Necroptosis: When Apoptosis Meets Necrosis

 

Contributed by Focus Biomolecules  

Among the many ways in which cells can die, necroptosis is a caspase-independent form of programmed cell death induced by certain changes in cellular homeostasis and when apoptosis is blocked1. It has roles in normal biological processes (inflammation, wound healing, combating infectious disease) as well as disease states (cancer, chronic inflammation)2. In fact, necroptosis can protect or kill tumor cells, depending on the context3.

Necroptosis can be viewed as a combination of apoptosis and necrosis4. It begins with external or internal triggers such as TNFα, TRAIL, interferon γ, genotoxic stress, viral DNA/RNA, bacterial LPS, or caspase 8 inhibition. These signals are transduced by receptors and binding proteins such as the toll-like receptor (TLR), tumor necrosis factor receptor 1, FAS, or Z-DNA binding protein 1 (ZBP1)2. The most understood pathway is the one beginning with TNF-α binding to its receptor, TNFR1 (See figure). Briefly, this results in the formation of complex I, which comprises RIPK1, TRADD, TRAF2 & -5, cIAP1 & -2, and LUBAC. If RIPK1 is polyubiquitinated by cIAP1/2 and LUBAC, cell survival is achieved by activation of the NF-κB pathway4. If instead RIPK1 is deubiquitinated by CYLD or A20, TRADD and RIPK1 are released and form either complex IIa (TRADD, FADD, and RIPK1) or complex IIb (FADD, RIPK1). If caspase 8 is present and active, apoptosis ensues via complex IIa or IIb. However, if caspase 8 is inhibited or absent, RIPK3 is recruited, causing RIPK3 oligomerization and autophosphorylation5. RIPK3 then phosphorylates MLKL, causing its oligomerization, which induces (among other things) Ca2+ influx via TRPM76, promoting cell membrane perforation and eventually necroptosis5. Alternatively, in the presence of reactive oxygen species (ROS) RIPK1 autophosphorylation recruits RIPK3 to form the necrosome, again leading to necroptosis7.
Necroptosis Pathway

Figure: Necroptosis pathway. Necroptosis can be induced by various stimuli such as TNFa. In short ubiquitination of RIPK1 leads to cell survival through activation of NF-kB, whereas deubiquitination of RIPK1 leads to the formation of complex II. The necrosome, a complex consisting of RIPK1, RIPK3, and MLKL is formed from complex IIa or IIb by inhibition of caspase 8. (Adapted from Chen et al., 2019, created with BioRender.com)

Components of necroptosis can overlap with other forms of cell death. For example, parthanatotic death is driven by DNA damage, and its pathways can involve RIPK1 and RIPK3 stimulation of PARP18. NETotic cell death can be blocked by RIPK1 or MLKL small molecule inhibitors9. Also, autophagy can mediate necroptosis via formation of necrosomes on autophagosomes10.

Necroptosis is a key player in pathologies such as neurodegeneration, inflammation, kidney damage, and cancer (proliferation, invasion, angiogenesis, metastasis)2, thus many small molecule modulators of necroptotic pathways have been developed for use as research tools and therapeutics4. For example, Necrostatin-1 (Nec-1) and RIPA-56 are potent and selective inhibitors of RIPK1, while Ponatinib inhibits both RIPK1 and RIPK3. Necrosulfonamide has a different mechanism of action, specifically blocking the interaction of MLKL with RIPK3. Compounds like these are important tools for necroptosis research, and many are currently in clinical trials for cancer, colitis, arthritis, psoriasis, Alzheimer’s, and ALS4.

 

Necroptosis Reagents (Provided by Focus Biomolecules)

Product Activity CAS Formula
7-Cl-O-Nec1   RIP1 Inhibitor 852391-15-2 C13H12ClN3O2
GSK872   Necroptosis Inhibitor, RIP3 Inhibitor 1346546-69-7 C19H17N3O2S2
Matrine   Necroptosis Inducer 519-02-8 C15H24N2O
Necrostatin-1   RIP1 Inhibitor 4311-88-0 C13H13N3OS
Necrosulfonamide   MLKL Inhibitor 1360614-48-7 C18H15N5O6S2
Ponatinib   Multikinase Inhibitor 943319-70-8 C29H27F32N6O
RIPA-56   RIPK1 Inhibitor 1956370-21-0 C13H19NO2

 

