Cullin Research Articles

Tangeretin maintains antioxidant activity by reducing CUL3 mediated NRF2 ubiquitination

To explore the antioxidant capacity of citrus flavonoids under different evaluation systems, chemical and biological methods were engaged to determine the antioxidant abilities of flavanones and polymethoxyflavones. Results showed that flavanones exhibited good antioxidant activity, while polymethoxyflavones had a weak ability to scavenge free radicals. Both flavanones and polymethoxyflavones exerted the ability to inhibit H2O2-induced oxidative stress, but the effective concentration of polymethoxyflavones was lower. Further exploration showed that neohesperidin and tangeretin selectively regulated antioxidant enzyme activity, both in vitro and in vivo. Tangeretin also maintained the expression of antioxidant enzymes in L02 cells and in ICR mice liver. The mechanism exploration showed that both neohesperidin and tangeretin promoted the expression of NRF2 and inhibit the expression of KEAP1, but tangeretin could inhibit the ubiquitination of NRF2 by inhibiting CUL3. The mechanism was verified by CUL3 gene silencing. This study demonstrates a novel antioxidant mechanism of natural products.

CUL4high Lung Adenocarcinomas Are Dependent on the CUL4-p21 Ubiquitin Signaling for Proliferation and Survival

Cullin (CUL) 4A and 4B ubiquitin ligases are often highly accumulated in human malignant neoplasms and are believed to possess oncogenic properties. However, the underlying mechanisms by which CUL4A and CUL4B promote pulmonary tumorigenesis remain largely elusive. This study reports that CUL4A and CUL4B are highly expressed in patients with non-small cell lung cancer (NSCLC), and their high expression is associated with disease progression, chemotherapy resistance, and poor survival in adenocarcinomas. Depletion of CUL4A (CUL4Ak/d) or CUL4B (CUL4Bk/d) leads to cell cycle arrest at G1 and loss of proliferation and viability of NSCLC cells in culture and in a lung cancer xenograft model, suggesting that CUL4A and 4B are oncoproteins required for tumor maintenance of certain NSCLCs. Mechanistically, increased accumulation of the cell cycle-dependent kinase inhibitor p21/Cip1/WAF1 was observed in lung cancer cells on CUL4 silencing. Knockdown of p21 rescued the G1 arrest of CUL4Ak/d or CUL4Bk/d NSCLC cells, and allowed proliferation to resume. These findings reveal that p21 is the primary downstream effector of lung adenocarcinoma dependence on CUL4, highlight the notion that not all substrates respond equally to abrogation of the CUL4 ubiquitin ligase in NSCLCs, and imply that CUL4Ahigh/CUL4Bhigh may serve as a prognostic marker and therapeutic target for patients with NSCLC.

DDB1 binds histone reader BRWD3 to activate the transcriptional cascade in adipogenesis and promote onset of obesity.

Obesity has become a global pandemic. Identification of key factors in adipogenesis helps to tackle obesity and related metabolic diseases. Here, we show that DDB1 binds the histone reader BRWD3 to promote adipogenesis and diet-induced obesity. Although typically recognized as a component of the CUL4-RING E3 ubiquitin ligase complex, DDB1 stimulates adipogenesis independently of CUL4. A DDB1 mutant that does not bind CUL4A or CUL4B fully restores adipogenesis in DDB1-deficient cells. Ddb1+/- mice show delayed postnatal development of white adipose tissues and are protected from diet-induced obesity. Mechanistically, by interacting with BRWD3, DDB1 is recruited to acetylated histones in the proximal promoters of ELK1 downstream immediate early response genes and facilitates the release of paused RNA polymerase II, thereby activating the transcriptional cascade in adipogenesis. Our findings have uncovered a CUL4-independent function of DDB1 in promoting the transcriptional cascade of adipogenesis, development of adipose tissues, and onset of obesity.

