The Experts below are selected from a list of 561 Experts worldwide ranked by ideXlab platform
John S. Lazo - One of the best experts on this subject based on the ideXlab platform.
-
Bleomycin Hydrolase Activity and Cytotoxicity
2013Co-Authors: John S. Lazo, Carl J. Bol, Peter E. Schwartz, Contact The Aacr Publications, In Human TumorsAbstract:A sensitive new method to assay Bleomycin (BLM) metabo lism was developed using an ion-paired, reverse-phase highpressure liquid chromatography technique in conjunction with fluorescence detection that allowed levels of BLM less than 20 ng/ml to be detected. The metabolism of Bleomycin B2 in homogenates from benign and malignant human tumors was studied, and all 14 tumors were capable of metabolizing bleo mycin to desamidoBleomycin B2. Metabolites other than desamidoBleomycin B2 were not detected. BLM Hydrolase activities in individual tumors varied more than 7-fold. The importance of BLM Hydrolase in limiting the therapeutic effectiveness of BLM was examined by measuring BLM Hydrolase activity and the response of human tumors to BLM in culture. Response to BLM in culture was measured by dissociating human tumors t
-
Advances in Brief Genomic Structure and Genetic Mapping of the Human Neutral Cysteine Protease
2013Co-Authors: Cysteine Protease, John S. Lazo, Susana E. Montoya, Robert E. Ferrell, Contact The Aacr Publications, Bleomycin HydrolaseAbstract:Bleomycin Hydrolase (BH) is the only known eukaryotic enzyme that inactivates the widely used antineoplastic agent Bleomycin (BLM) and is a primary candidate gene for protection against lethal BLM-induced pul monary fibrosis and for BLM resistance in tumors. Human B!! was found to exist as a single gene that was mapped to chromosome 17 using National Institute of General Medical Sciences human/rodent hybrid mapping panels and localized to 17q11.1—11.2 by linkage analysis using the Centre d'Etude du Polymorphisme Humain reference database. The human BH gene consisted of 11 exons ranging in size from 69—198bp separated by introns ofapproximately 1kb, reflectingthe archetypal genomicstructure of the cysteine protease family. A polymorphic site was identified in the eleventh exon at bp 1450 encodIng either valine or Isoleucine. These fmdings provide essential tools requlred to define the role of RH in BLM-induced pulmonary fibrosis and BLM resistance in tumors
-
Analysis of the role of Bleomycin Hydrolase in antigen presentation and the generation of CD8 T cell responses.
Journal of immunology (Baltimore Md. : 1950), 2007Co-Authors: Charles F. Towne, John S. Lazo, Ian A. York, Levi B. Watkin, Kenneth L. RockAbstract:Long oligopeptides (>10 residues) are generated during the catabolism of cellular proteins in the cytosol. To be presented to T cells, such peptides must be trimmed by aminopeptidases to the proper size (typically 8–10 residues) to stably bind to MHC class I molecules. Aminopeptidases also destroy epitopes by trimming them to even shorter lengths. Bleomycin Hydrolase (BH) is a cytosolic aminopeptidase that has been suggested to play a key role in generating MHC class I-presented peptides. We show that BH-deficient cells from mice are unimpaired in their ability to present epitopes from N-extended precursors or whole Ags and express normal levels of MHC class I molecules. Similarly, BH-deficient mice develop normal CD8+ T cell responses to eight epitopes from three different viruses in vivo. Therefore, BH by itself is not essential for the generation or destruction of MHC class I peptides. In contrast, when BH−/− mice are crossed to mice lacking another cytosolic aminopeptidase, leucine aminopeptidase, the resulting BH−/−leucine aminopeptidase−/− progeny show a selective increase in CD8+ T cell responses to the gp276 epitope from lymphocytic choriomeningitis virus, whereas the ability to present and respond to several other epitopes is unchanged. Therefore, BH does influence presentation of some Ags, although its role is largely redundant with other aminopeptidases.
