Formation of NPR1 Condensates Promotes Cell Survival during the Plant Immune Response.

In plants, pathogen effector-triggered immunity (ETI) often leads to programmed cell death, which is restricted by NPR1, an activator of systemic acquired resistance. However, the biochemical activities of NPR1 enabling it to promote defense and restrict cell death remain unclear. Here we show that NPR1 promotes cell survival by targeting substrates for ubiquitination and degradation through formation of salicylic acid-induced NPR1 condensates (SINCs). SINCs are enriched with stress response proteins, including nucleotide-binding leucine-rich repeat immune receptors, oxidative and DNA damage response proteins, and protein quality control machineries. Transition of NPR1 into condensates is required for formation of the NPR1-Cullin 3 E3 ligase complex to ubiquitinate SINC-localized substrates, such as EDS1 and specific WRKY transcription factors, and promote cell survival during ETI. Our analysis of SINCs suggests that NPR1 is centrally integrated into the cell death or survival decisions in plant immunity by modulating multiple stress-responsive processes in this quasi-organelle.

SPT5 stabilization of promoter-proximal RNA polymerase II.

Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression.

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.

The orphan nuclear receptor SHP regulates ER stress response by inhibiting XBP1s degradation.

The orphan nuclear receptor SHP (small heterodimer partner) is a well-known transcriptional corepressor of bile acid and lipid metabolism in the liver; however, its function in other tissues is poorly understood. Here, we report an unexpected role for SHP in the exocrine pancreas as a modulator of the endoplasmic reticulum (ER) stress response. SHP expression is induced in acinar cells in response to ER stress and regulates the protein stability of the spliced form of X-box-binding protein 1 (XBP1s), a key mediator of ER stress response. Loss of SHP reduces XBP1s protein level and transcriptional activity, which in turn attenuates the ER stress response during the fasting-feeding cycle. Consequently, SHP-deficient mice also are more susceptible to cerulein-induced pancreatitis. Mechanistically, we show that SHP physically interacts with the transactivation domain of XBP1s, thereby inhibiting the polyubiquitination and degradation of XBP1s by the Cullin3-SPOP (speckle-type POZ protein) E3 ligase complex. Together, our data implicate SHP in governing ER homeostasis and identify a novel posttranslational regulatory mechanism for the key ER stress response effector XBP1.

UBE2M Is a Stress-Inducible Dual E2 for Neddylation and Ubiquitylation that Promotes Targeted Degradation of UBE2F.

UBE2M and UBE2F are two family members of neddylation E2 conjugating enzyme that, together with E3s, activate CRLs (Cullin-RING Ligases) by catalyzing cullin neddylation. However, whether and how two E2s cross-talk with each other are largely unknown. Here, we report that UBE2M is a stress-inducible gene subjected to cis-transactivation by HIF-1 and AP1, and MLN4924, a small molecule inhibitor of E1 NEDD8-activating enzyme (NAE), upregulates UBE2M via blocking degradation of HIF-1α and c-JUN. UBE2M is a dual E2 for targeted ubiquitylation and degradation of UBE2F, acting as a neddylation E2 to activate CUL3-Keap1 E3 under physiological conditions but as a ubiquitylation E2 for Parkin-DJ-1 E3 under stressed conditions. UBE2M-induced UBE2F degradation leads to CRL5 inactivation and subsequent NOXA accumulation to suppress the growth of lung cancer cells. Collectively, our study establishes a negative regulatory axis between two neddylation E2s with UBE2M ubiquitylating UBE2F, and two CRLs with CRL3 inactivating CRL5.

Deficiency of Cullin 3, a Protein Encoded by a Schizophrenia and Autism Risk Gene, Impairs Behaviors by Enhancing the Excitability of Ventral Tegmental Area (VTA) DA Neurons.

