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1.
Cell ; 178(5): 1205-1221.e17, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31442408

RESUMO

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.


Assuntos
Dipeptidases/metabolismo , Neutrófilos/fisiologia , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Animais , Cilastatina/farmacologia , Cilastatina/uso terapêutico , Dipeptidases/antagonistas & inibidores , Dipeptidases/genética , Modelos Animais de Doenças , Endotoxemia/mortalidade , Endotoxemia/patologia , Endotoxemia/prevenção & controle , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Infiltração de Neutrófilos/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Taxa de Sobrevida
2.
Proc Natl Acad Sci U S A ; 115(32): 8161-8166, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30038027

RESUMO

Copper is an essential cofactor of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Inherited loss-of-function mutations in several genes encoding proteins required for copper delivery to CcO result in diminished CcO activity and severe pathologic conditions in affected infants. Copper supplementation restores CcO function in patient cells with mutations in two of these genes, COA6 and SCO2, suggesting a potential therapeutic approach. However, direct copper supplementation has not been therapeutically effective in human patients, underscoring the need to identify highly efficient copper transporting pharmacological agents. By using a candidate-based approach, we identified an investigational anticancer drug, elesclomol (ES), that rescues respiratory defects of COA6-deficient yeast cells by increasing mitochondrial copper content and restoring CcO activity. ES also rescues respiratory defects in other yeast mutants of copper metabolism, suggesting a broader applicability. Low nanomolar concentrations of ES reinstate copper-containing subunits of CcO in a zebrafish model of copper deficiency and in a series of copper-deficient mammalian cells, including those derived from a patient with SCO2 mutations. These findings reveal that ES can restore intracellular copper homeostasis by mimicking the function of missing transporters and chaperones of copper, and may have potential in treating human disorders of copper metabolism.


Assuntos
Antineoplásicos/farmacologia , Cobre/deficiência , Drogas em Investigação/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hidrazinas/farmacologia , Mitocôndrias/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Transporte Biológico/genética , Proteínas de Transporte/genética , Linhagem Celular , Coenzimas/deficiência , Cobre/uso terapêutico , Transportador de Cobre 1 , Suplementos Nutricionais , Modelos Animais de Doenças , Reposicionamento de Medicamentos , Drogas em Investigação/uso terapêutico , Fibroblastos , Humanos , Hidrazinas/uso terapêutico , Proteínas de Membrana Transportadoras/genética , Erros Inatos do Metabolismo/tratamento farmacológico , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Chaperonas Moleculares , Mutagênese Sítio-Dirigida , Mutação , Ratos , Saccharomyces cerevisiae , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
3.
Nucleic Acids Res ; 43(21): 10338-52, 2015 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-26519465

RESUMO

DNA polymerase gamma (POLG) is essential for replication and repair of mitochondrial DNA (mtDNA). Mutations in POLG cause mtDNA instability and a diverse range of poorly understood human diseases. Here, we created a unique Polg animal model, by modifying polg within the critical and highly conserved polymerase domain in zebrafish. polg(+/-) offspring were indistinguishable from WT siblings in multiple phenotypic and biochemical measures. However, polg(-/-) mutants developed severe mtDNA depletion by one week post-fertilization (wpf), developed slowly and had regenerative defects, yet surprisingly survived up to 4 wpf. An in vivo mtDNA polymerase activity assay utilizing ethidium bromide (EtBr) to deplete mtDNA, showed that polg(+/-) and WT zebrafish fully recover mtDNA content two weeks post-EtBr removal. EtBr further reduced already low levels of mtDNA in polg(-/-) animals, but mtDNA content did not recover following release from EtBr. Despite significantly decreased respiration that corresponded with tissue-specific levels of mtDNA, polg(-/-) animals had WT levels of ATP and no increase in lactate. This zebrafish model of mitochondrial disease now provides unique opportunities for studying mtDNA instability from multiple angles, as polg(-/-) mutants can survive to juvenile stage, rather than lose viability in embryogenesis as seen in Polg mutant mice.