-
Cysteine 73 in Bleomycin Hydrolase Is Critical for Amyloid Precursor Protein Processing
Biochemical and biophysical research communications, 2001Co-Authors: Iliya Lefterov, Radosveta Koldamova, Martina I. Lefterova, Donald R. Schwartz, John S. LazoAbstract:Abstract Human Bleomycin Hydrolase (hBH) is a neutral cysteine protease that may regulate the secretion of soluble amyloid precursor protein (APP) and amyloid beta (Aβ), which is a major constituent of the Alzheimer's disease-associated amyloid plaques. We have now determined that APP interacts with hBH by using yeast two hybrid methods and in vitro binding studies revealed that APP interacted with a 68 amino acid region that includes the catalytic domain of hBH. Ectopic expression of hBH increased the secretion of Aβ but not of a second secreted protein, apolipoprotein A-I. Expression of hBH in which the catalytic cysteine 73 was mutated to serine failed to increase Aβ secretion. These results indicate a critical role for cysteine 73 of hBH in mediating APP processing.
-
Human Bleomycin Hydrolase regulates the secretion of amyloid precursor protein
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2000Co-Authors: Iliya Lefterov, Radosveta Koldamova, John S. LazoAbstract:Human Bleomycin Hydrolase (hBH) is a neutral cysteine protease genetically associated with increased risk for Alzheimer disease. We show here that ectopic expression of hBH in 293APPwt and CHOAPPsw cells altered the processing of amyloid precursor protein (APP) and increased significantly the release of its proteolytic fragment, β amyloid (Aβ). We also found that hBH interacted and colocalized with APP as determined by subcellular fractionation, in vitro binding assay, and confocal immunolocalization. Metabolic labeling and pulse-chase experiments showed that ectopic hBH expression increased secretion of soluble APPα/β products without changing the half-life of cellular APP. We also observed that this increased Aβ secretion was independent of hBH isoforms. Our findings suggest a regulatory role for hBH in APP processing pathways.—Lefterov, I. M., Koldamova, R. P., Lazo, J. S. Human Bleomycin Hydrolase regulates the secretion of amyloid precursor protein.
Hieronim Jakubowski - One of the best experts on this subject based on the ideXlab platform.
-
Bleomycin Hydrolase and hyperhomocysteinemia modulate the expression of mouse proteins involved in liver homeostasis
Amino Acids, 2014Co-Authors: Joanna Suszyńska-zajczyk, Jacek Wróblewski, Olga Utyro, Magdalena Łuczak, Łukasz Marczak, Hieronim JakubowskiAbstract:The liver is the major contributor to homocysteine (Hcy) metabolism and fatty liver disease is associated with hyperhomocysteinemia. Bleomycin Hydrolase (Blmh) is an aminoHydrolase that also participates in Hcy metabolism by hydrolyzing Hcy-thiolactone. To gain insight into hepatic functions of Blmh, we analyzed the liver proteome of Blmh ^−/− and Blmh ^+/+ mice in the absence and presence of diet-induced (high methionine) hyperhomocysteinemia using 2D IEF/SDS-PAGE gel electrophoresis and MALDI–TOF mass spectrometry. We identified eleven liver proteins whose expression was significantly altered as a result of the Blmh gene inactivation. The differential expression ( Blmh ^−/− vs. Blmh ^+/+) of four liver proteins was lower, of two proteins was higher, and was further modified in mice fed with a hyperhomocysteinemic high-Met diet. The down-regulated proteins are involved in lipoprotein metabolism (ApoA1, ApoE), antigen processing (Psme1), energy metabolism (Atp5h, Gamt), methylglyoxal detoxification (Glo1), oxidative stress response (Sod1), and inactivation of catecholamine neurotransmitters (Comt). The two up-regulated proteins are involved in nitric oxide generation (Ddah1) and xenobiotic detoxification (Sult1c1). We also found that livers of Blmh ^−/− mice expressed a novel variant of glyoxalase domain-containing protein 4 (Glod4) by a post-transcriptional mechanism. Our findings suggest that Blmh interacts with diverse cellular processes—from lipoprotein metabolism, nitric oxide regulation, antigen processing, and energy metabolism to detoxification and antioxidant defenses—that are essential for liver homeostasis and that modulation of these interactions by hyperhomocysteinemia underlies the involvement of Hcy in fatty liver disease.
-
Methionine-induced hyperhomocysteinemia and Bleomycin Hydrolase deficiency alter the expression of mouse kidney proteins involved in renal disease.