Expression and purification of functional recombinant CUL2\u2022RBX1 from E. coli

Cullin-2 (CUL2) based cullin-RING ligases (CRL2s) comprise a family of ubiquitin E3 ligases that exist only in multi-cellular organisms and are crucial for cellular processes such as embryogenesis and viral pathogenesis. CUL2 is the scaffold protein that binds one of the interchangeable substrate receptor modules, which consists of adaptor proteins and the substrate receptor protein. The VHL protein is a substrate receptor known to target hypoxia-inducible factor α (HIF1α) for ubiquitination and degradation. Because of its critical role in the ubiquitination of important cellular factors such as HIF1α, CRL2s have been investigated for their biological functions and the development of novel therapeutics against diseases. Given the importance of CRL2s in biological and biomedical research, methods that efficiently produce functional CUL2 proteins will greatly facilitate studies on the mechanism and regulation of CRL2s. Here, we report two cost-effective systems for the expression and purification of recombinant human CUL2 from E. coli cells. The purified CUL2 proteins were ~ 95% pure, could bind their substrate receptor modules, and were enzymatically active in transferring ubiquitin or ubiquitin-like protein to the corresponding substrate in in vitro assays. The presented methodological advancements will help advance research in CRL2 function and regulation.

Long non‑coding RNA XIST inhibits osteoblast differentiation and promotes osteoporosis via Nrf2 hyperactivation by targeting CUL3.

Osteoporosis (OP) is a common skeletal disorder characterized by a low bone mass and the deterioration of bone structure. Long non-coding (lnc)RNA X inactive-specific transcript (XIST) is highly expressed in the serum and monocytes of patients with OP. Thus, the purpose of the present study was to explore the mechanisms underlying the role of XIST in the progression of OP. To establish animal models of OP, female rats underwent a bilateral ovariectomy. The bone mineral density of individual rats was measured using dual-energy X-ray absorptiometry. The combination of XIST and cullin-3 (CUL3) was analyzed using a dual-luciferase reporter assay. Bone histopathological changes were assessed by hematoxylin and eosin staining. Alkaline phosphatase activity was examined by ALP staining. Finally, a series of functional experiments were performed to examine the effects of XIST on cellular behaviors. In the present study, XIST promoted OP and inhibited bone formation by regulating the expression levels of CUL3 and nuclear factor erythroid 2-related factor 2 (Nrf2) in the rats with OP. Moreover, XIST directly targeted CUL3 and negatively regulated its expression. Of note, CUL3 downregulation reversed the effects of XIST silencing on cell viability, differentiation and mineralization, as well as the expression of Nrf2 and CUL3 in MC3T3-E1 cells. Collectively, XIST was demonstrated to inhibit the differentiation of osteoblasts and promote OP by inhibiting the degradation of Nrf2 via targeting CUL3.

Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development

De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to autism spectrum disorder (ASD). In mouse, constitutive Cul3 haploinsufficiency leads to motor coordination deficits as well as ASD-relevant social and cognitive impairments. However, induction of Cul3 haploinsufficiency later in life does not lead to ASD-relevant behaviors, pointing to an important role of Cul3 during a critical developmental window. Here we show that Cul3 is essential to regulate neuronal migration and, therefore, constitutive Cul3 heterozygous mutant mice display cortical lamination abnormalities. At the molecular level, we found that Cul3 controls neuronal migration by tightly regulating the amount of Plastin3 (Pls3), a previously unrecognized player of neural migration. Furthermore, we found that Pls3 cell-autonomously regulates cell migration by regulating actin cytoskeleton organization, and its levels are inversely proportional to neural migration speed. Finally, we provide evidence that cellular phenotypes associated with autism-linked gene haploinsufficiency can be rescued by transcriptional activation of the intact allele in vitro, offering a proof of concept for a potential therapeutic approach for ASDs.

A case report of pseudohypoaldosteronism type II with a homozygous KLHL3 variant accompanied by hyperthyroidism

BackgroundPseudohypoaldosteronism type II (PHAII), also called Gordon syndrome, is a rare hereditary disease caused by variants in the WNK1, WNK4, KLHL3 and CUL3 genes. The combination of PHAII with hyperthyroidism and secondary hyperparathyroidism has not been reported previously.Case presentationA 54-year-old female with recently diagnosed Graves’ disease presented hyperkalemia, hypertension, hypercalciuria, elevated levels of parathyroid hormone (PTH) and normal renal function. PHAII was established based on the finding of a homozygous variant (c.328 A > G, T110A) in the KLHL3 gene. Low-dose thiazide diuretics normalized her potassium, calcium and PTH.ConclusionsPHAII caused by a KLHL3 variant can affect adults later in life. This diagnosis should be considered in patients with hypertension, consistent hyperkalemia, and normal eGFR and can be corrected by thiazides. The patient also had hyperthyroidism and secondary hyperparathyroidism. The latter was also corrected by thiazide treatment. The hyperthyroidism was assumed to be unrelated to PHAII.

