Profiling Cullin4-E3 ligases interactomes and their rewiring in influenza A virus infection.

Understanding the integrated regulation of cellular processes during viral infection is crucial for developing host-targeted approaches. We have previously reported that an optimal in vitro infection by influenza A (IAV) requires three components of Cullin 4-RING E3 ubiquitin ligases (CRL4) complexes, namely the DDB1 adaptor and two Substrate Recognition Factors (SRF), DCAF11 and DCAF12L1, which mediate non-degradative poly-ubiquitination of the PB2 subunit of the viral polymerase. However, the impact of IAV infection on the CRL4 interactome remains elusive. Here, using Affinity Purification coupled with Mass Spectrometry (AP-MS) approaches, we identified cellular proteins interacting with these CRL4 components in IAV-infected and non-infected contexts. IAV infection induces significant modulations in protein interactions, resulting in a global loss of DDB1 and DCAF11 interactions, and an increase in DCAF12L1-associated proteins. The distinct rewiring of CRL4's associations upon infection impacted cellular proteins involved in protein folding, ubiquitination, translation, splicing, and stress responses. Using a split-nanoluciferase-based assay, we identified direct partners of CRL4 components and via siRNA-mediated silencing validated their role in IAV infection, representing potential substrates or regulators of CRL4 complexes. Our findings unravel the dynamic remodeling of the proteomic landscape of CRL4's E3 ubiquitin ligases during IAV infection, likely involved in shaping a cellular environment conducive to viral replication and offer potential for the exploration of future host-targeted antiviral therapeutic strategies.

SARS-CoV-2 ORF10 hijacking ubiquitination machinery reveals potential unique drug targeting sites.

Viruses often manipulate ubiquitination pathways to facilitate their replication and pathogenesis. CUL2ZYG11B known as the substrate receptor of cullin-2 RING E3 ligase, is bound by SARS-CoV-2 ORF10 to increase its E3 ligase activity, leading to degradation of IFT46, a protein component of the intraflagellar transport (IFT) complex B. This results in dysfunctional cilia, which explains certain symptoms that are specific to COVID-19. However, the precise molecular mechanism of how ORF10 recognizes CUL2ZYG11B remains unknown. Here, we determined the crystal structure of CUL2ZYG11B complexed with the N-terminal extension (NTE) of SARS-CoV-2 ORF10 (2.9 Å). The structure reveals that the ORF10 N-terminal heptapeptide (NTH) mimics the Gly/N-degron to bind CUL2ZYG11B. Mutagenesis studies identified key residues within ORF10 that are key players in its interaction with CUL2ZYG11B both in ITC assay and in vivo cells. In addition, we prove that enhancement of CUL2ZYG11B activity for IFT46 degradation by which ORF10-mediated correlates with the binding affinity between ORF10 and CUL2ZYG11B. Finally, we used a Global Protein Stability system to show that the NTH of ORF10 mimics the Gly/N-degron motif, thereby binding competitively to CUL2ZYG11B and inhibiting the degradation of target substrates bearing the Gly/N-degron motif. Overall, this study sheds light on how SARS-CoV-2 ORF10 exploits the ubiquitination machinery for proteasomal degradation, and offers valuable insights for optimizing PROTAC-based drug design based on NTH CUL2ZYG11B interaction, while pinpointing a promising target for the development of treatments for COVID-19.

FBXO38 is dispensable for PD-1 regulation.

SKP1-CUL1-F-box protein (SCF) ubiquitin ligases are versatile protein complexes that mediate the ubiquitination of protein substrates. The direct substrate recognition relies on a large family of F-box-domain-containing subunits. One of these substrate receptors is FBXO38, which is encoded by a gene found mutated in families with early-onset distal motor neuronopathy. SCFFBXO38 ubiquitin ligase controls the stability of ZXDB, a nuclear factor associated with the centromeric chromatin protein CENP-B. Loss of FBXO38 in mice results in growth retardation and defects in spermatogenesis characterized by deregulation of the Sertoli cell transcription program and compromised centromere integrity. Moreover, it was reported that SCFFBXO38 mediates the degradation of PD-1, a key immune-checkpoint inhibitor in T cells. Here, we have re-addressed the link between SCFFBXO38 and PD-1 proteolysis. Our data do not support the notion that SCFFBXO38 directly or indirectly controls the abundance and stability of PD-1 in T cells.

