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IntroductionHemidesmosomes (HDs) are multi-protein complexes that promote epithelial-stromal cohesion in stratified and complex epithelia, and connect the intermediate filament system of basal epithelial cells to proteins of the extracellular matrix. These complexes, which ultrastructurally appear as tripartite structures along the plasma membrane of basal cells, are composed of at least five different proteins: the laminin-5 receptor α6β4, the bullous pemphigoid antigens 180 (BP180, BPAG2 or type XVII collagen) and 230 (BP230 or BPAG1-e), CD151 and plectin Borradori and Sonnenberg, 1999;Sterk et al., 2000). In certain tissues, such as intestinal epithelia, and cultured epithelial cells, a second type of HD has been identified, which is composed of α6β4 and plectin (Uematsu et al., 1994;Orian-Rousseau et al., 1996). These type II HDs, in contrast to the classical or type I HDs, do not exhibit the typical tripartite structure.BP230 and plectin are cytoplasmic proteins that belong to the plakin protein family, which also includes desmoplakin, envoplakin and periplakin. These proteins are crucially involved in the organization of the cytoskeleton (Ruhrberg and Watt, 1997;Leung et al., 2001). They are composed of domains that have considerable sequence homology. Their N-terminus consists of a plakin domain containing a number of subdomains of high α-helical content, designated NN, Z, Y, X, W and V, whereas the central coiled-coil rod domain is composed of heptad repeats thought to be involved in the dimerization of the plakin (Green et al., 1992). Finally, their C-terminal end exhibits one or more homologous repeat sequences designated A, B or C. In plectin as well as in neuronal and muscular isoforms of BP230 (BPAG1-a and BPAG1-b, respectively), a calponin-type actin-binding domain (ABD) precedes the plakin domain (Brown et al., 1995;McLean et al., 1996;Leung et al., 2001). The C-terminal end of plakins has intermediate filament binding properties (Meng et al., 1997;Wiche et al., 1993;Yang et al., 1996;Leung et al., 1999), whereas the N-terminal end harbors specific sequences that target the proteins to distinct membrane sites, such as HDs or desmosomes, cell-cell adhesion complexes in a variety of epithelia (Kowalczyk et al., 1997;Rezniczek et al., 1998; Geerts et al., 1999;Hopkinson and Jones, 2000).The α6β4 integrin plays a central role in the assembly of HDs. Loss of α6β4 due to mutations in the genes for either the α6 or β4 subunit causes a distinct form of pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB), and is characterized by fragility and extensive blistering of the skin. In affected patients HDs are rudimentary or completely absent (Vidal et al., 1995;Niessen et al., 1996;Ruzzi et al., 1997). A similar phenotype is observed in α6 or β4 null mutant mice (van der Neut et al., 1996; Georges-Labouesse et al., Dowling et al., 1996). The large cytoplasmic domain of the integrin β4 subunit is essential for the formation of HDs Murgia et al., 1998). It is over 1000 amino acids long ...

The bullous pemphigoid antigen 1 (BP230) and desmoplakin (DP) are members of the plakin protein family of cytolinkers. Despite their homology, their COOH termini selectively bind distinct intermediate filaments (IFs). We studied sequences within their COOH termini required for their interaction with the epidermal keratins K5/K14, the simple epithelial keratins K8/K18, and type III IF vimentin by yeast three-hybrid, cell transfection, and overlay assays. The results indicate that BP230 interacts with K5/K14 but not with K8/K18 or vimentin via a region encompassing both the B and C subdomains and the COOH extremity, including a COOH-terminal eight-amino-acid stretch. In contrast, the C subdomain with the COOH-terminal extremity of DP interacts with K5/K14 and K8/K18, and its linker region is able to associate with K8/K18 and vimentin. Furthermore, the potential of DP to interact with IF proteins in yeast seems to be regulated by phosphorylation of Ser 2849 within its COOH terminus. Strikingly, BP230 and DP interacted with cytokeratins only when both type I and type II keratins were present. The head and tail domains of K5/K14 keratins were dispensable for their interaction with BP230 or DP. On the basis of our findings, we postulate that (1) the binding specificity of plakins for various IF proteins depends on their linker region between the highly homologous B and C subdomains and their COOH extremity and (2) the association of DP and BP230 with both epidermal and simple keratins is critically affected by the tertiary structure induced by heterodimerization and involves recognition sites located primarily in the rod domain of these keratins.

