Sterol regulatory element-binding protein 2 (SREBP-2) is a central regulator of cholesterol homeostasis and is canonically defined by the nuclear activity of its N-terminal transcription factor released upon proteolytic activation. While the metabolic role of the N-terminal domain is well characterized, the biological function of the complementary C-terminal fragment has remained largely unclear. Here, we demonstrate that the SREBP-2 C-terminal (C-term) fragment functions as a stress-inducible, secreted proapoptotic signaling mediator.

In our prior study, we first reported that the SREBP-2 C-term fragment is secreted into the extracellular milieu, where it can be detected in patient plasma, culture supernatants, and peripheral blood from COVID-19 patients.1 Canonically, cholesterol depletion triggers the SREBP-2–SCAP complex to translocate to the Golgi, where proteolytic cleavage releases the active N-terminal domain that enters the nucleus and induces genes required for cholesterol biosynthesis and uptake.2,3,4,5 Because this processing generates the C-term in parallel and because we can detect the C-term outside cells, we hypothesized that it may act beyond the intracellular environment under stress conditions.

We validated stress-associated induction in vivo via a cecal ligation and puncture (CLP) model of sepsis. The SREBP-2 C-term abundance increased progressively in the lung, liver, and kidney tissues, with the lung showing the most prominent and statistically significant increase (Fig. 1a). Sepsis severity was defined by clinical scoring and survival time post-CLP. Sepsis severity was defined by clinical scoring and survival time post-CLP. To minimize confounding by cell lysis, tissue damage was assessed in parallel by LDH release and histological scoring, both of which are correlated with fragment abundance (S Fig. 1a). Circulating C-term levels also increased progressively during sepsis (Fig. 1a). While the spleen was not analyzed due to sample limitations, we acknowledge its potential relevance to systemic inflammation.

SREBP-2 C-terminal fragment promotes apoptosis via IRAK1 interaction and extracellular signaling. a Left: Quantification of SREBP-2 C-term levels in lung, liver, and kidney tissues from CLP (cecal ligation and puncture) mice across sepsis severity stages. ***p < 0.001 vs normal lung, ## < 0.01 vs normal liver, & <0.05 vs normal kidney. Right upper: SREBP-2 C-term levels measured in the blood of septic mice. b Left: Construction and generation of human dermal fibroblast (HDF) cell lines stably overexpressing full-length SREBP-2 (full) or the C-term fragment (C-term), along with a scramble control (SC). Left bottom: immunoblotting of cell lysates and supernatants in time course analyses of SREBP-2 C-term expression in HDFs treated with lovastatin for 24, 36, or 48 h. A Venn diagram identified proteins that bind to the C-term after proteomic analysis. c Left upper: Co-IP assay confirming the interaction between C-term and IRAK1. Left bottom: Structural modeling of the SREBP-2 C-term and IRAK1 complex with the predicted binding interface zoomed in. Middle: Immunofluorescence microscopy revealed the colocalization of C-term and IRAK1, shown in green and red, respectively; scale bars correspond to 50 μm. Left: The colocalization quantitative analysis revealed colocalization coefficients and Pearson’s coefficients. For fluorescence-based assays (colocalization and TUNEL), the data represent n = 4–5 independent experiments. d Left and Middle: TUNEL-positive areas or colocalization coefficients (Pearson’s r) were calculated and then averaged per experiment for statistical analysis. TUNEL staining was performed under five conditions to assess apoptosis, including quantification of the TUNEL-positive area under each condition. Right: Protein expression was examined in HDFs following treatment with LPS (10 μg/ml) and TQ (25 μM) for 24 h Data are shown as the means ± S.D., n = 4–5, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA (Tukey); SC_CON (control), C-term_CON (SREBP-2 C-term overexpression), C-term_TQ (cotreatment with thymoquinone, an IRAK1 inhibitor), C-term_LPS (LPS stimulation), and C-term_LPS + TQ (LPS + TQ)

Because circulating C-term was observed in COVID-19 patients, we tested whether it can interact with SARS-CoV-2 proteins. In cultured cells, the coexpression of GFP–SREBP-2 C-term and mCherry–Orf3a, an accessory protein of SARS-CoV-2, demonstrated cytoplasmic colocalization (Fig. 1a). To biochemically confirm this interaction, we performed streptavidin pull-down assays in HEK293T cells expressing Orf3a fused to either the N-terminal or C-terminal fragments of SREBP-2. A robust interaction was detected between Orf3a and the C-term fragment, whereas only minimal binding was observed with the N-term fusion. Under nonpermeabilizing biotinylation conditions, the C-term was preferentially recovered in streptavidin pull-downs only when Orf3a was coexpressed, which is consistent with its association with accessible complexes. In silico docking further supported direct binding, with the strongest predicted affinity at the Glu348 site of the SREBP-2 C-term (supplementary figure deposited at Figshare; https://doi.org/10.6084/m9.figshare.31120696). Together, these results support the concept that the stress-induced and secreted SREBP-2 C-term fragment can engage a viral accessory protein.

