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1.
J Cell Sci ; 136(1)2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36594662

RESUMO

Desmosome diseases are caused by dysfunction of desmosomes, which anchor intermediate filaments (IFs) at sites of cell-cell adhesion. For many decades, the focus of attention has been on the role of actin filament-associated adherens junctions in development and disease, especially cancer. However, interference with the function of desmosomes, their molecular constituents or their attachments to IFs has now emerged as a major contributor to a variety of diseases affecting different tissues and organs including skin, heart and the digestive tract. The first Alpine desmosome disease meeting (ADDM) held in Grainau, Germany, in October 2022 brought together international researchers from the basic sciences with clinical experts from diverse fields to share and discuss their ideas and concepts on desmosome function and dysfunction in the different cell types involved in desmosome diseases. Besides the prototypic desmosomal diseases pemphigus and arrhythmogenic cardiomyopathy, the role of desmosome dysfunction in inflammatory bowel diseases and eosinophilic esophagitis was discussed.


Assuntos
Desmossomos , Doença , Humanos , Adesão Celular , Desmossomos/fisiologia , Pênfigo
2.
Int J Mol Sci ; 25(10)2024 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-38791277

RESUMO

Succinic semialdehyde dehydrogenase (SSADH) is a mitochondrial enzyme involved in the catabolism of the neurotransmitter γ-amino butyric acid. Pathogenic variants in the gene encoding this enzyme cause SSADH deficiency, a developmental disease that manifests as hypotonia, autism, and epilepsy. SSADH deficiency patients usually have family-specific gene variants. Here, we describe a family exhibiting four different SSADH variants: Val90Ala, Cys93Phe, and His180Tyr/Asn255Asp (a double variant). We provide a structural and functional characterization of these variants and show that Cys93Phe and Asn255Asp are pathogenic variants that affect the stability of the SSADH protein. Due to the impairment of the cofactor NAD+ binding, these variants show a highly reduced enzyme activity. However, Val90Ala and His180Tyr exhibit normal activity and expression. The His180Tyr/Asn255Asp variant exhibits a highly reduced activity as a recombinant species, is inactive, and shows a very low expression in eukaryotic cells. A treatment with substances that support protein folding by either increasing chaperone protein expression or by chemical means did not increase the expression of the pathogenic variants of the SSADH deficiency patient. However, stabilization of the folding of pathogenic SSADH variants by other substances may provide a treatment option for this disease.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Succinato-Semialdeído Desidrogenase , Succinato-Semialdeído Desidrogenase/deficiência , Succinato-Semialdeído Desidrogenase/genética , Succinato-Semialdeído Desidrogenase/química , Succinato-Semialdeído Desidrogenase/metabolismo , Humanos , Erros Inatos do Metabolismo dos Aminoácidos/genética , Masculino , Feminino , Linhagem , Mutação , Variação Genética , Dobramento de Proteína , Deficiências do Desenvolvimento
3.
J Inherit Metab Dis ; 2023 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-37606592

RESUMO

Due to the low number of patients, rare genetic diseases are a special challenge for the development of therapies, especially for diseases that result from numerous, patient-specific pathogenic variants. Precision medicine makes use of various kinds of molecular information about a specific variant, so that the possibilities for an effective therapy based on the molecular features of the variants can be elucidated. The attention to personalized precision therapies has increased among scientists and clinicians, since the "single drug for all patients" approach does not allow the classification of individuals in subgroups according to the differences in the disease genotype or phenotype. This review article summarizes some approaches of personalized precision medicine that can be used for a cost-effective and fast development of therapies, even for single patients. We have focused on specific examples on inborn errors of metabolism, with special attention on drug repurposing. Furthermore, we provide an overview of cell culture models that are suitable for precision medicine approaches.

4.
Int J Mol Sci ; 24(6)2023 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-36982794

RESUMO

Novel treatment strategies are emerging for rare, genetic diseases, resulting in clinical trials that require adequate biomarkers for the assessment of the treatment effect. For enzyme defects, biomarkers that can be assessed from patient serum, such as enzyme activity, are highly useful, but the activity assays need to be properly validated to ensure a precise, quantitative measurement. Aspartylglucosaminuria (AGU) is a lysosomal storage disorder caused by the deficiency of the lysosomal hydrolase aspartylglucosaminidase (AGA). We have here established and validated a fluorometric AGA activity assay for human serum samples from healthy donors and AGU patients. We show that the validated AGA activity assay is suitable for the assessment of AGA activity in the serum of healthy donors and AGU patients, and it can be used for diagnostics of AGU and, potentially, for following a treatment effect.


