Plectin is a widely expressed high molecular weight protein that is involved in cytoskeleton-membrane attachment in epithelial cells, muscle, and other tissues. The human autosomal recessive disorder epidermolysis bullosa with muscular dystrophy (MD-EBS) shows epidermal blister formation at the level of the hemidesmosome and is associated with a myopathy of unknown etiology. Here, plectin was found to be absent in skin and cultured keratinocytes from an MD-EBS patient by immunofluorescence and immunoprecipitation, suggesting that plectin is a candidate gene/protein system for MD-EBS mutation. The 14800-bp human plectin cDNA was cloned and sequenced. The predicted 518-kD polypeptide has homology to the actin-binding domain of the dystrophin family at the amino terminus, a central rod domain, and homology to the intermediate filament-associated protein desmoplakin at the carboxyl terminus. The corresponding human gene (PLECl), consisting of 33 exons spanning >26 kb of genomic DNA was cloned, sequenced, and mapped to chromosomal band 8q24. Homozygosity by descent was observed in the consanguineous MD-EBS family with intragenic plectin polymorphisms. Direct sequencing of PCR-amplified plectin cDNA from the patient's keratinocytes revealed a homozygous 8-bp deletion in exon 32 causing a frameshift and a premature termination codon 42 bp downstream. The clinically unaffected parents of the proband were found to be heterozygous carriers of the mutation. These results establish the molecular basis of MD-EBS in this family and clearly demonstrate the important structural role for plectin in cytoskeleton-membrane adherence in both skin and muscle.

Epithelial cells maintained in culture medium containing low calcium proteolytically process laminin 5 (␣3␤3␥2) within the ␣3 and ␥2 chains (1). Experiments were designed to identify the enzyme(s) responsible for the laminin 5 processing and the sites of proteolytic cleavage. To characterize the nature of laminin 5 processing, we determined the N-terminal amino acid sequences of the proteolytic fragments produced by the processing events. The results indicate that the first ␣3 chain cleavage (200-l65 kDa ␣3) occurs within subdomain G4 of the G domain. The second cleavage (l65-l45 kDa ␣3) occurs within the lIla domain, 11 residues Nterminal to the start of domain II. The ␥ chain is cleaved within the second epidermal growth factor-like repeat of domain Ill. The sequence cleaved within the ␥2 chain matches the consensus sequence for the cleavage of type I, II, and III procollagens by bone morphogenetic protein-1 (BMP-1), also known as type I procollagen C-proteinase (2). Recombinant BMP-1 cleaves ␥2 in vitro, both within intact laminin 5 and at the predicted site of a recombinant ␥2 short arm. ␣3 is also cleaved by BMP-1 in vitro, but the cleavage site is yet to be determined. These results show the laminin ␣3 and ␥2 chains to be substrates for BMP-1 in vitro. We speculate that ␥2 cleavage is required for formation of the laminin 5-6 complex and that this complex is directly involved in assembly of the interhemidesmosomal basement membrane. This further suggests that BMP-1 activity facilitates basement membrane assembly, but not hemidesmosome assembly, in the laminin 5-rich dermalepidermal junction basement membrane in vivo.The occurrence of physiological, extracellular proteolytic processing of collagens is well documented, as is the important role that it plays in controlling the fibrillogenesis of banded collagen fibers (3). An enzyme responsible for removal of the C-terminal procollagen propeptides of the major fibrillar collagen types I-III has been identified as BMP-1 (2).1 BMP-1 was first identified in osteogenetic fractions of mammalian bone (4 -7) but was subsequently found to show substantial homology to proteins involved in morphogenetic patterning, such as the products of Drosophila genes tolloid (tld) and tlr-1 (12, 43) and of sea urchin gene products BP10 and SpAN (8, 9). Each contains an N-terminal astacin-like zinc-binding metalloendopeptidase domain (10) followed by varying numbers of epidermal growth factor-like (EGF-like) motifs and internal repeats termed CUB domains thought to be responsible for protein-protein interactions (44).There is abundant genetic and molecular evidence that Drosophila tld mediates dorsal-ventral patterning in the fly embryo (11-13), with null phenotypes of tld showing partial transformation of the dorsal ectoderm into ventral ectoderm (14). Genetic and developmental expression studies have also indicated that the tld gene product TLD participates within the same developmental pathway as the product of the decapentaplegic gene, DPP, the fly cognate of mammalian BMP-2 an...

Platinum-based chemotherapy, with cytoreductive surgery, is the cornerstone of treatment of advanced ovarian cancer; however, acquired drug resistance is a major clinical obstacle. It has been proposed that subpopulations of tumor cells with stem cell-like properties, such as so-called side populations (SP) that overexpress ABC drug transporters, can sustain the growth of drug-resistant tumor cells, leading to tumor recurrence following chemotherapy. The histone methyltransferase EZH2 is a key component of the polycomb-repressive complex 2 required for maintenance of a stem cell state, and overexpression has been implicated in drug resistance and shorter survival of ovarian cancer patients. We observed higher percentage SP in ascites from patients that have relapsed following chemotherapy compared with chemonaive patients, consistent with selection for this subpopulation during platinum-based chemotherapy. Furthermore, ABCB1 (P-glycoprotein) and EZH2 are consistently overexpressed in SP compared with non-SP from patients' tumor cells. The siRNA knockdown of EZH2 leads to loss of SP in ovarian tumor models, reduced anchorageindependent growth, and reduced tumor growth in vivo. Together, these data support a key role for EZH2 in the maintenance of a drug-resistant, tumor-sustaining subpopulation of cells in ovarian cancers undergoing chemotherapy. As such, EZH2 is an important target for anticancer drug development.