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.

Design of the polyQ-LDEL fusion and examination of its interaction with P65. (A) Schematic diagram of a designer polyQ fusion protein, Atx7_93Q-N172-LDEL. Atx7_93Q-N172, the N-terminal 172-residue fragment of Atx7_93Q. LDEL, a homologous peptide sequence, LDKALDELMDGDIIVFQK, from the UbL2 domain of USP7. (B) Examining the interaction between Atx7_10Q-N172-LDEL and P65 by co-IP assay. HEK 293T cells were co-transfected with FLAG-tagged Atx7_10Q-N172 or Atx7_10Q-N172-LDEL and HA-P65. At 48 h post-transfection, the cell lysates were subjected to co-IP assay using anti-FLAG agarose beads. Vec., vector; Atx7_10Q-N172, the N-terminal 172-residue fragment of Atx7_10Q; LDEL, Atx7_10Q-N172-LDEL, a polyQ fusion combined Atx7_10Q-N172 with LDEL.

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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.

The polyQ-LDEL fusion sequesters endogenous P65 into aggregates. (A) S/P fractionation for characterizing the distribution of endogenous P65 in supernatant and pellet affected by the polyQ-LDEL fusion. HEK 293T cells were transfected with FLAG-tagged Atx7_93Q-N172 or Atx7_93Q-N172-LDEL, followed by culturing for 48 h. The P65 levels in the supernatant and pellet fractions were then detected and analyzed. Sup., supernatant; Pel., pellet. Vec., vector; Atx7_93Q-N172, the N-terminal 172-residue fragment of Atx7_93Q; LDEL, Atx7_93Q-N172-LDEL, a polyQ fusion combined Atx7_93Q-N172 with LDEL. Data are shown as Mean ± SD (n = 3). *, p < 0.05; **, p < 0.01; N.S., no significance. (B) Immunofluorescence imaging for characterizing the co-localization of endogenous P65 with the inclusions formed by the polyQ-LDEL fusion. HEK 293T cells were transfected with FLAG-tagged Atx7_93Q-N172 or Atx7_93Q-N172-LDEL, and after cultured for 48 h, the cells were fixed and immunostained with anti-FLAG (green) and anti-P65 (red) antibodies. Nuclei were stained with Hoechst (blue). Scale bar = 10 μm. Bottom: co-localization analysis of the fluorescence signals for the distance represented by white lines.

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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.

The polyQ-LDEL fusion affects nuclear translocation of P65 in cells. (A) Immunofluorescence imaging for assessing the nuclear translocation of P65 affected by the polyQ-LDEL fusion. HEK 293T cells were transfected with FLAG-tagged Atx7_93Q-N172 or Atx7_93Q-N172-LDEL. After cultured for 48 h, the cells were treated with TNF-α (10 ng/mL) for 40 min. Subsequently, the cells were fixed and immunostained with anti-FLAG (green) and anti-P65 (red) antibodies. Nuclei were stained with Hoechst (blue). TNF-α, a cytokine for activating NF-κB signaling. Vec., vector; Atx7_93Q-N172, the N-terminal 172-residue fragment of Atx7_93Q; LDEL, Atx7_93Q-N172-LDEL. Scale bar = 10 μm. (B) Nuclear/cytoplasmic fractionation for assessing the nuclear translocation of P65 affected by the polyQ-LDEL fusion. HEK 293T cells were transfected with FLAG-tagged Atx7_93Q-N172 or Atx7_93Q-N172-LDEL. After cultured for 48 h, the cells were treated with TNF-α (10 ng/mL) for 40 min. Subsequently, the cells were collected and subjected to nuclear/cytoplasmic fractionation and detection of the P65 levels in the cytoplasm and nucleus. H3, histone 3. Data are presented as Mean ± SD (n = 3). *, p < 0.05; N.S., no significance.

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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.

The polyQ-LDEL fusion modulates the NF-κB signaling activity. (A) Schematic diagram of the NF-κB-FL reporter. The NF-κB response element harbors a DNA sequence of tandem GGGAATTTCC, which drives the expression of the Firefly luciferase gene through the mini-promoter on the plasmid. minP, mini-promoter. (B, C) Effects of the polyQ-LDEL fusion on the luciferase activities. The pGL3-NF-κB-FL plasmid as well as the pGL3 vector harboring a Renilla luciferase gene was co-transfected with FLAG-tagged Atx7_93Q-N172 or Atx7_93Q-N172-LDEL into HEK 293T cells. After cultured for 48 h, the cells were treated without (B) or with (C) TNF-α (10 ng/mL) for 40 min, and then the cell lysates were subjected to luciferase activity assay (FLuc/RLuc). The empty vector was set as a control. Insert: immunoblots showing the expression levels of Atx7_93Q-N172 and its LDEL-fused form. Vec., vector; Atx7_93Q-N172, the N-terminal 172-residue fragment of Atx7_93Q; LDEL, Atx7_93Q-N172-LDEL. Data are presented as Mean ± SD (n = 3). *, p < 0.05; ***, p < 0.001; ****, p < 0.001; N.S., no significance.

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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.

Effects of the polyQ-LDEL fusion on the downstream gene expression. TNF-α expression. (B) IL-6 expression. HEK 293T cells were transfected with FLAG-tagged Atx7_93Q-N172 or Atx7_93Q-N172-LDEL. After cultured for 30 h, the cells were treated with TNFα (10 ng/mL) for 40 min. The cell lysates were then subjected to immunoblotting with an anti-TNF-α or anti-IL6 antibody to assess the protein level of TNF-α (A) or IL-6 (B). Vec., vector; Atx7_93Q-N172, the N-terminal 172-residue fragment of Atx7_93Q; LDEL, Atx7_93Q-N172-LDEL. Data are presented as Mean ± SD (n = 3). *, p < 0.05; **, p < 0.01; N.S., no significance.

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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

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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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