Assuntos
Aspartilglucosaminúria , Aspartilglucosilaminase , Doenças por Armazenamento dos Lisossomos , Humanos , Aspartilglucosilaminase/genética , Aspartilglucosaminúria/genética , Doenças por Armazenamento dos Lisossomos/genética , Lisossomos
5.
Mol Ther ; 29(3): 989-1000, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33186692

RESUMO

Aspartylglucosaminuria (AGU) is an autosomal recessive lysosomal storage disease caused by loss of the enzyme aspartylglucosaminidase (AGA), resulting in AGA substrate accumulation. AGU patients have a slow but progressive neurodegenerative disease course, for which there is no approved disease-modifying treatment. In this study, AAV9/AGA was administered to Aga-/- mice intravenously (i.v.) or intrathecally (i.t.), at a range of doses, either before or after disease pathology begins. At either treatment age, AAV9/AGA administration led to (1) dose dependently increased and sustained AGA activity in body fluids and tissues; (2) rapid, sustained, and dose-dependent elimination of AGA substrate in body fluids; (3) significantly rescued locomotor activity; (4) dose-dependent preservation of Purkinje neurons in the cerebellum; and (5) significantly reduced gliosis in the brain. Treated mice had no abnormal neurological phenotype and maintained body weight throughout the whole experiment to 18 months old. In summary, these results demonstrate that treatment of Aga-/- mice with AAV9/AGA is effective and safe, providing strong evidence that AAV9/AGA gene therapy should be considered for human translation. Further, we provide a direct comparison of the efficacy of an i.v. versus i.t. approach using AAV9, which should greatly inform the development of similar treatments for other related lysosomal storage diseases.


Assuntos
Aspartilglucosaminúria/terapia , Aspartilglucosilaminase/fisiologia , Dependovirus/genética , Modelos Animais de Doenças , Terapia Genética/métodos , Células de Purkinje/metabolismo , Animais , Aspartilglucosaminúria/enzimologia , Aspartilglucosaminúria/genética , Aspartilglucosaminúria/patologia , Peso Corporal , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
6.
J Inherit Metab Dis ; 43(2): 318-325, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31415096

RESUMO

Aspartylglucosaminuria (AGU) is a rare, recessively inherited lysosomal disease with relatively high prevalence in Finnish population. This progressive disease has a vast impact on patient's cognition and physical health, leading to intellectual disability and shorter life expectancy. Cognitive functions of 21 7- to 14-year-old children with AGU were studied cross-sectionally using Wechsler's Intelligence Scale for Children IV and the results were compared with a standardized Finnish sample. In addition to overall cognitive performance, abilities in discrete cognitive domains, including verbal comprehension, perceptual reasoning, working memory, and processing speed, were examined. The results showed that despite the very low overall level of cognitive performance, there were notable differences between individuals. All those children whose performance was closer to their own age level were 7 to 10 years old. Processing speed appeared more compromised, as compared with verbal comprehension. Furthermore, examining the subtest raw scores, there were no significant positive correlations between age and subtest scores, suggesting that the developmental level of AGU children could be rather stable throughout ages 7 to 14. This study gives insight to the severe nature of AGU disease. Since younger children performed better compared to their age norms than older children, the results raise a question whether the highest peak in cognitive functions is reached at an earlier age than previously thought.


Assuntos
Aspartilglucosaminúria/psicologia , Disfunção Cognitiva/diagnóstico , Deficiência Intelectual/diagnóstico , Adolescente , Criança , Cognição , Disfunção Cognitiva/etiologia , Feminino , Finlândia , Humanos , Deficiência Intelectual/etiologia , Masculino , Escalas de Wechsler
7.
Int J Mol Sci ; 21(22)2020 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-33203024

