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1.
Blood ; 135(10): 713-723, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31951650

RESUMO

Chimeric antigen receptor (CAR) T cells have radically improved the treatment of B cell-derived malignancies by targeting CD19. The success has not yet expanded to treat acute myeloid leukemia (AML). We developed a Sequentially Tumor-Selected Antibody and Antigen Retrieval (STAR) system to rapidly isolate multiple nanobodies (Nbs) that preferentially bind AML cells and empower CAR T cells with anti-AML efficacy. STAR-isolated Nb157 specifically bound CD13, which is highly expressed in AML cells, and CD13 CAR T cells potently eliminated AML in vitro and in vivo. CAR T cells bispecific for CD13 and TIM3, which are upregulated in AML leukemia stem cells, eradicated patient-derived AML, with much reduced toxicity to human bone marrow stem cells and peripheral myeloid cells in mouse models, highlighting a promising approach for developing effective AML CAR T cell therapy.


Assuntos
Leucemia Mieloide Aguda , Receptores de Antígenos de Linfócitos T , Animais , Antígenos CD13 , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Imunoterapia Adotiva , Camundongos , Linfócitos T
2.
Nature ; 482(7386): 542-6, 2012 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-22327296

RESUMO

Menin is a tumour suppressor protein whose loss or inactivation causes multiple endocrine neoplasia 1 (MEN1), a hereditary autosomal dominant tumour syndrome that is characterized by tumorigenesis in multiple endocrine organs. Menin interacts with many proteins and is involved in a variety of cellular processes. Menin binds the JUN family transcription factor JUND and inhibits its transcriptional activity. Several MEN1 missense mutations disrupt the menin-JUND interaction, suggesting a correlation between the tumour-suppressor function of menin and its suppression of JUND-activated transcription. Menin also interacts with mixed lineage leukaemia protein 1 (MLL1), a histone H3 lysine 4 methyltransferase, and functions as an oncogenic cofactor to upregulate gene transcription and promote MLL1-fusion-protein-induced leukaemogenesis. A recent report on the tethering of MLL1 to chromatin binding factor lens epithelium-derived growth factor (LEDGF) by menin indicates that menin is a molecular adaptor coordinating the functions of multiple proteins. Despite its importance, how menin interacts with many distinct partners and regulates their functions remains poorly understood. Here we present the crystal structures of human menin in its free form and in complexes with MLL1 or with JUND, or with an MLL1-LEDGF heterodimer. These structures show that menin contains a deep pocket that binds short peptides of MLL1 or JUND in the same manner, but that it can have opposite effects on transcription. The menin-JUND interaction blocks JUN N-terminal kinase (JNK)-mediated JUND phosphorylation and suppresses JUND-induced transcription. In contrast, menin promotes gene transcription by binding the transcription activator MLL1 through the peptide pocket while still interacting with the chromatin-anchoring protein LEDGF at a distinct surface formed by both menin and MLL1.


Assuntos
Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Transcrição Gênica , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cromatina/metabolismo , Cristalografia por Raios X , Fibroblastos , Células HEK293 , Histona-Lisina N-Metiltransferase , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide/química , Fosforilação , Ligação Proteica , Multimerização Proteica , Proteínas Proto-Oncogênicas c-jun/química , Relação Estrutura-Atividade
3.
J Biol Chem ; 289(14): 9902-8, 2014 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-24563463

