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1.
Nat Commun ; 12(1): 62, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397936

RESUMO

Histones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is tightly controlled by epigenetic modulator enzymes and further recognized by so-called reader domains. Histone microarrays have been widely applied to investigate histone-reader interactions, but not the transient interactions of Zn2+-dependent histone deacetylase (HDAC) eraser enzymes. Here, we synthesize hydroxamic acid-modified histone peptides and use them in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. Follow-up functional assays in solution provide insights into their suitability to discover HDAC substrates and inhibitors with nanomolar potency and activity in cellular assays. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme-selective HDAC inhibitors and probes.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Células HEK293 , Histonas/metabolismo , Humanos , Isoenzimas/metabolismo , Análise em Microsséries , Peptídeos/química , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Zinco/metabolismo
3.
PLoS One ; 15(9): e0238810, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915856

RESUMO

Previous studies reported that sea buckthorn (Hippophae rhamnoides L., Elaeagnaceae, HRP) exhibits hepatoprotective effects via its anti-inflammatory and antioxidant properties as well as its inhibitory effects on collagen synthesis. However, it is unclear whether this hepatoprotective effect is also achieved by regulating liver drug metabolism enzyme pathways. Herein, we examined the regulatory effect of HRP on cytochrome P450 3A (CYP3A) in rats with immune liver injury, and explored the molecular mechanism of its hepatoprotective effect. Rat models of immunological liver injury were induced by intravenous injections of Bacillus Calmette-Guerin (BCG; 125 mg kg-1; 2 wks). Specific protein levels were detected by ELISA or western blot, and CYP3A mRNA expression was detected by RT-PCR. High-performance liquid chromatography (HPLC) detected relative changes in CYP3A metabolic activity based on the rates of 1-hydroxylation of the probe drug midazolam (MDZ). BCG pretreatment (125 mg kg-1) significantly down-regulated liver CYP3A protein expression compared with the control, metabolic activity, and transcription levels while up-regulating liver NF-κB, IL-1ß, TNF-α and iNOS. HRP intervention (ED50: 78 mg kg-1) moderately reversed NF-κB, inflammatory cytokines, and iNOS activation in a dose-dependent manner (P < 0.05), and suppressed CYP3A down-regulation (P < 0.05); thereby partially alleviating liver injury. During immune liver injury, HRP may reverse CYP3A down-regulation by inhibiting NF-κB signal transduction, and protect liver function, which involves regulation of enzymes transcriptionally, translationally and post-translationally. The discovery that NF-κB is a molecular target of HRP may initiate the development and optimization of a clinical therapeutic approach to mitigate hepatitis B and other immunity-related liver diseases.


Assuntos
Citocromo P-450 CYP3A/genética , Regulação para Baixo/efeitos dos fármacos , Elaeagnaceae/metabolismo , Mycobacterium bovis/fisiologia , NF-kappa B/metabolismo , Animais , Citocromo P-450 CYP3A/metabolismo , Interleucina-1beta/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/microbiologia , Fígado/patologia , Masculino , Óxido Nítrico Sintase Tipo II/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transcrição Genética/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
4.
PLoS One ; 15(8): e0237669, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32810137

RESUMO

Pancreatic beta cell death is a hallmark of type 1 and 2 diabetes (T1D/T2D), but the underlying molecular mechanisms are incompletely understood. Key proteins of the DNA damage response (DDR), including tumor protein P53 (P53, also known as TP53 or TRP53 in rodents) and Ataxia Telangiectasia Mutated (ATM), a kinase known to act upstream of P53, have been associated with T2D. Here we test and compare the effect of ATM and P53 ablation on beta cell survival in the rat beta cell line Ins1E. We demonstrate that ATM and P53 differentially regulate beta cell apoptosis induced upon fundamentally different types of diabetogenic beta cell stress, including DNA damage, inflammation, lipotoxicity and endoplasmic reticulum (ER) stress. DNA damage induced apoptosis by treatment with the commonly used diabetogenic agent streptozotocin (STZ) is regulated by both ATM and P53. We show that ATM is a key STZ induced activator of P53 and that amelioration of STZ induced cell death by inhibition of ATM mainly depends on P53. While both P53 and ATM control lipotoxic beta cell apoptosis, ATM but not P53 fails to alter inflammatory beta cell death. In contrast, tunicamycin induced (ER stress associated) apoptosis is further increased by ATM knockdown or inhibition, but not by P53 knockdown. Our results reveal differential roles for P53 and ATM in beta cell survival in vitro in the context of four key pathophysiological types of diabetogenic beta cell stress, and indicate that ATM can use P53 independent signaling pathways to modify beta cell survival, dependent on the cellular insult.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sobrevivência Celular/genética , Células Secretoras de Insulina/patologia , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Diabetes Mellitus/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Técnicas de Silenciamento de Genes , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Ratos , Estreptozocina/toxicidade , Tunicamicina/toxicidade
5.
PLoS One ; 15(8): e0237845, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813721

