Effects and mechanisms of FBXO31 on Taxol chemoresistance in esophageal squamous cell carcinoma.

Taxol is commonly used chemotherapy regimen for esophageal squamous cell carcinoma (ESCC). Study of the underlying mechanisms of Taxol chemoresistance provides better understanding of esophageal cancer treatment and may provide a rational molecular target for diagnosis and intervention. Here we showed FBXO31, which was reported to be highly expressed in ESCC and significantly associated with poor prognosis, could regulate ESCC chemosensitivity to Taxol. Silencing of FBXO31 in ESCC cells sensitized cells to Taxol treatment, evidenced by FACS analysis and TUNEL assay, showing as an increased apoptotic population in FBXO31-knockdown cells compared to the control cells. The mass spectrometry data and coimmunoprecipitation results showed FBXO31 could bind with cofilin-1. Cofilin-1 knockdown in FBXO31-overexpression cells reversed FBXO31-induced suppression of cell apoptosis, suggesting FBXO31-mediated Taxol chemoresistance is associated with cofilin-1. Furthermore, in vivo experiments confirmed that knockdown of FBXO31 sensitized ESCC to Taxol treatment. This finding substantiated a pivotal role of FBOX31 in ESCC chemoresistance, indicating that FBXO31 may be a potential indicator or target for drug resistance in ESCC.

Genome-wide screens uncover KDM2B as a modifier of protein binding to heparan sulfate.

Heparan sulfate (HS) proteoglycans bind extracellular proteins that participate in cell signaling, attachment and endocytosis. These interactions depend on the arrangement of sulfated sugars in the HS chains generated by well-characterized biosynthetic enzymes; however, the regulation of these enzymes is largely unknown. We conducted genome-wide CRISPR-Cas9 screens with a small-molecule ligand that binds to HS. Screening of A375 melanoma cells uncovered additional genes and pathways impacting HS formation. The top hit was the epigenetic factor KDM2B, a histone demethylase. KDM2B inactivation suppressed multiple HS sulfotransferases and upregulated the sulfatase SULF1. These changes differentially affected the interaction of HS-binding proteins. KDM2B-deficient cells displayed decreased growth rates, which was rescued by SULF1 inactivation. In addition, KDM2B deficiency altered the expression of many extracellular matrix genes. Thus, KDM2B controls proliferation of A375 cells through the regulation of HS structure and serves as a master regulator of the extracellular matrix.

The p105 NF-ĸB precursor is a pseudo substrate of the ubiquitin ligase FBXO7, and its binding to the ligase stabilizes it and results in stimulated cell proliferation.

The NF-κB transcription factor is involved in inflammation and cell proliferation, survival, and transformation. It is a heterodimer made of p50 or p52 and a member of the Rel family of proteins. p50 and p52 are derived from limited ubiquitin- and proteasome-mediated proteolytic processing of the larger precursors p105 and p100, respectively. Both precursors can be either processed or completely degraded by the ubiquitin-proteasome system. Previous work in our laboratory identified KPC1 as a ubiquitin ligase that mediates processing of p105 to the p50 subunit. Overexpression of the ligase leads to increased level of p50 with a resultant marked tumor-suppressive effect. In the present study, we identify FBXO7, a known ubiquitin ligase that binds to p105 and ubiquitinates it, but surprisingly, leads to its accumulation and to that of p65 - the Rel partner of p50 - and to increased cell proliferation. Importantly, a ΔF-Box mutant of FBXO7 which is inactive has similar effects on accumulation of p105 and cell proliferation, strongly suggesting that p105 is a pseudo substrate of FBXO7.

Chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing the mitochondrial kinase PINK1.

