Structural Dynamics of <i>Ec</i>DOS Heme Domain Revealed by Time-Resolved Ultraviolet Resonance Raman Spectroscopy

Protein dynamics on the subnanosecond to microsecond time scale was investigated for the isolated heme domain of a gas sensor protein, <i>Ec</i>DOS, with time-resolved ultraviolet resonance Raman (UVRR) spectroscopy. Rapid structural changes (<0.5 ns) due to CO dissociation and nanosecond structural relaxation following geminate recombination of CO were observed through a Raman band of Trp53 located near the heme. Microsecond transient UVRR spectra showed several phases of intensity changes in both Trp and Tyr bands. In hundreds of nanoseconds after CO photodissociation, the W18, W16, and W3 bands of Trp residues and Y8a band of Tyr residues decreased in intensity and were followed by the intensity recovery of Tyr band in 50 μs and of Trp bands in hundreds microseconds. This observation demonstrates that a change in the heme ligation triggers conformational changes in the protein moiety through heme side chains

Prior to TBI surgery (A), the number of errors on trial 1 (T1), trial 4 (T4), and the 30 minute retention trial (T5) for NT (yellow) and AD (blue) mice during the final two days of RAWM testing.

<p>Asterisk indicates significant improvement from T1 for that group at p<0.05 or higher level of significance. For Post-TBI testing, the number of errors on T1, T4, T5 for the RAWM at two weeks (<b>B</b>) and six weeks (<b>C</b>) after TBI. NT-Sham (hatched yellow, n = 9), NT-TBI (solid yellow, n = 10), AD-Sham (hatched blue, n = 8), and AD-TBI (solid blue, n = 7) mice. Single asterisk indicates the AD-TBI group is significantly different from all other groups at p<0.02. Double asterisk indicates the AD-TBI group is significantly different from AD-Sham at p<0.01. The error bars represent the SEM.</p

Experimental design is shown.

<p>For cohort 1, animals were trained in the RAWM prior to TBI surgery and then tested for cognitive deficits in the RAWM at two and six weeks post-TBI. For cohort 2, there was no pre-TBI behavioral training or post-TBI behavioral testing. All the animals were euthanized at six weeks post-TBI and the brains were harvested for histological analysis.</p

TBI accelerated extracellular Aβ deposits in the hippocampus of AD-TBI mice.

<p>Statistical analysis revealed a significant upregulation of extracellular Aβ deposits in the AD mice that received TBI compared to the NT mice that received TBI and the AD and NT mice that received sham surgery (Panel A). MAP2 staining revealed a significant decrease of MAP2 positive cells in the hippocampus of AD mice that received TBI compared to the AD and NT mice that received sham surgery (Panel B). Immunofluorescence for the detection of extracellular Aβ deposits and MAP2 (Panel C). Scale bars = 50 µm. The insets correspond to representative high magnifications of MAP2 images. Scale bars  = 100 µm. The brain illustration shows the location of the brain slices chosen for histological analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.</p

Kinetic Analysis of a Globin-Coupled Histidine Kinase, <i>Af</i>GcHK: Effects of the Heme Iron Complex, Response Regulator, and Metal Cations on Autophosphorylation Activity

The globin-coupled histidine kinase, <i>Af</i>GcHK, is a part of the two-component signal transduction system from the soil bacterium <i>Anaeromyxobacter</i> sp. Fw109-5. Activation of its sensor domain significantly increases its autophosphorylation activity, which targets the His183 residue of its functional domain. The phosphate group of phosphorylated <i>Af</i>GcHK is then transferred to the cognate response regulator. We investigated the effects of selected variables on the autophosphorylation reaction’s kinetics. The <i>k</i>_cat values of the heme Fe­(III)-OH^–, Fe­(III)-cyanide, Fe­(III)-imidazole, and Fe­(II)-O₂ bound active <i>Af</i>GcHK forms were 1.1–1.2 min^–1, and their <i>K</i>_m^ATP values were 18.9–35.4 μM. However, the active form bearing a CO-bound Fe­(II) heme had a <i>k</i>_cat of 1.0 min^–1 but a very high <i>K</i>_m^ATP value of 357 μM, suggesting that its active site structure differs strongly from the other active forms. The Fe­(II) heme-bound inactive form had <i>k</i>_cat and <i>K</i>_m^ATP values of 0.4 min^–1 and 78 μM, respectively, suggesting that its low activity reflects a low affinity for ATP relative to that of the Fe­(III) form. The heme-free form exhibited low activity, with <i>k</i>_cat and <i>K</i>_m^ATP values of 0.3 min^–1 and 33.6 μM, respectively, suggesting that the heme iron complex is essential for high catalytic activity. Overall, our results indicate that the coordination and oxidation state of the sensor domain heme iron profoundly affect the enzyme’s catalytic activity because they modulate its ATP binding affinity and thus change its <i>k</i>_cat/<i>K</i>_m^ATP value. The effects of the response regulator and different divalent metal cations on the autophosphorylation reaction are also discussed

