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Stabilizing mutations of KLHL24 ubiquitin ligase cause loss of keratin 14 and human skin fragility

Nature Genetics volume 48, pages 1508–1516 (2016)Cite this article

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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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Figure 1: Pedigree, clinical features and skin pathology in an inherited skin fragility disorder.
Figure 2: KLHL24 start-codon mutations identified in the five patients result in an N-terminally truncated protein.
Figure 3: Removal of the first 28 residues of KLHL24 increases protein stability as a result of abolished autoubiquitination.
Figure 4: KRT14 is a ubiquitination substrate of KLHL24 and is excessively ubiquitinated and degraded by KLHL24-ΔN28.
Figure 5: Klhl24 mRNA levels are upregulated during mouse keratinocyte differentiation, and mutation of its start codon causes stabilization of KLHL24 and loss of Krt14 in a knock-in mouse model.
Figure 6: Schematic of the disease-causing mechanism of KLHL24 mutations.

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References

  1. Uitto, J., Richard, G. & McGrath, J.A. Diseases of epidermal keratins and their linker proteins. Exp. Cell Res. 313, 1995–2009 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

  3. Has, C. & Bruckner-Tuderman, L. The genetics of skin fragility. Annu. Rev. Genomics Hum. Genet. 15, 245–268 (2014).

    Article  CAS  PubMed  Google Scholar 

  4. Bruckner-Tuderman, L. & Has, C. Molecular heterogeneity of blistering disorders: the paradigm of epidermolysis bullosa. J. Invest. Dermatol. 132, E2–E5 (2012).

    Article  PubMed  Google Scholar 

  5. Coulombe, P.A. & Lee, C.H. Defining keratin protein function in skin epithelia: epidermolysis bullosa simplex and its aftermath. J. Invest. Dermatol. 132, 763–775 (2012).

    Article  CAS  PubMed  Google Scholar 

  6. Bonifas, J.M., Rothman, A.L. & Epstein, E.H. Jr. Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities. Science 254, 1202–1205 (1991).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  11. Koss-Harnes, D. 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).

    Article  CAS  PubMed  Google Scholar 

  12. Groves, R.W. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Pan, X., Hobbs, R.P. & Coulombe, P.A. The expanding significance of keratin intermediate filaments in normal and diseased epithelia. Curr. Opin. Cell Biol. 25, 47–56 (2013).

    Article  CAS  PubMed  Google Scholar 

  16. Toivola, D.M., Boor, P., Alam, C. & Strnad, P. Keratins in health and disease. Curr. Opin. Cell Biol. 32, 73–81 (2015).

    Article  CAS  PubMed  Google Scholar 

  17. Ku, N.O. & Omary, M.B. Keratins turn over by ubiquitination in a phosphorylation-modulated fashion. J. Cell Biol. 149, 547–552 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Rogel, M.R., Jaitovich, A. & Ridge, K.M. The role of the ubiquitin proteasome pathway in keratin intermediate filament protein degradation. Proc. Am. Thorac. Soc. 7, 71–76 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  20. Dhanoa, B.S., Cogliati, T., Satish, A.G., Bruford, E.A. & Friedman, J.S. Update on the Kelch-like (KLHL) gene family. Hum. Genomics 7, 13 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Perez-Torrado, R., Yamada, D. & Defossez, P.A. Born to bind: the BTB protein–protein interaction domain. BioEssays 28, 1194–1202 (2006).

    Article  CAS  PubMed  Google Scholar 

  22. Stogios, P.J. & Privé, G.G. The BACK domain in BTB-kelch proteins. Trends Biochem. Sci. 29, 634–637 (2004).

    CAS  PubMed  Google Scholar 

  23. Bennett, E.J., Rush, J., Gygi, S.P. & Harper, J.W. Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics. Cell 143, 951–965 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Laezza, F., Wilding, T.J., Sequeira, S., Craig, A.M. & Huettner, J.E. The BTB/kelch protein, KRIP6, modulates the interaction of PICK1 with GluR6 kainate receptors. Neuropharmacology 55, 1131–1139 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wu, W. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Harper, J.W. & Tan, M.K. Understanding cullin-RING E3 biology through proteomics-based substrate identification. Mol. Cell. Proteomics 11, 1541–1550 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Schumacher, F.R., Sorrell, F.J., Alessi, D.R., Bullock, A.N. & Kurz, T. Structural and biochemical characterization of the KLHL3–WNK kinase interaction important in blood pressure regulation. Biochem. J. 460, 237–246 (2014).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Gadd, M.S., Bulatov, E. & Ciulli, A. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Bikle, D.D., Xie, Z. & Tu, C.L. Calcium regulation of keratinocyte differentiation. Expert Rev. Endocrinol. Metab. 7, 461–472 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Rosenbluth, J.M., Mays, D.J., Pino, M.F., Tang, L.J. & Pietenpol, J.A. A gene signature–based approach identifies mTOR as a regulator of p73. Mol. Cell. Biol. 28, 5951–5964 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Raj, A., van den Bogaard, P., Rifkin, S.A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Chen, K.H., Boettiger, A.N., Moffitt, J.R., Wang, S. & Zhuang, X. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

  40. Lloyd, C. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  42. Reichelt, J., Büssow, H., Grund, C. & Magin, T.M. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Arin, M.J. & Roop, D.R. Disease model: heritable skin blistering. Trends Mol. Med. 7, 422–424 (2001).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  45. Kuhn, D.J. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  47. Kumar, P., Henikoff, S. & Ng, P.C. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat. Protoc. 4, 1073–1081 (2009).

    CAS  PubMed  Google Scholar 

  48. Schwarz, J.M., Rödelsperger, C., Schuelke, M. & Seelow, D. MutationTaster evaluates disease-causing potential of sequence alterations. Nat. Methods 7, 575–576 (2010).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Lichti, U., Anders, J. & Yuspa, S.H. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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.).

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  1. Zhimiao Lin, Shuo Li and Cheng Feng: These authors contributed equally to this work.

Authors and 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, Department of Human Molecular Genetics and Biochemistry, Tel Aviv Sourasky Medical Centre, 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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  1. Zhimiao Lin
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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.

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Correspondence to Yong Yang or Xu Tan.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–17 and Supplementary Tables 1–3. (PDF 1846 kb)

Supplementary Table 4

Sequences of primers, sgRNAs, siRNAs and probes used in the study. (XLSX 39 kb)

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Lin, Z., Li, S., Feng, C. et al. Stabilizing mutations of KLHL24 ubiquitin ligase cause loss of keratin 14 and human skin fragility. Nat Genet 48, 1508–1516 (2016). https://doi.org/10.1038/ng.3701

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