DCAF13 promotes ovarian cancer progression by activating FRAS1-mediated FAK signaling pathway.

Cullin-RING ubiquitin ligase 4 (CRL4) is closely correlated with the incidence and progression of ovarian cancer. DDB1- and CUL4-associated factor 13 (DCAF13), a substrate-recognition protein in the CRL4 E3 ubiquitin ligase complex, is involved in the occurrence and development of ovarian cancer. However, its precise function and the underlying molecular mechanism in this disease remain unclear. In this study, we confirmed that DCAF13 is highly expressed in human ovarian cancer and its expression is negatively correlated with the overall survival rate of patients with ovarian cancer. We then used CRISPR/Cas9 to knockout DCAF13 and found that its deletion significantly inhibited the proliferation, colony formation, and migration of human ovarian cancer cells. In addition, DCAF13 deficiency inhibited tumor proliferation in nude mice. Mechanistically, CRL4-DCAF13 targeted Fraser extracellular matrix complex subunit 1 (FRAS1) for polyubiquitination and proteasomal degradation. FRAS1 influenced the proliferation and migration of ovarian cancer cell through induction of the focal adhesion kinase (FAK) signaling pathway. These findings collectively show that DCAF13 is an important oncogene that promotes tumorigenesis in ovarian cancer cells by mediating FRAS1/FAK signaling. Our findings provide a foundation for the development of targeted therapeutics for ovarian cancer.

Hormone-mediated disassembly and inactivation of a plant E3 ubiquitin ligase complex.

Phytohormone abscisic acid (ABA) regulates key plant development and environmental stress responses. The ubiquitin-proteasome system tightly controls ABA signaling. CULLIN4-RING (CRL4) E3 ubiquitin ligases use the substrate receptor module CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP10)-DDB1-DET1-DDA1 (CDDD) to target Arabidopsis ABA receptor PYL8, acting as negative regulators of ABA responses. Conversely, ABA treatment attenuates PYL8 receptor degradation, although the molecular mechanism remained elusive. Here, we show that ABA promotes the disruption of CRL4-CDDD complexes, leading to PYL8 stabilization. ABA-mediated CRL4-CDDD dissociation likely involves an altered association between DDA1-containing complexes and the COP9 signalosome (CSN), a master regulator of the assembly of cullin-based E3 ligases, including CRL4-CDDD. Indeed, treatment with CSN inhibitor CSN5i-3 suppresses the ABA effect on CRL4-CDDD assembly. Our findings indicate that ABA stabilizes PYL8 by altering the dynamics of the CRL4-CDDD-CSN complex association, showing a regulatory mechanism by which a plant hormone inhibits an E3 ubiquitin ligase to protect its own receptors from degradation.

Cullin-RING Ligase 4 in Cancer: Structure, Functions, and Mechanisms.

Cullin-RING ligase 4 (CRL4) has attracted enormous attentions because of its extensive regulatory roles in a wide variety of biological and pathological events, especially cancer-associated events. CRL4 exerts pleiotropic effects by targeting various substrates for proteasomal degradation or changes in activity through different internal compositions to regulate diverse events in cancer progression. In this review, we summarize the structure of CRL4 with manifold compositional modes and clarify the emerging functions and molecular mechanisms of CRL4 in a series of cancer-associated events.

CRL4-DCAF1 Ubiquitin Ligase Dependent Functions of HIV Viral Protein R and Viral Protein X.

The Human Immunodeficiency Virus (HIV) encodes several proteins that contort the host cell environment to promote viral replication and spread. This is often accomplished through the hijacking of cellular ubiquitin ligases. These reprogrammed complexes initiate or enhance the ubiquitination of cellular proteins that may otherwise act to restrain viral replication. Ubiquitination of target proteins may alter protein function or initiate proteasome-dependent destruction. HIV Viral Protein R (Vpr) and the related HIV-2 Viral Protein X (Vpx), engage the CRL4-DCAF1 ubiquitin ligase complex to target numerous cellular proteins. In this review we describe the CRL4-DCAF1 ubiquitin ligase complex and its interactions with HIV Vpr and Vpx. We additionally summarize the cellular proteins targeted by this association as well as the observed or hypothesized impact on HIV.

