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1.
Nat Commun ; 12(1): 4536, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315872

RESUMO

Despite the substantial impact of post-translational modifications on programmed cell death 1 ligand 1 (PD-L1), its importance in therapeutic resistance in pancreatic cancer remains poorly defined. Here, we demonstrate that never in mitosis gene A-related kinase 2 (NEK2) phosphorylates PD-L1 to maintain its stability, causing PD-L1-targeted pancreatic cancer immunotherapy to have poor efficacy. We identify NEK2 as a prognostic factor in immunologically "hot" pancreatic cancer, involved in the onset and development of pancreatic tumors in an immune-dependent manner. NEK2 deficiency results in the suppression of PD-L1 expression and enhancement of lymphocyte infiltration. A NEK binding motif (F/LXXS/T) is identified in the glycosylation-rich region of PD-L1. NEK2 interacts with PD-L1, phosphorylating the T194/T210 residues and preventing ubiquitin-proteasome pathway-mediated degradation of PD-L1 in ER lumen. NEK2 inhibition thereby sensitizes PD-L1 blockade, synergically enhancing the anti-pancreatic cancer immune response. Together, the present study proposes a promising strategy for improving the effectiveness of pancreatic cancer immunotherapy.


Assuntos
Antígeno B7-H1/metabolismo , Imunidade , Quinases Relacionadas a NIMA/antagonistas & inibidores , Neoplasias Pancreáticas/imunologia , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Modelos Biológicos , Quinases Relacionadas a NIMA/deficiência , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosforilação , Fosfosserina/metabolismo , Prognóstico , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteólise , Ubiquitinação
2.
Nat Commun ; 12(1): 4245, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253722

RESUMO

Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). Transcription factor EB (TFEB), a master regulator of lysosome biogenesis, is negatively regulated by mTORC1 through a RAG GTPase-dependent phosphorylation. Here we show that lysosomal biogenesis is increased in TSC-associated renal tumors, pulmonary lymphangioleiomyomatosis, kidneys from Tsc2+/- mice, and TSC1/2-deficient cells via a TFEB-dependent mechanism. Interestingly, in TSC1/2-deficient cells, TFEB is hypo-phosphorylated at mTORC1-dependent sites, indicating that mTORC1 is unable to phosphorylate TFEB in the absence of the TSC1/2 complex. Importantly, overexpression of folliculin (FLCN), a GTPase activating protein for RAGC, increases TFEB phosphorylation at the mTORC1 sites in TSC2-deficient cells. Overexpression of constitutively active RAGC is sufficient to relocalize TFEB to the cytoplasm. These findings establish the TSC proteins as critical regulators of lysosomal biogenesis via TFEB and RAGC and identify TFEB as a driver of the proliferation of TSC2-deficient cells.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Lisossomos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Biogênese de Organelas , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Carcinoma de Células Renais/patologia , Núcleo Celular/metabolismo , Proliferação de Células , Feminino , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Neoplasias Renais/patologia , Lisossomos/ultraestrutura , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Fosfosserina/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas/metabolismo , Transcrição Genética , Proteína 2 do Complexo Esclerose Tuberosa/deficiência , Proteínas Supressoras de Tumor/metabolismo
3.
Nature ; 596(7870): 138-142, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34290405

RESUMO

In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex1. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD212-4). Separase is activated by degradation of its inhibitors, securin5 and cyclin B6, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans7 and yeast8, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalytic sites of both separase and CDK19,10. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine6 that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.