The dopaminergic neuromodulator system is fundamental to brain functions. Abnormal dopamine (DA) pathway is implicated in psychiatric disorders, including schizophrenia (SZ) and autism spectrum disorder (ASD). Mutations in Cullin 3 (CUL3), a core component of the Cullin-RING ubiquitin E3 ligase complex, have been associated with SZ and ASD. However, little is known about the function and mechanism of CUL3 in the DA system. Here, we show that CUL3 is critical for the function of DA neurons and DA-relevant behaviors in male mice. CUL3-deficient mice exhibited hyperactive locomotion, deficits in working memory and sensorimotor gating, and increased sensitivity to psychostimulants. In addition, enhanced DA signaling and elevated excitability of the VTA DA neurons were observed in CUL3-deficient animals. Behavioral impairments were attenuated by dopamine D2 receptor antagonist haloperidol and chemogenetic inhibition of DA neurons. Furthermore, we identified HCN2, a hyperpolarization-activated and cyclic nucleotide-gated channel, as a potential target of CUL3 in DA neurons. Our study indicates that CUL3 controls DA neuronal activity by maintaining ion channel homeostasis and provides insight into the role of CUL3 in the pathogenesis of psychiatric disorders.SIGNIFICANCE STATEMENT This study provides evidence that Cullin 3 (CUL3), a core component of the Cullin-RING ubiquitin E3 ligase complex that has been associated with autism spectrum disorder and schizophrenia, controls the excitability of dopamine (DA) neurons in mice. Its DA-specific heterozygous deficiency increased spontaneous locomotion, impaired working memory and sensorimotor gating, and elevated response to psychostimulants. We showed that CUL3 deficiency increased the excitability of VTA DA neurons, and inhibiting D2 receptor or DA neuronal activity attenuated behavioral deficits of CUL3-deficient mice. We found HCN2, a hyperpolarization-activated channel, as a target of CUL3 in DA neurons. Our findings reveal CUL3's role in DA neurons and offer insights into the pathogenic mechanisms of autism spectrum disorder and schizophrenia.

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 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-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 utilized Slc12a3-Cre-ERT2 mice to delete Cul3 (DCT-Cul3-/-) or Jab1 (DCT-Jab1-/-) only in 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, SPAK, and pNCC abundance. However, neither model demonstrated changes in plasma K+, Cl-, or TCO2, even though no injury was present. Long-term DCT-Jab1-/- mice showed significantly lower NCC and parvalbumin abundance, and higher abundance of kidney injury molecule 1 (KIM-1), a marker of proximal tubule injury. No injury, or reduction in NCC or parvalbumin were 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.

Buffalo colostrum peptide mitigates Parkinson's disease pathophysiology through Cullin-3 inhibition.

Colostrum/Milk is a chief repertoire of antioxidant peptides. Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a viable target for Parkinson's Disease (PD), as this pathway deduced to be impaired in PD. Cullin-3 is one of the crucial E3 ligase responsible for its regulation. The present study screened peptide libraries of buffalo colostrum & milk peptides for Cullin-3 inhibition, thus ensuing activation of Nrf2 to alleviate the molecular etiopathology in PD using the C. elegans as a model. The structure was modelled, binding sites analyzed and peptide-interactions analyzed by docking. Among the 55 sequences (≤1 kDa), the peptide SFVSEVPEL having the highest dock score (-16.919) was synthesized and evaluated for its effects on oxidative stress markers, antioxidant enzymes, neurochemical marker and Nrf2/Skn-1 levels. The lead peptide alleviated the oxidative pathophysiology and behavioural deficits associated with PD in C. elegans.

Generation and analysis of pseudohypoaldosteronism type II knock-in mice caused by a nonsense KLHL3 mutation in the Kelch domain.

