RESUMEN
Cells respond to environmental cues by remodeling their inventories of multiprotein complexes. Cellular repertoires of SCF (SKP1-CUL1-F box protein) ubiquitin ligase complexes, which mediate much protein degradation, require CAND1 to distribute the limiting CUL1 subunit across the family of â¼70 different F box proteins. Yet, how a single factor coordinately assembles numerous distinct multiprotein complexes remains unknown. We obtained cryo-EM structures of CAND1-bound SCF complexes in multiple states and correlated mutational effects on structures, biochemistry, and cellular assays. The data suggest that CAND1 clasps idling catalytic domains of an inactive SCF, rolls around, and allosterically rocks and destabilizes the SCF. New SCF production proceeds in reverse, through SKP1-F box allosterically destabilizing CAND1. The CAND1-SCF conformational ensemble recycles CUL1 from inactive complexes, fueling mixing and matching of SCF parts for E3 activation in response to substrate availability. Our data reveal biogenesis of a predominant family of E3 ligases, and the molecular basis for systemwide multiprotein complex assembly.
Asunto(s)
Proteínas Cullin , Proteínas F-Box , Proteínas Ligasas SKP Cullina F-box , Factores de Transcripción , Humanos , Proteínas Cullin/química , Proteínas Cullin/metabolismo , Proteínas F-Box/metabolismo , Conformación Molecular , Proteínas Ligasas SKP Cullina F-box/química , Proteínas Ligasas SKP Cullina F-box/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Modular SCF (SKP1-CUL1-Fbox) ubiquitin E3 ligases orchestrate multiple cellular pathways in eukaryotes. Their variable SKP1-Fbox substrate receptor (SR) modules enable regulated substrate recruitment and subsequent proteasomal degradation. CAND proteins are essential for the efficient and timely exchange of SRs. To gain structural understanding of the underlying molecular mechanism, we reconstituted a human CAND1-driven exchange reaction of substrate-bound SCF alongside its co-E3 ligase DCNL1 and visualized it by cryo-EM. We describe high-resolution structural intermediates, including a ternary CAND1-SCF complex, as well as conformational and compositional intermediates representing SR- or CAND1-dissociation. We describe in molecular detail how CAND1-induced conformational changes in CUL1/RBX1 provide an optimized DCNL1-binding site and reveal an unexpected dual role for DCNL1 in CAND1-SCF dynamics. Moreover, a partially dissociated CAND1-SCF conformation accommodates cullin neddylation, leading to CAND1 displacement. Our structural findings, together with functional biochemical assays, help formulate a detailed model for CAND-SCF regulation.
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Proteínas Cullin , Proteínas Ligasas SKP Cullina F-box , Humanos , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas Cullin/metabolismo , Factores de Transcripción/metabolismo , Proteínas Portadoras/metabolismoRESUMEN
Cullin-RING E3 ligases (CRLs) are essential ubiquitylation enzymes that combine a catalytic core built around cullin scaffolds with â¼300 exchangeable substrate adaptors. To ensure robust signal transduction, cells must constantly form new CRLs by pairing substrate-bound adaptors with their cullins, but how this occurs at the right time and place is still poorly understood. Here, we show that formation of individual CRL complexes is a tightly regulated process. Using CUL3KLHL12 as a model, we found that its co-adaptor PEF1-ALG2 initiates CRL3 formation by releasing KLHL12 from an assembly inhibitor at the endoplasmic reticulum, before co-adaptor monoubiquitylation stabilizes the enzyme for substrate modification. As the co-adaptor also helps recruit substrates, its role in CRL assembly couples target recognition to ubiquitylation. We propose that regulators dedicated to specific CRLs, such as assembly inhibitors or co-adaptors, cooperate with target-agnostic adaptor exchange mechanisms to establish E3 ligase complexes that control metazoan development.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Cullin/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas Cullin/genética , Células HEK293 , Humanos , Manosiltransferasas/genética , Manosiltransferasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , UbiquitinaciónRESUMEN
Inducing protein degradation via small molecules is a transformative therapeutic paradigm. Although structural requirements of target degradation are emerging, mechanisms determining the cellular response to small-molecule degraders remain poorly understood. To systematically delineate effectors required for targeted protein degradation, we applied genome-scale CRISPR/Cas9 screens for five drugs that hijack different substrate receptors (SRs) of cullin RING ligases (CRLs) to induce target proteolysis. We found that sensitivity to small-molecule degraders is dictated by shared and drug-specific modulator networks, including the COP9 signalosome and the SR exchange factor CAND1. Genetic or pharmacologic perturbation of these effectors impairs CRL plasticity and arrests a wide array of ligases in a constitutively active state. Resulting defects in CRL decommissioning prompt widespread CRL auto-degradation that confers resistance to multiple degraders. Collectively, our study informs on regulation and architecture of CRLs amenable for targeted protein degradation and outlines biomarkers and putative resistance mechanisms for upcoming clinical investigation.
