Assuntos
Proteínas Culina , Transição Epitelial-Mesenquimal , Neoplasias da Bexiga Urinária , Via de Sinalização Wnt , Humanos , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/genética , Proteínas Culina/metabolismo , Proteínas Culina/genética , Metástase Neoplásica , beta Catenina/metabolismo , AnimaisRESUMO
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.
Assuntos
Proteínas de Ligação a DNA , Reparo por Excisão , Ubiquitina-Proteína Ligases , Humanos , Microscopia Crioeletrônica , Proteínas Culina/metabolismo , Proteínas Culina/genética , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Células HEK293 , Ligação Proteica , Receptores de Interleucina-17 , Transcrição Gênica , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
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.
Assuntos
Proteínas Culina , Leishmania infantum , Leishmania infantum/metabolismo , Leishmania infantum/enzimologia , Proteínas Culina/metabolismo , Proteínas Culina/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Ubiquitinação , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/metabolismo , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
The development of cell-type-specific dendritic arbors is integral to the proper functioning of neurons within their circuit networks. In this study, we examine the regulatory relationship between the cytosolic chaperonin CCT, key insulin pathway genes, and an E3 ubiquitin ligase (Cullin1) in dendritic development. CCT loss of function (LOF) results in dendritic hypotrophy in Drosophila Class IV (CIV) multi-dendritic larval sensory neurons, and CCT has recently been shown to fold components of the TOR (Target of Rapamycin) complex 1 (TORC1) in vitro. Through targeted genetic manipulations, we confirm that an LOF of CCT and the TORC1 pathway reduces dendritic complexity, while overexpression of key TORC1 pathway genes increases the dendritic complexity in CIV neurons. Furthermore, both CCT and TORC1 LOF significantly reduce microtubule (MT) stability. CCT has been previously implicated in regulating proteinopathic aggregation, thus, we examine CIV dendritic development in disease conditions as well. The expression of mutant Huntingtin leads to dendritic hypotrophy in a repeat-length-dependent manner, which can be rescued by Cullin1 LOF. Together, our data suggest that Cullin1 and CCT influence dendritic arborization through the regulation of TORC1 in both health and disease.
Assuntos
Proteínas Culina , Dendritos , Proteínas de Drosophila , Drosophila melanogaster , Animais , Proteínas Culina/metabolismo , Proteínas Culina/genética , Dendritos/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteína Huntingtina/metabolismo , Proteína Huntingtina/genética , Larva/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Microtúbulos/metabolismo , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais , Fatores de Transcrição , Chaperonina com TCP-1RESUMO
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.
Assuntos
Apoptose , Catarata , Proteínas Culina , Humanos , Catarata/metabolismo , Catarata/genética , Catarata/etiologia , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas Culina/biossíntese , Masculino , Feminino , Idoso , Western Blotting , Reação em Cadeia da Polimerase em Tempo Real , Pessoa de Meia-Idade , Envelhecimento , Regulação da Expressão Gênica , Cápsula do Cristalino/metabolismo , Cápsula do Cristalino/patologia , Marcação In Situ das Extremidades CortadasRESUMO
The ORF9b protein, derived from the nucleocapsid's open-reading frame in both SARS-CoV and SARS-CoV-2, serves as an accessory protein crucial for viral immune evasion by inhibiting the innate immune response. Despite its significance, the precise regulatory mechanisms underlying its function remain elusive. In the present study, we unveil that the ORF9b protein of SARS-CoV-2, including emerging mutant strains like Delta and Omicron, can undergo ubiquitination at the K67 site and subsequent degradation via the proteasome pathway, despite certain mutations present among these strains. Moreover, our investigation further uncovers the pivotal role of the translocase of the outer mitochondrial membrane 70 (TOM70) as a substrate receptor, bridging ORF9b with heat shock protein 90 alpha (HSP90α) and Cullin 5 (CUL5) to form a complex. Within this complex, CUL5 triggers the ubiquitination and degradation of ORF9b, acting as a host antiviral factor, while HSP90α functions to stabilize it. Notably, treatment with HSP90 inhibitors such as GA or 17-AAG accelerates the degradation of ORF9b, leading to a pronounced inhibition of SARS-CoV-2 replication. Single-cell sequencing data revealed an up-regulation of HSP90α in lung epithelial cells from COVID-19 patients, suggesting a potential mechanism by which SARS-CoV-2 may exploit HSP90α to evade the host immunity. Our study identifies the CUL5-TOM70-HSP90α complex as a critical regulator of ORF9b protein stability, shedding light on the intricate host-virus immune response dynamics and offering promising avenues for drug development against SARS-CoV-2 in clinical settings.
