A designer polyQ fusion protein modulates NF-κB signaling by sequestering P65/RelA into aggregates

Protein aggregation is often observed in various biological processes, characterized by the accumulation of improperly processed or misfolded proteins within cells^1,2. This aberrant occurrence is frequently found in neurodegenerative disorders, for example in polyglutamine (polyQ) diseases, which arise from the unstable expansion of CAG trinucleotide repeats in the specific gene sequences³. Previous research indicated that protein aggregation could serve a protective role by isolating misfolded or damaged proteins, thereby potentially maintaining cellular functions^4,5. During this process, polyQ-expanded proteins can effectively sequester cellular essential factors⁶ including transcription factors^7,8,9 ubiquitin adaptor proteins^10,11 and molecular chaperones^12,13,14 into aggregates or inclusions. Such sequestration may reduce the solubility and availability of the sequestered proteins, potentially compromising their biological functions and altering cellular fates^6,15.

The polyQ-tract protein is prone to aggregation, and its expansion exacerbates this tendency, but it may exhibit relative inertness within cells¹⁶. By leveraging these biochemical traits, we proposed a therapeutic strategy to design and construct a series of polyQ fusion proteins by combining a polyQ tract with a peptide sequence that specifically interacts with the target protein. These designer polyQ fusions effectively sequester target proteins into aggregates, diminishing their cellular availability and influencing their functionality¹⁷. In this study, we have applied a designer polyQ fusion protein to modulate nuclear factor kappa-B (NF-κB) signaling pathway by sequestering the transcription factor P65 into aggregates.

The NF-κB signaling is an important cellular pathway that regulates immune responses, inflammation, cell survival and apoptosis¹⁸. Regulation of the NF-κB pathway primarily occurs through the action of inhibitor of kappa-B (IκB) proteins, which then recruit NF-κB in the cytoplasm and prevent its activation^19,20. Upon stimulation by various signals, such as TNF-α, IL-1β and reactive oxygen species (ROS), the IκB kinase (IKK) complex phosphorylates IκB, leading to its degradation and the release of NF-κB^19,20,21,22. Subsequently, the P65 (RelA) subunit of NF-κB translocates into the nucleus, where it regulates the expression of target genes involved in inflammatory responses, immune functions, and cell survival^18,23,24. Excessive activation of the NF-κB signaling has been implicated in various diseases, such as inflammation^24,25, autoimmune disorder^26,27 and cancer^18,28. Therefore, maintaining the precise control of this pathway is crucial for regulating cellular responses and averting pathological outcomes²⁹.

Our previous study demonstrated that the designer polyQ fusion proteins, polyQ-IRF and polyQ-PMI, can specifically bind to and effectively sequester USP7/HDM2 into aggregates, leading to depleting the availabilities of USP7 and/or HDM2 in A549 cells and enhancing the P53 functionality in the USP7-HDM2-P53 axis¹⁷. This polyQ-fusion strategy is feasible to modulate the P53 stability and functionality, furnishing a therapeutic potential for cancers. Based on the polyQ-fusion technology, we have engineered a polyQ fusion protein to modulate the NF-κB signaling pathway by directly sequestering cellular P65 into aggregates or inclusions. This study provides a novel therapeutic approach for treating inflammation and autoimmune diseases, which are closely associated with excessive P65 activation.

Design and construction of the PolyQ fusion protein targeting P65 directly

It was reported that the deubiquitinating enzyme USP7 can directly bind to the P65 subunit of NF-κB via its LDEL motif (sequence: LDKALDELMDGDIIVFQK) within the UbL2 domain and deubiquitinate the P65 protein³⁰. We thus designed a polyQ fusion protein by combining the N-terminal fragment of ataxin-7 (Atx7)^12,17 with the LDEL peptide sequence, referred to as Atx7_93Q-N172-LDEL (Fig. 1A). This fusion protein was designed and constructed aiming to modulate the NF-κB signaling pathway by sequestering P65 directly.

We firstly characterized the interaction between polyQ-LDEL and P65 by using the Atx7_10Q-N172 template but without aggregation propensity¹². HEK 293T cells were co-transfected with FLAG-tagged Atx7_10Q-N172 or Atx7_10Q-N172-LDEL and HA-tagged P65, and the cell lysates were subjected to co-IP experiment. The data showed that Atx7_10Q-N172-LDEL could precipitate P65 remarkably compared to Atx7_10Q-N172 (Fig. 1B), indicating that Atx7_10Q-N172-LDEL specifically interacts with P65 via its fused LDEL peptide sequence. As an inference, our designed polyQ fusion protein Atx7_93Q-N172-LDEL can also interacts with cellular P65 in its aggregated form.