Mitochondrial quality control is mediated by the PTEN-induced kinase 1 (PINK1), a cytoprotective protein that is dysregulated in inflammatory lung injury and neurodegenerative diseases. Here, we show that a ubiquitin E3 ligase receptor component, FBXO7, targets PINK1 for its cellular disposal. FBXO7, by mediating PINK1 ubiquitylation and degradation, was sufficient to induce mitochondrial injury and inflammation in experimental pneumonia. A computational simulation-based screen led to the identification of a small molecule, BC1464, which abrogated FBXO7 and PINK1 association, leading to increased cellular PINK1 concentrations and activities, and limiting mitochondrial damage. BC1464 exerted antiinflammatory activity in human tissue explants and murine lung inflammation models. Furthermore, BC1464 conferred neuroprotection in primary cortical neurons, human neuroblastoma cells, and patient-derived cells in several culture models of Parkinson's disease. The data highlight a unique opportunity to use small molecule antagonists that disrupt PINK1 interaction with the ubiquitin apparatus to enhance mitochondrial quality, limit inflammatory injury, and maintain neuronal viability.

F-box proteins and cancer: an update from functional and regulatory mechanism to therapeutic clinical prospects.

E3 ubiquitin ligases play a critical role in cellular mechanisms and cancer progression. F-box protein is the core component of the SKP1-cullin 1-F-box (SCF)-type E3 ubiquitin ligase and directly binds to substrates by various specific domains. According to the specific domains, F-box proteins are further classified into three sub-families: 1) F-box with leucine rich amino acid repeats (FBXL); 2) F-box with WD 40 amino acid repeats (FBXW); 3) F-box only with uncharacterized domains (FBXO). Here, we summarize the substrates of F-box proteins, discuss the important molecular mechanism and emerging role of F-box proteins especially from the perspective of cancer development and progression. These findings will shed new light on malignant tumor progression mechanisms, and suggest the potential role of F-box proteins as cancer biomarkers and therapeutic targets for future cancer treatment.

Knockdown of FBXO22 inhibits melanoma cell migration, invasion and angiogenesis via the HIF-1α/VEGF pathway.

F-box proteins, a type of substrate-recognition complexes consisting of SKP1-cullin 1-F-box protein (SCF) E3 ligase, can critically affect many cellular processes because of the ubiquitylation and subsequent degradation of target proteins. This study investigated the effect of FBXO22 on melanoma angiogenesis, migration, and invasion. Results showed that FBXO22 staining intensity was increased in malignant melanoma (MM) compared with that in skin tissue (P˂0.001). The percentage of high FBXO22 expression in MM (74.3%) was markedly higher than that in paracancerous and skin tissues (0%) (P˂0.001). FBXO22 was also overexpressed in MM tissues compared with that in normal skin tissues. FBXO22 knockdown in vitro inhibited MM cell migration, invasion, and angiogenesis (P < 0.001). In vivo studies confirmed that using nude mice with knocked down FBXO22 reduced the formation of blood vessels and decreased the positive rate of CD31 (P < 0.05). HIF-1α expression varied with FBXO22, indicating that FBXO22 regulated the expression of HIF-1α and VEGFA and that FBXO22 was a regulator of HIF-1α and VEGF for the control of tumor angiogenesis. In conclusion, FBXO22 promoted the migration and invasion of tumor cells and melanoma angiogenesis via HIF-1α upregulation. This study demonstrated that FBXO22 knockdown suppressed tumor progression and metastasis, suggesting that FBXO22 might be developed as a novel target for treating patients with MM.

The Identification of Potential Therapeutic Targets for Cutaneous Squamous Cell Carcinoma.

We performed a small interfering RNA screen to identify targets for cutaneous squamous cell carcinoma (cSCC) therapy in the ubiquitin/ubiquitin-like system. We provide evidence for selective anti-cSCC activity of knockdown of the E3 ubiquitin ligase MARCH4, the ATPase p97/VCP, the deubiquitinating enzyme USP8, the cullin-RING ligase (CRL) 4 substrate receptor CDT2/DTL, and components of the anaphase-promoting complex/cyclosome (APC/C). Specifically attenuating CRL4CDT2 by CDT2 knockdown can be more potent in killing cSCC cells than targeting CRLs or CRL4s in general by RBX1 or DDB1 depletion. Suppression of the APC/C or forced APC/C activation by targeting its repressor EMI1 are both potential therapeutic approaches. We observed that cSCC cells can be selectively killed by small-molecule inhibitors of USP8 (DUBs-IN-3/compound 22c) and the NEDD8 E1 activating enzyme/CRLs (MLN4924/pevonedistat). A substantial proportion of cSCC cell lines are very highly MLN4924-sensitive. Pathways that respond to defects in proteostasis are involved in the anti-cSCC activity of p97 suppression. Targeting USP8 can reduce the expression of growth factor receptors that participate in cSCC development. EMI1 and CDT2 depletion can selectively cause DNA re-replication and DNA damage in cSCC cells.

miR-324-5p upregulation potentiates resistance to cisplatin by targeting FBXO11 signalling in non-small cell lung cancer cells.