RESUMO

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare, monogenic disorder affecting the degradation of the main inhibitory neurotransmitter γ-amino butyric acid (GABA). Pathogenic variants in the ALDH5A1 gene that cause an enzymatic dysfunction of succinic semialdehyde dehydrogenase (SSADH) lead to an accumulation of potentially toxic metabolites, including γ-hydroxybutyrate (GHB). Here, we present a patient with a severe phenotype of SSADHD caused by a novel genetic variant c.728T > C that leads to an exchange of leucine to proline at residue 243, located within the highly conserved nicotinamide adenine dinucleotide (NAD)+ binding domain of SSADH. Proline harbors a pyrrolidine within its side chain known for its conformational rigidity and disruption of protein secondary structures. We investigate the effect of this novel variant in vivo, in vitro, and in silico. We furthermore examine the mutational spectrum of all previously described disease-causing variants and computationally assess all biologically possible missense variants of ALDH5A1 to identify mutational hotspots.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Simulação por Computador , Deficiências do Desenvolvimento , Mutação de Sentido Incorreto , Succinato-Semialdeído Desidrogenase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/genética , Substituição de Aminoácidos , Deficiências do Desenvolvimento/enzimologia , Deficiências do Desenvolvimento/genética , Células HEK293 , Humanos , Domínios Proteicos , Succinato-Semialdeído Desidrogenase/genética , Succinato-Semialdeído Desidrogenase/metabolismo
8.
J Mol Cell Cardiol ; 126: 86-95, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30452906

RESUMO

BACKGROUND: The intercalated disc (ID) is important for cardiac remodeling and has become a subject of intensive research efforts. However, as yet the composition of the ID has still not been conclusively resolved and the role of many proteins identified in the ID, like Flotillin-2, is often unknown. The Flotillin proteins are known to be involved in the stabilization of cadherins and desmosomes in the epidermis and upon cancer development. However, their role in the heart has so far not been investigated. Therefore, in this study, we aimed at identifying the role of Flotillin-1 and Flotillin-2 in the cardiac ID. METHODS: Location of Flotillins in human and murine cardiac tissue was evaluated by fluorescent immunolabeling and co-immunoprecipitation. In addition, the effect of Flotillin knockout (KO) on proteins of the ID and in electrical excitation and conduction was investigated in cardiac samples of wildtype (WT), Flotillin-1 KO, Flotilin-2 KO and Flotilin-1/2 double KO mice. Consequences of Flotillin knockdown (KD) on cardiac function were studied (patch clamp and Multi Electrode Array (MEA)) in neonatal rat cardiomyocytes (NRCMs) transfected with siRNAs against Flotillin-1 and/or Flotillin-2. RESULTS: First, we confirmed presence in the ID and mutual binding of Flotillin-1 and Flotillin-2 in murine and human cardiac tissue. Flotillin KO mice did not show cardiac fibrosis, nor hypertrophy or changes in expression of the desmosomal ID proteins. However, protein expression of the cardiac sodium channel NaV1.5 was significantly decreased in Flotillin-1 and Flotillin-1/2 KO mice compared to WT mice. In addition, sodium current density showed a significant decrease upon Flotillin-1/2 KD in NRCMs as compared to scrambled siRNA-transfected NRCMs. MEA recordings of Flotillin-2 KD NRCM cultures showed a significantly decreased spike amplitude and a tendency of a reduced spike slope when compared to control and scrambled siRNA-transfected cultures. CONCLUSIONS: In this study, we demonstrate the presence of Flotillin-1, in addition to Flotillin-2 in the cardiac ID. Our findings indicate a modulatory role of Flotillins on NaV1.5 expression at the ID, with potential consequences for cardiac excitation.


Assuntos
Proteínas de Membrana/metabolismo , Miocárdio/metabolismo , Animais , Animais Recém-Nascidos , Conexina 43/metabolismo , Humanos , Ativação do Canal Iônico , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Ratos Wistar
9.
Int J Mol Sci ; 20(19)2019 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-31569533

RESUMO

Mitogen-activated protein kinases (MAPKs) are involved in signaling processes induced by various stimuli, such as growth factors, stress, or even autoantibodies [...].


Assuntos
Suscetibilidade a Doenças , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Animais , Humanos
10.
Int J Mol Sci ; 20(13)2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31247885