PolyQ-LDEL fusion sequesters P65 into aggregates or inclusions

We performed S/P fractionation to characterize whether the polyQ-LDEL fusion could sequester endogenous P65 protein. The results showed that over-expression of Atx7_93Q-N172-LDEL in HEK 293T cells significantly reduced the protein level of endogenous P65 in the supernatant fraction, while remarkably increased its accumulation in the pellet (Fig. 2A). We also assessed sequestration of exogenous P65 by this polyQ-LDEL fusion and observed a notable increase of the P65 protein sequestered into the pellet fraction (Fig. S1). We then used immunofluorescence imaging to visualize the sequestration and co-localization in HEK 293T cells. While Atx7_93Q-N172 formed inclusion bodies in the cytoplasm, it was not competent to sequester endogenous P65 into the inclusions. Conversely, the cytoplasmic inclusions formed by Atx7_93Q-N172-LDEL exhibited clear co-localization with the endogenous P65 protein (Fig. 2B). These results indicate that the polyQ-LDEL fusion can effectively sequester either endogenous or exogenous P65 into cytoplasmic inclusions in cells and substantially diminish the P65 solubility. This specific sequestration may thereby reduce the solubility and availability of the target protein, potentially interfering with its biological function in the cellular milieu.

PolyQ-LDEL fusion affects nuclear translocation of P65 in cells

To verify whether the sequestration of P65 into cytoplasmic aggregates or inclusions affects its nuclear translocation, we over-expressed FLAG-tagged Atx7_93Q-N172 or Atx7_93Q-N172-LDEL in HEK 293T cells, followed by treating with the TNF-α protein to induce P65 translocation into the nucleus. Immunofluorescence imaging revealed that both the free and LDEL-fused species could form cytoplasmic inclusions in HEK 293T cells. Over-expression of Atx7_93Q-N172-LDEL effectively sequestered endogenous P65 into cytoplasmic inclusions, preventing its nuclear translocation even upon TNF-α stimulation (Fig. 3A), but the free Atx7_93Q-N172 form did not affect the nuclear translocation of P65.

Nuclear/cytoplasmic fractionation exhibited a significant reduction in the nuclear P65 content upon TNF-α treatment, after over-expression of Atx7_93Q-N172-LDEL compared to the free Atx7_93Q-N172 group (Fig. 3B). These results demonstrate that sequestration of endogenous P65 by the polyQ-LDEL fusion significantly affects the translocation of P65 into the nucleus.

PolyQ-LDEL fusion modulates NF-κB signaling by sequestering P65 into aggregates

The transcriptional factor P65 enters the nucleus in response to external stimuli, such as TNF-α, then P65 binds to the NF-κB response elements for regulating inflammation and immune responses within cells^18,31. We next performed a dual-luciferase reporter assay by using an NF-κB-FL reporter plasmid (Fig. 4A) to investigate the effects of P65 sequestration by polyQ-LDEL on the NF-κB pathway³². The results showed that transfection of NF-κB-FL significantly increased the luciferase activity (FLuc/RLuc) (Fig. 4B), whether Atx7_93Q-N172 or its LDEL-fused form is present or not, suggesting that the reporter system is functional and suitable for NF-κB signaling assay in HEK 293T cells. We then treated the transfected cells with TNF-α to activate NF-κB signaling. As usual, transfection of NF-κB-FL remarkably increased the luciferase activity (Fig. 4C, column 2). However, co-transfection of Atx7_93Q-N172-LDEL significantly reduced the luciferase activity (Fig. 4C, column 4), whereas that of Atx7_93Q-N172 had little effect only. As a contrast, Atx7_10Q-N172 is not an aggregation-prone template, its fusion with the LDEL sequence specifically interacts with P65 (Fig. 1B). We also over-expressed the Atx7_10Q-N172-fused forms in HEK293T cells and observed that neither Atx7_10Q-N172 nor Atx7_10Q-N172-LDEL affected the luciferase activity (Fig. S2), indicating that the simple LDEL-P65 binding without aggregation and sequestration does not influence NF-κB signaling activity. Together, these findings suggest that polyQ-LDEL can suppress NF-κB signaling possibly by sequestering P65 into aggregates and impeding its nuclear translocation.

