Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex
Abstract: Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra-epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutat...
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| Autor*in: |
Sørensen, Charlotte B. [verfasserIn] Andresen, Brage S. [verfasserIn] Jensen, Uffe B. [verfasserIn] |
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E-Artikel |
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| Erschienen: |
Oxford, UK: Munksgaard International Publishers ; 2003 |
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| Umfang: |
Online-Ressource |
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| Reproduktion: |
2003 ; Blackwell Publishing Journal Backfiles 1879-2005 |
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| Übergeordnetes Werk: |
In: Experimental dermatology - Oxford : Wiley-Blackwell, 1992, 12(2003), 4, Seite 0 |
| Übergeordnetes Werk: |
volume:12 ; year:2003 ; number:4 ; pages:0 |
| Links: |
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| DOI / URN: |
10.1034/j.1600-0625.2002.120416.x |
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NLEJ242384447 |
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| 245 | 1 | 0 | |a Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex |
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| 520 | |a Abstract: Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra-epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene.Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3′ end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN).No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). Thus, although a background KFN perturbance was observed upon transfection with the wild-type K14 construct, the mutant protein associated with the most severe form of EBS worsened the KFN perturbation significantly compared with the mutant proteins associated with the milder forms of the disease and the normal K14 protein. This shows that the clinical severity of disease-associated mutations identified in patients can be tested using this expression system, although it can not at present be used to discriminate between the milder forms.Assessment of the endogenous K14 protein expression in NHK and HaCaT cells indicated that the higher level of endogenous keratin expression in NHK might make these cells more resistant to perturbation of the keratin cytoskeleton by overexpressed K14 protein than HaCaT cells. | ||
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| 650 | 4 | |a epidermolysis bullosa simplex | |
| 700 | 1 | |a Andresen, Brage S. |e verfasserin |4 aut | |
| 700 | 1 | |a Jensen, Uffe B. |e verfasserin |4 aut | |
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| 700 | 1 | |a Jensen, Peter K. A. |4 oth | |
| 700 | 1 | |a Gregersen, Niels |4 oth | |
| 700 | 1 | |a Bolund, Lars |4 oth | |
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10.1034/j.1600-0625.2002.120416.x doi (DE-627)NLEJ242384447 DE-627 ger DE-627 rakwb Sørensen, Charlotte B. verfasserin aut Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex Oxford, UK Munksgaard International Publishers 2003 Online-Ressource nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Abstract: Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra-epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene.Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3′ end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN).No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). Thus, although a background KFN perturbance was observed upon transfection with the wild-type K14 construct, the mutant protein associated with the most severe form of EBS worsened the KFN perturbation significantly compared with the mutant proteins associated with the milder forms of the disease and the normal K14 protein. This shows that the clinical severity of disease-associated mutations identified in patients can be tested using this expression system, although it can not at present be used to discriminate between the milder forms.Assessment of the endogenous K14 protein expression in NHK and HaCaT cells indicated that the higher level of endogenous keratin expression in NHK might make these cells more resistant to perturbation of the keratin cytoskeleton by overexpressed K14 protein than HaCaT cells. 2003 Blackwell Publishing Journal Backfiles 1879-2005 |2003|||||||||| epidermolysis bullosa simplex Andresen, Brage S. verfasserin aut Jensen, Uffe B. verfasserin aut Jensen, Thomas G. oth Jensen, Peter K. A. oth Gregersen, Niels oth Bolund, Lars oth In Experimental dermatology Oxford : Wiley-Blackwell, 1992 12(2003), 4, Seite 0 Online-Ressource (DE-627)NLEJ243926081 (DE-600)2026228-0 1600-0625 nnns volume:12 year:2003 number:4 pages:0 http://dx.doi.org/10.1034/j.1600-0625.2002.120416.x text/html Verlag Deutschlandweit zugänglich Volltext GBV_USEFLAG_U ZDB-1-DJB GBV_NL_ARTICLE AR 12 2003 4 0 |
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10.1034/j.1600-0625.2002.120416.x doi (DE-627)NLEJ242384447 DE-627 ger DE-627 rakwb Sørensen, Charlotte B. verfasserin aut Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex Oxford, UK Munksgaard International Publishers 2003 Online-Ressource nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Abstract: Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra-epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene.Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3′ end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN).No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). Thus, although a background KFN perturbance was observed upon transfection with the wild-type K14 construct, the mutant protein associated with the most severe form of EBS worsened the KFN perturbation significantly compared with the mutant proteins associated with the milder forms of the disease and the normal K14 protein. This shows that the clinical severity of disease-associated mutations identified in patients can be tested using this expression system, although it can not at present be used to discriminate between the milder forms.Assessment of the endogenous K14 protein expression in NHK and HaCaT cells indicated that the higher level of endogenous keratin expression in NHK might make these cells more resistant to perturbation of the keratin cytoskeleton by overexpressed K14 protein than HaCaT cells. 2003 Blackwell Publishing Journal Backfiles 1879-2005 |2003|||||||||| epidermolysis bullosa simplex Andresen, Brage S. verfasserin aut Jensen, Uffe B. verfasserin aut Jensen, Thomas G. oth Jensen, Peter K. A. oth Gregersen, Niels oth Bolund, Lars oth In Experimental dermatology Oxford : Wiley-Blackwell, 1992 12(2003), 4, Seite 0 Online-Ressource (DE-627)NLEJ243926081 (DE-600)2026228-0 1600-0625 nnns volume:12 year:2003 number:4 pages:0 http://dx.doi.org/10.1034/j.1600-0625.2002.120416.x text/html Verlag Deutschlandweit zugänglich Volltext GBV_USEFLAG_U ZDB-1-DJB GBV_NL_ARTICLE AR 12 2003 4 0 |
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10.1034/j.1600-0625.2002.120416.x doi (DE-627)NLEJ242384447 DE-627 ger DE-627 rakwb Sørensen, Charlotte B. verfasserin aut Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex Oxford, UK Munksgaard International Publishers 2003 Online-Ressource nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Abstract: Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra-epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene.Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3′ end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN).No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). Thus, although a background KFN perturbance was observed upon transfection with the wild-type K14 construct, the mutant protein associated with the most severe form of EBS worsened the KFN perturbation significantly compared with the mutant proteins associated with the milder forms of the disease and the normal K14 protein. This shows that the clinical severity of disease-associated mutations identified in patients can be tested using this expression system, although it can not at present be used to discriminate between the milder forms.Assessment of the endogenous K14 protein expression in NHK and HaCaT cells indicated that the higher level of endogenous keratin expression in NHK might make these cells more resistant to perturbation of the keratin cytoskeleton by overexpressed K14 protein than HaCaT cells. 2003 Blackwell Publishing Journal Backfiles 1879-2005 |2003|||||||||| epidermolysis bullosa simplex Andresen, Brage S. verfasserin aut Jensen, Uffe B. verfasserin aut Jensen, Thomas G. oth Jensen, Peter K. A. oth Gregersen, Niels oth Bolund, Lars oth In Experimental dermatology Oxford : Wiley-Blackwell, 1992 12(2003), 4, Seite 0 Online-Ressource (DE-627)NLEJ243926081 (DE-600)2026228-0 1600-0625 nnns volume:12 year:2003 number:4 pages:0 http://dx.doi.org/10.1034/j.1600-0625.2002.120416.x text/html Verlag Deutschlandweit zugänglich Volltext GBV_USEFLAG_U ZDB-1-DJB GBV_NL_ARTICLE AR 12 2003 4 0 |
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10.1034/j.1600-0625.2002.120416.x doi (DE-627)NLEJ242384447 DE-627 ger DE-627 rakwb Sørensen, Charlotte B. verfasserin aut Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex Oxford, UK Munksgaard International Publishers 2003 Online-Ressource nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Abstract: Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra-epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene.Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3′ end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN).No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). Thus, although a background KFN perturbance was observed upon transfection with the wild-type K14 construct, the mutant protein associated with the most severe form of EBS worsened the KFN perturbation significantly compared with the mutant proteins associated with the milder forms of the disease and the normal K14 protein. This shows that the clinical severity of disease-associated mutations identified in patients can be tested using this expression system, although it can not at present be used to discriminate between the milder forms.Assessment of the endogenous K14 protein expression in NHK and HaCaT cells indicated that the higher level of endogenous keratin expression in NHK might make these cells more resistant to perturbation of the keratin cytoskeleton by overexpressed K14 protein than HaCaT cells. 2003 Blackwell Publishing Journal Backfiles 1879-2005 |2003|||||||||| epidermolysis bullosa simplex Andresen, Brage S. verfasserin aut Jensen, Uffe B. verfasserin aut Jensen, Thomas G. oth Jensen, Peter K. A. oth Gregersen, Niels oth Bolund, Lars oth In Experimental dermatology Oxford : Wiley-Blackwell, 1992 12(2003), 4, Seite 0 Online-Ressource (DE-627)NLEJ243926081 (DE-600)2026228-0 1600-0625 nnns volume:12 year:2003 number:4 pages:0 http://dx.doi.org/10.1034/j.1600-0625.2002.120416.x text/html Verlag Deutschlandweit zugänglich Volltext GBV_USEFLAG_U ZDB-1-DJB GBV_NL_ARTICLE AR 12 2003 4 0 |
