Jinhyuk Lee

Mutations in DDX58, which Encodes RIG-I, Cause Atypical Singleton-Merten Syndrome

Singleton-Merten syndrome (SMS) is an autosomal-dominant multi-system disorder characterized by dental dysplasia, aortic calcification, skeletal abnormalities, glaucoma, psoriasis, and other conditions. Despite an apparent autosomal-dominant pattern of inheritance, the genetic background of SMS and information about its phenotypic heterogeneity remain unknown. Recently, we found a family affected by glaucoma, aortic calcification, and skeletal abnormalities. Unlike subjects with classic SMS, affected individuals showed normal dentition, suggesting atypical SMS. To identify genetic causes of the disease, we performed exome sequencing in this family and identified a variant (c.1118A>C [p.Glu373Ala]) of DDX58, whose protein product is also known as RIG-I. Further analysis of DDX58 in 100 individuals with congenital glaucoma identified another variant (c.803G>T [p.Cys268Phe]) in a family who harbored neither dental anomalies nor aortic calcification but who suffered from glaucoma and skeletal abnormalities. Cys268 and Glu373 residues of DDX58 belong to ATP-binding motifs I and II, respectively, and these residues are predicted to be located closer to the ADP and RNA molecules than other nonpathogenic missense variants by protein structure analysis. Functional assays revealed that DDX58 alterations confer constitutive activation and thus lead to increased interferon (IFN) activity and IFN-stimulated gene expression. In addition, when we transduced primary human trabecular meshwork cells with c.803G>T (p.Cys268Phe) and c.1118A>C (p.Glu373Ala) mutants, cytopathic effects and a significant decrease in cell number were observed. Taken together, our results demonstrate that DDX58 mutations cause atypical SMS manifesting with variable expression of glaucoma, aortic calcification, and skeletal abnormalities without dental anomalies.

Oleic acid and linoleic acid from Tenebrio molitor larvae inhibit BACE1 activity in vitro: molecular docking studies.

In our ongoing research to find therapeutic compounds for Alzheimers disease (AD) from natural resources, the inhibitory activity of the BACE1 enzyme by Tenebrio molitor larvae and its major compounds were evaluated. The T. molitor larvae extract and its fractions exhibited strong BACE1 suppression. The major components of hexane fraction possessing both high yield and strong BACE1 inhibition were determined by thin layer chromatography, gas chromatography, and nuclear magnetic resonance analysis. A remarkable composition of unsaturated long chain fatty acids, including oleic acid and linoleic acid, were identified. Oleic acid, in particular, noncompetitively attenuated BACE1 activity with a half-maximal inhibitory concentration (IC₅₀) value of 61.31 μM and Ki value of 34.3u2009μM. Furthermore, the fatty acids were stably interacted with BACE1 at different allosteric sites of the enzyme bound with the OH of CYS319 and the NH₃ of TYR320 for oleic acid and with the C=O group of GLN304 for linoleic acid. Here, we first revealed novel pharmacophore features of oleic acids and linoleic acid to BACE1 by in silico docking studies. The present findings would clearly suggest potential guidelines for designing novel BACE1 selective inhibitors.

Effects of osmolytes on human brain-type creatine kinase folding in dilute solutions and crowding systems

The effects of osmolytes on the unfolding and refolding process of recombinant human brain-type creatine kinase (rHBCK) were comparatively, quantitatively studied in dilute solutions and macromolecular crowding systems (simulated by 100 g/L polyethylene glycol 2000), respectively. The results showed that the osmolytes, including glycerol, sucrose, dimethylsulfoxide, mannitol, inositol, and xylitol, could both protect the rHBCK from denaturation induced by 0.8 M GdnHCl and aid in the refolding of denatured-rHBCK in macromolecular crowding systems. When we examined the effects of sucrose and xylitol on the parameters of residual activity, reaction kinetics and intrinsic fluorescence of rHBCK during unfolding, it was found that the protecting effects of osmolytes in a macromolecular crowding system were more significant compared with those in a dilute solution, which resulted in more residual activities, protected the conformational changes and greatly decreased the rates of both the fast and slow tracks. Regarding the effects of glycerol, sucrose and mannitol on the denatured-rHBCK refolding parameters of refolding yield, reaction kinetics and aggregation, the results indicated that the osmolytes could alleviate the aggregation of rHBCK during refolding in both dilute solutions and macromolecular crowding systems, and the refolding yields and reaction rates under macromolecular crowding environment could be increased by the addition of osmolytes, though higher yields were obtained in the dilute solution. For further insight, osmolyte docking simulations and rHBCK denaturation were conducted successfully and confirmed our experimental results. The predictions based on the docking simulations suggested that the deactivation of guanidine may be blocked by osmolytes because they share common binding sites on rHBCK, and the higher number of interactions with rHBCK by osmolytes than guanidine may be one of the causes of rHBCK refolding. In brief, the additive effects of the exclusive volume effect from the macromolecular crowding system and the osmophobic effects from the osmolytes resulted in better performance of the osmolytes in a macromolecular crowding system, which also led to a better understanding of protein folding in the intracellular environment.