Assuntos
DNA Mitocondrial/análise , DNA Polimerase Dirigida por DNA/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Trifosfato de Adenosina/metabolismo , Nadadeiras de Animais/fisiologia , Animais , DNA Polimerase gama , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Engenharia Genética , Glicólise , Modelos Animais , Mutação , Consumo de Oxigênio , Regeneração , Análise de Sobrevida , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo
4.
Hum Mol Genet ; 23(13): 3596-606, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24549041

RESUMO

Mitochondrial respiratory chain biogenesis is orchestrated by hundreds of assembly factors, many of which are yet to be discovered. Using an integrative approach based on clues from evolutionary history, protein localization and human genetics, we have identified a conserved mitochondrial protein, C1orf31/COA6, and shown its requirement for respiratory complex IV biogenesis in yeast, zebrafish and human cells. A recent next-generation sequencing study reported potential pathogenic mutations within the evolutionarily conserved Cx9CxnCx10C motif of COA6, implicating it in mitochondrial disease biology. Using yeast coa6Δ cells, we show that conserved residues in the motif, including the residue mutated in a patient with mitochondrial disease, are essential for COA6 function, thus confirming the pathogenicity of the patient mutation. Furthermore, we show that zebrafish embryos with zfcoa6 knockdown display reduced heart rate and cardiac developmental defects, recapitulating the observed pathology in the human mitochondrial disease patient who died of neonatal hypertrophic cardiomyopathy. The specific requirement of Coa6 for respiratory complex IV biogenesis, its intramitochondrial localization and the presence of the Cx9CxnCx10C motif suggested a role in mitochondrial copper metabolism. In support of this, we show that exogenous copper supplementation completely rescues respiratory and complex IV assembly defects in yeast coa6Δ cells. Taken together, our results establish an evolutionarily conserved role of Coa6 in complex IV assembly and support a causal role of the COA6 mutation in the human mitochondrial disease patient.


Assuntos
Cobre/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Humanos , Mutação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Pele/citologia , Peixe-Zebra
5.
Differentiation ; 89(3-4): 51-69, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25771346

RESUMO

The etiology of mitochondrial disease is poorly understood. Furthermore, treatment options are limited, and diagnostic methods often lack the sensitivity to detect disease in its early stages. Disrupted oxidative phosphorylation (OXPHOS) that inhibits ATP production is a common phenotype of mitochondrial disorders that can be induced in zebrafish by exposure to 2,4-dinitrophenol (DNP), a FDA-banned weight-loss agent and EPA-regulated environmental toxicant, traditionally used in research labs as an uncoupler of OXPHOS. Despite the DNP-induced OXPHOS inhibition we observed using in vivo respirometry, the development of the DNP-treated and control zebrafish were largely similar during the first half of embryogenesis. During this period, DNP-treated embryos induced gene expression of mitochondrial and nuclear genes that stimulated the production of new mitochondria and increased glycolysis to yield normal levels of ATP. DNP-treated embryos were incapable of sustaining this mitochondrial biogenic response past mid-embryogenesis, as shown by significantly lowered ATP production and ATP levels, decreased gene expression, and the onset of developmental defects. Examining neural tissues commonly affected by mitochondrial disease, we found that DNP exposure also inhibited motor neuron axon arbor outgrowth and the proper formation of the retina. We observed and quantified the molecular and physiological progression of mitochondrial dysfunction during development with this new model of OXPHOS dysfunction, which has great potential for use in diagnostics and therapies for mitochondrial disease.


Assuntos
Desenvolvimento Embrionário/genética , Metabolismo Energético/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , 2,4-Dinitrofenol/toxicidade , Trifosfato de Adenosina/biossíntese , Animais , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/induzido quimicamente , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Retina/metabolismo , Retina/patologia , Peixe-Zebra
6.
Kidney Int ; 88(6): 1336-1344, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26287315

RESUMO

Recent studies show the importance of mitochondrial dysfunction in the initiation and progression of acute kidney injury (AKI). However, no biomarkers exist linking renal injury to mitochondrial function and integrity. To this end, we evaluated urinary mitochondrial DNA (UmtDNA) as a biomarker of renal injury and function in humans with AKI following cardiac surgery. mtDNA was isolated from the urine of patients following cardiac surgery and quantified by quantitative PCR. Patients were stratified into no AKI, stable AKI, and progressive AKI groups based on Acute Kidney Injury Network (AKIN) staging. UmtDNA was elevated in progressive AKI patients and was associated with progression of patients with AKI at collection to higher AKIN stages. To evaluate the relationship of UmtDNA to measures of renal mitochondrial integrity in AKI, mice were subjected to sham surgery or varying degrees of ischemia followed by 24 h of reperfusion. UmtDNA increased in mice after 10-15 min of ischemia and positively correlated with ischemia time. Furthermore, UmtDNA was predictive of AKI in the mouse model. Finally, UmtDNA levels were negatively correlated with renal cortical mtDNA and mitochondrial gene expression. These translational studies demonstrate that UmtDNA is associated with recovery from AKI following cardiac surgery by serving as an indicator of mitochondrial integrity. Thus UmtDNA may serve as valuable biomarker for the development of mitochondrial-targeted therapies in AKI.

