Disruption of TUFT1, a Desmosome-Associated Protein, Causes Skin Fragility, Woolly Hair, and Palmoplantar Keratoderma.

Desmosomes are dynamic complex protein structures involved in cellular adhesion. Disruption of these structures by loss-of-function variants in desmosomal genes leads to a variety of skin- and heart-related phenotypes. In this study, we report TUFT1 as a desmosome-associated protein, implicated in epidermal integrity. In two siblings with mild skin fragility, woolly hair, and mild palmoplantar keratoderma but without a cardiac phenotype, we identified a homozygous splice-site variant in the TUFT1 gene, leading to aberrant mRNA splicing and loss of TUFT1 protein. Patients' skin and keratinocytes showed acantholysis, perinuclear retraction of intermediate filaments, and reduced mechanical stress resistance. Immunolabeling and transfection studies showed that TUFT1 is positioned within the desmosome and that its location is dependent on the presence of the desmoplakin carboxy-terminal tail. A Tuft1-knockout mouse model mimicked the patients' phenotypes. Altogether, this study reveals TUFT1 as a desmosome-associated protein, whose absence causes skin fragility, woolly hair, and palmoplantar keratoderma.

Oligomer-to-monomer transition underlies the chaperone function of AAGAB in AP1/AP2 assembly.

Assembly of protein complexes is facilitated by assembly chaperones. Alpha and gamma adaptin-binding protein (AAGAB) is a chaperone governing the assembly of the heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) involved in clathrin-mediated membrane trafficking. Here, we found that before AP1/2 binding, AAGAB exists as a homodimer. AAGAB dimerization is mediated by its C-terminal domain (CTD), which is critical for AAGAB stability and is missing in mutant proteins found in patients with the skin disease punctate palmoplantar keratoderma type 1 (PPKP1). We solved the crystal structure of the dimerization-mediating CTD, revealing an antiparallel dimer of bent helices. Interestingly, AAGAB uses the same CTD to recognize and stabilize the γ subunit in the AP1 complex and the α subunit in the AP2 complex, forming binary complexes containing only one copy of AAGAB. These findings demonstrate a dual role of CTD in stabilizing resting AAGAB and binding to substrates, providing a molecular explanation for disease-causing AAGAB mutations. The oligomerization state transition mechanism may also underlie the functions of other assembly chaperones.

Snapshots from within the cell: Novel trafficking and non trafficking functions of Snap29 during tissue morphogenesis.

Membrane trafficking is a core cellular process that supports diversification of cell shapes and behaviors relevant to morphogenesis during development and in adult organisms. However, how precisely trafficking components regulate specific differentiation programs is incompletely understood. Snap29 is a multifaceted Soluble N-ethylmaleimide-sensitive factor Attachment protein Receptor, involved in a wide range of trafficking and non-trafficking processes in most cells. A body of knowledge, accrued over more than two decades since its discovery, reveals that Snap29 is essential for establishing and maintaining the operation of a number of cellular events that support cell polarity and signaling. In this review, we first summarize established functions of Snap29 and then we focus on novel ones in the context of autophagy, Golgi trafficking and vesicle fusion at the plasma membrane, as well as on non-trafficking activities of Snap29. We further describe emerging evidence regarding the compartmentalisation and regulation of Snap29. Finally, we explore how the loss of distinct functions of human Snap29 may lead to the clinical manifestations of congenital disorders such as CEDNIK syndrome and how altered SNAP29 activity may contribute to the pathogenesis of cancer, viral infection and neurodegenerative diseases.

Olmsted syndrome causing point mutants of TRPV3 (G568C and G568D) show defects in intracellular Ca2+-mobilization and induce lysosomal defects.

TRPV3, a non-selective cation channel known to be activated by physiological temperature, is expressed in skin and is involved in different skin functions. Point mutations in TRPV3 cause severe pathological condition, known as Olmsted Syndrome (OS). Now we demonstrate that two OS-inducing point mutations (G568C and G568D) located at the lipid-water-interface region joining TM4 with the loop4 of TRPV3 cause reduced cell size and major defects in lysosomal numbers, and distribution. We detected these two mutants in the lysosome. However, G568C and G568D mutants differ from themselves and also from Wild-type in terms of Ca2+-influx in response to activation by agonist (FPP). These two mutants fail to mobilise Ca2+ from intracellular stores, especially when cytosolic Ca2+ is chelated and/or in absence of extracellular Ca2+. We demonstrate that OS-mutants cause defective pH-maintenance at the lysosomes. We propose that G568C and G568D mutants most-likely act as Ca2+-leaky channels from lysosomes with different abilities.

