Anti-Cathepsin L Monoclonal Antibodies That Distinguish Cathepsin L from Cathepsin V

Kopitar-Jerala, Nataša; Bevec, Tadeja; Barlič–Maganja, Darja; Gubenšek, Franc; Türk, Vito

doi:10.1515/bchm.2001.382.5.867

Cited by 8 publications

(8 citation statements)

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“…Materials-The following antibodies were used: anti-Bcl-x L (H-62), anti-Bcl-2 (C-2), and anti-Bak (H-211) from Santa Cruz Biotechnology, anti-Mcl-1 (RC13) from Abcam, anti-cytochrome c from BD PharMingen, anti-actin from Sigma, anti-PARP antibodies recognizing caspase-cleaved PARP from Promega, anti-Bid from R&D Systems, anti-XIAP from BD Transduction Laboratories, and anti-cathepsin L, which were prepared in the laboratory (41). All horseradish peroxidaseconjugated secondary antibodies were from Sigma.…”

Section: Methodsmentioning

confidence: 99%

Cysteine Cathepsins Trigger Caspase-dependent Cell Death through Cleavage of Bid and Antiapoptotic Bcl-2 Homologues

Droga-Mazovec

Bojić

Petelin

et al. 2008

Journal of Biological Chemistry

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338

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As a model for defining the role of lysosomal cathepsins in apoptosis, we characterized the action of the lysosomotropic agent LeuLeuOMe using distinct cellular models. LeuLeuOMe induces lysosomal membrane permeabilization, resulting in release of lysosomal cathepsins that cleave the proapoptotic Bcl-2 family member Bid and degrade the antiapoptotic member Bcl-2, Bcl-xL, or Mcl-1. The papain-like cysteine protease inhibitor E-64d largely prevented apoptosis, Bid cleavage, and Bcl-2/Bcl-xL/Mcl-1 degradation. The pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone failed to prevent Bid cleavage and degradation of anti-apoptotic Bcl-2 homologues but substantially decreased cell death, suggesting that cathepsin-mediated apoptosis in these cellular models mostly follows a caspase-dependent pathway. Moreover, in vitro experiments showed that one or more of the cysteine cathepsins B, L, S, K, and H could cleave Bcl-2, Bcl-xL, Mcl-1, Bak, and BimEL, whereas no Bax cleavage was observed. On the basis of inhibitor studies, we demonstrate that lysosomal disruption triggered by LeuLeuOMe occurs before mitochondrial damage. We propose that degradation of anti-apoptotic Bcl-2 family members by lysosomal cathepsins synergizes with cathepsin-mediated activation of Bid to trigger a mitochondrial pathway to apoptosis. Moreover, XIAP (X-chromosome-linked inhibitor of apoptosis) was also found to be a target of cysteine cathepsins, suggesting that cathepsins can mediate caspase-dependent apoptosis also downstream of mitochondria.

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Section: Methodsmentioning

confidence: 99%

Cysteine Cathepsins Trigger Caspase-dependent Cell Death through Cleavage of Bid and Antiapoptotic Bcl-2 Homologues

Droga-Mazovec

Bojić

Petelin

et al. 2008

Journal of Biological Chemistry

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338

262

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“…Anti-stefin B (ab53725), anti-iNOS (ab3523), anti-ERK 1/2 (ab17942), anti-phospho-STAT3 Y705 (ab76315), anti-STAT3 (ab50761), from Sigma (St. Louis, USA) anti-diphosphorylated ERK 1/2 (M8159), from Cell Signaling (Danvers, MA, USA) anti-phospho-p38 (9211) and anti-p38 (9212). Anti-cathepsin L antibodies were described in [20]. Recombinant mouse M-CSF was purchased from eBioscience (San Diego, CA, USA) and used at a final concentration of 40 ng/ml.…”

Section: Reagents and Cell Culturementioning

confidence: 99%

“…Cathepsin L was labeled with mouse anti-cathepsin L monoclonal antibody N135 [20] and stefin B with rabbit anti-stefin B polyclonal antibodies. Fluorescence microscopy was performed using Carl Zeiss LSM 510 confocal microscope.…”

Section: Confocal Microscopymentioning

confidence: 99%

Decreased IL‐10 expression in stefin B‐deficient macrophages is regulated by the MAP kinase and STAT‐3 signaling pathways

et al. 2014

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a b s t r a c tInnate immune responses are tightly regulated to avoid excessive activation and subsequent inflammatory damage to the host, and interleukin-10 (IL-10) plays a crucial role in preventing inflammation. Stefin B (cystatin B) is an endogenous inhibitor of cysteine proteinases. In stefin B-deficient bone marrow-derived macrophages (BMDMs), we detected an increase in the induction of the LPS-induced pro-inflammatory signal nitric oxide (NO) but decreased IL-10 expression. The phosphorylation of ERK and p38 MAP-kinases was significantly decreased in stefin B-deficient macrophages, as was STAT-3 phosphorylation. These findings show that stefin B influences the expression of anti-inflammatory IL-10 in response to the TLR4 agonist LPS.

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“…The enzyme preparation was dialyzed against 50 mM acetate buffer, pH 4.4, containing 1 mM EDTA and then applied to the column equilibrated with the same buffer. Cathepsin L was detected in cathepsin X eluate using cathepsin L-specific monoclonal antibodies (Kopitar-Jerala et al, 2001), which were used to avoid potential confusion due to the unclear substrate specificity of the two enzymes. Adjustment of the original method described by Nagler et al (1999b) was necessary, since the separation of active cathepsin X from cathepsin L, which was used to activate cathepsin X, was incomplete at pH 7.0 on a DEAE column.…”

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confidence: 99%

Recombinant human cathepsin X is a carboxymonopeptidase only: a comparison with cathepsins B and L

Puzer

Cotrin

Cezari

et al. 2005

Biological Chemistry

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The S1 and S2 subsite specificity of recombinant human cathepsins X was studied using fluorescence resonance energy transfer (FRET) peptides with the general sequences Abz-Phe-Xaa-Lys(Dnp)-OH and Abz-Xaa-Arg-Lys(Dnp)-OH, respectively (Abz=ortho-aminobenzoic acid and Dnp=2,4-dinitrophenyl; Xaa=various amino acids). Cathepsin X cleaved all substrates exclusively as a carboxymonopeptidase and exhibited broad specificity. For comparison, these peptides were also assayed with cathepsins B and L. Cathepsin L hydrolyzed the majority of them with similar or higher catalytic efficiency than cathepsin X, acting as an endopeptidase mimicking a carboxymonopeptidase (pseudo-carboxymonopeptidase). In contrast, cathepsin B exhibited poor catalytic efficiency with these substrates, acting as a carboxydipeptidase or an endopeptidase. The S1' subsite of cathepsin X was mapped with the peptide series Abz-Phe-Arg-Xaa-OH and the enzyme preferentially hydrolyzed substrates with hydrophobic residues in the P1' position.

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Anti-Cathepsin L Monoclonal Antibodies That Distinguish Cathepsin L from Cathepsin V

Cited by 8 publications

References 0 publications

Cysteine Cathepsins Trigger Caspase-dependent Cell Death through Cleavage of Bid and Antiapoptotic Bcl-2 Homologues

Cysteine Cathepsins Trigger Caspase-dependent Cell Death through Cleavage of Bid and Antiapoptotic Bcl-2 Homologues

Decreased IL‐10 expression in stefin B‐deficient macrophages is regulated by the MAP kinase and STAT‐3 signaling pathways

Recombinant human cathepsin X is a carboxymonopeptidase only: a comparison with cathepsins B and L

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