RESUMO

Aluminum (Al3+) toxicity is one of the most important limitations to agricultural production worldwide. The overall response of plants to Al3+ stress has been documented, but the contribution of protein phosphorylation to Al3+ detoxicity and tolerance in plants is unclear. Using a combination of tandem mass tag (TMT) labeling, immobilized metal affinity chromatography (IMAC) enrichment and liquid chromatography-tandem mass spectrometry (LC-MS/MS), Al3+-induced phosphoproteomic changes in roots of Tamba black soybean (TBS) were investigated in this study. The Data collected in this study are available via ProteomeXchange with the identifier PXD019807. After the Al3+ treatment, 189 proteins harboring 278 phosphosites were significantly changed (fold change > 1.2 or < 0.83, p < 0.05), with 88 upregulated, 96 downregulated and 5 up-/downregulated. Enrichment and protein interaction analyses revealed that differentially phosphorylated proteins (DPPs) under the Al3+ treatment were mainly related to G-protein-mediated signaling, transcription and translation, transporters and carbohydrate metabolism. Particularly, DPPs associated with root growth inhibition or citric acid synthesis were identified. The results of this study provide novel insights into the molecular mechanisms of TBS post-translational modifications in response to Al3+ stress.


Assuntos
Alumínio/toxicidade , Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Proteômica , Soja/metabolismo , Citratos/metabolismo , Fosforilação/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Soja/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
6.
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
7.
Nat Commun ; 11(1): 3660, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694521

RESUMO

High expression or aberrant activation of epidermal growth factor receptor (EGFR) is related to tumor progression and therapy resistance across cancer types, including non-small cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors (TKIs) are first-line therapy for NSCLC. However, patients eventually deteriorate after inevitable acquisition of EGFR TKI-resistant mutations, highlighting the need for therapeutics with alternative mechanisms of action. Here, we report that the elevated tribbles pseudokinase 3 (TRIB3) is positively associated with EGFR stability and NSCLC progression. TRIB3 interacts with EGFR and recruits PKCα to induce a Thr654 phosphorylation and WWP1-induced Lys689 ubiquitination in the EGFR juxtamembrane region, which enhances EGFR recycling, stability, downstream activity, and NSCLC stemness. Disturbing the TRIB3-EGFR interaction with a stapled peptide attenuates NSCLC progression by accelerating EGFR degradation and sensitizes NSCLC cells to chemotherapeutic agents. These findings indicate that targeting EGFR degradation is a previously unappreciated therapeutic option in EGFR-related NSCLC.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ciclo Celular/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Adulto , Animais , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Linhagem Celular Tumoral , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Fosforilação/efeitos dos fármacos , Proteína Quinase C-alfa/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise/efeitos dos fármacos , Taxa de Sobrevida , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Nat Commun ; 11(1): 3339, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620764

RESUMO

Chromosomal NUP98-PHF23 translocation is associated with an aggressive form of acute myeloid leukemia (AML) and poor survival rate. Here, we report the molecular mechanisms by which NUP98-PHF23 recognizes the histone mark H3K4me3 and is inhibited by small molecule compounds, including disulfiram that directly targets the PHD finger of PHF23 (PHF23PHD). Our data support a critical role for the PHD fingers of NUP98-PHF23, and related NUP98-KDM5A and NUP98-BPTF fusions in driving leukemogenesis, and demonstrate that blocking this interaction in NUP98-PHF23 expressing AML cells leads to cell death through necrotic and late apoptosis pathways. An overlap of NUP98-KDM5A oncoprotein binding sites and H3K4me3-positive loci at the Hoxa/b gene clusters and Meis1 in ChIP-seq, together with NMR analysis of the H3K4me3-binding sites of the PHD fingers from PHF23, KDM5A and BPTF, suggests a common PHD finger-dependent mechanism that promotes leukemogenesis by this type of NUP98 fusions. Our findings highlight the direct correlation between the abilities of NUP98-PHD finger fusion chimeras to associate with H3K4me3-enriched chromatin and leukemic transformation.