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Complejo del Señalosoma COP9/metabolismo , Proteínas Cullin/metabolismo , Proteolisis , Factores de Transcripción/metabolismo , Complejo del Señalosoma COP9/genética , Proteínas Cullin/genética , Humanos , Factores de Transcripción/genéticaRESUMEN
Skp1â Cul1â F-box (SCF) ubiquitin ligase assembly is regulated by the interplay of substrate binding, reversible Nedd8 conjugation on Cul1, and the F-box protein (FBP) exchange factors Cand1 and Cand2. Detailed investigations into SCF assembly and function in reconstituted systems and Cand1/2 knockout cells informed the development of a mathematical model for how dynamical assembly of SCF complexes is controlled and how this cycle is coupled to degradation of an SCF substrate. Simulations predicted an unanticipated hypersensitivity of Cand1/2-deficient cells to FBP expression levels, which was experimentally validated. Together, these and prior observations lead us to propose the adaptive exchange hypothesis, which posits that regulation of the koff of an FBP from SCF by the actions of substrate, Nedd8, and Cand1 molds the cellular repertoire of SCF complexes and that the plasticity afforded by this exchange mechanism may enable large variations in FBP expression during development and in FBP gene number during evolution.
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Proteínas F-Box , Regulación de la Expresión Génica , Modelos Biológicos , Modelos Químicos , Proteolisis , Factores de Transcripción , Animales , Proteínas Cullin/química , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Proteínas F-Box/biosíntesis , Proteínas F-Box/química , Proteínas F-Box/genética , Ratones , Proteína NEDD8/química , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Pathological cardiac hypertrophy is a process characterized by significant disturbance of protein turnover. Cullin-associated and Neddylation-dissociated 1 (CAND1) acts as a coordinator to modulate substrate protein degradation by promoting the formation of specific cullin-based ubiquitin ligase 3 complex in response to substrate accumulation, which thereby facilitate the maintaining of normal protein homeostasis. Accumulation of calcineurin is critical in the pathogenesis of cardiac hypertrophy and heart failure. However, whether CAND1 titrates the degradation of hypertrophy related protein eg. calcineurin and regulates cardiac hypertrophy remains unknown. Therefore, we aim to explore the role of CAND1 in cardiac hypertrophy and heart failure and the underlying molecular mechanism. Here, we found that the protein level of CAND1 was increased in cardiac tissues from heart failure (HF) patients and TAC mice, whereas the mRNA level did not change. CAND1-KO+ /- aggravated TAC-induced cardiac hypertrophic phenotypes; in contrast, CAND1-Tg attenuated the maladaptive cardiac remodeling. At the molecular level, CAND1 overexpression downregulated, whereas CAND1-KO+ /- or knockdown upregulated calcineurin expression at both in vivo and in vitro conditions. Mechanistically, CAND1 overexpression favored the assembly of Cul1/atrogin1/calcineurin complex and rendered the ubiquitination and degradation of calcineurin. Notably, CAND1 deficiency-induced hypertrophic phenotypes were partially rescued by knockdown of calcineurin, and application of exogenous CAND1 prevented TAC-induced cardiac hypertrophy. Taken together, our findings demonstrate that CAND1 exerts a protective effect against cardiac hypertrophy and heart failure partially by inducing the degradation of calcineurin.
