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1.
Enzymes ; 45: 99-138, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627884

RESUMO

Nucleotide excision repair (NER) is a versatile DNA repair pathway that eliminates various helix-distorting base lesions such as ultraviolet (UV)-induced photolesions. Several recessive human disorders, such as xeroderma pigmentosum (XP), are caused by hereditary defects in NER, implying that the pathway plays critical roles in suppressing diverse pathogenic processes, including carcinogenesis. In general, discrimination of lesion sites from intact DNA, which is present in vast excess, is a key determinant of the overall efficiency of DNA repair. In mammalian cells, global genomic NER lesion recognition is initiated by the XPC protein complex, which achieves broad DNA-binding specificity by sensing destabilized base pairs rather than lesions per se. To avert unnecessary incisions at lesion-free sites, and thereby ensure the fidelity of the repair system, transcription factor IIH and the XPA protein then verify the presence of relevant lesions at suspicious sites bound by XPC. In the case of UV-induced photolesions, a specialized lesion sensor called UV-damaged DNA-binding protein (UV-DDB) contributes to efficient lesion recognition and the recruitment of XPC to lesion sites. The ubiquitin-proteasome system plays a crucial role in the handoff of lesions from UV-DDB to XPC and the subsequent NER process. In addition, recognition of lesions targeted by global genomic NER is intricately regulated by higher-order chromatin structures, which play distinct roles depending on the type of lesion.


Assuntos
Dano ao DNA , Reparo do DNA , Animais , Proteínas de Ligação a DNA/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator de Transcrição TFIIH/metabolismo , Ubiquitina/metabolismo , Raios Ultravioleta/efeitos adversos , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo
2.
Chem Commun (Camb) ; 55(87): 13093-13095, 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31612161

RESUMO

Ubiquitin monomers functionalized with an azide or multiple alkynes were utilized for the assembly of branched ubiquitin oligomers (K6/K11, K11/K48, K11/K63, K6/K11/K48) by click chemistry. The oligomers resist deubiquitylase-catalysed hydrolysis and exhibit stability in eukaryotic cell lysates.


Assuntos
Ubiquitina/biossíntese , Alquinos/química , Azidas/química , Biocatálise , Química Click , Enzimas Desubiquitinantes/metabolismo , Células Eucarióticas/metabolismo , Humanos , Hidrólise , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinação
3.
Mol Biol (Mosk) ; 53(4): 638-647, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397437

RESUMO

The ubiquitin-proteasome system (UPS) performs proteolysis of most intracellular proteins. The key components of the UPS are the proteasomes, multi-subunit protein complexes, playing an important role in cellular adaptation to various types of stress. We analyzed the dynamics of the proteasome activity, the content of proteasome subunits, and the expression levels of genes encoding catalytic subunits of proteasomes in the human histiocytic lymphoma U937 cell line immediately, 2, 4, 6, 9, 24, and 48 h after a heat shock (HS). The initial decrease (up to 62%) in the proteasome activity in cellular lysates was revealed, then 10 h after HS the activity began to recover. The amount of proteasomal α-subunits in the cells decreased 2 h after HS, and was restored to 24-48 h after HS. Fluctuations in the levels of mRNAs encoding proteasome catalytic subunits with the maximum expression 2 h after HS and a gradual decrease to 48 h after HS were observed. The average estimated number of mRNA copies per cell ranged from 10 for weakly to 150 for highly expressed proteasome genes. Thus, the recovery efficiency of UPS functionality after HS, which reflects the important role of proteasomes in maintaining cell homeostasis, was evaluated.


Assuntos
Resposta ao Choque Térmico , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/genética , Proteólise , Células U937 , Ubiquitina/metabolismo
4.
Zhonghua Gan Zang Bing Za Zhi ; 27(6): 477-480, 2019 Jun 20.
Artigo em Chinês | MEDLINE | ID: mdl-31357769

RESUMO

Cylindromatosis gene is a kind of tumor suppressor genes, whose mutation or deletion will lead to the development of a cylindrical tumor. The deubiquitinating enzyme CYLD protein encoded by it is a member of the deubiquitinating enzyme family. CYLD alters the function of the target molecules by removing the ubiquitin chain linked to the substrate protein K63, and participates in the regulation of signaling pathways, such as NF-κB, JNK and Wnt. This article reviews the recent year's research progress of CYLD, especially its negative regulatory role in the progression of liver-related diseases.


