Locking mixed-lineage kinase domain-like protein in its auto-inhibited state prevents necroptosis

Rübbelke, Martin; Fiegen, Dennis; Bauer, Margit; Binder, Florian; Hamilton, James A.; King, Jim; Thamm, Sven; Nar, Herbert; Zeeb, Markus

doi:10.1073/pnas.2017406117

Cited by 32 publications

(65 citation statements)

References 43 publications

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“…Other MLKL inhibitors, the xanthine class, also covalently bind to Cys86 within the N-terminal executioner domain, but the subsequent biochemical changes in MLKL are different. Necrosulfonamide locks the executioner and the pseudokinase domain in an inactive domain–domain orientation, while the xanthine class strengthens the inhibitory effect of α-helix 6 via interaction with Phe148 [ 141 ].…”

Section: Anti-necroptotic Agents: Pharmacodynamic Features and Modulation Of Oxidative Stressmentioning

confidence: 99%

Interplay of Oxidative Stress and Necrosis-like Cell Death in Cardiac Ischemia/Reperfusion Injury: A Focus on Necroptosis

et al. 2022

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Extensive research work has been carried out to define the exact significance and contribution of regulated necrosis-like cell death program, such as necroptosis to cardiac ischemic injury. This cell damaging process plays a critical role in the pathomechanisms of myocardial infarction (MI) and post-infarction heart failure (HF). Accordingly, it has been documented that the modulation of key molecules of the canonical signaling pathway of necroptosis, involving receptor-interacting protein kinases (RIP1 and RIP3) as well as mixed lineage kinase domain-like pseudokinase (MLKL), elicit cardioprotective effects. This is evidenced by the reduction of the MI-induced infarct size, alleviation of myocardial dysfunction, and adverse cardiac remodeling. In addition to this molecular signaling of necroptosis, the non-canonical pathway, involving Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated regulation of mitochondrial permeability transition pore (mPTP) opening, and phosphoglycerate mutase 5 (PGAM5)–dynamin-related protein 1 (Drp-1)-induced mitochondrial fission, has recently been linked to ischemic heart injury. Since MI and HF are characterized by an imbalance between reactive oxygen species production and degradation as well as the occurrence of necroptosis in the heart, it is likely that oxidative stress (OS) may be involved in the mechanisms of this cell death program for inducing cardiac damage. In this review, therefore, several observations from different studies are presented to support this paradigm linking cardiac OS, the canonical and non-canonical pathways of necroptosis, and ischemia-induced injury. It is concluded that a multiple therapeutic approach targeting some specific changes in OS and necroptosis may be beneficial in improving the treatment of ischemic heart disease.

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Section: Anti-necroptotic Agents: Pharmacodynamic Features and Modulation Of Oxidative Stressmentioning

confidence: 99%

Interplay of Oxidative Stress and Necrosis-like Cell Death in Cardiac Ischemia/Reperfusion Injury: A Focus on Necroptosis

et al. 2022

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“…2a). Not surprisingly, the multiple sequence alignment of the cluster closely matched (HHpred [66, 67] probability above 98%) the sequence of human MLKL executioner domain with an experimentally solved spatial structure (pdb:6vzo [68], Fig. 2b).…”

Section: Resultsmentioning

confidence: 68%

Exploring a diverse world of effector domains and amyloid signaling motifs in fungal NLR proteins

Wojciechowski

Tekoglu

Gąsior-Głogowska

et al. 2022

Preprint

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NLR proteins are intracellular receptors constituting a conserved component of the innate immune system of multicellular organisms. In fungi, NLRs are characterized by high diversity of architectures and presence of amyloid signaling. Here, we explore the diverse world of effector and signaling domains of fungal NLRs using state-of-the-art bioinformatic methods including MMseqs2 for fast clustering, probabilistic context-free grammars for sequence analysis, and AlphaFold2 deep neural networks for structure prediction. In addition to substantially improving the overall annotation, especially in basidiomycetes, the study identifies novel domains and reveals the structural similarity of MLKL-related HeLo- and Goodbye-like domains forming the most abundant superfamily of fungal NLR effectors. Moreover, compared to previous studies, we found several times more amyloid motifs, including novel families, and validated aggregating and prion-forming properties of the most abundant of them in vitro and in vivo. Also, through an extensive in silico search, the NLR-associated amyloid signaling is for the first time identified in basidiomycetes. The emerging picture highlights similarities and differences in the NLR architectures and amyloid signaling in ascomycetes, basidiomycetes and other branches of life.

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“…The obstacles to observe MLKL 2 –154 in these two membrane systems are due to nanodiscs that are restricted by the scaffolding protein to make enough room for the MLKL 2–154 to insert into the bilayers, while the size of liposomes are too large for NMR studies. Detergent micelles have been used to monitor MLKL-NTR binding to inositol phosphates [ 15 , 24 ] and phosphatidylinositols [ 25 ], but no details about the membrane insertion of MLKL. Moreover, detergents form monolayer in micelles which do not provide a bilayer membrane mimetic environment.…”

Section: Resultsmentioning

confidence: 99%

“…The structural characterization of the membrane-associated MLKL is a long standing question in the necroptosis studies. Previous solution-state NMR studies have been applied to MLKL-NTR reconstituted in liposomes, nanodiscs [ 20 ] and micelles [ 15 , 24 , 25 ]. In the present study, we reconstituted MLKL 2 –154 into bicelles, a lipid-bilayer system with better flexibility than nanodiscs and a smaller size than liposomes, to investigate the activation of NTR.…”

Section: Discussionmentioning

confidence: 99%

Positive Charges in the Brace Region Facilitate the Membrane Disruption of MLKL-NTR in Necroptosis

Yang

Xie

et al. 2021

Molecules

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Necroptosis is a type of programmed cell death executed through the plasma membrane disruption by mixed lineage kinase domain-like protein (MLKL). Previous studies have revealed that an N-terminal four-helix bundle domain (NBD) of MLKL is the executioner domain for the membrane permeabilization, which is auto-inhibited by the first brace helix (H6). After necroptosis initiation, this inhibitory brace helix detaches and the NBD can integrate into the membrane, and hence leads to necroptotic cell death. However, how the NBD is released and induces membrane rupture is poorly understood. Here, we reconstituted MLKL2–154 into membrane mimetic bicelles and observed the structure disruption and membrane release of the first brace helix that is regulated by negatively charged phospholipids in a dose-dependent manner. Using molecular dynamics simulation we found that the brace region in an isolated, auto-inhibited MLKL2–154 becomes intrinsically disordered in solution after 7 ns dynamic motion. Further investigations demonstrated that a cluster of arginines in the C-terminus of MLKL2–154 is important for the molecular conformational switch. Functional mutagenesis showed that mutating these arginines to glutamates hindered the membrane disruption of full-length MLKL and thus inhibited the necroptotic cell death. These findings suggest that the brace helix also plays an active role in MLKL regulation, rather than an auto-inhibitory domain.

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Locking mixed-lineage kinase domain-like protein in its auto-inhibited state prevents necroptosis

Cited by 32 publications

References 43 publications

Interplay of Oxidative Stress and Necrosis-like Cell Death in Cardiac Ischemia/Reperfusion Injury: A Focus on Necroptosis

Interplay of Oxidative Stress and Necrosis-like Cell Death in Cardiac Ischemia/Reperfusion Injury: A Focus on Necroptosis

Exploring a diverse world of effector domains and amyloid signaling motifs in fungal NLR proteins

Positive Charges in the Brace Region Facilitate the Membrane Disruption of MLKL-NTR in Necroptosis

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