Rho-Kinase Inhibition Ameliorates Metabolic Disorders through Activation of AMPK Pathway in Mice

<div><p>Background</p><p>Metabolic disorders, caused by excessive calorie intake and low physical activity, are important cardiovascular risk factors. Rho-kinase, an effector protein of the small GTP-binding protein RhoA, is an important cardiovascular therapeutic target and its activity is increased in patients with metabolic syndrome. We aimed to examine whether Rho-kinase inhibition improves high-fat diet (HFD)-induced metabolic disorders, and if so, to elucidate the involvement of AMP-activated kinase (AMPK), a key molecule of metabolic conditions.</p><p>Methods and Results</p><p>Mice were fed a high-fat diet, which induced metabolic phenotypes, such as obesity, hypercholesterolemia and glucose intolerance. These phenotypes are suppressed by treatment with selective Rho-kinase inhibitor, associated with increased whole body O₂ consumption and AMPK activation in the skeletal muscle and liver. Moreover, Rho-kinase inhibition increased mRNA expression of the molecules linked to fatty acid oxidation, mitochondrial energy production and glucose metabolism, all of which are known as targets of AMPK in those tissues. In systemic overexpression of dominant-negative Rho-kinase mice, body weight, serum lipid levels and glucose metabolism were improved compared with littermate control mice. Furthermore, in AMPKα2-deficient mice, the beneficial effects of fasudil, a Rho-kinase inhibitor, on body weight, hypercholesterolemia, mRNA expression of the AMPK targets and increase of whole body O₂ consumption were absent, whereas glucose metabolism was restored by fasudil to the level in wild-type mice. In cultured mouse myocytes, pharmacological and genetic inhibition of Rho-kinase increased AMPK activity through liver kinase b1 (LKB1), with up-regulation of its targets, which effects were abolished by an AMPK inhibitor, compound C.</p><p>Conclusions</p><p>These results indicate that Rho-kinase inhibition ameliorates metabolic disorders through activation of the LKB1/AMPK pathway, suggesting that Rho-kinase is also a novel therapeutic target of metabolic disorders.</p></div

Metabolic Parameters of Wild-type Mouse Groups.

<p>Results are expressed as mean ± SEM. The data were obtained at 12 weeks of age after 6-week treatment, with 15 h of fasting (n = 8 each). HOMA-IR was calculated using the following formula: {<i>fasting glucose (mg/dl) × fasting insulin (ng/ml)/405</i>}. Adipose tissue weight was the sum of the epididymal and peri-renal fat. ND, normal diet; HFD, high-fat diet. *P<0.05 vs. ND group, ^†P<0.05 vs. HFD-cont group.</p><p>Metabolic Parameters of Wild-type Mouse Groups.</p

Summary of the Present Study.

<p>Rho-kinase inhibition activates AMPKα2 via LKB1 pathway with a resultant increase in energy consumption and improvement of metabolic disorders (e.g. hypertension, obesity and hyperlipidemia). Although Rho-kinase inhibition also improves insulin tolerance, this might not be mediated by AMPK activation, at least in the present study.</p

Metabolic parameters of HFD-DN-ROCK Tg group and HFD-littermate group.

<p>Results are expressed as mean ± SEM. The data were obtained at 12-week-old, with 15 h of fasting (n = 6 each). HOMA-IR was calculated using the following formula: {<i>fasting glucose (mg/dl) × fasting insulin (ng/ml)/405</i>}. Adipose tissue weight was the sum of the epididymal and peri-renal fat. *P<0.05 vs. HFD-DN-ROCK Tg group.</p><p>Metabolic parameters of HFD-DN-ROCK Tg group and HFD-littermate group.</p

Fasudil Activates AMPK in Vitro via LKB1 Pathway.

<p>(<b>A</b>) In C2C12 myotubes, hydroxyfasudil (30 µmol/L) increased AMPK phosphorylation with a peak at 6 h after incubation (n = 3 each). (<b>B</b>) In C2C12 myotubes, hydroxyfasudil increased AMPK phosphorylation in a concentration-dependent manner after 6 hours incubation (n = 3 each). (<b>C</b>) Hydroxyfasudil (10 µmol/L for 48 h) significantly up-regulated mRNA expression of <i>Cpt1b</i> and <i>Cox4il</i> as well as NAD⁺/NADH ratio, all of which were inhibited by compound C (50 µmol/L) (n = 6 each). Results are normalized by the expression of <i>Gapdh</i>. (<b>D</b>) AMPK activation by hydroxyfasudil was significantly inhibited by siRNA for LKB1 and there was no additional effect by TAK1 or STO-609 inhibition (n = 3 each). Results are expressed as mean ± SEM. *P<0.05 vs. 0 h, †P<0.05 vs. 0 µM, ‡P<0.05 vs. 1 µM, §P<0.05 vs. fasudil without compound C, ¶P<0.05 vs. without hydroxyfasudil, siLKB1, siTAK1 and STO-609, #P<0.05 vs. hydroxyfasudil.</p