Necroptosis Antibodies

Product Clonality Reactivity Applications
A20 Antibody Monoclonal Human WB, IHC, IF, IP, FC
CIAP Antibody Polyclonal Human, Mouse WB, IHC, IF, ELISA
FLIP Antibody Polyclonal Human, Mouse, Rat WB, IHC, IF, FC, ELISA
IKB alpha Antibody Polyclonal Human, Mouse, Rat WB, EMSA
IKK alpha Antibody Polyclonal Human WB, IHC, IF, ELISA
IKK alpha Antibody Polyclonal Human WB, IHC, IF, ChIP, IP, FC
IKK beta Antibody Polyclonal Human, Mouse WB, IHC, IF, IP, FC
IKK beta Antibody Polyclonal Human, Mouse, Rat WB, IHC
NEMO/IKK-gamma Antibody Polyclonal Human WB, IP
NFkB p65 Antibody Polyclonal Human WB, IHC, IF, EMSA, ELISA
NFkB p65 Antibody Polyclonal Human, Mouse WB, IHC, IF, ChIP, IP, EMSA, ELISA
Recombinant Anti-TNF alpha Fab Antibody Recombinant Monoclonal Human WB, ELISA
RIPK1 Antibody Polyclonal Human, Mouse, Rat WB, IHC, IF, ELISA
RIP3 Antibody Polyclonal Human, Mouse, Rat WB, IHC, IF, IP, ELISA
TAB1 Antibody Polyclonal Human, Mouse, Rat WB, IHC, IF, ELISA
TAB2 Antibody Polyclonal Human IHC, ELISA
TAK1 Antibody Polyclonal Human, Mouse, Rat WB, IF, ELISA
TLR3 Antibody Monoclonal Human, Mouse WB, IHC, IF, ELISA
TLR3 Antibody Polyclonal Human WB, IHC, IF, IP, FC
TLR4 Antibody Polyclonal Human WB, IHC, ELISA
TLR4 Antibody Monoclonal Human, Mouse, Rat WB, IHC, IF, ChIP, FC, EMSA, ELISA
TNF alpha Antibody Polyclonal Human WB, IHC, IF
TNF alpha Antibody Polyclonal Mouse WB, IHC
TRAF2 Antibody Polyclonal Human, Mouse, Rat WB, IHC, IP, ELISA

 

References

  1. Galluzzi L, Vitale L, Aaronson SA et al. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ. 2018 Mar;25(3):486-541.
  2. He R, Wang Z, Dong S, Chen Z, Zhou W. Understanding Necroptosis in Pancreatic Diseases. 2022 Jun 13;12(6):828.
  3. Qin X, Ma D, Tan Y.-X, Wang H.-Y, Cai Z. The role of necroptosis in cancer: A double-edged sword? Biochim Biophys Acta Rev Cancer. 2019 Apr;1871(2):259-266.
  4. Chen J, Kos R, Garssen J, Redegeld F. Molecular Insights into the Mechanism of Necroptosis: The Necrosome as Potential Therapeutic Target. 2019 Nov 21;8(12):1486.
  5. Wang Q, Fan D, Xia Y, Ye Q, Xi X, Zhang G, Xiao C. The latest information on the RIPK1 post-translational modifications and functions. Biomed Pharmacother. 2021 Oct;142:112082.
  6. Cai Z, Jitkaew S, Zhao J, Chiang H-C, Choksi S, Liu J, Ward Y, Wu L-G, Liu Z-G. Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol. 2014 Jan;16(1):55-65.
  7. Seo J, Nam YW, Kim S, Oh D-B, Song J. Necroptosis molecular mechanisms: Recent findings regarding novel necroptosis regulators. Exp Mol Med. 2021 Jun;53(6):1007-1017.
  8. Jouan-Lanhouet S, Arshad MI, Piquet-Pellorce C, Martin-Chouly C, Le Moigne-Muller G, Van Herreweghe F, Takahashi N, Sergent O, Lagadic-Gossmann D, Vandenabeele P, Samson M, Dimanche-Boitrel M-T. TRAIL induces necroptosis involving RIPK1/RIPK3-dependent PARP-1 activation. Cell Death Differ. 2012 Dec;19(12):2003-14.
  9. Desai J, Kumar SV, Mulay SR, Konrad L, Romoli S, Schauer C, Herrmann M, Bilyy M, Müller S, Popper B, Nakazawa D, Weidenbusch M, Thomasova D, Krautwald S, Linkermann A, Anders H-A. PMA and crystal-induced neutrophil extracellular trap formation involves RIPK1-RIPK3-MLKL signaling. Eur J Immunol. 2016 Jan;46(1):223-9.
  10. Basit F, Cristofanon S, Fulda S. Obatoclax (GX15-070) triggers necroptosis by promoting the assembly of the necrosome on autophagosomal membranes. Cell Death Differ. 2013;20:1161–73.