Assuntos
Proteína Quinase CDC2/química , Proteína Quinase CDC2/metabolismo , Ciclina B1/química , Ciclina B1/metabolismo , Securina/química , Securina/metabolismo , Separase/química , Separase/metabolismo , Motivos de Aminoácidos , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/ultraestrutura , Quinases relacionadas a CDC2 e CDC28/química , Quinases relacionadas a CDC2 e CDC28/metabolismo , Quinases relacionadas a CDC2 e CDC28/ultraestrutura , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Microscopia Crioeletrônica , Ciclina B1/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Fosfosserina/metabolismo , Ligação Proteica , Domínios Proteicos , Securina/ultraestrutura , Separase/antagonistas & inibidores , Separase/ultraestrutura , Especificidade por Substrato
4.
Biomolecules ; 11(3)2021 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805672

RESUMO

Mitochondrial fission and fusion cycles are integrated with cell cycle progression. Here we first re-visited how mitochondrial ETC inhibition disturbed mitosis progression, resulting in multipolar spindles formation in HeLa cells. Inhibitors of ETC complex I (rotenone, ROT) and complex III (antimycin A, AA) decreased the phosphorylation of Plk1 T210 and Aurora A T288 in the mitotic phase (M-phase), especially ROT, affecting the dynamic phosphorylation status of fission protein dynamin-related protein 1 (Drp1) and the Ser637/Ser616 ratio. We then tested whether specific Drp1 inhibitors, Mdivi-1 or Dynasore, affected the dynamic phosphorylation status of Drp1. Similar to the effects of ROT and AA, our results showed that Mdivi-1 but not Dynasore influenced the dynamic phosphorylation status of Ser637 and Ser616 in Drp1, which converged with mitotic kinases (Cdk1, Plk1, Aurora A) and centrosome-associated proteins to significantly accelerate mitotic defects. Moreover, our data also indicated that evoking mito-Drp1-Ser637 by protein kinase A (PKA) rather than Drp1-Ser616 by Cdk1/Cyclin B resulted in mitochondrial fission via the PINK1/Parkin pathway to promote more efficient mitophagy and simultaneously caused multipolar spindles. Collectively, this study is the first to uncover that mito-Drp1-Ser637 by PKA, but not Drp1-Ser616, drives mitophagy to exert multipolar spindles formation during M-phase.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinaminas/metabolismo , Dinâmica Mitocondrial , Mitofagia , Mitose , Proteínas Quinases/metabolismo , Fuso Acromático/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Antimicina A/farmacologia , Aurora Quinase A/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Células HeLa , Humanos , Hidrazonas/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Estresse Oxidativo , Fosforilação , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Quinazolinonas/metabolismo , Rotenona/farmacologia
5.
Plant Cell ; 33(5): 1813-1827, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-33665670

RESUMO

Light-induced stomatal opening stimulates CO2 uptake and transpiration in plants. Weak blue light under strong red light effectively induces stomatal opening. Blue light-dependent stomatal opening initiates light perception by phototropins, and the signal is transmitted to a plasma membrane H+-ATPase in guard cells via BLUE LIGHT SIGNALING 1 (BLUS1) kinase. However, it is unclear how BLUS1 transmits the signal to H+-ATPase. Here, we characterized BLUS1 signaling in Arabidopsis thaliana, and showed that the BLUS1 C-terminus acts as an auto-inhibitory domain and that phototropin-mediated Ser-348 phosphorylation within the domain removes auto-inhibition. C-Terminal truncation and phospho-mimic Ser-348 mutation caused H+-ATPase activation in the dark, but did not elicit stomatal opening. Unexpectedly, the plants exhibited stomatal opening under strong red light and stomatal closure under weak blue light. A decrease in intercellular CO2 concentration via red light-driven photosynthesis together with H+-ATPase activation caused stomatal opening. Furthermore, phototropins caused H+-ATPase dephosphorylation in guard cells expressing constitutive signaling variants of BLUS1 in response to blue light, possibly for fine-tuning stomatal opening. Overall, our findings provide mechanistic insights into the blue light regulation of stomatal opening.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Dióxido de Carbono/farmacologia , Luz , Fosfotransferases/metabolismo , Estômatos de Plantas/fisiologia , Estômatos de Plantas/efeitos da radiação , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/química , Modelos Biológicos , Mutação/genética , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Fosfosserina/metabolismo , Fosfotransferases/química , Fototropinas/metabolismo , Estômatos de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas , Domínios Proteicos , ATPases Translocadoras de Prótons/metabolismo
6.
Biochem Biophys Res Commun ; 553: 51-57, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33756345