The predictive value of ultrasound markers for pregnancy outcomes in recurrent pregnancy loss: a retrospective study.

Establishing prediction models of pregnancy outcomes for recurrent pregnancy loss women at specific gestational weeks will provide patients and physicians with more precise information, ultimately leading to time and cost savings associated with unnecessary revisits. Therefore, our aim was to develop a prediction model for first trimester pregnancy loss in RPL patients. We used ultrasound indices during the first trimester of pregnancy in combination with demographic characteristics and commonly used serum markers. The independent risk factors for each week were as follows: age and P in the fifth week; age, mGSD and CRL in the sixth week; age, hCG and CRL in the seventh week; CRL in the eighth week; mGSD and CRL in ninth week. The corresponding AUC was 0.671, 0.796, 0.872, 0.871, 0.813, respectively. There is a linear relationship between age and first trimester pregnancy loss. hCG < 69,636.6 mIU/ml was associated with a higher risk of pregnancy loss in the seventh gestation week. An mGSD < 18.3 mm, adjusted for age, BMI, and previous pregnancy loss in the sixth week, was linked to an increased risk of first trimester pregnancy loss. A small CRL measurement (less than 2.4 mm, 9.9 mm, 16.9 mm, and 18.6 mm) in the sixth, seventh, eighth and ninth week was closely correlated with higher risk of first trimester pregnancy loss. Furthermore, an mGSD < 33.3 mm and > 48.3 mm in ninth gestational week was associated with a higher risk of pregnancy loss. These models and thresholds may help physicians and patients make more informed decisions together. Further studies are needed to confirm the results.

Excitatory Projections of Wide Field Collicular Neurons to the Nucleus of the Optic Tract in the Rat.

The superficial layers of the mammalian superior colliculus (SC) contain neurons that are generally responsive to visual stimuli but can differ considerably in morphology and response properties. To elucidate the structure and function of these neurons, we combined extracellular recording and juxtacellular labeling, detailed anatomical reconstruction, and ultrastructural analysis of the synaptic contacts of labeled neurons, using transmission electron microscopy. Our labeled neurons project to different brainstem nuclei. Of particular importance are neurons that fit the morphological criteria of the wide field (WF) neurons and whose dendrites are horizontally oriented. They display a rather characteristic axonal projection pattern to the nucleus of optic tract (NOT); thus, we call them superior collicular WF projecting to the NOT (SCWFNOT) neurons. We corroborated the morphological characterization of this neuronal type as a distinct neuronal class with the help of unsupervised hierarchical cluster analysis. Our ultrastructural data demonstrate that SCWFNOT neurons establish excitatory connections with their targets in the NOT. Although, in rodents, the literature about the WF neurons has focused on their extensive projection to the lateral posterior nucleus of the thalamus, as a conduit for information to reach the visual association areas of the cortex, our data suggest that this subclass of WF neurons may participate in the optokinetic nystagmus.

The CRL3KCTD10 ubiquitin ligase-USP18 axis coordinately regulates cystine uptake and ferroptosis by modulating SLC7A11.