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10.1034/j.1600-0625.2002.120416.x doi (DE-627)NLEJ242384447 DE-627 ger DE-627 rakwb Sørensen, Charlotte B. verfasserin aut Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex Oxford, UK Munksgaard International Publishers 2003 Online-Ressource nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Abstract: Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra-epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene.Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3′ end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN).No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). Thus, although a background KFN perturbance was observed upon transfection with the wild-type K14 construct, the mutant protein associated with the most severe form of EBS worsened the KFN perturbation significantly compared with the mutant proteins associated with the milder forms of the disease and the normal K14 protein. This shows that the clinical severity of disease-associated mutations identified in patients can be tested using this expression system, although it can not at present be used to discriminate between the milder forms.Assessment of the endogenous K14 protein expression in NHK and HaCaT cells indicated that the higher level of endogenous keratin expression in NHK might make these cells more resistant to perturbation of the keratin cytoskeleton by overexpressed K14 protein than HaCaT cells. 2003 Blackwell Publishing Journal Backfiles 1879-2005 |2003|||||||||| epidermolysis bullosa simplex Andresen, Brage S. verfasserin aut Jensen, Uffe B. verfasserin aut Jensen, Thomas G. oth Jensen, Peter K. A. oth Gregersen, Niels oth Bolund, Lars oth In Experimental dermatology Oxford : Wiley-Blackwell, 1992 12(2003), 4, Seite 0 Online-Ressource (DE-627)NLEJ243926081 (DE-600)2026228-0 1600-0625 nnns volume:12 year:2003 number:4 pages:0 http://dx.doi.org/10.1034/j.1600-0625.2002.120416.x text/html Verlag Deutschlandweit zugänglich Volltext GBV_USEFLAG_U ZDB-1-DJB GBV_NL_ARTICLE AR 12 2003 4 0 |
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The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene.Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3′ end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN).No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). 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Blackwell Publishing Journal Backfiles 1879-2005 |
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Sørensen, Charlotte B. |
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Sørensen, Charlotte B. misc epidermolysis bullosa simplex Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex |
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Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex epidermolysis bullosa simplex |
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functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex |
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Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex |
| abstract |
Abstract: Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra-epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene.Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3′ end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN).No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). Thus, although a background KFN perturbance was observed upon transfection with the wild-type K14 construct, the mutant protein associated with the most severe form of EBS worsened the KFN perturbation significantly compared with the mutant proteins associated with the milder forms of the disease and the normal K14 protein. This shows that the clinical severity of disease-associated mutations identified in patients can be tested using this expression system, although it can not at present be used to discriminate between the milder forms.Assessment of the endogenous K14 protein expression in NHK and HaCaT cells indicated that the higher level of endogenous keratin expression in NHK might make these cells more resistant to perturbation of the keratin cytoskeleton by overexpressed K14 protein than HaCaT cells. |
| abstractGer |
Abstract: Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra-epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene.Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3′ end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN).No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). Thus, although a background KFN perturbance was observed upon transfection with the wild-type K14 construct, the mutant protein associated with the most severe form of EBS worsened the KFN perturbation significantly compared with the mutant proteins associated with the milder forms of the disease and the normal K14 protein. This shows that the clinical severity of disease-associated mutations identified in patients can be tested using this expression system, although it can not at present be used to discriminate between the milder forms.Assessment of the endogenous K14 protein expression in NHK and HaCaT cells indicated that the higher level of endogenous keratin expression in NHK might make these cells more resistant to perturbation of the keratin cytoskeleton by overexpressed K14 protein than HaCaT cells. |
| abstract_unstemmed |
Abstract: Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra-epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene.Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3′ end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN).No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). Thus, although a background KFN perturbance was observed upon transfection with the wild-type K14 construct, the mutant protein associated with the most severe form of EBS worsened the KFN perturbation significantly compared with the mutant proteins associated with the milder forms of the disease and the normal K14 protein. This shows that the clinical severity of disease-associated mutations identified in patients can be tested using this expression system, although it can not at present be used to discriminate between the milder forms.Assessment of the endogenous K14 protein expression in NHK and HaCaT cells indicated that the higher level of endogenous keratin expression in NHK might make these cells more resistant to perturbation of the keratin cytoskeleton by overexpressed K14 protein than HaCaT cells. |
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