Localization of the MLKL Protein to the Plasma Membrane and how it Affects TNF-induced Necroptosis

“In this study, the authors’ main focus is the MLKL protein properties such as; where it is translocated, what it induces upon translocation, and the possible complexes that if forms. The experiment as whole, shows the MLKL becomes phosphorylated by RIP3 and results in an engagement with the necrosome through its interaction with RIP 3. It is demonstrated that MLKL forms a homotrimer and then localizes to the plasma membrane with RIP3 which is required for mediating necroptosis. Interestingly enough, the article heavily noted the importance of the ?Ca?

(2+) influx that occurs with MLKL localization, as well as identified TRMP7 and its involvement with this influx and how it is a target for TNF induced necroptosis. This is all expressed to be a critically important mechanism for MLKL-mediated TNF-induced necroptosis.

Tumor necrosis factor (TNF) is well accepted and known for its role in apoptosis and necroptosis. TNF also has an array of various intercessions such as pathological conditions, autoimmune diseases, inflammation, and immunity1. TNF also functions as a machine in forming the TNFR1 complex as a result of TNF signaling. This mechanism is used to mediate a quantity of pathways through the recruitment of effectors. TRADD, RIP1, and TRAF2 are present in this complex to carry out these pathways and have also been observed to disassociate from the receptor in order to recruit other proteins needed to carry out secondary complexes for apoptosis and necroptosis. RIP3 (receptor interacting protein 3) and MLKL (mixed lineage kinase-domain like) are required in the necrosome in order for necroptosis to occur as well as other proteins. Most likely, the other proteins that are possibly involved in the formation of the necrosome include CYLD and SIRT2. What remains in question is the generation of signaling that causes the initiation of TNF necroptosis. It is known that MLKL has a role in TNF induced necroptosis and that it is recruited by RIP3 but the mechanism is largely unknown. In this article, “Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis”, the authors’ relay that MLKL forms homotrimers and locates to the cell plasma membrane during TNF-induced necroptosis. Furthermore, they claimed to have identified TRPM7 as a MLKL downstream target to mediate the ?Ca?

(2+) influx which MLKL membrane localization is responsible for. Affirmation for the key mechanism of MLKL-mediated necroptosis is revealed in their study1. If these findings are in fact correct, the signaled formation of necroptosis is further uncovered.

The topic of interest in this experiment was to investigate how TNF-induced necroptosis is mediated by MLKL and the signal that triggers the mechanism for MLKL. The first experiment conducted was to observe how the MLKL protein was expressed under certain conditions. Localization of MLKL to the plasma membrane is essential for its function. These findings suggests that the translocation of MLKL to the plasma membrane has to do with RIP3 which is localized to the membrane as well. This led to further experiments to scrutinize RIP3 and why it also promotes MLKL trimerization. Testing also shows that MLKL and RIP3 are at cause for the extracellular calcium influx and intracellular calcium release which resulted in further testing to note which types of calcium channels are involved in necroptosis1.

It was predicted that MLKL forms oligomers based on the two coiled coil domains found at the N terminus. On SDS-Page, there were detections of MLKL protein expressed as a monomer. With co-expression of MLKL with other antigens, it was confirmed that MLKL does in fact form oligomers and high chances it forms homotrimers as well. The next step was to observe if MLKL would form trimers in cells that had been induced to undergo necroptosis. The experiments showed that the control had MLKL proteins that formed monomers whereas the TSZ treated cells had MLKL proteins that formed trimers. The trimerized MLKL was also observed in other necrotic induced cells further suggesting that the protein trimerizes during necroptosis. The significance of this experiment shows the changed characteristics of MLKL and how it is expressed by the presence of necroptosis.

The membrane localization experiment was carried out on coimmunoprecipitating gels to prove that MLKL and RIP3 localize specifically upon necroptosis. TNF or TS treatment did not change their localizations in the cytoplasm and MLKL and RIP3 were both detected in the crude membrane fraction of treated cells as trimeric1. When the cell surface proteins were isolated from necrotic cells, MLKL and RIP3 were still observed to localize at the plasma membrane. The take-away of this experiment shows that the localization of MLKL at the plasma membrane may be critical for its ability to mediate TNF induced necroptosis.

The next step in the experiment was testing whether localization of MLKL to the plasma membrane is required for the response of the calcium influx. In this study, it is suggested that both the calcium influx and ROS (reactive oxygen species) are required for MLKL to mediate necroptosis. The presence of a calcium influx involved in necroptosis was confirmed in HT29 cells. To prove that the influx was occurring during necroptosis, a calcium indicator, Fluo4 was used and increased after four hours of TSZ treatment1. The non-voltage sensitive channel, TRPM7 has been previously seen to have involvement with necroptosis and may be involved in the calcium channels in the plasma membrane. It is still in debate whether the calcium influx has any involvement with MLKL in necroptosis demonstrated by differing results in similar articles.