Knockdown of cullin 3 inhibits progressive phenotypes and increases chemosensitivity in cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) is an extremely aggressive malignancy arising from the epithelial cells lining the bile ducts. It presents a substantial global health issue, with the highest incidence rates, ranging from 40­100 cases/100,000 individuals, found in Southeast Asia, where liver fluke infection is endemic. In Europe and America, incidence rates range from 0.4­2 cases/100,000 individuals. Globally, mortality rates range from 0.2­2 deaths/100,000 person­years and are increasing in most countries. Chemotherapy is the primary treatment for advanced CCA due to limited options from late­stage diagnosis, but its efficacy is hindered by drug­resistant phenotypes. In a previous study, proteomics analysis of drug­resistant CCA cell lines (KKU­213A­FR and KKU­213A­GR) and the parental KKU­213A line identified cullin 3 (Cul3) as markedly overexpressed in drug­resistant cells. Cul3, a scaffold protein within CUL3­RING ubiquitin ligase complexes, is crucial for ubiquitination and proteasome degradation, yet its role in drug­resistant CCA remains to be elucidated. The present study aimed to elucidate the role of Cul3 in drug­resistant CCA cell lines. Reverse transcription­quantitative PCR and western blot analyses confirmed significantly elevated Cul3 mRNA and protein levels in drug­resistant cell lines compared with the parental control. Short interfering RNA­mediated Cul3 knockdown sensitized cells to 5­fluorouracil and gemcitabine and inhibited cell proliferation, colony formation, migration and invasion. In addition, Cul3 knockdown induced G0/G1 cell cycle arrest and suppressed key cell cycle regulatory proteins, cyclin D, cyclin­dependent kinase (CDK)4 and CDK6. Bioinformatics analysis of CCA patient samples using The Cancer Genome Atlas data revealed Cul3 upregulation in CCA tissues compared with normal bile duct tissues. STRING analysis of upregulated proteins in drug­resistant CCA cell lines identified a highly interactive Cul3 network, including COMM Domain Containing 3, Ariadne RBR E3 ubiquitin protein ligase 1, Egl nine homolog 1, Proteasome 26S Subunit Non­ATPase 13, DExH­box helicase 9 and small nuclear ribonucleoprotein polypeptide G, which showed a positive correlation with Cul3 in CCA tissues. Knocking down Cul3 significantly suppressed the mRNA expression of these genes, suggesting that Cul3 may act as an upstream regulator of them. Gene Ontology analysis revealed that the majority of these genes were categorized under binding function, metabolic process, cellular anatomical entity, protein­containing complex and protein­modifying enzyme. Taken together, these findings highlighted the biological and clinical significance of Cul3 in drug resistance and progression of CCA.

Zika virus NS5 protein inhibits type I interferon signaling via CRL3 E3 ubiquitin ligase-mediated degradation of STAT2.

The ZIKA virus (ZIKV) evades the host immune response by degrading STAT2 through its NS5 protein, thereby inhibiting type I interferon (IFN)-mediated antiviral immunity. However, the molecular mechanism underlying this process has remained elusive. In this study, we performed a genome-wide CRISPR/Cas9 screen, revealing that ZSWIM8 as the substrate receptor of Cullin3-RING E3 ligase is required for NS5-mediated STAT2 degradation. Genetic depletion of ZSWIM8 and CUL3 substantially impeded NS5-mediated STAT2 degradation. Biochemical analysis illuminated that NS5 enhances the interaction between STAT2 and the ZSWIM8-CUL3 E3 ligase complex, thereby facilitating STAT2 ubiquitination. Moreover, ZSWIM8 knockout endowed A549 and Huh7 cells with partial resistance to ZIKV infection and protected cells from the cytopathic effects induced by ZIKV, which was attributed to the restoration of STAT2 levels and the activation of IFN signaling. Subsequent studies in a physiologically relevant model, utilizing human neural progenitor cells, demonstrated that ZSWIM8 depletion reduced ZIKV infection, resulting from enhanced IFN signaling attributed to the sustained levels of STAT2. Our findings shed light on the role of ZIKV NS5, serving as the scaffold protein, reprograms the ZSWIM8-CUL3 E3 ligase complex to orchestrate STAT2 proteasome-dependent degradation, thereby facilitating evasion of IFN antiviral signaling. Our study provides unique insights into ZIKV-host interactions and holds promise for the development of antivirals and prophylactic vaccines.

