Discovery and Structure-Based Optimization of Fragments Binding the Mixed Lineage Kinase Domain-like Protein Executioner Domain

Clostridium perfringens enterotoxin (CPE) is responsible for the symptoms of common intestinal infections due to C. perfringens type F isolates. CPE is a pore-forming toxin that uses certain claudins as a receptor. Previous studies showed that, in enterocyte-like Caco-2 cells, low CPE concentrations cause caspase 3-mediated apoptosis but high CPE concentrations cause necrosis. The recent work published in mBio by Shrestha, Mehdizadeh Gohari, and McClane determined that RIP1 and RIP3 are involved in both CPE-mediated apoptosis and necrosis in Caco-2 cells. Furthermore, mixed lineage kinase-domain (MLKL) oligomerization was shown to be important for necrosis caused by CPE, identifying this necrosis as programmed necroptosis. In addition, calpain activation due to Ca2+ influx through the CPE pore was identified as a critical intermediate step for MLKL oligomerization and, thus, CPE-induced necroptosis. These findings may have applicability to understand the action of some other pore-forming toxins that induce necroptosis and may also be important for understanding CPE action in vivo.

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Decrease in fat de novo synthesis and chemokine ligand expression in non‐alcoholic fatty liver disease caused by inhibition of mixed lineage kinase domain‐like pseudokinase

Journal of Gastroenterology and Hepatology ◽

10.1111/jgh.14740 ◽

2019 ◽

Vol 34 (12) ◽

pp. 2206-2218 ◽

Cited By ~ 4

Author(s):

Waqar Khalid Saeed ◽

Dae Won Jun ◽

Kiseok Jang ◽

Ju Hee Oh ◽

Yeon Ji Chae ◽

...

Keyword(s):

Liver Disease ◽

Fatty Liver Disease ◽

De Novo ◽

Kinase Domain ◽

De Novo Synthesis ◽

Alcoholic Fatty Liver ◽

Mixed Lineage Kinase ◽

Chemokine Ligand ◽

Ligand Expression ◽

Alcoholic Fatty Liver Disease

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The molecular mechanisms of MLKL-dependent and MLKL-independent necrosis

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjaa055 ◽

2020 ◽

Author(s):

Lu Li ◽

An Tong ◽

Qiangsheng Zhang ◽

Yuquan Wei ◽

Xiawei Wei

Keyword(s):

Molecular Mechanisms ◽

Death Receptor ◽

Kinase Domain ◽

External Stress ◽

Programmed Necrosis ◽

Interacting Protein ◽

Mixed Lineage Kinase ◽

Caspase Inhibition ◽

Regulated Necrosis ◽

Receptor Family

Abstract Necrosis, a type of unwanted and passive cell demise, usually occurs under the excessive external stress and is considered to be unregulated. However, under some special conditions such as caspase inhibition, necrosis is regulable in a well-orchestrated way. The term ‘regulated necrosis’ has been proposed to describe such programmed necrosis. Recently, several forms of necrosis, including necroptosis, pyroptosis, ferroptosis, parthanatos, oxytosis, NETosis, and Na+/K+-ATPase-mediated necrosis, have been identified, and some crucial regulators governing regulated necrosis have also been discovered. Mixed lineage kinase domain-like pseudokinase (MLKL), a core regulator in necroptosis, acts as an executioner in response to ligands of death receptor family. Its activation requires the receptor-interacting protein kinases, RIP1 and RIP3. However, MLKL is only involved in necroptosis, that is, MLKL is dispensable for necrosis. Therefore, this review is aimed at summarizing the molecular mechanisms of MLKL-dependent and MLKL-independent necrosis.

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Loss of MLKL (Mixed Lineage Kinase Domain-Like Protein) Decreases Necrotic Core but Increases Macrophage Lipid Accumulation in Atherosclerosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.119.313640 ◽

2020 ◽

Vol 40 (5) ◽

pp. 1155-1167 ◽

Cited By ~ 2

Author(s):

Adil Rasheed ◽

Sabrina Robichaud ◽

My-Anh Nguyen ◽

Michele Geoffrion ◽

Hailey Wyatt ◽

...