7.
Sci Adv ; 8(5): eabm0142, 2022 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-35108057

RESUMO

The mechanisms that drive leukocyte recruitment to the kidney are incompletely understood. Dipeptidase-1 (DPEP1) is a major neutrophil adhesion receptor highly expressed on proximal tubular cells and peritubular capillaries of the kidney. Renal ischemia reperfusion injury (IRI) induces robust neutrophil and monocyte recruitment and causes acute kidney injury (AKI). Renal inflammation and the AKI phenotype were attenuated in Dpep1-/- mice or mice pretreated with DPEP1 antagonists, including the LSALT peptide, a nonenzymatic DPEP1 inhibitor. DPEP1 deficiency or inhibition primarily blocked neutrophil adhesion to peritubular capillaries and reduced inflammatory monocyte recruitment to the kidney after IRI. CD44 but not ICAM-1 blockade also decreased neutrophil recruitment to the kidney during IRI and was additive to DPEP1 effects. DPEP1, CD44, and ICAM-1 all contributed to the recruitment of monocyte/macrophages to the kidney following IRI. These results identify DPEP1 as a major leukocyte adhesion receptor in the kidney and potential therapeutic target for AKI.


Assuntos
Injúria Renal Aguda , Dipeptidases/metabolismo , Traumatismo por Reperfusão , Injúria Renal Aguda/etiologia , Animais , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Inflamação/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL
8.
PLoS Biol ; 6(11): e289, 2008 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-19067488

RESUMO

The multifunctional signaling protein p75 neurotrophin receptor (p75(NTR)) is a central regulator and major contributor to the highly invasive nature of malignant gliomas. Here, we show that neurotrophin-dependent regulated intramembrane proteolysis (RIP) of p75(NTR) is required for p75(NTR)-mediated glioma invasion, and identify a previously unnamed process for targeted glioma therapy. Expression of cleavage-resistant chimeras of p75(NTR) or treatment of animals bearing p75(NTR)-positive intracranial tumors with clinically applicable gamma-secretase inhibitors resulted in dramatically decreased glioma invasion and prolonged survival. Importantly, proteolytic processing of p75(NTR) was observed in p75(NTR)-positive patient tumor specimens and brain tumor initiating cells. This work highlights the importance of p75(NTR) as a therapeutic target, suggesting that gamma-secretase inhibitors may have direct clinical application for the treatment of malignant glioma.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Neoplasias Encefálicas/metabolismo , Inibidores Enzimáticos/uso terapêutico , Glioma/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Encéfalo/metabolismo , Neoplasias Encefálicas/terapia , Glioma/terapia , Humanos , Invasividade Neoplásica/fisiopatologia , Fatores de Crescimento Neural/metabolismo , Proteínas Recombinantes de Fusão
9.
PLoS Biol ; 5(8): e212, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17696644

RESUMO

The invasive nature of cancers in general, and malignant gliomas in particular, is a major clinical problem rendering tumors incurable by conventional therapies. Using a novel invasive glioma mouse model established by serial in vivo selection, we identified the p75 neurotrophin receptor (p75(NTR)) as a critical regulator of glioma invasion. Through a series of functional, biochemical, and clinical studies, we found that p75(NTR) dramatically enhanced migration and invasion of genetically distinct glioma and frequently exhibited robust expression in highly invasive glioblastoma patient specimens. Moreover, we found that p75(NTR)-mediated invasion was neurotrophin dependent, resulting in the activation of downstream pathways and producing striking cytoskeletal changes of the invading cells. These results provide the first evidence for p75(NTR) as a major contributor to the highly invasive nature of malignant gliomas and identify a novel therapeutic target.