Genomic profiling of esophageal squamous cell carcinoma to reveal actionable genetic alterations.

e16042 Background: Esophageal cancer is the eighth most common cancer in the world and more than half of global cases occur in China. Studies demonstrated that esophageal squamous cell carcinomas (ESCC) and esophageal adenocarcinomas (EAC) are two distinct disease entities. Due to the lack of effective therapies, the five-year survival rates of ESCC patients remain dismal. Therefore, there is an urgent need to establish a framework through genomic profiling to facilitate the development of precision therapies for ESCC. Methods: To characterize therapeutic targets in 118 Chinese ESCC patients, deep panel sequencing of 831 cancer genes (OncoPanscan, Genetronhealth) was performed on their tumor tissues and paired genomic DNA samples. Results: The most frequently mutated genes in our ESCC cohort were TP53 (97%), PIK3CA (19%), CDKN2A (18%), NOTCH1 (17%), KMT2D (15%), LRP1B (15%) NOTCH3 (15%) NFE2L2 (13%), and EP300 (12%). Consistent with previous reports, we found significantly elevated mutations in cancer-related genes including NOTCH1 (16.9%), NOTCH2 (3.2%), NOTCH3 (15.3%) and RB1 (9.3%). Importantly, 17.8% (21/118) patients in our cohort harbored the 11q13 amplicon ( CCND1, FGF3, FGF4 and FGF19). The median copy number was 8.19 (range 6.07-42.3). These patients can participate in clinical trials with FGFR inhibitor alone or in combination with CDK4/6 inhibitors. Additionally, we also observed frequent genetic alterations in the KEAP1 (Kelch-like ECH-associated protein 1)-NFE2L2 (nuclear factor erythroid 2 like 2)-CUL3 (cullin 3) pathway. 80% (12/15) of missense mutations in NFE2L2 were located at the KEAP1 binding domain of NRF2 protein. These mutations were either around the ETGE motif (D77G, E79Q, G81V/D and E82D) or the DLG motif (D27V, I28T, D29G, L30F, G31E, V32E, R34G). We also identified four missense mutations of KEAP1 and one alternation of CUL3 in splicing site. Taken together, 17% (20/118) of ESCC patients harbored mutations in the NFE2L2/KEAP1/CUL3 pathway, which may be eligible for clinical trials of glutaminase inhibitor telaglenastat. Two patients had high level of ERBB2 amplification which can be targeted with anti-HER2 therapy. Furthermore, 11.9% (14/118) patients carried activating PIK3CA mutations including N345K, E542K, E545K, M1043I and H1047R which may be targeted by PIK3CA inhibitor alpelisib. Lastly, patients with loss-of-function mutation in NF1 (n = 4), STK11 (n = 1) and PTEN (n = 3) can be respectively targeted with MEK inhibitor and mTOR inhibitor. Overall, 43% of patients in our ESCC cohort had actionable genetic mutations with corresponding precision therapy options. Conclusions: Our findings indicated that amplification of the 11q13 amplicon and dysfunction of the KEAP1-NRF2-CUL3 axis are the major driving events of ESCC. The results of genomic profiling can guide physicians to enroll a significant portion of ESCC patients into genomically matched clinical trials.

CUL3 (cullin 3)-mediated ubiquitination and degradation of BECN1 (beclin 1) inhibit autophagy and promote tumor progression

