Synthetic Biology Reveals the Uniqueness of the RIP Kinase Domain

Chirieleison, Steven M.; Kertesy, Sylvia B.; Abbott, Derek W.

doi:10.4049/jimmunol.1502631

Cited by 21 publications

(33 citation statements)

References 34 publications

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“…The GSDMD expression/ reconstitution vectors described in Figs. 1B and 2A were Gibson subcloned into the lentiviral expression plasmid previously described (27), which originally utilized LentiCRISPRv2 (Addgene) as a template with GSDMD separated from the neomycin resistance marker by a P2A self-cleaving peptide. GSDMD expression/reconstitution vectors were made CRISPR-Cas9 cut resistant through mutation of the PAM sequence via site-directed mutagenesis while conserving the amino acid sequence.…”

Section: Visualizing Gasdermin D-driven Pyroptotic Cell Deathmentioning

confidence: 99%

Live-cell visualization of gasdermin D-driven pyroptotic cell death

Rathkey¹,

Benson²,

Chirieleison³

et al. 2017

Journal of Biological Chemistry

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Pyroptosis is a form of cell death important in defenses against pathogens that can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, GSDMD (gasdermin D), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and -11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis but also enable its direct visualization.

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Section: Visualizing Gasdermin D-driven Pyroptotic Cell Deathmentioning

confidence: 99%

Live-cell visualization of gasdermin D-driven pyroptotic cell death

Rathkey¹,

Benson²,

Chirieleison³

et al. 2017

Journal of Biological Chemistry

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“…Mutant reconstitution in primary cells from XIAP Ϫ/Ϫ mice and subsequent study of those cells is difficult because of the limited proliferative capacity of primary cells, and the study of XIAPmutated patient cells is limited by both the availability of primary patient cells and the limited amount of blood that can be drawn from these young patients. Given these difficulties and the need to reconcile variable reports of inflammatory cell death phenotypes in XIAP-mutant cells, we coupled CRISPR/ Cas9 with our recently published myeloid lentiviral expression system (50) to generate a cell culture system in which the function of XIAP could be directly assessed. Stable knockout cell lines were generated using the LentiCrisprV2 system (51) with efficient loss of XIAP in HEK293Ts, a murine dendritic cell line (DC2.4s), and immortalized bone marrow-derived macrophages (iBMDMs) with two distinct single-guide RNA (sgRNA)-targeting constructs (G1 and G2) ( Fig.…”

Section: Genetic Xiap Loss Causes a Blunted Nod1/2 Responsementioning

confidence: 99%

“…Given that loss of XIAP led to a combined NOD2 and cell death phenotype, we next wanted to determine whether patient-derived mutations exhibited a defect in NOD2, a lack of inhibition of cell death, or both of these phenotypes. To test this, we reconstituted WT XIAP, ubiquitin ligase-dead XIAP, or a disease-associated XIAP variant to the XIAP-null cell lines using a lentiviral construct recently developed by our laboratory (50). XIAP contains three BIR domains, an ubiquitin-binding domain, and a RING domain.…”

Section: Reconstitution Of Xiap-null Stable Cell Lines With Patientdementioning

confidence: 99%

“…pcDNA3-HA-NOD1 and pcDNA3-HA-NOD2 were a generous gift from Dr. Christine McDonald (Department of Pathology, Cleveland Clinic Foundation, Cleveland, OH). Generation of stable XIAP-expressing reconstitution cell lines was achieved using a novel lentiviral vector we developed recently (50). To increase the range of use of the LentiCrisprV2 with our reconstitution construct, the original puromycin resistance gene was replaced with the hygromycin resistance gene, yielding a new LentiCrisprV2 construct selectable with hygromycin.…”

Section: Cells Plasmids Reagents Antibodies and Western Blottingmentioning

confidence: 99%

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Nucleotide-binding oligomerization domain (NOD) signaling defects and cell death susceptibility cannot be uncoupled in X-linked inhibitor of apoptosis (XIAP)-driven inflammatory disease

