RESUMO
Mycobacterium tuberculosis is the causative agent of Tuberculosis. During the host response to infection, the bacterium is exposed to both reactive oxygen species and nitrogen intermediates that can cause DNA damage. It is becoming clear that the DNA damage response in Mtb and related actinobacteria function via distinct pathways as compared to well-studied model bacteria. For example, we have previously shown that the DNA repair helicase UvrD1 is activated for processive unwinding via redox-dependent dimerization. In addition, mycobacteria contain a homo-dimeric Ku protein, homologous to the eukaryotic Ku70/Ku80 dimer, that plays roles in double-stranded break repair via non-homologous end-joining. Kuhas been shown to stimulate the helicase activity of UvrD1, but the molecular mechanism, as well as which redox form of UvrD1 is activated, is unknown. We show here that Ku specifically stimulates multi-round unwinding by UvrD1 monomers which are able to slowly unwind DNA, but at rates 100-fold slower than the dimer. We also demonstrate that the UvrD1 C-terminal Tudor domain is required for the formation of a Ku-UvrD1 protein complex and activation. We show that Mtb Ku dimers bind with high nearest neighbor cooperativity to duplex DNA and that UvrD1 activation is observed when the DNA substrate is bound with two or three Ku dimers. Our observations reveal aspects of the interactions between DNA, Mtb Ku, and UvrD1 and highlight the potential role of UvrD1 in multiple DNA repair pathways through different mechanisms of activation.
Assuntos
Proteínas de Bactérias , Reparo do DNA por Junção de Extremidades , DNA Helicases , Autoantígeno Ku , Mycobacterium tuberculosis , DNA/metabolismo , DNA Helicases/metabolismo , Autoantígeno Ku/metabolismo , Mycobacterium tuberculosis/genética , Proteínas de Bactérias/metabolismoRESUMO
Mycobacterium tuberculosis (Mtb) causes tuberculosis and, during infection, is exposed to reactive oxygen species and reactive nitrogen intermediates from the host immune response that can cause DNA damage. UvrD-like proteins are involved in DNA repair and replication and belong to the SF1 family of DNA helicases that use ATP hydrolysis to catalyze DNA unwinding. In Mtb, there are two UvrD-like enzymes, where UvrD1 is most closely related to other family members. Previous studies have suggested that UvrD1 is exclusively monomeric; however, it is well known that Escherichia coli UvrD and other UvrD family members exhibit monomer-dimer equilibria and unwind as dimers in the absence of accessory factors. Here, we reconcile these incongruent studies by showing that Mtb UvrD1 exists in monomer, dimer, and higher-order oligomeric forms, where dimerization is regulated by redox potential. We identify a 2B domain cysteine, conserved in many Actinobacteria, that underlies this effect. We also show that UvrD1 DNA-unwinding activity correlates specifically with the dimer population and is thus titrated directly via increasing positive (i.e., oxidative) redox potential. Consistent with the regulatory role of the 2B domain and the dimerization-based activation of DNA unwinding in UvrD family helicases, these results suggest that UvrD1 is activated under oxidizing conditions when it may be needed to respond to DNA damage during infection.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Helicases/metabolismo , Reparo do DNA/fisiologia , Mycobacterium tuberculosis/genética , Proteínas de Bactérias/genética , Cisteína/química , DNA/genética , DNA/metabolismo , Dano ao DNA , DNA Helicases/genética , Reparo do DNA/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples , Dimerização , Oxirredução , Ligação Proteica , Domínios Proteicos/genéticaRESUMO
Conditions of metabolic stress dysregulate the NAD metabolome. By restoring NAD, nicotinamide riboside (NR) provides resistance to such conditions. We tested the hypotheses that postpartum might dysregulate maternal NAD and that increasing systemic NAD with NR might benefit mothers and offspring. In postpartum mothers, the liver NAD metabolome is depressed while blood increases circulation of NAD metabolites to enable a >20-fold increase in mammary NAD+ and NADP+. Lactation and NR synergize in stimulating prolactin synthesis and mammary biosynthetic programs. NR supplementation of new mothers increases lactation and nursing behaviors and stimulates maternal transmission of macronutrients, micronutrients, and BDNF into milk. Pups of NR-supplemented mothers are advantaged in glycemic control, size at weaning, and synaptic pruning. Adult offspring of mothers supplemented during nursing retain advantages in physical performance, anti-anxiety, spatial memory, delayed onset of behavioral immobility, and promotion of adult hippocampal neurogenesis. Thus, postgestational maternal micronutrition confers lasting advantages to offspring.
