High Yield Production of a Soluble Human Interleukin-3 Variant from E. coli with Wild-Type Bioactivity and Improved Radiolabeling Properties

Hercus, Timothy R.; Barry, Emma F.; Dottore, Mara; McClure, Barbara J.; Webb, Andrew I.; Lopez, Angel F.; Young, Ian G.; Murphy, James M.

doi:10.1371/journal.pone.0074376

Cited by 15 publications

(12 citation statements)

References 41 publications

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“…Recombinant mouse MLKL pseudokinase domain (residues 179-464) and mRIPK3 kinase domain were expressed and purified from Sf21 cells as described previously (1,28). MLKL (1-169) was expressed and purified from Escherichia coli using an established strategy (29).…”

Section: Methodsmentioning

confidence: 99%

Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death

Hildebrand

Tanzer

Lucet

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein . How MLKL causes cell death is unclear, however RIPK3-mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecularweight, membrane-localized complex and cell death. Using alaninescanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.pseudoenzyme | RIP kinase | ATP mimetic | programmed necrosis P rogrammed necrosis or "necroptosis" has emerged in the past 5 years as a cell death mechanism that complements the conventional cell death pathway, apoptosis, in multicellular organisms. In contrast to apoptosis, necroptosis does not appear to serve an important role in multicellular organism development (1-3) but participates in the defense against pathogens and is a likely culprit in destructive inflammatory conditions (4-7). Receptor Interacting Protein Kinase-3 (RIPK3) was identified as a key effector of necroptosis in 2009 (4, 5) and its substrate, the pseudokinase Mixed Lineage Kinase Domain-Like (MLKL), in 2012 (8, 9), but the molecular events following RIPK3-mediated phosphorylation of MLKL required to induce cell death are unclear. The RIPK1/ RIPK3/MLKL necrosome has been proposed to activate PGAM5 (phosphoglycerate mutase 5) and Drp1 (Dynamin-related protein 1) to cause mitochondrial fragmentation and cell death (10), but the requirement for PGAM5, Drp1, and mitochondria for necroptosis has been questioned (1, 11-13).We described the structure of mouse MLKL revealing that MLKL contains a C-terminal pseudokinase domain and an N-terminal four-helix bundle (4HB) domain connected by a two-helix linker (the "brace" helices) (1). Based on our mutational and biochemical analyses, we proposed that the catalytically inactive pseudokinase domain functions as a molecular switch and that RIPK3-mediated phosphorylat...

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Section: Methodsmentioning

confidence: 99%

Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death

Hildebrand

Tanzer

Lucet

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

498

628

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“…Insert DNA was amplified from a mouse MLKL template (synthesised by DNA2.0, CA) by PCR using manuallydesigned oligonucleotides (Integrated DNA Technologies). For recombinant protein constructs, DNA inserts were cloned into vector (Kan r ) pETNusH HTB (derived from pETM60) [37,38], using BamHI and EcoRI restriction sites as an inframe fusion C-terminal to a TEV-protease cleavable NusA-His 6 tag. The mouse MLKL (1-169) construct is as described previously [23], and harbours a C169S substitution.…”

Section: Expression Constructsmentioning

confidence: 99%

The brace helices of MLKL mediate interdomain communication and oligomerisation to regulate cell death by necroptosis

et al. 2018

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The programmed cell death pathway, necroptosis, relies on the pseudokinase, Mixed Lineage Kinase domain-Like (MLKL), for cellular execution downstream of death receptor or Toll-like receptor ligation. Receptor-interacting protein kinase-3 (RIPK3)-mediated phosphorylation of MLKL's pseudokinase domain leads to MLKL switching from an inert to activated state, where exposure of the N-terminal four-helix bundle (4HB) 'executioner' domain leads to cell death. The precise molecular details of MLKL activation, including the stoichiometry of oligomer assemblies, mechanisms of membrane translocation and permeabilisation, remain a matter of debate. Here, we dissect the function of the two 'brace' helices that connect the 4HB to the pseudokinase domain of MLKL. In addition to establishing that the integrity of the second brace helix is crucial for the assembly of mouse MLKL homotrimers and cell death, we implicate the brace helices as a device to communicate pseudokinase domain phosphorylation event(s) to the N-terminal executioner 4HB domain. Using mouse:human MLKL chimeras, we defined the first brace helix and adjacent loop as key elements of the molecular switch mechanism that relay pseudokinase domain phosphorylation to the activation of the 4HB domain killing activity. In addition, our chimera data revealed the importance of the pseudokinase domain in conferring host specificity on MLKL killing function, where fusion of the mouse pseudokinase domain converted the human 4HB + brace from inactive to a constitutive killer of mouse fibroblasts. These findings illustrate that the brace helices play an active role in MLKL regulation, rather than simply acting as a tether between the 4HB and pseudokinase domains.

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“…Fractions containing purified hIL-3 were lyophilised, resuspended in PBS and sterilised using Corning Spin-X filters (Sigma-Aldrich). We, and others, have previously demonstrated that compared to full-length recombinant IL-3, the truncated IL-3 has equivalent bioactivity 45 .…”

Section: Methodsmentioning

confidence: 82%

“…3c, d ). Wild-type or K116W versions of human IL-3, comprising residues W13 to Q125 of the mature peptide with a W13Y mutation and a GAMGS N-terminal tail arising from the expression plasmid, were purified from E. coli 45 . BL21 CodonPlus (DE3)-RIPL cells transformed with pETNusH expression vectors encoding Nus-6xHis-TEV:IL-3 fusion proteins, were cultured to an OD 600 of 0.6 and induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 23 °C for 16 h. Cells were lysed and Nus-6xHis-TEV:IL-3 fusion proteins affinity-purified using HisTrap FF columns (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

A dual role for the N-terminal domain of the IL-3 receptor in cell signalling

et al. 2018

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The interleukin-3 (IL-3) receptor is a cell-surface heterodimer that links the haemopoietic, vascular and immune systems and is overexpressed in acute and chronic myeloid leukaemia progenitor cells. It belongs to the type I cytokine receptor family in which the α-subunits consist of two fibronectin III-like domains that bind cytokine, and a third, evolutionarily unrelated and topologically conserved, N-terminal domain (NTD) with unknown function. Here we show by crystallography that, while the NTD of IL3Rα is highly mobile in the presence of IL-3, it becomes surprisingly rigid in the presence of IL-3 K116W. Mutagenesis, biochemical and functional studies show that the NTD of IL3Rα regulates IL-3 binding and signalling and reveal an unexpected role in preventing spontaneous receptor dimerisation. Our work identifies a dual role for the NTD in this cytokine receptor family, protecting against inappropriate signalling and dynamically regulating cytokine receptor binding and function.

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High Yield Production of a Soluble Human Interleukin-3 Variant from E. coli with Wild-Type Bioactivity and Improved Radiolabeling Properties

Cited by 15 publications

References 41 publications

Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death

Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death

The brace helices of MLKL mediate interdomain communication and oligomerisation to regulate cell death by necroptosis

A dual role for the N-terminal domain of the IL-3 receptor in cell signalling

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