Biochemical Basis of Skin Disease Mal de Meleda: SLURP-1 Mutants Differently Affect Keratinocyte Proliferation and Apoptosis.

Mal de Meleda is an autosomal recessive palmoplantar keratoderma associated with mutations in a gene encoding SLURP-1. SLURP-1 controls growth, differentiation, and apoptosis of keratinocytes by interaction with α7-type nicotinic acetylcholine receptors. SLURP-1 has a three-finger structure with a ß-structural core (head) and three prolonged loops (fingers). To determine the role of SLURP-1 mutations, we produced 22 mutant variants of the protein, including those involved in Mal de Meleda pathogenesis. All mutants except R71H, R71P, T52A, R96P, and L98P were produced in the folded form. SLURP-1 reduces the growth of Het-1A keratinocytes; thus, we studied the influence of the mutations on its antiproliferative activity. Mutations in loops I and III led to the protein inactivation, whereas most mutations in loop II increased SLURP-1 antiproliferative activity. Alanine substitutions of R96 and L98 residues located in the protein head resulted in the appearance of additional pro-apoptotic activity. Our results agree with the diversity of Mal de Meleda phenotypes. Using obtained functional data, the SLURP-1/α7 type nicotinic acetylcholine receptor complex was modeled in silico. Our study provides functional and structural information about the role of the SLURP-1 mutations in Mal de Meleda pathogenesis and predicts SLURP-1 variants, which could drive the disease.

AP1S1 missense mutations cause a congenital enteropathy via an epithelial barrier defect.

Congenital diarrheal disorders (CDD) comprise > 50 monogenic entities featuring chronic diarrhea of early-onset, including defects in nutrient and electrolyte absorption, enterocyte polarization, enteroendocrine cell differentiation, and epithelial integrity. Diarrhea is also a predominant symptom in many immunodeficiencies, congenital disorders of glycosylation, and in some defects of the vesicular sorting and transporting machinery. We set out to identify the etiology of an intractable diarrhea in 2 consanguineous families by whole-exome sequencing, and identified two novel AP1S1 mutations, c.269T>C (p.Leu90Pro) and c.346G>A (p.Glu116Lys). AP1S1 encodes the small subunit of the adaptor protein 1 complex (AP-1), which plays roles in clathrin coat-assembly and trafficking between trans-Golgi network, endosomes and the plasma membrane. An AP1S1 knock-out (KO) of a CaCo2 intestinal cell line was generated to characterize intestinal AP1S1 deficiency as well as identified mutations by stable expression in KO background. Morphology and prototype transporter protein distribution were comparable between parental and KO cells. We observed altered localization of tight-junction proteins ZO-1 and claudin 3, decreased transepithelial electrical resistance and an increased dextran permeability of the CaCo2-AP1S1-KO monolayer. In addition, lumen formation in 3D cultures of these cells was abnormal. Re-expression of wild-type AP1S1 in CaCo2-AP1S1-KO cells reverted these abnormalities, while expression of AP1S1 containing either missense mutation did not. Our data indicate that loss of AP1S1 function causes an intestinal epithelial barrier defect, and that AP1S1 mutations can cause a non-syndromic form of congenital diarrhea, whereas 2 reported truncating AP1S1 mutations caused MEDNIK syndrome, characterized by mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratodermia.

AAGAB Mutations in 18 Canadian Families With Punctate Palmoplantar Keratoderma and a Possible Link to Cancer.