SLC7A11 is a cystine transporter and ferroptosis inhibitor. How the stability of SLC7A11 is coordinately regulated in response to environmental cystine by which E3 ligase and deubiquitylase (DUB) remains elusive. Here, we report that neddylation inhibitor MLN4924 increases cystine uptake by causing SLC7A11 accumulation, via inactivating Cullin-RING ligase-3 (CRL-3). We identified KCTD10 as the substrate-recognizing subunit of CRL-3 for SLC7A11 ubiquitylation, and USP18 as SLC7A11 deubiquitylase. Upon cystine deprivation, the protein levels of KCTD10 or USP18 are decreased or increased, respectively, contributing to SLC7A11 accumulation. By destabilizing or stabilizing SLC7A11, KCTD10, or USP18 inversely regulates the cystine uptake and ferroptosis. Biologically, MLN4924 combination with SLC7A11 inhibitor Imidazole Ketone Erastin (IKE) enhanced suppression of tumor growth. In human breast tumor tissues, SLC7A11 levels were negatively or positively correlated with KCTD10 or USP18, respectively. Collectively, our study defines how SLC7A11 and ferroptosis is coordinately regulated by the CRL3KCTD10/E3-USP18/DUB axis, and provides a sound rationale of drug combination to enhance anticancer efficacy.

The Mucus-Binding Factor Mediates Lacticaseibacillus rhamnosus CRL1505 Adhesion but Not Immunomodulation in the Respiratory Tract.

Lacticaseibacillus rhamnosus CRL1505 possesses immunomodulatory activities in the gastrointestinal and respiratory tracts when administered orally. Its adhesion to the intestinal mucosa does not condition its beneficial effects. The intranasal administration of L. rhamnosus CRL1505 is more effective than the oral route at modulating immunity in the respiratory tract. Nonetheless, it has not yet been established whether the adherence of the CRL1505 strain to the respiratory mucosa is needed to provide the immune benefits to the host. In this study, we evaluated the role of adhesion to the respiratory mucosa of the mucus-binding factor (mbf) knock-out L. rhamnosus CRL1505 mutant (Δmbf CRL1505) in the context of a Toll-like receptor 3 (TLR3)-triggered innate immunity response. In vitro adhesion studies in porcine bronchial epitheliocytes (PBE cells) indicated that L. rhamnosus Δmbf CRL1505 adhered weakly compared to the wild-type strain. However, in vivo studies in mice demonstrated that the Δmbf CRL1505 also reduced lung damage and modulated cytokine production in the respiratory tract after the activation of TLR3 to a similar extent as the wild-type strain. In addition, the mutant and the wild-type strains modulated the production of cytokines and antiviral factors by alveolar macrophages in the same way. These results suggest that the Mbf protein is partially involved in the ability of L. rhamnosus CRL1505 to adhere to the respiratory epithelium, but the protein is not necessary for the CRL1505 strain to exert its immunomodulatory beneficial effects. These findings are a step forward in the understanding of molecular interactions that mediate the beneficial effects of nasally administered probiotics.

Comparison of Fetal Crown-Rump Length Measurements between Thawed and Fresh Embryo Transfer.

Background and Objectives: Neonates born from thawed embryo transfers tend to have a significantly higher birthweight compared to those from fresh embryo transfers. The aim of this study was to compare the crown-rump length (CRL) between thawed and fresh embryos to investigate the potential causes of different growth patterns between them. Materials and Methods: This was a retrospective study (July 2010-December 2023) conducted at the Third Department of Obstetrics and Gynecology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Greece. In total, 3082 assisted reproductive technology (ART) pregnancies (4044 embryos) underwent a routine scan at 11+0-13+6 gestational weeks and were included in the study. Maternal age, the type of embryo transfer (thawed vs. fresh, donor vs. their own oocytes), CRL, twin and singleton gestations were analyzed. Results: The mean maternal age in thawed was significantly higher than in fresh embryos (39.8 vs. 35.8 years, p-value < 0.001). The mean CRL z-score was significantly higher in thawed compared to fresh embryo transfers (0.309 vs. 0.199, p-value < 0.001). A subgroup analysis on singleton gestations showed that the mean CRL z-score was higher in thawed blastocysts compared to fresh (0.327 vs. 0.215, p-value < 0.001). Accordingly, an analysis on twins revealed that the mean CRL z-score was higher in thawed blastocysts (0.285 vs. 0.184, p-value: 0.015) and in oocytes' recipients compared to own oocytes' cases (0.431 vs. 0.191, p-value: 0.002). Conclusions: The difference in CRL measurements between thawed and fresh embryos may be a first indication of the subsequent difference in sonographically estimated fetal weight and birthweight. This finding highlights the need for additional research into the underlying causes, including maternal factors and the culture media used.