To characterize C-term dynamics in a controlled system, full-length SREBP-2 (Full) or the C-term fragment (residues 641–1018, lacking the transmembrane domain) was constructed and stably overexpressed in human dermal fibroblasts (HDFs), alongside scramble control (SC) cells, deposited at Figshare. Following lovastatin treatment, full-length cells primarily displayed full-length-derived species without clear accumulation of a free C-terminal fragment. In contrast, direct C-term overexpression led to detectable C-term accumulation in both lysates and supernatants, peaking at 24 h and decreasing thereafter (Fig. 1b). Untreated controls confirmed that accumulation was not due to baseline expression. These data suggest that the endogenous C-term is tightly regulated and rapidly degraded, which is consistent with prior work identifying a degradation signal at the SREBP-2 C-term that targets this region for ER-associated, proteasome-dependent degradation.5 To identify host protein partners, we performed coimmunoprecipitation (Co-IP) in SC- and C-term-overexpressing HDFs via an anti–C-term antibody. A specific band corresponding to the C-term was detected exclusively in C-term-overexpressing cells, confirming successful enrichment. Co-IP products were subjected to mass spectrometry. Fifty-eight proteins were uniquely enriched in the SREBP-2 C-term pull-down, whereas 598 proteins were shared with the scramble controls (Fig. 1b Venn diagram). KEGG pathway analysis of the differentially enriched proteins revealed significant associations with neurodegenerative disease-related pathways, including prion disease and Parkinson’s disease pathways. Proteomic analysis identified 18 unique interactors deposited at Figshare. While overexpression imposes inherent limitations, these data suggest that the C-term may engage multiple partners involved in ER and mitochondrial quality control, potentially linking stress responses to degenerative signaling networks.

Among the candidates, we focused on interleukin-1 receptor-associated kinase 1 (IRAK1), a central node in TLR and IL-1R signaling. Co-IP confirmed a specific interaction between SREBP-2 C-term and IRAK1 (Fig. 1c). Structural modeling and molecular docking predicted a strong binding interface, with the highest affinity at the Thr360 site of the SREBP-2 C-term (Fig. 1c). Immunofluorescence further revealed that cytoplasmic colocalization between C-term and IRAK1 occurred in a construct-dependent manner (SC < Full < C-term). LPS did not significantly alter colocalization, which is consistent with enhanced binding rather than a change in bulk spatial overlap (Fig. 1c).

We then tested whether C promotes apoptosis through IRAK1. TUNEL assays were performed under SC_CON, C-term_CON, C-term_TQ (thymoquinone; an IRAK1 inhibitor), C-term_LPS, and C-term_LPS + TQ conditions (Fig. 1d). Compared with SC_CON cells, C-term_CON cells presented a marked increase in TUNEL-positive cells, indicating that C-term expression alone is sufficient to trigger apoptosis. Thymoquinone partially attenuated TUNEL positivity in C-term-expressing cells, supporting an IRAK1-dependent component of C-term-induced apoptosis. LPS produced, at most, a modest increase, with a trend toward reduction with thymoquinone cotreatment. Consistent with the findings of imaging, Western blot analysis revealed increased cleaved PARP and cleaved caspase-9 in C-term-expressing cells relative to those in SC_CON-expressing cells; compared with SC_CON, thymoquinone reproducibly reduced cleaved PARP, whereas changes in cleaved caspase-9 were less pronounced (Fig. 1d). These results indicate that the SREBP-2 C-terminal fragment is sufficient to activate IRAK1-dependent apoptotic signaling under stress conditions.

In summary, we identified a previously unrecognized role of the SREBP-2 C-term fragment as a stress-inducible, secreted proapoptotic signaling molecule distinct from the canonical cholesterol-regulatory function of the N-terminus. The fragment accumulates in tissues and the circulation during CLP sepsis, associates with SARS-CoV-2 Orf3a in accessible complexes, and binds IRAK1 with a predicted high-affinity interface. The extracellular release of a transcription factor fragment is unusual and suggests an autocrine or paracrine mechanism that may modulate cell fate during tissue stress. Further validation in infection-based and patient-derived models will be important to define the physiological scope of this pathway.