**Introduction**

**Chapter 1**

**Provisional chapter**

**Introductory Chapter: Keratins - What to Do with Too**

Keratin, from the Greek word for horn, **κέρατο**, denotes the proteinaceous covering layers and structures produced by chordates, including mammals, birds, fish, reptiles, and amphibians. The dead outermost layer of the epidermis, hair and wool, horns, claws, hooves, feathers, and scales is composed of keratin. Keratin is completely insoluble in water and is resistant to proteases that degrade other proteins—1000-year-old Egyptian and other ancient mummies often have full head of hair, virtually undamaged keratin. Keratin proteins can be either alpha-helical in structure, in the skin, hair, and wool of mammals, or parallel sheets of betapleated polypeptide chains found in the feathers of birds and scales of reptiles. Rich in amino acid cysteine, keratins become covalently crosslinked via disulfide bonds, which confers a great chemical and biochemical stability to keratin. Thus, keratin serves as important resilient

Importantly, *keratin* is also the resilient structural intracellular protein that protects living epithelial cells from mechanical damage or stress. In cytoplasm, keratin constitutes a filamentous cytoskeletal protein network, extending from the nucleus to the cell periphery, the intermediate filaments, thicker than the actin filaments but thinner than microtubules [1]. Two large families of keratin genes encode multiple proteins with both common and cell-type-specific

The indispensable fundamental intracellular keratin functions are revealed in congenital human skin diseases caused by mutations in keratin genes, for example, Epidermolysis bullosa simplex and Epidermolytic hyperkeratosis or in Meesmann's Corneal Dystrophy, the disease caused by a mutation in the gene specifically encoding a corneal keratin [3]. Most

**Introductory Chapter: Keratins - What to Do with Too** 

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.79998

**Much? What to Do with Too Little?**

**Much? What to Do with Too Little?**

Miroslav Blumenberg and Sidra Younis

Miroslav Blumenberg and Sidra Younis

http://dx.doi.org/10.5772/intechopen.79998

**1. Introduction**

functions [2].

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

structural and protective functions for the organism.

### **Introductory Chapter: Keratins - What to Do with Too Much? What to Do with Too Little? Introductory Chapter: Keratins - What to Do with Too Much? What to Do with Too Little?**

DOI: 10.5772/intechopen.79998

Miroslav Blumenberg and Sidra Younis Miroslav Blumenberg and Sidra Younis

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79998

### **1. Introduction**

Keratin, from the Greek word for horn, **κέρατο**, denotes the proteinaceous covering layers and structures produced by chordates, including mammals, birds, fish, reptiles, and amphibians. The dead outermost layer of the epidermis, hair and wool, horns, claws, hooves, feathers, and scales is composed of keratin. Keratin is completely insoluble in water and is resistant to proteases that degrade other proteins—1000-year-old Egyptian and other ancient mummies often have full head of hair, virtually undamaged keratin. Keratin proteins can be either alpha-helical in structure, in the skin, hair, and wool of mammals, or parallel sheets of betapleated polypeptide chains found in the feathers of birds and scales of reptiles. Rich in amino acid cysteine, keratins become covalently crosslinked via disulfide bonds, which confers a great chemical and biochemical stability to keratin. Thus, keratin serves as important resilient structural and protective functions for the organism.

Importantly, *keratin* is also the resilient structural intracellular protein that protects living epithelial cells from mechanical damage or stress. In cytoplasm, keratin constitutes a filamentous cytoskeletal protein network, extending from the nucleus to the cell periphery, the intermediate filaments, thicker than the actin filaments but thinner than microtubules [1]. Two large families of keratin genes encode multiple proteins with both common and cell-type-specific functions [2].

The indispensable fundamental intracellular keratin functions are revealed in congenital human skin diseases caused by mutations in keratin genes, for example, Epidermolysis bullosa simplex and Epidermolytic hyperkeratosis or in Meesmann's Corneal Dystrophy, the disease caused by a mutation in the gene specifically encoding a corneal keratin [3]. Most

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

keratin gene mutations have a dominant-negative effect, disrupting the filamentous structure formation even from the natural allele and leaving the cell with a deficient cytoskeleton.

**Author details**

**References**

Miroslav Blumenberg1

(NUMS), Rawalpindi, Pakistan

PubMed PMID: 25594948

\* and Sidra Younis2

Pharmacology, NYU Langone Medical Center, New York, USA

cshperspect.a018275. PubMed PMID: 29610398

651-656. PubMed PMID: 11157990

231-235. PubMed PMID: 16873051

\*Address all correspondence to: miroslav.blumenberg@nyumc.org

1 The R. O. Perelman Department of Dermatology, Biochemistry and Molecular

2 Department of Molecular Biology/Biochemistry, National University of Medical Sciences

Introductory Chapter: Keratins - What to Do with Too Much? What to Do with Too Little?

