[Method of Inducible Knockdown of Essential Genes in OSC Cell Culture of Drosophila melanogaster].

An RNA interference-based method was proposed to achieve an inducible knockdown of genes essential for cell viability. In the method, a genetic cassette in which a copper ion-dependent inducible metallothionein promoter controls expression of a siRNA precursor is inserted into a genomic pre-integrated transgene by CRIPSR/Cas9 technology. The endogenous siRNA source allows the gene knockdown in cell cultures that are refractory to conventional transfection with exogenous siRNA. The efficiency of the method was demonstrated in Drosophila ovarian somatic cell culture (OSC) for two genes that are essential for oogenesis: Cul3, encoding a component of the multiprotein ubiquitin-ligase complex with versatile functions in proteostasis, and cut, encoding a transcription factor regulating differentiation of ovarian follicular cells.

An Anti-Invasive Role for Mdmx through the RhoA GTPase under the Control of the NEDD8 Pathway.

Mdmx (Mdm4) is established as an oncogene mainly through repression of the p53 tumour suppressor. On the other hand, anti-oncogenic functions for Mdmx have also been proposed, but the underlying regulatory pathways remain unknown. Investigations into the effect of inhibitors for the NEDD8 pathway in p53 activation, human cell morphology, and in cell motility during gastrulation in Xenopus embryos revealed an anti-invasive function of Mdmx. Through stabilisation and activation of the RhoA GTPase, Mdmx is required for the anti-invasive effects of NEDDylation inhibitors. Mechanistically, through its Zn finger domain, Mdmx preferentially interacts with the inactive GDP-form of RhoA. This protects RhoA from degradation and allows for RhoA targeting to the plasma membrane for its subsequent activation. The effect is transient, as prolonged NEDDylation inhibition targets Mdmx for degradation, which subsequently leads to RhoA destabilisation. Surprisingly, Mdmx degradation requires non-NEDDylated (inactive) Culin4A and the Mdm2 E3-ligase. This study reveals that Mdmx can control cell invasion through RhoA stabilisation/activation, which is potentially linked to the reported anti-oncogenic functions of Mdmx. As inhibitors of the NEDD8 pathway are in clinical trials, the status of Mdmx may be a critical determinant for the anti-tumour effects of these inhibitors.

Neddylation and Its Target Cullin 3 Are Essential for Adipocyte Differentiation.

The ongoing obesity epidemic has raised awareness of the complex physiology of adipose tissue. Abnormal adipocyte differentiation results in the development of systemic metabolic disorders such as insulin resistance and diabetes. The conjugation of NEDD8 (neural precursor cell expressed, developmentally downregulated 8) to target protein, termed neddylation, has been shown to mediate adipogenesis. However, much remains unknown about its role in adipogenesis. Here, we demonstrated that neddylation and its targets, the cullin (CUL) family members, are differentially regulated during mouse and human adipogenesis. Inhibition of neddylation by MLN4924 significantly reduced adipogenesis of 3T3-L1 and human stromal vascular cells. Deletion of NAE1, a subunit of the only NEDD8 E1 enzyme, suppressed neddylation and impaired adipogenesis. Neddylation deficiency did not affect mitotic cell expansion. Instead, it disrupted CREB/CEBPß/PPARγ signaling, essential for adipogenesis. Interestingly, among the neddylation-targeted CUL family members, deletion of CUL3, but not CUL1, CUL2, or CUL4A, largely replicated the adipogenic defects observed with neddylation deficiency. A PPARγ agonist minimally rescued the adipogenic defects caused by the deletion of NAE1 and CUL3. In conclusion, our study demonstrates that neddylation and its targeted CUL3 are crucial for adipogenesis. These findings provide potential targets for therapeutic intervention in obesity and metabolic disorders.

Scaled and efficient derivation of loss-of-function alleles in risk genes for neurodevelopmental and psychiatric disorders in human iPSCs.

Translating genetic findings for neurodevelopmental and psychiatric disorders (NPDs) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, we developed a pipeline for clonal loss-of-function (LoF) allele mutagenesis in human induced pluripotent stem cells (hiPSCs) by introducing premature stop codons (iSTOP) that lead to mRNA nonsense-mediated decay (NMD) or protein truncation. We tested the pipeline for 23 NPD genes on 3 hiPSC lines and achieved highly reproducible, efficient iSTOP editing in 22 genes. Using RNA sequencing (RNA-seq), we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Despite high editing efficiency, three schizophrenia risk genes (SETD1A, TRIO, and CUL1) only had heterozygous LoF alleles, suggesting their essential roles for cell growth. We found that CUL1-LoF reduced neurite branches and synaptic puncta density. This iSTOP pipeline enables a scaled and efficient LoF mutagenesis of NPD genes, yielding an invaluable shareable resource.