RESUMO

The tumor suppressor gene HIC1 (Hypermethylated in Cancer 1) encodes a transcriptional repressor involved in the DNA-damage response. A SUMOylation increase on HIC1 Lysine314 favors the direct transcriptional repression of SIRT1 and thus the P53-dependent apoptotic response to irreparable DNA double strand breaks (DSBs). HIC1 is also essential for DSBs repair but in a SUMOylation-independent manner. Here, we show that repairable DSBs induced by a 1 h Etoposide treatment results in three specific posttranslational modifications (PTMs) of HIC1. Two of these PTMs, phosphorylation of Serine 694 and Acetylation of Lysine 623 are located in the conserved HIC1 C-terminal region located downstream of the Zinc Finger DNA-binding domain. By contrast, phosphorylation of Serine 285 found in the poorly conserved central region is unique to the human protein. We showed that Ser694 phosphorylation is mediated mainly by the PIKK kinase ATM and is essential for the DNA repair activity of HIC1 as demonstrated by the lack of efficiency of the S694A point mutant in Comet assays. Thus, our results provide the first evidence for a functional role of the conserved HIC1 C-terminal region as a novel ATM substrate that plays an essential role in the cellular HIC1-mediated cellular response to repairable DSBs.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo do DNA , Fatores de Transcrição Kruppel-Like/química , Fatores de Transcrição Kruppel-Like/metabolismo , Fosfosserina/metabolismo , Animais , Linhagem Celular , Ensaio Cometa , Sequência Conservada , Dano ao DNA , Humanos , Fosforilação
7.
Nucleic Acids Res ; 49(6): 3109-3126, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33684220

RESUMO

Recruitment of the mRNA capping enzyme (CE/RNGTT) to the site of transcription is essential for the formation of the 5' mRNA cap, which in turn ensures efficient transcription, splicing, polyadenylation, nuclear export and translation of mRNA in eukaryotic cells. The CE GTase is recruited and activated by the Serine-5 phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II. Through the use of molecular dynamics simulations and enhanced sampling techniques, we provide a systematic and detailed characterization of the human CE-CTD interface, describing the effect of the CTD phosphorylation state, length and orientation on this interaction. Our computational analyses identify novel CTD interaction sites on the human CE GTase surface and quantify their relative contributions to CTD binding. We also identify, for the first time, allosteric connections between the CE GTase active site and the CTD binding sites, allowing us to propose a mechanism for allosteric activation. Through binding and activity assays we validate the novel CTD binding sites and show that the CDS2 site is essential for CE GTase activity stimulation. Comparison of the novel sites with cocrystal structures of the CE-CTD complex in different eukaryotic taxa reveals that this interface is considerably more conserved than previous structures have indicated.


Assuntos
Nucleotidiltransferases/química , RNA Polimerase II/química , Regulação Alostérica , Animais , Sítios de Ligação , Domínio Catalítico , Ativação Enzimática , Humanos , Camundongos , Simulação de Dinâmica Molecular , Nucleotidiltransferases/metabolismo , Fosforilação , Fosfosserina/química , Fosfosserina/metabolismo , Phycodnaviridae/enzimologia , Ligação Proteica , Conformação Proteica , Domínios Proteicos , RNA Polimerase II/metabolismo
8.
J Mol Biol ; 433(8): 166875, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33556408

RESUMO

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association could regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, and could also hijack cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.


