Keratin 14-dependent disulfides regulate epidermal homeostasis and barrier function via 14-3-3σ and YAP1

The intermediate filament protein keratin 14 (K14) provides vital structural support in basal keratinocytes of epidermis. Recent studies evidenced a role for K14-dependent disulfide bonding in the organization and dynamics of keratin IFs in skin keratinocytes. Here we report that knock-in mice harboring a cysteine-to-alanine substitution at Krt14’s codon 373 (C373A) exhibit alterations in disulfide-bonded K14 species and a barrier defect secondary to enhanced proliferation, faster transit time and altered differentiation in epidermis. A proteomics screen identified 14-3-3 as K14 interacting proteins. Follow-up studies showed that YAP1, a transcriptional effector of Hippo signaling regulated by 14-3-3sigma in skin keratinocytes, shows aberrant subcellular partitioning and function in differentiating Krt14 C373A keratinocytes. Residue C373 in K14, which is conserved in a subset of keratins, is revealed as a novel regulator of keratin organization and YAP function in early differentiating keratinocytes, with an impact on cell mechanics, homeostasis and barrier function in epidermis.

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Keratin 14-dependent disulfides regulate epidermal homeostasis and barrier function via 14-3-3σ and YAP1

10.1101/824219 ◽

2019 ◽

Author(s):

Yajuan Guo ◽

Krystynne A. Leacock ◽

Catherine Redmond ◽

Vinod Jaskula-Ranga ◽

Pierre A. Coulombe

Keyword(s):

Barrier Function ◽

Type I ◽

Hippo Signaling ◽

Disulfide Bonding ◽

Structural Support ◽

Skin Keratinocytes ◽

Keratin 14 ◽

Barrier Defect ◽

Epidermal Homeostasis ◽

And Function

SummaryThe type I intermediate filament (IF) keratin 14 (K14) provides vital structural support in basal keratinocytes of epidermis. Recent studies evidenced a role for K14-dependent disulfide bonding in the organization and dynamics of keratin IFs in skin keratinocytes. Here we report that knock-in mice harboring a cysteine-to-alanine substitution at codon 373 (C373A) in Krt14 exhibit alterations in disulfide-bonded K14 species and a barrier defect secondary to enhanced proliferation, faster transit time and altered differentiation in the epidermis. A proteomics screen identified 14-3-3 as major K14 interacting proteins. Follow-up studies showed that YAP1, a transcriptional effector of Hippo signaling regulated by 14-3-3sigma in skin keratinocytes, shows aberrant subcellular partitioning and function in differentiating Krt14C373A keratinocytes. Residue C373 in K14, which is conserved in several other type I IFs, is thus revealed as a novel regulator of keratin organization and YAP function in early differentiating keratinocytes, with an impact on cell mechanics, homeostasis and barrier function in the epidermis.

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692 Keratin-14 dependent disulfide bonding contributes to epidermal homeostasis and barrier function through 14-3-3 and Hippo signaling in mouse skin in vivo

Journal of Investigative Dermatology ◽

10.1016/j.jid.2018.03.701 ◽

2018 ◽

Vol 138 (5) ◽

pp. S118

Author(s):

Y. Guo ◽

K. Leacock ◽

V. Ranganathan ◽

P. Coulombe

Keyword(s):

Barrier Function ◽

Mouse Skin ◽

Hippo Signaling ◽

Disulfide Bonding ◽

Keratin 14 ◽

Epidermal Homeostasis

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Purification of desmin from adult mammalian skeletal muscle

Biochemical Journal ◽

10.1042/bj1950345 ◽

1981 ◽

Vol 195 (2) ◽

pp. 345-356 ◽

Cited By ~ 29

Author(s):

J M O'Shea ◽

R M Robson ◽

M K Hartzer ◽

T W Huiatt ◽

W E Rathbun ◽

...

Keyword(s):

Skeletal Muscle ◽

Average Diameter ◽

Intermediate Filament Protein ◽

Mammalian Skeletal Muscle ◽

Cytoskeletal Function ◽

10 Nm Filaments ◽

And Function ◽

Filament Protein

A method has been developed for preparation of purified desmin from mature mammalian (porcine) skeletal muscle. A crude desmin-containing fraction was prepared by modification of procedures used for isolation of smooth-muscle intermediate-filament protein [Small & Sobieszek (1977) J. Cell Sci. 23, 243-268]. The desmin was extracted in 1 M-acetic acid/20 mM-NaCl at 4 degrees C for 15h from the residue remaining after actomyosin extraction from washed myofibrils. Successive chromatography on hydroxyapatite and DEAE-Sepharose CL-6B in 6M-urea yielded desmin that was routinely more than 97% 55 000-dalton protein and that had no detectable actin contamination. Removal of urea by dialysis against 10mM-Tris/acetate (pH 8.5)/1 mM dithioerythritol and subsequent clarification at 134 000 g (rav. 5.9 cm) for 1 h results in a clear desmin solution. Dialysis of purified desmin against 100 mM-NaCl/1 mM-MgCl2/10 mM-imidazole/HCl, pH 7.0, at 2 degrees C resulted in the formation of synthetic desmin filaments have an average diameter of 9-11.5 nm. The present studies demonstrate that the relatively small amount of desmin in mature skeletal muscle can be isolated in sufficient quantity and purity to permit detailed studies of its properties and function. Although 10nm filaments have not been unequivocally demonstrated in mature muscle in vivo, that the purified skeletal-muscle desmin will form 10 nm filaments in vitro lends support to their possible existence and cytoskeletal function in mature skeletal-muscle cells.