Searching for unknown targets of MLKL helps break down and understand the machinery of MLKL. In this particular article, “Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis”, conveys data that suggest the role of MLKL in TNF-induced necrosis is actually just a general phenomenon, regardless of cell types or if ROS generation are involved2. There is no mention of a calcium influx being affiliated with MLKL mediation which greatly differs from the previous article. These experiments are both still recent so evidence to back their claims are still sparse since there are limited studies to refer to that have investigated these findings.

Another piece of evidence that questions the accuracy of this data comes from the article, “Mixed Lineage Kinase Domain-like Protein MLKL Causes Necrotic Membrane In this study, the authors’ main focus is the MLKL protein properties such as; where it is translocated, what it induces upon translocation, and the possible complexes that if forms. The experiment as whole, shows the MLKL becomes phosphorylated by RIP3 and results in an engagement with the necrosome through its interaction with RIP 3. It is demonstrated that MLKL forms a homotrimer and then localizes to the plasma membrane with RIP3 which is required for mediating necroptosis. Interestingly enough, the article heavily noted the importance of the ?Ca?

(2+) influx that occurs with MLKL localization, as well as identified TRMP7 and its involvement with this influx and how it is a target for TNF induced necroptosis. This is all expressed to be a critically important mechanism for MLKL-mediated TNF-induced necroptosis.

Tumor necrosis factor (TNF) is well accepted and known for its role in apoptosis and necroptosis. TNF also has an array of various intercessions such as pathological conditions, autoimmune diseases, inflammation, and immunity1. TNF also functions as a machine in forming the TNFR1 complex as a result of TNF signaling. This mechanism is used to mediate a quantity of pathways through the recruitment of effectors. TRADD, RIP1, and TRAF2 are present in this complex to carry out these pathways and have also been observed to disassociate from the receptor in order to recruit other proteins needed to carry out secondary complexes for apoptosis and necroptosis. RIP3 (receptor interacting protein 3) and MLKL (mixed lineage kinase-domain like) are required in the necrosome in order for necroptosis to occur as well as other proteins. Most likely, the other proteins that are possibly involved in the formation of the necrosome include CYLD and SIRT2. What remains in question is the generation of signaling that causes the initiation of TNF necroptosis. It is known that MLKL has a role in TNF induced necroptosis and that it is recruited by RIP3 but the mechanism is largely unknown. In this article, “Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis”, the authors’ relay that MLKL forms homotrimers and locates to the cell plasma membrane during TNF-induced necroptosis. Furthermore, they claimed to have identified TRPM7 as a MLKL downstream target to mediate the ?Ca?

(2+) influx which MLKL membrane localization is responsible for. Affirmation for the key mechanism of MLKL-mediated necroptosis is revealed in their study1. If these findings are in fact correct, the signaled formation of necroptosis is further uncovered.

The topic of interest in this experiment was to investigate how TNF-induced necroptosis is mediated by MLKL and the signal that triggers the mechanism for MLKL. The first experiment conducted was to observe how the MLKL protein was expressed under certain conditions. Localization of MLKL to the plasma membrane is essential for its function. These findings suggests that the translocation of MLKL to the plasma membrane has to do with RIP3 which is localized to the membrane as well. This led to further experiments to scrutinize RIP3 and why it also promotes MLKL trimerization. Testing also shows that MLKL and RIP3 are at cause for the extracellular calcium influx and intracellular calcium release which resulted in further testing to note which types of calcium channels are involved in necroptosis1.

It was predicted that MLKL forms oligomers based on the two coiled coil domains found at the N terminus. On SDS-Page, there were detections of MLKL protein expressed as a monomer. With co-expression of MLKL with other antigens, it was confirmed that MLKL does in fact form oligomers and high chances it forms homotrimers as well. The next step was to observe if MLKL would form trimers in cells that had been induced to undergo necroptosis. The experiments showed that the control had MLKL proteins that formed monomers whereas the TSZ treated cells had MLKL proteins that formed trimers. The trimerized MLKL was also observed in other necrotic induced cells further suggesting that the protein trimerizes during necroptosis. The significance of this experiment shows the changed characteristics of MLKL and how it is expressed by the presence of necroptosis.

The membrane localization experiment was carried out on coimmunoprecipitating gels to prove that MLKL and RIP3 localize specifically upon necroptosis. TNF or TS treatment did not change their localizations in the cytoplasm and MLKL and RIP3 were both detected in the crude membrane fraction of treated cells as trimeric1. When the cell surface proteins were isolated from necrotic cells, MLKL and RIP3 were still observed to localize at the plasma membrane. The take-away of this experiment shows that the localization of MLKL at the plasma membrane may be critical for its ability to mediate TNF induced necroptosis.