Assuntos
Enzima Desubiquitinante CYLD , Hepatopatias , NF-kappa B , Proteínas Supressoras de Tumor , Enzima Desubiquitinante CYLD/metabolismo , Fígado/enzimologia , Hepatopatias/enzimologia , Pesquisa/tendências , Transdução de Sinais/fisiologia , Ubiquitina/metabolismo
5.
Nat Methods ; 16(8): 771-777, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31308549

RESUMO

Ubiquitin (Ub) conjugation is an essential post-translational modification that affects nearly all proteins in eukaryotes. The functions and mechanisms of ubiquitination are areas of extensive study, and yet the dynamics and regulation of even free (that is, unconjugated) Ub are poorly understood. A major impediment has been the lack of simple and robust techniques to quantify Ub levels in cells and to monitor Ub release from conjugates. Here, we describe avidity-based fluorescent sensors that address this need. The sensors bind specifically to free Ub, have dissociation constant Kd values down to 60 pM and, together with a newly developed workflow, allow us to distinguish and quantify the pools of free, protein-conjugated and thioesterified forms of Ub from cell lysates. Alternatively, free Ub in fixed cells can be visualized microscopically by staining with a sensor. Real-time assays using the sensors afford unprecedented flexibility and precision to measure deubiquitination of virtually any (poly)Ub conjugate.


Assuntos
Técnicas Biossensoriais , Homeostase , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Células HeLa , Humanos , Ligação Proteica , Conformação Proteica , Proteínas/química
6.
J Chem Theory Comput ; 15(8): 4318-4331, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31241940

RESUMO

The relative prevalence of native protein-protein interactions (PPIs) are the cornerstone for understanding the structure, dynamics and mechanisms of function of protein complexes. In this study, we develop a scheme for scaling the protein-water interaction in the CHARMM36 force field, in order to better fit the solvation free energy of amino acids side-chain analogues. We find that the molecular dynamics simulation with the scaled force field, CHARMM36s, as well as a recently released version, CHARMM36m, effectively improve on the overly sticky association of proteins, such as ubiquitin. We investigate the formation of a heterodimer protein complex between the SAM domains of the EphA2 receptor and the SHIP2 enzyme by performing a combined total of 48 µs simulations with the different potential functions. While the native SAM heterodimer is only predicted at a low rate of 6.7% with the original CHARMM36 force field, the yield is increased to 16.7% with CHARMM36s, and to 18.3% with CHARMM36m. By analyzing the 25 native SAM complexes formed in the simulations, we find that their formation involves a preorientation guided by Coulomb interactions, consistent with an electrostatic steering mechanism. In 12 cases, the complex could directly transform to the native protein interaction surfaces with only small adjustments in domain orientation. In the other 13 cases, orientational and/or translational adjustments are needed to reach the native complex. Although the tendency for non-native complexes to dissociate has nearly doubled with the modified potential functions, a dissociation followed by a reassociation to the correct complex structure is still rare. Instead, the remaining non-native complexes undergo configurational changes/surface searching, which, however, rarely leads to native structures on a time scale of 250 ns. These observations provide a rich picture of the mechanisms of protein-protein complex formation and suggest that computational predictions of native complex PPIs could be improved further.


Assuntos
Mapas de Interação de Proteínas , Proteínas/metabolismo , Humanos , Simulação de Dinâmica Molecular , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/química , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas/química , Receptor EphA2/química , Receptor EphA2/metabolismo , Eletricidade Estática , Termodinâmica , Ubiquitina/química , Ubiquitina/metabolismo , Água/metabolismo
7.
Microb Pathog ; 132: 362-368, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31054366

RESUMO

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. The cellular factors required for DTMUV replication have been poorly studied. The ubiquitin-proteasome system (UPS), the major intracellular proteolytic pathway, mediates diverse cellular processes, including endocytosis and signal transduction, which may be involved in the entry of virus. In the present study, we explored the interplay between DTMUV replication and the UPS in BHK-21 cells and found that treatment with proteasome inhibitor (MG132 and lactacystin) significantly decreased the DTMUV progency at the early infection stage. We further revealed that inhibition of the UPS mainly occurs on the level of viral protein expression and RNA transcription. In addition, using specific siRNAs targeting ubiquitin reduces the production of viral progeny. In the presence of MG132 the staining for the envelope protein of DTMUV was dramatically reduced in comparison with the untreated control cells. Overall, our observations reveal an important role of the UPS in multiple steps of the DTMUV infection cycle and identify the UPS as a potential drug target to modulate the impact of DTMUV infection.