There has only been one clinically confirmed case of terbinafine resistance in dermatophytes, where six sequential Trichophyton rubrum isolates from the same patient were found to be resistant to terbinafine and cross-resistant to other squalene epoxidase (SE) inhibitors. Microsomal SE activity from these resistant isolates was insensitive to terbinafine, suggesting a target-based mechanism of resistance (B. Favre, M. Ghannoum, and N. S. Ryder, Med. Mycol. 42:525-529, 2004). In this study, we have characterized at the molecular level the cause of the resistant phenotype of these clinical isolates. Cloning and sequencing of the SE gene and cDNA from T. rubrum revealed the presence of an intron in the gene and an open reading frame encoding a protein of 489 residues, with an equivalent similarity (57%) to both yeast and mammalian SEs. The nucleotide sequences of SE from two terbinafine-susceptible strains were identical whereas those of terbinafine-resistant strains, serially isolated from the same patient, each contained the same single missense introducing the amino acid substitution L393F. Introduction of the corresponding substitution in the Candida albicans SE gene (L398F) and expression of this gene in Saccharomyces cerevisiae conferred a resistant phenotype to the transformants when compared to those expressing the wild-type sequence. Terbinafine resistance in these T. rubrum clinical isolates appears to be due to a single amino acid substitution in SE.Dermatophytosis is a common infection of the keratinized tissues skin, hair, and nails caused by dermatophytes. Among the three known genera of dermatophytes, Epidermophyton, Microsporum, and Trichophyton, Trichophyton species, especially T. mentagrophytes, T. tonsurans, and T. rubrum, are the most common pathogens, with T. rubrum being the most prevalent isolated organism. T. rubrum is particularly involved in tinea pedis and tinea unguium (onychomychosis). While most superficial infections can be effectively cured with different topical agents with various mechanisms of action, tinea capitis and onychomychosis require the use of oral drugs such as fluconazole, itraconazole, griseofulvin, and terbinafine to be sucessfully treated. Nail infections require extended periods of therapy with at least 3 months of daily or intermittent dosing regimens.Despite the high incidence of dermatophytosis and the difficult and long-term treatment of some of these infections, with associated uneven patient compliance, antifungal resistance in dermatophytes appears to be rare. This is in contrast with candidiasis and aspergillosis, where numerous isolates resistant to various antifungals have been identified and then characterized (1,18,24,27,29,30). Systematic susceptibility testing of clinical isolates from patients with onychomychosis who failed on therapy with terbinafine did not reveal any correlation between the MIC of terbinafine against these isolates and clinical failure (20). Nevertheless, in one case terbinafine-resistant T. rubrum was identified (20). All isolates...

BackgroundParaneoplastic pemphigus (PNP) is a devastating autoimmune blistering disease, involving mucocutaneous and internal organs, and associated with underlying neoplasms. PNP is characterized by the production of autoantibodies targeting proteins of the plakin and cadherin families involved in maintenance of cell architecture and tissue cohesion. Nevertheless, the identity of an antigen of Mr 170,000 (p170), thought to be critical in PNP pathogenesis, has remained unknown.Methodology/Principal FindingsUsing an immunoprecipitation and mass spectrometry based approach, we identified p170 as alpha-2-macroglobuline-like-1, a broad range protease inhibitor expressed in stratified epithelia and other tissues damaged in the PNP disease course. We demonstrate that 10 PNP sera recognize alpha-2-macroglobuline-like-1 (A2ML1), while none of the control sera obtained from patients with bullous pemphigoid, pemphigus vulgaris, pemphigus foliaceus and normal subjects does.Conclusions/SignificanceOur study unravels a broad range protease inhibitor as a new class of target antigens in a paraneoplastic autoimmune multiorgan syndrome and opens a new challenging investigation avenue for a better understanding of PNP pathogenesis.