RESUMO
BACKGROUND: According to the Global Cancer Statistics in 2020, the incidence and mortality of colorectal cancer (CRC) rank third and second among all tumors. The disturbance of ubiquitination plays an important role in the initiation and development of CRC, but the ubiquitinome of CRC cells and the survival-relevant ubiquitination are poorly understood. METHODS: The ubiquitinome of CRC patients (n = 6) was characterized using our own data sets of proteomic and ubiquitin-proteomic examinations. Then, the probable survival-relevant ubiquitination was searched based on the analyses of data sets from public databases. RESULTS: For the ubiquitinomic examination, we identified 1690 quantifiable sites and 870 quantifiable proteins. We found that the highly-ubiquitinated proteins (n ≥ 10) were specifically involved in the biological processes such as G-protein coupling, glycoprotein coupling, and antigen presentation. Also, we depicted five motif sequences frequently recognized by ubiquitin. Subsequently, we revealed that the ubiquitination content of 1172 proteins were up-regulated and 1700 proteins were down-regulated in CRC cells versus normal adjacent cells. We demonstrated that the differentially ubiquitinated proteins were relevant to the pathways including metabolism, immune regulation, and telomere maintenance. Then, integrated with the proteomic datasets from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) (n = 98), we revealed that the increased ubiquitination of FOCAD at Lys583 and Lys587 was potentially associated with patient survival. Finally, we depicted the mutation map of FOCAD and elucidated its potential functions on RNA localization and translation in CRC. CONCLUSIONS: The findings of this study described the ubiquitinome of CRC cells and identified abnormal ubiquitination(s) potentially affecting the patient survival, thereby offering new probable opportunities for clinical treatment.
Assuntos
Neoplasias Colorretais , Proteínas Ubiquitinadas , Neoplasias Colorretais/patologia , Humanos , Proteômica , RNA/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/metabolismo , UbiquitinaçãoRESUMO
BACKGROUND: Gastric cancer remains the most prevalent and highly lethal disease worldwide. MAP4K4, a member of Ste20, plays an important role in various pathologies, including cancer. However, its role in gastric cancer is not yet fully elucidated. Therefore, this study aims to determine the tumor-promoting role of MAP4K4 in gastric cancer and whether it can be used as a new and reliable biomarker to predict the prognosis of gastric cancer. For this purpose, we divide the samples into high- and low-expression groups according to the expression level of MAP4K4. The association of MAP4K4 expression with prognosis is assessed using the Kaplan-Meier survival analysis. Furthermore, immune infiltration analysis using ESTIMATE is conducted to evaluate the tumor immune scores of the samples. RESULTS: The findings reveal a significantly higher expression of MAP4K4 in tumor samples than in adjacent samples. The high-expression group was significantly enriched in tumor-related pathways, such as the PI3K-Akt signaling pathway. In addition, immune infiltration analysis revealed a positive correlation between immune scores and MAP4K4 expression. We also observed that miRNAs, such as miR-192-3p (R = -0.317, p-value 3.111 × 10-9), miR-33b-5p (R= -0.238, p-value 1.166 × 10-5), and miR-582-3p (R = -0.214, p-value 8.430 × 10-5), had potential negative regulatory effects on MAP4K4. Moreover, we identified several transcription factors, ubiquitinated proteins, and interacting proteins that might regulate MAP4K4. The relationship between MAP4K4 and DNA methylation was also identified. Finally, we verified the high expression of MAP4K4 and its effect on promoting cancer. CONCLUSION: MAP4K4 might be closely related to gastric cancer's progression, invasion, and metastasis. Its high expression negatively impacts the prognosis of gastric cancer patients. This suggests MAP4K4 as an important prognostic factor for gastric cancer and could be regarded as a new potential prognostic detection and therapeutic target.