Dysregulation of microRNAs (miRNAs) plays a key role during the pathogenesis of chemoresistance in lung cancer (LCa). Previous study suggests that miR-324-5p may serve as a unique miRNA signature for LCa, but its role and the corresponding molecular basis remain largely explored. Herein, we report that miR-324-5p expression was significantly increased in cisplatin (CDDP)-resistant LCa tissues and cells, and this upregulation predicted a poor post-chemotherapy prognosis in LCa patients. miR-324-5p was further shown to impact CDDP response: Ectopic miR-324-5p expression in drug-naïve LCa cells was sufficient to attenuate sensitivity to CDDP and to confer more robust tumour growth in CDDP-challenged nude mice. Conversely, ablation of miR-324-5p expression in resistant cells effectively potentiated CDDP-suppressed cell growth in vitro and in vivo. Using multiple approaches, we further identified the tumour suppressor FBXO11 as the direct down-stream target of miR-324-5p. Stable expression of FBXO11 could abrogate the pro-survival effects of miR-324-5p in CDDP-challenged LCa cells. Together, these findings suggest that miR-324-5p upregulation mediates, at least partially, the CDDP resistance by directly targeting FBXO11 signalling in LCa cells. In-depth elucidation of the molecular basis underpinning miR-324-5p action bears potential implications for mechanism-based strategies to improve CDDP responses in LCa.

Nrf2 Activation Promotes Lung Cancer Metastasis by Inhibiting the Degradation of Bach1.

Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis.

FBXW5 reduction alleviates spinal cord injury (SCI) by blocking microglia activity: A mechanism involving p38 and JNK.

Traumatic spinal cord injury (SCI) is a major cause of death and lifelong disability in the world. However, the pathological process of SCI has not been fully understood. F-box/WD repeat-containing protein 5 (FBXW5), a subunit of the SCF-type E3 ubiquitin ligase complex, plays an essential role in regulating various pathologies. However, little is known about the effects of FBXW5 on the progression of SCI. In this study, using a rodent model with SCI, we found that FBXW5 expression was markedly down-regulated in spinal dorsal horn of rats after SCI surgery. Rats with FBXW5 knockdown showed the improved paw withdrawal latency responding to thermal stimuli on the ipsilateral side while showed no significant influence on the basal threshold on the contralateral side. In addition, SCI-induced increase of pro-inflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and IL-6, was obviously decreased by FBXW5 knockdown, along with microglia inactivation as evidenced by the reduced expression of Iba-1. Moreover, immunofluorescent staining suggested that FBXW5 was co-localized with Iba-1 in spinal cord tissues of SCI rats. Furthermore, p38, Jun kinase (JNK) and extracellular signal-regulated kinase (ERK)-1/2 activation was significantly increased by SCI in spinal dosal horn of rats. Notably, FBXW5 knockdown markedly reduced the expression of phosphorylated p38 and JNK without affecting ERK1/2 activity in SCI rats. What's more, suppressing p38 and JNK activation significantly alleviated SCI-induced abnormal behavior in rats, along with reduced expression of pro-inflammatory cytokines. Taken together, these results provided evidence that down-regulation of FBXW5 was involved in the prevention of SCI.

FBX8 degrades GSTP1 through ubiquitination to suppress colorectal cancer progression.