Mutations in the Kelch-like 3 (KLHL3) gene are the most common cause of inherited pseudohypoaldosteronism type II (PHAII) featuring thiazide-sensitive hypertension and hyperkalemic metabolic acidosis. Although Klhl3R528H/+ knock-in (KI) mice carrying a missense mutation in the Kelch repeat domain have been reported, nonsense KLHL3 mutations in the same domain that cause PHAII have not been fully investigated in vivo. We generated and analyzed Klhl3 KI mice harboring a nonsense W523X mutation (corresponding to the human KLHL3 W470X mutation). Both heterozygous and homozygous Klhl3W523X/+ KI mice exhibited typical PHAII with low-renin hypertension, hyperkalemia with reduced renal potassium excretion, and hyperchloremic metabolic acidosis. Their kidney tissues showed the presence of Klhl3 mRNA and increased Klhl3 protein levels along with enhanced downstream Wnk1/4-Spak/Osr1-N(k)cc phosphorylation. Increased protein expression of total Spak, phosphor(p-)Spak, total Ncc, and p-Ncc from urinary extracellular vesicles (uEVs) also confirmed the activation of the Wnk-mediated Ncc pathway. In vitro studies showed that the human KLHL3 W470X mutation resulted in increased KLHL3 protein stability and disrupted its binding affinity for WNK1/4, leading to the attenuated degradation and increased abundance of total WNKs. In conclusion, nonsense Klhl3W523X/+ mice recapitulating PHAII phenotypes exhibit Klhl3 protein stability, abrogating its binding to Wnks, with enhanced Ncc expression in the kidney tissue and even in uEVs. Activation of the WNK-mediated Na+ -Cl- co-transporter reiterated the in vivo pathogenic role of nonsense KLHL3 mutations in PHAII.

Combined Kelch-like 3 and Cullin 3 Degradation is a Central Mechanism in Familial Hyperkalemic Hypertension in Mice.

BACKGROUND: Mutations in the ubiquitin ligase scaffold protein Cullin 3 (CUL3) gene cause the disease familial hyperkalemic hypertension (FHHt). In the kidney, mutant CUL3 (CUL3-Δ9) increases abundance of With-No-Lysine (K) Kinase 4 (WNK4), inappropriately activating sterile 20/SPS-1-related proline/alanine-rich kinase (SPAK), which then phosphorylates and hyperactivates the Na+Cl- cotransporter (NCC). The precise mechanism by which CUL3-Δ9 causes FHHt is unclear. We tested the hypothesis that reduced abundance of CUL3 and of Kelch-like 3 (KLHL3), the CUL3 substrate adaptor for WNK4, is mechanistically important. Because JAB1, an enzyme that inhibits CUL3 activity by removing the ubiquitin-like protein NEDD8, cannot interact with CUL3-Δ9, we also determined whether Jab1 disruption mimicked the effects of CUL3-Δ9 expression. METHODS: We used an inducible renal tubule-specific system to generate several mouse models expressing CUL3-Δ9, mice heterozygous for both CUL3 and KLHL3 (Cul3+/-/Klhl3+/- ), and mice with short-term Jab1 disruption (to avoid renal injury associated with long-term disruption). RESULTS: Renal KLHL3 was higher in Cul3-/- mice, but lower in Cul3-/-/Δ9 mice and in the Cul3+/-/Δ9 FHHt model, suggesting KLHL3 is a target for both WT and mutant CUL3. Cul3+/-/Klhl3+/- mice displayed increased WNK4-SPAK activation and phospho-NCC abundance and an FHHt-like phenotype with increased plasma [K+] and salt-sensitive blood pressure. Short-term Jab1 disruption in mice lowered the abundance of CUL3 and KLHL3 and increased the abundance of WNK4 and phospho-NCC. CONCLUSIONS: Jab1-/- mice and Cul3+/-/Klhl3+/- mice recapitulated the effects of CUL3-Δ9 expression on WNK4-SPAK-NCC. Our data suggest degradation of both KLHL3 and CUL3 plays a central mechanistic role in CUL3-Δ9-mediated FHHt.

Cullin 3-Based Ubiquitin Ligases as Master Regulators of Mammalian Cell Differentiation.