Assuntos
MicroRNAs , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Biologia Computacional , Regulação Neoplásica da Expressão Gênica , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Biomarcadores , Fatores de Transcrição/genética , Proteínas Serina-Treonina Quinases , Peptídeos e Proteínas de Sinalização Intracelular/genéticaRESUMO
Protein aggregation and degradation via autophagy (aggrephagy) are major strategies adopted by cells to remove misfolded polypeptides when there is proteasome dysfunction. The functional protein complex consisting of heat shock protein 70 (Hsp70), cochaperone ubiquitin ligase carboxyl-terminal of Hsp70/Hsp90 interacting protein (CHIP), and co-chaperone Bcl-2-associated athanogene 3 (BAG3) has been associated with the activation of protein aggregation. However, data on the mechanisms of action of the complex in the protein degradation remains scant. Here, we report that upon proteasome stress, the M2 isoform of pyruvate kinase (PKM2) promotes the aggregation of ubiquitinated proteins and its knockout or knockdown aggravates the sensitivity of cells to proteasome inhibitors. Besides, following proteasome inhibition, PKM2 promotes the interaction of BAG3 with CHIP and HSP70. Interestingly, re-expression of loss-of-function mutants in PKM2-knockout cells showed that the regulatory function of PKM2 in this progress does not depend on the activity of glycolytic enzymes or protein kinases. Taken together, these findings demonstrate that PKM2 mediates the formation of the CHIP-HSP70-BAG3 protein complex and promotes the aggregation of ubiquitinated misfolded proteins, thus compensating for proteasome stress in cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Complexos Multiproteicos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos , Piruvato Quinase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Ubiquitinadas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Células HEK293 , Proteínas de Choque Térmico HSP70/genética , Células Hep G2 , Humanos , Complexos Multiproteicos/genética , Complexo de Endopeptidases do Proteassoma/genética , Piruvato Quinase/genética , Ubiquitina-Proteína Ligases/genética , Proteínas Ubiquitinadas/genéticaRESUMO
The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C terminus through the central pore of the hexameric double ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.
Assuntos
Poliubiquitina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas Ubiquitinadas/metabolismo , Proteína com Valosina/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transporte Proteico , Desdobramento de Proteína , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Ubiquitinadas/genética , Ubiquitinação , Proteína com Valosina/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
The autophagic degradation of misfolded and ubiquitinated proteins is important for cellular homeostasis. In this process, which is governed by cargo receptors, ubiquitinated proteins are condensed into larger structures and subsequently become targets for the autophagy machinery. Here we employ in vitro reconstitution and cell biology to define the roles of the human cargo receptors p62/SQSTM1, NBR1 and TAX1BP1 in the selective autophagy of ubiquitinated substrates. We show that p62 is the major driver of ubiquitin condensate formation. NBR1 promotes condensate formation by equipping the p62-NBR1 heterooligomeric complex with a high-affinity UBA domain. Additionally, NBR1 recruits TAX1BP1 to the ubiquitin condensates formed by p62. While all three receptors interact with FIP200, TAX1BP1 is the main driver of FIP200 recruitment and thus the autophagic degradation of p62-ubiquitin condensates. In summary, our study defines the roles of all three receptors in the selective autophagy of ubiquitin condensates.
Assuntos
Autofagia/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Ubiquitina/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Transporte , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/genética , Domínios Proteicos , Proteínas de Ligação a RNA/metabolismo , Proteína Sequestossoma-1/metabolismo , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/metabolismoRESUMO
TRIM17 is a member of the TRIM family, a large class of RING-containing E3 ubiquitin-ligases. It is expressed at low levels in adult tissues, except in testis and in some brain regions. However, it can be highly induced in stress conditions which makes it a putative stress sensor required for the triggering of key cellular responses. As most TRIM members, TRIM17 can act as an E3 ubiquitin-ligase and promote the degradation by the proteasome of substrates such as the antiapoptotic protein MCL1. Intriguingly, TRIM17 can also prevent the ubiquitination of other proteins and stabilize them, by binding to other TRIM proteins and inhibiting their E3 ubiquitin-ligase activity. This duality of action confers several pivotal roles to TRIM17 in crucial cellular processes such as apoptosis, autophagy or cell division, but also in pathological conditions as diverse as Parkinson's disease or cancer. Here, in addition to recent data that endorse this duality, we review what is currently known from public databases and the literature about TRIM17 gene regulation and expression, TRIM17 protein structure and interactions, as well as its involvement in cell physiology and human disorders.
