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1.
PLoS Pathog ; 16(8): e1008780, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866188

RESUMO

Ubiquitin like protein 5 (UBL5) interacts with other proteins to regulate their function but differs from ubiquitin and other UBLs because it does not form covalent conjugates. Ubiquitin and most UBLs mediate the degradation of target proteins through the 26S proteasome but it is not known if UBL5 can also do that. Here we found that the UBL5s of rice and Nicotiana benthamiana interacted with rice stripe virus (RSV) p3 protein. Silencing of NbUBL5s in N. benthamiana facilitated RSV infection, while UBL5 overexpression conferred resistance to RSV in both N. benthamiana and rice. Further analysis showed that NbUBL5.1 impaired the function of p3 as a suppressor of silencing by degrading it through the 26S proteasome. NbUBL5.1 and OsUBL5 interacted with RPN10 and RPN13, the receptors of ubiquitin in the 26S proteasome. Furthermore, silencing of NbRPN10 or NbRPN13 compromised the degradation of p3 mediated by NbUBL5.1. Together, the results suggest that UBL5 mediates the degradation of RSV p3 protein through the 26S proteasome, a previously unreported plant defense strategy against RSV infection.


Assuntos
Proteínas de Plantas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Repressoras/metabolismo , Tenuivirus/metabolismo , Tabaco/metabolismo , Ubiquitinas/metabolismo , Proteínas Virais/metabolismo , Proteínas de Plantas/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Repressoras/genética , Tenuivirus/genética , Tabaco/genética , Ubiquitinas/genética , Proteínas Virais/genética
2.
Nat Commun ; 11(1): 4580, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917864

RESUMO

Proteasomal machinery performs essential regulated protein degradation in eukaryotes. Classic proteasomes are symmetric, with a regulatory ATPase docked at each end of the cylindrical 20S. Asymmetric complexes are also present in cells, either with a single ATPase or with an ATPase and non-ATPase at two opposite ends. The mechanism that populates these different proteasomal complexes is unknown. Using archaea homologs, we construct asymmetric forms of proteasomes. We demonstrate that the gate conformation of the two opposite ends of 20S are coupled: binding one ATPase opens a gate locally, and also opens the opposite gate allosterically. Such allosteric coupling leads to cooperative binding of proteasomal ATPases to 20S and promotes formation of proteasomes symmetrically configured with two identical ATPases. It may also promote formation of asymmetric complexes with an ATPase and a non-ATPase at opposite ends. We propose that in eukaryotes a similar mechanism regulates the composition of the proteasomal population.


Assuntos
Archaea/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Adenosina Trifosfatases/metabolismo , Archaea/genética , Microscopia Crioeletrônica , Cinética , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/genética , Conformação Proteica , Thermoplasma/genética , Thermoplasma/metabolismo
3.
Nat Commun ; 11(1): 4382, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873802

RESUMO

Fusarium graminearum is a causal agent of Fusarium head blight (FHB) and a deoxynivalenol (DON) producer. In this study, OSP24 is identified as an important virulence factor in systematic characterization of the 50 orphan secreted protein (OSP) genes of F. graminearum. Although dispensable for growth and initial penetration, OSP24 is important for infectious growth in wheat rachis tissues. OSP24 is specifically expressed during pathogenesis and its transient expression suppresses BAX- or INF1-induced cell death. Osp24 is translocated into plant cells and two of its 8 cysteine-residues are required for its function. Wheat SNF1-related kinase TaSnRK1α is identified as an Osp24-interacting protein and shows to be important for FHB resistance in TaSnRK1α-overexpressing or silencing transgenic plants. Osp24 accelerates the degradation of TaSnRK1α by facilitating its association with the ubiquitin-26S proteasome. Interestingly, TaSnRK1α also interacts with TaFROG, an orphan wheat protein induced by DON. TaFROG competes against Osp24 for binding with the same region of TaSnRKα and protects it from degradation. Overexpression of TaFROG stabilizes TaSnRK1α and increases FHB resistance. Taken together, Osp24 functions as a cytoplasmic effector by competing against TaFROG for binding with TaSnRK1α, demonstrating the counteracting roles of orphan proteins of both host and fungal pathogens during their interactions.


Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/patogenicidade , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Triticum/microbiologia , Fatores de Virulência/metabolismo , Resistência à Doença , Fusarium/imunologia , Fusarium/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteólise , Tricotecenos/metabolismo , Triticum/imunologia
4.
PLoS Comput Biol ; 16(9): e1007740, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32881861

RESUMO

The circadian clock is a complex system that plays many important roles in most organisms. Previously, many mathematical models have been used to sharpen our understanding of the Arabidopsis clock, which brought to light the roles of each transcriptional and post-translational regulations. However, the presence of both regulations, instead of either transcription or post-translation, raised curiosity of whether the combination of these two regulations is important for the clock's system. In this study, we built a series of simplified oscillators with different regulations to study the importance of post-translational regulation (specifically, 26S proteasome degradation) in the clock system. We found that a simple transcriptional-based oscillator can already generate sustained oscillation, but the oscillation can be easily destroyed in the presence of transcriptional leakage. Coupling post-translational control with transcriptional-based oscillator in a feed-forward loop will greatly improve the robustness of the oscillator in the presence of basal leakage. Using these general models, we were able to replicate the increased variability observed in the E3 ligase mutant for both plant and mammalian clocks. With this insight, we also predict a plausible regulator of several E3 ligase genes in the plant's clock. Thus, our results provide insights into and the plausible importance in coupling transcription and post-translation controls in the clock system.


Assuntos
Relógios Circadianos/genética , Modelos Biológicos , Processamento de Proteína Pós-Traducional/genética , Transcrição Genética/genética , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biologia Computacional , Retroalimentação Fisiológica , Regulação da Expressão Gênica de Plantas/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
5.
Nat Commun ; 11(1): 3904, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764536

RESUMO

A major challenge in chemotherapy is chemotherapy resistance in cells lacking p53. Here we demonstrate that NIP30, an inhibitor of the oncogenic REGγ-proteasome, attenuates cancer cell growth and sensitizes p53-compromised cells to chemotherapeutic agents. NIP30 acts by binding to REGγ via an evolutionarily-conserved serine-rich domain with 4-serine phosphorylation. We find the cyclin-dependent phosphatase CDC25A is a key regulator for NIP30 phosphorylation and modulation of REGγ activity during the cell cycle or after DNA damage. We validate CDC25A-NIP30-REGγ mediated regulation of the REGγ target protein p21 in vivo using p53-/- and p53/REGγ double-deficient mice. Moreover, Phosphor-NIP30 mimetics significantly increase the growth inhibitory effect of chemotherapeutic agents in vitro and in vivo. Given that NIP30 is frequently mutated in the TCGA cancer database, our results provide insight into the regulatory pathway controlling the REGγ-proteasome in carcinogenesis and offer a novel approach to drug-resistant cancer therapy.


Assuntos
Autoantígenos/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Proteína Supressora de Tumor p53/deficiência , Animais , Autoantígenos/genética , Ciclo Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Resistencia a Medicamentos Antineoplásicos , Células HEK293 , Xenoenxertos , Humanos , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/deficiência , Complexo de Endopeptidases do Proteassoma/genética , Proteína Supressora de Tumor p53/genética , Fosfatases cdc25/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(32): 19190-19200, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723828

RESUMO

The 26S proteasome, a self-compartmentalized protease complex, plays a crucial role in protein quality control. Multiple levels of regulatory systems modulate proteasomal activity for substrate hydrolysis. However, the destruction mechanism of mammalian proteasomes is poorly understood. We found that inhibited proteasomes are sequestered into the insoluble aggresome via HDAC6- and dynein-mediated transport. These proteasomes colocalized with the autophagic receptor SQSTM1 and cleared through selective macroautophagy, linking aggresomal segregation to autophagic degradation. This proteaphagic pathway was counterbalanced with the recovery of proteasomal activity and was critical for reducing cellular proteasomal stress. Changes in associated proteins and polyubiquitylation on inhibited 26S proteasomes participated in the targeting mechanism to the aggresome and autophagosome. The STUB1 E3 Ub ligase specifically ubiquitylated purified human proteasomes in vitro, mainly via Lys63-linked chains. Genetic and chemical inhibition of STUB1 activity significantly impaired proteasome processing and reduced resistance to proteasomal stress. These data demonstrate that aggresomal sequestration is the crucial upstream event for proteasome quality control and overall protein homeostasis in mammals.