ABSTRACT Macroautophagy/autophagy plays an important role during the development of human cancer. BECN1 (beclin 1), a core player in autophagy regulation, is downregulated in many kinds of malignancy. The underlying mechanism, however, has not been fully illuminated. Here, we found that CUL3 (cullin 3), an E3 ubiquitin ligase, could interact with BECN1 and promote the K48-linked ubiquitination and degradation of this protein; In addition, CUL3 led to a decrease in autophagic activity through downregulating BECN1. We also found that KLHL38 was a substrate adaptor of the CUL3 E3 ligase complex-mediated ubiquitination and degradation of BECN1. In breast and ovarian cancer, CUL3 could promote the proliferation of tumor cells, and the expression of CUL3 was related to poor prognosis in patients. Our study reveals the underlying mechanism of BECN1 ubiquitination and degradation that affects autophagic activity and subsequently leads to tumor progression, providing a novel therapeutic strategy that regulates autophagy to combat cancer. Abbreviations: ATG: autophagy-related BECN1: beclin 1 CHX: cycloheximide CoIP: co-immunoprecipitation CUL3: cullin 3 IP: immunoprecipitation MS: mass spectrometry PtdIns3K: phosphatidylinositol3-kinase UPS: ubiquitin-proteasome system

A Rare Case of Pseudohypoaldosteronism Type II or Gordon Syndrome

Introduction: Pseudohypoaldosteronism type II (PHA II) or Gordon Syndrome is a rare, autosomally inherited disease with unknown prevalence. It is caused by mutations in the WNK1, WNK4, CUL3, or KLHL3 gene. It is characterized by hypertension, hyperkalemia, hyperchloremic metabolic acidosis and low plasma aldosterone levels, but otherwise normal kidney function. The age of onset of PHA2 is variable, ranging from infancy or childhood to adolescence and adultdood. The electrolyte and blood pressure abnormalities of PHA II is often managed with salt restriction and hydrochlorthiazide (HCTZ). Here we report a rare case of Pseudohypoaldosteronism type II in an adolescent patient.Case Presentation: A 16-yo female with past medical history of asthma and anemia presented to the emergency department with acute severe abdominal/suprapubic pain, associated with diaphoresis, non bloody diarrhea and non bilious non bloody vomiting. The patient also reported daily headaches relieved with Tylenol. In the ED, she was found to be hypertensive at 190/118 mmHg. Blood count showed mild anemia but normal white count and platelets. Comprehensive metabolic panel showed sodium 140, potassium 6.6, chloride 115, bicarbonate 16, creatinine 0.5, and normal liver enzymes. Urine electrolytes were as follows: sodium 189, potassium 20.8 and chloride 140. Arterial Blood Gas ahowed pH of 7.32. Plasma renin activity was low normal at 0.34 and aldosterone level was 2. CT scan of abdomen and pelvis was unremarkable. The blood work was consistent with pseudohypoaldosteronism type II or Gordon syndrome. The patient was adopted so there was no family history. She was started on hydrochlorothiazide. Later, she developed severe itching reaction with hydrochlorthiazide. She is currently being treated with Indapamide, with well controlled blood pressure and normal electrolytes.Conclusion: Pseudohypoaldosteronism type II or Gordon’s Syndrome is a rare disease, with usually autosomal dominant inheritance, with no specific diagnostic criteria for diagnosis. It should be suspected in adolescent or adult patients with hyperkalemia with normal glomerular filtartion, accompanied by hypertension (can be absent), metabolic acidosis, hyperchloremia, decreased plasma renin, relatively suppressed aldosteronism and family history of similar findings.

Expression and purification of recombinant human CUL2 from Escherichia coli cells

Cullin-2 (CUL2) based cullin RING ligases (CRL2) comprise a family of ubiquitin E3 ligases that exist only in multi-cellular organisms and are crucial for cellular processes such as embryogenesis and viral pathogenesis. In the CRL2 complex, CUL2 serves as a docking platform for the substrate receptor module, which consists of the adaptor proteins Elongin B and Elongin C (EloB/C) as well as the tumor suppressor protein VHL, where VHL is known to target hypoxia-inducible factor α (HIF1α) for ubiquitination and degradation. Abnormalities in HIF1α degradation can result in vascular tumor cell proliferation and cause a rare disease known as von-Hippel-Lindau (VHL) syndrome. Because of its critical role in HIF1α ubiquitination, CRL2VHL has been investigated for the development of novel therapeutics and treatments against disease. Given the importance of CRL2s in biological and biomedical research, a method that efficiently produces functional recombinant human CUL2 proteins will greatly facilitate studies on CRL2-dependent protein ubiquitination and degradation. Previous studies have generated recombinant human CUL2 using a baculovirus expression system. However, this method is very costly and time consuming for optimal protein expression in insect cells. To achieve a more cost-effective way of producing the human CUL2 protein, we propose to generate recombinant CUL2 in E. coli cells. We suggest that removal of unstructured regions in CUL2 would increase its solubility in E. coli. Additionally, we explored and applied the “Split-n-Co-express” strategy as previously used for CUL1 and CUL3. We report that each of our strategies resulted in soluble CUL2 expressed in E. coli cells, and following a series of purification by chromatography, the purity of the recombinant protein reached ~95%. Using in vitro assays, we found that our purified CUL2 could conjugate to NEDD8, a key activator of CRLs, and that the neddylated CUL2 could bind to VHL•EloB/C to ubiquitinate the HIF1α degron peptide. The binding and enzymatic activity of our recombinant protein demonstrate that we have established a new system for the expression and purification of functional human CUL2 from E. coli cells.