F-box only protein 8 (FBX8), as a critical component of the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases, has been associated with several malignancies through interacting with a member of proteins. However, the substrates of FBX8 for destruction in the progression of colorectal carcinoma (CRC) need to be explored. Here, we show that loss of FBX8 accelerates chemical-induced colon tumorigenesis. FBX8 directly targets GSTP1 for ubiquitin-mediated proteasome degradation in CRC. GSTP1 promotes the proliferation, invasion, and metastasis of CRC cells. Furthermore, GSTP1 is upregulated in CRC tissue samples and predicts poor prognosis of CRC patients. The inactivation of FBX8 negatively correlated with increased levels and stability of GSTP1 in clinical CRC tissues and FBX8 knockout transgenic mice. These findings identify a novel ubiquitination pathway as FBX8-GSTP1 axis that regulates the progression of CRC, which might be a potential prognostic biomarker for CRC patients.

MicroRNA­3666 suppresses the growth and migration of glioblastoma cells by targeting KDM2A.

MicroRNAs (miRNAs) are acknowledged as essential regulators in human cancer types, including glioblastoma (GBM). However, the functions of microRNA­3666 (miR­3666) in GBM remain unclear. In the present study, it was identified that the expression of miR­3666 was significantly downregulated in GBM tissues compared with adjacent normal tissues by reverse transcription­quantitative polymerase chain reaction. Additionally, miR­3666 was downregulated in GBM cell lines. Furthermore, it was observed that the miR­3666 expression level in patients with GBM was associated with prognosis. With functional experiments, it was identified that overexpression of miR­3666 significantly inhibited the proliferation, migration and invasion of GBM cells in vitro by Cell Counting kit­8 and Transwell assays. Ectopic expression of miR­3666 significantly arrested GBM cells in the G0 phase by fluorescence activated cell sorting. In terms of the underlying mechanism, it was identified that lysine­specific demethylase 2A (KDM2A) is a direct target of miR­3666 in GBM cells. Overexpression of miR­3666 significantly decreased the expression of KDM2A in GBM cells. Furthermore, it was observed that knockdown of KDM2A significantly suppressed the proliferation, migration and invasion of GBM cells. Collectively, the present results demonstrated that the miR­3666/KDM2A axis serves an important role in the progression of GBM, which provides novel insight into the development of therapeutic strategies for GBM treatment.

miR-146b promotes cell proliferation and increases chemosensitivity, but attenuates cell migration and invasion via FBXL10 in ovarian cancer.

Epithelial ovarian carcinoma (EOC) is the most lethal gynecologic malignancy. However, the molecular mechanisms remain unclear. In this study, we found that miR-146b was downregulated in EOC and its expression level was negatively correlated with the pathological staging. Follow-up functional experiments illustrated that overexpression of miR-146b significantly inhibited cell migration and invasion, and increased cell proliferation, but it also improved the response to chemotherapeutic agents. Mechanistically, we demonstrated that miR-146b exerted its function mainly through inhibiting F-box and leucine-rich repeat protein 10 (FBXL10), and upregulated the Cyclin D1, vimentin (VIM), and zona-occludens-1 (ZO-1) expression in EOC. These findings indicate that miR-146b-FBXL10 axis is an important epigenetic regulation pathway in EOC. Low miR-146b may contribute to cancer progression from primary stage to advanced stage, and may be the promising therapeutic target of EOC.

Spinal TNF-α impedes Fbxo45-dependent Munc13-1 ubiquitination to mediate neuropathic allodynia in rats.

Presynaptic active zone proteins play a crucial role in regulating synaptic plasticity. Although the ubiquitin-proteasome system underlying the degradation of the presynaptic active zone protein is well established, the contribution of this machinery to regulating spinal plasticity during neuropathic pain development remains unclear. Here, using male Sprague Dawley rats, we demonstrated along with behavioral allodynia, neuropathic injury induced a marked elevation in the expression levels of an active zone protein Munc13-1 in the homogenate and synaptic plasma membrane of the ipsilateral dorsal horn. Moreover, nerve injury-increased Munc13-1 expression was associated with an increase in the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in ipsilateral dorsal horn neurons. This neuropathic injury-induced accumulation of Munc13-1 colocalized with synaptophysin but not homer1 in the dorsal horn. Focal knockdown of spinal Munc13-1 expression attenuated behavioral allodynia and the increased frequency, not the amplitude, of mEPSCs in neuropathic rats. Remarkably, neuropathic injury decreased spinal Fbxo45 expression, Fbxo45-Munc13-1 co-precipitation, and Munc13-1 ubiquitination in the ipsilateral dorsal horn. Conversely, focal knockdown of spinal Fbxo45 expression in naive animals resulted in behavioral allodynia in association with similar protein expression and ubiquitination in the dorsal horn as observed with neuropathic injury rats. Furthermore, both neuropathic insults and intrathecal injection of tumor necrosis factor-α (TNF-α) impeded spinal Fbxo45-dependent Munc13-1 ubiquitination, which was reversed by intrathecal TNF-α-neutralizing antibody. Our data revealed that spinal TNF-α impedes Fbxo45-dependent Munc13-1 ubiquitination that accumulates Munc13-1 in the presynaptic area and hence facilitates the synaptic excitability of nociceptive neurotransmission underlying neuropathic pain.