Molecular genetics and metabolism, 2014Co-Authors: Joanna Suszyńska-zajczyk, Olga Utyro, Hieronim JakubowskiAbstract:Abstract Scope Hyperhomocysteinemia (HHcy) induced by dietary or genetic factors is linked to kidney disease. Bleomycin Hydrolase (Blmh) metabolizes Hcy-thiolactone to Hcy. We aimed to explain the role of dietary HHcy in kidney disease. Methods and results We examined kidney proteome in dietary HHcy and Blmh-knockout mouse models using 2D IEF/SDS-PAGE gel electrophoresis and MALDI-TOF mass spectrometry. We found that the kidney proteome was altered by dietary HHcy and the Blmh −/− genotype. Proteins involved in metabolism of lipoprotein (ApoA1), amino acid and protein (Acy1, Hspd1), carbohydrate (Pdhb, Fbp1-isoform 1, Eno1), and energy metabolism (Ndufs8, Ldhd) were down-regulated. Proteins involved in carbohydrate metabolism (Fbp1-isoform 2), oxidative stress response (Prdx2), and detoxification (Glod4) were up-regulated. The Blmh −/− genotype down-regulated Glod4 isoform 3 mRNA but did not affect isoform 1 mRNA expression in mouse kidneys, suggesting post-transcriptional regulation of the Glod4 protein by the Blmh +/+ genotype. Responses of ApoA1, Acy1, Hspd1, Ndufs8, Fbp1, Eno1, and Prdx2 to HHcy and/or Blmh deficiency mimic their responses to renal disease. Conclusion Our findings indicate that Blmh interacts with diverse cellular processes – lipoprotein, amino acid and protein, carbohydrate, and energy metabolisms, detoxification, antioxidant defenses – that are essential for normal kidney homeostasis and that deregulation of these processes can account for the involvement of HHcy in kidney disease.
-
Hyperhomocysteinemia and Bleomycin Hydrolase modulate the expression of mouse brain proteins involved in neurodegeneration.
Journal of Alzheimer's disease : JAD, 2014Co-Authors: Joanna Suszyńska-zajczyk, Łukasz Marczak, Magdalena Łuczak, Hieronim JakubowskiAbstract:Homocysteine (Hcy) is a risk factor for Alzheimer's disease (AD). Bleomycin Hydrolase (BLMH) participates in Hcy metabolism and is also linked to AD. The inactivation of the Blmh gene in mice causes accumulation of Hcy-thiolactone in the brain and increases susceptibility to Hcy-thiolactone-induced seizures. To gain insight into brain-related Blmh function, we used two-dimensional IEF/SDS-PAGE gel electrophoresis and MALDI-TOF/TOF mass spectrometry to examine brain proteomes of Blmh -/- mice and their Blmh +/+ littermates fed with a hyperhomocysteinemic high-Met or a control diet. We found that: 1) proteins involved in brain-specific function (Ncald, Nrgn, Stmn1, Stmn2), antioxidant defenses (Aop1), cell cycle (RhoGDI1, Ran), and cytoskeleton assembly (Tbcb, CapZa2) were differentially expressed in brains of Blmh-null mice; 2) hyperhomocysteinemia amplified effects of the Blmh -/- genotype on brain protein expression; 3) proteins involved in brain- specific function (Pebp1), antioxidant defenses (Sod1, Prdx2, DJ-1), energy metabolism (Atp5d, Ak1, Pgam-B), and iron metabolism (Fth) showed differential expression in Blmh-null brains only in hyperhomocysteinemic animals; 4) most proteins regulated by the Blmh -/- genotype were also regulated by high-Met diet, albeit in the opposite direction; and 5) the differentially expressed proteins play important roles in neural development, learning, plasticity, and aging and are linked to neurodegenerative diseases, including AD. Taken together, our findings suggest that Blmh interacts with diverse cellular processes from energy metabolism and anti-oxidative defenses to cell cycle, cytoskeleton dynamics, and synaptic plasticity essential for normal brain homeostasis and that modulation of these interactions by hyperhomocysteinemia underlies the involvement of Hcy in AD.