Assuntos
Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Escherichia coli , Humanos , Mutação , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , RNA Viral/metabolismo , Especificidade por Substrato
9.
Nat Commun ; 12(1): 1072, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594057

RESUMO

In addition to nucleosomes, chromatin contains non-histone chromatin-associated proteins, of which the high-mobility group proteins are the most abundant. Chromatin-mediated regulation of transcription involves DNA methylation and histone modifications. However, the order of events and the precise function of high-mobility group proteins during transcription initiation remain unclear. Here we show that high-mobility group AT-hook 2 protein (HMGA2) induces DNA nicks at the transcription start site, which are required by the histone chaperone FACT complex to incorporate nucleosomes containing the histone variant H2A.X. Further, phosphorylation of H2A.X at S139 (γ-H2AX) is required for repair-mediated DNA demethylation and transcription activation. The relevance of these findings is demonstrated within the context of TGFB1 signaling and idiopathic pulmonary fibrosis, suggesting therapies against this lethal disease. Our data support the concept that chromatin opening during transcriptional initiation involves intermediates with DNA breaks that subsequently require DNA repair mechanisms to ensure genome integrity.


Assuntos
Desmetilação do DNA , Nucleossomos/metabolismo , Iniciação da Transcrição Genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Cromatina/química , Cromatina/metabolismo , Células HEK293 , Proteína HMGA2/metabolismo , Histonas/metabolismo , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Camundongos , Fosforilação , Fosfosserina/metabolismo , RNA Polimerase II/metabolismo , Sítio de Iniciação de Transcrição , Ativação Transcricional/genética , Fator de Crescimento Transformador beta1/metabolismo
10.
Oxid Med Cell Longev ; 2021: 8836058, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33574981

RESUMO

A random-pattern skin flap plays an important role in the field of wound repair; the mechanisms that influence the survival of random-pattern skin flaps have been extensively studied but little attention has been paid to endogenous counterinjury substances and mechanism. Previous reports reveal that the apelin-APJ axis is an endogenous counterinjury mechanism that has considerable function in protecting against infection, inflammation, oxidative stress, necrosis, and apoptosis in various organs. As an in vivo study, our study proved that the apelin/APJ axis protected the skin flap by alleviating vascular oxidative stress and the apelin/APJ axis works as an antioxidant stress factor dependent on CaMKK/AMPK/GSK3ß signaling. In addition, the apelin/APJ-manipulated CaMKK/AMPK/GSK3ß-dependent mechanism improves HUVECs' resistance to oxygen and glucose deprivation/reperfusion (OGD/R), reduces ROS production and accumulation, maintained the normal mitochondrial membrane potential, and suppresses oxidative stress in vitro. Besides, activation of the apelin/APJ axis promotes vascular migration and angiogenesis under relative hypoxia condition through CaMKK/AMPK/GSK3ß signaling. In a word, we provide new evidence that the apelin/APJ axis is an effective antioxidant and can significantly improve the vitality of random flaps, so it has potential be a promising clinical treatment.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Apelina/farmacologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Transdução de Sinais , Pele/patologia , Cicatrização , Animais , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glucose , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fator 2 Relacionado a NF-E2/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Oxigênio , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Cicatrização/efeitos dos fármacos
11.
Mol Cancer ; 20(1): 16, 2021 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-33461590

RESUMO

BACKGROUND: The IκB kinase (IKK) complex, comprising the two enzymes IKKα and IKKß, is the main activator of the inflammatory transcription factor NF-κB, which is constitutively active in many cancers. While several connections between NF-κB signaling and the oncogene c-Myc have been shown, functional links between the signaling molecules are still poorly studied. METHODS: Molecular interactions were shown by co-immunoprecipitation and FRET microscopy. Phosphorylation of c-Myc was shown by kinases assays and its activity by improved reporter gene systems. CRISPR/Cas9-mediated gene knockout and chemical inhibition were used to block IKK activity. The turnover of c-Myc variants was determined by degradation in presence of cycloheximide and by optical pulse-chase experiments.. Immunofluorescence of mouse prostate tissue and bioinformatics of human datasets were applied to correlate IKKα- and c-Myc levels. Cell proliferation was assessed by EdU incorporation and apoptosis by flow cytometry. RESULTS: We show that IKKα and IKKß bind to c-Myc and phosphorylate it at serines 67/71 within a sequence that is highly conserved. Knockout of IKKα decreased c-Myc-activity and increased its T58-phosphorylation, the target site for GSK3ß, triggering polyubiquitination and degradation. c-Myc-mutants mimicking IKK-mediated S67/S71-phosphorylation exhibited slower turnover, higher cell proliferation and lower apoptosis, while the opposite was observed for non-phosphorylatable A67/A71-mutants. A significant positive correlation of c-Myc and IKKα levels was noticed in the prostate epithelium of mice and in a variety of human cancers. CONCLUSIONS: Our data imply that IKKα phosphorylates c-Myc on serines-67/71, thereby stabilizing it, leading to increased transcriptional activity, higher proliferation and decreased apoptosis.