Downregulation of Cullin 3 Ligase Signaling Pathways Contributes to Hypertension in Preeclampsia.

Background: Preeclampsia (PE) is a leading cause of maternal and perinatal morbidity and mortality; however, its etiology and pathophysiology remain obscure. PE is initiated by inadequate spiral artery remodeling and subsequent placental ischemia/hypoxia, which stimulates release of bioactive factors into maternal circulation, leading to hypertension and renal damage.Methods and Results: Abundance of key components of cullin 3-ring ubiquitin ligase (CRL3), including cullin 3 (CUL3) and its neddylated modification, and adaptors including Kelch-like 2 (KLHL2) and Rho-related BTB domain containing protein 1 was all decreased in spiral arteries and placentas of PE patients. Similar changes were found in aortic tissues and renal distal tubules of pregnant mice treated with Nω-nitro-l-arginine methyl ester hydrochloride. The downregulation of CRL3 function led to accumulation of with-no-lysine kinases, phosphodiesterase 5, and RhoA in vessels and renal distal tubules, which promoted vasoconstriction and Na–Cl cotransporter activation in the distal convoluted tubule (DCT), as well as vascular and DCT structure remodeling. Proton pump inhibitor esomeprazole partially restored CRL3 function. In vitro studies have shown that increased abundance of JAB1, a component of the COP9 signalosome, inhibited CUL3 neddylation and promoted the expression of hypoxia-inducible factor 1α, which downregulated peroxisome proliferator–activated receptor γ and further promoted CUL3 inactivation. KLHL3/2 was degraded by increased autophagy.Conclusion: These findings support that the downregulation of CRL3 function disrupts the balance of vasoconstriction and vasodilation and aggravates excess reabsorption of sodium in PE.

Role of KLHL3 and dietary K+ in regulating KS-WNK1 expression.

The physiological role of the shorter isoform of with no lysine kinase (WNK)1 that is exclusively expressed in the kidney (KS-WNK1), with particular abundance in the distal convoluted tubule, remains elusive. KS-WNK1, despite lacking the kinase domain, is nevertheless capable of stimulating the NaCl cotransporter, apparently through activation of WNK4. It has recently been shown that a less severe form of familial hyperkalemic hypertension featuring only hyperkalemia is caused by missense mutations in the WNK1 acidic domain that preferentially affect cullin 3 (CUL3)-Kelch-like protein 3 (KLHL3) E3-induced degradation of KS-WNK1 rather than that of full-length WNK1. Here, we show that full-length WNK1 is indeed less impacted by the CUL3-KLHL3 E3 ligase complex compared with KS-WNK1. We demonstrated that the unique 30-amino acid NH2-terminal fragment of KS-WNK1 is essential for its activating effect on the NaCl cotransporter and recognition by KLHL3. We identified specific amino acid residues in this region critical for the functional effect of KS-WNK1 and KLHL3 sensitivity. To further explore this, we generated KLHL3-R528H knockin mice that mimic human mutations causing familial hyperkalemic hypertension. These mice revealed that the KLHL3 mutation specifically increased expression of KS-WNK1 in the kidney. We also observed that in wild-type mice, the expression of KS-WNK1 was only detectable after exposure to a low-K+ diet. These findings provide new insights into the regulation and function of KS-WNK1 by the CUL3-KLHL3 complex in the distal convoluted tubule and indicate that this pathway is regulated by dietary K+ levels.NEW & NOTEWORTHY In this work, we demonstrated that the kidney-specific isoform of with no lysine kinase 1 (KS-WNK1) in the kidney is modulated by dietary K+ and activity of the ubiquitin ligase protein Kelch-like protein 3. We analyzed the role of different amino acid residues of KS-WNK1 in its activity against the NaCl cotransporter and sensitivity to Kelch-like protein 3.