Assuntos
Cromatina/metabolismo , Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Doença Aguda , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Cromatina/genética , Dissulfiram/farmacologia , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Dedos de Zinco PHD/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteína 2 de Ligação ao Retinoblastoma/genética , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Translocação Genética/efeitos dos fármacos , Translocação Genética/genética
9.
Nucleic Acids Res ; 48(12): 6799-6810, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32484546

RESUMO

Structure and/or function of proteins are frequently affected by oxidative/nitrosative stress via posttranslational modifications. Aminoacyl-tRNA synthetases (aaRSs) constitute a class of ubiquitously expressed enzymes that control cellular protein homeostasis. Here, we found the activity of human mitochondrial (mt) threonyl-tRNA synthetase (hmtThrRS) is resistant to oxidative stress (H2O2) but profoundly sensitive to nitrosative stress (S-nitrosoglutathione, GSNO). Further study showed four Cys residues in hmtThrRS were modified by S-nitrosation upon GSNO treatment, and one residue was one of synthetic active sites. We analyzed the effect of modification at individual Cys residue on aminoacylation and editing activities of hmtThrRS in vitro and found that both activities were decreased. We further confirmed that S-nitrosation of mtThrRS could be readily detected in vivo in both human cells and various mouse tissues, and we systematically identified dozens of S-nitrosation-modified sites in most aaRSs, thus establishing both mitochondrial and cytoplasmic aaRS species with S-nitrosation ex vivo and in vivo, respectively. Interestingly, a decrease in the S-nitrosation modification level of mtThrRS was observed in a Huntington disease mouse model. Overall, our results establish, for the first time, a comprehensive S-nitrosation-modified aaRS network and a previously unknown mechanism on the basis of the inhibitory effect of S-nitrosation on hmtThrRS.


Assuntos
Mitocôndrias/genética , Nitrosação/genética , Estresse Nitrosativo/genética , Treonina-tRNA Ligase/genética , Aminoacil-tRNA Sintetases/genética , Aminoacilação/genética , Animais , Domínio Catalítico/efeitos dos fármacos , Células HeLa , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Cinética , Camundongos , Mitocôndrias/enzimologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Treonina-tRNA Ligase/química
10.
Proc Natl Acad Sci U S A ; 117(22): 12029-12040, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32404427

RESUMO

Hutchinson-Gilford progeria syndrome (HGPS) is a uniformly fatal condition that is especially prevalent in skin, cardiovascular, and musculoskeletal systems. A wide gap exists between our knowledge of the disease and a promising treatment or cure. The aim of this study was to first characterize the musculoskeletal phenotype of the homozygous G608G BAC-transgenic progeria mouse model, and to determine the phenotype changes of HGPS mice after a five-arm preclinical trial of different treatment combinations with lonafarnib, pravastatin, and zoledronic acid. Microcomputed tomography and CT-based rigidity analyses were performed to assess cortical and trabecular bone structure, density, and rigidity. Bones were loaded to failure with three-point bending to assess strength. Contrast-enhanced µCT imaging of mouse femurs was performed to measure glycosaminoglycan content, thickness, and volume of the femoral head articular cartilage. Advanced glycation end products were assessed with a fluorometric assay. The changes demonstrated in the cortical bone structure, rigidity, stiffness, and modulus of the HGPS G608G mouse model may increase the risk for bending and deformation, which could result in the skeletal dysplasia characteristic of HGPS. Cartilage abnormalities seen in this HGPS model resemble changes observed in the age-matched WT controls, including early loss of glycosaminoglycans, and decreased cartilage thickness and volume. Such changes might mimic prevalent degenerative joint diseases in the elderly. Lonafarnib monotherapy did not improve bone or cartilage parameters, but treatment combinations with pravastatin and zoledronic acid significantly improved bone structure and mechanical properties and cartilage structural parameters, which ameliorate the musculoskeletal phenotype of the disease.