Assuntos
Quinase I-kappa B/metabolismo , Inflamação/enzimologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Células HEK293 , Humanos , Quinase I-kappa B/química , Inflamação/patologia , Masculino , Camundongos , Modelos Biológicos , Mutação/genética , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Próstata/metabolismo , Ligação Proteica , Estabilidade Proteica , Transcrição Genética
12.
Biochem J ; 478(3): 553-578, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33459343

RESUMO

Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson's mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Fibroblastos , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/imunologia , Camundongos , Camundongos Knockout , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Fosfosserina/metabolismo , Proteínas Quinases/deficiência , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Organismos Livres de Patógenos Específicos , Acetato de Tetradecanoilforbol/farmacologia
13.
Nat Commun ; 12(1): 20, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397932

RESUMO

Drug resistance and tumor recurrence are major challenges in cancer treatment. Cancer cells often display centrosome amplification. To maintain survival, cancer cells achieve bipolar division by clustering supernumerary centrosomes. Targeting centrosome clustering is therefore considered a promising therapeutic strategy. However, the regulatory mechanisms of centrosome clustering remain unclear. Here we report that KIFC1, a centrosome clustering regulator, is positively associated with tumor recurrence. Under DNA damaging treatments, the ATM and ATR kinases phosphorylate KIFC1 at Ser26 to selectively maintain the survival of cancer cells with amplified centrosomes via centrosome clustering, leading to drug resistance and tumor recurrence. Inhibition of KIFC1 phosphorylation represses centrosome clustering and tumor recurrence. This study identified KIFC1 as a prognostic tumor recurrence marker, and revealed that tumors can acquire therapeutic resistance and recurrence via triggering centrosome clustering under DNA damage stresses, suggesting that blocking KIFC1 phosphorylation may open a new vista for cancer therapy.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Centrossomo/metabolismo , Cinesina/metabolismo , Recidiva Local de Neoplasia/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Instabilidade Cromossômica , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos , Humanos , Cinesina/química , Camundongos , Recidiva Local de Neoplasia/patologia , Fosforilação , Fosfosserina/metabolismo
14.
Arch Biochem Biophys ; 698: 108716, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33309545

RESUMO

The effects of phosphorylation of histone H3 at serine 10 have been studied in the context of other posttranslational modifications such as lysine methylation. We set out to investigate the impact of phosphoserine-10 on arginine-8 methylation. We performed methylation reactions using peptides based on histone H3 that contain a phosphorylated serine and compared the extent of arginine methylation with unmodified peptides. Results obtained via fluorography indicate that peptides containing a phosphorylated serine-10 inhibit deposition of methyl groups to arginine-8 residues. To further explore the effects of phosphoserine on neighboring arginine residues, we physically characterized the non-covalent interactions between histone H3 phosphoserine-10 and arginine-8 using 31P NMR spectroscopy. A salt bridge was detected between the negatively charged phosphoserine-10 and the positively charged unmodified arginine-8 residue. This salt bridge was not detected when arginine-8 was symmetrically dimethylated. Finally, molecular simulations not only confirm the presence of a salt bridge but also identify a subset of electrostatic interactions present when arginine is replaced with alanine. Taken together, our work suggests that the negatively charged phosphoserine maximizes its interactions. By limiting its exposure and creating new contacts with neighboring residues, it will inhibit deposition of neighboring methyl groups, not through steric hindrance, but by forming intrapeptide interactions that may mask substrate recognition. Our work provides a mechanistic framework for understanding the role of phosphoserine on nearby amino acid residues and arginine methylation.