Assuntos
COVID-19 , Proteínas Culina , Proteínas de Choque Térmico HSP90 , SARS-CoV-2 , Ubiquitinação , Replicação Viral , Humanos , Proteínas Culina/genética , Proteínas Culina/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , COVID-19/virologia , COVID-19/genética , COVID-19/metabolismo , COVID-19/imunologia , Ubiquitinação/genética , Células HEK293 , Benzoquinonas/farmacologia , Estabilidade Proteica , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismo , Lactamas MacrocíclicasRESUMO
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.
Assuntos
Biomarcadores Tumorais , Neoplasias da Mama , Proteínas Culina , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Culina/metabolismo , Proteínas Culina/genética , Feminino , Prognóstico , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Células MCF-7 , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
BACKGROUND: As a member of the Cullin family, Cullin2 (CUL2) is involved in the development and spread of different types of cancers. However, the precise role of CUL2 in human cancer remains largely elusive. METHODS: In this study, various databases were applied to observe the CUL2 expression. Kaplan-Meier and Spearman correlation analyses were employed to investigate the potential links between CUL2 level, patient prognosis, and the infiltration of immune cells. In addition, the association between CUL2 and the efficacy of immunotherapy in an immunotherapy cohort was investigated. Moreover, the expression and distribution of CUL2 in cells were observed using the Human Protein Atlas (THPA) database. Finally, clinical tissue specimens and in vitro function assays were conducted to validate the expressions and effects of CUL2 on the biological functions in hepatocellular carcinoma (HCC) cells. RESULTS: While there are variations in CUL2 expression across different organs and cell types, it is notably upregulated in a majority of tumor tissues. In addition, CUL2 gene mutations are common in multiple cancers with low mutation rates and CUL2 is closely related to the prognosis of some cancer's patients, some immune regulatory factors, TMB, MSI, MMR genes, and DNA methylation. Further, our results found that downregulating CUL2 inhibits the proliferation, and migration abilities. CONCLUSIONS: The expression of CUL2 has an impact on the prognosis of various tumors, and this correlation is particularly noteworthy due to its significant association with the infiltration of immune cells within tumors. CUL2 was an oncogene contributing to the progression of HCC.
Assuntos
Carcinoma Hepatocelular , Proteínas Culina , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas Culina/genética , Proteínas Culina/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Prognóstico , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Proliferação de Células/genética , Mutação , Movimento Celular/genéticaRESUMO
The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through dimerization. However, how and why such a dimeric assembly is required for its ligase activity remains elusive. Here, we report the cryo-EM structure of the dimeric CRL3KLHL22 complex and reveal a conserved N-terminal motif in CUL3 that contributes to the dimerization assembly and the E3 ligase activity of CRL3KLHL22. We show that deletion of the CUL3 N-terminal motif impairs dimeric assembly and the E3 ligase activity of both CRL3KLHL22 and several other CRL3s. In addition, we found that the dynamics of dimeric assembly of CRL3KLHL22 generates a variable ubiquitination zone, potentially facilitating substrate recognition and ubiquitination. These findings demonstrate that a CUL3 N-terminal motif participates in the assembly process and provide insights into the assembly and activation of CRL3s.
Assuntos
Motivos de Aminoácidos , Microscopia Crioeletrônica , Proteínas Culina , Receptores de Interleucina-17 , Ubiquitina-Proteína Ligases , Ubiquitinação , Proteínas Culina/metabolismo , Proteínas Culina/química , Proteínas Culina/genética , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Células HEK293 , Multimerização Proteica , Sequência Conservada , Ligação Proteica , Modelos MolecularesRESUMO
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.