Effect of Ca 2+ on the activity and structure of α-glucosidase: Inhibition kinetics and molecular dynamics simulations

Understanding the mechanism of inhibition of α-glucosidase (EC 3.2.1.20) is clinically important because of the involvement of this enzyme in type 2 diabetes mellitus. In this study, we conducted inhibition kinetics of α-glucosidase with Ca(2+) and 10-ns molecular dynamics simulations. We found that direct binding of Ca(2+) to the enzyme induced structural changes and inhibited enzyme activity. Ca(2+) inhibited α-glucosidase in a mixed-type reaction (Kixa0=xa027.0xa0±xa02.0xa0mM) and directly induced the unfolding of α-glucosidase, which resulted in the exposure of hydrophobic residues. The simulations suggest that thirteen Ca(2+) ions may interact with α-glucosidase residues and that the Ca(2+) binding sites are associated with the structural changes in α-glucosidase. Our study provides insight into the mechanism of the Ca(2+)-induced structural changes in α-glucosidase and the inhibition of ligand binding. These results suggest that Ca(2+) could act as a potent inhibitor of α-glucosidase for the treatment of type 2 diabetes mellitus.

Effects of osmolytes on Pelodiscus sinensis creatine kinase: a study on thermal denaturation and aggregation.

The protective effect of osmolytes on the thermal denaturation and aggregation of Pelodiscus sinensis muscle creatine kinase (PSCK) was investigated by a combination of spectroscopic methods and thermodynamic analysis. Our results demonstrated that the addition of osmolytes, such as glycine and proline, could prevent thermal denaturation and aggregation of PSCK in a concentration-dependent manner. When the concentration of glycine and proline increased in the denatured system, the relative activation was significantly enhanced; meanwhile, the aggregation of PSCK during thermal denaturation was decreased. Spectrofluorometer results showed that glycine and proline significantly decreased the tertiary structural changes of PSCK and that thermal denaturation directly induced PSCK aggregation. In addition, we also built the 3D structure of PSCK and osmolytes by homology models. The results of computational docking simulations showed that the docking energy was relatively low and that the clustering groups were spread to the surface of PSCK, indicating that osmolytes directly protect the surface of the protein. P. sinensis are poikilothermic and quite sensitive to the change of ambient temperature; however, there were few studies on the thermal denaturation of reptile CK. Our study provides important insight into the protective effects of osmolytes on thermal denaturation and aggregation of PSCK.

Caspase and mitogen activated protein kinase pathways are involved in Solanum lyratum herba induced apoptosis

AIM OF STUDYnSolanum lyratum herba (SLH) has been traditionally used for the treatment of febrifuge, diarrhea, eye disease and cancer with little scientific evidences. Thus, in the present study, to elucidate the antitumor mechanism of SLH: in vitro and in vivo experiments were performed with hexane fraction of Solanum lyratum herba (HSLH).nnnMATERIALS AND METHODSnCytotoxicity assay, 4-6-diamidino-2-phenylindole (DAPI) staining, flow cytometric analysis for sub-G1 peaks, Western blot analysis were used with the antibodies of apoptosis related proteins in vitro. In addition, the effect of HSLH on in vivo tumor growth was evaluated in Lewis lung carcinoma (LLC) tumor model and immunohistochemistry also was performed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in tumor section.nnnRESULTSnHSLH exhibited cytotoxicity against LLC cells most effectively among its solvent fractions. Ladder like DNA fragmentation and apoptotic features such as chromatin condensation and apoptotic bodies were observed in HSLH treated LLC cells by 4-6-diamidino-2-phenylindole staining. HSLH also significantly increased sub-G(1) peaks, activated caspase-8, -9 and -3 proteins and cleaved poly(ADP-ribose) polymerase (PARP). Furthermore, HSLH increased the phosphorylation of extracellular signal-regulated kinase (ERK), transiently activated phospho-JNK (c-jun N-terminal kinase) and downregulated phospho-p38 MAPK. In addition, we have found for the first time HSLH treatment effectively suppressed the in vivo growth of LLC to up to approximately 30% of untreated control at 50mg/kg and significantly increased apoptotic expression in tumor section by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Taken together, these findings strongly demonstrate that hexane fraction of Solanum lyratum herba exerts antitumor activity via caspase activation and MAPK regulation and can be effectively applied to lung cancer as a cancer chemopreventive agent.