RESUMO

Multiple endocrine neoplasia type I (MEN1) is an inherited syndrome that includes susceptibility to pancreatic islet hyperplasia. This syndrome results from mutations in the MEN1 gene, which encodes menin protein. Menin interacts with several transcription factors, including JunD, and inhibits their activities. However, the precise mechanism by which menin suppresses gene expression is not well understood. Here, we show that menin interacts with arsenite-resistant protein 2 (ARS2), a component of the nuclear RNA CAP-binding complex that is crucial for biogenesis of certain miRNAs including let-7a. The levels of primary-let-7a (pri-let-7a) are not affected by menin; however, the levels of mature let-7a are substantially decreased upon Men1 excision. Let-7a targets, including Insr and Irs2, pro-proliferative genes that are crucial for insulin-mediated signaling, are up-regulated in Men1-excised cells. Inhibition of let-7a using anti-miRNA in wild type cells is sufficient to enhance the expression of insulin receptor substrate 2 (IRS2) to levels observed in Men1-excised cells. Depletion of menin does not affect the expression of Drosha and CBP80, but substantially impairs the processing of pri-miRNA to pre-miRNA. Ars2 knockdown decreased let-7a processing in menin-expressing cells but had little impact on let-7a levels in menin-excised cells. As IRS2 is known to mediate insulin signaling and insulin/mitogen-induced cell proliferation, these findings collectively unravel a novel mechanism whereby menin suppresses cell proliferation, at least partly by promoting the processing of certain miRNAs, including let-7a, leading to suppression of Irs2 expression and insulin signaling.


Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/biossíntese , Neoplasia Endócrina Múltipla Tipo 1/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Processamento Pós-Transcricional do RNA , RNA Neoplásico/biossíntese , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Camundongos , MicroRNAs/genética , Neoplasia Endócrina Múltipla Tipo 1/genética , Neoplasia Endócrina Múltipla Tipo 1/patologia , Proteínas de Neoplasias/genética , Complexo Proteico Nuclear de Ligação ao Cap/genética , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA Neoplásico/genética , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Ribonuclease III/biossíntese , Ribonuclease III/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Proc Natl Acad Sci U S A ; 107(47): 20358-63, 2010 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-21059956

RESUMO

A hallmark of diabetes is an absolute or relative reduction in the number of functional ß cells. Therapies that could increase the number of endogenous ß cells under diabetic conditions would be desirable. Prevalent gene targeting mouse models for assessing ß-cell proliferation and diabetes pathogenesis only address whether deletion of a gene prevents the development of diabetes. Models testing whether acute excision of a single gene can ameliorate or reverse preexisting hyperglycemia in established diabetes remain to be explored, which could directly validate the effect of gene excision on treating diabetes. Here, we report that acute and temporally controlled excision of the Men1 gene, which encodes menin, ameliorated preexisting hyperglycemia in streptozotocin-treated mice. Moreover, Men1 excision also improved the preexisting hyperglycemia and glucose intolerance in genetic db/db diabetic mice. Furthermore, acute Men1 excision reversed preexisting glucose intolerance in high-fat diet-fed mice. Men1 excision improved glucose metabolism at least partly through increasing proliferation of endogenous ß cells and islet size. Acute Men1 excision up-regulated a group of proproliferative genes in pancreatic islets. Together, these findings demonstrate that established hyperglycemia can be reversed through repression of a single gene, Men1, in diabetic conditions, and suggest that menin is a vital regulator in pathogenesis of diabetes.


Assuntos
Diabetes Mellitus Experimental , Deleção de Genes , Regulação da Expressão Gênica/fisiologia , Hiperglicemia/genética , Células Secretoras de Insulina/citologia , Proteínas Proto-Oncogênicas/genética , Animais , Western Blotting , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Teste de Tolerância a Glucose , Imuno-Histoquímica , Insulina/sangue , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
J Endocrinol ; 244(1): 41-52, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31539871

RESUMO

Protein arginine methyltransferase 5 (PRMT5), a symmetric arginine methyltransferase, regulates cell functions by influencing gene transcription through posttranslational modification of histones and non-histone proteins. PRMT5 interacts with multiple partners including menin, which controls beta cell homeostasis. However, the role of Prmt5 in pancreatic islets, particularly in beta cells, remains unclear. A mouse model with an islet-specific knockout (KO) of the Prmt5 gene was generated, and the influence of the Prmt5 excision on beta cells was investigated via morphologic and functional studies. Beta cell function was evaluated by glucose tolerance test (GTT) and glucose-stimulated insulin secretion (GSIS) test. Beta cell proliferation was evaluated by immunostaining. Gene expression change was determined by real-time qPCR. Molecular mechanisms were investigated in beta cells in vitro and in vivo in Prmt5 KO mice. The results show that islet-specific KO of Prmt5 reduced expression of the insulin gene and impaired glucose tolerance and GSIS in vivo. The mechanistic study indicated that PRMT5 is involved in the regulation of insulin gene transcription, likely via histone methylation-related chromatin remodeling. The reduced expression of insulin in beta cells in the Prmt5 KO mice may contribute to impaired glucose tolerance (IGT) and deficient GSIS in the mouse model. These results will provide new insights into exploring novel strategies to treat diabetes caused by insulin insufficiency.


