Fluorescent tags influence the enzymatic activity and subcellular localization of procaspase-1

Heymann, Michael C.; Rabe, S; Ruß, Susanne; Kapplusch, Franz; Schulze, Felix; Stein, Robert; Winkler, Stefan; Hedrich, Christian M.; Rösen‐Wolff, Angela; Hofmann, Sigrun R.

doi:10.1016/j.clim.2015.05.011

Cited by 8 publications

(6 citation statements)

References 25 publications

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“…The aggregates of the eGFP-containing VP7 proteins were electron dense particulate structures, lacking any well-defined crystalline shape. It has been shown that the attachment of a fluorescent eGFP tag to a protein can have an effect on its cellular distribution (Heymann et al 2015) whereas our results indicated that the site of eGFP insertion can also play a role. From our further results we concluded that the observed differences in localisation may be linked to differences in the proportion of misfolded versus correctly folded VP7-eGFP fusions proteins.…”

Section: Discussioncontrasting

confidence: 69%

A Dual Laser Scanning Confocal and Transmission Electron Microscopy Analysis of the Intracellular Localization, Aggregation and Particle Formation of African Horse Sickness Virus Major Core Protein VP7

et al. 2017

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The bulk of African horse sickness virus (AHSV) major core protein VP7 selfassembles into flat, hexagonal crystalline particles in a process appearing unrelated to viral replication. Why this unique characteristic of AHSV VP7 is genetically conserved, and whether VP7 aggregation and particle formation have an effect on cellular biology or the viral life cycle, is unknown. Here we investigated how different small peptide and enhanced green fluorescent protein (eGFP) insertions into the VP7 top domain affected VP7 localisation, aggregation and particle formation. This was done using a dual laser scanning confocal and transmission electron microscopy approach in conjunction with analyses of the solubility, aggregation and fluorescence profiles of the proteins. VP7 top domain modifications did not prevent trimerisation, or intracellular trafficking to one or two discrete sites in the cell. However, modifications that resulted in a misfolded and insoluble VP7-eGFP component blocked trafficking, and precluded protein accumulation at a single cellular site, perhaps by interfering with normal trimer-trimer interactions. Furthermore, the modifications disrupted the stable layering of the trimers into characteristic AHSV VP7 crystalline particles. It was concluded that VP7 trafficking is driven by a balance between VP7 solubility, trimer forming ability and trimer-trimer interactions.

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Section: Discussioncontrasting

confidence: 69%

A Dual Laser Scanning Confocal and Transmission Electron Microscopy Analysis of the Intracellular Localization, Aggregation and Particle Formation of African Horse Sickness Virus Major Core Protein VP7

et al. 2017

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“…It is possible that the fused C-terminal gyrase-GFP on caspase-1 is inhibiting its pyroptotic functions. 37 Alternatively, our immortalized MEFs may express reduced levels of important pyroptotic effector molecules required for efficient cell death downstream of inflammatory caspase activation, such as gasdermin D. 38,39 Regardless of the cause, our system has fortuitously allowed us to separate the cell death function of caspase-1 from its ability to cleave cytosolic IL-1β and cause its secretion.…”

Section: Discussionmentioning

confidence: 99%

Cell death is not essential for caspase-1-mediated interleukin-1β activation and secretion

et al. 2016

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Caspase-1 cleaves and activates the pro-inflammatory cytokine interleukin-1 beta (IL-1β), yet the mechanism of IL-1β release and its dependence on cell death remains controversial. To address this issue, we generated a novel inflammasome independent system in which we directly activate caspase-1 by dimerization. In this system, caspase-1 dimerization induced the cleavage and secretion of IL-1β, which did not require processing of caspase-1 into its p20 and p10 subunits. Moreover, direct caspase-1 dimerization allowed caspase-1 activation of IL-1β to be separated from cell death. Specifically, we demonstrate at the single cell level that IL-1β can be released from live, metabolically active, cells following caspase-1 activation. In addition, we show that dimerized or endogenous caspase-8 can also directly cleave IL-1β into its biologically active form, in the absence of canonical inflammasome components. Therefore, cell death is not obligatory for the robust secretion of bioactive IL-1β.