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Calcineurina , Cardiomegalia , Proteínas Cullin , Insuficiencia Cardíaca , Animales , Calcineurina/metabolismo , Cardiomegalia/genética , Proteínas Cullin/química , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Insuficiencia Cardíaca/genética , Humanos , Ratones , Factores de TranscripciónRESUMEN
Cullin-RING ubiquitin ligases (CRLs) determine the substrate specificity of ubiquitination reactions, and substrates are recruited to the cullin core through binding to their cognate substrate receptor modules. Because a family of substrate receptors compete for the same cullin core, the assembly and activity of CRLs are dynamically regulated to fulfill the needs of the cell to adapt to the changing pool of proteins demanding ubiquitination. Cullins are modified by NEDD8, a ubiquitin-like protein. This process, referred to as neddylation, promotes the E3 activity of CRLs by inducing conformational rearrangement in the Cullin-RING catalytic core. Cand1 is a cullin-associated protein whose binding is excluded by cullin neddylation. Although early biochemical studies suggested that Cand1 inhibits CRL activity, genetic studies revealed its positive role in ubiquitination. Emerging evidence from kinetic and quantitative proteomic studies demonstrated that Cand1 stimulates assembly of new Skp1-Cul1-F-box protein (SCF) complexes by exchanging the Skp1-F-box protein substrate receptor modules. Furthermore, aided by refined experimental design as well as computational simulation, an attractive model has been developed in which substrate, neddylation cycle and Cand1-mediated "adaptive exchange" collaborate to maintain the dynamics of the cellular SCF repertoire. Here, we review and discuss recent advances that have deepened our understanding of CRL regulation.
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Proteínas Cullin/química , Proteínas Cullin/metabolismo , Animales , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Humanos , Proteómica , Especificidad por Sustrato , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
PURPOSE: Accumulated evidence has shown that microRNAs (miRNAs) are closely related to the pathogenesis and progression of senile cataracts. Here we investigate the effect of miR-29a-3p in cataractogenesis and determined the potential molecular mechanism involved. METHODS: In this study, we constructed a selenite cataract model in rats and obtained the miRNAs related to cataracts by whole transcriptome sequencing. To investigate the effect and mechanism of miR-29a-3p on cataracts, we performed several in vivo and in vitro experiments, including CCK8 assay, flow cytometry, luciferase reporter assay, Edu assay, and western blot analysis. RESULT: Sequencing data showed downregulation of miR-29a-3p in rats with selenite cataracts. Down-regulation of miR-29a-3p could promote lens epithelial cells (SRA01/04) proliferation and inhibit cell apoptosis, and miR-29a-3p silence could inhibit the development of cataracts. Additionally, CAND1 was a direct target gene for miR-29a-3p. CONCLUSION: These data demonstrate that miR-29a-3p inhibits apoptosis of lens epithelial cells by regulating CAND1, which may be a potential target for senile cataracts.