Assuntos
Macroautofagia , Organelas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Células A549 , Humanos , Organelas/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
7.
Nucleic Acids Res ; 48(14): 7818-7833, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32609828

RESUMO

The tumor suppressor BRCA2 plays a key role in initiating homologous recombination by facilitating RAD51 filament formation on single-stranded DNA. The small acidic protein DSS1 is a crucial partner to BRCA2 in this process. In vitro and in cells (1,2), BRCA2 associates into oligomeric complexes besides also existing as monomers. A dimeric structure was further characterized by electron microscopic analysis (3), but the functional significance of the different BRCA2 assemblies remains to be determined. Here, we used biochemistry and electron microscopic imaging to demonstrate that the multimerization of BRCA2 is counteracted by DSS1 and ssDNA. When validating the findings, we identified three self-interacting regions and two types of self-association, the N-to-C terminal and the N-to-N terminal interactions. The N-to-C terminal self-interaction of BRCA2 is sensitive to DSS1 and ssDNA. The N-to-N terminal self-interaction is modulated by ssDNA. Our results define a novel role of DSS1 to regulate BRCA2 in an RPA-independent fashion. Since DSS1 is required for BRCA2 function in recombination, we speculate that the monomeric and oligomeric forms of BRCA2 might be active for different cellular events in recombinational DNA repair and replication fork stabilization.


Assuntos
Proteína BRCA2/metabolismo , DNA de Cadeia Simples/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Proteína BRCA2/química , Proteína BRCA2/genética , Proteína BRCA2/ultraestrutura , Linhagem Celular , Cricetulus , Humanos , Multimerização Proteica
8.
Biochem Biophys Res Commun ; 529(2): 251-256, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32703419

RESUMO

The nucleocapsid protein is significant in the formation of viral RNA of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), accounting for the largest proportion of viral structural proteins. Here, we report for the first time that the 11S proteasomal activator PA28γ regulates the intracellular abundance of the SARS-CoV-2 N protein (nCoV N). Furthermore, we have identified proteasome activator PA28γ as a nCoV N binding protein by co-immunoprecipitation assay. As a result of their interaction, nCoV N could be degraded by PA28γ-20S in vitro degradation assay. This was also demonstrated by blocking de novo protein synthesis with cycloheximide. The stability of nCoV N in PA28γ-knockout cells was greater than in PA28γ-wildtype cells. Notably, immunofluorescence staining revealed that knockout of the PA28γ gene in cells led to the transport of nCoV N from the nucleus to the cytoplasm. Overexpression of PA28γ enhanced proteolysis of nCoV N compared to that in PA28γ-N151Y cells containing a dominant-negative PA28γ mutation, which reduced this process. These results suggest that PA28γ binding is important in regulating 20S proteasome activity, which in turn regulates levels of the critical nCoV N nucleocapsid protein of SARS-CoV-2, furthering our understanding of the pathogenesis of COVID-19.


Assuntos
Autoantígenos/metabolismo , Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Pneumonia Viral/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Infecções por Coronavirus/virologia , Células HEK293 , Humanos , Técnicas In Vitro , Pandemias , Pneumonia Viral/virologia , Ligação Proteica , Estabilidade Proteica , Transporte Proteico
9.
Life Sci ; 257: 118041, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32622945

RESUMO

AIM: Transcription factor CCAAT/Enhancer binding protein alpha (C/EBPα) is a key regulator of myeloid differentiation, granulopoiesis in particular. Although CEBPA mutations are found in more than 10% in AML, functional inhibition of C/EBPα protein is also widely observed in AML. Here, we sought to examine if SKP2, an aberrantly enhanced E3 ubiquitin ligase in primary AMLs inhibits C/EBPα stability to induce differentiation block. MAIN METHODS: Here we employed cell based assays such as transfections, immunoblotting, co-immunoprecipitation, luciferase and gel shift assays along with differentiation assays to investigate SKP2 regulated C/EBPα protein stability in acute myeloid leukemia. KEY FINDINGS: Here we discovered that oncogenic E3 ubiquitin ligase SCFskp2 ubiquitinates and destabilizes C/EBPα in a proteasome-dependent manner. Our data demonstrates that SKP2 physically interacts with C-terminal of C/EBPα and promotes its K48-linked ubiquitination-mediated degradation leading to its reduced transactivation potential, DNA binding ability and cellular functions. We further show that while overexpression of SKP2 inhibits both ectopic as well as endogenous C/EBPα in heterologous (HEK293T) as well as myeloid leukemia cells respectively, SKP2 depletion restores endogenous C/EBPα leading to reduced colony formation and enhanced myeloid differentiation of myeloid leukemia cells. Using Estradiol-inducible K562-C/EBPα-ER cells as yet another model of granulocytic differentiation, we further confirmed that SKP2 overexpression indeed inhibits granulocytic differentiation by mitigating C/EBPα stability. SIGNIFICANCE: Our findings identify SKP2 as a potential negative regulator of C/EBPα stability and function in AML which suggests that SKP2 can be potentially targeted in AML to restore C/EBPα and overcome differentiation block.


Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Transfecção , Células U937 , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
10.
PLoS One ; 15(7): e0236403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32716961

RESUMO

Autophagy, a self-degradative physiological process, is critical for homeostasis maintenance and energy source balancing in response to various stresses, including nutrient deprivation. It is a highly conserved catabolic process in eukaryotes and is indispensable for cell survival as it involves degradation of unessential or excessive components and their subsequent recycling as building blocks for the synthesis of necessary molecules. Although the dysregulation of autophagy has been reported to broadly contribute to various diseases, including cancers and neurodegenerative diseases, the molecular mechanisms underlying the epigenetic regulation of autophagy are poorly elucidated. Here, we report that the level of lysine demethylase 3B (KDM3B) increases in nutrient-deprived HCT116 cells, a colorectal carcinoma cell line, resulting in transcriptional activation of the autophagy-inducing genes. KDM3B was found to enhance the transcription by demethylating H3K9me2 on the promoter of these genes. Furthermore, we observed that the depletion of KDM3B inhibited the autophagic flux in HCT116 cells. Collectively, these data suggested the critical role of KDM3B in the regulation of autophagy-related genes via H3K9me2 demethylation and induction of autophagy in nutrient-starved HCT116 cells.


Assuntos
Autofagia/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Ativação Transcricional/genética , Aminoácidos/deficiência , Autofagia/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Células HCT116 , Células HEK293 , Histonas/metabolismo , Humanos , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Sirolimo/farmacologia , Ativação Transcricional/efeitos dos fármacos , Proteína com Valosina/metabolismo
11.
Ecotoxicol Environ Saf ; 203: 110975, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32678756

RESUMO

Manganese (Mn) produces cholinergic neuronal loss in basal forebrain (BF) region that was related to cognitive dysfunction induced after single and repeated Mn treatment. All processes that generate cholinergic neuronal loss in BF remain to be understood. Mn exposure may produce the reduction of BF cholinergic neurons by increasing amyloid beta (Aß) and phosphorylated Tau (pTau) protein levels, altering heat shock proteins' (HSPs) expression, disrupting proteasome P20S activity and generating oxidative stress. These mechanisms, described to be altered by Mn in regions different than BF, could lead to the memory and learning process alteration produced after Mn exposure. The research performed shows that single and repeated Mn treatment of SN56 cholinergic neurons from BF induces P20S inhibition, increases Aß and pTau protein levels, produces HSP90 and HSP70 proteins expression alteration, and oxidative stress generation, being the last two effects mediated by NRF2 pathway alteration. The increment of Aß and pTau protein levels was mediated by HSPs and proteasome dysfunction. All these mechanisms mediated the cell decline observed after Mn treatment. Our results are relevant because they may assist to reveal the processes leading to the neurotoxicity and cognitive alterations observed after Mn exposure.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Prosencéfalo Basal/efeitos dos fármacos , Neurônios Colinérgicos/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Manganês/toxicidade , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas tau/metabolismo , Animais , Prosencéfalo Basal/metabolismo , Prosencéfalo Basal/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/patologia , Relação Dose-Resposta a Droga , Poluentes Ambientais/metabolismo , Manganês/metabolismo , Camundongos , Estresse Oxidativo/efeitos dos fármacos
12.
Mol Cell Biol ; 40(18)2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32631902