Assuntos
Arginina/metabolismo , Histonas/metabolismo , Fosfosserina/metabolismo , Processamento de Proteína Pós-Traducional , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Arginina/química , Histonas/química , Humanos , Metilação , Simulação de Dinâmica Molecular , Fosfosserina/química , Eletricidade Estática , Xenopus laevis
15.
J Cell Biol ; 220(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33351100

RESUMO

Centrosome duplication occurs under strict spatiotemporal regulation once per cell cycle, and it begins with cartwheel assembly and daughter centriole biogenesis at the lateral sites of the mother centrioles. However, although much of this process is understood, how centrosome duplication is initiated remains unclear. Here, we show that cartwheel assembly followed by daughter centriole biogenesis is initiated on the NEDD1-containing layer of the pericentriolar material (PCM) by the recruitment of SAS-6 to the mother centriole under the regulation of PLK4. We found that PLK4-mediated phosphorylation of NEDD1 at its S325 amino acid residue directly promotes both NEDD1 binding to SAS-6 and recruiting SAS-6 to the centrosome. Overexpression of phosphomimicking NEDD1 mutant S325E promoted cartwheel assembly and daughter centriole biogenesis initiations, whereas overexpression of nonphosphorylatable NEDD1 mutant S325A abolished the initiations. Collectively, our results demonstrate that PLK4-regulated NEDD1 facilitates initiation of the cartwheel assembly and of daughter centriole biogenesis in mammals.


Assuntos
Centríolos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Centrossomo/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Tubulina (Proteína)/metabolismo
16.
Int J Mol Sci ; 21(23)2020 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-33256213

RESUMO

Numerous studies have shown that following retrieval, a previously consolidated memory requires increased transcriptional regulation in order to be reconsolidated. Previously, it was reported that histone H3 lysine-4 trimethylation (H3K4me3), a marker of active transcription, is increased in the hippocampus after the retrieval of contextual fear memory. However, it is currently unknown how this epigenetic mark is regulated during the reconsolidation process. Furthermore, though recent evidence suggests that neuronal activity triggers DNA double-strand breaks (DSBs) in some early-response genes, it is currently unknown if DSBs contribute to the reconsolidation of a memory following retrieval. Here, using chromatin immunoprecipitation (ChIP) analyses, we report a significant overlap between DSBs and H3K4me3 in area CA1 of the hippocampus during the reconsolidation process. We found an increase in phosphorylation of histone H2A.X at serine 139 (H2A.XpS139), a marker of DSB, in the Npas4, but not c-fos, promoter region 5 min after retrieval, which correlated with increased H3K4me3 levels, suggesting that the two epigenetic marks may work in concert during the reconsolidation process. Consistent with this, in vivo siRNA-mediated knockdown of topoisomerase II ß, the enzyme responsible for DSB, prior to retrieval, reduced Npas4 promoter-specific H2A.XpS139 and H3K4me3 levels and impaired long-term memory, indicating an indispensable role of DSBs in the memory reconsolidation process. Collectively, our data propose a novel mechanism for memory reconsolidation through increases in epigenetic-mediated transcriptional control via DNA double-strand breaks.