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Catarata , MicroARNs , Animales , Ratas , Regulación hacia Arriba , Proliferación Celular , MicroARNs/genética , Células Epiteliales/patología , Catarata/genética , Catarata/patología , Apoptosis/genética , Ácido SeleniosoRESUMEN
The highly conserved ribosomal protein L34 (RPL34) has been reported to play an essential role in the progression of diverse malignancies. RPL34 is aberrantly expressed in multiple cancers, although its significant in colorectal cancer (CRC) is currently unclear. Here, we demonstrated that RPL34 expression was higher in CRC tissues than in normal tissues. Upon RPL34 overexpression, the ability of proliferation, migration, invasion, and metastasis of CRC cells were significantly enhanced in vitro and in vivo. Furthermore, high expression of RPL34 accelerated cell cycle progression, activated the JAK2/STAT3 signaling pathway, and induced the epithelial-to-mesenchymal transition (EMT) program. Conversely, RPL34 silencing inhibited the CRC malignant progression. Utilizing immunoprecipitation assays, we identified the RPL34 interactor, the cullin-associated NEDD8-dissociated protein 1 (CAND1), which is a negative regulator of cullin-RING ligases. CAND1 overexpression reduced the ubiquitin level of RPL34 and stabilized RPL34 protein. CAND1 silencing in CRC cells resulted in a decrease in the ability of proliferation, migration, and invasion. CAND1 overexpression promoted CRC malignant phenotypes and induced EMT, and RPL34 knockdown rescued CAND1-induced CRC progression. In summary, our study indicates that RPL34 acts as a mediator, is stabilized by CAND1, and promotes proliferation and metastasis, in part, through the activation of the JAK2/STAT3 signaling pathway and induction of EMT in CRC.
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Neoplasias Colorrectales , Proteínas Cullin , Humanos , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Regulación hacia Abajo , Movimiento Celular/genética , Transducción de Señal , Proliferación Celular/genética , Neoplasias Colorrectales/metabolismo , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Metástasis de la Neoplasia , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
The identification of novel prognostic biomarkers for breast cancer is an unmet clinical need. Cullin-associated and neddylation-dissociated 1 (CAND1) has been implicated in mediating carcinogenesis in prostate and lung cancers. In addition, CAND1 is an established prognostic biomarker for worse prognosis in liver cancer. However, the prognostic significance of CAND1 in breast cancer has not yet been explored. In this study, Breast Cancer Gene-Expression Miner (Bc-GenExMiner) and TIMER2.0 were utilized to explore the mRNA expression of CAND1 in ERα-positive breast cancer patients. The Kaplan-Meier plotter was used to explore the relationship between CAND1 expression and several prognostic indicators. The Gene Set Cancer Analysis (GSCA) web server was then used to explore the pathways of the genes that correlate with CAND1 in ERα-positive breast cancer. Immune infiltration was investigated using Bc-GenExMiner. Our bioinformatics analysis illustrates that breast cancer patients have higher CAND1 compared to normal breast tissue and that ERα-positive breast cancer patients with a high expression of CAND1 have poor overall survival (OS), distant metastasis-free survival (DMFS), and relapse-free survival (RFS) outcomes. Higher CAND1 expression was observed in histologic grade 3 compared to grades 2 and 1. Our results revealed that CAND1 positively correlates with lymph nodes and negatively correlates with the infiltration of immune cells, which is in agreement with published reports. Our findings suggest that CAND1 might mediate invasion and metastasis in ERα-positive breast cancer, possibly through the activation of estrogen and androgen signaling pathways; however, experiments should be carried out to further explore the role of CAND1 in activating the androgen and estrogen signaling pathways. In conclusion, the results suggest that CAND1 could be used as a potential novel biomarker for worse prognosis in ERα-positive breast cancer.