HBV PreC interacts with SUV39H1 to induce viral replication by blocking the proteasomal degradation of viral polymerase.

Hepatitis B e antigen (HBeAg) seropositivity during the natural history of chronic hepatitis B (CHB) is known to coincide with significant increases in serum and intrahepatic HBV DNA levels. However, the precise underlying mechanism remains unclear. In this study, we found that PreC (HBeAg precursor) genetic ablation leads to reduced viral replication both in vitro and in vivo. Furthermore, PreC impedes the proteasomal degradation of HBV polymerase, promoting viral replication. We discovered that PreC interacts with SUV39H1, a histone methyltransferase, resulting in a reduction in the expression of Cdt2, an adaptor protein of CRL4 E3 ligase targeting HBV polymerase. SUV39H1 induces H3K9 trimethylation of the Cdt2 promoter in a PreC-induced manner. CRISPR-mediated knockout of endogenous SUV39H1 or pharmaceutical inhibition of SUV39H1 decreases HBV loads in the mouse liver. Additionally, genetic depletion of Cdt2 in the mouse liver abrogates PreC-related HBV replication. Interestingly, a negative correlation of intrahepatic Cdt2 with serum HBeAg and HBV DNA load was observed in CHB patient samples. Our study thus sheds light on the mechanistic role of PreC in inducing HBV replication and identifies potential therapeutic targets for HBV treatment.

The UBE2F-CRL5ASB11-DIRAS2 axis is an oncogene and tumor suppressor cascade in pancreatic cancer cells.

UBE2F, a neddylation E2, neddylates CUL5 to activate cullin-RING ligase-5, upon coupling with neddylation E3 RBX2/SAG. Whether and how UBE2F controls pancreatic tumorigenesis is previously unknown. Here, we showed that UBE2F is essential for the growth of human pancreatic cancer cells with KRAS mutation. In the mouse KrasG12D pancreatic ductal adenocarcinoma (PDAC) model, Ube2f deletion suppresses cerulein-induced pancreatitis, and progression of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia. Mechanistically, Ube2f deletion inactivates the Mapk-c-Myc signals via blocking ubiquitylation of Diras2, a substrate of CRL5Asb11 E3 ligase. Biologically, DIRAS2 suppresses growth and survival of human pancreatic cancer cells harboring mutant KRAS, and Diras2 deletion largely rescues the phenotypes induced by Ube2f deletion. Collectively, Ube2f or Diras2 plays a tumor-promoting or tumor-suppressive role in the mouse KrasG12D PDAC model, respectively. The UBE2F-CRL5ASB11 axis could serve as a valid target for pancreatic cancer, whereas the levels of UBE2F or DIRAS2 may serve as prognostic biomarkers for PDAC patients.

Enhanced triolein and ethyl ferulate interesterification performance by CRL-AuNPs.

This study established a Candida rugosa lipase (CRL) system to catalyze triolein and ethyl ferulate interesterification. The products were identified, and the binding mode between the substrates and CRL was predicted through molecular docking. Three methods for preparing CRL-AuNPs were proposed and characterized. It was found that the addition of 40 mL of 15 nm gold nanoparticles increased the CRL activity from 3.05 U/mg to 4.75 U/mg, but the hybridization efficiency was only 32.7 %. By using 4 mL of 0.1 mg/mL chloroauric acid, the hybridization efficiency was improved to 50.7 %, but the enzyme activity was sharply decreased. However, when the molar ratio of Mb to HAuCl4 was 0.2, the hybridization efficiency increased to 71.8 %, and the CRL activity was also enhanced to 5.98 U/mg. Under optimal conditions, the enzyme activity of CRL-AuNPs③ was maintained at 95 % after 6 repetitions and 85.6 % after 30 days at room temperature.

