Белорусская экономическая модель с позиций макроэкономического анализа

МОДЕЛИ ЭКОНОМИЧЕСКИЕЭКОНОМИКАСОЦИОЛОГИЯБЕЛОРУССИЯСОЦИОЭКОНОМИЧЕСКИЕ ФАКТОРЫПОЛИТИКА ПРАВИТЕЛЬСТВЕННАЯПРАВИТЕЛЬСТВЕННЫЕ ПРОГРАММЫМАКРОЭКОНОМИК

Human Copper Chaperone Atox1 Translocates to the Nucleus but does not Bind DNA In Vitro.

After Ctr1-mediated cell uptake, copper (Cu) is transported by the cytoplasmic Cu chaperone Atox1 to P1B type ATPases ATP7A and ATP7B in the Golgi network, for incorporation into Cudependent enzymes. Atox1 is a small 68-residue protein that binds Cu in a conserved CXXC motif; it delivers Cu to target domains in ATP7A/B via direct protein-protein interactions. Specific transcription factors regulating expression of the human Cu transport proteins have not been reported although Atox1 was recently suggested to have dual functionality such that it, in addition to its cytoplasmic chaperone function, acts as a transcription factor in the nucleus. To examine this hypothesis, here we investigated the localization of Atox1 in HeLa cells using fluorescence imaging in combination with in vitro binding experiments to fluorescently labeled DNA duplexes harboring the proposed promotor sequence. We found that whereas Atox1 is present in the nucleus in HeLa cells, it does not bind to DNA in vitro. It appears that Atox1 mediates transcriptional regulation via additional (unknown) proteins

Human Copper Chaperone Atox1 Translocates to the Nucleus but does not Bind DNA In Vitro.

After Ctr1-mediated cell uptake, copper (Cu) is transported by the cytoplasmic Cu chaperone Atox1 to P1B type ATPases ATP7A and ATP7B in the Golgi network, for incorporation into Cudependent enzymes. Atox1 is a small 68-residue protein that binds Cu in a conserved CXXC motif; it delivers Cu to target domains in ATP7A/B via direct protein-protein interactions. Specific transcription factors regulating expression of the human Cu transport proteins have not been reported although Atox1 was recently suggested to have dual functionality such that it, in addition to its cytoplasmic chaperone function, acts as a transcription factor in the nucleus. To examine this hypothesis, here we investigated the localization of Atox1 in HeLa cells using fluorescence imaging in combination with in vitro binding experiments to fluorescently labeled DNA duplexes harboring the proposed promotor sequence. We found that whereas Atox1 is present in the nucleus in HeLa cells, it does not bind to DNA in vitro. It appears that Atox1 mediates transcriptional regulation via additional (unknown) proteins

Enthalpy-entropy compensation at play in human copper ion transfer

Copper (Cu) is an essential trace element but toxic in free form. After cell uptake, Cu is transferred, via direct protein-protein interactions, from the chaperone Atox1 to the Wilson disease protein (WD) for incorporation into Cu-dependent enzymes. Cu binds to a conserved C1XXC2 motif in the chaperone as well as in each of the cytoplasmic metal-binding domains of WD. Here, we dissect mechanism and thermodynamics of Cu transfer from Atox1 to the fourth metal binding domain of WD. Using chromatography and calorimetry together with single Cys-to-Ala variants, we demonstrate that Cu-dependent protein heterocomplexes require the presence of C-1 but not C-2. Comparison of thermodynamic parameters for mutant versus wild type reactions reveals that the wild type reaction involves strong entropy-enthalpy compensation. This property is explained by a dynamic inter-conversion of Cu-Cys coordinations in the wild type ensemble and may provide functional advantage by protecting against Cu mis-ligation and bypassing enthalpic traps

Illustration of Scheme 1.

<p>Upon mixing Cu-loaded Atox1 (purple) and apo-WD (green), the proteins interact and form a hetero-complex, Atox1-Cu-WD4 (shown with Cu coordinating one Cys in Atox1 and both Cys in WD4; however, there are other possible Cu coordinations in the hetero-complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036102#pone.0036102-RodriguezGranillo1" target="_blank">[17]</a>), and also products, apo-Atox1 and Cu-WD4, according to the equilibrium constants K₁ and K₂.</p

Computer simulations mimic ITC titration.

<p><b>A.</b> Concentrations of the five species as a function of progress of the titration experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036102#pone-0036102-g004" target="_blank">Figure 4A</a> using the K values that were derived from the SEC data. <b>B.</b> ITC experimental data (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036102#pone-0036102-g004" target="_blank">Figure 4A</a>) together with the best fit to the data (see Supplement for details).</p

SEC probed at 2 wavelengths is used to separate equilibrium species in Scheme 1.

<p><b>A.</b> SEC analysis of a mixture of the two apo proteins (300 µM each) at 280 and 254 nm. <b>B.</b> SEC analysis of holo Atox1 (300 µM) at 280 and 254 nm. <b>C.</b> SEC analysis at 280 nm of a mixture of 300 µM Cu-Atox1 and 300 µM apo-WD4. For comparison, the 280 nm elution trace for the mixture of the two apo-proteins is shown. <b>D.</b> SEC analysis at 280 nm and 254 nm of a mixture of 300 µM Cu-Atox1 and 300 µM apo-WD4. <b>E.</b> De-convolution of the underlying peaks in the elution trace of the Cu-Atox1+apo-WD4 mixture.</p

SEC analysis of concentrations of species.

<p>Concentrations of the five species in <i>scheme 1</i> determined from SEC measurements as described in the text using different initial concentrations (1∶1:1 of Atox1:Cu:WD4) as indicated. Also, the % of the total copper found in hetero-complex is reported. The equilibrium concentrations established are used to derive K₁ and K₂ and from this the copper exchange factor K₁*K₂ is calculated. Two experiments with 150 µM starting concentrations are reported. For 75 µM, also the opposite reaction, mixing Cu-WD4 with apo-Atox1 was performed.</p

Thermodynamic parameters for steps 1 and 2, and overall reaction.

<p>The parameters are based on <i>scheme 1</i> and equilibrium constants from SEC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036102#pone-0036102-t001" target="_blank">Table 1</a>) and ITC data, as described in the text.</p

ITC reveals interaction energetics.

<p>A. A solution of 666 µM Cu-Atox1 (in the syringe) is titrated into a solution of 52 µM apoWD4 (in the reaction chamber) at 3°C. B. A solution of 666 µM apo-Atox1 (in the syringe) is titrated into a solution of 62 µM apoWD4 (in the reaction chamber) at 3°C. The top plots are the raw data of heats versus time and the bottom plots are integrated heats as a function of molar ratio of Atox1/WD4. Noise estimation based on the data in B predicts uncertainties for individual ITC points of 0.03 kcal/mol.</p