A folding study of Antarctic krill (Euphausia superba) alkaline phosphatase using denaturants

To gain insight into the structural and folding mechanisms of Antarctic krill alkaline phosphatase (ALP), the enzyme was properly purified by (NH4)2SO4 fractionation and by both Sephadex G-75 and DEAE anion exchange chromatography. The purified enzyme (62.6 kDa; 2.62 unit/mg) was unstable at temperatures exceeding 30°C. Denaturants, such as sodium dodecyl sulfate (SDS), guanidine HCl, and urea, were applied to evaluate the folding mechanism, including kinetics and thermodynamics, of krill ALP. Sodium dodecyl sulfate elicited no significant effect on ALP activity even at excessively high concentrations (300 mM), whereas guanidine HCl and urea effectively inactivated the enzyme at concentrations of 2 and 3.5 M, respectively. Kinetic studies showed that the enzymatic inhibition by guanidine HCl and urea represented a first-order reaction that was a monophasic unfolding process. This process was found to be associated with conformational changes without significant transient free-energy changes. Additionally, the overall structural changes occurred proximally to the active site pocket. Our study provides new insight into ALP of the Antarctic krill, which lives in extreme environmental conditions.

Effects of L-Malic Acid on Alpha-Glucosidase: Inhibition Kinetics and Computational Molecular Dynamics Simulations

The inhibitory effect of L-malic acid (MA) on alpha-glucosidase (EC 3.2.1.20) was investigated by combination study between inhibition kinetics and computational simulations. The results from the serial kinetics demonstrated that MA could directly inactivate the enzyme activity in a dose-dependent manner and a typical non-competitive type, as well as in a fast inactivate process without detectable time course. The tertiary conformation study with an application of spectrofluorimetry showed that MA modulated the tertiary structural conformation of alpha-glucosidase both on the overall and on regional active site pocket, which monitored by red-shift intrinsic fluorescence peak with decreases intensities, and the significant intensity increasing of 1-anilinonaphthalene-8-sulfonate (ANS)-binding fluorescence, respectively. To have more insight, we also adapted the computational molecular dynamics (MD) simulations. The results showed that MA was located in the entrance of active pocket for the catalytic reaction and blocked the passage of substrate. It confirmed that MA inhibits as a non-competitive type, not direct docking to the glucose binding site. Our study provides important molecular mechanisms to figure out alpha-glucosidase inhibition that might associate to development of type 2 diabetes mellitus drug.

The effect of validamycin A on tyrosinase: inhibition kinetics and computational simulation.

In this study, we investigated validamycin A as a tyrosinase inhibitor based on its structural properties. We found that the reversible inhibition of tyrosinase by validamycin A occurred in a mixed-type manner with Ki=5.893±0.038mM, as determined by integrating kinetics studies and computational simulations. Time-interval tyrosinase studies showed that the inhibition followed first-order kinetics with two phases. Fluorescence measurements of ANS binding showed that validamycin A induced changes in the tertiary protein structure of tyrosinase. To obtain further insight, computational docking and molecular dynamics were applied, and the results indicated that HIS85, HIS244, GLU256, HIS259, and ASN260 of tyrosinase interacted with validamycin A. This strategy of predicting tyrosinase inhibition based on hydroxyl group numbers might be useful in the design and screening of potential tyrosinase inhibitors.

Inhibitory effect of hesperetin on α-glucosidase: Molecular dynamics simulation integrating inhibition kinetics

The α-glucosidase inhibitor is of interest to researchers due to its association with type-2 diabetes treatment. Hesperetin is a flavonoid with natural antioxidant properties. This paper presents an evaluation on the effects of hesperetin on α-glucosidase via inhibitory kinetics using a Molecular Dynamics (MD) simulation integration method. Due to the antioxidant properties of hesperetin, it reversibly inhibits α-glucosidase in a slope-parabolic mixed-type manner (IC50=0.38±0.05mM; Kslope=0.23±0.01mM), accompanied by tertiary structural changes. Based on computational MD and docking simulations, two hesperetin rings interact with several residues near the active site on the α-glucosidase, such as Lys155, Asn241, Glu304, Pro309, Phe311 and Arg312. This study provides insight into the inhibition of α-glucosidase by binding hesperetin onto active site residues and accompanying structural changes. Hesperetin presents as a potential agent for treating α-glucosidase-associated type-2 diabetes based on its α-glucosidase-inhibiting effect and its potential as a natural antioxidant.