BACKGROUND: Punctate palmoplantar keratoderma type 1 (PPPK1) presents in late childhood to adulthood with multiple small discrete hyperkeratotic papules on palms and soles. PPPK1 is an autosomal dominant skin disease caused by AAGAB mutations. It has been suggested that PPPK1 may be associated with an increased predisposition to systemic malignancies. OBJECTIVES: To evaluate the presence of AAGAB mutations in Canadian families with PPPK1 and the possible increased predisposition to systemic malignancies. METHODS: Eighteen unrelated Canadian families with PPPK1 were recruited for this study. Genomic DNA was extracted from saliva and PCR amplification was performed for all AAGAB exons and exon/intron junctions. PCR products were sequenced and analyzed for mutations. A family history of malignancy was obtained from the index case and, when possible, from other family members. RESULTS: We have identified 5 heterozygous AAGAB loss of function mutations in 11 families. The mutation c.370 C>T, p.Arg124* was the most prevalent and was identified in 6 families. A splice site mutation, c.451+3delAAGT, was identified in 2 families. The other mutations c.473delG, p.Gly158Glufs*0; c.550-551insAAT, p.Gly183*; and c.505-506 dupAA, p.Asn169Lysfs*6 were each identified in 1 family. Different cancers were reported in 11 families (Table 1 and Supplemental Figure S1). CONCLUSIONS: AAGAB mutations were found in 11 of 18 families with PPPK1. In some families there appears to be an association with cancer.

A genetic model of CEDNIK syndrome in zebrafish highlights the role of the SNARE protein Snap29 in neuromotor and epidermal development.

Homozygous mutations in SNAP29, encoding a SNARE protein mainly involved in membrane fusion, cause CEDNIK (Cerebral Dysgenesis, Neuropathy, Ichthyosis and Keratoderma), a rare congenital neurocutaneous syndrome associated with short life expectancy, whose pathogenesis is unclear. Here, we report the analysis of the first genetic model of CEDNIK in zebrafish. Strikingly, homozygous snap29 mutant larvae display CEDNIK-like features, such as microcephaly and skin defects. Consistent with Snap29 role in membrane fusion during autophagy, we observe accumulation of the autophagy markers p62 and LC3, and formation of aberrant multilamellar organelles and mitochondria. Importantly, we find high levels of apoptotic cell death during early development that might play a yet uncharacterized role in CEDNIK pathogenesis. Mutant larvae also display mouth opening problems, feeding impairment and swimming difficulties. These alterations correlate with defective trigeminal nerve formation and excess axonal branching. Since the paralog Snap25 is known to promote axonal branching, Snap29 might act in opposition with, or modulate Snap25 activity during neurodevelopment. Our vertebrate genetic model of CEDNIK extends the description in vivo of the multisystem defects due to loss of Snap29 and could provide the base to test compounds that might ameliorate traits of the disease.

Collagenous and elastotic marginal plaques of the hand: A potential clue to the diagnosis of alkaptonuria.

Collagenous and elastotic marginal plaques of the hand (CEMPH) is a rare, chronic keratoderma characterized by hyperkeratotic linear plaques located along the radial and ulnar aspects of the hands bilaterally. As an isolated finding, CEMPH occurs secondarily to chronic trauma and photodamage. Herein, CEMPH is described as a manifestation of alkaptonuria (AKU). In addition to keloidal collagen, ochronotic fibers and fragmented, thickened elastic fibers were observed. Additionally, mucin deposition-not previously described in this clinical context-was also identified. Given their overlapping clinicopathologic features, CEMPH due to AKU should be distinguished from the acquired variant as well as acrokeratoelastoidosis.

Structural Basis for Mutations of Human Aquaporins Associated to Genetic Diseases.

Aquaporins (AQPs) are among the best structural-characterized membrane proteins, fulfilling the role of allowing water flux across cellular membranes. Thus far, 34 single amino acid polymorphisms have been reported in HUMSAVAR for human aquaporins as disease-related. They affect AQP2, AQP5 and AQP8, where they are associated with nephrogenic diabetes insipidus, keratoderma and colorectal cancer, respectively. For half of these mutations, although they are mostly experimentally characterized in their dysfunctional phenotypes, a structural characterization at a molecular level is still missing. In this work, we focus on such mutations and discuss what the structural defects are that they appear to cause. To achieve this aim, we built a 3D molecular model for each mutant and explored the effect of the mutation on all of their structural features. Based on these analyses, we could collect the structural defects of all the pathogenic mutations (here or previously analysed) under few main categories, that we found to nicely correlate with the experimental phenotypes reported for several of the analysed mutants. Some of the structural analyses we present here provide a rationale for previously experimentally observed phenotypes. Furthermore, our comprehensive overview can be used as a reference frame for the interpretation, on a structural basis, of defective phenotypes of other aquaporin pathogenic mutants.