Assuntos
Adenoma , Proteínas Culina , Modelos Animais de Doenças , Células Supressoras Mieloides , Animais , Proteínas Culina/genética , Proteínas Culina/metabolismo , Camundongos , Células Supressoras Mieloides/metabolismo , Células Supressoras Mieloides/patologia , Adenoma/patologia , Adenoma/genética , Adenoma/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Humanos , Microambiente Tumoral/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/etiologia , Deleção de Genes , Mucosa Intestinal/patologia , Mucosa Intestinal/metabolismoRESUMO
Lung adenocarcinoma (LUAD) is the most prevalent histological type of lung cancer. Previous studies have reported that specific long noncoding RNAs (lncRNA) are involved in cancer development and progression. The phenotype and mechanism of ENST00000440028, named MSL3P1, an lncRNA referred to as a cancer-testis gene with potential roles in tumorigenesis and progression, have not been reported. MSL3P1 is overexpressed in LUAD tumor tissues, which is significantly associated with clinical characteristics, metastasis, and poor clinical prognosis. MSL3P1 promotes the metastasis of LUAD in vitro and in vivo. The enhancer reprogramming in LUAD tumor tissue is the major driver of the aberrant expression of MSL3P1. Mechanistically, owing to the competitive binding to CUL3 mRNA with ZFC3H1 protein (a protein involved in targeting polyadenylated RNA to exosomes and promoting the degradation of target mRNA), MSL3P1 can prevent the ZFC3H1-mediated RNA degradation of CUL3 mRNA and transport it to the cytoplasm. This activates the downstream epithelial-to-mesenchymal transition signaling pathway and promotes tumor invasion and metastasis. Implications: This study indicates that lncRNA MSL3P1 regulates CUL3 mRNA stability and promotes metastasis and holds potential as a prognostic biomarker and therapeutic target in LUAD.
Assuntos
Adenocarcinoma de Pulmão , Proteínas Culina , Neoplasias Pulmonares , Metástase Neoplásica , Estabilidade de RNA , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas Culina/metabolismo , Proteínas Culina/genética , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Animais , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Masculino , Linhagem Celular Tumoral , Citoplasma/metabolismo , Prognóstico , Regulação Neoplásica da Expressão Gênica , Camundongos Nus , Feminino , Transição Epitelial-Mesenquimal/genéticaRESUMO
The DNA methylation is gradually acquired during oogenesis, a process sustained by successful follicle development. However, the functional roles of methyl-CpG-binding protein 2 (MeCP2), an epigenetic regulator displaying specifical binding with methylated DNA, remains unknown in oogenesis. In this study, we found MeCP2 protein was highly expressed in primordial and primary follicle, but was almost undetectable in secondary follicles. However, in aged ovary, MeCP2 protein is significantly increased in both oocyte and granulosa cells. Overexpression of MeCP2 in growing oocyte caused transcription dysregulation, DNA hypermethylation, and genome instability, ultimately leading to follicle growth arrest and apoptosis. MeCP2 is targeted by DCAF13, a substrate recognition adaptor of the Cullin 4-RING (CRL4) E3 ligase, and polyubiquitinated for degradation in both cells and oocytes. Dcaf13-null oocyte exhibited an accumulation of MeCP2 protein, and the partial rescue of follicle growth arrest induced by Dcaf13 deletion was observed following MeCP2 knockdown. The RNA-seq results revealed that large amounts of genes were regulated by the DCAF13-MeCP2 axis in growing oocytes. Our study demonstrated that CRL4DCAF13 E3 ubiquitin ligase targets MeCP2 for degradation to ensure normal DNA methylome and transcription in growing oocytes. Moreover, in aged ovarian follicles, deceased DCAF13 and DDB1 protein were observed, indicating a potential novel mechanism that regulates ovary aging.
Assuntos
Proteína 2 de Ligação a Metil-CpG , Ubiquitina-Proteína Ligases , Feminino , Humanos , Proteínas Culina/genética , Proteínas Culina/metabolismo , DNA/metabolismo , Metilação de DNA , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Oócitos/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Heterotrimeric G proteins can be regulated by posttranslational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain, engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3KCTD5) that ubiquitylates Gßγ and reduces Gßγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/Gß1γ2 assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5BTB/CUL3NTD and KCTD5CTD/Gßγ moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate Gßγ in an E3-E3 superassembly, and extension of the structure to include full-length CUL3 with RBX1 and an ARIH1~ubiquitin conjugate reveals that some conformational states position the ARIH1~ubiquitin thioester bond to within 10 Å of lysine-23 of Gß and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD/Gßγ complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex.