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The Endothelial Cell-Specific Antibody PAL-E Identifies a Secreted Form of Vimentin in the Blood Vasculature

Molecular and Cellular Biology ◽

10.1128/mcb.24.20.9198-9206.2004 ◽

2004 ◽

Vol 24 (20) ◽

pp. 9198-9206 ◽

Cited By ~ 79

Author(s):

Bin Xu ◽

Robert M. deWaal ◽

Nirit Mor-Vaknin ◽

Chris Hibbard ◽

David M. Markovitz ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Lymphatic Vessels ◽

Mass Spectrometry Analysis ◽

Intermediate Filament Protein ◽

Spectrometry Analysis ◽

Tandem Mass Spectrometry Analysis ◽

And Function ◽

Filament Protein ◽

Specific Mrna

ABSTRACT During mammalian vascular development, endothelial cells form a complex array of vessels that differ markedly in structure and function, but the molecular basis for this vascular complexity is poorly understood. Recent insights into endothelial diversity have come from the identification of molecular markers expressed on distinct endothelial cell populations. One such marker, the PAL-E antibody, has been used for almost 20 years to distinguish blood and lymphatic vessels, but the identity of the protein recognized by PAL-E has been unknown. In the present study we have used protein purification and tandem mass spectrometry analysis of tryptic peptides to identify the PAL-E antigen as a secreted form of vimentin. Vimentin has been well characterized as an intracellular intermediate filament protein expressed broadly in mesenchymal cells. In contrast, PAL-E-reactive vimentin is secreted extracellularly, its synthesis is restricted to a distinct population of blood endothelial cells and activated macrophages, and PAL-E-reactive vimentin is found in circulating human blood. PAL-E-reactive vimentin does not arise from an endothelial cell-specific mRNA transcript but is the product of cell-specific posttranslational modification. The PAL-E antibody therefore defines secretion of vimentin as a molecular distinction among endothelial cells and exposes a novel, extracellular role for vimentin in the blood vasculature.

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Decision letter: Keratin 14-dependent disulfides regulate epidermal homeostasis and barrier function via 14-3-3σ and YAP1

10.7554/elife.53165.sa1 ◽

2019 ◽

Keyword(s):

Barrier Function ◽

Keratin 14 ◽

Epidermal Homeostasis

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Faculty Opinions recommendation of SUMO regulates the assembly and function of a cytoplasmic intermediate filament protein in C. elegans.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1282028.749119 ◽

2009 ◽

Author(s):

Yosef Gruenbaum ◽

Daniel Bar

Keyword(s):

Intermediate Filament ◽

Intermediate Filament Protein ◽

C Elegans ◽

And Function ◽

Filament Protein

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Keratin 8 protection of placental barrier function

The Journal of Cell Biology ◽

10.1083/jcb.200210004 ◽

2003 ◽

Vol 161 (4) ◽

pp. 749-756 ◽

Cited By ~ 52

Author(s):

Daniel Jaquemar ◽

Sergey Kupriyanov ◽

Miriam Wankell ◽

Jacqueline Avis ◽

Kurt Benirschke ◽

...

Keyword(s):

Giant Cell ◽

Barrier Function ◽

Genetic Background ◽

Cell Layer ◽

Giant Cells ◽

Intermediate Filament Protein ◽

Trophoblast Giant Cell ◽

Primary Defect ◽

Keratin 8 ◽

Filament Protein

The intermediate filament protein keratin 8 (K8) is critical for the development of most mouse embryos beyond midgestation. We find that 68% of K8−/− embryos, in a sensitive genetic background, are rescued from placental bleeding and subsequent death by cellular complementation with wild-type tetraploid extraembryonic cells. This indicates that the primary defect responsible for K8−/− lethality is trophoblast giant cell layer failure. Furthermore, the genetic absence of maternal but not paternal TNF doubles the number of viable K8−/− embryos. Finally, we show that K8−/− concepti are more sensitive to a TNF-dependent epithelial apoptosis induced by the administration of concanavalin A (ConA) to pregnant mothers. The ConA-induced failure of the trophoblast giant cell barrier results in hematoma formation between the trophoblast giant cell layer and the embryonic yolk sac in a phenocopy of dying K8-deficient concepti in a sensitive genetic background. We conclude the lethality of K8−/− embryos is due to a TNF-sensitive failure of trophoblast giant cell barrier function. The keratin-dependent protection of trophoblast giant cells from a maternal TNF-dependent apoptotic challenge may be a key function of simple epithelial keratins.

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