The Proteasome and Cul3-Dependent Protein Ubiquitination Is Required for Gli Protein-Mediated Activation of Gene Expression in the Hedgehog Pathway.

Many cellular processes are regulated by proteasome-mediated protein degradation, including regulation of signaling pathways and gene expression. Among the pathways regulated by the ubiquitin-proteasome system is the Hedgehog pathway and its downstream effectors, the Gli transcription factors. Here we provide evidence that proteasomal activity is necessary for maintaining the activation of the Hedgehog pathway, and this crucial event takes place at the level of Gli proteins. We undertook extensive work to demonstrate the specificity of the observed phenomenon by ruling out the involvement of primary cilium, impaired nuclear import, failed dissociation from Sufu, microtubule stabilization, and stabilization of Gli repressor forms. Moreover, we showed that proteasomal-inhibition-mediated Hedgehog pathway downregulation is not restricted to the NIH-3T3 cell line. We demonstrated, using CRISPR/Ca9 mutagenesis, that neither Gli1, Gli2, nor Gli3 are solely responsible for the Hedgehog pathway downregulation upon proteasome inhibitor treatment, and that Cul3 KO renders the same phenotype. Finally, we report two novel E3 ubiquitin ligases, Btbd9 and Kctd3, known Cul3 interactors, as positive Hedgehog pathway regulators. Our data pave the way for a better understanding of the regulation of gene expression and the Hedgehog signaling pathway.

Photoreceptor-targeted extracellular vesicles-mediated delivery of Cul7 siRNA for retinal degeneration therapy.

Rationale: Photoreceptor loss is a primary pathological feature of retinal degeneration (RD) with limited treatment strategies. RNA interference (RNAi) has emerged as a promising method of gene therapy in regenerative medicine. However, the transfer of RNAi therapeutics to photoreceptors and the deficiency of effective therapeutic targets are still major challenges in the treatment of RD. Methods: In this study, photoreceptor-derived extracellular vesicles (PEVs) conjugated with photoreceptor-binding peptide MH42 (PEVsMH42) were prepared using the anchoring peptide CP05. Transcriptome sequencing was applied to investigate the potential therapeutic target of RD. We then engineered PEVsMH42 with specific small-interfering RNAs (siRNAs) through electroporation and evaluated their therapeutic efficacy in N-methyl-N-nitrosourea (MNU)-induced RD mice and Pde6ßrd1/rd1 mutant mice. Results: PEVsMH42 were selectively accumulated in photoreceptors after intravitreal injection. Cullin-7 (Cul7) was identified as a novel therapeutic target of RD. Taking advantage of the established PEVsMH42, siRNAs targeting Cul7 (siCul7) were efficiently delivered to photoreceptors and consequently blocked the expression of Cul7. Moreover, suppression of Cul7 effectively protected photoreceptors to alleviate RD both in MNU-induced mouse model and Pde6ßrd1/rd1 mutant mouse model. Mechanistically, PEVsMH42 loaded with siCul7 (PEVsMH42-siCul7)-induced Cul7 downregulation was responsible for preventing Cul7-mediated glutathione peroxidase 4 (Gpx4) ubiquitination and degradation, resulting in the inhibition of photoreceptor ferroptosis. Conclusions: In summary, PEVsMH42-siCul7 attenuate photoreceptor ferroptosis to treat RD by inhibiting Cul7-induced ubiquitination of Gpx4. Our study develops a PEVs-based platform for photoreceptor-targeted delivery and highlights the potential of PEVsMH42-siCul7 as effective therapeutics for RD.

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.

Cullin 3/with No Lysine [K] Kinase/Ste20/SPS-Related Proline Alanine Rich Kinase Signaling: Impact on NaCl Cotransporter Activity in BP Regulation.

The sodium chloride cotransporter (NCC) fine-tunes Na + balance and indirectly affects the homeostasis of other ions including K + , Mg 2+ , and Ca 2+ . Owing to its effects on Na + balance, BP is significantly affected by alterations in NCC activity. Several factors have been reported to influence the expression and activity of NCC. One critical factor is NCC phosphorylation/dephosphorylation that occurs at key serine-threonine amino acid residues of the protein. Phosphorylation, which results in increased NCC activity, is mediated by the with no lysine [K] (WNK)-SPS-related proline alanine rich kinase (SPAK)/OSR1 kinases. NCC activation stimulates reabsorption of Na + , increasing extracellular fluid volume and hence BP. On the other hand, proteasomal degradation of WNK kinases after ubiquitination by the Cullin 3-Kelch-like 3 E3 ubiquitin ligase complex and dephosphorylation pathways oppose WNK-SPAK/OSR1-mediated NCC activation. Components of the Cullin 3/Kelch-like 3-WNK-SPAK/OSR1 regulatory pathway may be targets for novel antihypertensive drugs. In this review, we outline the impact of these regulators on the activity of NCC and the consequent effect on BP.