RESUMO

Pemphigus Vulgaris is an autoimmune disease that results in blister formation in the epidermis and in mucosal tissues due to antibodies recognizing desmosomal cadherins, mainly desmoglein-3 and -1. Studies on the molecular mechanisms of Pemphigus have mainly been carried out using the spontaneously immortalized human keratinocyte cell line HaCaT or in primary keratinocytes. However, both cell systems have suboptimal features, with HaCaT cells exhibiting a large number of chromosomal aberrations and mutated p53 tumor suppressor, whereas primary keratinocytes are short-lived, heterogeneous and not susceptible to genetic modifications due to their restricted life-span. We have here tested the suitability of the commercially available human keratinocyte cell line hTert/KER-CT as a model system for research on epidermal cell adhesion and Pemphigus pathomechanisms. We here show that hTert cells exhibit a calcium dependent expression of desmosomal cadherins and are well suitable for typical assays used for studies on Pemphigus, such as sequential detergent extraction and Dispase-based dissociation assay. Treatment with Pemphigus auto-antibodies results in loss of monolayer integrity and altered localization of desmoglein-3, as well as loss of colocalization with flotillin-2. Our findings demonstrate that hTert cells are well suitable for studies on epidermal cell adhesion and Pemphigus pathomechanisms.


Assuntos
Desmossomos/genética , Desmossomos/metabolismo , Queratinócitos/metabolismo , Pênfigo/etiologia , Pênfigo/metabolismo , Telomerase/genética , Autoanticorpos/imunologia , Biomarcadores , Adesão Celular , Linhagem Celular , Linhagem Celular Transformada , Desmossomos/imunologia , Imunofluorescência , Expressão Gênica , Humanos , Queratinócitos/imunologia , Modelos Biológicos , Pênfigo/patologia
11.
Biochim Biophys Acta Mol Basis Dis ; 1864(3): 668-675, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29247835

RESUMO

Aspartylglucosaminuria (AGU) is a lysosomal storage disorder caused by mutations in the gene for aspartylglucosaminidase (AGA). This enzyme participates in glycoprotein degradation in lysosomes. AGU results in progressive mental retardation, and no curative therapy is currently available. We have here characterized the consequences of AGA gene mutations in a compound heterozygous patient who exhibits a missense mutation producing a Ser72Pro substitution in one allele, and a nonsense mutation Trp168X in the other. Ser72 is not a catalytic residue, but is required for the stabilization of the active site conformation. Thus, Ser72Pro exchange impairs the autocatalytic activation of the AGA precursor, and results in a considerable reduction of the enzyme activity and in altered AGA precursor processing. Betaine, which can partially rescue the AGA activity in AGU patients carrying certain missense mutations, turned out to be ineffective in the case of Ser72Pro substitution. The Trp168X nonsense allele results in complete lack of AGA polypeptide due to nonsense-mediated decay (NMD) of the mRNA. Amlexanox, which inhibits NMD and causes a translational read-through, facilitated the synthesis of a full-length, functional AGA protein from the nonsense allele. This could be demonstrated as presence of the AGA polypeptide and increased enzyme activity upon Amlexanox treatment. Furthermore, in the Ser72Pro/Trp168X expressing cells, Amlexanox induced a synergistic increase in AGA activity and polypeptide processing due to enhanced processing of the Ser72Pro polypeptide. Our data show for the first time that Amlexanox might provide a valid therapy for AGU.


Assuntos
Aminopiridinas/uso terapêutico , Aspartilglucosaminúria/tratamento farmacológico , Aspartilglucosaminúria/genética , Aspartilglucosilaminase/genética , Códon sem Sentido , Substituição de Aminoácidos , Aminopiridinas/farmacologia , Células Cultivadas , Criança , Códon sem Sentido/efeitos dos fármacos , Feminino , Células HEK293 , Células HeLa , Humanos , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Doenças por Armazenamento dos Lisossomos/genética , Mutação de Sentido Incorreto
12.
Int J Mol Sci ; 19(2)2018 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-29470438

RESUMO

Juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene. Most JNCL patients exhibit a 1.02 kb genomic deletion removing exons 7 and 8 of this gene, which results in a truncated CLN3 protein carrying an aberrant C-terminus. A genetically accurate mouse model (Cln3Δex7/8 mice) for this deletion has been generated. Using cerebellar precursor cell lines generated from wildtype and Cln3Δex7/8 mice, we have here analyzed the consequences of the CLN3 deletion on levels of cellular gangliosides, particularly GM3, GM2, GM1a and GD1a. The levels of GM1a and GD1a were found to be significantly reduced by both biochemical and cytochemical methods. However, quantitative high-performance liquid chromatography analysis revealed a highly significant increase in GM3, suggesting a metabolic blockade in the conversion of GM3 to more complex gangliosides. Quantitative real-time PCR analysis revealed a significant reduction in the transcripts of the interconverting enzymes, especially of ß-1,4-N-acetyl-galactosaminyl transferase 1 (GM2 synthase), which is the enzyme converting GM3 to GM2. Thus, our data suggest that the complex a-series gangliosides are reduced in Cln3Δex7/8 mouse cerebellar precursor cells due to impaired transcription of the genes responsible for their synthesis.