Assuntos
Proteínas de Transporte , Ubiquitina-Proteína Ligases , Ligação Proteica , Ubiquitinação , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte/metabolismo , Ubiquitina/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismoRESUMO
CUL4B, a crucial scaffolding protein in the largest E3 ubiquitin ligase complex CRL4B, is involved in a broad range of physiological and pathological processes. While previous research has shown that CUL4B participates in maintaining intestinal homeostasis and function, its involvement in facilitating intestinal recovery following ionizing radiation (IR) damage has not been fully elucidated. Here, we utilized in vivo and in vitro models to decipher the role of CUL4B in intestinal repair after IR-injury. Our findings demonstrated that prior to radiation exposure, CUL4B inhibited the ubiquitination modification of PSME3, which led to the accumulation of PSME3 and subsequent negative regulation of p53-mediated apoptosis. In contrast, after radiation, CUL4B dissociated from PSME3 and translocated into the nucleus at phosphorylated histones H2A (γH2AX) foci, thereby impeding DNA damage repair and augmenting p53-mediated apoptosis through inhibition of BRCA1 phosphorylation and RAD51. Our study elucidated the dynamic role of CUL4B in the repair of radiation-induced intestinal damage and uncovered novel molecular mechanisms underlying the repair process, suggesting a potential therapeutic strategy of intestinal damage after radiation therapy for cancers.
Assuntos
Apoptose , Proteínas Culina , Intestinos , Regeneração , Proteína Supressora de Tumor p53 , Animais , Humanos , Camundongos , Apoptose/efeitos da radiação , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteínas Culina/metabolismo , Proteínas Culina/genética , Dano ao DNA , Reparo do DNA , Histonas/metabolismo , Intestinos/efeitos da radiação , Intestinos/patologia , Camundongos Endogâmicos C57BL , Fosforilação/efeitos da radiação , Rad51 Recombinase/metabolismo , Radiação Ionizante , Regeneração/efeitos da radiação , Proteína Supressora de Tumor p53/metabolismo , UbiquitinaçãoRESUMO
Diabetic retinopathy (DR) is a highly hazardous and widespread complication of diabetes mellitus (DM). The accumulated reactive oxygen species (ROS) play a central role in DR development. The aim of this research was to examine the impact and mechanisms of mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEV) on regulating ROS and retinal damage in DR. Intravitreal injection of sEV inhibited Cullin3 neddylation, stabilized Nrf2, decreased ROS, reduced retinal inflammation, suppressed Müller gliosis, and mitigated DR. Based on MSC-sEV miRNA sequencing, bioinformatics software, and dual-luciferase reporter assay, miR-143-3p was identified to be the key effector for MSC-sEV's role in regulating neural precursor cell expressed developmentally down-regulated 8 (NEDD8)-mediated neddylation. sEV were able to be internalized by Müller cells. Compared to advanced glycation end-products (AGEs)-induced Müller cells, sEV coculture decreased Cullin3 neddylation, activated Nrf2 signal pathway to combat ROS-induced inflammation. The barrier function of endothelial cells was impaired when endothelial cells were treated with the supernatant of AGEs-induced Müller cells, but was restored when treated with supernatant of AGEs-induced Müller cells cocultured with sEV. The protective effect of sEV was, however, compromised when miR-143-3p was inhibited in sEV. Moreover, the protective efficacy of sEV was diminished when NEDD8 was overexpressed in Müller cells. These findings showed MSC-sEV delivered miR-143-3p to inhibit Cullin3 neddylation, stabilizing Nrf2 to counteract ROS-induced inflammation and reducing vascular leakage. Our findings suggest that MSC-sEV may be a potential nanotherapeutic agent for DR, and that Cullin3 neddylation could be a new target for DR therapy.