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.

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.

Identification and Characterization of Novel Small-Molecule Enhancers of the CUL3LZTR1 E3 Ligase KRAS Complex.

The RAS family of GTPases is among the most frequently mutated proteins in human cancer, creating a high clinical demand for therapies that counteract their signaling activity. An important layer of regulation that could be therapeutically exploited is the proteostatic regulation of the main RAS GTPases KRAS, NRAS, and HRAS, as well as the closely related members, MRAS and RIT1, by the leucine zipper-like transcriptional regulator 1 cullin 3 RING E3 ubiquitin ligase complex (CUL3LZTR1). Genetic inactivation of LZTR1, as observed in different cancer entities and Noonan syndrome leads to enhanced RAS GTPase abundance and altered MAPK pathway activation state. Novel therapeutic approaches to interfere with hyperactive RAS signaling, thereby complementing existing treatments, are highly sought after. Motivated by the growing arsenal of molecular glue degraders, we report the identification of novel chemical fragments that enhance the protein-protein interaction (PPI) of the KRAS-LZTR1 complex. We established a split-luciferase-based reporter assay that monitors the RAS GTPase-LZTR1 interaction in a scalable format, capable of capturing chemical, as well as mutational perturbations. Using this screening system, in combination with a small fragment library, we identified two fragments, C53 and Z86, that enhance the interaction of the KRAS-LZTR1 complex in a dose-dependent manner. Further orthogonal validation experiments using proximity biotinylation (BioID), thermal shift assays, and NMR spectroscopy demonstrated fragment-dependent enhanced recruitment of endogenous LZTR1 and physical engagement of KRAS. The two fragments, which potentiate the KRAS-LZTR1 interaction, serve as starting points for fragment-based drug discovery. Additionally, the assay we introduced is amenable to high-throughput screening to further explore the pharmacological modulation of the CUL3LZTR1-RAS GTPase complex.

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.

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.

Diversity of structure and function in Cullin E3 ligases.

The cellular process by which the protein ubiquitin (Ub) is covalently attached to a protein substrate involves Ub activating (E1s) and conjugating enzymes (E2s) that work together with a large variety of E3 ligases that impart substrate specificity. The largest family of E3s is the Cullin-RING ligase (CRL) family which utilizes a wide variety of substrate receptors, adapter proteins, and cooperating ligases. Cryo-electron microscopy (cryoEM) has revealed a wide variety of structures which suggest how Ub transfer occurs. Hydrogen deuterium exchange mass spectrometry (HDXMS) has revealed the role of dynamics and expanded our knowledge of how covalent NEDD8 modification (neddylation) activates the CRLs, particularly by facilitating cooperation with additional RING-between-RING ligases to transfer Ub.

The small CRL4CSA ubiquitin ligase component DDA1 regulates transcription-coupled repair dynamics.

Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4CSA). Although ubiquitination of several TC-NER proteins by CRL4CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we apply a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis shows that DDA1 is an integral component of the CRL4CSA complex. Functional analysis reveals that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.

KCTD proteins regulate morphine dependence via heterologous sensitization of adenylyl cyclase 1 in mice.

Heterologous sensitization of adenylyl cyclase (AC) results in elevated cAMP signaling transduction that contributes to drug dependence. Inhibiting cullin3-RING ligases by blocking the neddylation of cullin3 abolishes heterologous sensitization, however, the modulating mechanism remains uncharted. Here, we report an essential role of the potassium channel tetramerization domain (KCTD) protein 2, 5, and 17, especially the dominant isoform KCTD5 in regulating heterologous sensitization of AC1 and morphine dependence via working with cullin3 and the cullin-associated and neddylation-dissociated 1 (CAND1) protein. In cellular models, we observed enhanced association of KCTD5 with Gß and cullin3, along with elevated dissociation of Gß from AC1 as well as of CAND1 from cullin3 in heterologous sensitization of AC1. Given binding of CAND1 inhibits the neddylation of cullin3, we further elucidated that the enhanced interaction of KCTD5 with both Gß and cullin3 promoted the dissociation of CAND1 from cullin3, attenuated the inhibitory effect of CAND1 on cullin3 neddylation, ultimately resulted in heterologous sensitization of AC1. The paraventricular thalamic nucleus (PVT) plays an important role in mediating morphine dependence. Through pharmacological and biochemical approaches, we then demonstrated that KCTD5/cullin3 regulates morphine dependence via modulating heterologous sensitization of AC, likely AC1 in PVT in mice. In summary, the present study revealed the underlying mechanism of heterologous sensitization of AC1 mediated by cullin3 and discovered the role of KCTD proteins in regulating morphine dependence in mice.