Specificity of the ubiquitin proteasome system is controlled by ubiquitin E3 ligases, including their major representatives, the multisubunit cullin-RING ubiquitin (Ub) ligases (CRLs). More than 200 different CRLs are divided into seven families according to their cullin scaffolding proteins (CUL1-7) around which they are assembled. Research over two decades has revealed that different CRL families are specialized to fulfill specific cellular functions. Whereas many CUL1-based CRLs (CRL1s) ubiquitylate cell cycle regulators, CRL4 complexes often associate with chromatin to control DNA metabolism. Based on studies about differentiation programs of mesenchymal stem cells (MSCs), including myogenesis, neurogenesis, chondrogenesis, osteogenesis and adipogenesis, we propose here that CRL3 complexes evolved to fulfill a pivotal role in mammalian cell differentiation.

A conserved acetylation switch enables pharmacological control of tubby-like protein stability.

Tubby-like proteins (TULPs) are characterized by a conserved C-terminal domain that binds phosphoinositides. Collectively, mammalian TULP1-4 proteins play essential roles in intracellular transport, cell differentiation, signaling, and motility. Yet, little is known about how the function of these proteins is regulated in cells. Here, we present the protein-protein interaction network of TULP3, a protein that is responsible for the trafficking of G-protein-coupled receptors to cilia and whose aberrant expression is associated with severe developmental disorders and polycystic kidney disease. We identify several protein interaction nodes linked to TULP3 that include enzymes involved in acetylation and ubiquitination. We show that acetylation of two key lysine residues on TULP3 by p300 increases TULP3 protein abundance and that deacetylation of these sites by HDAC1 decreases protein levels. Furthermore, we show that one of these sites is ubiquitinated in the absence of acetylation and that acetylation inversely correlates with ubiquitination of TULP3. This mechanism is evidently conserved across species and is active in zebrafish during development. Finally, we identify this same regulatory module in TULP1, TULP2, and TULP4 and demonstrate that the stability of these proteins is similarly modulated by an acetylation switch. This study unveils a signaling pathway that links nuclear enzymes to ciliary membrane receptors via TULP3, describes a dynamic mechanism for the regulation of all tubby-like proteins, and explores how to exploit it pharmacologically using drugs.

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.

Cullin 3 mutant causing familial hyperkalemic hypertension lacks normal activity in the kidney.

Mutations in the ubiquitin ligase scaffold protein cullin 3 (CUL3) cause the disease familial hyperkalemic hypertension (FHHt). We recently reported that in the kidney, aberrant mutant CUL3 (CUL3-Δ9) activity lowers the abundance of CUL3-Δ9 and Kelch-like 3, the CUL3 substrate adaptor for with-no-lysine kinase 4 (WNK4) and that this is mechanistically important. However, whether CUL3-Δ9 exerts additional effects on other targets that may alter renal function is unclear. Here, we sought to determine 1) whether CUL3-Δ9 expression can rescue the phenotype of renal tubule-specific Cul3 knockout mice, and 2) whether CUL3-Δ9 expression affects other CUL3 substrates. Using an inducible renal tubule-specific system, we studied two CUL3-Δ9-expressing mouse models: Cul3 knockout (Cul3-/-/Δ9) and Cul3 heterozygous background (Cul3+/-/Δ9, FHHt model). The effects of CUL3-Δ9 in these mice were compared with Cul3-/- and Cul3+/- mice. Similar to Cul3-/- mice, Cul3-/-/Δ9 mice displayed polyuria with loss of aquaporin 2 and collecting duct injury; proximal tubule injury also occurred. CUL3-Δ9 did not promote degradation of two CUL3 targets that accumulate in the Cul3-/- kidney: high-molecular-weight (HMW) cyclin E and NAD(P)H:quinone oxidoreductase 1 (NQO1) [a surrogate for the CUL3-Kelch-like ECH-associated protein 1 (KEAP1) substrate nuclear factor erythroid-2-related factor 2]. Since CUL3-Δ9 expression cannot rescue the Cul3-/- phenotype, our data suggest that CUL3-Δ9 cannot normally function in ubiquitin ligase complexes. In Cul3+/-/Δ9 mice, KEAP1 abundance did not differ but NQO1 abundance was higher, suggesting adaptor sequestration by CUL3-Δ9 in vivo. Together, our results provide evidence that in the kidney, CUL3-Δ9 completely lacks normal activity and can trap CUL3 substrate adaptors in inactive complexes.NEW & NOTEWORTHY CUL3 mutation (CUL3-Δ9) causes familial hyperkalemic hypertension (FHHt) by reducing adaptor KLHL3, impairing substrate WNK4 degradation. Whether CUL3-Δ9 affects other targets in kidneys remains unclear. We found that CUL3-Δ9 cannot degrade two CUL3 targets, cyclin E and nuclear factor erythroid-2-related factor 2 (NRF2; using a surrogate marker NQO1), or rescue injury or polyuria caused by Cul3 disruption. In an FHHt model, CUL3-Δ9 impaired NRF2 degradation without reduction of its adaptor KEAP1. Our data provide additional insights into CUL3-Δ9 function in the kidney.