-
metabolism and neurotoxicity of homocysteine thiolactone in mice protective role of Bleomycin Hydrolase
Amino Acids, 2012Co-Authors: Joanna Tisonczyk, Hieronim Jakubowski, Kamila BorowczykAbstract:Genetic or nutritional disorders in homocysteine (Hcy) metabolism elevate Hcy-thiolactone and cause heart and brain diseases. Hcy-thiolactone has been implicated in these diseases because it has the ability to modify protein lysine residues and generate toxic N-Hcy-proteins with auto-immunogenic, pro-thrombotic, and amyloidogenic properties. Bleomycin Hydrolase (Blmh) has the ability to hydrolyze l-Hcy-thiolactone (but not d-Hcy-thiolactone) to Hcy in vitro, but whether this reflects a physiological function has been unknown. Here, we show that Blmh−/− mice excreted in urine 1.8-fold more Hcy-thiolactone than wild-type Blmh+/+ animals (P = 0.02). Hcy-thiolactone was elevated 2.3-fold in brains (P = 0.004) and 2.0-fold in kidneys (P = 0.047) of Blmh−/− mice relative to Blmh+/+ animals. Plasma N-Hcy-protein was elevated in Blmh−/− mice fed a normal (2.3-fold, P < 0.001) or hyperhomocysteinemic diet (1.5-fold, P < 0.001), compared with Blmh+/+ animals. More intraperitoneally injected l-Hcy-thiolactone was recovered in plasma in Blmh−/− mice than in wild-type Blmh+/+ animals (83.1 vs. 39.3 μM, P < 0.0001). In Blmh+/+ mice injected intraperitoneally with d-Hcy-thiolactone, d,l-Hcy-thiolactone, or l-Hcy-thiolactone, 88, 47, or 6.3%, respectively, of the injected dose was recovered in plasma. The incidence of seizures induced by l-Hcy-thiolactone injections (3,700 nmol/g body weight) was higher in Blmh−/− than in Blmh+/+ mice (93.8 vs. 29.5%, P < 0.001). Using the Blmh null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself, is neurotoxic in vivo. Taken together, our findings indicate that Blmh protects mice against l-Hcy-thiolactone toxicity by metabolizing it to Hcy and suggest a mechanism by which Blmh might protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer’s disease.
-
Protective mechanisms against homocysteine toxicity : The role of Bleomycin Hydrolase
The Journal of biological chemistry, 2006Co-Authors: Jarosław Zimny, Marta Sikora, Andrzej Guranowski, Hieronim JakubowskiAbstract:Homocysteine (Hcy) editing by methionyl-tRNA synthetase results in the formation of Hcy-thiolactone and initiates a pathway that has been implicated in human disease. In addition to being cleared from the circulation by urinary excretion, Hcy-thiolactone is detoxified by the serum Hcy-thiolactonase/paraoxonase carried on high density lipoprotein. Whether Hcy-thiolactone is detoxified inside cells was unknown. Here we show that Hcy-thiolactone is hydrolyzed by an intracellular enzyme, which we have purified to homogeneity from human placenta and identified by proteomic analyses as human Bleomycin Hydrolase (hBLH). We have also purified an Hcy-thiolactonase from the yeast Saccharomyces cerevisiae and identified it as yeast Bleomycin Hydrolase (yBLH). BLH belongs to a family of evolutionarily conserved cysteine aminopeptidases, and its only known biologically relevant function was deamidation of the anticancer drug Bleomycin. Recombinant hBLH or yBLH, expressed in Escherichia coli, exhibits Hcy-thiolactonase activity similar to that of the native enzymes. Active site mutations, C73A for hBLH and H369A for yBLH, inactivate Hcy-thiolactonase activities. Yeast blh1 mutants are deficient in Hcy-thiolactonase activity in vitro and in vivo, produce more Hcy-thiolactone, and exhibit greater sensitivity to Hcy toxicity than wild type yeast cells. Our data suggest that BLH protects cells against Hcy toxicity by hydrolyzing intracellular Hcy-thiolactone.
Atsushi Takeda - One of the best experts on this subject based on the ideXlab platform.
-
Expression of Bleomycin Hydrolase in keratinization disorders
Archives of Dermatological Research, 2012Co-Authors: Yayoi Kamata, Akira Watarai, Norimitsu Saito, Hideki Maejima, Kensei Katsuoka, Atsushi Takeda, Kazuhiko IshiharaAbstract:A neutral cysteine protease, Bleomycin Hydrolase (BH), is widely expressed in mammalian tissues, with the skin seeming to contain the highest level. Our previous study revealed that BH transcription is modulated both during differentiation and by cytokines. However, BH involvement in keratinization disorder is not well known. In the present study, we performed immunohistochemical studies of BH and other serine/cysteine proteases in human normal skin and lesional skin with keratinization disorders. BH-positive cells were detected in granular layers of orthokeratotic and hyperkeratotic skin diseases, such as erythrokeratoderma and lichen planus. In parakeratotic skin diseases with porokeratosis, pityriasis rubra pilaris and psoriasis, BH staining was decreased in lesional skins compared to that in normal skin. Similar results were obtained for cysteine proteases, caspase-14 and calpain I. On the other hand, cells positive for serine proteases kallikrein 5 and 7 were increased in parakeratotic and inflammatory skin diseases, such as psoriasis. Semi-quantification analysis revealed that BH- and caspase-14-positive staining had higher intensity than those of the other proteases in normal epidermis. As BH is the major citrulline aminopeptidase in normal granular layer, the alternation would have a significant effect on terminal differentiation processes, such as aberrant processing of deiminated peptides. Therefore, BH may play an important role during the late stage of epidermal differentiation.