The herpesvirus UL49.5 protein hijacks a cellular C-degron pathway to drive TAP transporter degradation.

The transporter associated with antigen processing (TAP) is a key player in the major histocompatibility class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and triggers its proteasomal degradation. How UL49.5 promotes TAP degradation has, so far, remained unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal. We propose that the C terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the cullin-RING E3 ligase in endoplasmic reticulum-associated degradation.

Cul-4 inhibition rescues spastin levels and reduces defects in hereditary spastic paraplegia models.

Hereditary spastic paraplegias (HSPs) are degenerative motor neuron diseases characterized by progressive spasticity and weakness in the lower limbs. The most common form of HSP is due to SPG4 gene haploinsufficiency. SPG4 encodes the microtubule severing enzyme spastin. Although, there is no cure for SPG4-HSP, strategies to induce a spastin recovery are emerging as promising therapeutic approaches. Spastin protein levels are regulated by poly-ubiquitination and proteasomal-mediated degradation, in a neddylation-dependent manner. However, the molecular players involved in this regulation are unknown. Here, we show that the Cullin-4-RING E3 ubiquitin ligase complex (CRL4) regulates spastin stability. Inhibition of CRL4 increases spastin levels by preventing its poly-ubiquitination and subsequent degradation in spastin-proficient and in patient derived SPG4 haploinsufficient cells. To evaluate the role of CRL4 complex in spastin regulation in vivo, we developed a Drosophila melanogaster model of SPG4 haploinsufficiency which show alterations of synapse morphology and locomotor activity, recapitulating phenotypical defects observed in patients. Downregulation of the CRL4 complex, highly conserved in Drosophila, rescues spastin levels and the phenotypical defects observed in flies. As a proof of concept of possible pharmacological treatments, we demonstrate a recovery of spastin levels and amelioration of the SPG4-HSP-associated defects both in the fly model and in patient-derived cells by chemical inactivation of the CRL4 complex with NSC1892. Taken together, these findings show that CRL4 contributes to spastin stability regulation and that it is possible to induce spastin recovery and rescue of SPG4-HSP defects by blocking the CRL4-mediated spastin degradation.

A Postbiotic Formulation of Lactiplantibacillus plantarum CRL 759 Attenuates Endotoxin Induced Uveitis.

PURPOSE: To evaluate the anti-inflammatory activity of a cell-free supernatant from Lactiplantibacillus plantarum CRL 759, in phosphate buffer modified according to Sorensen called POF-759. METHODS: The activity of POF-759 administered by means of eye drops was evaluated on animals subcutaneously injected with the lipopolysaccharide animals in which uveitis was induced by a subcutaneous injection of lipopolysaccharide (EIU). Clinical signs of ocular inflammation, cytokines and proteins were examined in the aqueous humor. Additionally, cellular infiltration was evaluated by histopathological analysis. RESULTS: The new postbiotic administered locally decreases signs of ocular damage, the number of infiltrating cells in the anterior and posterior chambers, the proinflammatory mediators and the proteins in the aqueous humor on mice with EIU. CONCLUSIONS: Our results provide an impetus to relieve ocular inflammation and to identify and develop preventive and therapeutic approaches, to avoid deterioration and to maintain healthy eyes on inflammatory processes.

Modulation of Cullin-RING E3 ubiquitin ligase-dependent ubiquitination by small molecule compounds.