Keyword(s):

Lipid Accumulation ◽

Kinase Domain ◽

Necrotic Core ◽

Mixed Lineage Kinase

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Atypical Gasdermin D and Mixed Lineage Kinase Domain-like Protein Leakage Aggravates Tetrachlorobenzoquinone-Induced Nod-like Receptor Protein 3 Inflammasome Activation

Chemical Research in Toxicology ◽

10.1021/acs.chemrestox.8b00306 ◽

2018 ◽

Vol 31 (12) ◽

pp. 1418-1425 ◽

Cited By ~ 5

Author(s):

Xiaomin Xia ◽

Bin Lu ◽

Wenjing Dong ◽

Bingwei Yang ◽

Yawen Wang ◽

...

Keyword(s):

Kinase Domain ◽

Receptor Protein ◽

Inflammasome Activation ◽

Mixed Lineage Kinase ◽

Protein Leakage

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Analysis of the N-terminal region of human MLKL, as well as two distinct MLKL isoforms, reveals new insights into necroptotic cell death

Bioscience Reports ◽

10.1042/bsr20150246 ◽

2016 ◽

Vol 36 (1) ◽

Cited By ~ 9

Author(s):

Katja Hrovat Arnež ◽

Michaela Kindlova ◽

Nilesh J. Bokil ◽

James M. Murphy ◽

Matthew J. Sweet ◽

...

Keyword(s):

Cell Death ◽

Human Monocyte ◽

Kinase Domain ◽

Terminal Region ◽

Activation Loop ◽

Phosphorylation Sites ◽

Mixed Lineage Kinase

We show that mixed lineage kinase domain-like (MLKL) isoform 2, which lacks the pseudokinase domain and activation loop phosphorylation sites, is a more potent activator of cell death compared with MLKL isoform 1. Both MLKL isoforms are expressed in human monocyte-derived macrophages.

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Diverse Sequence Determinants Control Human and Mouse Receptor Interacting Protein 3 (RIP3) and Mixed Lineage Kinase domain-Like (MLKL) Interaction in Necroptotic Signaling

Journal of Biological Chemistry ◽

10.1074/jbc.m112.435545 ◽

2013 ◽

Vol 288 (23) ◽

pp. 16247-16261 ◽

Cited By ~ 138

Author(s):

Wanze Chen ◽

Zhenru Zhou ◽

Lisheng Li ◽

Chuan-Qi Zhong ◽

Xinru Zheng ◽

...

Keyword(s):

Protein Kinase ◽

Kinase Domain ◽

Phosphorylation Sites ◽

Flanking Sequence ◽

Interacting Protein ◽

Mixed Lineage Kinase ◽

Signaling Complex ◽

Evolutionarily Conserved ◽

Mouse Cells ◽

Human And Mouse

Receptor interacting protein 3 (RIP3) is a protein kinase essential for TNF-induced necroptosis. Phosphorylation on Ser-227 in human RIP3 (hRIP3) is required for its interaction with human mixed lineage kinase domain-like (MLKL) in the necrosome, a signaling complex induced by TNF stimulation. RIP1 and RIP3 mediate necrosome aggregation leading to the formation of amyloid-like signaling complexes. We found that TNF induces Thr-231 and Ser-232 phosphorylation in mouse RIP3 (mRIP3) and this phosphorylation is required for mRIP3 to interact with mMLKL. Ser-232 in mRIP3 corresponds to Ser-227 in hRIP3, whereas Thr-231 is not conserved in hRIP3. Although the RIP3-MLKL interaction is required for necroptosis in both human and mouse cells, hRIP3 does not interact with mMLKL and mRIP3 cannot bind to hMLKL. The species specificity of the RIP3-MLKL interaction is primarily determined by the sequence differences in the phosphorylation sites and the flanking sequence around the phosphorylation sites in hRIP3 and mRIP3. It appears that the RIP3-MLKL interaction has been selected as an evolutionarily conserved mechanism in mediating necroptosis signaling despite that differing structural and mechanistic bases for this interaction emerged simultaneously in different organisms. In addition, we further revealed that the interaction of RIP3 with MLKL prevented massive abnormal RIP3 aggregation, and therefore should be crucial for formation of the amyloid signaling complex of necrosomes. We also found that the interaction between RIP3 and MLKL is required for the translocation of necrosomes to mitochondria-associated membranes. Our data demonstrate the importance of the RIP3-MLKL interaction in the formation of functional necrosomes and suggest that translocation of necrosomes to mitochondria-associated membranes is essential for necroptosis signaling.

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