The keratin 16 null phenotype is modestly impacted by genetic strain background in mice.

The type I intermediate filament keratin 16 (K16) is constitutively expressed in ectoderm-derived appendages and is inducibly expressed in the epidermis upon barrier-compromising challenges. Dominantly acting missense alleles in KRT16 are causative for pachyonychia congenita (PC), a genodermatosis involving debilitating palmoplantar keratoderma (PPK), nail dystrophy, oral lesions and, frequently, alterations in glands and hair. C57Bl/6;Krt16-/- mice develop oral lesions early after birth and PC-like PPK lesions as young adults. These PPK lesions have a marked dysregulation of skin barrier-related genes and innate immunity effectors (eg danger-associated molecular patterns) and are preceded by oxidative stress secondary to hypoactive Nrf2 signalling. These molecular features are present in PPK lesions of PC patients. Here, we report that all components of the C57Bl/6;Krt16-/- mouse phenotype occur as well in the FVB strain background, albeit less severely so, a significant observation in the light of variations in the clinical presentation of individuals harbouring disease-causing mutations in the KRT16 gene.

NEK3-mediated SNAP29 phosphorylation modulates its membrane association and SNARE fusion dependent processes.

Intracellular membrane fusion depends on the presence of specific mediators, the vesicle (v-) and the target (t-) SNAREs (Soluble N-ethylmaleimide-sensitive factor, NSF, attachment protein SNAP receptors), whose interaction brings apposing membranes to close proximity and initiates their fusion. SNAP29 (synaptosomal-associated protein 29), a t-SNARE protein, is involved in multiple fusion events during intracellular transport and affects structure of organelles such as the Golgi apparatus and focal adhesions. Mutations in SNAP29 gene result in CEDNIK (Cerebral dysgenesis, neuropathy, ichthyosis and palmoplantar keratoderma) syndrome. In the present study, we show that NEK3 (NIMA-never in mitosis gene A-related kinase 3)-mediated serine 105 (S105) phosphorylation of SNAP29 directs its membrane association, without which cells present defective focal adhesion formation, impaired Golgi structure and attenuated cellular recycling. In contrast to a phosphorylation-defective serine 105 to alanine (S105A) mutant, wildtype SNAP29, partially rescued the abnormal morphology of a CEDNIK patient derived fibroblasts. Our results highlight the importance of NEK3-mediated S105 phosphorylation of SNAP29 for its membrane localization and for membrane fusion dependent processes.

Exome sequencing identifies a TCF4 mutation in a Chinese pedigree with symmetrical acral keratoderma.

BACKGROUND: Symmetrical acral keratoderma (SAK) is a rare skin disorder and its pathogenesis and inheritability are unknown. OBJECTIVES: To investigate the inheritance and pathogenesis of SAK. METHODS: Four SAK cases occurred in a four-generation Chinese family. Exome sequencing identified SNPs with potential SAK-related mutations, and a potentially responsible gene transcription factor 4 (TCF4) was identified. TCF4 was then sequenced in all 11 family members, and pedigree analysis was performed. Histopathology and immunohistochemistry evaluated TCF4 expression in skin lesions. The gene mutation was investigated in human keratinocytes for keratin-related protein expression. RESULTS: A novel heterozygous missense mutation, c.85C>A (p.Pro29Thr) was found in TCF4. The mutation showed autosomal dominant inheritance and perfectly cosegregated with the SAK phenotype in all family members. In skin lesions, TCF4 was present in the cytoplasm and membranes of the basal layer, the stratum spinosum and the stratum granulosum of the epidermis. The mutant TCF4 induced overexpression of differentiation markers including KRT1, KRT14, loricrin and involucrin. CONCLUSIONS: A SAK-related gene mutation in TCF4 may function through transcriptional regulation of keratin.

Disease-linked connexin26 S17F promotes volar skin abnormalities and mild wound healing defects in mice.