Cullin (CUL)-RING (Really Interesting New Gene) E3 ubiquitin (Ub) ligases (CRLs) are the largest E3 family. The E3 CRL core ligase is a subcomplex formed by the CUL C-terminal domain bound with the ROC1/RBX1 RING finger protein, which acts as a hub that mediates and organizes multiple interactions with E2, Ub, Nedd8, and the ARIH family protein, thereby resulting in Ub transfer to the E3-bound substrate. This report describes the modulation of CRL-dependent ubiquitination by small molecule compounds including KH-4-43, #33, and suramin, which target the CRL core ligases. We show that both KH-4-43 and #33 inhibit the ubiquitination of CK1α by CRL4CRBN. However, either compound's inhibitory effect on this reaction is significantly reduced when a neddylated form of CRL4CRBN is used. On the other hand, both #33 and KH-4-43 inhibit the ubiquitination of ß-catenin by CRL1ß-TrCP and Nedd8-CRL1ß-TrCP almost equally. Thus, neddylation of CRL1ß-TrCP does not negatively impact the sensitivity to inhibition by #33 and KH-4-43. These findings suggest that the effects of neddylation to alter the sensitivity of CRL inhibition by KH-4-43/#33 is dependent upon the specific CRL type. Suramin, a compound that targets CUL's basic canyon, can effectively inhibit CRL1/4-dependent ubiquitination regardless of neddylation status, in contrast to the results observed with KH-4-43/#33. This observed differential drug sensitivity of KH-4-43/#33 appears to echo CUL-specific Nedd8 effects on CRLs as revealed by recent high-resolution structural biology efforts. The highly diversified CRL core ligase structures may provide opportunities for specific targeting by small molecule modulators.

A methylation-phosphorylation switch controls EZH2 stability and hematopoiesis.

The Polycomb Repressive Complex 2 (PRC2) methylates H3K27 to regulate development and cell fate by transcriptional silencing. Alteration of PRC2 is associated with various cancers. Here, we show that mouse Kdm1a deletion causes a dramatic reduction of PRC2 proteins, whereas mouse null mutation of L3mbtl3 or Dcaf5 results in PRC2 accumulation and increased H3K27 trimethylation. The catalytic subunit of PRC2, EZH2, is methylated at lysine 20 (K20), promoting EZH2 proteolysis by L3MBTL3 and the CLR4DCAF5 ubiquitin ligase. KDM1A (LSD1) demethylates the methylated K20 to stabilize EZH2. K20 methylation is inhibited by AKT-mediated phosphorylation of serine 21 in EZH2. Mouse Ezh2K20R/K20R mutants develop hepatosplenomegaly associated with high GFI1B expression, and Ezh2K20R/K20R mutant bone marrows expand hematopoietic stem cells and downstream hematopoietic populations. Our studies reveal that EZH2 is regulated by methylation-dependent proteolysis, which is negatively controlled by AKT-mediated S21 phosphorylation to establish a methylation-phosphorylation switch to regulate the PRC2 activity and hematopoiesis.

Can Lacticaseibacillus rhamnosus CRL1505 postbiotic improve emergency myelopoiesis in immunocompromised mice?

We evaluated whether viable and non-viable Lacticaseibacillus rhamnosus CRL1505 (Lr05V or Lr05NV, respectively) was able to improve emergency myelopoiesis induced by Streptococcus pneumoniae (Sp) infection. Adult Swiss-mice were orally treated with Lr05V or Lr05NV during five consecutive days. The Lr05V and Lr05NV groups and untreated control group received an intraperitoneal dose of cyclophosphamide (Cy-150 mg/kg). Then, the mice were nasally challenged with Sp (107 UFC/mice) on day 3 post-Cy injection. After the pneumococcal challenge, the innate and myelopoietic responses were evaluated. The control group showed a high susceptibility to pneumococcal infection, an impaired innate immune response and a decrease of hematopoietic stem cells (HSCs: Lin-Sca-1+c-Kit+), and myeloid multipotent precursors (MMPs: Gr-1+Ly6G+Ly6C-) in bone marrow (BM). However, lactobacilli treatments were able to significantly increase blood neutrophils and peroxidase-positive cells, while improving cytokine production and phagocytic activity of alveolar macrophages. This, in turn, led to an early Sp lung clearance compared to the control group. Furthermore, Lr05V was more effective than Lr05NV to increase growth factors in BM, which allowed an early HSCs and MMPs recovery with respect to the control group. Both Lr05V and Lr05NV were able to improve BM emergency myelopiesis and protection against respiratory pathogens in mice undergoing chemotherapy.

