RESUMO
Constraint-based modeling has been shown, in many instances, to be useful for metabolic engineering by allowing the prediction of the metabolic phenotype resulting from genetic manipulations. But the basic premise of constraint-based modeling-that of applying constraints to preclude certain behaviors-only makes sense for certain genetic manipulations (such as knockouts and knockdowns). In particular, when genes (such as those associated with a heterologous pathway) are introduced under artificial control, it is unclear how to predict the correct behavior. In this paper, we introduce a modeling method that we call proportional flux forcing (PFF) to model artificially induced enzymatic genes. The model modifications introduced by PFF can be transformed into a set of simple mass balance constraints, which allows computational methods for strain optimization based on flux balance analysis (FBA) to be utilized. We applied PFF to the metabolic engineering of Escherichia coli (E. coli) for free fatty acid (FFA) production-a metabolic engineering problem that has attracted significant attention because FFAs are a precursor to liquid transportation fuels such as biodiesel and biogasoline. We show that PFF used in conjunction with FBA-based computational strain optimization methods can yield non-obvious genetic manipulation strategies that significantly increase FFA production in E. coli. The two mutant strains constructed and successfully tested in this work had peak fatty acid (FA) yields of 0.050 g FA/g carbon source (17.4% theoretical yield) and 0.035 g FA/g carbon source (12.3% theoretical yield) when they were grown using a mixed carbon source of glucose and casamino acids in a ratio of 2-to-1. These yields represent increases of 5.4- and 3.8-fold, respectively, over the baseline strain.
Assuntos
Escherichia coli/enzimologia , Ácidos Graxos/metabolismo , Engenharia Metabólica , Sequência de Bases , Simulação por Computador , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácidos Graxos/genética , Regulação Enzimológica da Expressão Gênica , Genes Bacterianos , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , MutaçãoRESUMO
Mitochondria are the powerhouse of the cell and their disruption leads to cell death. We have used a peptide trivalent arsenical, 4-(N-(S-glutathionylacetyl)amino) phenylarsenoxide (GSAO), to inactivate the adenine nucleotide translocator (ANT) that exchanges matrix ATP for cytosolic ADP across the inner mitochondrial membrane and is the key component of the mitochondrial permeability transition pore (MPTP). GSAO triggered Ca(2+)-dependent MPTP opening by crosslinking Cys(160) and Cys(257) of ANT. GSAO treatment caused a concentration-dependent increase in superoxide levels, ATP depletion, mitochondrial depolarization, and apoptosis in proliferating, but not growth-quiescent, endothelial cells. Endothelial cell proliferation drives new blood vessel formation, or angiogenesis. GSAO inhibited angiogenesis in the chick chorioallantoic membrane and in solid tumors in mice. Consequently, GSAO inhibited tumor growth in mice with no apparent toxicity at efficacious doses.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Arsenicais/uso terapêutico , Células Endoteliais/metabolismo , Amarelo de Eosina-(YS)/análogos & derivados , Mitocôndrias/efeitos dos fármacos , Neovascularização Patológica , Translocador 1 do Nucleotídeo Adenina/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Aorta/citologia , Apoptose , Biotina/farmacologia , Cálcio/metabolismo , Cálcio/farmacologia , Bovinos , Divisão Celular , Sobrevivência Celular , Embrião de Galinha , Citosol/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/uso terapêutico , Amarelo de Eosina-(YS)/farmacologia , Feminino , Imuno-Histoquímica , Canais Iônicos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Modelos Químicos , Peptídeos/química , Fatores de TempoAssuntos
Bioquímica/métodos , Membrana Celular/metabolismo , Glutationa/análogos & derivados , Oxirredução , Arsenicais/farmacologia , Dissulfetos , Relação Dose-Resposta a Droga , Glutationa/metabolismo , Glutationa/farmacologia , Concentração de Íons de Hidrogênio , Indicadores e Reagentes/farmacologia , Lisina/análogos & derivados , Lisina/farmacologia , Maleimidas/farmacologia , Modelos Químicos , Oxigênio/metabolismoRESUMO
CD4, a member of the immunoglobulin superfamily of receptors that mediates cell-cell interactions in the immune system, is the primary receptor for HIV-1. The extracellular portion of CD4 is a concatenation of four immunoglobulin-like domains, D1 to D4. The D1, D2 and D4 domains each contain a disulfide bond. We show here that the D2 disulfide bond is redox-active. The redox state of the thiols (disulfide versus dithiol) appeared to be regulated by thioredoxin, which is secreted by CD4(+) T cells. Locking the CD4 and the thioredoxin active-site dithiols in the reduced state with a hydrophilic trivalent arsenical blocked entry of HIV-1 into susceptible cells. These findings indicate that redox changes in CD4 D2 are important for HIV-1 entry and represent a new target for HIV-1 entry inhibitors.