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5

[1] Loschke F, Seltmann K, Bouameur JE, Magin TM. Regulation of keratin network organization. Current Opinion in Cell Biology. 2015;**32**:56-64. DOI: 10.1016/j.ceb.2014.12.006.

[2] Jacob JT, Coulombe PA, Kwan R, Omary MB. Types I and II keratin intermediate filaments. Cold Spring Harbor Perspectives in Biology. 2018;**10**(4):pii: a018275. DOI: 10.1101/

[3] Chamcheu JC, Siddiqui IA, Syed DN, Adhami VM, Liovic M, Mukhtar H. Keratin gene mutations in disorders of human skin and its appendages. Archives of Biochemistry and Biophysics. 2011;**508**(2):123-137. DOI: 10.1016/j.abb.2010.12.019. PubMed PMID: 21176769

[4] Cao T, Longley MA, Wang XJ, Roop DR. An inducible mouse model for epidermolysis bullosa simplex: Implications for gene therapy. Journal of Cell Biology. 2001;**152**(3):

[5] Dykxhoorn DM, Lieberman J. Knocking down disease with siRNAs. Cell. 2006;**126**(2):

[6] Leachman SA, Hickerson RP, Hull PR, Smith FJ, Milstone LM, Lane EB, Bale SJ, Roop DR, McLean WH, Kaspar RL. Therapeutic siRNAs for dominant genetic skin disorders including pachyonychia congenita. Journal of Dermatological Science. 2008;**51**(3):151-157.

[7] Werner NS, Windoffer R, Strnad P, Grund C, Leube RE, Magin TM. Epidermolysis bullosa simplex-type mutations alter the dynamics of the keratin cytoskeleton and reveal a contribution of actin to the transport of keratin subunits. Molecular Biology of the Cell.

[8] Leachman SA, Hickerson RP, Schwartz ME, Bullough EE, Hutcherson SL, Boucher KM, et al. First-in-human mutation-targeted siRNA phase Ib trial of an inherited skin disorder. Molecular Therapy. 2010;**18**(2):442-446. DOI: 10.1038/mt.2009.273. PubMed PMID: 19935778

DOI: 10.1016/j.jdermsci.2008.04.003. PubMed PMID: 18495438

2004;**15**(3):990-1002. PubMed PMID: 14668478

### **2. What to do with too little?**

Several chapters in this volume address the diseases associated with keratin deficiencies (see manuscripts by Komine et al., Zhang et al.). Corrective gene therapy approaches attempt to specifically target the mutant keratin gene allele, thus allowing the normal keratin protein to decrease cell fragility [4]. Short inhibitory RNA (siRNA) technology was effectively used to downregulate mutant K6a and K14 allele expressions in cultured PC and EBS cells, respectively [5–7]. This mutation-specific siRNA therapy has been used in a human clinical trial, resulting in effective siRNA treatment of a skin disorder [8]. The functional redundancy of keratins in tissues affected by keratin mutation allows for a possibility to use gene-specific silencing, rather than allele-specific siRNA. Spliceosome-mediated RNA trans-splicing uses the endogenous spliceosome machinery to excise mutant exons and was used to replace the first seven exons of the KRT14 gene in an EBS cell line [9].

Induced pluripotent stem cells and even patient-specific-induced pluripotent stem cells have been generated for use in treatment of inherited keratinopathies [10–12]. Such cell-based therapies have been proposed in conjunction with CRISPR/Cas9- and TALEN-based gene-editing techniques for targeting mutations in the keratin genes [13–15].

A naturally occurring phenomenon, whereby a subpopulation of mutant cells spontaneously reverts to the wild-type phenotype, "revertant mosaicism," has been observed in several patients with EB [16–20]. Revertant mosaicism keratinopathies have two major advantages: (1) the revertant skin is visible and easily accessible and (2) the revertant keratinocytes often have a growth advantage over their mutant progenitors, and so may outgrow and correct the patient's ichthyotic phenotype [21]. Harvesting, expanding, and autologous re-grafting the revertant tissue therefore may be feasible in a clinical setting.