PolyQ-LDEL fusion down-regulates expression of the P65-regulated genes

When cells are stimulated by TNF-α, it may bind to TNF-α receptors on the cell surface and activate the NF-κB pathway¹⁸. Once NF-κB signaling is activated, P65/P50 may translocate into nucleus and binds to the DNA elements, which facilitates the transcription of a series of inflammation-related genes, including TNF-α, IL-6, IL-1β, and COX-2³³. Thus, we examined the expression of two NF-κB downstream genes, TNF-α and IL-6, which might be influenced by polyQ-LDEL. The results showed that, without TNF-α treatment, over-expression of either the free or LDEL-fused form did not affect the expression levels of TNF-α (Fig. 5A) and IL-6 (Fig. 5B) in HEK 293T cells. However, upon TNF-α treatment, the protein levels of TNF-α (Fig. 5A, column 4) and IL-6 (Fig. 5B, column 4) increased remarkably. Over-expression of Atx7_93Q-N172-LDEL significantly reduced the protein levels of TNF-α (Fig. 5A, column 6) and IL-6 (Fig. 5B, column 6), whereas that of Atx7_93Q-N172 exhibited little effect on their expression. These findings suggest that the polyQ-LDEL fusion down-regulates the expression of inflammatory factors in response to TNF-α activation.

P65 (RelA) is a critical subunit of the NF-κB transcription factor family³⁴ which regulates the expression of numerous cytokines associated with inflammation and immunity¹⁸. Dysregulation of the NF-κB pathway has been implicated in the pathologies of inflammation and autoimmune disorders³⁵. Thus, the NF-κB pathway, particularly involving P65, becomes a significant target for therapeutic intervention.

Our previous studies established a polyQ fusion strategy to modulate the biological processes and thereby enable therapeutic application¹⁷. A polyQ fusion protein may be constructed by combination of a polyQ tract and a peptide sequence that specifically binds to the target protein. The LDEL motif within the UBL2 domain of USP7 mediates the interaction between USP7 and P65³⁰. Based on the interacting motif, we have engineered a polyQ fusion protein that can specifically sequester P65 into inclusions, affect its nuclear translocation, reduce the P65 abundancy in the nucleus, and thereby prevent activation of the NF-κB signaling. Thus, this polyQ fusion technology may provide an alternative approach for modulating the NF-κB signaling pathway, highlighting its potential for therapeutic application in inflammation and autoimmune disorders caused by the excessive P65 activity.

However, it should be mentioned that polyQ-expanded protein aggregation has been shown to induce impairments of some cellular pathways, such as ubiquitin-proteasome pathway (UPS), at least in cell overexpression systems^36,37. As known, UPS dysfunction can alter expression of the protein components including IκB and IKKβ^38,39 both of which play key roles in NF-κB signaling. Thus, while applying the polyQ fusion technology in future studies, it is important to verify the expression and degradation of these protein components to rule out any potential UPS-related effects on the NF-κB signaling.

In some aspects, the sequestration effect of polyQ fusion is similar to that of the small-molecule inhibitors and interference RNAs^40,41. Some small-molecule inhibitors, such as parthenolide and BAY 11-7082, can inhibit the phosphorylation of IκBα and the subsequent nuclear translocation of P65^42,43; while silencing P65 by siRNA or shRNA can directly reduce its expression and thereby inhibit NF-κB signaling^44,45. However, compared to the off-target effects of small-molecule inhibitors and siRNAs, this approach by sequestering P65 into inclusions may offer a higher specificity for directly targeting the transcription factor. Further research will focus on the sequestration effects of polyQ fusions in animal models and examine the feasibility of this technology in therapeutic application to inflammation and autoimmune diseases.

In summary, a polyQ fusion protein has been developed to modulate the NF-κB signaling pathway by sequestering the transcription factor P65 into aggregates. This sequestration effect impedes the nuclear entry process of P65 and consequently suppresses NF-κB signaling, leading to down-regulation of the downstream genes, especially TNF-α and IL-6. This technical approach is potentially applicable to therapeutic intervention of inflammation and autoimmune diseases.

Plasmids, antibodies, and reagents

The DNA sequences encoding Atx7_10Q-N172 and Atx7_93Q-N172 were cloned in the FLAG-pcDNA3.1 vector¹². The cDNAs encoding Atx7_10Q-N172-LDEL and Atx7_93Q-N172-LDEL were inserted into the FLAG-pcDNA3.1 vector via EcoR I/Xba I sites. The DNA sequences for P65 were cloned into the HA-pcDNA3.1 vector using BamH I/Xba I sites. All the DNA sequences were verified by DNA sequencing (Table S1), and the PCR primers used in this study were listed in nucleotide sequences (Table S2).