RESUMO

hRpn13/ADRM1 links substrate recruitment with deubiquitination at the proteasome through its proteasome- and ubiquitin-binding Pru domain and DEUBAD domain, which binds and activates deubiquitinating enzyme (DUB) UCHL5/Uch37. Here, we edit the HCT116 colorectal cancer cell line to delete part of the hRpn13 Pru, producing cells that express truncated hRpn13 (trRpn13), which is competent for UCHL5 binding but defective for proteasome interaction. trRpn13 cells demonstrate reduced levels of proteasome-bound ubiquitinated proteins, indicating that the loss of hRpn13 function at proteasomes cannot be fully compensated for by the two other dedicated substrate receptors (hRpn1 and hRpn10). Previous studies indicated that the loss of full-length hRpn13 causes a corresponding reduction of UCHL5. We find UCHL5 levels unaltered in trRpn13 cells, but hRpn11 is elevated in ΔhRpn13 and trRpn13 cells, perhaps from cell stress. Despite the ∼90 DUBs in human cells, including two others in addition to UCHL5 at the proteasome, we found deletion of UCHL5 from HCT116 cells to cause increased levels of ubiquitinated proteins in whole-cell extract and at proteasomes, suggesting that UCHL5 activity cannot be fully assumed by other DUBs. We also report anticancer molecule RA190, which binds covalently to hRpn13 and UCHL5, to require hRpn13 Pru and not UCHL5 for cytotoxicity.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Chaperonas Moleculares/metabolismo , Ubiquitina Tiolesterase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Citoplasma/metabolismo , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/genética , Proteínas Ubiquitinadas/metabolismo
13.
Life Sci ; 257: 118021, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32621919

RESUMO

AIMS: Tribbles homolog 3 (TRIB3) is emerging as a multifunctional oncoprotein associated with various cellular events in different tumors. However, the regulatory mechanism of TRIB3 in acute myeloid leukemia (AML) remains unknown. This study aims to investigate the molecular mechanisms and uncover the functions of TRIB3 in AML. METHODS: Western blotting and quantitative real-time PCR were used to analyze the expression levels of TRIB3, peroxisome proliferator-activated receptor α (PPARα), apoptosis markers and autophagy markers in AML cells. Flow cytometry was used to assess cell apoptosis. The interaction of TRIB3 and PPARα was evaluated by immunofluorescence, coimmunoprecipitation, and in vivo ubiquitination assays. KEY FINDINGS: We demonstrated that downregulating TRIB3 in leukemic cells effectively induced apoptosis and autophagy by regulating the degradation of PPARα. Mechanistically, TRIB3 interacted with PPARα and contributed to its destabilization by promoting its ubiquitination. When PPARα was activated by its specific agonist clofibrate, the apoptosis and autophagy of AML cells were significantly enhanced. These results were confirmed by rescue experiments. Blocking PPARα expression using the PPARα inhibitor GW6471 reversed the functional influence of TRIB3 on AML cells. SIGNIFICANCE: The aim of this study is to provide evidence of the degradation of PPARα by TRIB3 via ubiquitin-dependent proteasomal degradation. This process meditates the progression of AML and prolongs the survival of leukemic cells. As a result, these data indicate that TRIB3 is a novel and promising therapeutic target for AML treatment.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Leucemia Mieloide Aguda/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Apoptose/fisiologia , Autofagia/fisiologia , Bases de Dados Genéticas , Humanos , Leucemia Mieloide Aguda/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteostase/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina/metabolismo , Ubiquitinação
14.
Mol Cell ; 79(2): 320-331.e9, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32645369

RESUMO

Valosin-containing protein (VCP)/p97 is an AAA-ATPase that extracts polyubiquitinated substrates from multimeric macromolecular complexes and biological membranes for proteasomal degradation. During p97-mediated extraction, the substrate is largely deubiquitinated as it is threaded through the p97 central pore. How p97-extracted substrates are targeted to the proteasome with few or no ubiquitins is unknown. Here, we report that p97-extracted membrane proteins undergo a second round of ubiquitination catalyzed by the cytosolic ubiquitin ligase RNF126. RNF126 interacts with transmembrane-domain-specific chaperone BAG6, which captures p97-liberated substrates. RNF126 depletion in cells diminishes the ubiquitination of extracted membrane proteins, slows down their turnover, and dramatically stabilizes otherwise transient intermediates in the cytosol. We reconstitute the reubiquitination of a p97-extracted, misfolded multispanning membrane protein with purified factors. Our results demonstrate that p97-extracted substrates need to rapidly engage ubiquitin ligase-chaperone pairs that rebuild the ubiquitin signal for proteasome targeting to prevent harmful accumulation of unfolded intermediates.