### **3. What to do with too much?**

The increased importance of ecological dangers and ways to alleviate them focused attention on the very large volume of keratin industrial waste. Several chapters in this volume address the incipient remediation efforts (see manuscripts by Ningthoujam et al., Sharma et al., and Nugroho et al.). The mechanical and chemical methodology, cumbersome and inadequate, seems to be giving in to the new biological technology. We can expect deeper understanding of the microbiome and its efficient biodegradation capabilities to play ever more important role. Especially promising are the studies of complex microbiomes, and we can expect in the not-so-distant future that combinations, communities of specific microbes, will be able to convert the obnoxious keratin waste into delightful new materials [22, 23].

### **Author details**

keratin gene mutations have a dominant-negative effect, disrupting the filamentous structure formation even from the natural allele and leaving the cell with a deficient cytoskeleton.

Several chapters in this volume address the diseases associated with keratin deficiencies (see manuscripts by Komine et al., Zhang et al.). Corrective gene therapy approaches attempt to specifically target the mutant keratin gene allele, thus allowing the normal keratin protein to decrease cell fragility [4]. Short inhibitory RNA (siRNA) technology was effectively used to downregulate mutant K6a and K14 allele expressions in cultured PC and EBS cells, respectively [5–7]. This mutation-specific siRNA therapy has been used in a human clinical trial, resulting in effective siRNA treatment of a skin disorder [8]. The functional redundancy of keratins in tissues affected by keratin mutation allows for a possibility to use gene-specific silencing, rather than allele-specific siRNA. Spliceosome-mediated RNA trans-splicing uses the endogenous spliceosome machinery to excise mutant exons and was used to replace the

Induced pluripotent stem cells and even patient-specific-induced pluripotent stem cells have been generated for use in treatment of inherited keratinopathies [10–12]. Such cell-based therapies have been proposed in conjunction with CRISPR/Cas9- and TALEN-based gene-editing

A naturally occurring phenomenon, whereby a subpopulation of mutant cells spontaneously reverts to the wild-type phenotype, "revertant mosaicism," has been observed in several patients with EB [16–20]. Revertant mosaicism keratinopathies have two major advantages: (1) the revertant skin is visible and easily accessible and (2) the revertant keratinocytes often have a growth advantage over their mutant progenitors, and so may outgrow and correct the patient's ichthyotic phenotype [21]. Harvesting, expanding, and autologous re-grafting the

The increased importance of ecological dangers and ways to alleviate them focused attention on the very large volume of keratin industrial waste. Several chapters in this volume address the incipient remediation efforts (see manuscripts by Ningthoujam et al., Sharma et al., and Nugroho et al.). The mechanical and chemical methodology, cumbersome and inadequate, seems to be giving in to the new biological technology. We can expect deeper understanding of the microbiome and its efficient biodegradation capabilities to play ever more important role. Especially promising are the studies of complex microbiomes, and we can expect in the not-so-distant future that combinations, communities of specific microbes, will be able to convert the obnoxious keratin waste into delightful new materials [22, 23].

**2. What to do with too little?**

4 Keratin

first seven exons of the KRT14 gene in an EBS cell line [9].

techniques for targeting mutations in the keratin genes [13–15].

revertant tissue therefore may be feasible in a clinical setting.

**3. What to do with too much?**

Miroslav Blumenberg1 \* and Sidra Younis2

\*Address all correspondence to: miroslav.blumenberg@nyumc.org

1 The R. O. Perelman Department of Dermatology, Biochemistry and Molecular Pharmacology, NYU Langone Medical Center, New York, USA

2 Department of Molecular Biology/Biochemistry, National University of Medical Sciences (NUMS), Rawalpindi, Pakistan

### **References**


[9] Wally V, Brunner M, Lettner T, Wagner M, Koller U, Trost A, Murauer EM, et al. K14 mRNA reprogramming for dominant epidermolysis bullosa simplex. Human Molecular Genetics. 2010;**19**(23):4715-4725. DOI: 10.1093/hmg/ddq405. PubMed PMID: 20861136

[20] Lai-Cheong JE, McGrath JA, Uitto J. Revertant mosaicism in skin: Natural gene therapy. Trends in Molecular Medicine. 2011;**17**(3):140-148. DOI: 10.1016/j.molmed.2010.11.003.

Introductory Chapter: Keratins - What to Do with Too Much? What to Do with Too Little?

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[21] van den Akker PC, Pasmooij AMG, Joenje H, Hofstra RMW, TeMeerman GJ, Jonkman MF. A late-but-fitter revertant cell explains the high frequency of revertant mosaicism in epidermolysis bullosa. PLoS One. 2018;**13**(2):e0192994. DOI: 10.1371/journal.