Assuntos
Glioma , Receptor de Fator de Crescimento Neural/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Feminino , Perfilação da Expressão Gênica , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Camundongos SCID , Invasividade Neoplásica , Transplante de Neoplasias , Fatores de Crescimento Neural/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Receptor de Fator de Crescimento Neural/genética , Proteína rhoA de Ligação ao GTP/metabolismo
10.
Biomaterials ; 252: 120105, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32417652

RESUMO

Despite extensive molecular characterization, human glioblastoma remains a fatal disease with survival rates measured in months. Little improvement is seen with standard surgery, radiotherapy and chemotherapy. Clinical progress is hampered by the inability to detect and target glioblastoma disease reservoirs based on a diffuse invasive pattern and the presence of molecular and phenotypic heterogeneity. The goal of this study was to target the invasive and stem-like glioblastoma cells that evade first-line treatments using agents capable of delivering imaging enhancers or biotherapeutic cargo. To accomplish this, a combinatorial phage display library was biopanned against glioblastoma cell model systems that accurately recapitulate the intra- and inter-tumor heterogeneity and infiltrative nature of the disease. Candidate peptides were screened for specificity and ability to target glioblastoma cells in vivo. Cargo-conjugated peptides delivered contrast-enhancing agents to highly infiltrative tumor populations in intracranial xenograft models without the obvious need for blood brain barrier disruption. Simultaneous use of five independent targeting peptides provided greater coverage of this complex tumor and selected peptides have the capacity to deliver a therapeutic cargo (oncolytic virus VSVΔM51) to the tumor cells in vivo. Herein, we have identified a series of peptides with utility as an innovative platform to assist in targeting glioblastoma for the purpose of diagnostic or prognostic imaging, image-guided surgery, and/or improved delivery of therapeutic agents to glioblastoma cells implicated in disease relapse.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Vírus Oncolíticos , Animais , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Humanos , Peptídeos
11.
Mol Cancer Res ; 6(4): 555-67, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18403635

RESUMO

MUC1, a transmembrane glycoprotein of the mucin family, when aberrantly expressed on breast cancer cells is correlated with increased lymph node metastases. We have previously shown that MUC1 binds intercellular adhesion molecule-1 (ICAM-1) on surrounding accessory cells and facilitates transendothelial migration of MUC1-bearing cells. Nevertheless, the underlying molecular mechanism is still obscure. In the present study, we used a novel assay of actin cytoskeletal reorganization to show that by ligating ICAM-1, MUC1 triggers Rac1- and Cdc42-dependent actin cytoskeletal protrusive activity preferentially at the heterotypic cell-cell contact sites. Further, we show that these MUC1/ICAM-1 interaction-initiated lamellipodial and filopodial protrusions require Src family kinase and CT10 regulator of kinase like (CrkL) accompanied by the rapid formation of a Src-CrkL signaling complex at the MUC1 cytoplasmic domain. Through inhibition of Src kinase activity, we further revealed that Src is required for recruiting CrkL to the MUC1 cytoplasmic domain as well as mediating the observed actin cytoskeleton dynamics. These findings suggest a novel MUC1-Src-CrkL-Rac1/Cdc42 signaling cascade following ICAM-1 ligation, through which MUC1 regulates cytoskeletal reorganization and directed cell motility during cell migration.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Citoesqueleto/enzimologia , Molécula 1 de Adesão Intercelular/metabolismo , Mucina-1/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Células NIH 3T3 , Ligação Proteica , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo
12.
Clin Exp Metastasis ; 22(6): 475-83, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16320110

RESUMO

MUC1 is a transmembrane glycoprotein expressed by normal breast epithelium and virtually all breast cancers. MUC1 is normally restricted to the apical surface of epithelia and loss of this polarized distribution in breast carcinomas is associated with lymph node metastasis. Our previous work found that MUC1 can bind intercellular adhesion molecule-1 (ICAM-1), mediating adhesion of breast cancer cells to a simulated blood vessel wall, and also triggering a calcium-based signal in the MUC1-bearing cells. It is possible that the depolarized membrane distribution of MUC1 in breast carcinomas may facilitate interactions with stromal/endothelial ICAM-1 leading to adhesion and subsequent migration through the vessel wall. In the current study, we provide evidence that ICAM-1 can influence the migration of cells that express endogenous or transfected MUC1. Migration across a gelatin-coated Transwell membrane could be increased in a step-wise manner by the sequential addition of ICAM-1-expressing cells (endothelial cells and fibroblasts), and ICAM-1-inducing inflammatory cytokines (tumour necrosis factor-alpha and interleukin-1 beta). Antibodies against MUC1 or ICAM-1, but not a control antibody, could abrogate migratory increases. Cells that did not express MUC1 were unresponsive to ICAM-1. Our current findings build on our earlier work, by suggesting that the end result of the MUC1/ICAM-1-mediated cell-cell adhesion and calcium-based signal is migration. This has implications for the exit of circulating tumour cells from the vasculature, as well as tumour cell migration through fibroblast-containing stroma underlying the endothelial wall.


