PAICS ubiquitination recruits UBAP2 to trigger phase separation for purinosome assembly.

Purinosomes serve as metabolons to enhance de novo purine synthesis (DNPS) efficiency through compartmentalizing DNPS enzymes during stressed conditions. However, the mechanism underpinning purinosome assembly and its pathophysiological functions remains elusive. Here, we show that K6-polyubiquitination of the DNPS enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) by cullin-5/ankyrin repeat and SOCS box containing 11 (Cul5/ASB11)-based ubiquitin ligase plays a driving role in purinosome assembly. Upon several purinosome-inducing cues, ASB11 is upregulated by relieving the H3K9me3/HP1α-mediated transcriptional silencing, thus stimulating PAICS polyubiquitination. The polyubiquitinated PAICS recruits ubiquitin-associated protein 2 (UBAP2), a ubiquitin-binding protein with multiple stretches of intrinsically disordered regions, thereby inducing phase separation to trigger purinosome assembly for enhancing DNPS pathway flux. In human melanoma, ASB11 is highly expressed to facilitate a constitutive purinosome formation to which melanoma cells are addicted for supporting their proliferation, viability, and tumorigenesis in a xenograft model. Our study identifies a driving mechanism for purinosome assembly in response to cellular stresses and uncovers the impact of purinosome formation on human malignancies.

Long noncoding RNA BCRP3 stimulates VPS34 and autophagy activities to promote protein homeostasis and cell survival.

BACKGROUND: Autophagy plays important roles in cell homeostasis and protein quality control. Long non-coding RNAs (lncRNAs) have been revealed as an emerging class of autophagy regulators, but the majority of them function in regulating the expression of autophagy-related genes. LncRNAs that directly act on the core autophagic proteins remain to be explored. METHODS: Immunofluorescence staining and Western blotting were used to evaluate the function of BCRP3 in autophagy and aggrephagy. RNA immunoprecipitation and in vitro RNA-protein binding assay were used to evaluate the interaction of BCRP3 with its target proteins. Phosphatidylinositol 3-phosphate ELISA assay was used to quantify the enzymatic activity of VPS34 complex. qRT-PCR analysis was used to determine BCRP3 expression under stresses, whereas mass spectrometry and Gene Ontology analyses were employed to evaluate the effect of BCRP3 deficiency on proteome changes. RESULTS: We identified lncRNA BCRP3 as a positive regulator of autophagy. BCRP3 was mainly localized in the cytoplasm and bound VPS34 complex to increase its enzymatic activity. In response to proteotoxicity induced by proteasome inhibition or oxidative stress, BCRP3 was upregulated to promote aggrephagy, thereby facilitating the clearance of ubiquitinated protein aggregates. Proteomics analysis revealed that BCRP3 deficiency under proteotoxicity resulted in a preferential accumulation of proteins acting in growth inhibition, cell death, apoptosis, and Smad signaling. Accordingly, BCRP3 deficiency in proteotoxic cells compromised cell proliferation and survival, which was mediated in part through the upregulation of TGF-ß/Smad2 pathway. CONCLUSIONS: Our study identifies BCRP3 as an RNA activator of the VPS34 complex and a key role of BCRP3-mediated aggrephagy in protein quality control and selective degradation of growth and survival inhibitors to maintain cell fitness.

Retro-labyrinthine Lesion Site Detected by Galvanic Vestibular Stimulation Elicited Vestibular-evoked Myogenic Potentials in Patients with Auditory Neuropathy.