Assuntos
Conservadores da Densidade Óssea/uso terapêutico , Modelos Animais de Doenças , Lamina Tipo A/genética , Progéria , Envelhecimento/efeitos dos fármacos , Envelhecimento/patologia , Animais , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/patologia , Cartilagem/efeitos dos fármacos , Cartilagem/patologia , Fêmur/efeitos dos fármacos , Fêmur/patologia , Glicosaminoglicanos/análise , Articulações/efeitos dos fármacos , Articulações/patologia , Lamina Tipo A/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Osteoartrite/tratamento farmacológico , Osteoartrite/patologia , Fenótipo , Piperidinas/uso terapêutico , Pravastatina/uso terapêutico , Progéria/tratamento farmacológico , Progéria/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Piridinas/uso terapêutico , Microtomografia por Raio-X , Ácido Zoledrônico/uso terapêutico
11.
Nat Commun ; 11(1): 2396, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32409666

RESUMO

Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.


Assuntos
Arginina/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Estresse Fisiológico , Animais , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Metilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9
12.
Nat Commun ; 11(1): 2174, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358582

RESUMO

Poly-ADP-ribosylation (PARylation) is a fully reversible post-translational modification with key roles in cellular physiology. Due to the multi-domain structure of poly(ADP-ribose) polymerase-1 (PARP1) and the highly dynamic nature of the PARylation reaction, studies on the biochemical mechanism and structural dynamics remain challenging. Here, we report label-free, time-resolved monitoring of PARP1-dependent PARylation using ATR-FTIR spectroscopy. This includes PARP1 activation by binding to DNA strand break models, NAD+ substrate binding, PAR formation, and dissociation of automodified PARP1 from DNA. Analyses of PARP1 activation at different DNA models demonstrate a strong positive correlation of PARylation and PARP1 dissociation, with the strongest effects observed for DNA nicks and 3' phosphorylated ends. Moreover, by examining dynamic structural changes of PARP1, we reveal changes in the secondary structure of PARP1 induced by NAD+ and PARP inhibitor binding. In summary, this approach enables holistic and dynamic insights into PARP1-dependent PARylation with molecular and temporal resolution.


Assuntos
Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação/genética , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Humanos , Cinética , NAD/análogos & derivados , NAD/biossíntese , NAD/metabolismo , Oligonucleotídeos/química , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Poli ADP Ribosilação/efeitos dos fármacos , Poli ADP Ribosilação/fisiologia , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier/métodos
13.
Oncogene ; 39(22): 4436-4449, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32350443

RESUMO

Activator protein (AP)-1 transcription factors are essential elements of the pro-oncogenic functions of transforming growth factor-ß (TGFß)-SMAD signaling. Here we show that in multiple HER2+ and/or EGFR+ breast cancer cell lines these AP-1-dependent tumorigenic properties of TGFß critically rely on epidermal growth factor receptor (EGFR) activation and expression of the ΔN isoform of transcriptional regulator p63. EGFR and ΔNp63 enabled and/or potentiated the activation of a subset of TGFß-inducible invasion/migration-associated genes, e.g., ITGA2, LAMB3, and WNT7A/B, and enhanced the recruitment of SMAD2/3 to these genes. The TGFß- and EGF-induced binding of SMAD2/3 and JUNB to these gene loci was accompanied by p63-SMAD2/3 and p63-JUNB complex formation. p63 and EGFR were also found to strongly potentiate TGFß induction of AP-1 proteins and, in particular, FOS family members. Ectopic overexpression of FOS could counteract the decrease in TGFß-induced gene activation after p63 depletion. p63 is also involved in the transcriptional regulation of heparin binding (HB)-EGF and EGFR genes, thereby establishing a self-amplification loop that facilitates and empowers the pro-invasive functions of TGFß. These cooperative pro-oncogenic functions of EGFR, AP-1, p63, and TGFß were efficiently inhibited by clinically relevant chemical inhibitors. Our findings may, therefore, be of importance for therapy of patients with breast cancers with an activated EGFR-RAS-RAF pathway.


