligand screen and Western immunoblotting revealed that the N-terminal A domain of SpsD bound fibrinogen, fibronectin, elastin and cytokeratin 10. SpsD also interfered with thrombin-induced fibrinogen coagulation and blocked ADP-induced platelet aggregation. The binding site for SpsD was mapped to residues 395-411 in the fibrinogen γ-chain, while binding sites in fibronectin were localized to the N- and C-terminal regions. SpsD also bound to glycine- and serine-rich omega loops within the C-terminal tail region of cytokeratin 10. Ligand binding studies using SpsD variants lacking the C-terminal segment or containing an amino-acid substitution in the putative ligand binding site provided insights into interaction mechanism of SpsD with the different ligands. Together these data demonstrate the multi-ligand binding properties of SpsD and illustrate some interesting differences in the variety of ligands bound by SpsD and related proteins from S. aureus.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Interações Hospedeiro-Patógeno/genética , Staphylococcus/metabolismo , Animais , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Cães , Elastina/química , Elastina/metabolismo , Proteínas da Matriz Extracelular/química , Fibrinogênio/química , Fibrinogênio/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Humanos , Queratina-10/química , Queratina-10/metabolismo , Ligantes , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Mycobacteria are known to induce autoimmune response in the host. Anti-host keratrin antibodies (AkAbs) might be responsible for the autoimmune phenomena in leprosy patients as majority of leprosy lesions are manifested in the skin and occurrence of keratosis is not an uncommon feature. The aim of this study was to find out the level of AkAbs in leprosy patients across the spectrum and to explore its correlation with the clinical manifestation of the disease. Further, mimicking epitopes of keratin and Mycobacterium leprae components were characterized. We screened 140 leprosy patients (27 BT, 28 BL, 41 LL, 25 T1R, 19 ENL), 74 healthy controls (HC) and 3 psoriasis patients as positive control. Highest AkAbs level was observed in the psoriasis patients followed by T1R, LL, BL, ENL, TT/BT. AkAbs level was significantly (p<0.05) higher in all the groups of leprosy patients except TT/BT in comparison to HC. Significant positive correlation was found between number of lesions and level of AkAbs in leprosy patients. Highest lympho-proliferation for keratin protein was observed in T1R, followed by BL/LL, TT/BT, ENL. Lympho-proliferation was significantly (p<0.05) higher in all groups of leprosy patients except ENL in comparison to HC. Interestingly, it was noted that hyperimmunization of inbred strains of female BALB/c mice and rabbit with M. leprae soluble antigen (MLSA) induce higher level of AkAbs. The percentage of FoxP3(+) expressing Treg cells (total CD4(+)CD25(+)FoxP3(+) andCD4(+)CD25(+hi)FoxP3(+)) in splenocytes and lymph nodes of hyperimmunized mice were declined in comparison to control mice. Further, it was found that this autoimmune response can be adoptively transferred in naïve mice by splenocytes and lymph node cells as well as T cells. Comparative molecular characterization between keratin and MLSA noted a cross-reactivity/similarity between these two antigens. The cross-reactive protein of keratin was found to be in molecular weight range ≈74-51kDa and at pI 4.5 while the cross-reactive protein of MLSA was found to be in molecular weight ≈65kDa and at pI 4-4.5. Cross-reactive protein of keratin and MLSA was identified and characterized by MALDI-TOF/TOF analysis and Mascot software. It was found that the keratin (host protein) which reacted with anti-M. leprae sera is cytokeratin-10 and MLSA which reacted with anti-keratin sera is heat shock protein 65 (HSP 65). Seven B-cell epitopes of cytokeratin-10 and HSP 65 was found to be similar by multiple sequence alignment using ClustalW server and out of which 6 B-cell epitopes were found to be on the surface of HSP 65. In conclusion, our study provides evidence for the existence of molecular mimicry between cytokeratin-10 of keratin (host protein) and 65kDa HSP (groEL2) of M. leprae. Presence of heightened CMI response of leprosy patients to keratin and positive correlation of AkAbs level with number of lesions of leprosy patients showed the clinical evidence for its role in the pathogenesis in leprosy.
Assuntos
Proteínas de Bactérias/química , Chaperonina 60/química , Queratina-10/química , Hanseníase/imunologia , Hanseníase/prevenção & controle , Mycobacterium leprae/imunologia , Transferência Adotiva , Animais , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/imunologia , Autoanticorpos/biossíntese , Autoanticorpos/imunologia , Proteínas de Bactérias/imunologia , Estudos de Casos e Controles , Chaperonina 60/imunologia , Reações Cruzadas , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Expressão Gênica , Humanos , Imunização , Queratina-10/imunologia , Hanseníase/microbiologia , Linfonodos/citologia , Linfonodos/imunologia , Camundongos , Mimetismo Molecular , Coelhos , Índice de Gravidade de Doença , Pele/imunologia , Pele/microbiologia , Baço/citologia , Baço/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/microbiologiaRESUMO
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.
Assuntos
Queratinas/análise , Metionina/química , Pele/química , Sequência de Aminoácidos , Artefatos , Humanos , Peróxido de Hidrogênio/química , Queratina-1/química , Queratina-10/química , Queratina-2/química , Queratina-9/química , Queratinas/química , Queratinas/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Oxirredução , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem , Tripsina/metabolismoRESUMO
Somatic loss of wild-type alleles can produce disease traits such as neoplasia. Conversely, somatic loss of disease-causing mutations can revert phenotypes; however, these events are infrequently observed. Here we show that ichthyosis with confetti, a severe, sporadic skin disease in humans, is associated with thousands of revertant clones of normal skin that arise from loss of heterozygosity on chromosome 17q via mitotic recombination. This allowed us to map and identify disease-causing mutations in the gene encoding keratin 10 (KRT10); all result in frameshifts into the same alternative reading frame, producing an arginine-rich C-terminal peptide that redirects keratin 10 from the cytokeratin filament network to the nucleolus. The high frequency of somatic reversion in ichthyosis with confetti suggests that revertant stem cell clones are under strong positive selection and/or that the rate of mitotic recombination is elevated in individuals with this disorder.