Assuntos
Flavivirus/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Replicação Viral/fisiologia , Acetilcisteína/análogos & derivados , Acetilcisteína/antagonistas & inibidores , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Patos , Flavivirus/efeitos dos fármacos , Flavivirus/patogenicidade , Técnicas de Silenciamento de Genes , Leupeptinas/antagonistas & inibidores , Doenças das Aves Domésticas/virologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , RNA Interferente Pequeno , Transfecção , Ubiquitina/efeitos dos fármacos , Ubiquitina/genética , Proteínas do Envelope Viral , Internalização do Vírus
8.
Plant Physiol Biochem ; 140: 78-87, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31085449

RESUMO

Because of their sessile nature, plants have evolved complex and robust mechanisms to respond to adverse environments. Stress conditions trigger an increase in protein turnover and degradation. Proteasomes are essential to the cell for removing, in a highly regulated manner, partially denatured or oxidized proteins thus minimizing their cytotoxicity. We observed that suspension cells of Arabidopsis thaliana treated with high temperature (37 °C) directed the assembly of high molecular mass proteasomes. The removal of a 75% of the original ubiquitin conjugates and the maintenance of protein carbonyls at basal levels correlated with a specific proteasome profiles. The profiles obtained by the separation of different proteasomes populations by Blue-Native Polyacrylamide Gel Electrophoresis and western blot analysis suggest that synthesis, assembly, and heavy ubiquitination of 20S (CP) subunits are promoted by heat stress.


Assuntos
Arabidopsis/metabolismo , Arabidopsis/fisiologia , Resposta ao Choque Térmico , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/genética , Ubiquitinação/fisiologia
9.
Nature ; 570(7759): 117-121, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31068692

RESUMO

Aneuploidy, which refers to unbalanced chromosome numbers, represents a class of genetic variation that is associated with cancer, birth defects and eukaryotic micro-organisms1-4. Whereas it is known that each aneuploid chromosome stoichiometry can give rise to a distinct pattern of gene expression and phenotypic profile4,5, it remains a fundamental question as to whether there are common cellular defects that are associated with aneuploidy. Here we show the existence in budding yeast of a common aneuploidy gene-expression signature that is suggestive of hypo-osmotic stress, using a strategy that enables the observation of common transcriptome changes of aneuploidy by averaging out karyotype-specific dosage effects in aneuploid yeast-cell populations with random and diverse chromosome stoichiometry. Consistently, aneuploid yeast exhibited increased plasma-membrane stress that led to impaired endocytosis, and this defect was also observed in aneuploid human cells. Thermodynamic modelling showed that hypo-osmotic-like stress is a general outcome of the proteome imbalance that is caused by aneuploidy, and also predicted a relationship between ploidy and cell size that was observed in yeast and aneuploid cancer cells. A genome-wide screen uncovered a general dependency of aneuploid cells on a pathway of ubiquitin-mediated endocytic recycling of nutrient transporters. Loss of this pathway, coupled with the endocytic defect inherent to aneuploidy, leads to a marked alteration of intracellular nutrient homeostasis.


Assuntos
Aneuploidia , Pressão Osmótica , Proteoma/genética , Proteoma/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Estresse Fisiológico , Membrana Celular/metabolismo , Membrana Celular/patologia , Proteínas de Ligação a DNA/metabolismo , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Homeostase , Humanos , Cariótipo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Termodinâmica , Fatores de Transcrição/metabolismo , Transcriptoma/genética , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo
10.
Nat Commun ; 10(1): 1973, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036822