The anti-FLAG antibody was purchased from Sigma, and the anti-P65 was purchased from Santa Cruz. Anti-TNF-α, anti-IL-6, and anti-GAPDH antibodies were purchased from Proteintech, anti-H3 was from Cell Signaling Technology, and anti-HA was from Sigma-Aldrich Corporation. All secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (Table S3). Recombinant human soluble TNF-α protein was purchased from Beyotime Corporation.

Cell culture and transfection

HEK 293T cells, sourced from the Cell Bank of the Chinese Academy of Sciences (Shanghai), were cultured in Dulbecco’s Modified Eagle’s medium (HyClone) supplemented with 10% fetal bovine serum (Gibco) and penicillin-streptomycin. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Plasmid transfections were carried out using PolyJet reagent (SignaGen) according to the manufacturer’s instructions.

Co-immunoprecipitation

Co-immunoprecipitation (co-IP) was performed as previously described⁴⁶. Approximately 48 h post-transfection, the HEK 293T cells were harvested and lysed with a RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and protease inhibitor cocktail (Roche)). After cell lysis, the lysates were centrifuged at 13,000 rpm for 20 min at 4 °C. Following centrifugation, the supernatants were collected and incubated with anti-FLAG agarose beads (Abmart) overnight at 4 °C. The beads were then washed three times with the RIPA buffer before boiling in 40 µL of 2x loading buffer (4% SDS). The protein eluates from the beads were analyzed by immunoblotting.

Supernatant/pellet fractionation

The supernatant/pellet (S/P) fractionation experiment was performed according to the previously established protocol⁴⁷. Approximately 48 h post-transfection, the HEK 293T cells were harvested and lysed in 90 µL of the RIPA buffer on ice for 30 min. The lysates were centrifuged at 13,000 rpm for 15 min at 4 °C. Subsequently, 80 µL of the supernatant was mixed with 20 µL of 4x loading buffer (8% SDS). The pellet was thoroughly washed thrice with the RIPA buffer at 4 °C and re-suspended in 40 µL of 4x loading buffer (8% SDS). Equal volumes of both supernatant and pellet fractions were analyzed by SDS-PAGE and Western blotting.

Nuclear and cytoplasmic protein extraction and Western blotting

The nuclear and cytoplasmic proteins were obtained separately from HEK 293 cells by using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotech., Shanghai, P0028) following the manufacturer’s instructions. For protein analysis, the cell lysates were subjected to SDS-PAGE and then transferred onto PVDF membranes (Millipore). Before antibody incubation, the blots were sectioned as required and blocked with 5% skim milk in a PBS buffer (10 mM Na₂HPO₄, 2 mM KH₂PO₄,137 mM NaCl, 2.7mM KCl, ) containing 0.1% Tween-20 for 1 h at room temperature. Specific protein detection was accomplished using the appropriate primary and secondary antibodies, followed by visualization using an ECL detection kit (Thermo Fisher Scientific). Quantitative analysis of the protein bands was carried out by using the integral grayscale values recorded with Sage Capture V3.2.2 software (Sage Science, http://www.sagecreation.com.cn/en/).

Dual-luciferase reporter assay

The pGL3-NF-κB-FL reporter plasmid was purchased from Bioscien (Shanghai). The dual-luciferase reporter assay was conducted using a Luciferase Reporter Assay kit (YEASEN, Shanghai) following the manufacturer’s instructions⁴⁸. Briefly, HEK 293T cells were co-transfected with pGL3-NF-κB-FL and FLAG-tagged Atx7_93Q-N172 or Atx7_93Q-N172-LDEL, as well as the pGL3 vector harboring a Renilla luciferase gene for recording control. To verify the transactivation function of endogenous P65, the recombinant human soluble protein TNF-α (Beyotime, Shanghai) was included in the cell culture (10 ng/ml) to activate the NF-κB signaling pathway. After treated for 40 min, the cells were collected and the luciferase activity was measured. The relative luciferase activity was calculated as the ratio of Firefly luciferase over Renilla luciferase activities (FLuc/RLuc).