PTEN counteracts FBXL2 to promote IP3R3- and Ca2+-mediated apoptosis limiting tumour growth.

In response to environmental cues that promote IP3 (inositol 1,4,5-trisphosphate) generation, IP3 receptors (IP3Rs) located on the endoplasmic reticulum allow the 'quasisynaptical' feeding of calcium to the mitochondria to promote oxidative phosphorylation. However, persistent Ca2+ release results in mitochondrial Ca2+ overload and consequent apoptosis. Among the three mammalian IP3Rs, IP3R3 appears to be the major player in Ca2+-dependent apoptosis. Here we show that the F-box protein FBXL2 (the receptor subunit of one of 69 human SCF (SKP1, CUL1, F-box protein) ubiquitin ligase complexes) binds IP3R3 and targets it for ubiquitin-, p97- and proteasome-mediated degradation to limit Ca2+ influx into mitochondria. FBXL2-knockdown cells and FBXL2-insensitive IP3R3 mutant knock-in clones display increased cytosolic Ca2+ release from the endoplasmic reticulum and sensitization to Ca2+-dependent apoptotic stimuli. The phosphatase and tensin homologue (PTEN) gene is frequently mutated or lost in human tumours and syndromes that predispose individuals to cancer. We found that PTEN competes with FBXL2 for IP3R3 binding, and the FBXL2-dependent degradation of IP3R3 is accelerated in Pten-/- mouse embryonic fibroblasts and PTEN-null cancer cells. Reconstitution of PTEN-null cells with either wild-type PTEN or a catalytically dead mutant stabilizes IP3R3 and induces persistent Ca2+ mobilization and apoptosis. IP3R3 and PTEN protein levels directly correlate in human prostate cancer. Both in cell culture and xenograft models, a non-degradable IP3R3 mutant sensitizes tumour cells with low or no PTEN expression to photodynamic therapy, which is based on the ability of photosensitizer drugs to cause Ca2+-dependent cytotoxicity after irradiation with visible light. Similarly, disruption of FBXL2 localization with GGTi-2418, a geranylgeranyl transferase inhibitor, sensitizes xenotransplanted tumours to photodynamic therapy. In summary, we identify a novel molecular mechanism that limits mitochondrial Ca2+ overload to prevent cell death. Notably, we provide proof-of-principle that inhibiting IP3R3 degradation in PTEN-deregulated cancers represents a valid therapeutic strategy.

Deubiquitinase USP13 maintains glioblastoma stem cells by antagonizing FBXL14-mediated Myc ubiquitination.

Glioblastoma is the most lethal brain tumor and harbors glioma stem cells (GSCs) with potent tumorigenic capacity. The function of GSCs in tumor propagation is maintained by several core transcriptional regulators including c-Myc. c-Myc protein is tightly regulated by posttranslational modification. However, the posttranslational regulatory mechanisms for c-Myc in GSCs have not been defined. In this study, we demonstrate that the deubiquitinase USP13 stabilizes c-Myc by antagonizing FBXL14-mediated ubiquitination to maintain GSC self-renewal and tumorigenic potential. USP13 was preferentially expressed in GSCs, and its depletion potently inhibited GSC proliferation and tumor growth by promoting c-Myc ubiquitination and degradation. In contrast, overexpression of the ubiquitin E3 ligase FBXL14 induced c-Myc degradation, promoted GSC differentiation, and inhibited tumor growth. Ectopic expression of the ubiquitin-insensitive mutant T58A-c-Myc rescued the effects caused by FBXL14 overexpression or USP13 disruption. These data suggest that USP13 and FBXL14 play opposing roles in the regulation of GSCs through reversible ubiquitination of c-Myc.

