Modes of Programmed Necrosis

Phosphorylation-Driven Assembly of the RIP1-RIP3 Complex Regulates Programmed Necrosis and Virus-Induced Inflammation was a revolutionary research paper as it revealed the importance of a specific regulatory factor to a newly discovered form of cell death, necroptosis, and how the entire process is vital to inflammatory antiviral responses. The term “necroptosis” was recently developed at this time, and many researchers were focusing their work towards better understanding of the process and its importance. This paper, along with others, were extremely important as they simultaneously revealed the function and regulatory mechanisms of the kinase, RIP3, within TNF-induced necrotic cells. Cho and others discovered that for a cell to undergo necroptosis, RIP3 was required to form a complex with RIP1 via phosphorylation, and that necroptosis was the preferred form of cell death to initiate an immunological response to viral infections.

This specific study was performed due to the uncertainties surrounding the regulatory mechanisms and components defining programmed necrosis. Programmed necrosis had been defined in a few previous papers as an alternative form of the already well-known apoptosis. The most unique, defining features of programmed necrosis provided from this research were that it occurred via caspase-independent pathways and induced pro-inflammatory responses, two large contrasts of apoptosis. These were breakthrough discoveries in the field of cell death research because both the intrinsic and extrinsic pathways of apoptosis involve a variety of caspases. These two interesting features of programmed necrosis led Cho and his team of researchers to explore the detailed mechanisms involved in signaling for necrotic cell death and how it signaled for inflammatory responses.

This paper successfully discovered and defined one of the most important signaling complexes involved in programmed necrosis. In extrinsic apoptosis, an internal complex of protein molecules forms in response to pro-death signaling. The major components of the complexes commonly include FADD, TRADD, pro-caspase-8, and RIP1. This paper and others introduced a new kinase, RIP3, which can become activated and bind to the factor RIP1 to form a second complex which signals for programmed necrosis. Cho and his team provided the specific mechanism of how these two RIP molecules interact when signaling for programmed necrosis. The paper provided the composition of RIP1 and RIP3, with two regions of specific importance. These two regions were the kinase domain (KD) and the RIP homotypic interaction motif (RHIM). It was determined through separate mutations within these two regions that the RHIM is vital for proper RIP1 and RIP3 interaction and at least one functional KD within the complex is required for cleavage and subsequent activation of the molecules. They also tested the effects of a vaccinia viral infection on wild type or mutant mice. The mutant mice did not express RIP3. From previous in vitro cellular viral infections, they determined that RIP3 is required for an immunological response because the cells prefer to undergo necroptosis in response to the virus. As expected, the RIP3-deficient mice had severe defects in fighting off the viral infection. This paper was important to the field as it described two important modes of programmed necrosis: the requirement of a second signaling complex composed of RIP1 and RIP3 and the fact that necrotic cells are able to induce an inflammatory response against viral infections.

Two other scientific papers were published in the same year as this paper and helped to explain further questions about the importance and activity of RIP3 in programmed necrosis. The paper by He and others introduced the mechanism and role of RIP3 in necrotic cell death in response to TNF? signaling. TNF signaling has long been used in cell death research to induce apoptotic cell death in many cell lines. TNF signaling induces the formation of a pro-apoptotic complex containing caspase-8 and RIP1. In this paper, they show that the presence of a RIP3 molecule (among other factors) within the cells can induce the formation of the important second signaling complex composed of RIP1 and RIP3. They further prove the importance of RIP3 to necrotic cell death through RIP3-deficient mice being unable to perform programmed necrosis and induce an inflammatory response. Both papers share many characteristics. They both provide the importance of RIP3 to programmed necrosis and inflammatory responses. Cho and his team provided a little more detail on the inflammatory mechanisms while He primarily focused on the activity of RIP3 in necrotic cells in response to TNF? signaling. Cho showed that the inflammatory responses from necrotic cells consisted of high levels of recruitment of neutrophils and macrophages to the site of infection.

Zhang’s paper3 appears extremely similar in the sense that it was another important supporter of the identification of RIP3 as a required molecule for programmed necrosis. Zhang and others focused their paper3 on how RIP3 was able to switch TNF signaling for apoptosis to programmed necrosis by stimulating specific intracellular pathways and through its association with RIP. The main finding in Zhang’s research3 was that when RIP3 was expressed within cells responding to TNF signaling and zVAD caspase inhibition, programmed necrosis was executed due to a significant production of reactive oxygen species. They3 concluded that through RIP3’s activation of specific enzymatic pathways within these cells, more reactive oxygen species could be generated and subsequent programmed necrosis could ensue. Again, Cho’s paper incorporates these important findings as well as adding an additional systemic response to programmed response. Cho shows the similar mechanism of RIP3 associating with RIP in cells responding to TNF signaling to induce programmed necrosis through the downstream production of reactive oxygen species. The research performed by Zhang3 appeared to be a major influence for Cho as his paper integrated Zhang’s programmed necrosis approach while exploring the mechanism of programmed necrosis in response to tumor formation.

All three of these research papers were extremely pieces to the field of study surrounding programmed necrosis. They successfully identified the factor of RIP3, which is absolutely required for cells responding to TNF signaling to induce necroptosis. Cho and his team were able to take this finding one step further by showing the importance of programmed necrosis as the cell death mechanism involved in proper anti-tumor growth response.

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