Assuntos
Quebras de DNA de Cadeia Dupla , Medo/fisiologia , Memória/fisiologia , Animais , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/metabolismo , Técnicas de Silenciamento de Genes , Hipocampo/metabolismo , Histonas , Masculino , Rememoração Mental , Fosfosserina/metabolismo , Ratos Sprague-Dawley
17.
Int J Mol Sci ; 22(1)2020 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-33379356

RESUMO

Hepatocellular carcinoma is one of the most common cancer types worldwide. In cases of advanced-stage disease, sorafenib is considered the treatment of choice. However, resistance to sorafenib remains a major obstacle for effective clinical application. Based on integrated phosphoproteomic and The Cancer Genome Atlas (TCGA) data, we identified a transcription factor, Y-box binding protein-1 (YB-1), with elevated phosphorylation of Ser102 in sorafenib-resistant HuH-7R cells. Phosphoinositide-3-kinase (PI3K) and protein kinase B (AKT) were activated by sorafenib, which, in turn, increased the phosphorylation level of YB-1. In functional analyses, knockdown of YB-1 led to decreased cell migration and invasion in vitro. At the molecular level, inhibition of YB-1 induced suppression of zinc-finger protein SNAI1 (Snail), twist-related protein 1 (Twist1), zinc-finger E-box-binding homeobox 1 (Zeb1), matrix metalloproteinase-2 (MMP-2) and vimentin levels, implying a role of YB-1 in the epithelial-mesenchymal transition (EMT) process in HuH-7R cells. Additionally, YB-1 contributes to morphological alterations resulting from F-actin rearrangement through Cdc42 activation. Mutation analyses revealed that phosphorylation at S102 affects the migratory and invasive potential of HuH-7R cells. Our collective findings suggest that sorafenib promotes YB-1 phosphorylation through effect from the EGFR/PI3K/AKT pathway, leading to significant enhancement of hepatocellular carcinoma (HCC) cell metastasis. Elucidation of the specific mechanisms of action of YB-1 may aid in the development of effective strategies to suppress metastasis and overcome resistance.


Assuntos
Carcinoma Hepatocelular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Neoplasias Hepáticas/metabolismo , Sorafenibe/farmacologia , Proteína 1 de Ligação a Y-Box/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Invasividade Neoplásica , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Prognóstico , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Proteína 1 de Ligação a Y-Box/genética , Proteína cdc42 de Ligação ao GTP/metabolismo
18.
Mol Cancer ; 19(1): 171, 2020 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-33308223

RESUMO

BACKGROUND: N6-methyladenosine (m6A) is the most prevalent messenger RNA modification in mammalian cells. However, the disease relevant function of m6A on specific oncogenic long non-coding RNAs (ncRNAs) is not well understood. METHODS: We analyzed the m6A status using patients samples and bone metastatic PDXs. Through m6A high-throughput sequencing, we identified the m6A sites on NEAT1-1 in prostate bone metastatic PDXs. Mass spec assay showed interaction among NEAT1-1, CYCLINL1 and CDK19. RNA EMSA, RNA pull-down, mutagenesis, CLIP, western blot, ChIP and ChIRP assays were used to investigate the molecular mechanisms underlying the functions of m6A on NEAT1-1. Loss-of function and rescued experiments were executed to detect the biological roles of m6A on NEAT1-1 in the PDX cell phenotypes in vivo. RESULTS: In this study, we identified 4 credible m6A sites on long ncRNA NEAT1-1. High m6A level of NEAT1-1 was related to bone metastasis of prostate cancer and m6A level of NEAT1-1 was a powerful predictor of eventual death. Transcribed NEAT1-1 served as a bridge to facility the binding between CYCLINL1 and CDK19 and promoted the Pol II ser2 phosphorylation. Importantly, depletion of NEAT1-1or decreased m6A of NEAT1-1 impaired Pol II Ser-2p level in the promoter of RUNX2. Overexpression of NEAT1-1 induced cancer cell metastasis to lung and bone; xenograft growth and shortened the survival of mice, but NEAT1-1 with m6A site mutation failed to do these. CONCLUSION: Collectively, the findings indicate that m6A on ncRNA NEAT1-1 takes critical role in regulating Pol II ser2 phosphorylation and may be novel specific target for bone metastasis cancer therapy and diagnosis. New complex CYCLINL1/CDK19/NEAT1-1 might provide new insight into the potential mechanism of the pathogenesis and development of bone metastatic prostate cancer.