Deficiency of Parkinson's disease-related gene Fbxo7 is associated with impaired mitochondrial metabolism by PARP activation.

The Parkinson's disease (PD)-related protein F-box only protein 7 (Fbxo7) is the substrate-recognition component of the Skp1-Cullin-F-box protein E3 ubiquitin ligase complex. We have recently shown that PD-associated mutations in Fbxo7 disrupt mitochondrial autophagy (mitophagy), suggesting a role for Fbxo7 in modulating mitochondrial homeostasis. Here we report that Fbxo7 deficiency is associated with reduced cellular NAD+ levels, which results in increased mitochondrial NADH redox index and impaired activity of complex I in the electron transport chain. Under these conditions of compromised respiration, mitochondrial membrane potential and ATP contents are reduced, and cytosolic reactive oxygen species (ROS) production is increased. ROS activates poly (ADP-ribose) polymerase (PARP) activity in Fbxo7-deficient cells. PARP inhibitor restores cellular NAD+ content and redox index and ATP pool, suggesting that PARP overactivation is cause of decreased complex I-driven respiration. These findings bring new insight into the mechanism of Fbxo7 deficiency, emphasising the importance of mitochondrial dysfunction in PD.

The pseudophosphatase STYX targets the F-box of FBXW7 and inhibits SCFFBXW7 function.

The F-box protein FBXW7 is the substrate-recruiting subunit of an SCF ubiquitin ligase and a major tumor-suppressor protein that is altered in several human malignancies. Loss of function of FBXW7 results in the stabilization of numerous proteins that orchestrate cell proliferation and survival. Little is known about proteins that directly regulate the function of this protein. In the current work, we have mapped the interactome of the enigmatic pseudophosphatase STYX We reasoned that a catalytically inactive phosphatase might have adopted novel mechanisms of action. The STYX interactome contained several F-box proteins, including FBXW7. We show that STYX binds to the F-box domain of FBXW7 and disables its recruitment into the SCF complex. Therefore, STYX acts as a direct inhibitor of FBXW7, affecting the cellular levels of its substrates. Furthermore, we find that levels of STYX and FBXW7 are anti-correlated in breast cancer patients, which affects disease prognosis. We propose the STYX-FBXW7 interaction as a promising drug target for future investigations.

RSK1 protects P-glycoprotein/ABCB1 against ubiquitin-proteasomal degradation by downregulating the ubiquitin-conjugating enzyme E2 R1.

P-glycoprotein (P-gp) is a critical determinant of multidrug resistance in cancer. We previously reported that MAPK inhibition downregulates P-gp expression and that P-gp undergoes ubiquitin-proteasomal degradation regulated by UBE2R1 and SCFFbx15. Here, we investigated the crosstalk between MAPK inhibition and the ubiquitin-proteasomal degradation of P-gp. Proteasome inhibitors or knockdown of FBXO15 and/or UBE2R1 cancelled MEK inhibitor-induced P-gp downregulation. RSK1 phosphorylated Thr162 on UBE2R1 but did not phosphorylate FBXO15. MEK and RSK inhibitors increased UBE2R1-WT but not UBE2R1-T162D and -T162A expression. UBE2R1-T162D showed higher self-ubiquitination and destabilisation than UBE2R1-WT and -T162A. Unlike UBE2R1-WT and -T162A, UBE2R1-T162D did not induce P-gp ubiquitination. UBE2R1-WT or -T162A downregulated P-gp expression and upregulated rhodamine 123 level and sensitivity to vincristine and doxorubicin. However, UBE2R1-T162D did not confer any change in P-gp expression, rhodamine 123 accumulation and sensitivity to the drugs. These results suggest that RSK1 protects P-gp against ubiquitination by reducing UBE2R1 stability.

Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1-F-box interface.

Skp1-Cul1-F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.