Immunofluorescence imaging

HEK 293T cells cultured on glass coverslips were processed approximately 48 h post-transfection or for the specified culture duration. Initially, the cells were washed with a PBS buffer (10 mM Na₂HPO₄, 1.8 mM KH₂PO4, 140 mM NaCl, 2.7 mM KCl, pH 7.3) and fixed with 4% paraformaldehyde for 15 min. After three washes with the PBS buffer, the cells were permeabilized with 0.1% Triton X-100 and then blocked in a solution containing 5% bovine serum albumin (BSA) in the PBS buffer for 1 h at room temperature. Subsequently, the specific primary antibodies were applied, and the cells were incubated overnight at 4 °C. Following primary antibody incubation, the cells were washed with the PBS buffer and incubated with either fluorescein isothiocyanate (FITC)-conjugated or tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). The nuclei were stained with Hoechst 33,342 (Thermo Fisher Scientific). Imaging was performed using a Leica TCS SP8 WLL confocal microscope (Leica Microsystems), and the images were captured using LAS X V3.5 software (Leica, https://www.leica-microsystems.com/) and presented without modification. For co-localization analysis, the fluorescence intensities from the respective channels were quantified using ImageJ V1.53 software (NIH, https://imagej.net/) and visualized as plots generated with GraphPad Prism V8.0.2 (GraphPad Software, https://www.graphpad.com/).

Quantification and statistical analysis

Quantitative analysis of the Western blots was conducted using ImageJ V1.53 software (NIH, https://imagej.net/). The integral grayscale values of the indicated protein bands were normalized against those of the respective controls. Data were derived from three independent experiments and are presented as Mean ± SD. Statistical analyses were conducted using GraphPad Prism 8.0.2 (GraphPad software, https://www.graphpad.com/). One-way ANOVA algorithm was used to compare differences among three or more experimental groups, followed by Tukey’s multiple comparisons test to assess significance between group pairs. Statistical significance was defined as p < 0.05, where the p-values were indicated in graphs as ∗ (p < 0.05), ∗∗ (p < 0.01), ∗∗∗ (p < 0.001), **** (p < 0.0001) or N.S. (no significance).

Atx7:

Ataxin-7

co-IP:

Co-immunoprecipitation

HDM2:

Human double minute 2 homolog

IκB:

Inhibitor of kappa-B

IKK:

IκB kinase

LDEL:

A P65-binding motif from the UbL2 domain of USP7

NF-κB:

Nuclear factor kappa-B

PMI:

P53/MDM2 inhibitor

polyQ:

Polyglutamine

ROS:

Reactive oxygen species

S/P:

Supernatant/pellet

UPS:

Ubiquitin-proteasome pathway

USP7:

Ubiquitin-specific protease 7

vIRF1:

Viral interferon regulatory factor 1

1. Housmans, J. A. J., Wu, G., Schymkowitz, J. & Rousseau, F. A guide to studying protein aggregation. FEBS J. 290, 554–583. https://doi.org/10.1111/febs.16312 (2023).

   Article  CAS  Google Scholar 

2. Chiti, F. & Dobson, C. M. Protein misfolding amyloid formation, and human disease: a summary of progress over the last decade. Annu. Rev. Biochem. 86, 27–68. https://doi.org/10.1146/annurev-biochem-061516-045115 (2017).

   Article  CAS  Google Scholar 

3. Lieberman, A. P., Shakkottai, V. G. & Albin, R. L. Polyglutamine repeats in neurodegenerative diseases. Annu. Rev. Pathol. 14, 1–27. https://doi.org/10.1146/annurev-pathmechdis-012418-012857 (2019).

   Article  CAS  Google Scholar 

4. Arrasate, M., Mitra, S., Schweitzer, E. S., Segal, M. R. & Finkbeiner, S. Inclusion body formation reduces levels of mutant Huntingtin and the risk of neuronal death. Nature 431, 805–810. https://doi.org/10.1038/nature02998 (2004).

   Article  ADS  CAS  Google Scholar 

5. Mahul-Mellier, A. L. et al. The process of lewy body formation, rather than simply alpha-synuclein fibrillization, is one of the major drivers of neurodegeneration. Proc. Natl. Acad. Sci. U S A. 117, 4971–4982. https://doi.org/10.1073/pnas.1913904117 (2020).

   Article  ADS  CAS  Google Scholar 

6. Yang, H. & Hu, H. Y. Sequestration of cellular interacting partners by protein aggregates: implication in a loss-of-function pathology. FEBS J. 283, 3705–3717. https://doi.org/10.1111/febs.13722 (2016).

   Article  CAS  Google Scholar 

7. Jobe, F. et al. Respiratory syncytial virus sequesters NF-κB subunit p65 to cytoplasmic inclusion bodies to inhibit innate immune signaling. J. Virol. 94, 563. https://doi.org/10.1128/jvi.01380-20 (2020).

