Detecting Necroptosis in Virus-Infected Cells
Necroptosis has been implicated as a critical cell death pathway in cancers, Alzheimer’s and other neurodegenerative diseases, and virus-infected cells. Necroptosis occurs when mixed-lineage kinase domain-like protein (MLKL) punctures the cytoplasmic memb
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Introduction Necroptosis is a relatively newly identified form of programmed cell death that, unlike apoptosis and pyroptosis, is independent of caspases [1, 2]. Necroptotic cell death has been implicated in the response to infection, death receptor activation, and activation of toll-like receptors 3 and 4 (TLR 3 and TLR 4). Viruses have evolved necroptotic inhibitor proteins to retain pathogenesis during infection [3, 4]. Additionally, necroptosis has been detected in brains of patients with Alzheimer’s disease and Parkinson’s disease and has been implicated in age-related macular degeneration and in skin inflammation [5–7]. Additionally, a key regulator of necroptosis, receptor-interacting protein kinase 3 (RIPK3), has been implicated in tumor invasion during colorectal cancers and in response to chemotherapeutic agents [8, 9]. While necroptosis inhibits viral pathogenesis in animal models, its involvement in human cancers and neurological diseases has not been elucidated. By studying the regulation of necroptosis during viral infection and other human diseases, we hope to identify targets for oncolytic viruses and viral protein therapies.
Alexandra R. Lucas (ed.), Viruses as Therapeutics: Methods and Protocols, Methods in Molecular Biology, vol. 2225, https://doi.org/10.1007/978-1-0716-1012-1_11, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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Samantha M. Cotsmire et al.
While independent of caspase activation, necroptosis is dependent on the serine/threonine protein kinase, RIPK3. In fact, active caspase 8, one key initiator caspase for apoptosis, can inhibit necroptosis by cleaving RIPK3 [10]. Once activated, RIPK3 can phosphorylate mixed-lineage kinase domain like pseudokinase (MLKL), the executioner of necroptosis [11]. Phosphorylation of MLKL on serine 358 in humans and S345 in mice is necessary but not sufficient to induce necroptotic cell death [12, 13]. A second step, trimerization, is dependent on highly phosphorylated inositol phosphates and on tyrosine phosphorylation (T357) of MLKL by the tyro3-Axl-Myr (TAM) kinases [14–16]. There are three sensors, receptor-interacting serine/threonine protein kinase 1 (RIPK1), DNA-dependent activator of interferonregulatory factors ((DAI, also known as Z-DNA binding protein 1 (ZBP1) and DLM-1), (Note: DLM-1 does not have a definition, it is a protein expressed from the Dlm1 gene), and TIR-domaincontaining adapter-inducing interferon-β (TRIF) that act upstream of RIPK3 that can lead to RIPK3 activation. RIPK1 senses activation of death receptors [17]. DAI senses Z-form nucleic acid, associated with viral infection, while TRIF senses signaling by TRL3 and TLR4 [18, 19]. RIPK1, DAI, and TRIF can bind to RIPK3 through interaction of mutual RIP, receptor interacting protein, is defined above. Homotypic interaction motifs, (RHIM) RHIMs, leading to RIPK3 phosphorylation and activation [20]. Once activated, RIPK3 can phosphorylate MLKL, initiating the pathway leading to necroptotic cell death. In this manuscript, we describe methods to d
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