Cullin-5 neddylation-mediated NOXA degradation is enhanced by PRDX1 oligomers in colorectal cancer

NOXA, a BH3-only proapoptotic protein involved in regulating cell death decisions, is highly expressed but short-lived in colorectal cancer (CRC). Neddylated cullin-5 (CUL5)-mediated ubiquitination and degradation of NOXA is crucial to prevent its overaccumulation and maintain an appropriate action time. However, how this process is manipulated by CRC cells commonly exposed to oxidative stress remain unknown. The peroxiredoxin PRDX1, a conceivable antioxidant overexpressed in CRC tissues, has been shown to inhibit apoptosis and TRAF6 ubiquitin-ligase activity. In this study, we found that PRDX1 inhibits CRC cell apoptosis by downregulating NOXA. Mechanistically, PRDX1 promotes NOXA ubiquitination and degradation, which completely depend on CUL5 neddylation. Further studies have demonstrated that PRDX1 oligomers bind with both the Nedd8-conjugating enzyme UBE2F and CUL5 and that this tricomplex is critical for CUL5 neddylation, since silencing PRDX1 or inhibiting PRDX1 oligomerization greatly dampens CUL5 neddylation and NOXA degradation. An increase in reactive oxygen species (ROS) is not only a hallmark of cancer cells but also the leading driving force for PRDX1 oligomerization. As shown in our study, although ROS play a role in upregulating NOXA mRNA transcription, ROS scavenging in CRC cells by N-acetyl-L-cysteine (NAC) can significantly reduce CUL5 neddylation and extend the NOXA protein half-life. Therefore, in CRC, PRDX1 plays a key role in maintaining intracellular homeostasis under conditions of high metabolic activity by reinforcing UBE2F-CUL5-mediated degradation of NOXA, which is also evidenced in the resistance of CRC cells to etoposide treatment. Based on these findings, targeting PRDX1 could be an effective strategy to overcome the resistance of CRC to DNA damage-inducing chemotherapeutics.

Decreased KLHL3 expression is involved in the activation of WNK-OSR1/SPAK-NCC cascade in type 1 diabetic mice.

Familial hyperkalemic hypertension (FHHt; also called pseudohypoaldosteronism type II) is a hereditary hypertensive disease which can be caused by mutations in four genes: WNK1 [with no lysine (K) 1], WNK4, Kelch-like3 (KLHL3), and cullin3 (CUL3). Decreased KLHL3 expression was identified as being involved in the pathogenesis of FHHt caused by cullin 3 disease mutations. Recent studies have revealed an increased WNK4 and hence Na-Cl cotransporter (NCC) activity in the db/db mice, resulting from PKC-mediated KLHL3 phosphorylation, which impairs the degradation of its substrate, WNK4. However, whether WNK4 and NCC were activated in type 1 diabetes still remains unclear. We created streptozotocin-induced type 1 diabetic mice and revealed that renal WNK-oxidative stress response kinase-1/STE20/SPS1-related proline alanine-rich kinase (OSR1/SPAK)-NCC cascade was activated, whereas KLHL3 expression was markedly decreased and CUL3 was heavily neddylated. Moreover, decreased KLHL3 was reversed and WNK1 and WNK4 abundance increased by MLN4924, a neddylation inhibitor. In vitro, our study also showed decreased KLHL3 abundance without any significant change in phosphorylated KLHL3 under high glucose exposure. These results indicate that decreased KLHL3 likely plays a role in the pathogenesis of renal sodium reabsorption in hyperglycemic conditions.

Dimerization of the ETO1 family proteins plays a crucial role in regulating ethylene biosynthesis in Arabidopsis thaliana.