Assuntos
Cerebelo/enzimologia , Cerebelo/patologia , Gangliosídeo G(M3)/metabolismo , Lipofuscinoses Ceroides Neuronais/enzimologia , Lipofuscinoses Ceroides Neuronais/patologia , Animais , Toxina da Cólera/metabolismo , Modelos Animais de Doenças , Gangliosídeo G(M3)/química , Lisossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Chaperonas Moleculares/metabolismo
13.
Int J Mol Sci ; 18(4)2017 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-28346360

RESUMO

Aspartylglucosaminidase (AGA) is a lysosomal hydrolase that participates in the breakdown of glycoproteins. Defects in the AGA gene result in a lysosomal storage disorder, aspartylglucosaminuria (AGU), that manifests mainly as progressive mental retardation. A number of AGU missense mutations have been identified that result in reduced AGA activity. Human variants that contain either Ser or Thr in position 149 have been described, but it is unknown if this affects AGA processing or activity. Here, we have directly compared the Ser149/Thr149 variants of AGA and show that they do not differ in terms of relative specific activity or processing. Therefore, Thr149 AGA, which is the rare variant, can be considered as a neutral or benign variant. Furthermore, we have here produced codon-optimized versions of these two variants and show that they are expressed at significantly higher levels than AGA with the natural codon-usage. Since optimal AGA expression is of vital importance for both gene therapy and enzyme replacement, our data suggest that use of codon-optimized AGA may be beneficial for these therapy options.


Assuntos
Aspartilglucosilaminase/metabolismo , Aspartilglucosilaminase/química , Aspartilglucosilaminase/genética , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Frequência do Gene , Genótipo , Células HEK293 , Células HeLa , Humanos , Doenças por Armazenamento dos Lisossomos/enzimologia , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/patologia , Lisossomos/química , Lisossomos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Polimorfismo de Nucleotídeo Único , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transfecção
14.
Int J Mol Sci ; 16(3): 6447-63, 2015 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-25803106

RESUMO

Acetylcholine and its receptors regulate numerous cellular processes in keratinocytes and other non-neuronal cells. Muscarinic acetylcholine receptors are capable of transactivating the epidermal growth factor receptor (EGFR) and, downstream thereof, the mitogen-activated protein kinase (MAPK) cascade, which in turn regulates transcription of genes involved in cell proliferation and migration. We here show that cholinergic stimulation of human HaCaT keratinocytes results in increased transcription of matrix metalloproteinase MMP-3 as well as several ligands of the epidermal growth factor family. Since both metalloproteinases and the said ligands are involved in the transactivation of the EGFR, this transcriptional upregulation may provide a positive feed-forward loop for EGFR/MAPK activation. We here also show that the cholinergic EGFR and MAPK activation and the upregulation of MMP-3 and EGF-like ligands are dependent on the expression of flotillin-1 which we have previously shown to be a regulator of MAPK signaling.


Assuntos
Receptores ErbB/metabolismo , Queratinócitos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Western Blotting , Linhagem Celular , Humanos
15.
Basic Res Cardiol ; 109(6): 439, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25204797

RESUMO

Endothelial cells are important elements in the vascular response to danger-associated molecules signaling through toll-like receptors (TLRs). Flotillin-1 and -2 are markers of membrane rafts but their true endothelial function is unknown. We hypothesized that flotillins are required for TLR signaling in human umbilical vein endothelial cells (HUVECs). Knockdown of flotillin-1 by shRNA decreased the TLR3-mediated poly-I:C-induced but not the TLR4-mediated LPS-induced inflammatory activation of HUVEC. As TLR3 but not TLR4 signals through the endosomal compartment, flotillin-1 might be involved in the transport of poly-I:C to its receptor. Consistently, uptake of poly-I:C was attenuated by flotillin-1 knockdown and probably involved the scavenger receptor SCARA4 as revealed by knockdown of this receptor. To determine the underlying mechanism, SILAC proteomics was performed. Down-regulation of flotillin-1 led to a reduction of the structural caveolae proteins caveolin-1, cavin-1 and -2, suggesting a role of flotillin-1 in caveolae formation. Flotillin-1 and caveolin-1 colocalized within the cell, and knockdown of flotillin-1 decreased caveolin-1 expression in an endoplasmic reticulum stress-dependent manner. Importantly, downregulation of caveolin-1 also attenuated TLR3-induced signaling. To demonstrate the importance of this finding, cell adhesion was studied. Flotillin-1 shRNA attenuated the poly-I:C-mediated induction of the adhesion molecules VCAM-1 and ICAM-1. As a consequence, the poly-I:C-induced adhesion of peripheral blood mononuclear cells onto HUVECs was significantly attenuated by flotillin-1 shRNA. Collectively, these data suggest that interaction between flotillin-1 and caveolin-1 may facilitate the transport of TLR3-ligands to its intracellular receptor and enables inflammatory TLR3 signaling.