Assuntos
Adenosina/análogos & derivados , Neoplasias Ósseas/genética , Neoplasias Ósseas/secundário , Neoplasias da Próstata/patologia , RNA Longo não Codificante/metabolismo , Adenosina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Humanos , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Fosforilação , Fosfosserina/metabolismo , Prognóstico , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/genética , RNA Polimerase II/metabolismo , RNA Longo não Codificante/genética
19.
Cells ; 9(11)2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33153033

RESUMO

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by decreased levels of the survival of motoneuron (SMN) protein. Post-translational mechanisms for regulation of its stability are still elusive. Thus, we aimed to identify regulatory phosphorylation sites that modulate function and stability. Our results show that SMN residues S290 and S292 are phosphorylated, of which SMN pS290 has a detrimental effect on protein stability and nuclear localization. Furthermore, we propose that phosphatase and tensin homolog (PTEN), a novel phosphatase for SMN, counteracts this effect. In light of recent advancements in SMA therapies, a significant need for additional approaches has become apparent. Our study demonstrates S290 as a novel molecular target site to increase the stability of SMN. Characterization of relevant kinases and phosphatases provides not only a new understanding of SMN function, but also constitutes a novel strategy for combinatorial therapeutic approaches to increase the level of SMN in SMA.


Assuntos
Aminoácidos/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/química , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosforilação , Fosfosserina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteólise , Relação Estrutura-Atividade
20.
Cells ; 9(11)2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33138150

RESUMO

The synucleinopathy underlying multiple system atrophy (MSA) is characterized by the presence of abundant amyloid inclusions containing fibrillar α-synuclein (α-syn) aggregates in the brains of the patients and is associated with an extensive neurodegeneration. In contrast to Parkinson's disease (PD) where the pathological α-syn aggregates are almost exclusively neuronal, the α-syn inclusions in MSA are principally observed in oligodendrocytes (OLs) where they form glial cytoplasmic inclusions (GCIs). This is intriguing because differentiated OLs express low levels of α-syn, yet pathogenic amyloid α-syn seeds require significant amounts of α-syn monomers to feed their fibrillar growth and to eventually cause the buildup of cytopathological inclusions. One of the transgenic mouse models of this disease is based on the targeted overexpression of human α-syn in OLs using the PLP promoter. In these mice, the histopathological images showing a rapid emergence of S129-phosphorylated α-syn inside OLs are considered as equivalent to GCIs. Instead, we report here that they correspond to the accumulation of phosphorylated α-syn monomers/oligomers and not to the appearance of the distinctive fibrillar α-syn aggregates that are present in the brains of MSA or PD patients. In spite of a propensity to co-sediment with myelin sheath contaminants, the phosphorylated forms found in the brains of the transgenic animals are soluble (>80%). In clear contrast, the phosphorylated species present in the brains of MSA and PD patients are insoluble fibrils (>95%). Using primary cultures of OLs from PLP-αSyn mice we observed a variable association of S129-phosphorylated α-syn with the cytoplasmic compartment, the nucleus and with membrane domains suggesting that OLs functionally accommodate the phospho-α-syn deriving from experimental overexpression. Yet and while not taking place spontaneously, fibrillization can be seeded in these primary cultures by challenging the OLs with α-syn preformed fibrils (PFFs). This indicates that a targeted overexpression of α-syn does not model GCIs in mice but that it can provide a basis for seeding aggregation using PFFs. This approach could help establishing a link between α-syn aggregation and the development of a clinical phenotype in these transgenic animals.


Assuntos
Atrofia de Múltiplos Sistemas/metabolismo , Atrofia de Múltiplos Sistemas/patologia , Oligodendroglia/metabolismo , Agregados Proteicos , alfa-Sinucleína/metabolismo , Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Células Cultivadas , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Proteína Básica da Mielina/metabolismo , Proteína Proteolipídica de Mielina/genética , Neurônios/metabolismo , Doença de Parkinson/patologia , Fosforilação , Fosfosserina/metabolismo , Regiões Promotoras Genéticas/genética , Multimerização Proteica
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