Ribosomal Protein S4 X-Linked as a Novel Modulator of MDM2 Stability by Suppressing MDM2 Auto-Ubiquitination and SCF Complex-Mediated Ubiquitination.

Mouse double minute 2 (MDM2) is an oncoprotein that is frequently overexpressed in tumors and enhances cellular transformation. Owing to the important role of MDM2 in modulating p53 function, it is crucial to understand the mechanism underlying the regulation of MDM2 levels. We identified ribosomal protein S4X-linked (RPS4X) as a novel binding partner of MDM2 and showed that RPS4X promotes MDM2 stability. RPS4X suppressed polyubiquitination of MDM2 by suppressing homodimer formation and preventing auto-ubiquitination. Moreover, RPS4X inhibited the interaction between MDM2 and Cullin1, a scaffold protein of the Skp1-Cullin1-F-box protein (SCF) complex and an E3 ubiquitin ligase for MDM2. RPS4X expression in cells enhanced the steady-state level of MDM2 protein. RPS4X was associated not only with MDM2 but also with Cullin1 and then blocked the MDM2/Cullin1 interaction. This is the first report of an interaction between ribosomal proteins (RPs) and Cullin1. Our results contribute to the elucidation of the MDM2 stabilization mechanism in cancer cells, expanding our understanding of the new functions of RPs.

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.

Discovery of small molecule inhibitors of neddylation catalyzing enzymes for anticancer therapy.

Protein neddylation, a type of post-translational modifications, involves the transfer of the ubiquitin-like protein NEDD8 to the lysine residues of a target substrate, which is catalyzed by the NEDD8 activating enzyme (E1), NEDD8 conjugating enzyme (E2), and NEDD8 ligase (E3). Cullin family proteins, core components of Cullin-RING E3 ubiquitin ligases (CRLs), are the most well-known physiological substrates of neddylation. CRLs, activated upon cullin neddylation, promote the ubiquitination of a variety of key signaling proteins for proteasome degradation, thereby regulating many critical biological functions. Abnormal activation of neddylation enzymes as well as CRLs has been frequently observed in various human cancers and is associated with poor prognosis for cancer patients. Consequently, targeting neddylation has emerged as a promising strategy for the development of novel anticancer therapeutics. This review first briefly introduces the properties of protein neddylation and its role in cancer, and then systematically summarizes all reported chemical inhibitors of the three neddylation enzymes, providing a focused, up to date, and comprehensive resource in the discovery and development of these small molecule inhibitors.

A BTB extension and ion-binding domain contribute to the pentameric structure and TFAP2A binding of KCTD1.

KCTD family proteins typically assemble into cullin-RING E3 ligases. KCTD1 is an atypical member that functions instead as a transcriptional repressor. Mutations in KCTD1 cause developmental abnormalities and kidney fibrosis in scalp-ear-nipple syndrome. Here, we present unexpected mechanistic insights from the structure of human KCTD1. Disease-causing mutation P20S maps to an unrecognized extension of the BTB domain that contributes to both its pentameric structure and TFAP2A binding. The C-terminal domain (CTD) shares its fold and pentameric assembly with the GTP cyclohydrolase I feedback regulatory protein (GFRP) despite lacking discernible sequence similarity. Most surprisingly, the KCTD1 CTD establishes a central channel occupied by alternating sodium and iodide ions that restrict TFAP2A dissociation. The elucidation of the structure redefines the KCTD1 BTB domain fold and identifies an unexpected ion-binding site for future study of KCTD1's function in the ectoderm, neural crest, and kidney.

Distal convoluted tubule-specific disruption of the COP9 signalosome but not its regulatory target cullin 3 causes tubular injury.