Functional characterization of Cullin-1-RING ubiquitin ligase (CRL1) complex in Leishmania infantum.

Cullin-1-RING ubiquitin ligases (CRL1) or SCF1 (SKP1-CUL1-RBX1) E3 ubiquitin ligases are the largest and most extensively investigated class of E3 ligases in mammals that regulate fundamental processes, such as the cell cycle and proliferation. These enzymes are multiprotein complexes comprising SKP1, CUL1, RBX1, and an F-box protein that acts as a specificity factor by interacting with SKP1 through its F-box domain and recruiting substrates via other domains. E3 ligases are important players in the ubiquitination process, recognizing and transferring ubiquitin to substrates destined for degradation by proteasomes or processing by deubiquitinating enzymes. The ubiquitin-proteasome system (UPS) is the main regulator of intracellular proteolysis in eukaryotes and is required for parasites to alternate hosts in their life cycles, resulting in successful parasitism. Leishmania UPS is poorly investigated, and CRL1 in L. infantum, the causative agent of visceral leishmaniasis in Latin America, is yet to be described. Here, we show that the L. infantum genes LINF_110018100 (SKP1-like protein), LINF_240029100 (cullin-like protein-like protein), and LINF_210005300 (ring-box protein 1 -putative) form a LinfCRL1 complex structurally similar to the H. sapiens CRL1. Mass spectrometry analysis of the LinfSkp1 and LinfCul1 interactomes revealed proteins involved in several intracellular processes, including six F-box proteins known as F-box-like proteins (Flp) (data are available via ProteomeXchange with identifier PXD051961). The interaction of LinfFlp 1-6 with LinfSkp1 was confirmed, and using in vitro ubiquitination assays, we demonstrated the function of the LinfCRL1(Flp1) complex to transfer ubiquitin. We also found that LinfSKP1 and LinfRBX1 knockouts resulted in nonviable L. infantum lineages, whereas LinfCUL1 was involved in parasite growth and rosette formation. Finally, our results suggest that LinfCul1 regulates the S phase progression and possibly the transition between the late S to G2 phase in L. infantum. Thus, a new class of E3 ubiquitin ligases has been described in L. infantum with functions related to various parasitic processes that may serve as prospective targets for leishmaniasis treatment.

The cullin Rtt101 promotes ubiquitin-dependent DNA-protein crosslink repair across the cell cycle.

DNA-protein crosslinks (DPCs) challenge faithful DNA replication and smooth passage of genomic information. Our study unveils the cullin E3 ubiquitin ligase Rtt101 as a DPC repair factor. Genetic analyses demonstrate that Rtt101 is essential for resistance to a wide range of DPC types including topoisomerase 1 crosslinks, in the same pathway as the ubiquitin-dependent aspartic protease Ddi1. Using an in vivo inducible Top1-mimicking DPC system, we reveal the significant impact of Rtt101 ubiquitination on DPC removal across different cell cycle phases. High-throughput methods coupled with next-generation sequencing specifically highlight the association of Rtt101 with replisomes as well as colocalization with DPCs. Our findings establish Rtt101 as a main contributor to DPC repair throughout the yeast cell cycle.

Prognostic potential of CUL3 ligase with differential roles in luminal A and basal type breast cancer tumors.

Breast cancer is a prevalent and significant cause of mortality in women, and manifests as six molecular subtypes. Its further histologic classification into non-invasive ductal or lobular carcinoma (DCIS) and invasive carcinoma (ILC or IDC) underscores its heterogeneity. The ubiquitin-proteasome system plays a crucial role in breast cancer, with inhibitors targeting the 26S proteasome showing promise in clinical treatment. The Cullin-RING ubiquitin ligases, including CUL3, have direct links to breast cancer. This study focuses on CUL3 as a potential biomarker, leveraging high-throughput sequencing, gene expression profiling, experimental and data analysis tools. Through comprehensive analysis using databases like GEPIA2 and UALCAN, as well as TCGA datasets, CUL3's expression and its association with prognostic values were assessed. Additionally, the impact of CUL3 overexpression was explored in MCF-7 and MDA-MB-231 breast cancer cell lines, revealing distinct differences in molecular and phenotypic characteristics. We further profiled its expression and localization in breast cancer tissues identifying prominent differences between luminal A and TNBC tumors. Conclusively, CUL3 was found to be associated with cell cycle progression, and DNA damage response, exhibiting diverse roles depending on the tumor's molecular type. It exhibits a tendency to act as an oncogene in triple-negative tumors and as a tumor suppressor in luminal A types, suggesting a potential significance in breast cancer progression and therapeutic directions.