RESUMEN
Gastric cancer (GC) is one of the most severe gastric diseases worldwide. However, the molecular basis that drives tumorigenesis and progression is not completely understood, which hinders the efficacy and development of therapeutic options. Glutathione-S-transferases (GSTs) are a group of phase II detoxification enzymes that maintain redox homeostasis; however, their roles in cancers are not well defined. Here, we revealed that the expression of GST family members is significantly impaired in GC tissues. Glutathione-S-transferase mu 3 (GSTM3), a member of GST family, is dramatically downregulated in cancerous tissues and has been identified as an independent prognostic factor in GC associated with tumor differentiation, inhibiting GC cell proliferation and migration in vitro and in vivo. Mechanistically, GSTM3 is transcriptionally activated by NRF2/KEAP1 signaling. As a feedback loop, GSTM3 binds to Cullin-associated and neddylation-dissociated 1 protein (CAND1), an exchange factor for integrating Kelch-like ECH-associated protein 1 (KEAP1) into Cul3-RING ubiquitin ligases (CRL3), to disrupt nuclear factor-erythroid factor 2-related factor 2 (NRF2)/KEAP1 binding and prevent NRF2 ubiquitination and degradation, leading to its activation. A deficiency in glutathione S-Transferase Mu 3 (GSTM3) reduces DNA mismatch repair (MMR) gene expression and increases mutagenesis via CAND1/NRF2 binding. Importantly, GSTM3/NRF2 and KEAP1 were negatively and positively associated with the genomic signature for microsatellite instability, respectively. Clinically, GSTM3, NRF2, and MutS homolog 6 (MSH6) were positively correlated in the GC specimens. This study uncovered a reciprocal regulation between GSTM3 and NRF2 and established a functional and clinical link between GSTM3-NRF2/KEAP1 and MMR during GC cell proliferation and progression, thus providing potential therapeutic targets for GC.
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Proteínas Cullin , Factor 2 Relacionado con NF-E2 , Carcinogénesis/genética , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Reparación de la Incompatibilidad de ADN , Glutatión/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Factores de TranscripciónRESUMEN
The dynamic assembly of SKP1â¢CUL1â¢F-box protein (SCF) ubiquitin ligases is important for protein ubiquitination and degradation. This process is enabled by CAND1, which exchanges F-box proteins associated with the common CUL1 scaffold, and thereby, recycles the limited CUL1 core and allows diverse F-box proteins to assemble active SCFs. Previous human cell biological and computational studies have led to the adaptive exchange hypothesis, which suggests that the CAND1-mediated exchange confers plasticity on the SCF system, allowing cells to tolerate large variations in F-box protein expression. Here, we tested this hypothesis using Arabidopsis thaliana, a multicellular organism expressing hundreds of F-box protein genes at variable levels in different tissues. The cand1 null mutant in Arabidopsis is viable but produce almost no seeds. Bioinformatic, cell biological, and developmental analyses revealed that the low fertility in the cand1 mutant is associated with cell death in pollen, where the net expression of F-box protein genes is significantly higher than any other Arabidopsis tissue. In addition, we show that the transmission efficiency of the cand1 null allele was reduced through the male but not the female gametophyte. Our results suggest that CAND1 activity is essential in cells or tissues expressing high levels of F-box proteins. This finding is consistent with the proposed adaptive exchange hypothesis, demonstrating the necessity of the evolutionarily conserved CAND1-mediated exchange system in the development of a multicellular organism.
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The proliferation and migration of Schwann cells (SCs) promote nerve regeneration after facial nerve injury. In recent years, the role of long noncoding RNAs (lncRNAs) in regulating SC proliferation and migration has been gradually uncovered. However, there is little evidence on the function of lncRNA RMRP (lnc-RMRP) in SC growth. In the present study, we performed loss-of-function and overexpression assays to explore the function of lnc-RMRP in SCs. The relationships between lnc-RMRP, miR-766-5p and CAND1 (cullin-associated and neddylation-dissociated 1) were analyzed using bioinformatics analysis, luciferase detection, RNA binding protein immunoprecipitation and RNA pulldown methods. CCK-8, EdU, Transwell and wound healing assays were utilized for the detections of cell proliferation and migration. We found that lnc-RMRP silencing enhanced cell proliferation and migration of SCs, while lnc-RMRP overexpression showed the opposite effect. Mechanistically, lnc-RMRP directly bound to and negatively modulated the expression of miR-766-5p. MiR-766-5p knockdown decreased cell viability, proliferation and migration of SCs, and also reversed the effects of lnc-RMRP silencing. In addition, lnc-RMRP positively regulated CAND1 expression by sponging miR-766-5p. Upregulation of CAND1 rescued the function of lnc-RMRP knockdown in regulating SC proliferation and migration. These data suggested that lnc-RMRP played a significant role in SC proliferation and migration, indicating that lnc-RMRP might be a potential therapeutic target for the treatment of facial nerve injury.