Assuntos
Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação , Animais , Morte Celular , Sobrevivência Celular , Estabilidade Enzimática , Humanos , Estabilidade Proteica , Proteólise , Transdução de Sinais , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Proteínas Ubiquitinadas/genéticaRESUMO
Long non-coding RNAs (lncRNAs) are a class of RNA transcripts longer than 200 nucleotides and mostly cannot be translated into proteins. Next-generation transcriptome sequencing of various cell types has enabled the annotation of tens of thousands of lncRNAs in human genome. Varying levels of evidence supports the implications of lncRNAs in the onset and progression of cancers. Ubiquitin is an evolutionarily conserved protein and could post-translationally mark a number of proteins. The most important proteolytic role of ubiquitination is degradation of substrate proteins by the 26S proteasome. Compiling evidences demonstrated that lncRNAs are involved in the accurate execution of protein stability programs via the ubiquitin-proteasome system. In the current review, we systematically summarize the detailed mechanisms how lncRNAs modulate ubiquitination of target proteins, regulate cancerous signaling pathways and control tumorigenesis of gastrointestinal cancers. Although there are still considerable studies on unraveling the complicated interactions between lncRNAs and proteins, we believe that lncRNAs are promising but challenging molecules which may strongly facilitate precision cancer therapeutics in the future.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Gastrointestinais/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação/fisiologia , Animais , Biomarcadores Tumorais/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Neoplasias Gastrointestinais/genética , Regulação Neoplásica da Expressão Gênica , Humanos , RNA Longo não Codificante/genética , Proteínas Ubiquitinadas/genéticaRESUMO
We previously demonstrated that cisplatin administration in mice induces muscle atrophy and an increase in the expression of two muscle-specific ubiquitin E3 ligase genes, muscle ring finger protein 1 (MuRF1), and atrophy gene-1 (atrogin-1), in skeletal muscle. Ubiquitination serves as a degradation signal in both the ubiquitin-proteasome and selective autophagy pathways. In the present study, we investigated changes in the expression of ubiquitin and ubiquitinated proteins and their degradation pathways. Ubiquitin and ubiquitinated protein levels were increased by cisplatin compared with those in the vehicle and dietary restriction (DR) groups. To quantify the levels of ubiquitin and ubiquitinated proteins, we conducted a dot blot assay using an anti-ubiquitin antibody. The expression of ubiquitin was also significantly increased by cisplatin compared with that in the vehicle and DR groups. Since the ubiquitin proteins were upregulated by cisplatin, we measured the mRNA levels of the ubiquitin genes: Ubb, Ubc, Rps27a, and Uba52. All these four genes were increased by cisplatin administration compared with those in both the vehicle-treated and DR groups in quadriceps muscle tissue. The anti-ubiquitin antibody-sensitive bands increased when C2C12 myotubes were treated with cisplatin. Furthermore, MG-132 (26 s proteasome inhibitor), but not bafilomycin A1 (autophagy inhibitor), caused a further increase in expression. In conclusion, ubiquitin and ubiquitinated proteins are upregulated in cisplatin-induced muscle atrophy. Cisplatin-induced ubiquitinated proteins are degraded by the 26 s proteasome pathway.
Assuntos
Cisplatino/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Atrofia Muscular/induzido quimicamente , Proteínas Ubiquitinadas/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Antineoplásicos/toxicidade , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mioblastos/efeitos dos fármacos , Proteínas Ubiquitinadas/genéticaRESUMO
The autosomal dominant neuronal ceroid lipofuscinoses (NCL) CLN4 is caused by mutations in the synaptic vesicle (SV) protein CSPα. We developed animal models of CLN4 by expressing CLN4 mutant human CSPα (hCSPα) in Drosophila neurons. Similar to patients, CLN4 mutations induced excessive oligomerization of hCSPα and premature lethality in a dose-dependent manner. Instead of being localized to SVs, most CLN4 mutant hCSPα accumulated abnormally, and co-localized with ubiquitinated proteins and the prelysosomal markers HRS and LAMP1. Ultrastructural examination revealed frequent abnormal membrane structures in axons and neuronal somata. The lethality, oligomerization and prelysosomal accumulation induced by CLN4 mutations was attenuated by reducing endogenous wild type (WT) dCSP levels and enhanced by increasing WT levels. Furthermore, reducing the gene dosage of Hsc70 also attenuated CLN4 phenotypes. Taken together, we suggest that CLN4 alleles resemble dominant hypermorphic gain of function mutations that drive excessive oligomerization and impair membrane trafficking.