Assuntos
Intolerância à Glucose/genética , Secreção de Insulina/genética , Ilhotas Pancreáticas/metabolismo , Proteína-Arginina N-Metiltransferases/deficiência , Animais , Proliferação de Células/genética , Teste de Tolerância a Glucose , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Knockout
6.
Cancer Biol Ther ; 16(2): 219-24, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25482929

RESUMO

Pancreatic neuroendocrine tumors (PNETs) are rare, indolent tumors that may occur sporadically or develop in association with well-recognized hereditary syndromes, particularly multiple endocrine neoplasia type 1 (MEN-1). We previously demonstrated that the hedgehog (HH) signaling pathway was aberrantly up-regulated in a mouse model that phenocopies the human MEN-1 syndrome, Men1l/l;RipCre, and that inhibition of this pathway suppresses MEN-1 tumor cell proliferation. We hypothesized that the HH signaling pathway is similarly upregulated in human PNETs. We performed immunohistochemical (IHC) staining for PTCH1 in human fresh and archival PNET specimens to examine whether human sporadic and MEN-1-associated PNETs revealed similar abnormalities as in our mouse model and correlated the results with clinical and demographic factors of the study cohort. PTCH1 staining was positive in 12 of 22 PNET patients (55%). Four of 5 MEN-1 patients stained for PTCH1 (p = 0.32 as compared with sporadic disease patients). Nine of 16 patients with metastatic disease stained for PTCH1 as compared with zero of 3 with localized disease only (p = 0.21). No demographic or clinical features appeared to be predictive of PTCH 1 positivity and PTCH 1 positivity per se was not predictive of clinical outcome. PTCH1, a marker of HH pathway up regulation, is detectable in both primary and metastatic tumors in more than 50% of PNET patients. Although no clinical or demographic factors predict PTCH1 positivity and PTCH1 positivity does not predict clinical outcome, the frequency of expression alone indicates that perturbation of this pathway with agents such as Vismodegib, an inhibitor of Smoothened (SMO), should be examined in future clinical trials.


Assuntos
Neoplasia Endócrina Múltipla/metabolismo , Tumores Neuroendócrinos/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores de Superfície Celular/metabolismo , Adulto , Estudos de Coortes , Terapia Combinada , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasia Endócrina Múltipla/diagnóstico , Neoplasia Endócrina Múltipla/terapia , Gradação de Tumores , Metástase Neoplásica , Tumores Neuroendócrinos/diagnóstico , Tumores Neuroendócrinos/terapia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/terapia , Receptores Patched , Receptor Patched-1 , Resultado do Tratamento , Carga Tumoral
7.
Cancer Cell ; 28(4): 472-485, 2015 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-26461093

RESUMO

Human epidermal growth factor receptor 2 (HER2) is upregulated in a subset of human breast cancers. However, the cancer cells often quickly develop an adaptive response to HER2 kinase inhibitors. We found that an epigenetic pathway involving MLL2 is crucial for growth of HER2(+) cells and MLL2 reduces sensitivity of the cancer cells to a HER2 inhibitor, lapatinib. Lapatinib-induced FOXO transcription factors, normally tumor-suppressing, paradoxically upregulate c-Myc epigenetically in concert with a cascade of MLL2-associating epigenetic regulators to dampen sensitivity of the cancer cells to lapatinib. An epigenetic inhibitor suppressing c-Myc synergizes with lapatinib to suppress cancer growth in vivo, partly by repressing the FOXO/c-Myc axis, unraveling an epigenetically regulated FOXO/c-Myc axis as a potential target to improve therapy.


Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética , Fatores de Transcrição Forkhead/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-myc/genética , Receptor ErbB-2/antagonistas & inibidores , Animais , Benzodiazepinas/administração & dosagem , Benzodiazepinas/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Epigênese Genética/efeitos dos fármacos , Feminino , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lapatinib , Camundongos , Proteínas de Neoplasias/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Quinazolinas/administração & dosagem , Quinazolinas/farmacologia , Receptor ErbB-2/genética , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Oncoscience ; 1(9): 562-6, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25594065

RESUMO

Menin, a protein encoded by the MEN1 gene, is mutated in patients with multiple endocrine neoplasia type 1 (MEN1). Menin acts as a tumor suppressor in endocrine organs while it is also required for transformation of a subgroup of leukemia. The recently solved crystal structure of menin with different binding partners reveals that menin is a key scaffold protein that cross-talks with various partners, including transcription factors, to regulate gene transcription. Our recent findings unravel a previously undiscovered mechanism for menin-mediated control of gene expression via processing of certain microRNA's, thus adding to the plethora of ways in which menin regulates gene expression. By interacting with ARS2, an RNA binding protein, menin facilitates the processing of pri-let 7a and pri-miR155 to pre-let 7a and pre-miR155 respectively. Consistently, excision of the Men1 gene results in upregulation of IRS2, a let-7a target. As IRS2 is known to mediate both insulin signaling and insulin-induced cell proliferation, and let-7a targets include oncogenes like RAS and HMGA2, a deeper understanding of the menin-ARS2 complex in regulating miRNA biogenesis will yield further insights into the pathogenesis of the MEN1 syndrome and other menin-associated malignancies.

9.
Mol Cancer Res ; 11(10): 1215-22, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23928057

RESUMO

UNLABELLED: Multiple endocrine neoplasia type 1 (MEN-1), is a familial tumor syndrome resulting from mutations in the tumor suppressor gene menin (MEN1). Menin plays an essential role in both repressing and activating gene expression. However, it is not well understood how menin represses expression of multiple genes. Upon MEN1 excision, the transcription factor Gli1 and its target genes, including Ptch1 and c-Myc, were shown to be elevated in the absence of an apparent Hedgehog) pathway-activating ligand or when Smoothened (SMO), a key component of the pathway, is inhibited. Menin binds to the GLI1 promoter and recruits PRMT5, a histone arginine methyltransferase associated with transcriptional repression. Both PRMT5 binding and histone H4 arginine 3 methylation (H4R3m2s) are decreased at the GLI1 promoter in MEN1-excised cells. Moreover, MEN1 ablation resulted in increased binding of transcriptionally active Gli1 at the GLI1 promoter in a manner not influenced by the canonical Hedgehog signaling pathway. Inhibition of Gli1 by the small-molecule inhibitor GANT-61 led to decreased expression of Gli1 and its target genes in MEN1-depeleted cells. Furthermore, GANT-61 potently suppressed proliferation of MEN1-excised cells as compared with control cells. These findings uncover a novel epigenetic link whereby menin directly represses Gli1 expression, independent of the canonical Hedgehog signaling pathway, via PRMT5 and its repressive H4R3m2s mark. IMPLICATIONS: Inhibition of GLI1 suppresses neuroendocrine tumors harboring mutations in the MEN1 gene.