Assuntos
Células Endoteliais/fisiologia , Proteínas de Membrana/fisiologia , Transdução de Sinais/fisiologia , Receptor 3 Toll-Like/fisiologia , Humanos
16.
Int J Mol Sci ; 15(11): 21433-54, 2014 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-25421240

RESUMO

Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP) kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR), which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation.


Assuntos
Receptores ErbB/genética , Queratinócitos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Receptores Muscarínicos/genética , Ativação Transcricional/genética , Linhagem Celular , Endocitose/genética , Ativação Enzimática/genética , Fator de Crescimento Epidérmico/genética , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosforilação/genética , Proteína Quinase C/genética , Transdução de Sinais/genética , Ubiquitinação/genética , Quinases da Família src/genética
17.
J Invest Dermatol ; 144(2): 263-272.e8, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37717934

RESUMO

Pemphigus vulgaris (PV) is an autoimmune blistering disorder of the skin and/or mucous membranes caused by IgG autoantibodies that predominantly target two transmembrane desmosomal cadherins: desmoglein (DSG)1 and DSG3. DSG-specific T cells play a central role in PV pathogenesis because they provide help to autoreactive B cells for autoantibody production. In this study, we characterized DSG3-specific peripheral T cells in a cohort of 52 patients with PV and 41 healthy controls with regard to cytokine profile and epitope specificity. By ELISpot analysis, type 2 T cells reactive with the DSG3 ectodomain were significantly increased in patients with PV compared with those in healthy controls. By dextramer analysis, CD4+ T cells specific for an epitope within the extracellular domain of DSG3, DSG3(206-220), were found at significantly higher frequencies in patients with PV than in HLA-matched healthy controls. T-cell recognition of two distinct DSG3 epitopes, that is, DSG3(206-220) and DSG3(378-392), correlated significantly, suggesting a synergistic effect in B-cell help. Immunization of HLA-DRB1∗04:02-transgenic mice with PV with the same set of DSG3 peptides induced pathogenic DSG3-specific IgG antibodies, which induced loss of keratinocyte adhesion in vitro. Thus, DSG3 peptide-specific T cells are of particular interest as surrogate markers of disease activity and potential therapeutic targets in PV.


Assuntos
Pênfigo , Animais , Humanos , Camundongos , Autoanticorpos , Desmogleína 1 , Desmogleína 3/genética , Epitopos , Imunoglobulina G , Camundongos Transgênicos , Peptídeos
18.
J Biol Chem ; 287(10): 7265-78, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22232557

RESUMO

Our previous work has shown that the membrane microdomain-associated flotillin proteins are potentially involved in epidermal growth factor (EGF) receptor signaling. Here we show that knockdown of flotillin-1/reggie-2 results in reduced EGF-induced phosphorylation of specific tyrosines in the EGF receptor (EGFR) and in inefficient activation of the downstream mitogen-activated protein (MAP) kinase and Akt signaling. Although flotillin-1 has been implicated in endocytosis, its depletion affects neither the endocytosis nor the ubiquitination of the EGFR. However, EGF-induced clustering of EGFR at the cell surface is altered in cells lacking flotillin-1. Furthermore, we show that flotillins form molecular complexes with EGFR in an EGF/EGFR kinase-independent manner. However, knockdown of flotillin-1 appears to affect the activation of the downstream MAP kinase signaling more directly. We here show that flotillin-1 forms a complex with CRAF, MEK1, ERK, and KSR1 (kinase suppressor of RAS) and that flotillin-1 knockdown leads to a direct inactivation of ERK1/2. Thus, flotillin-1 plays a direct role during both the early phase (activation of the receptor) and late (activation of MAP kinases) phase of growth factor signaling. Our results here unveil a novel role for flotillin-1 as a scaffolding factor in the regulation of classical MAP kinase signaling. Furthermore, our results imply that other receptor-tyrosine kinases may also rely on flotillin-1 upon activation, thus suggesting a general role for flotillin-1 as a novel factor in receptor-tyrosine kinase/MAP kinase signaling.