Assuntos
Drosophila melanogaster/genética , Mutação com Ganho de Função , Lipofuscinoses Ceroides Neuronais/genética , Neurônios/metabolismo , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Drosophila melanogaster/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/genética , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Lipofuscinoses Ceroides Neuronais/metabolismo , Neurônios/ultraestrutura , Vesículas Sinápticas/metabolismo , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/metabolismoRESUMO
The proteasome-associated deubiquitinating enzyme Usp14/Ubp6 inhibits protein degradation by catalyzing substrate deubiquitination and by poorly understood allosteric actions. However, upon binding a ubiquitin chain, Usp14 enhances proteasomal degradation by stimulating ATP and peptide degradation. These studies were undertaken to clarify these seemingly opposite regulatory roles of Usp14 and their importance. To learn how the presence of Usp14 on 26S proteasomes influences its different activities, we compared enzymatic and regulatory properties of 26S proteasomes purified from wild-type mouse embryonic fibroblast cells and those lacking Usp14. The proteasomes lacking Usp14 had higher basal peptidase activity than WT 26S, and this activity was stimulated to a greater extent by adenosine 5'-O-(thiotriphosphate) (ATPγS) than with WT particles. These differences were clear even though Usp14 is present on only a minor fraction (30-40%) of the 26S in WT mouse embryonic fibroblast cells. Addition of purified Usp14 to the WT and Usp14-defficient proteasomes reduced both their basal peptidase activity and the stimulation by ATPγS. Usp14 inhibits these processes allosterically because a catalytically inactive Usp14 mutant also inhibited them. Proteasomes lacking Usp14 also exhibited greater deubiquitinating activity by Rpn11 and greater basal ATPase activity than WT particles. ATP hydrolysis by WT proteasomes is activated if they bind a ubiquitinated protein, which is loosely folded. Surprisingly, proteasomes lacking Usp14 could be activated by such proteins even without a ubiquitin chain present. Furthermore, proteasomes lacking Usp14 are much more active in degrading non-ubiquitinated proteins (e.g. Sic1) than WT particles. Thus, without a ubiquitinated substrate present, Usp14 suppresses multiple proteasomal activities, especially basal ATP consumption and degradation of non-ubiquitinated proteins. These allosteric effects thus reduce ATP hydrolysis by inactive proteasomes and nonspecific proteolysis and enhance proteasomal specificity for ubiquitinated proteins.
Assuntos
Embrião de Mamíferos/enzimologia , Fibroblastos/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina Tiolesterase/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/fisiologia , Animais , Células Cultivadas , Humanos , Camundongos , Camundongos Knockout , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Transativadores , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/genética , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação/fisiologiaRESUMO
Acyl-CoA:1,2-diacylglycerol acyltransferase (DGAT)-2 is one of the two DGAT enzymes that catalyzes the synthesis of triacylglycerol, which is an important form of stored energy for eukaryotic organisms. There is currently limited information available regarding how DGAT2 and triacylglycerol synthesis are regulated. Recent studies have indicated that DGAT2 can be regulated by changes in gene expression. How DGAT2 is regulated post-transcriptionally remains less clear. In this study, we demonstrated that DGAT2 is a very unstable protein and is rapidly degraded in an ubiquitin-dependent manner via the proteasome. Many of the 25 lysines present in DGAT2 appeared to be involved in promoting its degradation. However, the six C-terminal lysines were the most important in regulating stability. We also demonstrated that acyl-CoA:monoacylglycerol acyltransferase (MGAT)-2, an enzyme with extensive sequence homology to DGAT2 that catalyzes the synthesis of diacylglycerol, was also ubiquitinated. However, MGAT2 was found to be much more stable than DGAT2. Interestingly, when co-expressed, MGAT2 appeared to stabilize DGAT2. Finally, we found that both DGAT2 and MGAT2 are substrates of the endoplasmic reticulum-associated degradation pathway.