Assuntos
Exposição Materna/efeitos adversos , Neurogênese/efeitos dos fármacos , Niacinamida/análogos & derivados , Período Pós-Parto/metabolismo , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Redução de Peso/efeitos dos fármacos , Animais , Feminino , Lactação/efeitos dos fármacos , Lactação/metabolismo , Fígado/metabolismo , Fígado/patologia , Camundongos , NAD/metabolismo , Niacinamida/efeitos adversos , Niacinamida/farmacologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/patologia , Compostos de PiridínioRESUMO
Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a new approach to the problem, as individual subunits adopt a stable and functionally verifiable fold that constrains the system to two states - monomer or dimer. Here, we use single-molecule photobleaching analysis to measure the probability of ClC-ec1 subunit capture into liposomes during extrusion of large, multilamellar membranes. The capture statistics describe a monomer to dimer transition that is dependent on the subunit/lipid mole fraction density and follows an equilibrium dimerization isotherm. This allows for the measurement of the free energy of ClC-ec1 dimerization in lipid bilayers, revealing that it is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes.
Assuntos
Canais de Cloreto/metabolismo , Bicamadas Lipídicas , Multimerização Proteica , Imagem Individual de MoléculaRESUMO
Male C57BL/6J mice raised on high fat diet (HFD) become prediabetic and develop insulin resistance and sensory neuropathy. The same mice given low doses of streptozotocin are a model of type 2 diabetes (T2D), developing hyperglycemia, severe insulin resistance and diabetic peripheral neuropathy involving sensory and motor neurons. Because of suggestions that increased NAD(+) metabolism might address glycemic control and be neuroprotective, we treated prediabetic and T2D mice with nicotinamide riboside (NR) added to HFD. NR improved glucose tolerance, reduced weight gain, liver damage and the development of hepatic steatosis in prediabetic mice while protecting against sensory neuropathy. In T2D mice, NR greatly reduced non-fasting and fasting blood glucose, weight gain and hepatic steatosis while protecting against diabetic neuropathy. The neuroprotective effect of NR could not be explained by glycemic control alone. Corneal confocal microscopy was the most sensitive measure of neurodegeneration. This assay allowed detection of the protective effect of NR on small nerve structures in living mice. Quantitative metabolomics established that hepatic NADP(+) and NADPH levels were significantly degraded in prediabetes and T2D but were largely protected when mice were supplemented with NR. The data justify testing of NR in human models of obesity, T2D and associated neuropathies.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Neuropatias Diabéticas/prevenção & controle , Hipoglicemiantes/farmacologia , Niacinamida/análogos & derivados , Obesidade/tratamento farmacológico , Estado Pré-Diabético/tratamento farmacológico , Animais , Glicemia/metabolismo , Córnea/efeitos dos fármacos , Córnea/inervação , Córnea/patologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Neuropatias Diabéticas/induzido quimicamente , Neuropatias Diabéticas/metabolismo , Neuropatias Diabéticas/patologia , Dieta Hiperlipídica , Insulina/sangue , Resistência à Insulina , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Niacinamida/farmacologia , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Estado Pré-Diabético/etiologia , Estado Pré-Diabético/metabolismo , Estado Pré-Diabético/patologia , Compostos de Piridínio , EstreptozocinaRESUMO
The 5' untranslated region (5'UTR) of the hepatitis A virus (HAV) genomic RNA contains an internal ribosome entry site (IRES) which interacts with various cellular proteins and facilitates cap-independent translation. We report the interaction of a 25kDa protein (p25), present in certain murine tissues and most abundantly in mouse kidney, with the HAV 5'UTR. This protein was found to be a cleavage product of the polypyrimidine tract-binding protein (PTB) and competed with it for binding to the HAV 5'UTR RNA. The binding site of p25 overlapped with the reported binding site of PTB. Exogenous addition of partially purified p25 to in vitro translation reactions resulted in the inhibition of HAV IRES-mediated translation, which could be rescued by the addition of purified PTB. These results suggest that p25 is a cleavage product of PTB which binds to the HAV IRES and antagonizes the translation-stimulating activity of PTB. The presence of the 25kDa cleavage product of PTB may therefore play a role in the inhibition of HAV IRES-mediated translation in mouse tissues.