OBJECTIVE: Auditory neuropathy (AN) is a unique pattern of hearing loss with preservation of hair cell function. The condition is characterized by the presence of otoacoustic emissions (OAE) or cochlear microphonic (CM) responses with severe abnormalities of the auditory brainstem response (ABR). The vestibular branches of the VIII cranial nerve and the structures innervated by it can also be affected. However, the precise lesion sites in the vestibular system are not well characterized in patients with AN. METHODS: The air-conducted sound (ACS) vestibular-evoked myogenic potentials (VEMPs) and galvanic vestibular stimuli (GVS)-VEMPs were examined in 14 patients with AN. RESULTS: On examination of VEMPs (n=14, 28 ears), the absent rates of ACS-cervical VEMP (cVEMP), ACS-ocular VEMP (oVEMP), GVS-cVEMP, GVS-oVEMP and caloric test were 92.9% (26/28), 85.7% (24/28), 67.9% (19/28), 53.6% (15/28), and 61.5% (8/13), respectively. Impaired functions of the saccule, inferior vestibular nerve, utricle, superior vestibular nerve, and horizontal semicircular canal were found in 25.0% (7/28), 67.9% (19/28), 32.1% (9/28), 53.6% (15/28) and 61.5% (8/13) patients, respectively. On comparing the elicited VEMPs parameters of AN patients with those of normal controls, both ACS-VEMPs and GVS-VEMPs showed abnormal results in AN patients (such as, lower presence rates, elevated thresholds, prolonged latencies, and decreased amplitudes). CONCLUSION: The study suggested that patients with AN often have concomitant vestibular disorders. Retro-labyrinthine lesions were more frequently observed in this study. GVS-VEMPs combined with ACS-VEMPs may help identify the lesion sites and facilitate detection of areas of vestibular dysfunction in these patients.

BIK ubiquitination by the E3 ligase Cul5-ASB11 determines cell fate during cellular stress.

The BH3-only pro-apoptotic protein BIK is regulated by the ubiquitin-proteasome system. However, the mechanism of this regulation and its physiological functions remain elusive. Here, we identify Cul5-ASB11 as the E3 ligase targeting BIK for ubiquitination and degradation. ER stress leads to the activation of ASB11 by XBP1s during the adaptive phase of the unfolded protein response, which stimulates BIK ubiquitination, interaction with p97/VCP, and proteolysis. This mechanism of BIK degradation contributes to ER stress adaptation by promoting cell survival. Conversely, genotoxic agents down-regulate this IRE1α-XBP1s-ASB11 axis and stabilize BIK, which contributes in part to the apoptotic response to DNA damage. We show that blockade of this BIK degradation pathway by an IRE1α inhibitor can stabilize a BIK active mutant and increase its anti-tumor activity. Our study reveals that different cellular stresses regulate BIK ubiquitination by ASB11 in opposing directions, which determines whether or not cells survive, and that blocking BIK degradation has the potential to be used as an anti-cancer strategy.

Long non-coding RNA HOXB-AS3 promotes myeloid cell proliferation and its higher expression is an adverse prognostic marker in patients with acute myeloid leukemia and myelodysplastic syndrome.

BACKGROUND: Long non-coding RNAs (lncRNAs) represent the majority of cellular transcripts and play pivotal roles in hematopoiesis. However, their clinical relevance in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) remains largely unknown. Here, we investigated the functions of HOXB-AS3, a lncRNA located at human HOXB cluster, in the myeloid cells, and analyzed the prognostic significances in patients with AML and MDS. METHODS: shRNAs were used to downregulate HOXB-AS3 in the cell lines and the effect was evaluated by quantitative polymerase chain reaction. The proliferation of the cell lines was illustrated by proliferation and BrdU flow assays. Further, we retrospectively analyzed the HOXB-AS3 expression in 193 patients with AML and 157 with MDS by microarray analysis, and evaluated its clinical importance. RESULTS: Downregulation of HOXB-AS3 suppressed cell proliferation. Mechanistically, HOXB-AS3 potentiated the expressions of several key factors in cell cycle progression and DNA replication without affecting the expressions of HOX genes. In AML, patients with higher HOXB-AS3 expression had shorter survival than those with lower HOXB-AS3 expression (median overall survival (OS), 17.7 months versus not reached, P <  0.0001; median relapse-free survival, 12.9 months versus not reached, P = 0.0070). In MDS, patients with higher HOXB-AS3 expression also had adverse prognosis compared with those with lower HOXB-AS3 expression (median OS, 14.6 months versus 42.4 months, P = 0.0018). The prognostic significance of HOXB-AS3 expression was validated in the TCGA AML cohort and another MDS cohort from our institute. The subgroup analyses in MDS patients showed that higher HOXB-AS3 expressions could predict poor prognosis only in lower-risk (median OS, 29.2 months versus 77.3 months, P = 0.0194), but not higher-risk group. CONCLUSIONS: This study uncovers a promoting role of HOXB-AS3 in myeloid malignancies and identifies the prognostic value of HOXB-AS3 expression in AML and MDS patients, particularly in the lower-risk group.

Activation of aryl hydrocarbon receptor reduces carbendazim-induced cell death.