The next step in the experiment was testing whether localization of MLKL to the plasma membrane is required for the response of the calcium influx. In this study, it is suggested that both the calcium influx and ROS (reactive oxygen species) are required for MLKL to mediate necroptosis. The presence of a calcium influx involved in necroptosis was confirmed in HT29 cells. To prove that the influx was occurring during necroptosis, a calcium indicator, Fluo4 was used and increased after four hours of TSZ treatment1. The non-voltage sensitive channel, TRPM7 has been previously seen to have involvement with necroptosis and may be involved in the calcium channels in the plasma membrane. It is still in debate whether the calcium influx has any involvement with MLKL in necroptosis demonstrated by differing results in similar articles.

Searching for unknown targets of MLKL helps break down and understand the machinery of MLKL. In this particular article, “Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis”, conveys data that suggest the role of MLKL in TNF-induced necrosis is actually just a general phenomenon, regardless of cell types or if ROS generation are involved2. There is no mention of a calcium influx being affiliated with MLKL mediation which greatly differs from the previous article. These experiments are both still recent so evidence to back their claims are still sparse since there are limited studies to refer to that have investigated these findings.

Another piece of evidence that questions the accuracy of this data comes from the article, “Mixed Lineage Kinase Domain-like Protein MLKL Causes Necrotic Membrane Disruption

upon Phosphorylation by RIP3”. As mentioned above, MLKL favors the oligomer formation and is expressed as a tetramer on SDS-Page. In this more current article, the authors present their findings on SDS-PAGE gel under nonreducing conditions and gel ?ltration chromatography, indicates that the migration they observe shows it is most likely a hexamer3. This concludes in their studies that MLKL gains the ability to translocate to a variety of plasma membranes.

It has been continuously proven that MLKL is an important component in the mediation of TNF induced necrosis. The next step is additional studies on the MLKL targets, what they consist of, what exactly triggers the association of the complex, and its regulatory aspects. The goal in most cases is to further understand the machinery that makes us necrosis and what induces it to surface. Understanding this process can lead to the management of this phenomenon or could possibly be used as an advantage in medical applications such as preventing diseases if not rendering them curable.

In this study, the authors’ main focus is the MLKL protein properties such as; where it is translocated, what it induces upon translocation, and the possible complexes that if forms. The experiment as whole, shows the MLKL becomes phosphorylated by RIP3 and results in an engagement with the necrosome through its interaction with RIP 3. It is demonstrated that MLKL forms a homotrimer and then localizes to the plasma membrane with RIP3 which is required for mediating necroptosis. Interestingly enough, the article heavily noted the importance of the ?Ca?

(2+) influx that occurs with MLKL localization, as well as identified TRMP7 and its involvement with this influx and how it is a target for TNF induced necroptosis. This is all expressed to be a critically important mechanism for MLKL-mediated TNF-induced necroptosis.

Tumor necrosis factor (TNF) is well accepted and known for its role in apoptosis and necroptosis. TNF also has an array of various intercessions such as pathological conditions, autoimmune diseases, inflammation, and immunity1. TNF also functions as a machine in forming the TNFR1 complex as a result of TNF signaling. This mechanism is used to mediate a quantity of pathways through the recruitment of effectors. TRADD, RIP1, and TRAF2 are present in this complex to carry out these pathways and have also been observed to disassociate from the receptor in order to recruit other proteins needed to carry out secondary complexes for apoptosis and necroptosis. RIP3 (receptor interacting protein 3) and MLKL (mixed lineage kinase-domain like) are required in the necrosome in order for necroptosis to occur as well as other proteins. Most likely, the other proteins that are possibly involved in the formation of the necrosome include CYLD and SIRT2. What remains in question is the generation of signaling that causes the initiation of TNF necroptosis. It is known that MLKL has a role in TNF induced necroptosis and that it is recruited by RIP3 but the mechanism is largely unknown. In this article, “Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis”, the authors’ relay that MLKL forms homotrimers and locates to the cell plasma membrane during TNF-induced necroptosis. Furthermore, they claimed to have identified TRPM7 as a MLKL downstream target to mediate the ?Ca?

(2+) influx which MLKL membrane localization is responsible for. Affirmation for the key mechanism of MLKL-mediated necroptosis is revealed in their study1. If these findings are in fact correct, the signaled formation of necroptosis is further uncovered.