The disease familial hyperkalemic hypertension (FHHt; also known as Gordon syndrome) is caused by aberrant accumulation of with-no-lysine kinase (WNK4) activating the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney. Mutations in cullin 3 (CUL3) cause FHHt by disrupting interaction with the deneddylase COP9 signalosome (CSN). Deletion of Cul3 or Jab1 (the catalytically active CSN subunit) along the entire nephron causes a partial FHHt phenotype with activation of the WNK4-STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NCC pathway. However, progressive kidney injury likely prevents hypertension, hyperkalemia, and hyperchloremic metabolic acidosis associated with FHHt. We hypothesized that DCT-specific deletion would more closely model the disease. We used Slc12a3-Cre-ERT2 mice to delete Cul3 (DCT-Cul3-/-) or Jab1 (DCT-Jab1-/-) only in the DCT and examined the mice after short- and long-term deletion. Short-term DCT-specific knockout of both Cul3 and Jab1 mice caused elevated WNK4, pSPAKS373, and pNCCT53 abundance. However, neither model demonstrated changes in plasma K+, Cl-, or total CO2, even though no injury was present. Long-term DCT-Jab1-/- mice showed significantly lower NCC and parvalbumin abundance and a higher abundance of kidney injury molecule-1, a marker of proximal tubule injury. No injury or reduction in NCC or parvalbumin was observed in long-term DCT-Cul3-/- mice. In summary, the prevention of injury outside the DCT did not lead to a complete FHHt phenotype despite activation of the WNK4-SPAK-NCC pathway, possibly due to insufficient NCC activation. Chronically, only DCT-Jab1-/- mice developed tubule injury and atrophy of the DCT, suggesting a direct JAB1 effect or dysregulation of other cullins as mechanisms for injury.NEW & NOTEWORTHY CUL3 degrades WNK4, which prevents activation of NCC in the DCT. CSN regulation of CUL3 is impaired in the disease FHHt, causing accumulation of WNK4. Short-term DCT-specific disruption of CUL3 or the CSN in mice resulted in activation of the WNK4-SPAK-NCC pathway but not hyperkalemic metabolic acidosis found in FHHt. Tubule injury was observed only after long-term CSN disruption. The data suggest that disruption of other cullins may be the cause for the injury.

Proteomic analysis reveals a PLK1-dependent G2/M degradation program and a role for AKAP2 in coordinating the mitotic cytoskeleton.

Ubiquitination is an essential regulator of cell division. The kinase Polo-like kinase 1 (PLK1) promotes protein degradation at G2/M phase through the E3 ubiquitin ligase Skp1-Cul1-F box (SCF)ßTrCP. However, the magnitude to which PLK1 shapes the mitotic proteome is uncharacterized. Combining quantitative proteomics with pharmacologic PLK1 inhibition revealed a widespread, PLK1-dependent program of protein breakdown at G2/M. We validated many PLK1-regulated proteins, including substrates of the cell-cycle E3 SCFCyclin F, demonstrating that PLK1 promotes proteolysis through at least two distinct E3 ligases. We show that the protein-kinase-A-anchoring protein A-kinase anchor protein 2 (AKAP2) is cell-cycle regulated and that its mitotic degradation is dependent on the PLK1/ßTrCP signaling axis. Expression of a non-degradable AKAP2 mutant resulted in actin defects and aberrant mitotic spindles, suggesting that AKAP2 degradation coordinates cytoskeletal organization during mitosis. These findings uncover PLK1's far-reaching role in shaping the mitotic proteome post-translationally and have potential implications in malignancies where PLK1 is upregulated.

Stabilization of GTSE1 by cyclin D1-CDK4/6 promotes cell proliferation: relevance in cancer prognosis.

Cyclin D1 is the activating subunit of the cell cycle kinases CDK4 and CDK6, and its dysregulation is a well-known oncogenic driver in many human cancers. The biological function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G2 and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unknown substrate of cyclin D1-CDK4/6. The phosphorylation of GTSE1 mediated by cyclin D1-CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 also during the G1 phase of the cell cycle. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1's role in cell cycle control and oncogenesis beyond RB phosphorylation.

TRAF2 associates with cullin neddylation complex assembly.

Cullin-based RING ligases (CRLs) comprise the largest family of ubiquitin E3 ligases. CRL activity is tightly regulated by cullin neddylation, which has been associated with various diseases. Although inhibitors of CRLs neddylation have been reported, there is a lack of small molecules that can selectively target individual cullins. Here, we identified a natural product, liquidambaric acid (LDA), with relatively selective inhibition properties against cullin (Cul) 2 neddylation, and found that its target, Tumor Necrosis Factor receptor-associated factor 2 (TRAF2) was required for the activity. TRAF2 associates with the Cul2 neddylation complex and regulates the machinery assembly, especially that of E2 (UBC12) and E3 (RBX1) enzymes. In addition, we demonstrated that by intervention of the associations between TRAF2 and the neddylation machinery, LDA disturbed NEDD8 transfer from E1 to E2, therefore blocking Cul2 neddylation. Taken together, we show that TRAF2 plays a positive role in neddylation cascades, and we have identified a small molecule capable of selective modulation of cullin neddylation.