Assuntos
Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina/metabolismo , Catálise , Citosol/metabolismo , Células HEK293 , Humanos , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteólise , Solubilidade , Ubiquitinação
15.
Nat Commun ; 11(1): 3284, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601292

RESUMO

The inner nuclear membrane (INM) selectively accumulates proteins that are essential for nuclear functions; however, overaccumulation of INM proteins results in a range of rare genetic disorders. So far, little is known about how defective, mislocalized, or abnormally accumulated membrane proteins are actively removed from the INM, especially in plants and animals. Here, via analysis of a proximity-labeling proteomic profile of INM-associated proteins in Arabidopsis, we identify critical components for an INM protein degradation pathway. We show that this pathway relies on the CDC48 complex for INM protein extraction and 26S proteasome for subsequent protein degradation. Moreover, we show that CDC48 at the INM may be regulated by a subgroup of PUX proteins, which determine the substrate specificity or affect the ATPase activity of CDC48. These PUX proteins specifically associate with the nucleoskeleton underneath the INM and physically interact with CDC48 proteins to negatively regulate INM protein degradation in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Membrana Nuclear/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Proteoma/genética , Coloração e Rotulagem/métodos , Espectrometria de Massas em Tandem/métodos , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
16.
Nucleic Acids Res ; 48(14): 7944-7957, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32667666

RESUMO

Circadian clocks are endogenous oscillators that control ∼24-hour physiology and behaviors in virtually all organisms. The circadian oscillator comprises interconnected transcriptional and translational feedback loops, but also requires finely coordinated protein homeostasis including protein degradation and maturation. However, the mechanisms underlying the mammalian clock protein maturation is largely unknown. In this study, we demonstrate that necdin, one of the Prader-Willi syndrome (PWS)-causative genes, is highly expressed in the suprachiasmatic nuclei (SCN), the pacemaker of circadian clocks in mammals. Mice deficient in necdin show abnormal behaviors during an 8-hour advance jet-lag paradigm and disrupted clock gene expression in the liver. By using yeast two hybrid screening, we identified BMAL1, the core component of the circadian clock, and co-chaperone SGT1 as two necdin-interactive proteins. BMAL1 and SGT1 associated with the N-terminal and C-terminal fragments of necdin, respectively. Mechanistically, necdin enables SGT1-HSP90 chaperone machinery to stabilize BMAL1. Depletion of necdin or SGT1/HSP90 leads to degradation of BMAL1 through the ubiquitin-proteasome system, resulting in alterations in both clock gene expression and circadian rhythms. Taken together, our data identify the PWS-associated protein necdin as a novel regulator of the circadian clock, and further emphasize the critical roles of chaperone machinery in circadian clock regulation.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Relógios Circadianos , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Animais , Relógios Circadianos/genética , Expressão Gênica , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Ubiquitina/metabolismo
17.
Cell Mol Biol (Noisy-le-grand) ; 66(3): 221-229, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: covidwho-603065

RESUMO

It can be misleading to think that the new severe acute respiratory syndrome coronavirus (SARS-CoV2) which has a very strong mutation and adaptation capabilities, uses only the angiotensin-converting enzyme II (ACE2) pathway to reach target cells. Despite all the precautions taken, the pandemic attack continues and the rapid increase in the number of deaths suggest that this virus has entered the cell through different pathways and caused damage through different mechanisms. The main reason why the ACE2 pathway comes to the fore in all scientific studies is that this receptor is located at the entry point of basic mechanisms that provide alveolo-capillary homeostasis. SARS-CoV-2 has to use nuclear factor-κB (NF-kB), caveloae, clathrin, lipoxin, serine protease and proteasome pathways in addition to ACE2 to enter the target cell and initiate damage. For this reason, while new drug development studies are continuing, in order to be beneficial to patients in their acute period, it is imperative that we are able to come up with drugs that activate or inhibit these pathways and are currently in clinical use. It is also critical that we adopt these new pathways to the treatment of pregnant women affected by SARS-CoV-2, based on the scientific data we use to treat the general population.


