Cystatin E/M suppresses legumain activity and invasion of human melanoma

<p>Abstract</p> <p>Background</p> <p>High activity of cysteine proteases such as legumain and the cathepsins have been shown to facilitate growth and invasion of a variety of tumor types. In breast cancer, several recent studies have indicated that loss of the cysteine protease inhibitor cystatin E/M leads to increased growth and metastasis. Although cystatin E/M is normally expressed in the skin, its role in cysteine protease regulation and progression of malignant melanoma has not been studied.</p> <p>Methods</p> <p>A panel of various non-melanoma and melanoma cell lines was used. Cystatin E/M and C were analyzed in cell media by immunoblotting and ELISA. Legumain, cathepsin B and L were analyzed in cell lysates by immunoblotting and their enzymatic activities were analyzed by peptide substrates. Two melanoma cell lines lacking detectable secretion of cystatin E/M were transfected with a cystatin E/M expression plasmid (pCST6), and migration and invasiveness were studied by a Matrigel invasion assay.</p> <p>Results</p> <p>Cystatin E/M was undetectable in media from all established melanoma cell lines examined, whereas strong immunobands were detected in two of five primary melanoma lines and in two of six lines derived from patients with metastatic disease. Among the four melanoma lines secreting cystatin E/M, the glycosylated form (17 kD) was predominant compared to the non-glycosylated form (14 kD). Legumain, cathepsin B and L were expressed and active in most of the cell lines, although at low levels in the melanomas expressing cystatin E/M. In the melanoma lines where cystatin E/M was secreted, cystatin C was generally absent or expressed at a very low level. When melanoma cells lacking secretion of cystatin E/M were transfected with pCST6, their intracellular legumain activity was significantly inhibited. In contrast, cathepsin B activity was not affected. Furthermore, invasion was suppressed in cystatin E/M over-expressing melanoma cell lines as measured by the transwell Matrigel assay.</p> <p>Conclusions</p> <p>These results suggest that the level of cystatin E/M regulates legumain activity and hence the invasive potential of human melanoma cells.</p

The Protease Inhibitor Alpha-2-Macroglobuline-Like-1 Is the p170 Antigen Recognized by Paraneoplastic Pemphigus Autoantibodies in Human

Paraneoplastic 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

Deficiency of the human cysteine protease inhibitor cystatin M/E causes hypotrichosis and dry skin

Purpose: We aimed to assess the biological and clinical significance of the human cysteine protease inhibitor cystatin M/E, encoded by the CTS6 gene, in diseases of human hair and skin. Methods: Exome and Sanger sequencing was performed to reveal the genetic cause in two related patients with hypotrichosis. Immunohistochemical, biophysical, and biochemical measurements were performed on patient skin and 3D-reconstructed skin from patient-derived keratinocytes. Results: We identified a homozygous variant c.361C>T (p.Gln121*), resulting in a premature stop codon in exon 2 of CST6 associated with hypotrichosis, eczema, blepharitis, photophobia and impaired sweating. Enzyme assays using recombinant mutant cystatin M/E protein, generated by site-directed mutagenesis, revealed that this p.Gln121* variant was unable to inhibit any of its three target proteases (legumain and cathepsins L and V). Three-dimensional protein structure prediction confirmed the disturbance of the protease/inhibitor binding sites of legumain and cathepsins L and V in the p.Gln121* variant. Conclusion: The herein characterized autosomal recessive hypotrichosis syndrome indicates an important role of human cystatin M/E in epidermal homeostasis and hair follicle morphogenesis

Sexually dimorphic characteristics of the small intestine and colon of prepubescent C57BL/6 mice

Background There is increasing appreciation for sexually dimorphic effects, but the molecular mechanisms underlying these effects are only partially understood. In the present study, we explored transcriptomics and epigenetic differences in the small intestine and colon of prepubescent male and female mice. In addition, the microbiota composition of the colonic luminal content has been examined. Methods At postnatal day 14, male and female C57BL/6 mice were sacrificed and the small intestine, colon and content of luminal colon were isolated. Gene expression of both segments of the intestine was analysed by microarray analysis. DNA methylation of the promoter regions of selected sexually dimorphic genes was examined by pyrosequencing. Composition of the microbiota was explored by deep sequencing. Results Sexually dimorphic genes were observed in both segments of the intestine of 2-week-old mouse pups, with a stronger effect in the small intestine. Amongst the total of 349 genes displaying a sexually dimorphic effect in the small intestine and/or colon, several candidates exhibited a previously established function in the intestine (i.e. Nts, Nucb2, Alox5ap and Retnlγ). In addition, differential expression of genes linked to intestinal bowel disease (i.e. Ccr3, Ccl11 and Tnfr) and colorectal cancer development (i.e. Wt1 and Mmp25) was observed between males and females. Amongst the genes displaying significant sexually dimorphic expression, nine genes were histone-modifying enzymes, suggesting that epigenetic mechanisms might be a potential underlying regulatory mechanism. However, our results reveal no significant changes in DNA methylation of analysed CpGs within the selected differentially expressed genes. With respect to the bacterial community composition in the colon, a dominant effect of litter origin was found but no significant sex effect was detected. However, a sex effect on the dominance of specific taxa was observed. Conclusions This study reveals molecular dissimilarities between males and females in the small intestine and colon of prepubescent mice, which might underlie differences in physiological functioning and in disease predisposition in the two sexes

Mutations in Protein-Binding Hot-Spots on the Hub Protein Smad3 Differentially Affect Its Protein Interactions and Smad3-Regulated Gene Expression

Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses.We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression.Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for specific biological responses

Pattern recognition receptors in immune disorders affecting the skin.

Contains fulltext : 109004.pdf (publisher's version ) (Open Access)Pattern recognition receptors (PRRs) evolved to protect organisms against pathogens, but excessive signaling can induce immune responses that are harmful to the host. Putative PRR dysfunction is associated with numerous immune disorders that affect the skin, such as systemic lupus erythematosus, cryopyrin-associated periodic syndrome, and primary inflammatory skin diseases including psoriasis and atopic dermatitis. As yet, the evidence is often confined to genetic association studies without additional proof of a causal relationship. However, insight into the role of PRRs in the pathophysiology of some disorders has already resulted in new therapeutic approaches based on immunomodulation of PRRs

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

Skin-derived antileukoproteinase (SKALP) and epidermal fatty acid-binding protein (E-FABP) : two novel markers of the psoriatic phenotype that respond differentially to topical steroid

Contains fulltext : 24892.pdf (publisher's version ) (Open Access