Carbendazim inhibits microtubule assembly, thus blocking mitosis and inhibiting cancer cell proliferation. Accordingly, carbendazim is being explored as an anticancer drug. Data show that carbendazim increased mRNA and protein expressions and promoter activity of CYP1A1. In addition, carbendazim activated transcriptional activity of the aryl hydrocarbon response element, and induced nuclear translocation of the aryl hydrocarbon receptor (AhR), a sign the AhR is activated. Carbendazim-induced CYP1A1 expression was blocked by AhR antagonists, and was abolished in AhR signal-deficient cells. Results demonstrated that carbendazim activated the AhR, thereby stimulating CYP1A1 expression. In order to understand whether AhR-induced metabolic enzymes turn carbendazim into less-toxic metabolites, Hoechst 33342 staining to reveal carbendazim-induced nuclear changes and flow cytometry to reveal the subG0/G1 population were applied to monitor carbendazim-induced cell apoptosis. Carbendazim induced less apoptosis in Hepa-1c1c7 cells than in AhR signal-deficient Hepa-1c1c7 mutant cells. Pretreatment with ß-NF, an AhR agonist that highly induces CYP1A1 expression, decreased carbendazim-induced cell death. In addition, the lower the level of AhR was, the lower the vitality present in carbendazim-treated cells, including hepatoma cells and their derivatives with AhR RNA interference, also embryonic kidney cells, bladder carcinoma cells, and AhR signal-deficient Hepa-1c1c7 cells. In summary, carbendazim is an AhR agonist. The toxicity of carbendazim was lower in cells with the AhR signal. This report provides clues indicating that carbendazim is more potent at inducing cell death in tissues without than in those with the AhR signal, an important reference for applying carbendazim in cancer chemotherapy.

AKT mediates actinomycin D-induced p53 expression.

At high cytotoxic concentrations, actinomycin D (ActD) blocks transcription, decreasing levels of MDM2 and thus causing p53 stabilization. At low cytostatic concentrations, ActD causes ribosomal stress, which decreases MDM2 activity, resulting in p53 stabilization and activation. ActD can thus be used for p53-based cyclotherapy. We analyzed pathways mediating ActD-induced p53 expression. Inhibitors (LY294002, wortmannin, and deguelin) of phosphatidylinositol 3-kinases (PI3K) and AKT, but not inhibitors of MEK1/2, JNK, and p38-MAPK abolished the ActD-induced p53 expression in diverse cell types. RNA interference further supported these results. When AKT was downregulated by small hairpin RNA-AKTs, ActD-induced p53 expression was significantly decreased. ActD caused AKT phosphorylation at Ser473, indicating full activation of AKT. The potential for cancer therapy is discussed.

[Objective characteristics of nystagmus in horizontal semicircular canal benign paroxysmal positional vertigo].

OBJECTIVE: To discuss the objective characteristics and mechanism of nystagmus direction, intensity and time in horizontal semicircular canal benign paroxysmal positional vertigo (HSC-BPPV) . METHODS: A total of 233 patients with HSC-BPPV, whereas 179 horizontal semicircular canalithasis (HSC-Can) and 54 horizontal semicircular cupulolithiasis (HSC-Cup) were involved respectively. The induced nystagmus in roll tests recorded by video-nystagmograph(VNG) , whose direction, intensity and time characteristics were compared in various BPPV. RESULTS: Horizontal nystagmus was both induced by turning left or right in HSC-BPPV roll tests. The direction of the induced nystagmus was the same with turning in HSC-Can. The latency, duration time and intensity ([AKx(-)D] ± s) turning to lesion and normal side were (1.922 ± 1.501)s and (1.447 ± 0.855)s, (25.620 ± 10.409)s, and (22.110 ± 10.931)s, (56.441 ± 33.168)°/s and (24.239 ± 13.892) °/s in HSC-Can. The latency, duration time and intensity turning to lesion side were larger than normal side (t = 3.715, 15.219 and 4.070, P < 0.01) , the difference was statistically significant, and the intensity rate was about 2: 1. The direction of the induced nystagmus was opposite to turning in HSC-Cup. The intensity turning to normal side was larger than lesion side obviously. The intensity rate was about 2: 1, the difference was statistically significant (t = -7.634, P < 0.01) . While the latency and intensity of turning to lesion side in HSC-Can were larger than turn to normal side in HSC-Cup, and the difference detected no statistically significant difference (t = 1.554 and 0.305, P > 0.05). CONCLUSIONS: The induced nystagmus intensity of head to two sides in roll tests for HSC-BPPV both follow Ewald's law, and the ratio between stronger and weaker are both 2: 1. These nystagmus parameters of VNG in roll tests are an objective guideline for BPPV diagnosis.

