The Preparation of Catalytically Active Human Cathepsin B from Its Precursor Expressed in <i>Escherichia coli</i> in the Form of Inclusion Bodies

Kuhelj, Robert; Dolinar, Marko; Pungerčar, Jože; Türk, Vito

doi:10.1111/j.1432-1033.1995.0533k.x

Cited by 111 publications

(60 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cysteine cathepsin protein levels were determined by ELISAs as reported previously [51]. Human recombinant CtsB [64], CtsS (donated by Dr. Boris Turk, Jožef Stefan Institute, SI), and CtsX [65] were used as standards.…”

Section: Determination Of Levels Of Cysteine Cathepsin Protein and Acmentioning

confidence: 99%

Three‐dimensional invasion of macrophages is mediated by cysteine cathepsins in protrusive podosomes

et al. 2012

View full text Add to dashboard Cite

IntroductionThe ability of macrophages (Mfs) to progress to specific tissues, increase matrix remodeling, and induce angiogenesis is essential for their normal physiological function [1,2]. In addition, tissue infiltration of Mfs is also involved in pathological processes such as chronic inflammation, atherosclerosis, neurodegenerative disorders, and cancer progression [3]. The presence of Mfs Correspondence: Dr. Bojana Mirković e-mail: bojana.mirkovic@ffa.uni-lj.si within tumors is generally a marker of poor prognosis since they enhance angiogenesis and metastasis [4]. Furthermore, tumorassociated Mfs are emerging as essential matrix-remodeling cells during tumor-cell invasion. They have been reported to be present at high frequency at the invasive front of the tumor, where the breakdown of extracellular matrix (ECM) occurs [5]. Therefore, there is a great need to specifically control tissue infiltration of Mfs by targeting Mf migration-related molecules [6]. * These authors contributed equally to the present work. Eur. J. Immunol. 2012. 42: 3429-3441 Mf migration has been studied extensively in two dimensions (2D) on artificial substrates; however, in vivo Mfs migrate through physiological and pathological tissue and interact with the surrounding three-dimensional (3D) ECM, including basement membrane, collagen-rich interstitial matrices, and dense tissues such as tumors. Cells of different origins and/or at different differentiation stages use distinct mechanisms of cell motility in the 3D environment. In the mesenchymal mode, cells wider than the matrix pore size degrade the matrix via proteolytic and force-based matrix remodeling, thus migrating in a "path-generating" manner, while amoeboid cells squeeze through pre-existing pores in a proteaseindependent fashion in a "path-finding" mode [7,8]. The first 3D migration studies performed on cells of the immune system suggested that these cells migrate in a protease-independent fashion [7,9]. In contrast, a recent study demonstrated that Mfs adapt their migration strategy depending on the architecture of the 3D environment. In this study, Mfs were found to use the amoeboid migration mode in fibrillar collagen I and the mesenchymal migration mode in dense substrates, such as Matrigel and gelled collagen I [10].The invasion of cancer cells is facilitated by ECM-degrading invadopodia [11]. Podosomes are structures functionally similar to the invadopodia that are found in Mfs, osteoclasts, and DCs [12,13]. During migration on 2D surfaces [14], dot or ringlike podosomes form at the ventral surface of the migrating cells where they establish direct contact with the substratum and are also involved in matrix degradation [11]. Structurally, podosomes comprise a concentrated core of F-actin and actin-regulatory proteins [14]. Surrounding the core is a ring region that contains scaffolding-, signaling-, and integrin-binding proteins, including the typical focal adhesion proteins vinculin, talin, paxillin, and FAK [15]. When Mfs migrate in a 3D environment (i.e., migra...

show abstract

Section: Determination Of Levels Of Cysteine Cathepsin Protein and Acmentioning

confidence: 99%

Three‐dimensional invasion of macrophages is mediated by cysteine cathepsins in protrusive podosomes

et al. 2012

View full text Add to dashboard Cite

show abstract

“…Papain (2 Â crystallized) was purchased from Sigma (St. Louis, USA) and further purified by affinity chromatography as described by Blumberg et al 38 Other enzymes were either purified from various sources (human cathepsin C, 39 porcine cathepsin H, 40 human cathepsin X 37 and porcine legumain 41 ), or expressed in E. coli (human cathepsin B, 42 human cathepsin L 43 and murine caspases 1, 3, 6, 7, 8 and 11 44,45 ) or in P. pastoris (human cathepsin F, 46 human cathepsin K 47 and human cathepsin V). 48 Papain-like cysteine proteases were active-site titrated by E-64, stefin A 49 or cystatin C 39 and caspases by Z-VAD-fmk.…”

Section: Enzymesmentioning

confidence: 99%

Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity

et al. 2003

View full text Add to dashboard Cite

We report here that a number of commonly used small peptide caspase inhibitors consisting of a caspase recognition sequence linked to chloromethylketone, fluoromethylketone or aldehyde reactive group efficiently inhibit other cysteine proteases than caspases. The in vitro studies included cathepsins B, H, L, S, K, F, V, X and C, papain and legumain. Z-DEVD-cmk was shown to be the preferred irreversible inhibitor of most of the cathepsins in vitro, followed by Z-DEVD-fmk, Ac-YVAD-cmk, Z-YVAD-fmk and Z-VAD-fmk. Inactivation of legumain by all the inhibitors investigated was moderate, whereas cathepsins H and C were poorly inhibited or not inhibited at all. Inhibition by aldehydes was not very potent. All the three fluoromethylketones efficiently inhibited cathepsins in Jurkat and human embryonic kidney 293 cells at concentrations of 100 lM. Furthermore, they completely inhibited cathepsins B and X activity in tissue extracts at concentrations as low as 1 lM. These results suggest that data based on the use of these inhibitors should be taken with caution and that other proteases may be implicated in the processes previously ascribed solely to caspases.

show abstract

“…7) suggest that the recombinant cathepsin B is functionally equivalent to the native enzyme. In addition, recombinant cathepsin B from rat, expressed and secreted by S. cerevisiae, as well as human cathepsin B, expressed in E. coli, renaturated, and pepsinactivated, were both fully functional (25,45).…”

Section: Purification Of Secreted Recombinant Procathepsin B-thementioning

confidence: 99%

Cathepsin B of Schistosoma mansoni

Lipps

Füllkrug

Beck

1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

Procathepsin B from the parasitic trematode Schistosoma mansoni was expressed as a glycosylation-minus mutant in yeast cells and purified by means of a histidine affinity tag which was added to the carboxyl terminus of the recombinant protein. The purified zymogen underwent autoprocessing but required an assisting protease for activation. Pepsin-activated schistosomal cathepsin B was further characterized with the cathepsin B-specific substrates N-benzyloxycarbonyl (Z)-Arg-Arg-p-nitroanilide, Z-Arg-Arg-7-amido-4-methylcoumarin, and Z-Phe-Arg-7-amido-4-methylcoumarin. A proteolytic activity comparable to mammalian cathepsin B was observed. In addition, we analyzed the degradation of human hemoglobin by schistosomal cathepsin B, which has been suggested to be the physiological target of the protease.

show abstract

The Preparation of Catalytically Active Human Cathepsin B from Its Precursor Expressed in Escherichia coli in the Form of Inclusion Bodies

Cited by 111 publications

References 35 publications

Three‐dimensional invasion of macrophages is mediated by cysteine cathepsins in protrusive podosomes

Three‐dimensional invasion of macrophages is mediated by cysteine cathepsins in protrusive podosomes

Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity

Cathepsin B of Schistosoma mansoni

Contact Info

Product

Resources

About