Assuntos
Aciltransferases/metabolismo , Diacilglicerol O-Aciltransferase/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Ubiquitinadas/metabolismo , Aciltransferases/genética , Animais , Células COS , Diacilglicerol O-Aciltransferase/genética , Diglicerídeos/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Proteínas Ubiquitinadas/genéticaRESUMO
Elevated valosin containing protein (VCP/p97) levels promote the progression of non-small cell lung carcinoma (NSCLC). Although many VCP inhibitors are available, most of these therapeutic compounds have low specificity for targeted tumor cell delivery. Hence, the primary aim of this study was to evaluate the in vitro efficacy of dendrimer-encapsulated potent VCP-inhibitor drug in controlling non-small cell lung carcinoma (NSCLC) progression. The VCP inhibitor(s) (either in their pure form or encapsulated in generation-4 PAMAM-dendrimer with hydroxyl surface) were tested for their in vitro efficacy in modulating H1299 (NSCLC cells) proliferation, migration, invasion, apoptosis and cell cycle progression. Our results show that VCP inhibition by DBeQ was significantly more potent than NMS-873 as evident by decreased cell proliferation (p<0.0001, MTT-assay) and migration (p<0.05; scratch-assay), and increased apoptosis (p<0.05; caspase-3/7-assay) as compared to untreated control cells. Next, we found that dendrimer-encapsulated DBeQ (DDNDBeQ) treatment increased ubiquitinated-protein accumulation in soluble protein-fraction (immunoblotting) of H1299 cells as compared to DDN-control, implying the effectiveness of DBeQ in proteostasis-inhibition. We verified by immunostaining that DDNDBeQ treatment increases accumulation of ubiquitinated-proteins that co-localizes with an ER-marker, KDEL. We observed that proteostasis-inhibition with DDNDBeQ, significantly decreased cell migration rate (scratch-assay and transwell-invasion) as compared to the control-DDN treatment (p<0.05). Moreover, DDNDBeQ treatment showed a significant decrease in cell proliferation (p<0.01, MTT-assay) and increased caspase-3/7 mediated apoptotic cell death (p<0.05) as compared to DDN-control. This was further verified by cell cycle analysis (propidium-iodide-staining) that demonstrated significant cell cycle arrest in the G2/M-phase (p<0.001) by DDNDBeQ treatment as compared to control-DDN. Moreover, we confirmed by clonogenic-assay that DDNDBeQ treatment significantly (p<0.001) inhibits H1299 colony-formation as compared to control/DDN. Overall, encapsulation of potent VCP-inhibitor DBeQ into a dendrimer allows selective VCP-mediated proteostasis-inhibition for controlling NSCLC-tumor growth and progression to allow tumor-targeted sustained drug delivery.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Quinazolinas/farmacologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Antineoplásicos/química , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Brônquios/patologia , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dendrímeros/química , Dendrímeros/metabolismo , Portadores de Fármacos , Composição de Medicamentos , Inibidores Enzimáticos/química , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Proteólise/efeitos dos fármacos , Quinazolinas/química , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/metabolismo , Proteína com ValosinaRESUMO
Ubiquitination proteasome pathway (UPP) is the most important and selective way to degrade proteins in vivo. Here, a novel proteolysis targeting peptide (PROTAP) strategy, composed of a target protein binding peptide, a linker and a ubiquitin E3 ligase recognition peptide, was designed to recruit both target protein and E3 ligase and then induce polyubiquitination and degradation of the target protein through UPP. In our study, the PROTAP strategy was proved to be a general method with high specificity using Bcl-xL protein as model target in vitro and in cells, which indicates that the strategy has great potential for in vivo application.