Assuntos
Antígenos/metabolismo , Neoplasias da Mama/fisiopatologia , Movimento Celular , Endotélio Vascular/metabolismo , Glicoproteínas/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Mucinas/metabolismo , Anticorpos/farmacologia , Antígenos/genética , Antígenos de Neoplasias , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/genética , Humanos , Interleucina-1/farmacologia , Mucina-1 , Mucinas/antagonistas & inibidores , Mucinas/genética , Metástase Neoplásica , Fator de Necrose Tumoral alfa/farmacologia
13.
Sci Rep ; 5: 13989, 2015 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-26365306

RESUMO

Mitochondria are involved in key cellular functions including energy production, metabolic homeostasis, and apoptosis. Normal mitochondrial function is preserved by several interrelated mechanisms. One mechanism - intramitochondrial quality control (IMQC) - is represented by conserved proteases distributed across mitochondrial compartments. Many aspects and physiological roles of IMQC components remain unclear. Here, we show that the IMQC protease Oma1 is required for the stability of the respiratory supercomplexes and thus balanced and tunable bioenergetic function. Loss of Oma1 activity leads to a specific destabilization of respiratory supercomplexes and consequently to unbalanced respiration and progressive respiratory decline in yeast. Similarly, experiments in cultured Oma1-deficient mouse embryonic fibroblasts link together impeded supercomplex stability and inability to maintain proper respiration under conditions that require maximal bioenergetic output. Finally, transient knockdown of OMA1 in zebrafish leads to impeded bioenergetics and morphological defects of the heart and eyes. Together, our biochemical and genetic studies in yeast, zebrafish and mammalian cells identify a novel and conserved physiological role for Oma1 protease in fine-tuning of respiratory function. We suggest that this unexpected physiological role is important for cellular bioenergetic plasticity and may contribute to Oma1-associated disease phenotypes in humans.


Assuntos
Metaloproteases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Linhagem Celular , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Metabolismo Energético , Larva/metabolismo , Metaloproteases/química , Metaloproteases/genética , Camundongos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Morfolinos/farmacologia , Fenótipo , Estabilidade Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
14.
PLoS One ; 8(3): e59218, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23516612

RESUMO

Opa1 catalyzes fusion of inner mitochondrial membranes and formation of the cristae. OPA1 mutations in humans lead to autosomal dominant optic atrophy. OPA1 knockout mice lose viability around embryonic day 9 from unknown reasons, indicating that OPA1 is essential for embryonic development. Zebrafish are an attractive model for studying vertebrate development and have been used for many years to describe developmental events that are difficult or impractical to view in mammalian models. In this study, Opa1 was successfully depleted in zebrafish embryos using antisense morpholinos, which resulted in disrupted mitochondrial morphology. Phenotypically, these embryos exhibited abnormal blood circulation and heart defects, as well as small eyes and small pectoral fin buds. Additionally, startle response was reduced and locomotor activity was impaired. Furthermore, Opa1 depletion caused bioenergetic defects, without impairing mitochondrial efficiency. In response to mitochondrial dysfunction, a transient upregulation of the master regulator of mitochondrial biogenesis, pgc1a, was observed. These results not only reveal a new Opa1-associated phenotype in a vertebrate model system, but also further elucidates the absolute requirement of Opa1 for successful vertebrate development.


Assuntos
Mitocôndrias/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Western Blotting , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Morfolinos/farmacologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética
15.
Methods Mol Biol ; 920: 445-70, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22941622

RESUMO

The study of gene function has been greatly facilitated by the development of strategies to modify genomic DNA. Gene targeting is one of the most successfully applied techniques used to examine the roles of specific genes in a wide variety of model systems from yeast to mammals. Our laboratory has pioneered the use of the Chinese hamster ovary (CHO) cell culture model system to study pathways of DNA repair and recombination at the hemizygous CHO APRT locus. By using a simple and effective gene targeting method, we have generated a number of DNA repair-deficient cell lines that have been used in targeted recombination experiments to investigate pathways of recombinational repair in somatic mammalian cells. These methods can be readily customized to generate a variety of cell lines deficient in specific genes of interest and can be applied to study the roles of other DNA repair proteins in pathways of mammalian recombinational repair.


