- Structural basis for the glycosyltransferase activity of the Salmonella effector SseK3. [Journal Article]
- JBJ Biol Chem 2018 Feb 15
- The Salmonella secreted effector SseK3 translocates into host cells, targeting innate immune responses including NF-κB activation. SseK3 is a glycosyltransferase that transfers an N-acetylglucosamine...
The Salmonella secreted effector SseK3 translocates into host cells, targeting innate immune responses including NF-κB activation. SseK3 is a glycosyltransferase that transfers an N-acetylglucosamine (GlcNAc) moiety onto the guanidino group of a target arginine, modulating host cell function. However, a lack of structural information has precluded elucidation of the molecular mechanisms in arginine and GlcNAc selection. We report here the crystal structure of SseK3 in its apo form and in complex with hydrolysed UDP-GlcNAc. SseK3 possesses the typical glycosyltransferase type-A (GT-A)-family fold and the metal-coordinating DXD motif essential for ligand binding and enzymatic activity. Several conserved residues were essential for arginine-GlcNAcylation and SseK3-mediated inhibition of NF-κB activation. Isothermal titration calorimetry revealed SseK3's preference for manganese coordination. The pattern of interactions in the substrate-bound SseK3 structure explained the selection of the primary ligand. Structural re-arrangement of the C-terminal residues upon ligand binding was crucial for SseK3's catalytic activity and NMR analysis indicated that SseK3 has limited UDP-GlcNAc hydrolysis activity. The release of free N-acetyl α-D-glucosamine, and the presence of the same molecule in the SseK3 active site, classified it as a retaining glycosyltransferase. A glutamate residue in the active site suggested a double-inversion mechanism for the arginine N-glycosylation reaction. Homology models of SseK1, SseK2, and the Escherichia coli orthologue NleB1, reveal differences in the surface electrostatic charge distribution possibly accounting for their diverse activities. This first structure of a retaining GT-A arginine N-glycosyltransferase provides an important step towards a better understanding of this enzyme class and their roles as bacterial effectors.
- Low apolipoprotein A-I levels in Friedreich's ataxia and in frataxin-deficient cells: Implications for therapy. [Journal Article]
- PlosPLoS One 2018; 13(2):e0192779
- Friedreich's ataxia (FA) is an autosomal recessive neurodegenerative disorder, which results primarily from reduced expression of the mitochondrial protein frataxin. FA has an estimated prevalence of...
Friedreich's ataxia (FA) is an autosomal recessive neurodegenerative disorder, which results primarily from reduced expression of the mitochondrial protein frataxin. FA has an estimated prevalence of one in 50,000 in the population, making it the most common hereditary ataxia. Paradoxically, mortality arises most frequently from cardiomyopathy and cardiac failure rather than from neurological effects. Decreased high-density lipoprotein (HDL) and apolipoprotein A-I (ApoA-l) levels in the general population are associated with an increased risk of mortality from cardiomyopathy and heart failure. However, the pathophysiology of heart disease in FA is non-vascular and there are conflicting data on HDL-cholesterol in FA. Two studies have shown a decrease in HDL-cholesterol compared with controls and two have shown there was no difference between FA and controls. One also showed that there was no difference in serum Apo-A-I levels in FA when compared with controls. Using a highly specific stable isotope dilution mass spectrometry-based assay, we demonstrated a 21.6% decrease in serum ApoA-I in FA patients (134.8 mg/dL, n = 95) compared with non-affected controls (172.1 mg/dL, n = 95). This is similar to the difference in serum ApoA-I levels between non-smokers and tobacco smokers. Knockdown of frataxin by > 70% in human hepatoma HepG2 cells caused a 20% reduction in secreted ApoA-I. Simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor caused a 200% increase in HMG-CoA in the control HepG2 cells with a similar increase in the frataxin knockdown HepG2 cells, back to levels found in the control cells. There was a concomitant 20% increase in secreted ApoA-I to levels found in the control cells that were treated with simvastatin. This study provides compelling evidence that ApoA-I levels are reduced in FA patients compared with controls and suggest that statin treatment would normalize the ApoA-I levels.
- Effects of Peutz-Jeghers syndrome (PJS) causing missense mutations L67P, L182P, G242V and R297S on the structural dynamics of LKB1 (Liver kinase B1) protein. [Journal Article]
- JBJ Biomol Struct Dyn 2018 Feb 15; :1-35
- The Liver kinase B1 (LKB1) is encoded by LKB1 gene. Several pathogenic mutations of LKB1 causing Peutz-Jeghers syndrome (PJS) and also cancers in breast, gastric, pancreas and colon have been reporte...
The Liver kinase B1 (LKB1) is encoded by LKB1 gene. Several pathogenic mutations of LKB1 causing Peutz-Jeghers syndrome (PJS) and also cancers in breast, gastric, pancreas and colon have been reported. The present study is focused to analyze the effects on the structural dynamics of LKB1 caused by the 4 pathogenic missense mutations (L67P, L182P, G242V and R297S), which are reported to reduce the catalytic activity. In this study, the structural changes of LKB1 in apo- and in heterotrimeric complex (LKB1-STRADα-MO25α) form with wild and mutated LKB1 are investigated using all atomistic molecular dynamic simulation. The present study reveals that these four mutations initiate local structural distortions and the solvent accessibility of the surrounding regions of ATP binding pocket such as Glycine rich loop, αB and αC loop, activation and catalytic loops. The mutations of L67P, L182P and G242V induce distortions of the secondary structure of β1-β3 sheets, π-π interaction (observed between Phe204 of LKB1 and Phe243 of MO25α) and increase the helical properties (both helical twist and length) of the adjacent αH-helix, respectively. The active kinase features like the conformation of catalytic and activation loops, salt bridge and finally the formation of stable R- and C-hydrophobic spines are also found to be perturbed by these mutations. Hence, the observed mutation induced structural distortions fail to coordinate the essential binding nature of LKB1 with STRADα and MO25α, which eventually affects the native function of LKB1. These observations are in line with the experimentally reported reduced kinase activity of LKB1.
