Search

Arginine kinase is a crucial phosphagen kinase in invertebrates, which is associated to the environmental stress response, plays a key role in cellular energy metabolism. In this study, we investigated the Pb 2+ -induced inhibition and aggregation of Euphausia superba arginine kinase (ESAK) and found that significantly inactivated ESAK in a dose-dependent manner ( IC 50 = 0.058 ± 0.002 mM). Spectrofluorimetry results showed that Pb 2+ induced tertiary structural changes via the internal polarity increased and the non-polarity decreased in ESAK and directly induced ESAK aggregation. The ESAK aggregation process induced by Pb 2+ occurred with multi-phase kinetics. The addition of osmolytes did not show protective effect on Pb 2+ -induced inactivation of ESAK. The computational molecular dynamics (MD) simulation showed that three Pb 2+ interrupt the entrance of the active site of ESAK and it could be the reason on the loss of activity of ESAK. Several important residues of ESAK were detected that were importantly contributed the conformation and catalytic function of ESAK. Our study showed that Pb 2+ -induced misfolding of ESAK and the complete loss of activity irreversibly, which cannot be recovered by osmolytes.Communicated by Ramaswamy H. Sarma.

The function of acetaldehyde dehydrogenase 1 (ALDH1) has been gradually elucidated in several diseases, especially in various cancers. However, the role of ALDH1 in skin-related diseases has been mostly unknown. Previously, we found that ALDH1 is involved in the pathogenesis of atopic dermatitis (AD). In this study, we used high-throughput screening (HTS) approaches to identify critical factors associated with ALDH1 in human keratinocytes to reveal its functions in skin. We overexpressed ALDH1 in human HaCaT keratinocytes and then conducted serial HTS studies, a DNA microarray and antibody array integrated with bioinformatics algorithms. Together, those tests identified several novel genes associated with the function of ALDH1 in keratinocytes, as well as AD, including CTSG and CCL11. In particular, GNB3, GHSR, TAS2R9, FFAR1, TAS2R16, CCL21, GPR32, NPFFR1, GPR15, FBXW12, CCL19, EDNRA, FFAR3, and RXFP3 proteins were consistently detected as hub proteins in the PPI maps. By integrating the datasets obtained from these HTS studies and using the strengths of each method, we obtained new insights into the functional role of ALDH1 in skin keratinocytes. The approach used here could contribute to the clinical understanding of ALDH1-associated applications for the treatment of AD.Communicated by Ramaswamy H. Sarma.

The inhibition of α-glucosidase is used as a key clinical approach to treat type 2 diabetes mellitus and thus, we assessed the inhibitory effect of α-ketoglutaric acid (AKG) on α-glucosidase with both an enzyme kinetic assay and computational simulations. AKG bound to the active site and interacted with several key residues, including ASP68, PHE157, PHE177, PHE311, ARG312, TYR313, ASN412, ILE434 and ARG439, as detected by protein-ligand docking and molecular dynamics simulations. Subsequently, we confirmed the action of AKG on α-glucosidase as mixed-type inhibition with reversible and rapid binding. The relevant kinetic parameter IC 50 was measured (IC 50 = 1.738 ± 0.041 mM), and the dissociation constant was determined ( K i Slope = 0.46 ± 0.04 mM). Regarding the relationship between structure and activity, a high AKG concentration induced the slight modulation of the shape of the active site, as monitored by hydrophobic exposure. This tertiary conformational change was linked to AKG inhibition and mostly involved regional changes in the active site. Our study provides insight into the functional role of AKG due to its structural property of a hydroxyphenyl ring that interacts with the active site. We suggest that similar hydroxyphenyl ring-containing compounds targeting key residues in the active site might be potential α-glucosidase inhibitors. AbbreviationsAKGalpha-ketoglutaric acidpNPG4-nitrophenyl-α-d-glucopyranosideANS1-anilinonaphthalene-8-sulfonateMDmolecular dynamics.Communicated by Ramaswamy H. Sarma.

Arginine kinase is closely associated with adaptation to environmental stresses such as high salinity and heavy metal ion levels in marine invertebrates. In this study, the effects of Cd(2+) on the cuttlefish Sepia pharaonis' arginine kinase (SPAK) were investigated. SPAK was isolated from the muscles of S. pharaonis and upon further purification, showed a single band on SDS-PAGE. Cd(2+) effectively inactivated SPAK, and the double-reciprocal kinetics indicated that Cd(2+) induced non-competitive inhibition of arginine and ATP. Spectrofluorometry results showed that Cd(2+) induced tertiary structure changes in SPAK with the exposure of hydrophobic surfaces that directly induced SPAK aggregation. The addition of osmolytes, glycine, and proline successfully blocked SPAK aggregation and restored the conformation and activity of SPAK. Molecular dynamics simulations involving SPAK and Cd(2+) showed that Cd(2+) partly blocks the entrance of ATP to the active site, and this result is consistent with the experimental results showing Cd(2+)-induced inactivation of SPAK. These results demonstrate the effect of Cd(2+) on SPAK enzymatic function and unfolding, including aggregation and the protective effects of osmolytes on SPAK folding. This study provides concrete evidence of the toxicity of Cd(2+) in the context of the metabolic enzyme SPAK, and it illustrates the toxic effects of heavy metals and detoxification mechanisms in cuttlefish.

