Scientists discover the molecular switch that lets TNF trigger cell death

Tumor necrosis factor, or TNF, has long been one of the most paradoxical molecules in immunology. It is a proinflammatory cytokine that helps orchestrate the body’s defenses, yet when its signaling goes awry it can drive lethal inflammation. For decades, researchers have known that TNF can instruct a cell either to survive or to die, and that the same protein, a kinase called RIPK1, sits at the heart of both decisions. What they have not known is how the cell flips that switch. A new study published in Nature Structural & Molecular Biology now identifies the missing piece: an E3 ubiquitin ligase named HERC4, which the authors show is the molecular determinant that licenses RIPK1 to initiate TNF-induced cell death.

To understand why this matters, it helps to picture the two signaling complexes that form after TNF binds its receptor, TNFR1. At the plasma membrane, TNFR1 recruits RIPK1 through its death domain to build complex I, a platform that activates the IKK/NF-κB and MAPK pathways and thereby promotes cell survival and inflammatory gene transcription. When complex I is destabilized, RIPK1 can instead nucleate cytosolic complexes: complex IIa, containing RIPK1, FADD and caspase 8, which drives apoptosis; or, when caspase 8 is inhibited, complex IIb, the RIPK1–RIPK3 necrosome that initiates necroptosis, a lytic form of cell death in which activated RIPK3 phosphorylates MLKL, sending it to the plasma membrane to rupture the cell. RIPK1 is a component of all these assemblies, but the biochemical event that converts it from a prosurvival scaffold in complex I into a prodeath kinase in complex II has remained elusive.

The search began with an unbiased genetic approach. The team, led by Haohao Lu and Tongde Du in the laboratory of Sudan He at the Suzhou Institute of Systems Medicine, together with She Chen and Henning Walczak, conducted a CRISPR–Cas9 screen in human HT-29 colon cancer cells using a guide RNA library targeting genes encoding ubiquitin E3 ligases, deubiquitinating enzymes and other ubiquitination-associated proteins. The cells were challenged with a necroptosis-inducing cocktail of TNF, a Smac mimetic and the pan-caspase inhibitor z-VAD, a combination known as TSZ. After four rounds of selection, the single guide RNA targeting HERC4 emerged as the most enriched hit among all the E3 ligases and deubiquitinases tested, marking the enzyme as a prime suspect in the cell death machinery.

Validation followed swiftly across cell types. HERC4-knockout lines generated in HT-29, LS 174T, THP-1 and Jurkat cells all showed marked resistance to necroptosis, surviving doses of TSZ that killed their wild-type counterparts. Reintroducing HERC4, even at subphysiological levels, restored sensitivity to the death stimulus. Crucially, the loss of HERC4 did not merely blunt a downstream effector: phosphorylation of RIPK1, RIPK3 and MLKL, the sequential activations that define the necroptosis pathway, were all significantly reduced in HERC4-deficient cells. MLKL oligomerization and its translocation to the plasma membrane, the final steps before necroptotic rupture, were likewise suppressed. The same protection appeared in mouse embryonic fibroblasts and in a second necroptosis model using a TAK1 inhibitor, establishing HERC4 as a driver acting upstream of the canonical RIPK1–RIPK3–MLKL axis in both human and mouse cells.

Because HERC4 is a ubiquitin ligase, the next question was which protein it decorates. Coexpression experiments showed that HERC4 increased polyubiquitination of RIPK1 but not of RIPK3. Using mutant ubiquitin restricted to single linkage types, the researchers found that HERC4 specifically catalyzes K63-linked chains, a modification classically associated with signal transduction rather than protein degradation, and indeed HERC4-mediated ubiquitination did not lead to RIPK1’s destruction. In vitro assays with recombinant proteins confirmed that HERC4 directly ubiquitinates RIPK1, and mapping experiments showed that the intermediate domain of RIPK1 binds the HECT domain of HERC4, the region that carries the ligase’s catalytic activity.

Mass spectrometry then pinpointed the exact modification site. Among the ubiquitinated peptides recovered from RIPK1, one, EubKVYQMLQK, appeared exclusively when HERC4 was present and was absent with a catalytically inactive HERC4 mutant. The fragmentation spectrum unmasked lysine 627 on human RIPK1 as the ubiquitination site, a residue within the death domain that is highly conserved across species and corresponds to K612 in the mouse protein. This was a striking finding, because earlier work had shown that substituting this very lysine prevents TNF-induced cell death, hinting that ubiquitination there might be a necessary licensing step. A catalytically inactive HERC4 mutant, engineered on the basis of AlphaFold3 predictions by mutating H1023, C1025 and D1030, still bound RIPK1 but could no longer ubiquitinate it, and cells expressing this mutant were markedly resistant to necroptosis, proving that the ligase activity itself is what matters.