The topic of interest in this experiment was to investigate how TNF-induced necroptosis is mediated by MLKL and the signal that triggers the mechanism for MLKL. The first experiment conducted was to observe how the MLKL protein was expressed under certain conditions. Localization of MLKL to the plasma membrane is essential for its function. These findings suggests that the translocation of MLKL to the plasma membrane has to do with RIP3 which is localized to the membrane as well. This led to further experiments to scrutinize RIP3 and why it also promotes MLKL trimerization. Testing also shows that MLKL and RIP3 are at cause for the extracellular calcium influx and intracellular calcium release which resulted in further testing to note which types of calcium channels are involved in necroptosis1.

It was predicted that MLKL forms oligomers based on the two coiled coil domains found at the N terminus. On SDS-Page, there were detections of MLKL protein expressed as a monomer. With co-expression of MLKL with other antigens, it was confirmed that MLKL does in fact form oligomers and high chances it forms homotrimers as well. The next step was to observe if MLKL would form trimers in cells that had been induced to undergo necroptosis. The experiments showed that the control had MLKL proteins that formed monomers whereas the TSZ treated cells had MLKL proteins that formed trimers. The trimerized MLKL was also observed in other necrotic induced cells further suggesting that the protein trimerizes during necroptosis. The significance of this experiment shows the changed characteristics of MLKL and how it is expressed by the presence of necroptosis.

The membrane localization experiment was carried out on coimmunoprecipitating gels to prove that MLKL and RIP3 localize specifically upon necroptosis. TNF or TS treatment did not change their localizations in the cytoplasm and MLKL and RIP3 were both detected in the crude membrane fraction of treated cells as trimeric1. When the cell surface proteins were isolated from necrotic cells, MLKL and RIP3 were still observed to localize at the plasma membrane. The take-away of this experiment shows that the localization of MLKL at the plasma membrane may be critical for its ability to mediate TNF induced necroptosis.

The next step in the experiment was testing whether localization of MLKL to the plasma membrane is required for the response of the calcium influx. In this study, it is suggested that both the calcium influx and ROS (reactive oxygen species) are required for MLKL to mediate necroptosis. The presence of a calcium influx involved in necroptosis was confirmed in HT29 cells. To prove that the influx was occurring during necroptosis, a calcium indicator, Fluo4 was used and increased after four hours of TSZ treatment1. The non-voltage sensitive channel, TRPM7 has been previously seen to have involvement with necroptosis and may be involved in the calcium channels in the plasma membrane. It is still in debate whether the calcium influx has any involvement with MLKL in necroptosis demonstrated by differing results in similar articles.

Searching for unknown targets of MLKL helps break down and understand the machinery of MLKL. In this particular article, “Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis”, conveys data that suggest the role of MLKL in TNF-induced necrosis is actually just a general phenomenon, regardless of cell types or if ROS generation are involved2. There is no mention of a calcium influx being affiliated with MLKL mediation which greatly differs from the previous article. These experiments are both still recent so evidence to back their claims are still sparse since there are limited studies to refer to that have investigated these findings.

Another piece of evidence that questions the accuracy of this data comes from the article, “Mixed Lineage Kinase Domain-like Protein MLKL Causes Necrotic Membrane Disruption

upon Phosphorylation by RIP3”. As mentioned above, MLKL favors the oligomer formation and is expressed as a tetramer on SDS-Page. In this more current article, the authors present their findings on SDS-PAGE gel under nonreducing conditions and gel ?ltration chromatography, indicates that the migration they observe shows it is most likely a hexamer3. This concludes in their studies that MLKL gains the ability to translocate to a variety of plasma membranes.

It has been continuously proven that MLKL is an important component in the mediation of TNF induced necrosis. The next step is additional studies on the MLKL targets, what they consist of, what exactly triggers the association of the complex, and its regulatory aspects. The goal in most cases is to further understand the machinery that makes us necrosis and what induces it to surface. Understanding this process can lead to the management of this phenomenon or could possibly be used as an advantage in medical applications such as preventing diseases if not rendering them curable.

Disruption

upon Phosphorylation by RIP3”. As mentioned above, MLKL favors the oligomer formation and is expressed as a tetramer on SDS-Page. In this more current article, the authors present their findings on SDS-PAGE gel under nonreducing conditions and gel ?ltration chromatography, indicates that the migration they observe shows it is most likely a hexamer3. This concludes in their studies that MLKL gains the ability to translocate to a variety of plasma membranes.

It has been continuously proven that MLKL is an important component in the mediation of TNF induced necrosis. The next step is additional studies on the MLKL targets, what they consist of, what exactly triggers the association of the complex, and its regulatory aspects. The goal in most cases is to further understand the machinery that makes us necrosis and what induces it to surface. Understanding this process can lead to the management of this phenomenon or could possibly be used as an advantage in medical applications such as preventing diseases if not rendering them curable.”

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