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Movimiento Celular , Proliferación Celular , Células de Schwann , Factores de Transcripción , Animales , Ratas , Línea Celular Tumoral , Células de Schwann/metabolismo , Células de Schwann/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: The current study set out to identify the miRNA-mRNA regulatory networks that influence the radiosensitivity in esophageal cancer based on the The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. METHODS: Firstly, esophageal cancer-related miRNA-seq and mRNA-seq data were retrieved from the TCGA database, and the mRNA dataset of esophageal cancer radiotherapy was downloaded from the GEO database to analyze the differential expressed miRNAs (DEmiRNAs) and mRNAs (DEmRNAs) in radiosensitive and radioresistant samples, followed by the construction of the miRNA-mRNA regulatory network and Gene Ontology and KEGG enrichment analysis. Additionally, a prognostic risk model was constructed, and its accuracy was evaluated by means of receiver operating characteristic analysis. RESULTS: A total of 125 DEmiRNAs and 42 DEmRNAs were closely related to the radiosensitivity in patients with esophageal cancer. Based on 47 miRNA-mRNA interactions, including 21 miRNAs and 21 mRNAs, the miRNA-mRNA regulatory network was constructed. The prognostic risk model based on 2 miRNAs (miR-132-3p and miR-576-5p) and 4 mRNAs (CAND1, ZDHHC23, AHR, and MTMR4) could accurately predict the prognosis of esophageal cancer patients. Finally, it was verified that miR-132-3p/CAND1/ZDHHC23 and miR-576-5p/AHR could affect the radiosensitivity in esophageal cancer. CONCLUSION: Our study demonstrated that miR-132-3p/CAND1/ZDHHC23 and miR-576-5p/AHR were critical molecular pathways related to the radiosensitivity of esophageal cancer.
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Neoplasias Esofágicas , MicroARNs , Humanos , MicroARNs/genética , ARN Mensajero/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/radioterapia , Tolerancia a Radiación/genética , Bases de Datos FactualesRESUMEN
OBJECTIVE: MiR-BART16 is a newly discovered Epstein-Barr Virus-encoded microRNA (miRNA). We aimed to explore the role of EBV-miR-BART16 in infectious mononucleosis (IM). METHODS: Peripheral blood lymphocyte subsets were analyzed in 30 IM and 10 healthy children by flow cytometry. MiR-BART16 and its targets were measured by real-time PCR, western blot, ELISA, and dual-luciferase assay. RESULTS: Serum miR-BART16 expression was significantly higher in the IM children than that in the healthy children, and was positively correlated with EBV copy number. Receiver operating characteristic analysis revealed serum miR-BART16 could differentiate IM and healthy individuals (P=0.0041). CAND1 was targeted and downregulated by miR-BART16 in an EBV infection-dependent way. CONCLUSIONS: These results highlight that EBV-miR-BART16 plays an important role in regulating the expression of CAND1 to affect pediatric IM.
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Herpesvirus Humano 4/genética , Mononucleosis Infecciosa/genética , MicroARNs/genética , Niño , Infecciones por Virus de Epstein-Barr/sangre , Infecciones por Virus de Epstein-Barr/virología , Femenino , Citometría de Flujo/métodos , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Herpesvirus Humano 4/patogenicidad , Humanos , Mononucleosis Infecciosa/metabolismo , Subgrupos Linfocitarios , Masculino , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: Stroke is a severe complication of atrial fibrillation (AF). We aimed to discover key genes and microRNAs related to stroke risk in patients with AF using bioinformatics analysis. METHODS: GSE66724 microarray data, including peripheral blood samples from eight patients with AF and stroke and eight patients with AF without stroke, were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between AF patients with and without stroke were identified using the GEO2R online tool. Functional enrichment analysis was performed using the DAVID database. A protein-protein interaction (PPI) network was obtained using the STRING database. MicroRNAs (miRs) targeting these DEGs were obtained from the miRNet database. A miR-DEG network was constructed using Cytoscape software. RESULTS: We identified 165 DEGs (141 upregulated and 24 downregulated). Enrichment analysis showed enrichment of certain inflammatory processes. The miR-DEG network revealed key genes, including MEF2A, CAND1, PELI1, and PDCD4, and microRNAs, including miR-1, miR-1-3p, miR-21, miR-21-5p, miR-192, miR-192-5p, miR-155, and miR-155-5p. CONCLUSION: Dysregulation of certain genes and microRNAs involved in inflammation may be associated with a higher risk of stroke in patients with AF. Evaluating these biomarkers could improve prediction, prevention, and treatment of stroke in patients with AF.