Chirieleison

Marsh

Kumar

et al. 2017

Journal of Biological Chemistry

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The X-linked inhibitor of apoptosis (XIAP) protein has been identified as a key genetic driver of two distinct inflammatory disorders, X-linked lymphoproliferative syndrome 2 (XLP-2) and very-early-onset inflammatory bowel disease (VEO-IBD). Molecularly, the role of XIAP mutations in the pathogenesis of these disorders is unclear. Recent work has consistently shown XIAP to be critical for signaling downstream of the Crohn's disease susceptibility protein nucleotide-binding oligomerization domain-containing 2 (NOD2); however, the reported effects of XLP-2 and VEO-IBD XIAP mutations on cell death have been inconsistent. In this manuscript, we describe a CRISPR-mediated genetic system for cells of the myeloid lineage in which XIAP alleles can be replaced with disease-associated XIAP variants expressed at endogenous levels to simultaneously study inflammation-related cell death and NOD2 signaling. We show that, consistent with previous studies, NOD2 signaling is critically dependent on the BIR2 domain of XIAP. We further used this system to reconcile the aforementioned inconsistent XIAP cell death data to show that XLP-2 and VEO-IBD XIAP mutations that exhibit a loss-of-function NOD2 phenotype also lower the threshold for inflammatory cell death. Last, we identified and studied three novel patient XIAP mutations and used this system to characterize NOD2 and cell death phenotypes driven by XIAP. The results of this work support the role of XIAP in mediating NOD2 signaling while reconciling the role of XLP-2 and VEO-IBD XIAP mutations in inflammatory cell death and provide a set of tools and framework to rapidly test newly discovered XIAP variants.

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“…RIP-2 is the only member of the RIP family that besides phosphorylating serines and threonines is able to autophosphorylate tyrosine residues [13,14]. Its ATP- and substrate binding sites spread over much of the N-terminal kinase domain, and a caspase recruitment domain (CARD) is present in the C-terminal region [15] (isoform 1, Fig 1A).…”

Section: Introductionmentioning

confidence: 99%

Evidence of a noncoding transcript of theRIPK2gene overexpressed in head and neck tumor

Villagra

Cunha

Polachini

et al. 2018

Preprint

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39Receptor-interacting proteins are a family of serine/threonine kinases, which integrate 40 extra and intracellular stress signals caused by different factors, including infections, 41 inflammation and DNA damage. Receptor-interacting serine/threonine-protein kinase 2 (RIP-42 2) is a member of this family and an important component of the nuclear factor NF-kappa-B 43 signaling pathway. The corresponding human gene RIPK2 generates two transcripts by 44 alternative splicing, the full-length and a short transcript. The short transcript has a truncated 45 5' sequence, which results in a predicted isoform with a partial kinase domain but able to 46 transduce signals through its caspase recruitment domain. In this study, the expression of 47 RIPK2 was investigated in human tissue samples and, in order to determine if both transcripts 48 are similarly regulated at the transcriptional level, cancer cell lines were submitted to 49 temperature and acid stresses. We observed that both transcripts are expressed in all tissues 50 analyzed, with higher expression of the short one in tumor samples, and they are differentially 51 regulated following temperature stress. Despite transcription, no corresponding protein for the 52 short transcript was detected in tissues and cell lines analyzed. We propose that the shorter 53 transcript is a noncoding RNA and that its presence in the cell may play regulatory roles and 54 affect inflammation and other biological processes related to the kinase activity of RIP-2. Villagra et al 3 3 56 Introduction 57Unicellular and multicellular organisms are constantly exposed to stressful 58 environments. Chemical and physical stimuli trigger different adaptive responses, which will 59 determine the capability of the organism to maintain internal homeostasis [1]. 60 Receptor-interacting proteins (RIP) are a family of serine/threonine kinases, which 61 integrate extra-and intracellular stress signals and share a homologous kinase domain at the 62 N-terminus, but have different C-terminal functional domains [2-4]; RIP-2 (receptor-63 interacting serine/threonine-protein kinase 2) is a member of the RIP family, which has 64 received attention in the recent years for its role in modulating immune and inflammatory 65 processes [5], and as a sensor of cellular stress [6]. It is expressed at high levels in several 66 normal human tissues [7], as well as in pathological conditions, for example ulcerative colitis 67 [8], triple-negative breast cancers [9,10] and in stressful conditions, such as after 68 hypoxic/ischemic insults [11]. Conversely, lower levels of RIP-2 have been correlated with 69 tumor progression in squamous cell carcinoma (SCC) of the oral cavity [12]. 70RIP-2 is the only member of the RIP family that besides phosphorylating serines and 71 threonines is able to autophosphorylate tyrosine residues [13,14]. Its ATP-and substrate 72 binding sites spread over much of the N-terminal kinase domain, and a caspase recruitment Fig 1A). CARD 74 specifically interacts with the nucleotide-bind...

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Synthetic Biology Reveals the Uniqueness of the RIP Kinase Domain

Cited by 21 publications

References 34 publications

Live-cell visualization of gasdermin D-driven pyroptotic cell death

Live-cell visualization of gasdermin D-driven pyroptotic cell death

Nucleotide-binding oligomerization domain (NOD) signaling defects and cell death susceptibility cannot be uncoupled in X-linked inhibitor of apoptosis (XIAP)-driven inflammatory disease

Evidence of a noncoding transcript of theRIPK2gene overexpressed in head and neck tumor

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