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“…1, B-D). Hence, assuming that our method was sufficiently reproducing physiological macrophage functions in vitro and was not affected by the fluorescent tags, we conducted live cell imaging experiments under the aforementioned conditions (26). In response to stimulation with LPS or nigericin alone, cells did not show remarkable changes.…”

Section: Transduction Of Immortalized Murine Macrophages With Lentivimentioning

confidence: 99%

Enzymatically Inactive Procaspase 1 stabilizes the ASC Pyroptosome and Supports Pyroptosome Spreading during Cell Division

Stein

Kapplusch

Heymann

et al. 2016

Journal of Biological Chemistry

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Caspase-1 is a key player during the initiation of pro-inflammatory innate immune responses, activating pro-IL-1␤ in socalled inflammasomes. A subset of patients with recurrent febrile episodes and systemic inflammation of unknown origin harbor mutations in CASP1 encoding caspase-1. CASP1 variants result in reduced enzymatic activity of caspase-1 and impaired IL-1␤ secretion. The apparent paradox of reduced IL-1␤ secretion but systemic inflammation led to the hypothesis that CASP1 mutations may result in variable protein interaction clusters, thus activating alternative signaling pathways. To test this hypothesis, we established and characterized an in vitro system of transduced immortalized murine macrophages expressing either WT or enzymatically inactive (p.C284A) procaspase-1 fusion reporter proteins. Macrophages with variant p.C284A caspase-1 did not secrete IL-1␤ and exhibited reduced inflammatory cell death, referred to as pyroptosis. Caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) formed cytosolic macromolecular complexes (so-called pyroptosomes) that were significantly increased in number and size in cells carrying the p.C284A caspase-1 variant compared with WT caspase-1. Furthermore, enzymatically inactive caspase-1 interacted with ASC longer and with increased intensity compared with WT caspase-1. Applying live cell imaging, we documented for the first time that pyroptosomes containing enzymatically inactive variant p.C284A caspase-1 spread during cell division. In conclusion, variant p.C284A caspase-1 stabilizes pyroptosome formation, potentially enhancing inflammation by two IL-1␤-independent mechanisms: pyroptosomes convey an enhanced inflammatory stimulus through the recruitment of additional proteins (such as RIP2, receptor interacting protein kinase 2), which is further amplified through pyroptosome and cell division.Caspase-1 is a key player during innate immune responses. It aids to control pathogens and danger signals by triggering pyroptotic cell death. Together with caspase-11 and -12 in mice and caspase-4, -5, and -12 in humans, caspase-1 belongs to the family of inflammatory caspases, which are produced as inactive forms. After the detection of pathogens or cytoplasmic danger signals, inactive caspase-1 monomers recruit to multiprotein complexes referred to as inflammasomes (1-3). In such inflammasomes, inactive procaspases are autoprocessed into caspase activation and recruitment domains (CARDs), 2 the p10 and the p20 subunits (4). Two p10 and two p20 proteins subsequently form the active caspase-1 tetramer, which then cleaves and thereby activates IL-1␤ in the so-called canonical inflammasome pathway (5, 6). Furthermore, inflammatory caspases induce pro-inflammatory cell death, referred to as pyroptosis, which involves cell swelling, cell lysis, and the release of cytoplasmic contents (7,8). Pyroptosis depends on the activation of caspase-4, -5, or -11 and caspase-1 (9 -11). Caspase-1 or -11 cleaves gasdermin D, a gasdermin domain-containing protein, at amin...

show abstract

Fluorescent tags influence the enzymatic activity and subcellular localization of procaspase-1

Cited by 8 publications

References 25 publications

A Dual Laser Scanning Confocal and Transmission Electron Microscopy Analysis of the Intracellular Localization, Aggregation and Particle Formation of African Horse Sickness Virus Major Core Protein VP7

A Dual Laser Scanning Confocal and Transmission Electron Microscopy Analysis of the Intracellular Localization, Aggregation and Particle Formation of African Horse Sickness Virus Major Core Protein VP7

Cell death is not essential for caspase-1-mediated interleukin-1β activation and secretion

Enzymatically Inactive Procaspase 1 stabilizes the ASC Pyroptosome and Supports Pyroptosome Spreading during Cell Division

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