8. Cortes, C. J. et al. Polyglutamine-expanded androgen receptor interferes with TFEB to elicit autophagy defects in SBMA. Nat. Neurosci. 17, 1180–1189. https://doi.org/10.1038/nn.3787 (2014).

   Article  CAS  Google Scholar 

9. Yang, J. et al. A prion-like domain of TFEB mediates the co-aggregation of TFEB and mHTT. Autophagy 19, 544–550. https://doi.org/10.1080/15548627.2022.2083857 (2023).

   Article  CAS  Google Scholar 

10. Yang, H. et al. PolyQ-expanded Huntingtin and ataxin-3 sequester ubiquitin adaptors hHR23B and UBQLN2 into aggregates via conjugated ubiquitin. FASEB J. 32, 2923–2933. https://doi.org/10.1096/fj.201700801RR (2018).

    Article  CAS  Google Scholar 

11. Farrawell, N. E. et al. Ubiquitin Homeostasis Is Disrupted in TDP-43 and FUS Cell Models of ALS. iScience 23, 101700, (2020). https://doi.org/10.1016/j.isci.2020.101700

12. Yue, H. W., Hong, J. Y., Zhang, S. X., Jiang, L. L. & Hu, H. Y. PolyQ-expanded proteins impair cellular proteostasis of ataxin-3 through sequestering the co-chaperone HSJ1 into aggregates. Sci. Rep. 11, 7815. https://doi.org/10.1038/s41598-021-87382-w (2021).

    Article  ADS  CAS  Google Scholar 

13. Park, S. H. et al. PolyQ proteins interfere with nuclear degradation of cytosolic proteins by sequestering the Sis1p chaperone. Cell 154, 134–145. https://doi.org/10.1016/j.cell.2013.06.003 (2013).

    Article  CAS  Google Scholar 

14. Yu, A. et al. Tau protein aggregates inhibit the protein-folding and vesicular trafficking arms of the cellular proteostasis network. J. Biol. Chem. 294, 7917–7930. https://doi.org/10.1074/jbc.RA119.007527 (2019).

    Article  CAS  Google Scholar 

15. Hu, H. Y. & Liu, Y. J. Sequestration of cellular native factors by biomolecular assemblies: physiological or pathological? Biochim. Biophys. Acta Mol. Cell. Res. 1869, 119360. https://doi.org/10.1016/j.bbamcr.2022.119360 (2022).

    Article  CAS  Google Scholar 

16. Orr, H. T. & Zoghbi, H. Y. Trinucleotide repeat disorders. Annu. Rev. Neurosci. 30, 575–621. https://doi.org/10.1146/annurev.neuro.29.051605.113042 (2007).

    Article  CAS  Google Scholar 

17. Zhang, X. L. et al. Designer PolyQ fusion proteins sequester USP7/HDM2 for modulating P53 functionality. iScience 28, 112025. https://doi.org/10.1016/j.isci.2025.112025 (2025).

    Article  CAS  Google Scholar 

18. Hoesel, B. & Schmid, J. A. The complexity of NF-κB signaling in inflammation and cancer. Mol. Cancer. 12, 86. https://doi.org/10.1186/1476-4598-12-86 (2013).

    Article  CAS  Google Scholar 

19. Ghosh, S. & Baltimore, D. Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B. Nature 344, 678–682. https://doi.org/10.1038/344678a0 (1990).

    Article  ADS  CAS  Google Scholar 

20. Napetschnig, J. & Wu, H. Molecular basis of NF-kappaB signaling. Annu. Rev. Biophys. 42, 443–468. https://doi.org/10.1146/annurev-biophys-083012-130338 (2013).

    Article  CAS  Google Scholar 

21. Guo, Q. et al. NF-kappaB in biology and targeted therapy: new insights and translational implications. Signal. Transduct. Target. Ther. 9, 523. https://doi.org/10.1038/s41392-024-01757-9 (2024).

22. Jacobs, M. D. & Harrison, S. C. Structure of an IkappaBalpha/NF-kappaB complex. Cell 95, 749–758. https://doi.org/10.1016/s0092-8674(00)81698-0 (1998).

    Article  CAS  Google Scholar 

23. Vallabhapurapu, S. & Karin, M. Regulation and function of NF-kappaB transcription factors in the immune system. Annu. Rev. Immunol. 27, 693–733. https://doi.org/10.1146/annurev.immunol.021908.132641 (2009).

    Article  CAS  Google Scholar 

24. Liu, T., Zhang, L., Joo, D. & Sun, S. C. NF-kappaB signaling in inflammation. Signal. Transduct. Target. Ther. 2, 17023. https://doi.org/10.1038/sigtrans.2017.23 (2017).

