Abstract

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.

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NCBI Reference Sequence

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References

  1. 1.

    , & Diseases of epidermal keratins and their linker proteins. Exp. Cell Res. 313, 1995–2009 (2007).

  2. 2.

    et al. Inherited epidermolysis bullosa: updated recommendations on diagnosis and classification. J. Am. Acad. Dermatol. 70, 1103–1126 (2014).

  3. 3.

    & The genetics of skin fragility. Annu. Rev. Genomics Hum. Genet. 15, 245–268 (2014).

  4. 4.

    & Molecular heterogeneity of blistering disorders: the paradigm of epidermolysis bullosa. J. Invest. Dermatol. 132, E2–E5 (2012).

  5. 5.

    & Defining keratin protein function in skin epithelia: epidermolysis bullosa simplex and its aftermath. J. Invest. Dermatol. 132, 763–775 (2012).

  6. 6.

    , & Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities. Science 254, 1202–1205 (1991).

  7. 7.

    et al. Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses. Cell 66, 1301–1311 (1991).

  8. 8.

    et al. A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering. Nature 356, 244–246 (1992).

  9. 9.

    et al. Loss of plectin causes epidermolysis bullosa with muscular dystrophy: cDNA cloning and genomic organization. Genes Dev. 10, 1724–1735 (1996).

  10. 10.

    et al. Plectin deficiency results in muscular dystrophy with epidermolysis bullosa. Nat. Genet. 13, 450–457 (1996).

  11. 11.

    et al. A site-specific plectin mutation causes dominant epidermolysis bullosa simplex Ogna: two identical de novo mutations. J. Invest. Dermatol. 118, 87–93 (2002).

  12. 12.

    et al. A homozygous nonsense mutation within the dystonin gene coding for the coiled-coil domain of the epithelial isoform of BPAG1 underlies a new subtype of autosomal recessive epidermolysis bullosa simplex. J. Invest. Dermatol. 130, 1551–1557 (2010).

  13. 13.

    et al. Epidermolysis bullosa simplex in Scotland caused by a spectrum of keratin mutations. J. Invest. Dermatol. 127, 574–580 (2007).

  14. 14.

    , & The emerging family of CULLIN3-RING ubiquitin ligases (CRL3s): cellular functions and disease implications. EMBO J. 32, 2307–2320 (2013).

  15. 15.

    , & The expanding significance of keratin intermediate filaments in normal and diseased epithelia. Curr. Opin. Cell Biol. 25, 47–56 (2013).

  16. 16.

    , , & Keratins in health and disease. Curr. Opin. Cell Biol. 32, 73–81 (2015).

  17. 17.

    & Keratins turn over by ubiquitination in a phosphorylation-modulated fashion. J. Cell Biol. 149, 547–552 (2000).

  18. 18.

    , & The role of the ubiquitin proteasome pathway in keratin intermediate filament protein degradation. Proc. Am. Thorac. Soc. 7, 71–76 (2010).

  19. 19.

    , & Hsp40 regulates the amount of keratin proteins via ubiquitin–proteasome pathway in cultured human cells. Int. J. Mol. Med. 29, 165–168 (2012).

  20. 20.

    , , , & Update on the Kelch-like (KLHL) gene family. Hum. Genomics 7, 13 (2013).

  21. 21.

    , & Born to bind: the BTB protein–protein interaction domain. BioEssays 28, 1194–1202 (2006).

  22. 22.

    & The BACK domain in BTB-kelch proteins. Trends Biochem. Sci. 29, 634–637 (2004).

  23. 23.

    , , & Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics. Cell 143, 951–965 (2010).

  24. 24.

    et al. Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases. J. Biol. Chem. 288, 7803–7814 (2013).

  25. 25.

    et al. KRIP6: a novel BTB/kelch protein regulating function of kainate receptors. Mol. Cell. Neurosci. 34, 539–550 (2007).

  26. 26.

    , , , & The BTB/kelch protein, KRIP6, modulates the interaction of PICK1 with GluR6 kainate receptors. Neuropharmacology 55, 1131–1139 (2008).

  27. 27.

    et al. A microRNA encoded by HSV-1 inhibits a cellular transcriptional repressor of viral immediate early and early genes. Sci. China Life Sci. 56, 373–383 (2013).

  28. 28.

    , & The predator becomes the prey: regulating the ubiquitin system by ubiquitylation and degradation. Nat. Rev. Mol. Cell Biol. 12, 605–620 (2011).

  29. 29.

    , & Ubiquitin ligase activity of Cul3-KLHL7 protein is attenuated by autosomal dominant retinitis pigmentosa causative mutation. J. Biol. Chem. 286, 33613–33621 (2011).

  30. 30.

    et al. Structures of SPOP–substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases. Mol. Cell 36, 39–50 (2009).

  31. 31.

    & Understanding cullin-RING E3 biology through proteomics-based substrate identification. Mol. Cell. Proteomics 11, 1541–1550 (2012).

  32. 32.

    , , , & Structural and biochemical characterization of the KLHL3–WNK kinase interaction important in blood pressure regulation. Biochem. J. 460, 237–246 (2014).

  33. 33.

    et al. Keratins as the main component for the mechanical integrity of keratinocytes. Proc. Natl. Acad. Sci. USA 110, 18513–18518 (2013).

  34. 34.

    , & Serendipitous SAD solution for DMSO-soaked SOCS2–elonginC–elonginB crystals using covalently incorporated dimethylarsenic: insights into substrate receptor conformational flexibility in cullin RING ligases. PLoS One 10, e0131218 (2015).