Casein kinase 1α mediates phosphorylation of the Merkel cell polyomavirus large T antigen for ß-TrCP destruction complex interaction and subsequent degradation.

Merkel cell polyomavirus (MCPyV) is a double-stranded tumor virus that is the main causative agent of Merkel cell carcinoma (MCC). The MCPyV large T antigen (LT), an essential viral DNA replication protein, maintains viral persistence by interacting with host Skp1-Cullin 1-F-box (SCF) E3 ubiquitin ligase complexes, which subsequently induces LT's proteasomal degradation, restricting MCPyV DNA replication. SCF E3 ubiquitin ligases require their substrates to be phosphorylated to bind them, utilizing phosphorylated serine residues as docking sites. The MCPyV LT unique region (MUR) is highly phosphorylated and plays a role in multiple host protein interactions, including SCF E3 ubiquitin ligases. Therefore, this domain highly governs LT stability. Though much work has been conducted to identify host factors that restrict MCPyV LT protein expression, the kinase(s) that cooperates with the SCF E3 ligase remains unknown. Here, we demonstrate that casein kinase 1 alpha (CK1α) negatively regulates MCPyV LT stability and LT-mediated replication by modulating interactions with the SCF ß-TrCP. Specifically, we show that numerous CK1 isoforms (α, δ, ε) localize in close proximity to MCPyV LT through in situ proximity ligation assays (PLA) and CK1α overexpression mainly resulted in decreased MCPyV LT protein expression. Inhibition of CK1α using short hairpin RNA (shRNA) and treatment of a CK1α inhibitor or an mTOR inhibitor, TORKinib, resulted in decreased ß-TrCP interaction with LT, increased LT expression, and enhanced MCPyV replication. The expression level of the CSNK1A1 gene transcripts is higher in MCPyV-positive MCC, suggesting a vital role of CK1α in limiting MCPyV replication required for establishing persistent infection. IMPORTANCE: Merkel cell polyomavirus (MCPyV) large tumor antigen is a polyphosphoprotein and the phosphorylation event is required to modulate various functions of LT, including viral replication. Therefore, cellular kinase pathways are indispensable for governing MCPyV polyomavirus infection and life cycle in coordinating with the immunosuppression environment at disease onset. Understanding the regulation mechanisms of MCPyV replication by viral and cellular factors will guide proper prevention strategies with targeted inhibitors for MCPyV-associated Merkel cell carcinoma (MCC) patients, who currently lack therapies.

Association of CUL4B with the pathogenesis of age-related cataract.

PURPOSE: Age-related cataract (ARC) is the most common cause of visual impairment and blindness in older adults. However, the role of CUL4B in the ARC remains unclear. Therefore, we investigated CUL4B expression and its effects on apoptosis. MATERIALS AND METHODS: CUL4B expression levels were detected by a quantitative real-time polymerase chain reaction from the anterior lens capsules of patients with ARC and HLE-B3 cells treated with different concentrations of H2O2. CUL4B expression was silenced by siRNA transfection to evaluate apoptosis. CUL4B and apoptotic proteins B cell lymphoma 2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), caspase-3, cleaved caspase-3, Bax, Bak, and Bid were assessed using western blot analysis. Apoptosis was monitored using the TUNEL assay. RESULTS: CUL4B expression was downregulated in the anterior lens capsules (P < 0.0001) and H2O2-treated HLE-B3 cells (P = 0.0405). CUL4B protein levels were significantly lower in 100 µmol/L (P = 0.0012) and 200 µmol/L (P = 0.0041) H2O2-treated HLE-B3 cells than in the untreated cells. CUL4B expression was significantly knocked down at the mRNA (P = 0.0043) and protein levels (P = 0.0002) in HLE-B3 cells. Bcl-2 (P = 0.0199), Mcl-1 (P = 0.0042), and caspase-3 (P = 0.0142) were significantly downregulated, whereas cleaved caspase-3 (P = 0.0089) and Bak (P = 0.009) were significantly upregulated in the knockdown group. The TUNEL assay showed a greater induction of apoptosis. CONCLUSIONS: CUL4B downregulation promotes the apoptosis of lens epithelial cells. Our study may help in understanding the role of CUL4B in ARC pathogenesis.