Assuntos
Betacoronavirus/metabolismo , Caveolina 1/metabolismo , Infecções por Coronavirus/metabolismo , Lipoxinas/metabolismo , NF-kappa B/metabolismo , Pneumonia Viral/metabolismo , Complicações Infecciosas na Gravidez/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Anticolesterolemiantes/uso terapêutico , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos/métodos , Feminino , Humanos , Transmissão Vertical de Doença Infecciosa/prevenção & controle , NF-kappa B/antagonistas & inibidores , Uso Off-Label , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Gravidez , Complicações Infecciosas na Gravidez/virologia , Inibidores de Proteassoma/uso terapêutico , Serina Endopeptidases/metabolismo , Inibidores de Serino Proteinase/uso terapêutico , Internalização do Vírus
18.
Nat Chem Biol ; 16(7): 716-724, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572259

RESUMO

Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription. Here we review the molecular mechanisms by which specific CDKs are thought to act at discrete steps in the transcription cycle and describe the recent emergence of transcriptional CDKs as promising drug targets in cancer. We emphasize recent advances in understanding the transcriptional CDK network that were facilitated by development and deployment of small-molecule inhibitors with increased selectivity for individual CDKs. Unexpectedly, several of these compounds have also shown selectivity in killing cancer cells, despite the seemingly universal involvement of their target CDKs during transcription in all cells. Finally, we describe remaining and emerging challenges in defining functions of individual CDKs in transcription and co-transcriptional processes and in leveraging CDK inhibition for therapeutic purposes.


Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , RNA Polimerase II/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antineoplásicos/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Modelos Animais de Doenças , Humanos , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/química , Proteólise , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/química , Transcrição Genética
19.
Mol Pharmacol ; 98(3): 222-233, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32587095

RESUMO

DNA topoisomerase II (TOP2) is required for the unwinding and decatenation of DNA through the induction of an enzyme-linked double-strand break (DSB) in one DNA molecule and passage of another intact DNA duplex through the break. Anticancer drugs targeting TOP2 (TOP2 poisons) prevent religation of the DSB and stabilize a normally transient intermediate of the TOP2 reaction mechanism called the TOP2-DNA covalent complex. Subsequently, TOP2 remains covalently bound to each end of the enzyme-bridged DSB, which cannot be repaired until TOP2 is removed from the DNA. One removal mechanism involves the proteasomal degradation of the TOP2 protein, leading to the liberation of a protein-free DSB. Proteasomal degradation is often regulated by protein ubiquitination, and here we show that inhibition of ubiquitin-activating enzymes reduces the processing of TOP2A- and TOP2B-DNA complexes. Depletion or inhibition of ubiquitin-activating enzymes indicated that ubiquitination was required for the liberation of etoposide-induced protein-free DSBs and is therefore an important layer of regulation in the repair of TOP2 poison-induced DNA damage. TOP2-DNA complexes stabilized by etoposide were shown to be conjugated to ubiquitin, and this was reduced by inhibition or depletion of ubiquitin-activating enzymes. SIGNIFICANCE STATEMENT: There is currently great clinical interest in the ubiquitin-proteasome system and ongoing development of specific inhibitors. The results in this paper show that the therapeutic cytotoxicity of DNA topoisomerase II (TOP2) poisons can be enhanced through combination therapy with ubiquitin-activating enzyme inhibitors or by specific inhibition of the BMI/RING1A ubiquitin ligase, which would lead to increased cellular accumulation or persistence of TOP2-DNA complexes.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , Nucleosídeos/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Sulfonamidas/farmacologia , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Linhagem Celular , DNA/metabolismo , DNA Topoisomerases Tipo II/química , Humanos , Células K562 , Proteínas de Ligação a Poli-ADP-Ribose/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos
20.
Nat Commun ; 11(1): 3234, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591519

RESUMO

In shotgun proteomics, the analysis of label-free quantification experiments is typically limited by the identification rate and the noise level in the quantitative data. This generally causes a low sensitivity in differential expression analysis. Here, we propose a quantification-first approach for peptides that reverses the classical identification-first workflow, thereby preventing valuable information from being discarded in the identification stage. Specifically, we introduce a method, Quandenser, that applies unsupervised clustering on both MS1 and MS2 level to summarize all analytes of interest without assigning identities. This reduces search time due to the data reduction. We can now employ open modification and de novo searches to identify analytes of interest that would have gone unnoticed in traditional pipelines. Quandenser+Triqler outperforms the state-of-the-art method MaxQuant+Perseus, consistently reporting more differentially abundant proteins for all tested datasets. Software is available for all major operating systems at https://github.com/statisticalbiotechnology/quandenser, under Apache 2.0 license.


Assuntos
Proteômica , Análise por Conglomerados , Bases de Dados de Proteínas , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Software , Ubiquitina/metabolismo
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