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Fibrilación Atrial/complicaciones , Susceptibilidad a Enfermedades , Inflamación/complicaciones , Accidente Cerebrovascular/etiología , Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Biomarcadores , Biología Computacional , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Ontología de Genes , Humanos , Inflamación/genética , Inflamación/metabolismo , Mapeo de Interacción de Proteínas , Medición de Riesgo , Factores de Riesgo , Accidente Cerebrovascular/metabolismoRESUMEN
Evidence has accumulated asserting the importance of cullin-RING (really interesting new gene) ubiquitin ligases (CRLs) and their regulator Cullin-associated neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) dissociated protein 1 (Cand1) in various cancer entities. However, the role of Cand1 in prostate cancer (PCa) has not been intensively investigated so far. Thus, in the present study, we aimed to assess the relevance of Cand1 in the clinical and preclinical setting. Immunohistochemical analyses of radical prostatectomy specimens of PCa patients showed that Cand1 protein levels are elevated in PCa compared to benign areas. In addition, high Cand1 levels were associated with higher Gleason Scores, as well as higher tumor recurrence and decreased overall survival. In line with clinical findings, in vitro experiments in different PCa cell lines revealed that knockdown of Cand1 reduced cell viability and proliferation and increased apoptosis, therefore underlining its role in tumor progression. We also found that the cyclin-dependent kinase inhibitor p21 is significantly upregulated upon downregulation of Cand1. Using bioinformatic tools, we detected genes encoding for proteins linked to mRNA turnover, protein polyubiquitination, and proteasomal degradation to be significantly upregulated in Cand1high tumors. Next generation sequencing of PCa cell lines resistant to the anti-androgen enzalutamide revealed that Cand1 is mutated in enzalutamide-resistant cells, however, with little functional and clinically relevant impact in the process of resistance development. To summarize the present study, we found that high Cand1 levels correlate with PCa aggressiveness.
RESUMEN
E3 cullin-RING ubiquitin ligase (CRL) complexes recognize specific substrates and are activated by covalent modification with ubiquitin-like Nedd8. Deneddylation inactivates CRLs and allows Cand1/A to bind and exchange substrate recognition subunits. Human as well as most fungi possess a single gene for the receptor exchange factor Cand1, which is split and rearranged in aspergilli into two genes for separate proteins. Aspergillus nidulans CandA-N blocks the neddylation site, and CandA-C inhibits the interaction to the adaptor/substrate receptor subunits similar to the respective N-terminal and C-terminal parts of single Cand1. The pathogen Aspergillus fumigatus and related species express a CandA-C with a 190-amino-acid N-terminal extension domain encoded by an additional exon. This extension corresponds in most aspergilli, including A. nidulans, to a gene directly upstream of candA-C encoding a 20-kDa protein without human counterpart. This protein was named CandA-C1, because it is also required for the cellular deneddylation/neddylation cycle and can form a trimeric nuclear complex with CandA-C and CandA-N. CandA-C and CandA-N are required for asexual and sexual development and control a distinct secondary metabolism. CandA-C1 and the corresponding domain of A. fumigatus control spore germination, vegetative growth, and the repression of additional secondary metabolites. This suggests that the dissection of the conserved Cand1-encoding gene within the genome of aspergilli was possible because it allowed the integration of a fungus-specific protein required for growth into the CandA complex in two different gene set versions, which might provide an advantage in evolution.IMPORTANCEAspergillus species are important for biotechnological applications, like the production of citric acid or antibacterial agents. Aspergilli can cause food contamination or invasive aspergillosis to immunocompromised humans or animals. Specific treatment is difficult due to limited drug targets and emerging resistances. The CandA complex regulates, as a receptor exchange factor, the activity and substrate variability of the ubiquitin labeling machinery for 26S proteasome-mediated protein degradation. Only Aspergillus species encode at least two proteins that form a CandA complex. This study shows that Aspergillus species had to integrate a third component into the CandA receptor exchange factor complex that is unique to aspergilli and required for vegetative growth, sexual reproduction, and activation of the ubiquitin labeling machinery. These features have interesting implications for the evolution of protein complexes and could make CandA-C1 an interesting candidate for target-specific drug design to control fungal growth without affecting the human ubiquitin-proteasome system.