Assuntos
Receptores ErbB/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Ativação Enzimática/fisiologia , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Proteínas de Membrana/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Complexos Multiproteicos/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo
19.
BMC Cancer ; 13: 575, 2013 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-24304721

RESUMO

BACKGROUND: Flotillin-1 and flotillin-2 are two homologous and ubiquitously expressed proteins that are involved in signal transduction and membrane trafficking. Recent studies have reported that flotillins promote breast cancer progression, thus making them interesting targets for breast cancer treatment. In the present study, we have investigated the underlying molecular mechanisms of flotillins in breast cancer. METHODS: Human adenocarcinoma MCF7 breast cancer cells were stably depleted of flotillins by means of lentivirus mediated short hairpin RNAs. Western blotting, immunofluorescence and quantitative real-time PCR were used to analyze the expression of proteins of the epidermal growth factor receptor (EGFR) family. Western blotting was used to investigate the effect of EGFR stimulation or inhibition as well as phosphatidylinositol 3-kinase (PI3K) inhibition on mitogen activated protein kinase (MAPK) signaling. Rescue experiments were performed by stable transfection of RNA intereference resistant flotillin proteins. RESULTS: We here show that stable knockdown of flotillin-1 in MCF7 cells resulted in upregulation of EGFR mRNA and protein expression and hyperactivation of MAPK signaling, whereas ErbB2 and ErbB3 expression were not affected. Treatment of the flotillin knockdown cells with an EGFR inhibitor reduced the MAPK signaling, demonstrating that the increased EGFR expression and activity is the cause of the increased signaling. Stable ectopic expression of flotillins in the knockdown cells reduced the increased EGFR expression, demonstrating a direct causal relationship between flotillin-1 expression and EGFR amount. Furthermore, the upregulation of EGFR was dependent on the PI3K signaling pathway which is constitutively active in MCF7 cells, and PI3K inhibition resulted in reduced EGFR expression. CONCLUSIONS: This study demonstrates that flotillins may not be suitable as cancer therapy targets in cells that carry certain other oncogenic mutations such as PI3K activating mutations, as unexpected effects are prone to emerge upon flotillin knockdown which may even facilitate cancer cell growth and proliferation.


Assuntos
Receptores ErbB/metabolismo , Proteínas de Membrana/genética , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Mama , Proliferação de Células , Cromonas/farmacologia , Endocitose , Fator de Crescimento Epidérmico/fisiologia , Receptores ErbB/genética , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Células MCF-7 , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Transporte Proteico , Regulação para Cima
20.
Nat Cell Biol ; 8(2): 163-9, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16429130

RESUMO

Proteins containing ubiquitin-binding domains (UBDs) interact with ubiquitinated targets and regulate diverse biological processes, including endocytosis, signal transduction, transcription and DNA repair. Many of the UBD-containing proteins are also themselves monoubiquitinated, but the functional role and the mechanisms that underlie this modification are less well understood. Here, we demonstrate that monoubiquitination of the endocytic proteins Sts1, Sts2, Eps15 and Hrs results in intramolecular interactions between ubiquitin and their UBDs, thereby preventing them from binding in trans to ubiquitinated targets. Permanent monoubiquitination of these proteins, mimicked by the fusion of ubiquitin to their carboxyl termini, impairs their ability to regulate trafficking of ubiquitinated receptors. Moreover, we mapped the in vivo monoubiquitination site in Sts2 and demonstrated that its mutation enhances the Sts2-mediated effects of epidermal-growth-factor-receptor downregulation. We propose that monoubiquitination of ubiquitin-binding proteins inhibits their capacity to bind to and control the functions of ubiquitinated targets in vivo.


Assuntos
Proteínas de Transporte/fisiologia , Ubiquitina/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/metabolismo , Receptores ErbB/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisina/genética , Lisina/metabolismo , Proteínas de Membrana , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Tirosina Fosfatases , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Transferrina/metabolismo , Ubiquitina/metabolismo
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