Assuntos
Reparo do DNA , Marcação de Genes/métodos , Mutação , Recombinação Genética , Adenina Fosforribosiltransferase/deficiência , Adenina Fosforribosiltransferase/genética , Animais , Células CHO , Técnicas de Cultura de Células , Células Clonais , Cricetinae , Cricetulus , Eletroporação , Técnicas de Inativação de Genes , Loci Gênicos/genética , Vetores Genéticos/genética
16.
PLoS One ; 6(9): e25652, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21980518

RESUMO

Many debilitating conditions are linked to bioenergetic defects. Developing screens to probe the genetic and/or chemical basis for such links has proved intractable. Furthermore, there is a need for a physiologically relevant assay of bioenergetics in whole organisms, especially for early stages in life where perturbations could increase disease susceptibility with aging. Thus, we asked whether we could screen bioenergetics and mitochondrial function in the developing zebrafish embryo. We present a multiplexed method to assay bioenergetics in zebrafish embryos from the blastula period (3 hours post-fertilization, hpf) through to hatching (48 hpf). In proof of principle experiments, we measured respiration and acid extrusion of developing zebrafish embryos. We quantified respiratory coupling to various bioenergetic functions by using specific pharmacological inhibitors of bioenergetic pathways. We demonstrate that changes in the coupling to ATP turnover and proton leak are correlated with developmental stage. The multiwell format of this assay enables the user to screen for the effects of drugs and environmental agents on bioenergetics in the zebrafish embryo with high sensitivity and reproducibility.


Assuntos
Embrião não Mamífero/metabolismo , Metabolismo Energético , Análise em Microsséries/métodos , Peixe-Zebra/embriologia , Trifosfato de Adenosina/metabolismo , Animais , Respiração Celular/efeitos dos fármacos , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Concentração de Íons de Hidrogênio , Modelos Lineares , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Prótons , Reprodutibilidade dos Testes , Peixe-Zebra/metabolismo
17.
DNA Repair (Amst) ; 10(2): 188-98, 2011 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-21123118

RESUMO

The ERCC1-XPF structure-specific endonuclease is necessary for correct processing of homologous recombination intermediates requiring the removal of end-blocking nonhomologies. We previously showed that targeting the endogenous CHO APRT locus with plasmids designed to generate such intermediates revealed defective recombination phenotypes in ERCC1 deficient cells, including suppression of targeted insertion and vector correction recombinants and the generation of a novel class of aberrant recombinants through a deletogenic mechanism. In the present study, we examined some of the mechanistic features of ERCC1-XPF in processing recombination intermediates by varying gene targeting parameters. These included altering the distance between the double-strand break (DSB) in the targeting vector and the inactivating mutation in the APRT target gene, and changing the position of the target gene mutation relative to the DSB to result in target mutations that were either upstream or downstream from the DSB. Increasing the distance from the DSB in the targeting vector to the chromosomal target gene mutation resulted in an ERCC1 dependent decrease in the efficiency of gene targeting from intermediates presenting lengthy end-blocking nonhomologies. This decrease was accompanied by a shift in the distribution of recombinant classes away from target gene conversions to targeted insertions in both wild-type and ERCC1 deficient cells, and a dramatic increase in the proportion of aberrant recombinants in ERCC1 deficient cells. Changing the position of the target gene mutation relative to the DSB in the plasmid also altered the distribution of targeted insertion subclasses recovered in wild-type cells, consistent with two-ended strand invasion followed by resolution into crossover-type products and vector integration. Our results confirm expectations from studies of Rad10-Rad1 in budding yeast that ERCC1-XPF activity affects conversion tract length, and provide evidence for the mechanism of generation of the novel, aberrant recombinant class first described in our previous study.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Endonucleases/fisiologia , Recombinação Genética , Adenina Fosforribosiltransferase/genética , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Marcação de Genes , Mutagênese , Mutação Puntual , Multimerização Proteica
18.
Environ Mol Mutagen ; 51(6): 567-81, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20658648