Assuntos
Peptídeos/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Ubiquitinadas/química , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação/fisiologia , Sítios de Ligação , Células HEK293 , Humanos , Peptídeos/química , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Engenharia de Proteínas/métodos , Proteólise , Ubiquitina-Proteína Ligases/genética , Proteínas Ubiquitinadas/genéticaRESUMO
Protein ubiquitination is an evolutionarily conserved post-translational modification process in eukaryotes, and it plays an important role in many biological processes. Aspergillus nidulans, a model filamentous fungus, contributes to our understanding of cellular physiology, metabolism and genetics, but its ubiquitination is not completely revealed. In this study, the ubiquitination sites in the proteome of A. nidulans were identified using a highly sensitive mass spectrometry combined with immuno-affinity enrichment of the ubiquitinated peptides. The 4816 ubiquitination sites were identified in 1913 ubiquitinated proteins, accounting for 18.1% of total proteins in A. nidulans. Bioinformatic analysis suggested that the ubiquitinated proteins associated with a number of biological functions and displayed various sub-cellular localisations. Meanwhile, seven motifs were revealed from the ubiquitinated peptides, and significantly over-presented in the different pathways. Comparison of the enriched functional catalogues indicated that the ubiquitination functions divergently during growth of A. nidulans and Saccharomyces cerevisiae. Additionally, the proteins in A. nidulans-specific sub-category (cell growth/morphogenesis) were subjected to the protein interaction analysis which demonstrated that ubiquitination is involved in the comprehensive protein interactions. This study presents a first proteomic view of ubiquitination in the filamentous fungus, and provides an initial framework for exploring the physiological roles of ubiquitination in A. nidulans.
Assuntos
Aspergillus nidulans/crescimento & desenvolvimento , Aspergillus nidulans/metabolismo , Proteínas Fúngicas/metabolismo , Lisina/metabolismo , Proteoma , Proteínas Ubiquitinadas/metabolismo , Motivos de Aminoácidos , Aspergillus nidulans/genética , Análise por Conglomerados , Biologia Computacional/métodos , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Anotação de Sequência Molecular , Peptídeos/metabolismo , Matrizes de Pontuação de Posição Específica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteômica/métodos , Proteínas Ubiquitinadas/genética , UbiquitinaçãoRESUMO
BACKGROUND: Our previous studies have demonstrated that autophagosome-enriched vaccine (named DRibbles: DRiPs-containing blebs) induce a potent anti-tumor efficacy in different murine tumor models, in which DRibble-containing ubiquitinated proteins are efficient tumor-specific antigen source for the cross-presentation after being loaded onto dendritic cells. In this study, we sought to detect whether ubiquitinated proteins enriched from tumor cells could be used directly as a novel cancer vaccine. METHODS: The ubiquitin binding protein Vx3(A7) was used to isolate ubiquitinated proteins from EL4 and B16-F10 tumor cells after blocking their proteasomal degradation pathway. C57BL/6 mice were vaccinated with different doses of Ub-enriched proteins via inguinal lymph nodes or subcutaneous injection and with DRibbles, Ub-depleted proteins and whole cell lysate as comparison groups, respectively. The lymphocytes from the vaccinated mice were re-stimulated with inactivated tumor cells and the levels of IFN-γ in the supernatant were detected by ELISA. Anti-tumor efficacy of Ub-enriched proteins vaccine was evaluated by monitoring tumor growth in established tumor mice models. Graphpad Prism 5.0 was used for all statistical analysis. RESULTS: We found that after stimulation with inactivated tumor cells, the lymphocytes from the Ub-enriched proteins-vaccinated mice secreted high level of IFN-γ in dose dependent manner, in which the priming vaccination via inguinal lymph nodes injection induced higher IFN-γ level than that via subcutaneous injection. Moreover, the level of secreted IFN-γ in the Ub-enriched proteins group was markedly higher than that in the whole cell lysate and Ub-depleted proteins. Interestingly, the lymphocytes from mice vaccinated with Ub-enriched proteins, but not Ub-depleted proteins