Assuntos
Retinopatia Diabética , Vesículas Extracelulares , Células-Tronco Mesenquimais , MicroRNAs , Proteína NEDD8 , Fator 2 Relacionado a NF-E2 , Espécies Reativas de Oxigênio , Animais , Humanos , Camundongos , Proteínas Culina/metabolismo , Proteínas Culina/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/genética , Retinopatia Diabética/patologia , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Produtos Finais de Glicação Avançada/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína NEDD8/metabolismo , Proteína NEDD8/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
The E3 ligase-degron interaction determines the specificity of the ubiquitinâproteasome system. We recently discovered that FEM1B, a substrate receptor of Cullin 2-RING ligase (CRL2), recognizes C-degrons containing a C-terminal proline. By solving several cryo-EM structures of CRL2FEM1B bound to different C-degrons, we elucidate the dimeric assembly of the complex. Furthermore, we reveal distinct dimerization states of unmodified and neddylated CRL2FEM1B to uncover the NEDD8-mediated activation mechanism of CRL2FEM1B. Our research also indicates that, FEM1B utilizes a bipartite mechanism to recognize both the C-terminal proline and an upstream aromatic residue within the substrate. These structural findings, complemented by in vitro ubiquitination and in vivo cell-based assays, demonstrate that CRL2FEM1B-mediated polyubiquitination and subsequent protein turnover depend on both FEM1B-degron interactions and the dimerization state of the E3 ligase complex. Overall, this study deepens our molecular understanding of how Cullin-RING E3 ligase substrate selection mediates protein turnover.
Assuntos
Microscopia Crioeletrônica , Proteína NEDD8 , Receptores de Interleucina-17 , Ubiquitina-Proteína Ligases , Ubiquitinação , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/química , Proteína NEDD8/metabolismo , Proteína NEDD8/genética , Prolina/metabolismo , Multimerização Proteica , Células HEK293 , Ligação Proteica , Especificidade por Substrato , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Modelos Moleculares , Proteínas Culina/metabolismo , Proteínas Culina/química , Proteínas Culina/genética , DegronsRESUMO
OBJECTIVE: Fibroblast-like synoviocytes (FLS) contribute to the pathogenesis of rheumatoid arthritis (RA), in part due to activation of the proinflammatory transcription factor NF-κB. Neddylation is modulated by the negative regulator of ubiquitin-like protein (NUB) 1. We determined whether NUB1 and neddylation are aberrant in the models with RA FLS, thereby contributing to their aggressive phenotype. METHODS: Models with RA or osteoarthritis (OA) FLS were obtained from arthroplasty synovia. Real-time quantitative polymerase chain reaction and Western blot analysis assessed gene and protein expression, respectively. NUB1 was overexpressed using an expression vector. NF-κB activation was assessed by stimulating FLS with interleukin (IL)-1ß. Neddylation inhibitor (MLN4924) and proteasome inhibitor were used in migration and gene expression assays. MLN4924 was used in the model with K/BxN serum-transfer arthritis. RESULTS: Enhanced H3K27ac and H3K27me3 peaks were observed in the NUB1 promoter in the OA FLS compared with the RA FLS. NUB1 was constitutively expressed by FLS, but induction by IL-1ß was significantly greater in the OA FLS. The ratio of neddylated cullin (CUL) 1 to nonneddylated CUL1 was lower in the OA FLS than the RA FLS. NUB1 overexpression decreased NF-κB nuclear translocation and IL-6 messenger RNA (mRNA) in IL-1ß-stimulated the RA FLS. MLN4924 decreased CUL1 neddylation, NF-κB nuclear translocation, and IL-6 mRNA in IL-1ß-stimulated the RA FLS. MLN4924 significantly decreased arthritis severity in the model with K/BxN serum-transfer arthritis. CONCLUSION: CUL1 neddylation and NUB1 induction is dysregulated in the models with RA, which increases FLS activation. Inhibition of neddylation is an effective therapy in an animal model of arthritis. These data suggest that the neddylation system contributes to the pathogenesis of RA and that regulation of neddylation could be a novel therapeutic approach.