The mechanism that emerges is elegant and precise. HERC4 does not act indiscriminately: its interaction with RIPK1 occurs only under apoptotic or necroptotic stimulation, not with TNF alone, and is abolished by the RIPK1 kinase inhibitor necrostatin 1s. The team showed that HERC4 selectively recognizes RIPK1 that has been autophosphorylated at serine 166, the hallmark of RIPK1 kinase activation, since an S166A mutant lost the interaction while phosphomimetic S166D and S166E variants enhanced it. Sequential immunoprecipitation of the TNFR1 signaling complexes revealed that HERC4 is absent from membrane-bound complex I and instead binds complex I-derived, S166-phosphorylated RIPK1 once it has been released into the cytosol. There, HERC4’s K63-linked ubiquitination of the death domain enables RIPK1 to oligomerize, both with itself and with FADD, nucleating the assembly of complex IIa and, when caspases are blocked, the RIPK1–RIPK3 necrosome. Gel-filtration analysis confirmed that intact HERC4, but not the catalytic mutant, permits the formation of high-molecular-weight complexes containing phosphorylated RIPK1, RIPK3, MLKL and caspase 8.

Importantly, HERC4’s role is confined to the death arm of TNF signaling. The enzyme is dispensable for NF-κB activation: HERC4 knockout left phosphorylation of P65, IκBα, TAK1 and IKKα/β untouched, and it did not alter RIPK1 ubiquitination at K377, the cIAP1/2-mediated modification in complex I that supports survival signaling. This separation is what makes HERC4 so attractive as a drug target. Blocking it would, in principle, disarm TNF’s lethal potential while leaving its beneficial inflammatory and prosurvival functions intact, an effect similar to direct RIPK1 kinase inhibition but achieved one step upstream, at the licensing event itself.

The in vivo evidence is compelling. Mice lacking HERC4 were born and bred normally, indicating the protein is not essential for development. But when challenged with an overdose of TNF, a model of systemic inflammatory response syndrome, all wild-type littermates succumbed within 28 hours, whereas 80 percent of HERC4-deficient mice survived. The protected animals showed lower serum IL-6, reduced caspase 3 and PARP cleavage in the intestine, less cecal tissue damage on histology, and markedly reduced RIPK1 autophosphorylation. In a second model of acute liver injury induced by TNF and D-galactosamine, nearly all wild-type mice reached humane endpoints within four to six hours, while more than 70 percent of HERC4-deficient mice survived, with lower ALT and AST liver enzymes, reduced IL-6, suppressed hepatic caspase 3 activation and diminished histological damage.

The discovery resolves a long-standing conundrum in cell death biology: why RIPK1’s kinase activity, whose only known substrate is RIPK1 itself, is essential for TNF-induced death. The answer is that autophosphorylation at S166 creates the molecular tag that HERC4 recognizes, and HERC4’s ubiquitination of the death domain is what converts the phosphorylated kinase into a competent nucleator of the death complexes. Beyond inflammatory diseases such as SIRS, colitis and liver injury, the finding may reach into oncology, where necroptosis’s immunogenic nature has been proposed as a way to stimulate antitumor immunity, and where HERC4 has previously been implicated as a tumor suppressor. By supplying RIPK1 with what the authors call its license to kill, HERC4 now stands as both a mechanistic keystone of TNF signaling and a promising target for diseases driven by aberrant cell death.

Subject of Research: HERC4-mediated ubiquitination of RIPK1 as the molecular switch initiating TNF-induced cell death and inflammation

Article Title: HERC4-mediated ubiquitination licenses RIPK1 to initiate TNF-induced cell death

Article References: Lu, H., Du, T., Li, L., Cao, D., Li, K., Liu, L., Li, R., Yu, X., Hou, S., Wang, X., Shi, M., Liu, Y., Ma, F., Chen, S., Walczak, H., & He, S. (2026). HERC4-mediated ubiquitination licenses RIPK1 to initiate TNF-induced cell death. Nature Structural & Molecular Biology. https://doi.org/10.1038/s41594-026-01871-y

Image Credits: AI Generated

DOI: 10.1038/s41594-026-01871-y

Keywords: HERC4, RIPK1, TNF, ubiquitination, necroptosis, apoptosis, RIPK3, MLKL, inflammation, E3 ligase, liver injury, cell death signaling