Cyprodinil as an activator of aryl hydrocarbon receptor.

Cyprodinil is a pyrimidinamine fungicide, used worldwide by agriculture. It is used to protect fruit plants and vegetables from a wide range of pathogens. Benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are toxic environmental pollutants and are prototypes of aryl hydrocarbon receptor (AHR) ligands. Although the structure of cyprodinil distinctly differs from those of BaP and TCDD, our results show that cyprodinil induced nuclear translocation of the AHR, and induced the transcriptional activity of aryl hydrocarbon response element (AHRE). Cyprodinil induced the expression of cytochrome P450 (CYP) 1A1, a well-known AHR-targeted gene, in ovarian granulosa cells, HO23, and hepatoma cells, Hepa-1c1c7. Its induction did not appear in AHR signal-deficient cells, and was blocked by the AHR antagonist, CH-223191. Cyprodinil decreased AHR expression in HO23 cells, resulting in CYP1A1 expression decreasing after it peaked at 9h of treatment in HO23 cells. Dexamethasone is a synthetic agonist of glucocorticoids. Cyprodinil enhanced dexamethasone-induced gene expression, and conversely, its induction of CYP1A1 expression was decreased by dexamethasone in HO23 cells, indicating its induction of crosstalk between the AHR and glucocorticoid receptor and its role as a potential endocrine disrupter. In addition to BaP, TCDD, and an AHR agonist, ß-NF, cyprodinil also phosphorylated extracellular signal-regulated kinase (ERK) in HO23 and Hepa-1c1c7 cells, indicating its deregulation of ERK activity. In summary, our results demonstrate that cyprodinil, similar to BaP, acts as an AHR activator, a potential endocrine disrupter, and an ERK disrupter.

Fluoranthene enhances p53 expression and decreases mutagenesis induced by benzo[a]pyrene.

Fluoranthene (Fla) is the most abundant polycyclic aromatic hydrocarbon (PAH) in diesel particulate extracts. Benzo[a]pyrene (BaP) is genotoxic and is a prototype of PAH carcinogens. Fla's toxicity and mutagenicity are minor relative to BaP's. Our data showed that Fla enhanced BaP-induced p53 expression and promoted BaP-induced cell death. In contrast, Fla decreased BaP-induced mutagenesis. Fla had almost no influence on the cell cycle. However, the effect of cotreatment with BaP (1µM) and Fla (10µM) in regulating the cell cycle was greater than that of BaP (2µM) alone. It is known that BaP activates the aryl hydrocarbon receptor (AhR), and, in turn, the AhR induces cytochrome P450 (Cyp)1a1 expression. The expression of Cyp1a1 corresponds well with the induction of apoptosis and mutagenesis by BaP. Fla did not activate the AhR or antagonize BaP's induction of AhR activity and Cyp1a1 expression. Therefore, the actions of Fla on BaP's toxicity were independent of the AhR signal and Cyp1a1. In summary, results indicated that Fla directs BaP-treated cells into death rather than mutagenesis, consequently preventing cells from being transformed. The novel cooperation between Fla and BaP provides valuable information for how to increase expression of the p53 tumor suppressor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin's suppression of 1-nitropyrene-induced p53 expression is mediated by cytochrome P450 1A1.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), 1-nitropyrene (1-NP), and benzo[a]pyrene (BaP) are toxic environmental pollutants. TCDD was shown to suppress p53 expression in response to genotoxic stress and hypoxic conditions. However, the mechanism of TCDD's actions is not clearly understood. Our data showed that pretreatment with TCDD abolished 1-NP- but not BaP-induced p53 and mouse double minute 2 (MDM2; HDM2 in humans) expressions. TCDD suppressed 1-NP- but not BaP-induced p53 activity, and in contrast, pifithrin-alpha (PFT-α), a p53 inhibitor, suppressed both 1-NP- and BaP-induced p53 activity. In the presence of nutlin-3, an HDM2 inhibitor, TCDD was still able to suppress 1-NP-induced p53 expression. However, TCDD-activated HDM2 did not distinctly cause the degradation of BaP- or nutlin-3-induced p53 expression. Accordingly, TCDD's suppression of 1-NP-induced p53 expression was compound-specific, and the contribution of HDM2 to the abolition of 1-NP-induced p53 was limited. ß-Naphthoflavon (ß-NF), an aryl hydrocarbon receptor (AHR) agonist, mimicked TCDD's action and abolished 1-NP-induced p53 expression. In the presence of CH-223191, an AHR antagonist, TCDD was unable to abolish 1-NP-induced p53 expression. Results indicate that activation of the AHR is required for TCDD's suppression of 1-NP's induction of p53. Cytochrome P450 (CYP) 1A1 is an AHR-targeting gene and a xenobiotic-metabolizing enzyme. TCDD was unable to abolish 1-NP's induction of p53 in CYP1A1-deficient cells, the CYP1A1 transcript of which was degraded by small hairpin RNA-CYP1A1. Both TCDD and PFT-α are potent CYP1A1 inducers and decreased 1-NP-induced cell death and mutagenesis. In summary, TCDD induced detoxification of 1-NP's toxicity, which was mediated by the CYP1A1 enzyme.