Targeted substrate degradation by Kelch controls the actin cytoskeleton during ring canal expansion.

During Drosophila oogenesis, specialized actin-based structures called ring canals form and expand to accommodate growth of the oocyte. Previous work demonstrated that Kelch and Cullin 3 function together in a Cullin 3-RING ubiquitin ligase complex (CRL3Kelch) to organize the ring canal cytoskeleton, presumably by targeting a substrate for proteolysis. Here, we use tandem affinity purification followed by mass spectrometry to identify HtsRC as the CRL3Kelch ring canal substrate. CRISPR-mediated mutagenesis of HtsRC revealed its requirement in the recruitment of the ring canal F-actin cytoskeleton. We present genetic evidence consistent with HtsRC being the CRL3Kelch substrate, as well as biochemical evidence indicating that HtsRC is ubiquitylated and degraded by the proteasome. Finally, we identify a short sequence motif in HtsRC that is necessary for Kelch binding. These findings uncover an unusual mechanism during development wherein a specialized cytoskeletal structure is regulated and remodeled by the ubiquitin-proteasome system.

KCTD10 Biology: An Adaptor for the Ubiquitin E3 Complex Meets Multiple Substrates: Emerging Divergent Roles of the cullin-3/KCTD10 E3 Ubiquitin Ligase Complex in Various Cell Lines.

Protein ubiquitination constitutes a post-translational modification mediated by ubiquitin ligases whereby ubiquitinated substrates are degraded through the proteasomal or lysosomal pathways, or acquire novel molecular functions according to their "ubiquitin codes." Dysfunction of the ubiquitination process in cells causes various diseases such as cancers along with neurodegenerative, auto-immune/inflammatory, and metabolic diseases. KCTD10 functions as a substrate recognition receptor for cullin-3 (CUL3), a scaffold protein in RING-type ubiquitin ligase complexes. Recently, studies by ourselves and others have identified new substrates that are ubiquitinated by the CUL3/KCTD10 ubiquitin ligase complex. Moreover, the type of polyubiquitination (e.g., K27-, K48-, or K63-chain) of various substrates (e.g., RhoB, CEP97, EIF3D, and TRIF) mediated by KCTD10 underlies its divergent roles in endothelial barrier formation, primary cilium formation, plasma membrane dynamics, cell proliferation, and immune response. Here, the physiological functions of KCTD10 are summarized and potential mechanisms are proposed.

Cullin 3, a cellular scripter of the non-proteolytic ubiquitin code.

Cullin-RING ubiquitin ligases (CRLs) represent the largest family of E3 ubiquitin ligases that control most if not all cellular processes. In CUL3-based CRLs, the substrate specificity is conferred by the interaction with one of around 183 existing BTB proteins, implying a broad spectrum of possible ubiquitylation signals and possible direct ubiquitylation substrates. Indeed, CUL3-based E3-ligases can catalyze various proteolytic and non-proteolytic ubiquitin signals regulating many physiological and pathophysiological states. Here, we discuss the recent studies focusing on the non-proteolytic CUL3-based signaling in mammalian cells, which emerge as important pathways during cell division, embryonic development as well as other biological processes. Mechanistically, non-proteolytic ubiquitin signals generated by CUL3 E3-ligases often regulate substrates' interactions with other downstream factors or their subcellular localization. Existing data also demonstrate an interplay with the proteolytic ubiquitylation catalyzed on the same substrates by different E3-ligases or by the same CUL3-BTB CRL3s on different substrates. In future, a deeper understanding of the upstream spatiotemporal regulatory mechanisms will help to dissect this fascinating CUL3 ubiquitin code.