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Aspergillus nidulans/enzimología , Aspergillus nidulans/genética , Proteínas Cullin/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Complejos Multiproteicos , Ubiquitina/metabolismoRESUMEN
BACKGROUND: Lung cancer continues to be one of the top five causes of cancer-related mortality. This study aims to identify down- and upregulated miRNAs and mRNA which can be used as potential biomarkers and/or therapeutic targets for lung cancer. METHODS: Integrated analysis of differential expression profiles of miRNA and mRNA in lung cancer was performed by searching Gene Expression Omnibus datasets. Based on miRNA expression profiles, direct mRNA targets of miRNAs with experimental support were identified through miRTarBase. The levels of representative miRNAs and mRNAs were confirmed through qualitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). RESULTS: The miR-33a was decreased in non-small cell lung cancer (NSCLC) tissues compared with the para-carcinoma tissues, whereas its target mRNA of cullin-associated NEDD8-dissociated protein 1 (CAND1) was increased in NSCLC tissues. Further research has shown that miR-33a can inhibit lung cancer cell proliferation, cell cycle progression, and migration by targeting CAND1. Moreover, the CAND1 knockout lung cancer cells showed similar results as cells transfected with miR-33a mimic. CONCLUSIONS: These results suggested that the data mining based on online databases was an effective method in finding novel target in cancer research, and the miR-33a and CAND1 played an important role in lung cancer proliferation and cell migration.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Pulmonares/patología , MicroARNs/genética , Factores de Transcripción/biosíntesis , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Movimiento Celular/genética , Proliferación Celular/genética , Minería de Datos/métodos , Humanos , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/genética , Factores de Transcripción/genéticaRESUMEN
Cullin-associated NEDD8-dissociated 1 (CAND1) plays a vital role in regulating the activity of Cullin-RING ubiquitin ligases (CRLs), which are frequently dysregulated in cancer. However, the role of CAND1 in hepatocellular carcinoma (HCC) remains unknown. Here, we found that CAND1 was overexpressed in HCC tissues compared to corresponding adjacent liver tissues (71.7% vs 16.7%); high expression of CAND1 was associated with poor overall survival (40.7 vs 57.3 months, P=0.0013); and CAND1 was an independent risk factor for the prognosis of HCC patients (N=138, P=0.018). Functional studies revealed that CAND1 knockdown efficiently suppressed the proliferation of liver cancer cells by activating caspase-8-dependent mitochondrial apoptosis. We also observed a mutual activation loop between caspase-8 and Receptor Interacting Protein 1 (RIP1), which amplified CAND1 knockdown-induced apoptotic signals in the cells. Furthermore, RIP1 inhibitor Necrostatin-1 eliminated the activation of caspase-8. In conclusion, our study pioneered in reporting high CAND1 expression as a predictor of poor prognosis for HCC patients. CAND1 silencing suppressed HCC cell proliferation by inducing caspase-8/RIP1-dependent apoptosis. These findings supported that CAND1 could be a new therapeutic target for liver cancer.