-
Bleomycin Hydrolase is regulated biphasically in a differentiation- and cytokine-dependent manner: relevance to atopic dermatitis.
The Journal of biological chemistry, 2010Co-Authors: Yayoi Kamata, Kazuhiko Ishihara, Atsushi Takeda, Mami Yamamoto, Fumitaka Kawakami, Ryoji Tsuboi, Toshihiko HibinoAbstract:Loss-of-function mutation in the profilaggrin gene is a major risk factor for atopic dermatitis (AD). Previously, we showed that a neutral cysteine protease, Bleomycin Hydrolase (BH), has a role in generating natural moisturizing factors, and calpain I is an upstream protease in the filaggrin degradation pathway. Here, we investigated the transcriptional regulatory mechanisms of BH and the relevance of BH to AD. First, we cloned the 5'-flanking region of BH. Deletion analyses identified a critical region for BH promoter activity within -216 bp upstream. Electrophoretic mobility shift assay revealed that MZF-1, Sp-1, and interferon regulatory factor-1/2 could bind to this region in vitro. Moreover, site-directed mutagenesis of the MZF-1 and Sp-1 motifs markedly reduced BH promoter activity. These data indicate that BH expression is up-regulated via MZF-1 and Sp-1. Interestingly, a Th1 cytokine, IFN-γ, significantly reduced the expression of BH. Analyses with site-directed mutagenesis and small interference RNA supported the suppressing effect of IFN-γ on BH expression. On the other hand, a Th2 cytokine, IL-4, did not show any direct effect on BH expression. However, it down-regulated MZF-1 and Sp-1 in cultured keratinocytes, indicating that IL-4 could work as a suppressor in BH regulation. Lastly, we examined expression of BH in skins of patients with AD. BH activity and expression were markedly decreased in AD lesional skin, suggesting a defect of the filaggrin degradation pathway in AD. Our results suggest that BH transcription would be modulated during both differentiation and inflammation.
-
Quantification of neutral cysteine protease Bleomycin Hydrolase and its localization in rat tissues.
Journal of biochemistry, 2006Co-Authors: Yayoi Kamata, Akane Kajiya, Yoshiko Itoh, Sachiyo Karasawa, Chie Sakatani, Susumu Takekoshi, R. Yoshiyuki Osamura, Atsushi TakedaAbstract:A neutral cysteine protease, Bleomycin Hydrolase (BH), was found to be present in the range 3.7-131.1 ng per mg of rat tissues by enzyme-lined immunosorbent assay (ELISA). Newborn rat skin contained the highest amount of BH, and relatively high levels of BH were detected in the kidney and liver of 6-week-old male rats. The tissue distribution of BH in female rats was similar to that in male rats. Moreover, BH was detected in the extracts of erythrocytes and leukocyte-rich cells as well as in those of rat hemo-lymphocytic lineage cell lines by Western blotting. The BH level was increased at 6 weeks after birth and then slightly decreased. By immunohistochemistry, BH was localized as granular staining in the distal and proximal tubular cells of the kidney, and it was also detected in hepatocytes of the liver, in the red pulpy region of the spleen and in neurons of the brain. An immunoelectron microscopic study showed that BH-immunoreactivity was essentially located in the cytoplasm and at the outer membrane of the rough endoplasmic reticulum of epithelial cells of the kidney, as well as in that of hepatocytes of the liver. These results suggest that BH may play ubiquitous and unique roles in rat tissues.