We studied the effects of Zn(2+) on creatine kinase from the Chinese soft-shelled turtle, Pelodiscus sinensis (PSCK). Zn(2+) inactivated the activity of PSCK (IC(50) = .079 ± .004 mM) following first-order kinetics consistent with multiple phases. The spectrofluorimetry results showed that Zn(2+) induced significant tertiary structural changes of PSCK with exposure to hydrophobic surfaces and that Zn(2+) directly induced PSCK aggregation. The addition of osmolytes such as glycine, proline, and liquaemin successfully blocked PSCK aggregation, recovering the conformation and activity of PSCK. We measured the ORF gene sequence of PSCK by rapid amplification of cDNA end and simulated the 3D structure of PSCK. The results of molecular dynamics simulations showed that eight Zn(2+) bind to PSCK and one Zn(2+) is predicted to bind in a plausible active site of creatine and ATP. The interaction of Zn(2+) with the active site could mostly block the activity of PSCK. Our study provides important insight into the action of Zn(2+) on PSCK as well as more insights into the PSCK folding and ligand-binding mechanisms, which could provide important insight into the metabolic enzymes of P. sinensis.

Fucoidan is a complex sulfated polysaccharide extracted from brown seaweed and has a wide variety of biological activities. In this study, we investigated the inhibitory effect of fucoidan on tyrosinase via a combination of inhibition kinetics and computational simulations. Fucoidan reversibly inhibited tyrosinase in a mixed-type manner. Time-interval kinetics showed that the inhibition was processed as first order with biphasic processes. For further insight, we simulated dockings with various sizes of molecular models (monomer to decamer) of fucoidan and showed that the best binding energy change results were obtained from the pentamer (-1.89 kcal/mol) and the hexamer (-1.97 kcal/mol) models of AutoDock Vina. The molecular dynamics simulation confirmed the binding mechanisms between tyrosinase and fucoidan and suggested that fucoidan mostly interacts with several residues including copper ions located in the active site. Our study suggests that fucoidan might be a potential natural antipigment agent.

Tyrosinase inhibition studies have recently gained the attention of researchers due to their potential application values. We simulated docking (binding energies for AutoDock Vina: -9.1 kcal/mol) and performed a molecular dynamics simulation to verify docking results between tyrosinase and rutin. The docking results suggest that rutin mostly interacts with histidine residues located in the active site. A 10 ns molecular dynamics simulation showed that one copper ion at the tyrosinase active site was responsible for the interaction with rutin. Kinetic analyses showed that rutin-mediated inactivation followed a first-order reaction and mono- and biphasic rate constants occurred with rutin. The inhibition was a typical competitive type with K(i) = 1.10±0.25 mM. Measurements of intrinsic and ANS-binding fluorescences showed that rutin showed a relatively strong binding affinity for tyrosinase and one possible binding site that could be a copper was detected accompanying with a hydrophobic exposure of tyrosinase. Cell viability testing with rutin in HaCaT keratinocytes showed that no toxic effects were produced. Taken together, rutin has the potential to be a potent anti-pigment agent. The strategy of predicting tyrosinase inhibition based on hydroxyl group number and computational simulation may prove useful for the screening of potential tyrosinase inhibitors.

Tyrosinase plays various roles in organisms and much research has focused on the regulation of tyrosinase activity. We studied the inhibitory effect of thiobarbituric acid (TBA) on tyrosinase. Our kinetic study showed that TBA inhibited tyrosinase in a reversible noncompetitive manner (K(i) 5 14.0 ± 8.5 mM and IC₅₀ 5 8.0 ± 1.0 mM). Intrinsic and ANS-binding fluorescences studies were also performed to gain more information regarding the binding mechanism. The results showed that no tertiary structural changes were obviously observed. For further insight, we predicted the 3D structure of tyrosinase and simulated the docking between tyrosinase and TBA. The docking simulation was successful with significant scores (binding energy for AutoDock4: -5.52 kcal/mol) and suggested that TBA was located in the active site. The 11 ns molecular dynamics simulation convinced that the four HIS residues (residue numbers: 57, 90, 250, and 282) were commonly responsible for the interaction with TBA. Our results provide a new inhibition strategy that works using an antioxidant rather than targeting the copper ions within the tyrosinase active site.

The tyrosinase gene was overexpressed in HEK293 cells, and then a DNA microarray and proteomic tools were applied to detect the dysregulated genes in highly pigmented cells. The candidate genes from the microarray were compared to the yeast two-hybridization results. Computational prediction via protein-protein interaction mapping suggested the existence of 66 hub genes in melanogenesis. Most importantly, RNA binding motif protein 9 is newly detected as a putative critical melanogenesis-associated gene in this study. The approach of combining the expression data analysis and predicted protein interaction partners performed in large scales can bring more reliable gene targets for understanding pigmentation.