ETHYLENE OVERPRODUCER1 (ETO1), ETO1-LIKE1 (EOL1), and EOL2 are members of the Broad complex, Tramtrack, Bric-a-brac (BTB) protein family that collectively regulate type-2 1-aminocyclopropane-1-carboxylic acid synthase (ACS) activity in Arabidopsis thaliana. Although ETO1 and EOL1/EOL2 encode structurally related proteins, genetic studies suggest that they do not play an equivalent role in regulating ethylene biosynthesis. The mechanistic details underlying the genetic analysis remain elusive. In this study, we reveal that ETO1 collaborates with EOL1/2 to play a key role in the regulation of type-2 ACS activity via protein-protein interactions. ETO1, EOL1, and EOL2 exhibit overlapping but distinct tissue-specific expression patterns. Nevertheless, neither EOL1 nor EOL2 can fully complement the eto1 phenotype under control of the ETO1 promoter, which suggests differential functions of ETO1 and EOL1/EOL2. ETO1 forms homodimers with itself and heterodimers with EOLs. Furthermore, CULLIN3 (CUL3) interacts preferentially with ETO1. The BTB domain of ETO1 is sufficient for interaction with CUL3 and is required for homodimerization. However, domain-swapping analysis in transgenic Arabidopsis suggests that the BTB domain of ETO1 is essential but not sufficient for a full spectrum of ETO1 function. The missense mutation in eto1-5 generates a substitution of phenylalanine with an isoleucine in ETO1F466I that impairs its dimerization and interaction with EOLs but does not affect binding to CUL3 or ACS5. Overexpression of ETO1F466I in Arabidopsis results in a constitutive triple response phenotype in dark-grown seedlings. Our findings reveal the mechanistic role of protein-protein interactions of ETO1 and EOL1/EOL2 that is crucial for their biological function in ethylene biosynthesis.

Th1 cytokine interferon gamma improves response in HER2 breast cancer by modulating the ubiquitin proteasomal pathway

HER2 breast cancer (BC) remains a significant problem in patients with locally advanced or metastatic BC. We investigated the relationship between T helper 1 (Th1) immune response and the proteasomal degradation pathway (PDP), in HER2-sensitive and -resistant cells. HER2 overexpression is partially maintained because E3 ubiquitin ligase Cullin5 (CUL5), which degrades HER2, is frequently mutated or underexpressed, while the client-protective co-chaperones cell division cycle 37 (Cdc37) and heat shock protein 90 (Hsp90) are increased translating to diminished survival. The Th1 cytokine interferon (IFN)-γ caused increased CUL5 expression and marked dissociation of both Cdc37 and Hsp90 from HER2, causing significant surface loss of HER2, diminished growth, and induction of tumor senescence. In HER2-resistant mammary carcinoma, either IFN-γ or Th1-polarizing anti-HER2 vaccination, when administered with anti-HER2 antibodies, demonstrated increased intratumor CUL5 expression, decreased surface HER2, and tumor senescence with significant therapeutic activity. IFN-γ synergized with multiple HER2-targeted agents to decrease surface HER2 expression, resulting in decreased tumor growth. These data suggest a novel function of IFN-γ that regulates HER2 through the PDP pathway and provides an opportunity to impact HER2 responses through anti-tumor immunity.

Cullin-5 (CUL5) as a potential prognostic marker in a pan-cancer analysis of human tumors

ABSTRACT There is some evidence supporting an association between Cullin-5 (CUL5) and cancer, but no research using pan-cancer analysis has been conducted previously. We therefore investigated the oncogenic role of CUL5 in 33 tumors from the Gene Expression Omnibus and The Cancer Genome Atlas databases. Many cancers reduce CUL5 levels, and the prognosis of certain cancers is vitally linked with CUL5 expression. CUL5 expression is associated with CD8 + T-cell infiltration levels in uveal melanomas and head and neck squamous cell carcinomas, and we observed a positive relationship between CUL5 and Tcm (T central memory) cells, and a negative relationship between T helper (Th) cells and pDC (plasmacytoid DC). CUL5 had negative associations with NK cells, NK CD56bright cells, NK CD56dim cells, Tregs, cytotoxic cells, and Th17 cells. Functions relating to protein processing and ubiquitin were included in the CUL5 functional mechanisms. The top 100 genes that are most strongly related to CUL5 were identified, and enrichment analysis indicated that the biological process with the closest relationship was neddylation, related pathways included the TGF-beta signaling pathway and intracellular receptor signaling pathway. CUL5 is related to biological cell behaviors such as chromosome segregation and positive regulation of chromosome organization. As the first study to perform a pan-cancer analysis of CUL5, the present findings will improve the understanding of the oncogenic role of CUL5 in different tumors.