Lysine Methylation-Dependent Proteolysis by the Malignant Brain Tumor (MBT) Domain Proteins.

Lysine methylation is a major post-translational protein modification that occurs in both histones and non-histone proteins. Emerging studies show that the methylated lysine residues in non-histone proteins provide a proteolytic signal for ubiquitin-dependent proteolysis. The SET7 (SETD7) methyltransferase specifically transfers a methyl group from S-Adenosyl methionine to a specific lysine residue located in a methylation degron motif of a protein substrate to mark the methylated protein for ubiquitin-dependent proteolysis. LSD1 (Kdm1a) serves as a demethylase to dynamically remove the methyl group from the modified protein. The methylated lysine residue is specifically recognized by L3MBTL3, a methyl-lysine reader that contains the malignant brain tumor domain, to target the methylated proteins for proteolysis by the CRL4DCAF5 ubiquitin ligase complex. The methylated lysine residues are also recognized by PHF20L1 to protect the methylated proteins from proteolysis. The lysine methylation-mediated proteolysis regulates embryonic development, maintains pluripotency and self-renewal of embryonic stem cells and other stem cells such as neural stem cells and hematopoietic stem cells, and controls other biological processes. Dysregulation of the lysine methylation-dependent proteolysis is associated with various diseases, including cancers. Characterization of lysine methylation should reveal novel insights into how development and related diseases are regulated.

CRL4B E3 ligase recruited by PRPF19 inhibits SARS-CoV-2 infection by targeting ORF6 for ubiquitin-dependent degradation.

The accessory protein ORF6 of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key interferon (IFN) antagonist that strongly suppresses the production of primary IFN as well as the expression of IFN-stimulated genes. However, how host cells respond to ORF6 remains largely unknown. Our research of ORF6-binding proteins by pulldown revealed that E3 ligase components such as Cullin 4B (CUL4B), DDB1, and RBX1 are potential ORF6-interacting proteins. Further study found that the substrate recognition receptor PRPF19 interacts with CUL4B, DDB1, and RBX1 to form a CRL4B-based E3 ligase, which catalyzes ORF6 ubiquitination and subsequent degradation. Overexpression of PRPF19 promotes ORF6 degradation, releasing ORF6-mediated IFN inhibition, which inhibits SARS-CoV-2 replication. Moreover, we found that activation of CUL4B by the neddylation inducer etoposide alleviates lung lesions in a SARS-CoV-2 mouse infection model. Therefore, targeting ORF6 for degradation may be an effective therapeutic strategy against SARS-CoV-2 infection.IMPORTANCEThe cellular biological function of the ubiquitin-proteasome pathway as an important modulator for the regulation of many fundamental cellular processes has been greatly appreciated. The critical role of the ubiquitin-proteasome pathway in viral pathogenesis has become increasingly apparent. It is a powerful tool that host cells use to defend against viral infection. Some cellular proteins can function as restriction factors to limit viral infection by ubiquitin-dependent degradation. In this research, we identificated of CUL4B-DDB1-PRPF19 E3 Ubiquitin Ligase Complex can mediate proteasomal degradation of ORF6, leading to inhibition of viral replication. Moreover, the CUL4B activator etoposide alleviates disease development in a mouse infection model, suggesting that this agent or its derivatives may be used to treat infections caused by SARS-CoV-2. We believe that these results will be extremely useful for the scientific and clinic communities in their search for cues and preventive measures to combat the COVID-19 pandemic.