Comprehensive analysis of the Cullin family of genes reveals that CUL7 and CUL9 are the significant prognostic biomarkers in colorectal cancer.

OBJECTIVES: The purpose of this study is to decipher the role of Cullin family genes in colorectal cancer (CRC), drawing insights from comprehensive analyses encompassing multiple databases and experimental validations. METHODS: UALCAN, GEPIA2, Human Protein Atlas (HPA), KM plotter, cBioPortal, TISIDB, DAVID, colon cancer cell lines culturing, gene knockdown, CCK8 assay, colony formation, and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) assays. RESULTS: Initial scrutiny of The Cancer Genome Atlas (TCGA) CRC datasets through the UALCAN and GEPIA databases unveiled significant alterations in Cullin family gene expressions. Elevations in CUL1, CUL2, CUL4A, CUL4B, CUL5, CUL7, and CUL9 were observed in CRC tissues compared to normal counterparts, while CUL3 demonstrated down-regulation consistently across datasets. Further exploration revealed notable correlations between Cullin gene expressions and various clinical parameters of CRC patients, substantiating the potential diagnostic and prognostic utility of these genes. Protein expression analyses conducted via the HPA corroborated the transcriptomic findings, indicating high levels of Cullin proteins in CRC tissues. Prognostic assessments identified CUL7 and CUL9 as significant predictors of poor survival outcomes in CRC patients, emphasizing their clinical relevance. Genetic alterations within the Cullin family genes were elucidated through the cBioPortal database, shedding light on the mutation landscape and prevalence of missense mutations in CRC. Immune subtype and tumor immune microenvironment analyses underscored the intricate interplay between Cullin family genes and immune processes in CRC. Experimental validation in CRC cell lines demonstrated the functional significance of CUL7 and CUL9 in promoting CRC growth, further solidifying their roles as potential therapeutic targets. CONCLUSION: Overall, these multifaceted analyses elucidated the intricate involvement of Cullin family genes in CRC pathogenesis and provided valuable insights for future diagnostic and therapeutic endeavors in CRC management.

CUL-6/cullin ubiquitin ligase-mediated degradation of HSP-90 by intestinal lysosomes promotes thermotolerance.

Heat shock can be a lethal stressor. Previously, we described a CUL-6/cullin-ring ubiquitin ligase complex in the nematode Caenorhabditis elegans that is induced by intracellular intestinal infection and proteotoxic stress and that promotes improved survival upon heat shock (thermotolerance). Here, we show that CUL-6 promotes thermotolerance by targeting the heat shock protein HSP-90 for degradation. We show that CUL-6-mediated lowering of HSP-90 protein levels, specifically in the intestine, improves thermotolerance. Furthermore, we show that lysosomal function is required for CUL-6-mediated promotion of thermotolerance and that CUL-6 directs HSP-90 to lysosome-related organelles upon heat shock. Altogether, these results indicate that a CUL-6 ubiquitin ligase promotes organismal survival upon heat shock by promoting HSP-90 degradation in intestinal lysosomes. Thus, HSP-90, a protein commonly associated with protection against heat shock and promoting degradation of other proteins, is itself degraded to protect against heat shock.

Enhanced ApcMin/+ adenoma formation after epithelial CUL4B deletion by recruitment of myeloid-derived suppressor cells.

Colorectal cancer (CRC) stands as a prevalent malignancy globally. A pivotal event in CRC pathogenesis involves the loss-of-function mutation in the APC gene, leading to the formation of benign polyps. Despite the well-established role of APC, the contribution of CUL4B to CRC initiation in the pre-tumorous stage remains poorly understood. In this investigation, we generated a murine model by crossing ApcMin/+ mice with Cul4bΔIEC mice to achieve specific deletion of Cul4b in the gut epithelium against an ApcMin/+ background. By employing histological methods, RNA-sequencing (RNA-seq), and flow cytometry, we assessed alterations and characterized the immune microenvironment. Our results unveiled that CUL4B deficiency in gut epithelium expedited ApcMin/+ adenoma formation. Notably, CUL4B in adenomas restrained the accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs). In vivo inhibition of MDSCs significantly delayed the growth of CUL4B deleted ApcMin/+ adenomas. Furthermore, the addition of MDSCs to in vitro cultured ApcMin/+; Cul4bΔIEC adenoma organoids mitigated their alterations. Mechanistically, CUL4B directly interacted with the promoter of Csf3, the gene encoding granulocyte-colony stimulating factor (G-CSF) by coordinating with PRC2. Inhibiting CUL4B epigenetically activated the expression of G-CSF, promoting the recruitment of MDSCs. These findings offer novel insights into the tumor suppressor-like roles of CUL4B in regulating ApcMin/+ adenomas, suggesting a potential therapeutic strategy for CRC initiation and progression in the context of activated Wnt signaling.