Cullin3 aggravates the inflammatory response of periodontal ligament stem cells via regulation of SHH signaling and Nrf2

ABSTRACT It is found that the activation of Sonic Hedgehog (SHH) signaling pathway is related to the degree of inflammation in patients suffering from periodontitis. Cullin3 (CUL3), an important ubiquitin ligase, can control SHH signaling. In this study, we were dedicated to clarify the roles of SHH and CUL3 in P. gingivalis-LPS (Pg-LPS)-treated periodontal ligament stem cells (PDLSCs). In this study, cell viability was detected using cell counting kit-8 (CCK-8). The inflammatory cytokines of PDLSCs were estimated by enzyme-linked immunosorbent assay (ELISA). With the application of western blots, the protein levels of SHH, Gli1 and NF-E2-related factor 2 (Nrf2) were determined. Alkaline phosphatase staining and Alizarin red staining were performed to evaluate the differentiation and mineralization capabilities of PDLSCs. The apoptotic cells were screened using TUNEL staining. The results showed that Pg-LPS inhibited cell viability and triggered inflammation of PDLSCs. Overexpression of CUL3 weakened the differentiation and mineralization capabilities of PDLSCs. Moreover, CUL3 overexpression aggravated inflammation and cell apoptosis induced by Pg-LPS. It is worth noting that although the protein levels of SHH, Gli1 and Nrf2 were elevated in PDLSCs treated with Pg-LPS, overexpression of CUL3 decreased the expressions of Gli1 and Nrf2. Overall, SHH/Gli1 and Nrf2 were involved in the inflammation and cell apoptosis of PDLSCs, which was dominated by CUL3.

Genetic ablation of Cullin-RING E3 ubiquitin ligase 7 restrains pressure overload-induced myocardial fibrosis.

Fibrosis is a pathognomonic feature of structural heart disease and counteracted by distinct cardioprotective mechanisms, e.g. activation of the phosphoinositide 3-kinase (PI3K) / AKT pro-survival pathway. The Cullin-RING E3 ubiquitin ligase 7 (CRL7) was identified as negative regulator of PI3K/AKT signalling in skeletal muscle, but its role in the heart remains to be elucidated. Here, we sought to determine whether CRL7 modulates to cardiac fibrosis following pressure overload and dissect its underlying mechanisms. For inactivation of CRL7, the Cullin 7 (Cul7) gene was deleted in cardiac myocytes (CM) by injection of adeno-associated virus subtype 9 (AAV9) vectors encoding codon improved Cre-recombinase (AAV9-CMV-iCre) in Cul7flox/flox mice. In addition, Myosin Heavy Chain 6 (Myh6; alpha-MHC)-MerCreMer transgenic mice with tamoxifen-induced CM-specific expression of iCre were used as alternate model. After transverse aortic constriction (TAC), causing chronic pressure overload and fibrosis, AAV9-CMV-iCre induced Cul7-/- mice displayed a ~50% reduction of interstitial cardiac fibrosis when compared to Cul7+/+ animals (6.7% vs. 3.4%, p<0.01). Similar results were obtained with Cul7flox/flox Myh6-Mer-Cre-MerTg(1/0) mice which displayed a ~30% reduction of cardiac fibrosis after TAC when compared to Cul7+/+ Myh6-Mer-Cre-MerTg(1/0) controls after TAC surgery (12.4% vs. 8.7%, p<0.05). No hemodynamic alterations were observed. AKTSer473 phosphorylation was increased 3-fold (p<0.01) in Cul7-/- vs. control mice, together with a ~78% (p<0.001) reduction of TUNEL-positive apoptotic cells three weeks after TAC. In addition, CM-specific expression of a dominant-negative CUL71152stop mutant resulted in a 16.3-fold decrease (p<0.001) of in situ end-labelling (ISEL) positive apoptotic cells. Collectively, our data demonstrate that CM-specific ablation of Cul7 restrains myocardial fibrosis and apoptosis upon pressure overload, and introduce CRL7 as a potential target for anti-fibrotic therapeutic strategies of the heart.