Assuntos
Neoplasias da Mama/patologia , Fator de Crescimento Epidérmico/fisiologia , Regulação Neoplásica da Expressão Gênica , Invasividade Neoplásica/genética , Proteínas de Neoplasias/fisiologia , Transdução de Sinais , Fator de Transcrição AP-1/genética , Fatores de Transcrição/genética , Transcrição Genética , Fator de Crescimento Transformador beta1/fisiologia , Proteínas Supressoras de Tumor/genética , Neoplasias da Mama/química , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Receptores ErbB/fisiologia , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas de Neoplasias/genética , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/patologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/fisiologia , Proteínas Proto-Oncogênicas c-jun/fisiologia , Receptor ErbB-2/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo I/fisiologia , Proteínas Smad/fisiologia
14.
Sci Rep ; 10(1): 6538, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32300132

RESUMO

Alzheimer's disease (AD) is the most common form of dementia globally and is characterized by aberrant accumulations of amyloid-beta (Aß) and tau proteins. Oligomeric forms of these proteins are believed to be most relevant to disease progression, with oligomeric amyloid-ß (oAß) particularly implicated in AD. oAß pathology spreads among interconnected brain regions, but how oAß induces pathology in these previously unaffected neurons requires further study. Here, we use well characterized iPSC-derived human neurons to study the early changes to the proteome and phosphoproteome after 24 h exposure to oAß 1-42. Using nLC-MS/MS and label-free quantification, we identified several proteins that are differentially regulated in response to acute oAß challenge. At this early timepoint, oAß induced the decrease of TDP-43, heterogeneous nuclear ribonucleoproteins (hnRNPs), and coatomer complex I (COPI) proteins. Conversely, increases were observed in 20 S proteasome subunits and vesicle associated proteins VAMP1/2, as well as the differential phosphorylation of tau at serine 208. These changes show that there are widespread alterations to the neuronal proteome within 24 h of oAß uptake, including proteins previously not shown to be related to neurodegeneration. This study provides new targets for the further study of early mediators of AD pathogenesis.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Células-Tronco Pluripotentes Induzidas/patologia , Neurônios/metabolismo , Multimerização Proteica , Proteoma/metabolismo , Regulação para Baixo/efeitos dos fármacos , Humanos , Mutação/genética , Neurônios/efeitos dos fármacos , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Espectrometria de Massas em Tandem , Regulação para Cima/efeitos dos fármacos , Proteínas tau/metabolismo
15.
Development ; 147(7)2020 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165492

RESUMO

Although the developing pancreas is exquisitely sensitive to nutrient supply in utero, it is not entirely clear how nutrient-driven post-translational modification of proteins impacts the pancreas during development. We hypothesized that the nutrient-sensing enzyme O-GlcNAc transferase (Ogt), which catalyzes an O-GlcNAc-modification onto key target proteins, integrates nutrient-signaling networks to regulate cell survival and development. In this study, we investigated the heretofore unknown role of Ogt in exocrine and endocrine islet development. By genetic manipulation in vivo and by using morphometric and molecular analyses, such as immunofluorescence imaging and single cell RNA sequencing, we show the first evidence that Ogt regulates pancreas development. Genetic deletion of Ogt in the pancreatic epithelium (OgtKOPanc) causes pancreatic hypoplasia, in part by increased apoptosis and reduced levels of of Pdx1 protein. Transcriptomic analysis of single cell and bulk RNA sequencing uncovered cell-type heterogeneity and predicted upstream regulator proteins that mediate cell survival, including Pdx1, Ptf1a and p53, which are putative Ogt targets. In conclusion, these findings underscore the requirement of O-GlcNAcylation during pancreas development and show that Ogt is essential for pancreatic progenitor survival, providing a novel mechanistic link between nutrients and pancreas development.


