Defining the Properties of the Nonhelical Tail Domain in Type II Keratin 5: Insight from a Bullous Disease-causing Mutation

Abstract

Inherited mutations in the intermediate filament (IF) proteins keratin 5 (K5) or keratin 14 (K14) cause epidermolysis bullosa simplex (EBS), in which basal layer keratinocytes rupture upon trauma to the epidermis. Most mutations are missense alleles affecting amino acids located in the central ␣-helical rod domain of K5 and K14. Here, we study the properties of an unusual EBS-causing mutation in which a nucleotide deletion (1649delG) alters the last 41 amino acids and adds 35 residues to the C terminus of K5. Relative to wild type, filaments coassembled in vitro from purified K5-1649delG and K14 proteins are shorter and exhibit weak viscoelastic properties when placed under strain. Loss of the C-terminal 41 residues contributes to these alterations. When transfected in cultured epithelial cells, K5-1649delG incorporates into preexisting keratin IFs and also forms multiple small aggregates that often colocalize with hsp70 in the cytoplasm. Aggregation is purely a function of the K5-1649delG tail domain; in contrast, the cloned 109 residue-long tail domain from wild type K5 is distributed throughout the cytoplasm and colocalizes partly with keratin IFs. These data provide a mechanistic basis for the cell fragility seen in individuals bearing the K5-1649delG allele, and point to the role of the C-terminal 41 residues in determining K5's assembly properties. INTRODUCTIONIntermediate filaments (IFs) are encoded by a large family of genes comprising Ͼ67 members in the human and mouse genomes Hesse et al., 2001). These 10-to 12-nm wide filaments are prominent structural constituents of the cytoplasm and nucleus in multicellular eukaryotes, but they are distinct from F-actin and microtubules in several key respects. Among them is their association with disease. Inherited mutations in IF proteins are associated with nearly 30 human diseases (Omary et al., 2004). These conditions are dominantly inherited with rare exceptions (Fuchs and Cleveland, 1998;Cassidy et al., 2002;Omary et al., 2004), consistent with the complexity of the assembly pathway and of the architecture of mature IF polymers (Herrmann and Aebi, 2004). Cell and tissue fragility underlies lesion pathogenesis in many of these disorders, reflecting the major function of structural scaffolding fulfilled by IFs in both the cytoplasm and nucleus (Omary et al., 2004;Worman and Courvalin, 2004). Types I and II keratin genes together represent ϳ75% of IF genes and are specifically expressed in epithelial cells Hesse et al., 2001). In part because of a strict heteropolymerization requirement at the protein level (Herrmann and Aebi, 2004), type I and type II keratin genes are tightly regulated in a pairwise and differentiation-related manner in epithelial tissues (Moll et al., 1982;O'Guin et al., 1990;Fuchs, 1995). Correlating with frequent exposure to mechanical stress, epithelia lining up the skin and oral mucosa are keratin rich and sensitive to mutations compromising the structural scaffolding function of keratin IFs. Accordingly, a large fraction of the known IF...

Identification of Novel Interaction between Annexin A2 and Keratin 17

“…The mammalian expression constructs pEGFP-C3 K5 (16) and pEGFP-C3 K17 (17) have been described. AnxA2 cDNA was cloned out of pOTB7 AnxA2 (ATCC) using PCR with the primers 5Ј-AGTCGACGATGTCTACTG TTCACG-3Ј (forward) (with a SalI restriction site underlined) and 5Ј-GTGGATCCTCAGTCA-TCTC CACC-3Ј (reverse) (with a BamHI restriction site underlined) and cloned into mCherry-C1 (Clontech, Mountain View, CA).…”

Epidermolysis bullosa simplex: a paradigm for disorders of tissue fragility

“…For the EBS-localized, EBS-generalized, and EBS-DM variants, a strong concordance exists among the position and nature of the mutation in the target keratin protein, the extent to which it disrupts keratin IF assembly and structure (when tested in vitro or through transfection in cultured cells), and, ultimately, the severity of clinical presentation (52)(53)(54)(55)(56)(57). A corollary to this principle is that the nature of the mutation affecting K5 or K14 defines the sensitivity threshold that the epithelium displays toward frictional trauma, thereby contributing to whether the symptoms are local (e.g., EBS-localized) or generalized (e.g., EBS-generalized and EBS-DM).…”

Intermediate filament scaffolds fulfill mechanical, organizational, and signaling functions in the cytoplasm

“…Additional studies confirmed that the cell fragility seen in EBS corresponds to a loss-of-function phenotype. Key among them were the severe epithelial blistering phenotype exhibited by K14-and K5-null mice (Lloyd et al 1995;Peters et al 2001), the finding that disease-causing mutations in either K5 or K14 markedly weaken the mechanical resilience of K5-K14 filament assemblies in vitro Gu and Coulombe 2005) and affect de novo IF network formation in live keratinocytes (Werner et al 2004), and the realization that EBS disease severity correlates with the impact of K5 or K14 mutations on IF assembly in vitro (e.g., see Letai et al 1993).…”