-
Processing of Amyloid β-Peptides by Neutral Cysteine Protease Bleomycin Hydrolase
Protein and peptide letters, 2006Co-Authors: Akane Kajiya, Hiroyuki Kaji, Toshiaki Isobe, Atsushi TakedaAbstract:We studied the processing of amyloid β-peptides (Aβs) including Aβ1-40, Aβ1-42 and pAβ3-42 by rat neutral cysteine protease Bleomycin Hydrolase (BH) according to the methods of SDS-PAGE, HPLC and matrix-assisted laser desorption/inonization time-of-flight mass spectrometry (MALDI-TOF MS). BH significantly processed them by novel features of its diverse activities. It initially cleaved at two sites, His14-Gln15 and Phe19-Phe20 bonds, in Aβ1-40 and Aβ1-42 by endopeptidase activity. The resultant peptides were degraded to short intermediates then to amino acids by aminopeptidase and/or carboxypeptidase activities. Also, full-length Aβs were clipped at the carboxyl(C)-terminal region. On the other hand, BH cleaved at only the His14-Gln15 bond in pβA3-42 within A short period of the reaction by endopeptidase activity, and processed the intermediates in order by carboxypeptidase activity. On processing by BH, it found that both fibrillar Aβ1-40 and Aβ1-42 were more resistant than non-fibrillar peptides. These results indicate that the processing specificity of BH depends upon the structure and sequence of Aβs.
-
Bleomycin Hydrolase immunoreactivity in senile plaque in the brains of patients with alzheimer s disease
Brain Research, 1999Co-Authors: Yoshio Namba, Atsushi Takeda, Yasuyoshi Ouchi, Akira Ueki, Kazuhiko IkedaAbstract:Bleomycin Hydrolase (BH), a cysteine protease belonging to the papain superfamily, is one of the candidate β secretases. We performed immunohistochemical studies of Alzheimer's disease (AD) brains using an antibody to BH. Polyclonal antibody to BH immunostained neocortical neurons. The immunoreactivity was also found in senile plaques in AD. These results may suggest a role of BH in amyloid formation.
Yayoi Kamata - One of the best experts on this subject based on the ideXlab platform.
-
Expression of Bleomycin Hydrolase in keratinization disorders
Archives of Dermatological Research, 2012Co-Authors: Yayoi Kamata, Akira Watarai, Norimitsu Saito, Hideki Maejima, Kensei Katsuoka, Atsushi Takeda, Kazuhiko IshiharaAbstract:A neutral cysteine protease, Bleomycin Hydrolase (BH), is widely expressed in mammalian tissues, with the skin seeming to contain the highest level. Our previous study revealed that BH transcription is modulated both during differentiation and by cytokines. However, BH involvement in keratinization disorder is not well known. In the present study, we performed immunohistochemical studies of BH and other serine/cysteine proteases in human normal skin and lesional skin with keratinization disorders. BH-positive cells were detected in granular layers of orthokeratotic and hyperkeratotic skin diseases, such as erythrokeratoderma and lichen planus. In parakeratotic skin diseases with porokeratosis, pityriasis rubra pilaris and psoriasis, BH staining was decreased in lesional skins compared to that in normal skin. Similar results were obtained for cysteine proteases, caspase-14 and calpain I. On the other hand, cells positive for serine proteases kallikrein 5 and 7 were increased in parakeratotic and inflammatory skin diseases, such as psoriasis. Semi-quantification analysis revealed that BH- and caspase-14-positive staining had higher intensity than those of the other proteases in normal epidermis. As BH is the major citrulline aminopeptidase in normal granular layer, the alternation would have a significant effect on terminal differentiation processes, such as aberrant processing of deiminated peptides. Therefore, BH may play an important role during the late stage of epidermal differentiation.
-
Bleomycin Hydrolase is regulated biphasically in a differentiation- and cytokine-dependent manner: relevance to atopic dermatitis.
The Journal of biological chemistry, 2010Co-Authors: Yayoi Kamata, Kazuhiko Ishihara, Atsushi Takeda, Mami Yamamoto, Fumitaka Kawakami, Ryoji Tsuboi, Toshihiko HibinoAbstract:Loss-of-function mutation in the profilaggrin gene is a major risk factor for atopic dermatitis (AD). Previously, we showed that a neutral cysteine protease, Bleomycin Hydrolase (BH), has a role in generating natural moisturizing factors, and calpain I is an upstream protease in the filaggrin degradation pathway. Here, we investigated the transcriptional regulatory mechanisms of BH and the relevance of BH to AD. First, we cloned the 5'-flanking region of BH. Deletion analyses identified a critical region for BH promoter activity within -216 bp upstream. Electrophoretic mobility shift assay revealed that MZF-1, Sp-1, and interferon regulatory factor-1/2 could bind to this region in vitro. Moreover, site-directed mutagenesis of the MZF-1 and Sp-1 motifs markedly reduced BH promoter activity. These data indicate that BH expression is up-regulated via MZF-1 and Sp-1. Interestingly, a Th1 cytokine, IFN-γ, significantly reduced the expression of BH. Analyses with site-directed mutagenesis and small interference RNA supported the suppressing effect of IFN-γ on BH expression. On the other hand, a Th2 cytokine, IL-4, did not show any direct effect on BH expression. However, it down-regulated MZF-1 and Sp-1 in cultured keratinocytes, indicating that IL-4 could work as a suppressor in BH regulation. Lastly, we examined expression of BH in skins of patients with AD. BH activity and expression were markedly decreased in AD lesional skin, suggesting a defect of the filaggrin degradation pathway in AD. Our results suggest that BH transcription would be modulated during both differentiation and inflammation.