Assuntos
Acetilglucosamina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Nutrientes/farmacologia , Pâncreas Exócrino/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Animais , Embrião de Mamíferos , Feminino , Ilhotas Pancreáticas/embriologia , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Acetilglucosaminiltransferases/efeitos dos fármacos , N-Acetilglucosaminiltransferases/metabolismo , Pâncreas Exócrino/embriologia , Pâncreas Exócrino/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
Hum Genet ; 139(5): 657-673, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32219518

RESUMO

GM1-gangliosidosis, a lysosomal storage disorder, is associated with ~ 161 missense variants in the GLB1 gene. Affected patients present with ß-galactosidase (ß-Gal) deficiency in lysosomes. Loss of function in ER-retained misfolded enzymes with missense variants is often due to subcellular mislocalization. Deoxygalactonojirimycin (DGJ) and its derivatives are pharmaceutical chaperones that directly bind to mutated ß-Gal in the ER promoting its folding and trafficking to lysosomes and thus enhancing its activity. An Emirati child has been diagnosed with infantile GM1-gangliosidosis carrying the reported p.D151Y variant. We show that p.D151Y ß-Gal in patient's fibroblasts retained < 1% residual activity due to impaired processing and trafficking. The amino acid substitution significantly affected the enzyme conformation; however, p.D151Y ß-Gal was amenable for partial rescue in the presence of glycerol or at reduced temperature where activity was enhanced with ~ 2.3 and 7 folds, respectively. The butyl (NB-DGJ) and nonyl (NN-DGJ) derivatives of DGJ chaperoning function were evaluated by measuring their IC50s and ability to stabilize the wild-type ß-Gal against thermal degradation. Although NN-DGJ showed higher affinity to ß-Gal, it did not show a significant enhancement in p.D151Y ß-Gal activity. However, NB-DGJ promoted p.D151Y ß-Gal maturation and enhanced its activity up to ~ 4.5% of control activity within 24 h which was significantly increased to ~ 10% within 6 days. NB-DGJ enhancement effect was sustained over 3 days after washing it out from culture media. We therefore conclude that NB-DGJ might be a promising therapeutic chemical chaperone in infantile GM1 amenable variants and therefore warrants further analysis for its clinical applications.


Assuntos
1-Desoxinojirimicina/farmacologia , Fibroblastos/metabolismo , Gangliosidose GM1/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , beta-Galactosidase/metabolismo , 1-Desoxinojirimicina/química , Pré-Escolar , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Gangliosidose GM1/tratamento farmacológico , Gangliosidose GM1/patologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Masculino , Chaperonas Moleculares/farmacologia , Proteínas Mutantes/química , Proteínas Mutantes/genética , Conformação Proteica , Transporte Proteico , beta-Galactosidase/química , beta-Galactosidase/genética
17.
Neoplasia ; 22(4): 179-191, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32145688

RESUMO

Cullin-RING E3 ligase (CRL) is the largest family of E3 ubiquitin ligase, responsible for ubiquitylation of ∼20% of cellular proteins. CRL plays an important role in many biological processes, particularly in cancers due to abnormal activation. CRL activation requires neddylation, an enzymatic cascade transferring small ubiquitin-like protein NEDD8 to a conserved lysine residue on cullin proteins. Recent studies have validated that neddylation is an attractive anticancer target. In this study, we report the establishment of an Alpha-Screen-based high throughput screen (HTS) assay for in vitro CUL5 neddylation, and screened a library of 17,000 compounds including FDA approved drugs, natural products and synthetic drug-like small-molecule compounds. Gossypol, a natural compound derived from cotton seed, was identified as an inhibitor of cullin neddylation. Biochemical studies showed that gossypol blocked neddylation of both CUL5 and CUL1 through direct binding to SAG-CUL5 or RBX1-CUL1 complex, and CUL5-H572 plays a key role for gossypol binding. On cellular level, gossypol inhibited cullin neddylation in a variety of cancer cell lines and selectively caused accumulation of NOXA and MCL1, the substrates of CUL5 and CUL1, respectively, in multiple cancer cell lines. Combination of gossypol with specific MCL1 inhibitor synergistically suppress growth of human cancer cells. Our study revealed a previously unknown anti-cancer mechanism of gossypol with potential to develop a new class of neddylation inhibitors.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Gossipol/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular Tumoral , Anticoncepcionais Masculinos , Proteínas Culina/genética , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Gossipol/química , Ensaios de Triagem em Larga Escala , Humanos , Modelos Biológicos , Modelos Moleculares , Estrutura Molecular , Complexos Multiproteicos , Ligação Proteica , Relação Estrutura-Atividade
18.
Neoplasia ; 22(4): 192-202, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32145689