RESUMO

Ubiquitin-mediated xenophagy, a type of selective autophagy, plays crucial roles in host defense against intracellular pathogens including Mycobacterium tuberculosis (Mtb). However, the exact mechanism by which host ubiquitin targets invaded microbes to trigger xenophagy remains obscure. Here we show that ubiquitin could recognize Mtb surface protein Rv1468c, a previously unidentified ubiquitin-binding protein containing a eukaryotic-like ubiquitin-associated (UBA) domain. The UBA-mediated direct binding of ubiquitin to, but not E3 ubiquitin ligases-mediated ubiquitination of, Rv1468c recruits autophagy receptor p62 to deliver mycobacteria into LC3-associated autophagosomes. Disruption of Rv1468c-ubiquitin interaction attenuates xenophagic clearance of Mtb in macrophages, and increases bacterial loads in mice with elevated inflammatory responses. Together, our findings reveal a unique mechanism of host xenophagy triggered by direct binding of ubiquitin to the pathogen surface protein, and indicate a diplomatic strategy adopted by Mtb to benefit its persistent intracellular infection through controlling intracellular bacterial loads and restricting host inflammatory responses.


Assuntos
Autofagossomos/metabolismo , Macrófagos/metabolismo , Mycobacterium tuberculosis/metabolismo , Ubiquitina/metabolismo , Imunidade Inata/fisiologia , RNA Mensageiro/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
11.
Georgian Med News ; (287): 29-35, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30958284

RESUMO

The aim of the study was to assess the activity of the ubiquitin-proteasome system in patients with chronic kidney disease stage 5D (CKD5D) and its relationship with clinical and laboratory parameters. We examined 80 patients with CKD5D on hemodialysis (HD). The mean age was 51.7±11.6 years, the duration of HD was 33.5 (19.7; 58.25) months. All patients underwent physical examination, bio-impedancemetry. Hypoxia-inducible factor 1-alpha (HIF-1α) and 20S-proteasome (20S-PSM) levels were determined in the blood by ELISA. The hemoglobin level as well as its fluctuation over the preceding 12 months did not differ in the groups with normal and elevated 20S-PSM levels, however, there were some features of ferrokinetics depending on the level of serum transferrin (p=0.04), its fluctuations over the preceding 12 months (p=0.03) and its saturation (p=0.03). It was shown that as the level of 20S-PSM in the blood increases, the probability of detecting protein-energy wasting (PEW) increases (χ2=4.8, p=0.029). This is probably due to the implementation of the catabolic link of protein metabolism involving the ubiquitin-proteasome system. There was a strong negative correlation between the HIF-1a and 20S-PSM parameters (r=-0.86, p<0.05), which was confirmed when building the logistic regression model (χ2=65.9, p<0.0001). Depending on the level of hemoglobin and HIF-1a, we divided all patients into groups with hemoglobin and hypoxia-dependent 20S-PSM increase mechanisms. The found interrelations of these molecular markers with ferrokinetics parameters, features of renal replacement therapy (RRT) require additional study.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Adulto , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Rim/efeitos dos fármacos , Rim/metabolismo , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Desnutrição Proteico-Calórica , Diálise Renal , Síndrome de Emaciação/patologia
12.
Methods Mol Biol ; 1977: 25-34, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980320

RESUMO

Protein homeostasis is essential for the survival of cells. It is closely related to the functioning of the ubiquitin-proteasome system, which utilizes the small protein ubiquitin as a posttranslational modifier (PTM). Clinically, the modification is of great importance as its disruption is the cause of many diseases. Unlike other PTMs, ubiquitin can encode several cellular signals by being attached as a single molecule or as a chain of several ubiquitins in various conformations. Thus, ubiquitin signaling is dependent not only on the site of attachment but also on the chain type, the so-called ubiquitin chain topology.The most reliable quantification method for the chain topology uses a bottom-up targeted mass spectrometry-based proteomics technique. While similar to other targeted proteomics techniques, the measurement of ubiquitination chain topology is complicated. First, the ubiquitin chains in the sample have to be biochemically stabilized. Second, the selection of peptides for the analysis is restricted to a given set harboring the PTMs and does not allow for optimization for amenability to mass spectrometry-based quantification. Instead, the topology-characteristic peptides are fixed. We here present such a methodology, including notes for a successful application.