Assuntos
Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/genética , Transativadores/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Epigênese Genética , Epigenômica , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Metilação , Camundongos , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Metiltransferases/genética , Proteínas Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases , Piridinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteína GLI1 em Dedos de Zinco
10.
Am J Cancer Res ; 3(1): 96-106, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23359867

RESUMO

Menin acts as contextual a tumor suppressor and a tumor promoter, partly via epigenetic regulation of gene transcription. While menin is phosphorylated, it remains unclear whether wild type menin has other post-translational modifications. Here, we report that menin is SUMOylated by SUMO1 in vivo and in vitro, and the SUMOylation is reduced by a SUMO protease. Lysine 591 of menin was covalently modified by SUMO1 and K591R mutation in menin blocked SUMOylation of the C-terminal part of menin in transfected cells. Full-length menin with K591 mutation was still SUMOylated in vivo, suggesting the existence of multiple SUMOylation sites. Menin K591R mutant or menin-SUMO fusion protein still retains the ability to regulate cell proliferation and the expression of the examined menin target genes.

11.
Cancer Res ; 73(8): 2650-8, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23580576

RESUMO

Multiple endocrine neoplasia type 1 (MEN1) is an inherited tumor syndrome that includes susceptibility to pancreatic islet tumors. This syndrome results from mutations in the MEN1 gene, encoding menin. Although menin acts as an oncogenic cofactor for mixed lineage leukemia (MLL) fusion protein-mediated histone H3 lysine 4 methylation, the precise basis for how menin suppresses gene expression and proliferation of pancreatic beta cells remains poorly understood. Here, we show that menin ablation enhances Hedgehog signaling, a proproliferative and oncogenic pathway, in murine pancreatic islets. Menin directly interacts with protein arginine methyltransferase 5 (PRMT5), a negative regulator of gene transcription. Menin recruits PRMT5 to the promoter of the Gas1 gene, a crucial factor for binding of Sonic Hedgehog (Shh) ligand to its receptor PTCH1 and subsequent activation of the Hedgehog signaling pathway, increases repressive histone arginine symmetric dimethylation (H4R3m2s), and suppresses Gas1 expression. Notably, MEN1 disease-related menin mutants have reduced binding to PRMT5, and fail to impart the repressive H4R3m2s mark at the Gas1 promoter, resulting in its elevated expression. Pharmacologic inhibition of Hedgehog signaling significantly reduces proliferation of insulinoma cells, and expression of Hedgehog signaling targets including Ptch1, in MEN1 tumors of mice. These findings uncover a novel link between menin and Hedgehog signaling whereby menin/PRMT5 epigenetically suppresses Hedgehog signaling, revealing it as a target for treating MEN1 tumors.


Assuntos
Epigênese Genética , Proteínas Hedgehog/metabolismo , Neoplasia Endócrina Múltipla Tipo 1/genética , Neoplasia Endócrina Múltipla Tipo 1/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Anilidas/farmacologia , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Metilação , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Piridinas/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened
12.
Cancer Cell ; 18(4): 329-40, 2010 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-20951943

RESUMO

Cyclin D1 elicits transcriptional effects through inactivation of the retinoblastoma protein and direct association with transcriptional regulators. The current work reveals a molecular relationship between cyclin D1/CDK4 kinase and protein arginine methyltransferase 5 (PRMT5), an enzyme associated with histone methylation and transcriptional repression. Primary tumors of a mouse lymphoma model exhibit increased PRMT5 methyltransferase activity and histone arginine methylation. Analyses demonstrate that MEP50, a PRMT5 coregulatory factor, is a CDK4 substrate, and phosphorylation increases PRMT5/MEP50 activity. Increased PRMT5 activity mediates key events associated with cyclin D1-dependent neoplastic growth, including CUL4 repression, CDT1 overexpression, and DNA rereplication. Importantly, human cancers harboring mutations in Fbx4, the cyclin D1 E3 ligase, exhibit nuclear cyclin D1 accumulation and increased PRMT5 activity.