1,10-phenanthroline stabilizes mRNA of the carcinogen-metabolizing enzyme, cytochrome P450 1a1.

1,10-phenanthroline (phen), flufenamic acid, and indomethacin are inhibitors of aldo-keto reductases 1C1 (AKR1C1), but only phen decreased the benzo[a]pyrene (BaP)-induced cytochrome P450 1a1 (Cyp1a1) protein level. Therefore the decrease in the BaP-induced Cyp1a1 protein level was not due to inhibition of Akr1c1, but to phen itself. Phen decreased the BaP-induced Cyp1a1 promoter activity and protein expression, and in contrast, it increased Cyp1a1 mRNA, resulting from an increase in mRNA stability. Phen is also known as a transition metal ion-chelator. Along with the phen study, we also found that Zn(2+), Fe(2+) and Cu(2+) increased Cyp1a1 mRNA and protein stability. Our results show that phen stabilized the mRNA of Cyp1a1, although it decreased cell viability. In addition, Zn(2+) and Fe(2+) highly neutralized phen's suppression of Cyp1a1 protein expression, but they only slightly neutralized phen's promotion of mRNA stability and suppression of cell viability, and had no effect on phen's suppression of promoter activity. Phen's effect on Cyp1a1 expression was reversible, which indicates that phen is non-covalently linked to its target. This report elucidates a new role for phen of stabilizing Cyp1a1 mRNA, and provides information for further studies on mRNA stabilization.

1-Nitropyrene stabilizes the mRNA of cytochrome P450 1a1, a carcinogen-metabolizing enzyme, via the Akt pathway.

Cytochrome P450 1a1 (Cyp1a1) is a phase I xenobiotic-metabolizing enzyme, the expression of which is mainly driven by the aryl hydrocarbon receptor (AhR). Cyp1a1 messenger (m)RNA is labile. Our study indicates that 1-nitropyrene (1-NP) highly induced Cyp1a1 protein expression, although its induction of AhR transactivation activity was negligible. The fact that the nuclear receptors, CAR, FXR LXR, or PXR, did not induce Cyp1a1 expression indicates that they do not mediate 1-NP's action. When the AhR transcript was degraded by small hairpin (sh)RNA-AhR, 1-NP-induced Cyp1a1 expression largely decreased. In addition, 1-NP did not induce Cyp1a1 in AhR pathway-deficient mutant cells, which indicates that the AhR is essential for 1-NP's action. When Cyp1a1's turnover was examined, 1-NP was able to stabilize the 1-NP- and benzo[a]pyrene (BaP)-induced Cyp1a1 mRNA, but not protein. 1-NP-induced Cyp1a1 mRNA stabilization was mediated by Akt, but not by p38 MAPK, MEK1/2, or JNK. Among aryl hydrocarbons with four annealed phenyl rings, including pyrene, 1-NP, fluoranthene, 3-nitrofluoranthene, chrysene, and 6-nitrochrysene, only 1-NP was able to stabilize Cyp1a1 mRNA. 1-NP's action was gene specific. In conclusion, stabilizing Cyp1a1 mRNA greatly contributed to 1-NP-induced Cyp1a1 expression, which provides new insight into gene regulation by the AhR ligand and mRNA stabilization.