and whole cell lysates, isolated from EL4 or B16-F10 tumor cells also produced an obvious level of IFN-γ when stimulated alternately with inactivated B16-F10 or EL4 tumor cells. Furthermore, Ub-enriched proteins vaccine showed a significant inhibitory effect on in vivo growth of homologous tumor, as well as allogeneic tumor, compared with Ub-depleted proteins and tumor cell lysate. Tumor growth was regressed after three times of vaccination with Ub-enriched proteins in contrast to other groups. CONCLUSION: These results indicated that Ub-enriched proteins isolated from tumor cells may have a potential as a potent vaccine for immunotherapy against cancer.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Neoplasias/imunologia , Proteínas Ubiquitinadas/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/isolamento & purificação , Linhagem Celular Tumoral , Feminino , Imunidade Celular , Interferon gama/metabolismo , Linfoma , Melanoma Experimental , Camundongos , Neoplasias/genética , Neoplasias/patologia , Neoplasias/terapia , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/isolamento & purificaçãoRESUMO
BACKGROUND AND AIMS: Hyperhomocysteinemia (HHcy) is associated with cardiovascular diseases and is thought to induce endogenous oxidative stress and causes many cellular damages. Proteasome that degrades oxidized and ubiquitinated proteins can regulate the cellular response to oxidative stress. We aimed to investigate whether hyperhomocysteinemia induces oxidative stress and alters proteasome function and composition in heart and aorta tissues of rat. METHODS AND RESULTS: To create hyperhomocysteinemia, male Wistar rats (Pasteur Institute-Algiers) were received daily intraperitoneal injections of dl-homocysteine (0.6-1.2µM/g body weight) for 3weeks. Biomarkers of oxidative stress (malondialdehyde (MDA), protein carbonyl (PC), superoxide dismutase (SOD) and catalase (CAT)) were first measured by biochemical methods and tissue damages by histological sections. Proteasome activities were quantitated using fluorogenic synthetic peptides; ubiquitinated proteins and proteasome subunits expression were then evaluated by SDS PAGE and Western blot analysis. We showed increased MDA and PC but decreased SOD and CAT levels both in plasma, heart and aorta accompanied by histological changes. A significant decrease of proteasome activities was observed in heart, whereas proteasome activity was not affected in aorta. However proteasome composition was altered in both tissues, as the accumulation of ubiquitinated proteins. CONCLUSION: Data demonstrated an alteration of the ubiquitin-proteasome system in hyperhomocysteinemia as a result of accumulating oxidized and ubiquitinated proteins in response to oxidative stress. Further studies must be conducted to better understanding mechanisms responsible of proteasome alterations in hyperhomocysteinemia.
Assuntos
Aorta/metabolismo , Hiper-Homocisteinemia/metabolismo , Miocárdio/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Aorta/patologia , Catalase/genética , Catalase/metabolismo , Regulação da Expressão Gênica , Glutationa/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Homocisteína/administração & dosagem , Hiper-Homocisteinemia/induzido quimicamente , Hiper-Homocisteinemia/genética , Hiper-Homocisteinemia/patologia , Injeções Intraperitoneais , Masculino , Malondialdeído/metabolismo , Miocárdio/patologia , Oxirredução , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Carbonilação Proteica , Ratos , Ratos Wistar , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/metabolismo , UbiquitinaçãoRESUMO
In the present study we have used a transgenic mice overexpressing an amyloidogenic protein, gamma-synuclein, in the nervous system to address the effect of dimebon on proteinopathy progression. Neuroprotective effect of chronic dimebon administration in these mice at organismal level was confirmed by the increased lifespan. Using histological and biochemical approaches we have demonstrated that dimebon reduced the number of amyloid inclusions in spinal cord of transgenic animals and decreased the content of ubiquitinated proteins in detergent-insoluble fractions. These effects are likely to occur at the level of aggregated protein species, since transgene expression was not altered. Thus, pathological protein aggregation serves as one of dimebon targets in neurodegeneration.