Assuntos
Espectrometria de Massas , Proteômica , Ubiquitina/química , Análise de Dados , Humanos , Espectrometria de Massas/métodos , Espectrometria de Massas/normas , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Coloração e Rotulagem , Ubiquitina/metabolismo , Ubiquitinação
13.
Int J Mol Sci ; 20(8)2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30999567

RESUMO

Ubiquitin-like/ubiquitin-associated proteins (UbL-UbA) are a well-studied family of non-proteasomal ubiquitin receptors that are evolutionarily conserved across species. Members of this non-homogenous family facilitate and support proteasomal activity by promoting different effects on proteostasis but exhibit diverse extra-proteasomal activities. Dysfunctional UbL-UbA proteins render cells, particularly neurons, more susceptible to stressors or aging and may cause earlier neurodegeneration. In this review, we summarized the properties and functions of UbL-UbA family members identified to date, with an emphasis on new findings obtained using Drosophila models showing a direct or indirect role in some neurodegenerative diseases.


Assuntos
Doenças Neurodegenerativas/metabolismo , Neurônios/patologia , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Drosophila , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteostase , Fatores de Transcrição/metabolismo
14.
Chin J Traumatol ; 22(2): 93-98, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30928194

RESUMO

The clinical treatment of joint contracture due to immobilization remains difficult. The pathological changes of muscle tissue caused by immobilization-induced joint contracture include disuse skeletal muscle atrophy and skeletal muscle tissue fibrosis. The proteolytic pathways involved in disuse muscle atrophy include the ubiquitin-proteasome-dependent pathway, caspase system pathway, matrix metalloproteinase pathway, Ca2+-dependent pathway and autophagy-lysosomal pathway. The important biological processes involved in skeletal muscle fibrosis include intermuscular connective tissue thickening caused by transforming growth factor-ß1 and an anaerobic environment within the skeletal muscle leading to the induction of hypoxia-inducible factor-1α. This article reviews the progress made in understanding the pathological processes involved in immobilization-induced muscle contracture and the currently available treatments. Understanding the mechanisms involved in immobilization-induced contracture of muscle tissue should facilitate the development of more effective treatment measures for the different mechanisms in the future.


Assuntos
Contratura/etiologia , Imobilização/efeitos adversos , Articulações , Músculo Esquelético , Transdução de Sinais/fisiologia , Atrofia , Autofagia , Cálcio/metabolismo , Caspases/metabolismo , Tecido Conjuntivo/metabolismo , Tecido Conjuntivo/patologia , Contratura/metabolismo , Contratura/patologia , Contratura/terapia , Fibrose , Humanos , Lisossomos/metabolismo , Metaloproteinases da Matriz/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Fator de Crescimento Transformador beta1/metabolismo , Ubiquitina/metabolismo
15.
PLoS Pathog ; 15(4): e1007541, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31017975

RESUMO

DNA damage response (DDR) and selective autophagy both can be activated by reactive oxygen/nitrogen species (ROS/RNS), and both are of paramount importance in cancer development. The selective autophagy receptor and ubiquitin (Ub) sensor p62 plays a key role in their crosstalk. ROS production has been well documented in latent infection of oncogenic viruses including Epstein-Barr Virus (EBV). However, p62-mediated selective autophagy and its interplay with DDR have not been investigated in these settings. In this study, we provide evidence that considerable levels of p62-mediated selective autophagy are spontaneously induced, and correlate with ROS-Keap1-NRF2 pathway activity, in virus-transformed cells. Inhibition of autophagy results in p62 accumulation in the nucleus, and promotes ROS-induced DNA damage and cell death, as well as downregulates the DNA repair proteins CHK1 and RAD51. In contrast, MG132-mediated proteasome inhibition, which induces rigorous autophagy, promotes p62 degradation but accumulation of the DNA repair proteins CHK1 and RAD51. However, pretreatment with an autophagy inhibitor offsets the effects of MG132 on CHK1 and RAD51 levels. These findings imply that p62 accumulation in the nucleus in response to autophagy inhibition promotes proteasome-mediated CHK1 and RAD51 protein instability. This claim is further supported by the findings that transient expression of a p62 mutant, which is constitutively localized in the nucleus, in B cell lines with low endogenous p62 levels recaptures the effects of autophagy inhibition on CHK1 and RAD51 protein stability. These results indicate that proteasomal degradation of RAD51 and CHK1 is dependent on p62 accumulation in the nucleus. However, small hairpin RNA (shRNA)-mediated p62 depletion in EBV-transformed lymphoblastic cell lines (LCLs) had no apparent effects on the protein levels of CHK1 and RAD51, likely due to the constitutive localization of p62 in the cytoplasm and incomplete knockdown is insufficient to manifest its nuclear effects on these proteins. Rather, shRNA-mediated p62 depletion in EBV-transformed LCLs results in significant increases of endogenous RNF168-γH2AX damage foci and chromatin ubiquitination, indicative of activation of RNF168-mediated DNA repair mechanisms. Our results have unveiled a pivotal role for p62-mediated selective autophagy that governs DDR in the setting of oncogenic virus latent infection, and provide a novel insight into virus-mediated oncogenesis.