Assuntos
Artrite Reumatoide , Ciclopentanos , Fibroblastos , NF-kappa B , Sinoviócitos , Artrite Reumatoide/metabolismo , Sinoviócitos/metabolismo , Sinoviócitos/efeitos dos fármacos , Humanos , Ciclopentanos/farmacologia , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , NF-kappa B/metabolismo , Osteoartrite/metabolismo , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Pirimidinas/farmacologia , Animais , Ubiquitinas/metabolismo , Ubiquitinas/genética , Inflamação/metabolismo , Proteínas Culina/metabolismo , Proteínas Culina/genética , Proteína NEDD8/metabolismo , Proteína NEDD8/genética , CamundongosRESUMO
BACKGROUND: Alternative splicing (AS) is a principal mode of genetic regulation and one of the most widely used mechanisms to generate structurally and functionally distinct mRNA and protein variants. Dysregulation of AS may result in aberrant transcription and protein products, leading to the emergence of human diseases. Although considered important for regulating gene expression, genome-wide AS dysregulation, underlying mechanisms, and clinical relevance in knee osteoarthritis (OA) remain unelucidated. Therefore, in this study, we elucidated and validated AS events and their regulatory mechanisms during OA progression. RESULTS: In this study, we identified differentially expressed genes between human OA and healthy meniscus samples. Among them, the OA-associated genes were primarily enriched in biological pathways such as extracellular matrix organization and ossification. The predominant OA-associated regulated AS (RAS) events were found to be involved in apoptosis during OA development. The expression of the apoptosis-related gene BCL2L13, XAF1, and NF2 were significantly different between OA and healthy meniscus samples. The construction of a covariation network of RNA-binding proteins (RBPs) and RAS genes revealed that differentially expressed RBP genes LAMA2 and CUL4B may regulate the apoptotic genes XAF1 and BCL2L13 to undergo AS events during OA progression. Finally, RT-qPCR revealed that CUL4B expression was significantly higher in OA meniscus samples than in normal controls and that the AS ratio of XAF1 was significantly different between control and OA samples; these findings were consistent with their expected expression and regulatory relationships. CONCLUSIONS: Differentially expressed RBPs may regulate the AS of apoptotic genes during knee OA progression. XAF1 and its regulator, CUL4B, may serve as novel biomarkers and potential therapeutic targets for this disease.
Assuntos
Osteoartrite do Joelho , Humanos , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/metabolismo , Processamento Alternativo , RNA Mensageiro/genética , Biomarcadores/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismoRESUMO
KRAS is a proto-oncogene encoding a small GTPase. Mutations contribute to â¼30% of human solid tumours, including lung adenocarcinoma, pancreatic, and colorectal carcinomas. Most KRAS activating mutations interfere with GTP hydrolysis, essential for its role as a molecular switch, leading to alterations in their molecular environment and oncogenic signalling. However, the precise signalling cascades these mutations affect are poorly understood. Here, APEX2 proximity labelling was used to profile the molecular environment of WT, G12D, G13D, and Q61H-activating KRAS mutants under starvation and stimulation conditions. Through quantitative proteomics, we demonstrate the presence of known KRAS interactors, including ARAF and LZTR1, which are differentially captured by WT and KRAS mutants. Notably, the KRAS mutations G12D, G13D, and Q61H abrogate their association with LZTR1, thereby affecting turnover. Elucidating the implications of LZTR1-mediated regulation of KRAS protein levels in cancer may offer insights into therapeutic strategies targeting KRAS-driven malignancies.
Assuntos
Neoplasias Colorretais , Proteínas Proto-Oncogênicas p21(ras) , Ubiquitina-Proteína Ligases , Humanos , Proteínas Culina/genética , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais/genética , Fatores de TranscriçãoRESUMO
Therapy resistance and metastatic progression jointly determine the fatal outcome of cancer, therefore, elucidating their crosstalk may provide new opportunities to improve therapeutic efficacy and prevent recurrence and metastasis in esophageal squamous cell carcinoma (ESCC). Here, we have established radioresistant ESCC cells with the remarkable metastatic capacity, and identified miR-494-3p (miR494) as a radioresistant activator. Mechanistically, we demonstrated that cullin 3 (CUL3) is a direct target of miR494, which is transcriptionally regulated by JunD, and highlighted that JunD-miR494-CUL3 axis promotes radioresistance and metastasis by facilitating epithelial-mesenchymal transition (EMT) and restraining programmed cell death 1 ligand 1 (PD-L1) degradation. In clinical specimens, miR494 is significantly up-regulated and positively associated with T stage and lymph node metastasis in ESCC tissues and serum. Notably, patients with higher serum miR494 expression have poor prognosis, and patients with higher CUL3 expression have more conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs), less cancer-associated fibroblasts (CAF2/4), and tumor endothelial cells (TEC2/3) infiltration than patients with lower CUL3 expression, suggesting that CUL3 may be involved in tumor microenvironment (TME). Overall, miR494 may serve as a potential prognostic predictor and therapeutic target, providing a promising strategy for ESCC treatment.