    Article  Google Scholar 

25. Zheng, X. et al. FAM76B regulates NF-κB-mediated inflammatory pathway by influencing the translocation of hnRNPA2B1. eLife 12, e85659. https://doi.org/10.7554/eLife.85659 (2023).

    Article  ADS  Google Scholar 

26. Chen, G. et al. The NF-kappaB transcription factor c-Rel is required for Th17 effector cell development in experimental autoimmune encephalomyelitis. J. Immunol. 187, 4483–4491. https://doi.org/10.4049/jimmunol.1101757 (2011).

    Article  CAS  Google Scholar 

27. Campbell, I. K., Gerondakis, S., O’Donnell, K. & Wicks, I. P. Distinct roles for the NF-kappaB1 (p50) and c-Rel transcription factors in inflammatory arthritis. J. Clin. Invest. 105, 1799–1806. https://doi.org/10.1172/JCI8298 (2000).

    Article  CAS  Google Scholar 

28. Caruana, B. T. & Byrne, F. L. The NF-kappaB signalling pathway regulates GLUT6 expression in endometrial cancer. Cell. Signal. 73, 109688. https://doi.org/10.1016/j.cellsig.2020.109688 (2020).

    Article  CAS  Google Scholar 

29. Yu, H., Lin, L., Zhang, Z., Zhang, H. & Hu, H. Targeting NF-kappaB pathway for the therapy of diseases: mechanism and clinical study. Signal. Transduct. Target. Ther. 5, 209. https://doi.org/10.1038/s41392-020-00312-6 (2020).

    Article  CAS  Google Scholar 

30. Mitxitorena, I. et al. The deubiquitinase USP7 uses a distinct ubiquitin-like domain to deubiquitinate NF-kB subunits. J. Biol. Chem. 295, 11754–11763. https://doi.org/10.1074/jbc.RA120.014113 (2020).

    Article  CAS  Google Scholar 

31. Kempe, S., Kestler, H., Lasar, A. & Wirth, T. NF-kappaB controls the global pro-inflammatory response in endothelial cells: evidence for the regulation of a pro-atherogenic program. Nucleic Acids Res. 33, 5308–5319. https://doi.org/10.1093/nar/gki836 (2005).

    Article  CAS  Google Scholar 

32. Pessara, U. & Koch, N. Tumor necrosis factor alpha regulates expression of the major histocompatibility complex class II-associated invariant chain by binding of an NF-kappa B-like factor to a promoter element. Mol. Cell. Biol. 10, 4146–4154. https://doi.org/10.1128/mcb.10.8.4146-4154.1990 (1990).

    Article  CAS  Google Scholar 

33. Wang, S., Liu, Z., Wang, L. & Zhang, X. NF-kappaB signaling pathway, inflammation and colorectal cancer. Cell. Mol. Immunol. 6, 327–334. https://doi.org/10.1038/cmi.2009.43 (2009).

    Article  CAS  Google Scholar 

34. Ashburner, B. P., Westerheide, S. D. & Baldwin, A. S. Jr The p65 (RelA) subunit of NF-kappaB interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression. Mol. Cell. Biol. 21, 7065–7077. https://doi.org/10.1128/MCB.21.20.7065-7077.2001 (2001).

    Article  CAS  Google Scholar 

35. Giridharan, S. & Srinivasan, M. Mechanisms of NF-kappaB p65 and strategies for therapeutic manipulation. J. Inflamm. Res. 11, 407–419. https://doi.org/10.2147/JIR.S140188 (2018).

    Article  CAS  Google Scholar 

36. Bence, N. F., Sampat, R. M. & Kopito, R. R. Impairment of the ubiquitin-proteasome system by protein aggregation. Sci. (New York N Y). 292, 1552–1555. https://doi.org/10.1126/science.292.5521.1552 (2001).

    Article  ADS  CAS  Google Scholar 

37. Jana, N. R., Zemskov, E. A., Wang, G. & Nukina, N. Altered proteasomal function due to the expression of polyglutamine-expanded truncated N-terminal Huntingtin induces apoptosis by caspase activation through mitochondrial cytochrome c release. Hum. Mol. Genet. 10, 1049–1059. https://doi.org/10.1093/hmg/10.10.1049 (2001).