Assuntos
Autofagia , Transformação Celular Viral , Dano ao DNA , Infecções por Vírus Epstein-Barr/patologia , Estresse Oxidativo , Proteínas de Ligação a RNA/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Linfoma de Burkitt/virologia , Cromatina , Reparo do DNA , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Ubiquitina/metabolismo , Latência Viral
16.
J Cancer Res Clin Oncol ; 145(6): 1449-1460, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30968255

RESUMO

BACKGROUND: A growing body of evidence suggests that exercise training has beneficial effects in cancer patients. The aim of the present study was to investigate the molecular basis underlying these beneficial effects in skeletal muscle from cancer patients. METHODS: We investigated expression of selected proteins involved in cellular processes known to orchestrate adaptation to exercise training by western blot. Skeletal muscle biopsies were sampled from ten cancer patients before and after 4-7 weeks of ongoing chemotherapy, and subsequently after 10 weeks of continued chemotherapy in combination with exercise training. Biopsies from ten healthy matched subjects served as reference. RESULTS: The expression of the insulin-regulated glucose transporter, GLUT4, increased during chemotherapy and continued to increase during exercise training. A similar trend was observed for ACC, a key enzyme in the biosynthesis and oxidation of fatty acids, but we did not observe any changes in other regulators of substrate metabolism (AMPK and PDH) or mitochondrial proteins (Cyt-C, COX-IV, SDHA, and VDAC). Markers of proteasomal proteolysis (MURF1 and ATROGIN-1) decreased during chemotherapy, but did not change further during chemotherapy combined with exercise training. A similar pattern was observed for autophagy-related proteins such as ATG5, p62, and pULK1 Ser757, but not ULK1 and LC3BII/LC3BI. Phosphorylation of FOXO3a at Ser318/321 did not change during chemotherapy, but decreased during exercise training. This could suggest that FOXO3a-mediated transcriptional regulation of MURF1 and ATROGIN-1 serves as a mechanism by which exercise training maintains proteolytic systems in skeletal muscle in cancer patients. Phosphorylation of proteins that regulate protein synthesis (mTOR at Ser2448 and 4EBP1 at Thr37/46) increased during chemotherapy and leveled off during exercise training. Finally, chemotherapy tended to increase the number of satellite cells in type 1 fibers, without any further change during chemotherapy and exercise training. Conversely, the number of satellite cells in type 2 fibers did not change during chemotherapy, but increased during chemotherapy combined with exercise training. CONCLUSIONS: Molecular signaling cascades involved in exercise training are disturbed during cancer and chemotherapy, and exercise training may prevent further disruption of these pathways. TRIAL REGISTRATION: The study was approved by the local Scientific Ethics Committee of the Central Denmark Region (Project ID: M-2014-15-14; date of approval: 01/27/2014) and the Danish Data Protection Agency (case number 2007-58-0010; date of approval: 01/28/2015). The trial was registered at http//www.clinicaltrials.gov (registration number: NCT02192216; date of registration 07/17-2014).


Assuntos
Exercício , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiopatologia , Neoplasias/fisiopatologia , Adulto , Feminino , Transportador de Glucose Tipo 4/biossíntese , Humanos , Pessoa de Meia-Idade , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/terapia , Complexo de Endopeptidases do Proteassoma/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/patologia , Ubiquitina/metabolismo
17.
Anim Sci J ; 90(6): 728-736, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31006927