Targeting Cul3-scaffold E3 ligase complex via KLHL substrate adaptors for cancer therapy.

Targeted therapy has become increasingly important and indispensable in cancer therapy. Cullin3-RING ligases (CRL3) serve as essential executors for regulating protein homeostasis in cancer development, highlighting that CRL3 might be promising targets in various cancer treatment. However, how to design new targeted therapies by disrupting the function of CRL3 is poorly understood. Here, we focus on the substrate adaptors of CRL3, and carry out a systematical research on the function of Kelch-like (KLHL) family proteins. We have identified twenty-four KLHL proteins with function of tumor promotion and thirteen KLHL proteins with high clinical significance on cancer therapy. Furthermore, we have clarified the novel biological function of KLHL13 as a vital factor that contributes to malignant progression in lung cancer. Taken together, our findings reveal multiple potential therapeutical targets and provide evidence for targeting CRL3 via KLHL substrate adaptors for cancer therapy.

Functional analysis of Cullin 3 E3 ligases in tumorigenesis.

Cullin 3-RING ligases (CRL3) play pivotal roles in the regulation of various physiological and pathological processes, including neoplastic events. The substrate adaptors of CRL3 typically contain a BTB domain that mediates the interaction between Cullin 3 and target substrates to promote their ubiquitination and subsequent degradation. The biological implications of CRL3 adaptor proteins have been well described where they have been found to play a role as either an oncogene, tumor suppressor, or can mediate either of these effects in a context-dependent manner. Among the extensively studied CRL3-based E3 ligases, the role of the adaptor protein SPOP (speckle type BTB/POZ protein) in tumorigenesis appears to be tissue or cellular context dependent. Specifically, SPOP acts as a tumor suppressor via destabilizing downstream oncoproteins in many malignancies, especially in prostate cancer. However, SPOP has largely an oncogenic role in kidney cancer. Keap1, another well-characterized CRL3 adaptor protein, likely serves as a tumor suppressor within diverse malignancies, mainly due to its specific turnover of its downstream oncogenic substrate, NRF2 (nuclear factor erythroid 2-related factor 2). In accordance with the physiological role the various CRL3 adaptors exhibit, several pharmacological agents have been developed to disrupt its E3 ligase activity, therefore blocking its potential oncogenic activity to mitigate tumorigenesis.

20-HETE downregulates Na/K-ATPase α1 expression via the ubiquitination pathway.

In this article, we found that 20-Hydroxyeicosatetraenoic acid (20-HETE) reduced Na/K-ATPase α1 expression via the ubiquitin-proteasome pathway. The ubiquitination level of Na/K-ATPase α1 protein was increased in 20-HETE-treated mouse cortical collecting duct cells and the kidney tissues of CYP4F2 transgenic mice. We also demonstrated that 20-HETE-induced high level phosphorylation of Na/K-ATPase α1 was necessary for its ubiquitination.The protein kinase C inhibitor sotrastaurin significantly reduced the phosphorylation of Na/K-ATPase α1 and increased the expression of Na/K-ATPase α1 although 20-HETE stimulus being applied at the same time. Moreover, high level of 20-HETE increased the expression and neddylation of Cullin3,which is an important ubiquitin E3 ligase in kidney. MLN4924, an inhibitor of NEDD8-activating enzyme, inhibited neddylation of Cullin3 and reversed the reduction of Na/K-ATPase α1 expression caused by 20-HETE. Thus, 20-HETE downregulates Na/K-ATPase α1 via the ubiquitination pathway, and phosphorylation of Na/K-ATPase α1 is a prerequisite to ubiquitination. Additionally, 20-HETE regulates Cullin3 expression via neddylation.