Autism risk gene Cul3 alters neuronal morphology via caspase-3 activity in mouse hippocampal neurons.

Autism Spectrum Disorders (ASDs) are neurodevelopmental disorders (NDDs) in which children display differences in social interaction/communication and repetitive stereotyped behaviors along with variable associated features. Cul3, a gene linked to ASD, encodes CUL3 (CULLIN-3), a protein that serves as a key component of a ubiquitin ligase complex with unclear function in neurons. Cul3 homozygous deletion in mice is embryonic lethal; thus, we examine the role of Cul3 deletion in early synapse development and neuronal morphology in hippocampal primary neuronal cultures. Homozygous deletion of Cul3 significantly decreased dendritic complexity and dendritic length, as well as axon formation. Synaptic spine density significantly increased, mainly in thin and stubby spines along with decreased average spine volume in Cul3 knockouts. Both heterozygous and homozygous knockout of Cul3 caused significant reductions in the density and colocalization of gephyrin/vGAT puncta, providing evidence of decreased inhibitory synapse number, while excitatory synaptic puncta vGulT1/PSD95 density remained unchanged. Based on previous studies implicating elevated caspase-3 after Cul3 deletion, we demonstrated increased caspase-3 in our neuronal cultures and decreased neuronal cell viability. We then examined the efficacy of the caspase-3 inhibitor Z-DEVD-FMK to rescue the decrease in neuronal cell viability, demonstrating reversal of the cell viability phenotype with caspase-3 inhibition. Studies have also implicated caspase-3 in neuronal morphological changes. We found that caspase-3 inhibition largely reversed the dendrite, axon, and spine morphological changes along with the inhibitory synaptic puncta changes. Overall, these data provide additional evidence that Cul3 regulates the formation or maintenance of cell morphology, GABAergic synaptic puncta, and neuronal viability in developing hippocampal neurons in culture.

Cullin 3 RING E3 ligase inactivation causes NRF2-dependent NADH reductive stress, hepatic lipodystrophy, and systemic insulin resistance.

Cullin RING E3 ligases (CRL) have emerged as key regulators of disease-modifying pathways and therapeutic targets. Cullin3 (Cul3)-containing CRL (CRL3) has been implicated in regulating hepatic insulin and oxidative stress signaling. However, CRL3 function in liver pathophysiology is poorly defined. Here, we report that hepatocyte Cul3 knockout results in rapid resolution of steatosis in obese mice. However, the remarkable resistance of hepatocyte Cul3 knockout mice to developing steatosis does not lead to overall metabolic improvement but causes systemic metabolic disturbances. Liver transcriptomics analysis identifies that CRL3 inactivation causes persistent activation of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant defense pathway, which also reprograms the lipid transcriptional network to prevent TG storage. Furthermore, global metabolomics reveals that NRF2 activation induces numerous NAD+-consuming aldehyde dehydrogenases to increase the cellular NADH/NAD+ ratio, a redox imbalance termed NADH reductive stress that inhibits the glycolysis-citrate-lipogenesis axis in Cul3 knockout livers. As a result, this NRF2-induced cellular lipid storage defect promotes hepatic ceramide accumulation, elevates circulating fatty acids, and worsens systemic insulin resistance in a vicious cycle. Hepatic lipid accumulation is restored, and liver injury and hyperglycemia are attenuated when NRF2 activation and NADH reductive stress are abolished in hepatocyte Cul3/Nrf2 double-knockout mice. The resistance to hepatic steatosis, hyperglycemia, and NADH reductive stress are observed in hepatocyte Keap1 knockout mice with NRF2 activation. In summary, our study defines a critical role of CRL3 in hepatic metabolic regulation and demonstrates that the CRL3 downstream NRF2 overactivation causes hepatic metabolic maladaptation to obesity and insulin resistance.