RESUMO

This study evaluated the effects of rice whole crop silage (RWCS) on growth, plasma levels of vitamin A, ß-carotene, vitamin E and IGF-1, and expression of genes involved in muscle protein degradation and synthesis in Japanese Black calves. Eleven calves were divided into RWCS (fed RWCS ad libitum and concentrate, n = 5) and control groups (fed hay ad libitum and concentrate, n = 6). Final body weight and dairy gain were significantly larger in the RWCS group compared with the control group. Plasma ß-carotene and vitamin E concentrations were significantly higher in the RWCS group compared with control group. Although plasma vitamin E concentration in the RWCS group significantly increased from 4 to 9 months of age, it did not increase in the control group. At 6 months of age in the RWCS group, ubiquitin B (p < 0.05) and calpain 1 (p = 0.097) mRNA expression were lower than control group, but they were not different between groups at 9 months of age. These results indicate that RWCS increases plasma ß-carotene level and promotes muscle growth because of a decrease in the rate of protein degradation, but the effect is lost with the increase in plasma vitamin E level.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal/fisiologia , Bovinos/crescimento & desenvolvimento , Bovinos/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Oryza , Proteólise , Silagem , Vitamina A/metabolismo , Vitamina E/metabolismo , beta Caroteno/metabolismo , Fenômenos Fisiológicos da Nutrição Animal/genética , Animais , Calpaína/genética , Calpaína/metabolismo , Expressão Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo
18.
Chem Biol Interact ; 306: 70-77, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980806

RESUMO

PURPOSE: Skeletal muscle is severely affected in diabetes leading to muscle atrophy. Previously we reported the role of ER stress in muscle atrophy due to hyperglycemia. Hence, in the present study, we investigated the effect of a classical ER stress inhibitor, 4-phenylbutyric acid (PBA), on muscle atrophy in diabetic rats. METHODS: Diabetes was induced in male rats by streptozotocin, and PBA was administered (40 mg/kg/day; intraperitoneal) after two months of diabetes for two more months. Gastrocnemius muscle is collected after four months of experimental period. The cross-sectional area of myocytes was measured on Hematoxylin and Eosin stained muscle sections. Protein levels of ER stress markers, ubiquitin-proteasome system (UPS) components, and apoptosis were analysed by immunoblot. Proteasomal activity and apoptotic cells were measured. RESULTS: ER stress markers (GRP78, ATF6, ATF4 and CHOP) that are elevated in diabetes are decreased with PBA treatment. PBA also averted diabetes-induced alterations in UPS (higher levels of E1, atrogin-1, UCHL1 and UCHL5, accumulation of ubiquitinated proteins and increased proteasomal activity). Apoptosis mediators-p53, BAX, and cleaved caspase-3 protein levels, and TUNEL positive cells were decreased in PBA treated diabetic rats. PBA notably improved the muscle-cross sectional area. CONCLUSIONS: Results highlighted the therapeutic potential of PBA in diabetes muscle wastage.


Assuntos
Diabetes Mellitus Experimental/prevenção & controle , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Atrofia Muscular/prevenção & controle , Fenilbutiratos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , Injeções Intraperitoneais , Masculino , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/patologia , Fenilbutiratos/administração & dosagem , Ratos , Ratos Sprague-Dawley , Estreptozocina
19.
Genes Dev ; 33(11-12): 620-625, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30923167

RESUMO

DOT1L is a histone H3 Lys79 methyltransferase whose activity is stimulated by histone H2B Lys120 ubiquitination, suggesting cross-talk between histone H3 methylation and H2B ubiquitination. Here, we present cryo-EM structures of DOT1L complexes with unmodified or H2B ubiquitinated nucleosomes, showing that DOT1L recognizes H2B ubiquitin and the H2A/H2B acidic patch through a C-terminal hydrophobic helix and an arginine anchor in DOT1L, respectively. Furthermore, the structures combined with single-molecule FRET experiments show that H2B ubiquitination enhances a noncatalytic function of the DOT1L-destabilizing nucleosome. These results establish the molecular basis of the cross-talk between H2B ubiquitination and H3 Lys79 methylation as well as nucleosome destabilization by DOT1L.


Assuntos
Histonas/química , Histonas/metabolismo , Metiltransferases/química , Metiltransferases/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Arginina/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Metilação , Modelos Moleculares , Estabilidade Proteica , Estrutura Secundária de Proteína , Ubiquitina/metabolismo , Ubiquitinação
20.
Nature ; 567(7747): 267-272, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30842657

RESUMO

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink1; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination2. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2-7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings3,4 establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Reparo do DNA , DNA/química , DNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , DNA/biossíntese , Replicação do DNA , Feminino , Humanos , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , N-Glicosil Hidrolases/metabolismo , Ligação Proteica , Ubiquitina/metabolismo , Ubiquitinação , Xenopus
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