Assuntos
Amiloidose/tratamento farmacológico , Indóis/farmacologia , Fármacos Neuroprotetores/farmacologia , RNA Mensageiro/genética , Proteínas Ubiquitinadas/genética , gama-Sinucleína/genética , Administração Oral , Amiloidose/genética , Amiloidose/metabolismo , Amiloidose/patologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Floculação , Expressão Gênica , Longevidade/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Terapia de Alvo Molecular , RNA Mensageiro/metabolismo , Solubilidade , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Proteínas Ubiquitinadas/antagonistas & inibidores , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação , gama-Sinucleína/metabolismoRESUMO
Monoubiquitination of histone H2B has emerged as an important chromatin modification with roles not only in transcription but also in cell differentiation, DNA repair or mRNA processing. Recently, the genome-wide distribution of histone H2B ubiquitination in different organisms has been reported. In this review we discuss the mechanisms regulating H2B ubiquitination and its downstream effectors as well as the suggested functions for this mark in light of these recent studies.:
Assuntos
Histonas/metabolismo , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação/fisiologia , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Reparo do DNA/genética , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Histonas/genética , Humanos , Transdução de Sinais , Fatores de Elongação da Transcrição/genética , Ubiquitina/genética , Proteínas Ubiquitinadas/genéticaRESUMO
In the past decade, ubiquitination has been well documented to have multifaceted roles in regulating NF-κB activation in mammals. However, its function, especially how deubiquitinating enzymes balance the NF-κB activation, remains largely elusive in invertebrates. Investigating bbtA20 and its binding proteins, bbt A20-binding inhibitor of NF-κB (bbtABIN1) and bbtABIN2, in Chinese amphioxus Branchiostoma belcheri tsingtauense, we found that bbtABIN2 can colocalize and compete with bbt TNF receptor-associated factor 6 to connect the K63-linked polyubiquitin chains, whereas bbtABIN1 physically links bbtA20 to bbt NF-κB essential modulator (bbtNEMO) to facilitate the K48-linked ubiquitination of bbtNEMO. Similar to human A20, bbtA20 is a dual enzyme that removes the K63-linked polyubiquitin chains and builds the K48-linked polyubiquitin chains on bbt receptor-interacting serine/threonine protein kinase 1b, leading to the inhibition of NF-κB signaling. Our study not only suggests that ubiquitination is an ancient strategy in regulating NF-κB activation but also provides the first evidence, to our knowledge, for ABINs/A20-mediated inhibition of NF-κB via modifying the ubiquitinated proteins in a basal chordate, adding information on the stepwise development of vertebrate innate immune signaling.
Assuntos
Anfioxos/metabolismo , NF-kappa B/antagonistas & inibidores , Proteínas Ubiquitinadas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Anfioxos/genética , Anfioxos/imunologia , Masculino , Dados de Sequência Molecular , NF-kappa B/imunologia , NF-kappa B/metabolismo , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Filogenia , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/imunologia , UbiquitinaçãoRESUMO
Heat shock proteins HSPA4L and HSPA4 are closely related members of the HSP110 family and act as cochaperones. We generated Hspa4l(-/-)Hspa4(-/-) mice to investigate a functional complementarity between HSPA4L and HSPA4 during embryonic development. Hspa4l(-/-)Hspa4(-/-) embryos exhibited marked pulmonary hypoplasia and neonatal death. Compared with lungs of wild-type, Hspa4l(-/-), and Hspa4(-/-) embryos, Hspa4l(-/-)Hspa4(-/-) lungs were characterized by diminished saccular spaces and increased mesenchymal septa. Mesenchymal hypercellularity was determined to be due to an increased cell proliferation index and decreased cell death. A significant increase in expression levels of prosurvival protein B cell leukemia/lymphoma 2 may be the cause for inhibition of apoptotic process in lungs of Hspa4(-/-)Hspa4l(-/-) embryos. Accumulation of glycogen and diminished expression of surfactant protein B, prosurfactant protein C, and aquaporin 5 in saccular epithelium suggested impaired maturation of type II and type I pneumocytes in the Hspa4l(-/-)Hspa4(-/-) lungs. Further experiments showed a significant accumulation of ubiquitinated proteins in the lungs of Hspa4l(-/-)Hspa4(-/-) embryos, indicating an impaired chaperone activity. Our study demonstrates that HSPA4L and HSPA4 collaborate in embryonic lung maturation, which is necessary for adaptation to air breathing at birth.