[22] Toju H, Peay KG, Yamamichi M, Narisawa K, Hiruma K, Naito K, Fukuda S, Ushio M, Nakaoka S, Onoda Y, Yoshida K, Schlaeppi K, Bai Y, Sugiura R, Ichihashi Y, Minamisawa K, Kiers ET. Core microbiomes for sustainable agroecosystems. Nature Plants.

[23] Zengler K, Zaramela LS. The social network of microorganisms—How auxotrophies shape complex communities. Nature Reviews. Microbiology. 2018;**16**(6):383-390. DOI:

2018;**4**(5):247-257. DOI: 10.1038/s41477-018-0139-4. PubMed PMID: 29725101

10.1038/s41579-018-0004-5. PubMed PMID: 29599459

PMID: 21195026

pone.0192994. PMID: 29470523


[20] Lai-Cheong JE, McGrath JA, Uitto J. Revertant mosaicism in skin: Natural gene therapy. Trends in Molecular Medicine. 2011;**17**(3):140-148. DOI: 10.1016/j.molmed.2010.11.003. PMID: 21195026

[9] Wally V, Brunner M, Lettner T, Wagner M, Koller U, Trost A, Murauer EM, et al. K14 mRNA reprogramming for dominant epidermolysis bullosa simplex. Human Molecular Genetics. 2010;**19**(23):4715-4725. DOI: 10.1093/hmg/ddq405. PubMed PMID: 20861136

[10] Uitto J, Christiano AM, McLean WH, McGrath JA. Novel molecular therapies for heritable skin disorders. The Journal of Investigative Dermatology. 2012;**132**(3 Pt 2):820-828.

[11] Itoh M, Kiuru M, Cairo MS, Christiano AM. Generation of keratinocytes from normal and recessive dystrophic epidermolysis bullosa-induced pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America. 2011;**108**(21):

[12] Umegaki-Arao N, Pasmooij AM, Itoh M, Cerise JE, Guo Z, Levy B, et al. Induced pluripotent stem cells from human revertant keratinocytes for the treatment of epidermolysis bullosa. Science Translational Medicine. 2014;**6**(264):264ra164. DOI: 10.1126/scitrans-

[13] Kocher T, Peking P, Klausegger A, Murauer EM, Hofbauer JP, Wally V, et al. Cut and paste: Efficient homology-directed repair of a dominant negative KRT14 mutation via CRISPR/ Cas9 nickases. Molecular Therapy. 2017;**25**(11):2585-2598. DOI: 10.1016/j.ymthe.2017.08.

[14] Courtney DG, Moore JE, Atkinson SD, Maurizi E, Allen EH, Pedrioli DM, et al. CRISPR/ Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting. Gene Therapy. 2016;**23**(1):108-112. DOI: 10.1038/gt.2015.82.

[15] Aushev M, Koller U, Mussolino C, Cathomen T, Reichelt J. Traceless targeting and isolation of gene-edited immortalized keratinocytes from epidermolysis bullosa simplex patients. Molecular Therapy—Methods and Clinical Development. 2017;**6**:112-123. DOI:

[16] Kiritsi D, Nanda A, Kohlhase J, Bernhard C, Bruckner-Tuderman L, Happle R, Has C. Extensive postzygotic mosaicism for a novel keratin 10 mutation in epidermolytic ichthyosis. Acta Dermato-Venereologica. 2014;**94**(3):346-348. DOI: 10.2340/00015555-1695.

[17] Smith FJ, Morley SM, McLean WH. Novel mechanism of revertant mosaicism in Dowling-Meara epidermolysis bullosa simplex. The Journal of Investigative Dermatology. 2004;

[18] Schuilenga-Hut PH, Scheffer H, Pas HH, Nijenhuis M, Buys CH, Jonkman MF. Partial revertant mosaicism of keratin 14 in a patient with recessive epidermolysis bullosa simplex. The Journal of Investigative Dermatology. 2002;**118**(4):626-630. PubMed PMID: 11918708

[19] Lim YH, Fisher JM, Choate KA. Revertant mosaicism in genodermatoses. Cellular and Molecular Life Sciences. 2017;**74**(12):2229-2238. DOI: 10.1007/s00018-017-2468-2. PMID:

8797-8802. DOI: 10.1073/pnas.1100332108. PubMed PMID: 21555586

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lmed.3009342. PMID: 25429057

10.1016/j.omtm.2017.06.008. PMID: 28765827

**122**(1):73-77. PubMed PMID: 14962092

015. PMID: 28888469

6 Keratin

PMID: 26289666

PubMed PMID: 24096702

28168442


**Section 2**

**Human Skin Keratins**