-
neutral cysteine protease Bleomycin Hydrolase is essential for the breakdown of deiminated filaggrin into amino acids
Journal of Biological Chemistry, 2009Co-Authors: Yayoi Kamata, Kazuhiko Ishihara, Aya Taniguchi, Mami Yamamoto, Junko Nomura, Hidenari Takahara, Toshihiko Hibino, A TakedaAbstract:Abstract 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 NH2 terminus of representative tryptic peptides perfectly matched the sequence of rat Bleomycin Hydrolase (BH). The enzyme released various amino acids except Pro from β-naphthylamide derivatives and hydrolyzed citrulline-β-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.
-
Quantification of neutral cysteine protease Bleomycin Hydrolase and its localization in rat tissues.
Journal of biochemistry, 2006Co-Authors: Yayoi Kamata, Akane Kajiya, Yoshiko Itoh, Sachiyo Karasawa, Chie Sakatani, Susumu Takekoshi, R. Yoshiyuki Osamura, Atsushi TakedaAbstract:A neutral cysteine protease, Bleomycin Hydrolase (BH), was found to be present in the range 3.7-131.1 ng per mg of rat tissues by enzyme-lined immunosorbent assay (ELISA). Newborn rat skin contained the highest amount of BH, and relatively high levels of BH were detected in the kidney and liver of 6-week-old male rats. The tissue distribution of BH in female rats was similar to that in male rats. Moreover, BH was detected in the extracts of erythrocytes and leukocyte-rich cells as well as in those of rat hemo-lymphocytic lineage cell lines by Western blotting. The BH level was increased at 6 weeks after birth and then slightly decreased. By immunohistochemistry, BH was localized as granular staining in the distal and proximal tubular cells of the kidney, and it was also detected in hepatocytes of the liver, in the red pulpy region of the spleen and in neurons of the brain. An immunoelectron microscopic study showed that BH-immunoreactivity was essentially located in the cytoplasm and at the outer membrane of the rough endoplasmic reticulum of epithelial cells of the kidney, as well as in that of hepatocytes of the liver. These results suggest that BH may play ubiquitous and unique roles in rat tissues.
Stephen Albert Johnston - One of the best experts on this subject based on the ideXlab platform.
-
Crystal structure of human Bleomycin Hydrolase, a self-compartmentalizing cysteine protease
Structure (London England : 1993), 1999Co-Authors: Paul A. O'farrell, Stephen Albert Johnston, Fernando Gonzalez, W. Zheng, Leemor Joshua-torAbstract:Abstract Background: Bleomycin Hydrolase (BH) is a cysteine protease that is found in all tissues in mammals as well as in many other eukaryotes and prokaryotes. Although its conserved cellular function is as yet unknown, human Bleomycin Hydrolase (hBH) has clinical significance in that it is thought to be the major cause of tumor cell resistance to Bleomycin chemotherapy. In addition, it has been reported that an allelic variant of hBH is genetically linked to Alzheimer's disease. Results: We have determined the crystal structures of wild-type hBH and of a mutant form of the enzyme. The overall structure is very similar to that of the previously determined yeast homolog, however, there is a striking difference in the charge distribution. The central channel, which has a strong positive electrostatic potential in the yeast protein, is slightly negative in hBH. We have determined that hBH does not have the DNA-binding activity of the yeast protein and that the enzyme is localized to the cytoplasm. Conclusions: The difference in charge distribution between the yeast and human BH enzymes is most likely responsible for the difference in DNA-binding activity. Nevertheless, the C-terminal autoprocessing activity and the role of the C terminus as a determinant for peptidase activity are conserved between the yeast and human forms. The structure of hBH suggests that the putative Alzheimer's disease linked variation does not directly after the intrinsic peptidase activity. Rather, the position of the mutation suggests that it could affect interactions with another protein, which may modulate peptidase activity through repositioning of the C terminus.