RESUMO

Androgen receptor (AR) and its constitutively active variants (AR-Vs) have been extensively implicated in the progression and recurrence of prostate cancer, making them attractive targets in the treatment of this disease. Whether and how neddylation modification regulates AR, and the therapeutic implications of this potential regulation, are relatively unexplored areas of investigation. Here we report that neddylation inactivation by the pharmacological inhibitor MLN4924 or Lenti-shRNA-based genetic knockdown of neddylation activating enzyme (NAE) selectively suppressed growth and survival of prostate cancer cells with minor, if any, effect on normal prostate epithelial cells. MLN4924 also significantly suppressed the invasive capacity of prostate cancer cells. Furthermore, compared to monotherapy, the combination of MLN4924 with AR antagonist or castration significantly enhanced growth suppression of prostate cancer cells in vitro, and tumor growth in an in vivo xenograft model. Mechanistically, MLN4924 repressed the transcription of AR/AR-V7 and its downstream targets, and blocked MMP2 and MMP9 expression. Taken together, our study reveals that the neddylation pathway positively regulates AR/AR-V7 transcription, and that the neddylation inhibitor MLN4924 has therapeutic potential for the treatment of aggressive prostate cancers.


Assuntos
Regulação Neoplásica da Expressão Gênica , Processamento de Proteína Pós-Traducional , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclopentanos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Modelos Biológicos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Pirimidinas/farmacologia , Transcrição Genética
19.
Biochim Biophys Acta Rev Cancer ; 1873(2): 188351, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32007596

RESUMO

Cancer is a multifaceted global disease. Transformation of a normal to a malignant cell takes several steps, including somatic mutations, epigenetic alterations, metabolic reprogramming and loss of cell growth control. Recently, the mevalonate pathway has emerged as a crucial regulator of tumor biology and a potential therapeutic target. This pathway controls cholesterol production and posttranslational modifications of Rho-GTPases, both of which are linked to several key steps of tumor progression. Inhibitors of the mevalonate pathway induce pleiotropic antitumor-effects in several human malignancies, identifying the pathway as an attractive candidate for novel therapies. In this review, we will provide an overview about the role and regulation of the mevalonate pathway in certain aspects of cancer initiation and progression and its potential for therapeutic intervention in oncology.


Assuntos
Antineoplásicos/farmacologia , Transformação Celular Neoplásica/metabolismo , Colesterol/biossíntese , Ácido Mevalônico/metabolismo , Neoplasias/metabolismo , Antineoplásicos/uso terapêutico , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Ensaios Clínicos como Assunto , Progressão da Doença , Geraniltranstransferase/antagonistas & inibidores , Geraniltranstransferase/metabolismo , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas de Ligação a Elemento Regulador de Esterol/antagonistas & inibidores , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Resultado do Tratamento , Proteínas rho de Ligação ao GTP/metabolismo
20.
Nutrients ; 12(2)2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32019055

RESUMO

This study used nanofluidic protein posttranslational modification (PTM) profiling to measure the effects of six cannabidiol (CBD) oils and isolated CBD on the signaling pathways of a cultured SH-SY5Y neuronal cell line. Chemical composition analysis revealed that all CBD oils met the label claims and legal regulatory limit regarding the CBD and tetrahydrocannabinol (THC) contents, respectively. Isolated CBD was cytotoxic, with an effective concentration (EC50) of 40 µM. In contrast, the CBD oils had no effect on cell viability at CBD concentrations exceeding 1.2 mM. Interestingly, only an unadulterated CBD oil had strong and statistically significant suppressive effects on the pI3K/Akt/mTOR signaling pathway with an EC50 value of 143 µM and a slow-acting timescale requiring hours. Systematic profiling of twenty-six proteins, which served as biomarkers for nine signaling pathways, revealed that the unadulterated CBD oil downregulated seven signaling pathways but had no measurable effect on the other two signaling pathways. The remaining CBD oils, which were adulterated, and isolated CBD had weak, variable, or undetectable effects on neuronal signaling pathways. Our data clearly showed that adulteration diminished the biological activities of CBD oils. In addition, nanofluidic protein PTM profiling provided a robust means for potency assessment of CBD oils.


Assuntos
Canabidiol/farmacologia , Óleos Vegetais/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos
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