    Article  CAS  Google Scholar 

38. Scherer, D. C., Brockman, J. A., Chen, Z., Maniatis, T. & Ballard, D. W. Signal-induced degradation of I kappa B alpha requires site-specific ubiquitination. Proc. Natl. Acad. Sci. U S A. 92, 11259–11263. https://doi.org/10.1073/pnas.92.24.11259 (1995).

    Article  ADS  CAS  Google Scholar 

39. Carter, R. S., Pennington, K. N., Arrate, P., Oltz, E. M. & Ballard, D. W. Site-specific monoubiquitination of IkappaB kinase IKKbeta regulates its phosphorylation and persistent activation. J. Biol. Chem. 280, 43272–43279. https://doi.org/10.1074/jbc.M508656200 (2005).

    Article  CAS  Google Scholar 

40. Wang, J. Y. et al. PolyQ-expanded ataxin-2 aggregation impairs cellular processing-body homeostasis via sequestering the RNA helicase DDX6. J. Biol. Chem. 300, 107413. https://doi.org/10.1016/j.jbc.2024.107413 (2024).

    Article  CAS  Google Scholar 

41. Liu, Y. J., Wang, J. Y., Zhang, X. L., Jiang, L. L. & Hu, H. Y. Ataxin-2 sequesters raptor into aggregates and impairs cellular mTORC1 signaling. FEBS J. 291, 1795–1812. https://doi.org/10.1111/febs.17081 (2024).

    Article  CAS  Google Scholar 

42. Hehner, S. P., Hofmann, T. G., Droge, W. & Schmitz, M. L. The antiinflammatory sesquiterpene lactone parthenolide inhibits NF-kappa B by targeting the I kappa B kinase complex. J. Immunol. 163, 5617–5623 (1999).

    CAS  Google Scholar 

43. Lee, J., Rhee, M. H., Kim, E. & Cho, J. Y. BAY 11-7082 is a broad-spectrum inhibitor with anti-inflammatory activity against multiple targets. Mediators Inflamm. 2012, 416036. https://doi.org/10.1155/2012/416036 (2012).

44. Guo, J., Fu, Y. C. & Becerra, C. R. Dissecting role of regulatory factors in NF-kappaB pathway with SiRNA. Acta Pharmacol. Sin. 26, 780–788. https://doi.org/10.1111/j.1745-7254.2005.00140.x (2005).

    Article  CAS  Google Scholar 

45. Yang, Q. et al. AAV-based ShRNA Silencing of NF-kappaB ameliorates muscle pathologies in Mdx mice. Gene Ther. 19, 1196–1204. https://doi.org/10.1038/gt.2011.207 (2012).

    Article  CAS  Google Scholar 

46. Liu, Y. J., Wang, J. Y., Zhang, X. L., Jiang, L. L. & Hu, H. Y. Ataxin-2 sequesters raptor into aggregates and impairs cellular mTORC1 signaling. FEBS J. https://doi.org/10.1111/febs.17081 (2024).

    Article  Google Scholar 

47. Jiang, L. L., Guan, W. L., Wang, J. Y., Zhang, S. X. & Hu, H. Y. RNA-assisted sequestration of RNA-binding proteins by cytoplasmic inclusions of the C-terminal 35-kDa fragment of TDP-43. J. Cell. Sci. 135, jcs259380. https://doi.org/10.1242/jcs.259380 (2022).

    Article  CAS  Google Scholar 

48. Solberg, N. & Krauss, S. Luciferase assay to study the activity of a cloned promoter DNA fragment. Methods Mol. Biol. 977, 65–78. https://doi.org/10.1007/978-1-62703-284-1_6 (2013).

    Article  CAS  Google Scholar 

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Authors and Affiliations

1. Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, People’s Republic of China

   Xiang-Le Zhang, Lei-Lei Jiang, Heng-Tong Duan & Hong-Yu Hu

2. First Affiliated Hospital, Zhejiang University School of Medicine, and Liangzhu Laboratory of Zhejiang University, Hangzhou, Zhejiang 310000; Institute of Hematology, Zhejiang University, Hangzhou, Zhejiang 310000, People’s Republic of China

   Xiang-Le Zhang

3. University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China

   Xiang-Le Zhang & Heng-Tong Duan

Contributions

H. Y. H. and K. L. Z. conceived and designed the investigation. X. L. Z, L. L. J. and H. T. D. performed the experiments and statistical analysis. L. L. J. analysed and stores the experimental data. X. L. Z. and H. Y. H. wrote and edited the manuscript. All authors reviewed and gave final approval to the manuscript.

Corresponding author

Correspondence to Hong-Yu Hu.

Competing interests

The authors declare no competing interests.

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