-
The nucleic acid binding activity of Bleomycin Hydrolase is involved in Bleomycin detoxification.
Molecular and Cellular Biology, 1998Co-Authors: Wenjin J Zheng, Stephen Albert JohnstonAbstract:Yeast Bleomycin Hydrolase, Gal6p, is a cysteine peptidase that detoxifies the anticancer drug Bleomycin. Gal6p is a dual-function protein capable of both nucleic acid binding and peptide cleavage. We now demonstrate that Gal6p exhibits sequence-independent, high-affinity binding to single-stranded DNA, nicked double-stranded DNA, and RNA. A region of the protein that is involved in binding both RNA and DNA substrates is delineated. Immunolocalization reveals that the Gal6 protein is chiefly cytoplasmic and thus may be involved in binding cellular RNAs. Variant Gal6 proteins that fail to bind nucleic acid also exhibit reduced ability to protect cells from Bleomycin toxicity, suggesting that the nucleic acid binding activity of Gal6p is important in Bleomycin detoxification and may be involved in its normal biological functions.
-
The cysteine-peptidase Bleomycin Hydrolase is a member of the galactose regulon in yeast.
The Journal of biological chemistry, 1997Co-Authors: Wenjin J Zheng, Stephen Albert JohnstonAbstract:Abstract Bleomycin Hydrolase is a cysteine peptidase discovered through its ability to detoxify the anti-cancer glycopeptide, Bleomycin. Although found in all tissues in mammals and in both eukaryotes and prokaryotes, the normal cellular function of this peptidase is not known. We had previously reported the purification of Bleomycin Hydrolase from yeast based on its unexpected ability to bind DNA. Recently we collaborated in solving the crystal structure of this protein, revealing a hexameric ring organization. We now report that the molecular characterization of the gene encoding yeast Bleomycin Hydrolase is also surprising. The transcription of the gene is regulated by galactose. Furthermore, this regulation is conveyed by a binding site for the Gal4 regulatory protein in its promoter, prompting the designation of this gene as GAL6. Gal6p also appears to have a negative effect on the GALsystem as a deletion of the gene leads to a 2–5-fold higher expression of the GAL1, GAL2, GAL7, and MEL1 genes. TheGAL6 deletion does not affect the expression of another inducible gene, HSP26. Neither the peptidase nor the nucleic acid binding activity of Gal6p as assayed is apparently required to convey this regulation, implying yet another function for this new member of the GAL regulon.
-
Yeast Bleomycin Hydrolase is a DNA-binding cysteine protease. Identification, purification, biochemical characterization.
The Journal of biological chemistry, 1994Co-Authors: Stephen Albert JohnstonAbstract:Abstract Bleomycin (BLM) is a DNA binding and damaging antibiotic produced by Streptomyces verticillus that has been used as an anti-tumor drug for various human cancers. The mammalian BLM Hydrolase is a cysteine protease that inactivates BLM to relieve the toxicity of BLM to normal and tumor cells. The normal physiological function of BLM Hydrolase is not known, but its activity limits the use of BLM in cancer chemotherapy. We have discovered a DNA binding activity for the yeast homolog of the mammalian BLM Hydrolase in the course of studying the interaction of GAL4, a DNA-binding transcription factor, with its DNA recognition sites. Using gel mobility shift assays, we have purified a protein from yeast that binds specifically to the GAL4 DNA-binding sites. The purified protein is a tetramer of a 48-kDa polypeptide. The gene encoding the 48-kDa polypeptide was cloned and has a high homology to rabbit BLM Hydrolase. The purified protein was confirmed to have a cysteine protease activity that can hydrolyze and inactivate BLM in vitro. We have established the optimal conditions for the protease activity of this protein and biochemically characterized its DNA binding activity. This protein binds both single- and double-stranded forms of the GAL4 DNA-binding sites with high affinity (10 nM to single strand and 1 microM to double strand). Its DNA binding activity is heat stable and resistant to various detergents. This protein may represent the first example of a eukaryotic DNA-binding protease. The discovery of a DNA binding activity for BLM Hydrolase suggests an in vivo interaction between it and BLM on DNA.