Skin integrity is essential for protection from external stress and trauma. Defects in structural proteins such as keratins cause skin fragility, epitomized by epidermolysis bullosa (EB), a life-threatening disorder. Here we show that dominant mutations of KLHL24, encoding a cullin 3-RBX1 ubiquitin ligase substrate receptor, cause EB. We have identified start-codon mutations in the KLHL24 gene in five patients with EB. These mutations lead to truncated KLHL24 protein lacking the initial 28 amino acids (KLHL24-ΔN28). KLHL24-ΔN28 is more stable than its wild-type counterpart owing to abolished autoubiquitination. We have further identified keratin 14 (KRT14) as a KLHL24 substrate and found that KLHL24-ΔN28 induces excessive ubiquitination and degradation of KRT14. Using a knock-in mouse model, we have confirmed that the Klhl24 mutations lead to stabilized Klhl24-ΔN28 and cause Krt14 degradation. Our findings identify a new disease-causing mechanism due to dysregulation of autoubiquitination and open new avenues for the treatment of related disorders.

BackgroundThe formation and development of bulblets are crucial to the Lilium genus since these processes are closely related to carbohydrate metabolism, especially to starch and sucrose metabolism. However, little is known about the transcriptional regulation of both processes. To gain insight into carbohydrate-related genes involved in bulblet formation and development, we conducted comparative transcriptome profiling of Lilium davidii var. unicolor bulblets at 0 d, 15 d (bulblets emerged) and 35 d (bulblets formed a basic shape with three or four scales) after scale propagation.ResultsAnalysis of the transcriptome revealed that a total of 52,901 unigenes with an average sequence size of 630 bp were generated. Based on Clusters of Orthologous Groups (COG) analysis, 8% of the sequences were attributed to carbohydrate transport and metabolism. The results of KEGG pathway enrichment analysis showed that starch and sucrose metabolism constituted the predominant pathway among the three library pairs. The starch content in mother scales and bulblets decreased and increased, respectively, with almost the same trend as sucrose content. Gene expression analysis of the key enzymes in starch and sucrose metabolism suggested that sucrose synthase (SuSy) and invertase (INV), mainly hydrolyzing sucrose, presented higher gene expression in mother scales and bulblets at stages of bulblet appearance and enlargement, while sucrose phosphate synthase (SPS) showed higher expression in bulblets at morphogenesis. The enzymes involved in the starch synthetic direction such as ADPG pyrophosphorylase (AGPase), soluble starch synthase (SSS), starch branching enzyme (SBE) and granule-bound starch synthase (GBSS) showed a decreasing trend in mother scales and higher gene expression in bulblets at bulblet appearance and enlargement stages while the enzyme in the cleavage direction, starch de-branching enzyme (SDBE), showed higher gene expression in mother scales than in bulblets.ConclusionsAn extensive transcriptome analysis of three bulblet development stages contributes considerable novel information to our understanding of carbohydrate metabolism-related genes in Lilium at the transcriptional level, and demonstrates the fundamentality of carbohydrate metabolism in bulblet emergence and development at the molecular level. This could facilitate further investigation into the molecular mechanisms underlying these processes in lily and other related species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0358-4) contains supplementary material, which is available to authorized users.

Isoform 2 of the ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. In the present study, two kinds of RyR2 cDNA were constructed, one encoding the wild type mouse RyR2 (RyR2 wt ) and the other encoding modified RyR2, into which was inserted a cDNA encoding green fluorescent protein (GFP). GFP was inserted into the divergent region 1 (DR1) of RyR2, after the Asp-4365 (RyR2 D4365-GFP ). HEK293 cells expressing both RyR2 wt and RyR2 D4365-GFP cDNAs showed caffeine-and ryanodine-sensitive calcium release, demonstrating that both wild type and modified RyR2s form functional calcium release channels. Cells expressing the fusion protein, RyR2 D4365-GFP , were readily identified by their fluorescence due to the presence of GFP, indicating that the inserted GFP folded properly. Both expressed RyR2s were purified from cell lysates in a single step by affinity chromatography using a GST-FKBP12.6 as the affinity ligand. Cryoelectron microscopy of purified RyR2s showed structurally intact receptors, and three-dimensional reconstructions were obtained by single particle image processing. The three-dimensional reconstruction of RyR2 wt appeared very similar to that of the native RyR2 purified from dog heart. The location of the inserted GFP, and consequently of DR1, was mapped on the three-dimensional structure of RyR2 to one of the subunit's characteristic domains, domain 3, also known as the "handle" domain. This study describes the first internal fusion of a protein into a ryanodine receptor, and it demonstrates the potential of this technology for localizing functional and structural domains on the threedimensional structure of RyR.

Ash1 is a Trithorax group protein that possesses H3K36-specific histone methyltransferase activity, which antagonizes Polycomb silencing. Here we report the identification of two Ash1 complex subunits, Mrg15 and Nurf55. In vitro, Mrg15 stimulates the enzymatic activity of Ash1. In vivo, Mrg15 is recruited by Ash1 to their common targets, and Mrg15 reinforces Ash1 chromatin association and facilitates the proper deposition of H3K36me2. To dissect the functional role of Mrg15 in the context of the Ash1 complex, we identify an Ash1 point mutation (Ash1-R1288A) that displays a greatly attenuated interaction with Mrg15. Knock-in flies bearing this mutation display multiple homeotic transformation phenotypes, and these phenotypes are partially rescued by overexpressing the Mrg15-Nurf55 fusion protein, which stabilizes the association of Mrg15 with Ash1. In summary, Mrg15 is a subunit of the Ash1 complex, a stimulator of Ash1 enzymatic activity and a critical regulator of the TrxG protein function of Ash1 in Drosophila.