Assuntos
Núcleo Celular/enzimologia , Proteínas Culina/metabolismo , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Neoplasias/enzimologia , Neoplasias/patologia , Proteínas Metiltransferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Proteínas Culina/genética , Metilação de DNA , Replicação do DNA , Ativação Enzimática , Proteínas F-Box/metabolismo , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Linfoma/enzimologia , Linfoma/genética , Linfoma/patologia , Camundongos , Neoplasias/genética , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estabilidade Proteica
14.
J Biol Chem ; 283(42): 28087-94, 2008 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-18701458

RESUMO

Stigmatellin, a Q(P) site inhibitor, inhibits electron transfer from iron-sulfur protein (ISP) to cytochrome c1 in the bc1 complex. Stigmatellin raises the midpoint potential of ISP from 290 mV to 540 mV. The binding of stigmatellin to the fully oxidized complex, oxidized completely by catalytic amounts of cytochrome c oxidase and cytochrome c, results in ISP reduction. The extent of ISP reduction is proportional to the amount of inhibitor used and reaches a maximum when the ratio of inhibitor to enzyme complex reaches unity. A g = 2.005 EPR peak, characteristic of an organic free radical, is also observed when stigmatellin is added to the oxidized complex, and its signal intensity depends on the amount of stigmatellin. Addition of ferricyanide, a strong oxidant, to the oxidized complex also generates a g = 2.005 EPR peak that is oxidant concentration-dependent. Oxygen radicals are generated when stigmatellin is added to the oxidized complex in the absence of the exogenous substrate, ubiquinol. The amount of oxygen radical formed is proportional to the amount of stigmatellin added. Oxygen radicals are not generated when stigmatellin is added to a mutant bc1 complex lacking the Rieske iron-sulfur cluster. Based on these results, it is proposed that ISP becomes a strong oxidant upon stigmatellin binding, extracting electrons from an organic compound, likely an amino acid residue. This results in the reduction of ISP and generation of organic radicals.


Assuntos
Antibacterianos/farmacologia , Complexo III da Cadeia de Transporte de Elétrons/química , Regulação da Expressão Gênica , Proteínas Ferro-Enxofre/metabolismo , Oxigênio/química , Animais , Cianobactérias/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Elétrons , Ferricianetos/farmacologia , Cavalos , Modelos Biológicos , Oxidantes/química , Polienos/farmacologia , Superóxidos/química
15.
J Biol Chem ; 280(26): 24895-902, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15878858

RESUMO

The destruction of the Rieske iron-sulfur cluster ([2Fe-2S]) in the bc(1) complex by hematoporphyrin-promoted photoinactivation resulted in the complex becoming proton-permeable. To study further the role of this [2Fe-2S] cluster in proton translocation of the bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with mutations at the histidine ligands of the [2Fe-2S] cluster were generated and characterized. These mutants lacked the [2Fe-2S] cluster and possessed no bc(1) activity. When the mutant complex was co-inlaid in phospholipid vesicles with intact bovine mitochondrial bc(1) complex or cytochrome c oxidase, the proton ejection, normally observed in intact reductase or oxidase vesicles during the oxidation of their corresponding substrates, disappeared. This indicated the creation of a proton-leaking channel in the mutant complex, whose [2Fe-2S] cluster was lacking. Insertion of the bc(1) complex lacking the head domain of the Rieske iron-sulfur protein, removed by thermolysin digestion, into PL vesicles together with mitochondrial bc(1) complex also rendered the vesicles proton-permeable. Addition of the excess purified head domain of the Rieske iron-sulfur protein partially restored the proton-pumping activity. These results indicated that elimination of the [2Fe-2S] cluster in mutant bc(1) complexes opened up an otherwise closed proton channel within the bc(1) complex. It was speculated that in the normal catalytic cycle of the bc(1) complex, the [2Fe-2S] cluster may function as a proton-exiting gate.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/química , Proteínas Ferro-Enxofre/química , Animais , Transporte Biológico , Fenômenos Biofísicos , Biofísica , Western Blotting , Catálise , Bovinos , Dicroísmo Circular , Dimerização , Espectroscopia de Ressonância de Spin Eletrônica , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Elétrons , Eletroforese em Gel de Poliacrilamida , Escherichia/metabolismo , Histidina/química , Ligantes , Luz , Mitocôndrias/metabolismo , Modelos Moleculares , Mutação , Oxigênio/química , Fosfolipídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Prótons , Rhodobacter sphaeroides/metabolismo , Espectrofotometria , Fatores de Tempo
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