Human caspases in vitro: Expression, purification and kinetic characterization

Roschitzki-Voser, Heidi; Schroeder, Thilo; Lenherr, Esther D.; Frölich, Franziska; Schweizer, Andreas; Donepudi, Mrudula; Ganesan, Rajkumar; Mittl, Peer R. E.; Baici, Antonio; Grütter, Markus G.

doi:10.1016/j.pep.2012.05.009

Cited by 38 publications

(31 citation statements)

References 48 publications

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“…1A ) in HEK293T cells and assessed the precipitated proteins for their catalytic activity in vitro in the presence of the kosmotropic salt ammonium citrate, which is known to activate initiator caspases by favoring their dimerization [37], [38]. As previously reported, wildtype MALT1 was highly active in ammonium citrate buffer [19], [22], [34], but this activity was completely lost in the E549A mutant ( Fig.…”

Section: Resultssupporting

confidence: 56%

Monoubiquitination and Activity of the Paracaspase MALT1 Requires Glutamate 549 in the Dimerization Interface

et al. 2013

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The mucosa-associated lymphoid tissue protein-1 (MALT1, also known as paracaspase) is a protease whose activity is essential for the activation of lymphocytes and the growth of cells derived from human diffuse large B-cell lymphomas of the activated B-cell subtype (ABC DLBCL). Crystallographic approaches have shown that MALT1 can form dimers via its protease domain, but why dimerization is relevant for the biological activity of MALT1 remains largely unknown. Using a molecular modeling approach, we predicted Glu 549 (E549) to be localized within the MALT1 dimer interface and thus potentially relevant. Experimental mutation of this residue into alanine (E549A) led to a complete impairment of MALT1 proteolytic activity. This correlated with an impaired capacity of the mutant to form dimers of the protease domain in vitro, and a reduced capacity to promote NF-κB activation and transcription of the growth-promoting cytokine interleukin-2 in antigen receptor-stimulated lymphocytes. Moreover, this mutant could not rescue the growth of ABC DLBCL cell lines upon MALT1 silencing. Interestingly, the MALT1 mutant E549A was unable to undergo monoubiquitination, which we identified previously as a critical step in MALT1 activation. Collectively, these findings suggest a model in which E549 at the dimerization interface is required for the formation of the enzymatically active, monoubiquitinated form of MALT1.

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Section: Resultssupporting

confidence: 56%

Monoubiquitination and Activity of the Paracaspase MALT1 Requires Glutamate 549 in the Dimerization Interface

et al. 2013

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“…The screen was designed in a 384-well format (Greiner 781207), using 33 μM compound, 1 μM recombinant human caspase-4 in high citrate buffer (50 mM Tris-HCl, pH 7.5, 1 M sodium citrate, 10 mM DTT, 10% sucrose), and 20 μM Promega Caspase-Glo® 9 Assay substrate (O’Brien et al, 2005; Roschitzki-Voser et al, 2012). While designed for use with caspase-9, the LEHD substrate is also recognized by caspase-4 (Roschitzki-Voser et al, 2012; Talanian et al, 1997). The assay Z’ factor is 0.66, recommending this method for high-throughput experimentation (Figure S1) (Zhang et al, 1999).…”

Section: Methodsmentioning

confidence: 99%

Non-steroidal Anti-inflammatory Drugs Are Caspase Inhibitors

Smith

Soti

Jones

et al. 2017

Cell Chemical Biology

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Summary Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used drugs in the world. While the role of NSAIDs as cyclooxygenase (COX) inhibitors is well established, other targets may contribute to anti-inflammation. Here we report caspases as a new pharmacological target for NSAID-family drugs such as ibuprofen, naproxen, and ketorolac at physiologic concentrations both in vitro and in vivo. We characterize caspase activity both in vitro and in cell culture, and combine computational modeling and biophysical analysis to determine the mechanism of action. We observe that inhibition of caspase catalysis reduces cell death and the generation of pro-inflammatory cytokines. Further, NSAID inhibition of caspases is COX-independent, representing a new anti-inflammatory mechanism. This finding expands upon existing NSAID anti-inflammatory behaviors, with implications for patient safety and next-generation drug design.

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“…; Denault & Salvesen ; Roschitzki‐Voser et al . ). These procedures can easily produce milligram amounts of active pure caspase per liter of E. coli culture using 1 or 2 chromatographic steps, whether or not the refolding reaction from bacterial inclusion bodies occurs.…”

Section: Discussionmentioning

confidence: 97%

Efficient expression and purification of recombinant human μ‐calpain using an Escherichia coli expression system

2013

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Calpains comprise a superfamily of Ca 2+ -regulated cysteine proteases that are indispensable for the regulation of various cellular functions. Of these, the mammalian l-and m-calpains are the best characterized isoforms. They are ubiquitously expressed and form heterodimers consisting of a distinct 80-kDa catalytic subunit (CAPN1 for l-calpain and CAPN2 for m-calpain) and a common 30-kDa regulatory subunit (CAPNS1). To date, various expression systems have been developed for producing recombinant calpains for structural and functional studies; however, no low-cost, simple and efficient bacterial expression system for l-calpain has been available, because the protein forms aggregates. Here, we established an efficient method for producing active recombinant human l-calpain using an Escherichia coli expression system. This was achieved by co-expressing CAPN1 and CAPNS1 lacking the N-terminal Gly-rich domain (CAPNS1DGR) in the SoluBL21 strain. From 1 L of E. coli culture, over 2 and 6 mg, respectively, of l-calpain and its active-site mutant l-calpain:C115S (CAPN1:C115S+CAP-NS1DGR) were purified by two successive column chromatographies. Compared to the native enzyme, the purified l-calpain showed almost identical properties, demonstrating its suitability for use in structural and functional studies. This is the first report of the bacterial expression and the simple and efficient purification of active recombinant l-calpain.

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Human caspases in vitro: Expression, purification and kinetic characterization

Cited by 38 publications

References 48 publications

Monoubiquitination and Activity of the Paracaspase MALT1 Requires Glutamate 549 in the Dimerization Interface

Monoubiquitination and Activity of the Paracaspase MALT1 Requires Glutamate 549 in the Dimerization Interface

Non-steroidal Anti-inflammatory Drugs Are Caspase Inhibitors

Efficient expression and purification of recombinant human μ‐calpain using an Escherichia coli expression system

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