Several mutant mice have been generated to model connexin (Cx)-linked skin diseases; however, the role of connexins in skin maintenance and during wound healing remains to be fully elucidated. Here we generated a novel, viable, and fertile mouse (Cx26CK14-S17F/+) with the keratitis-ichthyosis-deafness mutant (Cx26S17F) driven by the cytokeratin 14 promoter. This mutant mouse mirrors several Cx26-linked human skin pathologies suggesting that the etiology of Cx26-linked skin disease indeed stems from epidermal expression of the Cx26 mutant. Cx26CK14-S17F/+ foot pad epidermis formed severe palmoplantar keratoderma, which expressed elevated levels of Cx26 and filaggrin. Primary keratinocytes isolated from Cx26CK14-S17F/+ neonates exhibited reduced gap junctional intercellular communication and migration. Furthermore, Cx26CK14-S17F/+ mouse skin wound closure was normal but repaired epidermis appeared hyperplastic with elevated expression of cytokeratin 6. Taken together, we suggest that the Cx26S17F mutant disturbs keratinocyte differentiation and epidermal remodeling following wound closure. We further posit that Cx26 contributes to epidermal homeostasis by regulating keratinocyte differentiation, and that mice harboring a disease-linked Cx26 mutant display epidermal abnormalities yet retain most wound healing properties.

PIGO deficiency: palmoplantar keratoderma and novel mutations.

BACKGROUND: Several genetic defects have been identified in the glycosylphosphatidylinositol (GPI) anchor synthesis, including mutations in PIGO encoding phosphatidylinositol glycan anchor biosynthesis class O protein. These defects constitute a subgroup of the congenital disorders of glycosylation (CDG). Seven patients from five families have been reported carrying variants in PIGO that cause an autosomal recessive syndrome characterised by dysmorphism, psychomotor disability, epilepsy and hyperphosphatasemia. METHODS: Whole exome sequencing was performed in a boy with dysmorphism, psychomotor disability, epilepsy, palmoplantar keratoderma, hyperphosphatasemia and platelet dysfunction without a clinical bleeding phenotype. RESULTS: Two novel variants in PIGO were detected. The missense variant encoding p. His871Pro was inherited from the boy's father while the frameshift variant encoding p. Arg604ProfsTer40 was maternally inherited. CONCLUSION: A boy with two novel PIGO variants is reported. The skin phenotype and platelet dysfunction in this patient have not been described in previously reported patients with PIGO deficiency but it is of course uncertain whether these are caused by this disorder. The literature on PIGO deficiency is reviewed.

Decreases in 15-lipoxygenase metabolites in Olmsted syndrome model rats.

BACKGROUND: Olmsted syndrome (OS) is a congenital dermatosis characterized by palmoplantar keratoderma and periorificial keratotic plaque. TRPV3 (transient receptor potential vanilloid subtype 3) encodes a thermosensitive Ca2+ channel and is the causative gene of OS. However, the molecular mechanism that causes the pathological development of OS is unclear. OBJECTIVE: We aimed to investigate the molecular mechanisms underlying OS pathology from the perspective of lipid metabolism. METHODS: Comprehensive lipidomics and microarray analyses were conducted on tissue samples from a non-lesional skin area of OS model rats (Ht rats) and from wild type (WT) rats as the control. RESULTS: Infiltration of leukocytes such as eosinophils and neutrophils and an increase in the fibrotic region were detected in the unaffected skin area of Ht rats compared with the WT rats. Among about 600 lipid species examined, the levels of 15-lipoxygenase (LOX) metabolites, the precursors of anti-inflammatory and pro-resolving lipid mediators, and dihydroceramides decreased by ≥16-fold in Ht rats compared with WT rats. Consistent with the decreases in the 15-LOX metabolites, expression levels of the genes that encode the 15-LOXs, Alox15 and Alox15b, were largely reduced. Conversely, increased expression levels were detected of Il36b, Ccl20, Cxcl1, and Cxcl2, which encode cytokines/chemokines, and S100a8 and S100a9, which encode the Ca2+ binding proteins that are implicated in epidermal proliferation. CONCLUSION: The pro-inflammatory state in the unaffected skin of Ht rats caused by decreases in 15-LOX metabolites and increases in cytokines/chemokines may contribute to the pathogenesis of OS.