RESUMO

DNA interstrand crosslinks (ICLs) are among the most deleterious cytotoxic lesions encountered by cells, mainly due to the covalent linkage these lesions create between the two strands of DNA which effectively blocks replication and transcription. Although ICL repair in mammalian cells is not fully understood, processing of these lesions is thought to begin by "unhooking" at the site of the damaged base accompanied by the generation of a double strand break and ultimately repair through translesion synthesis and homologous recombination. A key player in this repair process is the heterodimeric protein complex ERCC1-XPF. Although some models of ICL repair restrict ERCC1-XPF activity to the unhooking step, recent data suggest that this protein complex acts in additional downstream steps. Here, we review the evidence implicating ERCC1-XPF in multiple steps of ICL repair.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Endonucleases/metabolismo , Animais , Humanos , Modelos Biológicos
19.
Mech Ageing Dev ; 131(9): 562-73, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20708636

RESUMO

Werner syndrome (WS) is a rare, segmental progeroid syndrome caused by defects in the WRN gene, which encodes a RecQ helicase. WRN has roles in many aspects of DNA metabolism including DNA repair and recombination. In this study, we exploited two different recombination assays previously used to describe a role for the structure-specific endonuclease ERCC1-XPF in mitotic and targeted homologous recombination. We constructed Chinese hamster ovary (CHO) cell lines isogenic with the cell lines used in these previous studies by depleting WRN using shRNA vectors. When intrachromosomal, mitotic recombination was assayed in WRN-depleted CHO cells, a hyperrecombination phenotype was observed, and a small number of aberrant recombinants were generated. Targeted homologous recombination was also examined in WRN-depleted CHO cells using a plasmid-chromosome targeting assay. In these experiments, loss of WRN resulted in a significant decrease in nonhomologous integration events and ablation of recombinants that required random integration of the corrected targeting vector. Aberrant recombinants were also recovered, but only from WRN-depleted cells. The pleiotropic recombination phenotypes conferred by WRN depletion, reflected in distinct homologous and nonhomologous recombination pathways, suggest a role for WRN in processing specific types of homologous recombination intermediates as well as an important function in nonhomologous recombination.


Assuntos
Exodesoxirribonucleases/genética , Exodesoxirribonucleases/fisiologia , Mitose , RecQ Helicases/genética , RecQ Helicases/fisiologia , Recombinação Genética , Animais , Células CHO , Cromossomos/ultraestrutura , Cricetinae , Cricetulus , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Humanos , Camundongos , Fenótipo , RNA Interferente Pequeno/metabolismo , RecQ Helicases/metabolismo , Helicase da Síndrome de Werner
20.
Comp Biochem Physiol C Toxicol Pharmacol ; 149(2): 129-33, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18692156

RESUMO

Genetic hybrids of the genus Xiphophorus have historically been useful models for study of the genetic aspects of tumor formation. In the most studied Xiphophorus tumor model, two-gene loci, XMRK and DIFF, are implicated as critical both to UV-induced and spontaneous melanoma formation in BC(1) hybrids of crosses between X. maculatus and X. helleri, with X. helleri as the recurrent backcross parent. In addition to UV, the direct-acting carcinogen N-methyl-N-nitrosourea (MNU) has been used to induce tumors in Xiphophorus BC(1) hybrids from several cross types. In the present study, we address the hypothesis that excess melanomas in MNU-treated BC(1) hybrids may have been generated by direct mutation of CDKN2AB, a candidate gene for DIFF. MNU treatment of F(1) and BC(1) hybrid fish significantly increased tumor incidence at 6 months; however, no association was found between MNU-induced tumor formation and zygosity of the candidate tumor tumor-suppressor CDKN2AB in BC(1) hybrids, consistent with previously reported results. Sequence analysis of the X. maculatus CDKN2AB locus of heterozygous individuals (both BC(1) and F(1) hybrids) did not reveal any mutations caused by MNU, suggesting that the mechanism of MNU-induced melanoma formation in this Xiphophorus model does not involve direct mutation of CDKN2AB but may result from mutation of other critical genes.


Assuntos
Alquilantes , Ciprinodontiformes/genética , Melanoma Experimental/etiologia , Metilnitrosoureia , Neoplasias Cutâneas/etiologia , Animais , Cruzamentos Genéticos , Ciprinodontiformes/classificação , Hibridização Genética , Melanoma Experimental/genética , Neoplasias Cutâneas/genética
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