MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis

Liu, Shuzhen; Liu, Hua; Johnston, Andrea N.; Hanna-Addams, Sarah; Reynoso, Eduardo; Xiang, Yougui; Wang, Zhigao

doi:10.1073/pnas.1707531114

Cited by 130 publications

(138 citation statements)

References 46 publications

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“…MLKL finally forms oligomers and incites binding of the NTD to the plasma membrane [61]. The phosphorylation-dependent oligomerization of MLKL starts from forming tetramers which serves as building blocks for larger polymers [62]. It has been reported that MLKL forms an octamer through two tetramers with disulphide bonds at α-helices 4 and 5 within the KLD [63].…”

Section: Mlkl Is the Effector Protein In Necroptosismentioning

confidence: 99%

Molecular Insights into the Mechanism of Necroptosis: The Necrosome as a Potential Therapeutic Target

Chen

Garssen

et al. 2019

Cells

128

115

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Necroptosis, or regulated necrosis, is an important type of programmed cell death in addition to apoptosis. Necroptosis induction leads to cell membrane disruption, inflammation and vascularization. It plays important roles in various pathological processes, including neurodegeneration, inflammatory diseases, multiple cancers, and kidney injury. The molecular regulation of necroptotic pathway has been intensively studied in recent years. Necroptosis can be triggered by multiple stimuli and this pathway is regulated through activation of receptor-interacting protein kinase 1 (RIPK1), RIPK3 and pseudokinase mixed lineage kinase domain-like (MLKL). A better understanding of the mechanism of regulation of necroptosis will further aid to the development of novel drugs for necroptosis-associated human diseases. In this review, we focus on new insights in the regulatory machinery of necroptosis. We further discuss the role of necroptosis in different pathologies, its potential as a therapeutic target and the current status of clinical development of drugs interfering in the necroptotic pathway.

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Section: Mlkl Is the Effector Protein In Necroptosismentioning

confidence: 99%

Molecular Insights into the Mechanism of Necroptosis: The Necrosome as a Potential Therapeutic Target

Chen

Garssen

et al. 2019

Cells

128

115

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“…With this evidence, it was concluded that MLKL was not dissociating from the fibers during the SDD-AGE process and that the MLKL-containing fibers seen in Figure 1 were indeed distinct from the RIPK1/RIPK3 fibers. Furthermore, when RIPK3 was immuno-depleted from the cell lysates, the MLKL fibers remained intact 4 , confirming that MLKL fibers and RIPK1/RIPK3 fibers are indeed distinct entities.…”

Section: Representative Resultsmentioning

confidence: 72%

“…More recently, the formation of amyloid or amyloid-like fibers has been shown to be a part of signaling pathways in humans, including during anti-viral innate immune response 2 and necroptosis 3,4 , and in lower organisms such as yeast 5,6 . Therefore, the ability to detect these fibers in the lab is important.…”

Section: Introductionmentioning

confidence: 99%

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Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Hanna-Addams

Wang

2018

JoVE

Self Cite

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Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

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“…Two inhibitors were employed to determine whether the stimulated death pathway was canonical MLKL-and RIP3-dependent necroptosis. GSK'872 binds to and inhibits the RIP3 kinase domain (31,32), whereas necrosulfonamide (NSA) inhibits downstream effector functions of MLKL via covalent reaction with the Cys86 residue of human but not murine MLKL (17,33). NSA and GSK'872 inhibited TBZ-induced cytotoxicity in HFFF-TERTs, indicating that these cells expressed sufficient RIP3 to induce measurable canonical necroptosis ( Figure 3C).…”

Section: Pul36 Protects Cells From Necroptosismentioning

confidence: 99%

Human cytomegalovirus protein pUL36: a dual cell death pathway inhibitor

Fletcher-Etherington

Nobre

Nightingale

et al. 2020

Preprint

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Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of intrinsic, innate and adaptive viral immune evasion. Here, we employed multiplexed tandem mass tag-based proteomics to characterise host proteins targeted for degradation late during HCMV infection. This approach revealed that mixed lineage kinase domain-like protein (MLKL), a key terminal mediator of cellular necroptosis, was rapidly and persistently degraded by the minimally passaged HCMV strain Merlin but not the extensively passaged strain AD169. The strain Merlin viral inhibitor of apoptosis pUL36 was necessary and sufficient both to degrade MLKL and to inhibit necroptosis. Furthermore, mutation of pUL36 Cys 131 abrogated MLKL degradation and restored necroptosis. As the same residue is also required for pUL36-mediated inhibition of apoptosis by preventing proteolytic activation of pro-caspase 8, we define pUL36 as a multifunctional inhibitor of both apoptotic and necroptotic cell death. SIGNIFICANCE STATEMENTCell death is a key defence against viral infection, preventing spread from infected to uninfected cells.Correspondingly, certain viruses encode inhibitors of apoptotic and necroptotic cell death pathways in order to facilitate their persistence. Human cytomegalovirus (HCMV) is an important human pathogen that can block apoptosis, but hitherto it has been unclear whether or how the virus blocks necroptosis.Here, we used a proteomic screen to identify human proteins targeted for destruction by HCMV, finding that the key necroptosis mediator MLKL is degraded throughout infection. MLKL is targeted for degradation by HCMV protein pUL36, which is also instrumental in inhibiting apoptosis. Thus, pUL36 is a dual cell death pathway inhibitor, and may represent an important therapeutic target.

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MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis

Cited by 130 publications

References 46 publications

Molecular Insights into the Mechanism of Necroptosis: The Necrosome as a Potential Therapeutic Target

Molecular Insights into the Mechanism of Necroptosis: The Necrosome as a Potential Therapeutic Target

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Human cytomegalovirus protein pUL36: a dual cell death pathway inhibitor

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