Quantification of Neutral Cysteine Protease Bleomycin Hydrolase and its Localization in Rat Tissues

Kamata, Yoichi; Itoh, Yoshiko; Kajiya, Akane; Karasawa, Sachiyo; Sakatani, Chie; Takekoshi, Susumu; Osamura, R. Yoshiyuki; Takeda, Atsushi

doi:10.1093/jb/mvm005

Cited by 30 publications

(12 citation statements)

References 35 publications

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“…Unfortunately, that particular study did not evaluate BH levels in older animals (ie, 1–2 years). 44 Others have shown alterations in BH associated with Alzheimer’s disease, which is a disease of older individuals. 45,46 Therefore, presumably, there could be some changes of BH in lungs of old mice compared with young ones.…”

Section: Discussionmentioning

confidence: 99%

Predisposition for Disrepair in the Aged Lung

Sueblinvong¹,

Neujahr²,

Mills³

et al. 2012

The American Journal of the Medical Sciences

114

125

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Introduction Idiopathic pulmonary fibrosis (IPF) is a devastating progressive lung disease with an average survival of only 3 to 5 years. The mechanisms underlying the initiation and progression of IPF are poorly understood, and treatments available have only modest effect on disease progression. Interestingly, the incidence of IPF is approximately 60 times more common in individuals aged 75 years and older, but the mechanism by which aging promotes fibrosis is unclear. The authors hypothesized that aged lungs have a profibrotic phenotype that render it susceptible to disrepair after injury. Methods Young and old mice were treated with bleomycin to examine disrepair in the aged lung. In addition, uninjured young and old mouse lungs were analyzed for transforming growth factor-beta 1 (TGF-β1) production, extracellular matrix composition and lung fibroblast phenotype. Lung fibroblasts were treated with a DNA methyltransferase inhibitor to examine the potential epigenetic mechanisms involved in age-associated phenotypic alterations. Results The lungs of old mice showed worse fibrosis after bleomycin-induced injury compared with the lungs from young mice. At baseline, aged lungs expressed a profibrotic phenotype characterized by increased mRNA expression for fibronectin extracellular domain A (Fn-EDA) and the matrix metalloproteinases (MMPs) MMP-2 and MMP-9. Old lungs also expressed higher levels of TGF-β receptor 1 and TGF-β1 mRNA, protein and activity as determined by increased Smad3 expression, protein phosphorylation and DNA binding. Lung fibroblasts harvested from aged lungs showed reduced expression of the surface molecule Thy-1, a finding also implicated in lung fibrosis; the latter did not seem related to Thy-1 gene methylation. Conclusion Altogether, aged lungs manifest a profibrotic phenotype characterized by enhanced fibronectin extracellular domain A and MMP expression and increased TGF-β1 expression and signaling and are populated by Thy-1–negative fibroblasts, all implicated in the pathogenesis of lung fibrosis.

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Section: Discussionmentioning

confidence: 99%

Predisposition for Disrepair in the Aged Lung

Sueblinvong¹,

Neujahr²,

Mills³

et al. 2012

The American Journal of the Medical Sciences

114

125

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“…However, it had no activity toward any protein substrates including myelin basic protein, azocasein, bovine serum albumin, myoglobin, and cystatin A. The enzyme was co-localized with filaggrin in the upper layers of the stratum corneum, where it was present in large amounts, in mammalian skin (31,(45)(46)(47). These results strongly suggested that BH participated in the release of free amino acids including citrulline in peptides processed from deiminated filaggrin in mammalian skin.…”

Section: Filaggrin Fragmentmentioning

confidence: 96%

Neutral Cysteine Protease Bleomycin Hydrolase Is Essential for the Breakdown of Deiminated Filaggrin into Amino Acids

Kamata

Taniguchi

Yamamoto

et al. 2009

Journal of Biological Chemistry

152

131

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Filaggrin is a component of the cornified cell envelope and the precursor of free amino acids acting as a natural moisturizing factor in the stratum corneum. Deimination is critical for the degradation of filaggrin into free amino acids. In this study, we tried to identify the enzyme(s) responsible for the cleavage of deiminated filaggrin in vitro. First, we investigated citrulline aminopeptidase activity in the extract of newborn rat epidermis by double layer fluorescent zymography and detected strong activity at neutral pH. Monitoring the citrulline-releasing activity, we purified an enzyme of 280 kDa, comprised of six identical subunits of 48 kDa. The NH 2 terminus of representative tryptic peptides perfectly matched the sequence of rat bleomycin hydrolase (BH). The enzyme released various amino acids except Pro from ␤-naphthylamide derivatives and hydrolyzed citrulline-␤-naphthylamide most effectively. Thus, to break down deiminated filaggrin, another protease would be required. Among proteases tested, calpain I degraded the deiminated filaggrin effectively into many peptides of different mass on the matrix-assisted laser desorption/ionization-time of flight mass spectrum. We confirmed that various amino acids including citrulline were released by BH from those peptides. On the other hand, caspase 14 degraded deiminated filaggrin into a few peptides of limited mass. Immunohistochemical analysis of normal human skin revealed co-localization of BH and filaggrin in the granular layer. Collectively, our results suggest that BH is essential for the synthesis of natural moisturizing factors and that calpain I would play a role as an upstream protease in the degradation of filaggrin.The mammalian epidermal keratinocytes arise from proliferating basal cells and move outward through a series of distinct differentiation events to form the stratum corneum (1, 2). During this progressive epidermal differentiation, keratinocytes express different proteins such as keratins, profilaggrin/filaggrin, involucrin, small proline-rich proteins, loricrin, cystatin A, and elafin, which form the cornified envelope of mature corneocytes (3-7). Profilaggrin is synthesized as a large, extremely insoluble phosphoprotein that consists of a unique NH 2 -terminal Ca 2ϩ -binding protein of the S-100 family, linked to 10 -20 tandem filaggrin monomer repeats (8 -10). Each individual filaggrin repeat is completely removed by proteolysis to generate the mature filaggrin monomer (a molecular mass of 37 kDa in human). Then, filaggrin is completely degraded in the uppermost layer of the stratum corneum to produce a mixture of free and modified hygroscopic amino acids that are important for maintaining epidermal hydration (2, 11-13). In addition, a number of proteins are subjected to various post-translational modifications such as disulfide bonding, N-(␥-glutamyl)-lysine isopeptide cross-linking, and deimination during the terminal differentiation of epidermal keratinocytes (4, 6, 14, 15). Deimination is catalyzed by peptidylarginine deiminase (P...

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“…Once in the cytosol, BLM can be degraded by BLM hydrolase [1]. Different cell lines could have different amount or activity of BLM hydrolase, which could lead to BLM degradation effectiveness [34,35]. Alternatively, different cell lines could show different BLM resistance due to drug accumulation properties or DNA reparation mechanisms [36,37].…”

Section: Different Cell Line Sensitivity To Blmmentioning

confidence: 99%