  35. 35.

    , & Calcium regulation of keratinocyte differentiation. Expert Rev. Endocrinol. Metab. 7, 461–472 (2012).

  36. 36.

    , , , & A gene signature–based approach identifies mTOR as a regulator of p73. Mol. Cell. Biol. 28, 5951–5964 (2008).

  37. 37.

    , , , & Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

  38. 38.

    , , , & Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015).

  39. 39.

    et al. Tissue-based map of the human proteome. Science 347, 1260419 (2015).

  40. 40.

    et al. The basal keratin network of stratified squamous epithelia: defining K15 function in the absence of K14. J. Cell Biol. 129, 1329–1344 (1995).

  41. 41.

    et al. The ubiquitin ligase CHIP/STUB1 targets mutant keratins for degradation. Hum. Mutat. 31, 466–476 (2010).

  42. 42.

    , , & Formation of a normal epidermis supported by increased stability of keratins 5 and 14 in keratin 10 null mice. Mol. Biol. Cell 12, 1557–1568 (2001).

  43. 43.

    & Disease model: heritable skin blistering. Trends Mol. Med. 7, 422–424 (2001).

  44. 44.

    et al. A phase 2 study of bortezomib in relapsed, refractory myeloma. N. Engl. J. Med. 348, 2609–2617 (2003).

  45. 45.

    et al. Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin–proteasome pathway, against preclinical models of multiple myeloma. Blood 110, 3281–3290 (2007).

  46. 46.

    et al. The Human Intermediate Filament Database: comprehensive information on a gene family involved in many human diseases. Hum. Mutat. 29, 351–360 (2008).

  47. 47.

    , & Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat. Protoc. 4, 1073–1081 (2009).

  48. 48.

    , , & MutationTaster evaluates disease-causing potential of sequence alterations. Nat. Methods 7, 575–576 (2010).

  49. 49.

    et al. A method and server for predicting damaging missense mutations. Nat. Methods 7, 248–249 (2010).

  50. 50.

    et al. Distribution and intensity of constraint in mammalian genomic sequence. Genome Res. 15, 901–913 (2005).

  51. 51.

    , & Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice. Nat. Protoc. 3, 799–810 (2008).

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Acknowledgements

We thank J. Jin, G. Ou, L. Yu, G. Xu and N. Zheng for advice and support, A. Elia, M. Emanuele and S. Elledge for critical reading of the manuscript, and all the patients and their families for participating in this study. This work was supported by the National Natural Science Foundation of China (grants 81201220 to Z.L., 81271744 to Y.Y. and 31670777 to X.T.), the Young Thousand Talents Program of China to X.T., China National Funds for Distinguished Young Scientists (grant 81425020 to Y.Y.), China National Funds for Excellent Young Scientists (grant 81522037 to Z.L.) and the Beijing Nova Program (grant Z151100000315044 to Z.L.).

Author information

Author notes

    • Zhimiao Lin
    • , Shuo Li
    •  & Cheng Feng

    These authors contributed equally to this work.

Affiliations

  1. Department of Dermatology, Peking University First Hospital, Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, China.

    • Zhimiao Lin
    • , Cheng Feng
    • , Huijun Wang
    • , Jing Zhang
    • , Dingfang Bu
    • , Xintong Wang
    •  & Yong Yang
  2. School of Pharmaceutical Sciences, Center for Infectious Disease Research, School of Medicine, Tsinghua University, Tsinghua-Peking Center for Life Sciences, Beijing, China.

    • Shuo Li
    • , Shang Yang
    • , Danhui Ma
    • , Mengting Gou
    • , Tengjiang Zhang
    • , Xiaohui Kong
    •  & Xu Tan
  3. Peking-Tsinghua Center for Life Sciences, Beijing, China.

    • Huijun Wang
    • , Jing Zhang
    •  & Yong Yang
  4. Department of Dermatology, Tel Aviv Sourasky Medical Centre, Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

    • Ofer Sarig
    • , Gilly Padalon-Brauch
    • , Dan Vodo
    •  & Eli Sprecher
  5. Laboratory of Electron Microscopy, Peking University First Hospital, Beijing, China.

    • Yali Ren
  6. BGI-Shenzhen, Shenzhen, China.

    • Lanlan Dai
    • , Hankui Liu
    •  & Jianguo Zhang
  7. National Institute of Biological Sciences, Beijing, China.

    • Fei Li
    •  & Ting Chen
  8. Laboratory Animal Facility, Peking University First Hospital, Beijing, China.

    • Yongyan Hu
  9. Institutes of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai, China.

    • Feng Zhou
  10. MOE Key Laboratory of Bioinformatics, School of Life Sciences and Centre of Biomedical Analysis, Tsinghua University, Beijing, China.

    • Haiteng Deng

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Contributions

Z.L., S.L., C.F., S.Y., H.W., D.M., Jing Zhang, M.G., X.K., D.B., T.Z., Y.R., X.W. and Y.H. performed the experiments under the supervision of X.T. and Y.Y. O.S., E.S., F.L., T.C., G.P.-B. and D.V. contributed critical reagents. L.D., H.L. and Jianguo Zhang performed the exome sequencing and data analysis. H.D. and F.Z. contributed to the proteomics data analysis. X.T., C.F., S.L., Z.L. and Y.Y. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Yong Yang or Xu Tan.

Supplementary information

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    Supplementary Figures 1–17 and Supplementary Tables 1–3.

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    Supplementary Table 4

    Sequences of primers, sgRNAs, siRNAs and probes used in the study.

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DOI

https://doi.org/10.1038/ng.3701

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