- The Structure of the Apo cAMP-Binding Domain of HCN4: A Stepping Stone towards Understanding the cAMP-Dependent Modulation of the Hyperpolarization-Activated Cyclic-Nucleotide-Gated (HCN) Ion Channels. [Review]
- FJFEBS J 2018 Feb 14
- The hyperpolarization-activated cyclic-nucleotide-gated (HCN) ion channels control nerve impulse transmission and cardiac pacemaker activity. The modulation by cAMP is critical for the regulatory fun...
The hyperpolarization-activated cyclic-nucleotide-gated (HCN) ion channels control nerve impulse transmission and cardiac pacemaker activity. The modulation by cAMP is critical for the regulatory function of HCN in both neurons and cardiomyocytes, but the underlying mechanism is not fully understood. Here, we show how the structure of the apo cAMP-binding domain of the HCN4 isoform has contributed to a model for the cAMP-dependent modulation of the HCN ion-channel. This model recapitulates the structural and dynamical changes that occur along the thermodynamic cycle arising from the coupling of cAMP-binding and HCN self-association equilibria. The proposed model addresses some of the questions previously open about the auto-inhibition of HCN and its cAMP-induced activation, while opening new opportunities for selectively targeting HCN through allosteric ligands. A remaining challenge is the investigation of HCN dimers and their regulatory role. Overcoming this challenge will require the integration of crystallography, cryo EM, NMR, electrophysiology and simulations. This article is protected by copyright. All rights reserved.
- Effects of MALAT1 on proliferation and apo- ptosis of human non-small cell lung cancer A549 cells in vitro and tumor xenograft growth in vivo by modulating autophagy. [Journal Article]
- CBCancer Biomark 2018 Jan 29
- CONCLUSIONS: Downregulation of MALAT1 may promote apoptosis and suppress proliferation, migration and invasion of human NSCLC A549 cells by inhibiting autophagy, thereby suppressing the development of NSCLC.
- Effect of curcumin on amyloid-like aggregates generated from methionine-oxidized apolipoprotein A-I. [Journal Article]
- FOFEBS Open Bio 2018; 8(2):302-310
- Curcumin is a polyphenolic phytonutrient that has antineurodegenerative properties. In this study, we investigated the anti-amyloidogenic properties of curcumin. Following incubation with curcumin, i...
Curcumin is a polyphenolic phytonutrient that has antineurodegenerative properties. In this study, we investigated the anti-amyloidogenic properties of curcumin. Following incubation with curcumin, intrinsic tryptophan fluorescence emission of apolipoprotein (apo) A-I was strongly quenched. At the same time, curcumin fluorescence emission was enhanced. The fluorescence emission spectra of curcumin in the presence of amyloid-like aggregates formed by methionine-oxidized (ox) apoA-I varied, depending on whether curcumin was added before, or after, aggregate formation. The impact of curcumin on the structure of the aggregating material was revealed by the lower amount of β-structure in ox-apoA-I amyloid-like aggregates formed in the presence of curcumin, compared to aggregates formed without curcumin. However, the kinetics of ox-apoA-I amyloid-like aggregate formation was not altered by the presence of curcumin. Moreover, electron microscopy analysis detected no discernable differences in amyloid morphology when ox-apoA-I amyloid-like aggregates were formed in the presence or absence of curcumin. In conclusion, curcumin interacts with apoA-I and alters the structure of ox-apoA-I amyloid-like aggregates yet does not diminish the propensity of ox-apoA-I to form aggregates.
- Applying pose clustering and MD simulations to eliminate false positives in molecular docking. [Journal Article]
- JCJ Chem Inf Model 2018 Feb 12
- In this work, we developed a computational protocol that employs multiple molecular docking experiments, followed by pose clustering, molecular dynamic simulations (10ns), and energy rescoring to pro...
In this work, we developed a computational protocol that employs multiple molecular docking experiments, followed by pose clustering, molecular dynamic simulations (10ns), and energy rescoring to produce reliable 3D models of antibody-carbohydrate complexes. The protocol was applied to 10 carbohydrate-antibody co-complexes and 3 unliganded (apo) antibodies. Pose clustering significantly reduced the number of potential poses. For each system 15 or fewer clusters, out of 100 initial poses, were generated and chosen for further analysis. MD simulations allowed the docked poses to either converge or disperse, and rescoring increased the likelihood that the best-ranked pose was an acceptable pose. This approach is amenable to automation, and can be a valuable aid in determining the structure of antibody-carbohydrate complexes provided there is no major side chain rearrangement or backbone conformational change in the H3 loop of the CDR regions. Further, the basic protocol of docking a small ligand to a known binding site, clustering the results, and performing MD with a suitable force field is applicable to any protein ligand system.
- Lipid-Modulating Effect of Black Lingzhi Medicinal Mushroom, Amauroderma rugosum (Agaricomycetes), on Oleate-Induced Human Hepatocellular Liver Carcinoma Cells. [Journal Article]
- IJInt J Med Mushrooms 2017; 19(12):1101-1111
- Dyslipidemia is the key precursor of atherosclerotic cardiovascular disease. The aim of this study was to investigate the lipid-modifying potential of organic solvent-partitioned extracts from fruiti...
Dyslipidemia is the key precursor of atherosclerotic cardiovascular disease. The aim of this study was to investigate the lipid-modifying potential of organic solvent-partitioned extracts from fruiting bodies of Amauroderma rugosum in vitro using oleate-induced human hepatocellular liver carcinoma (HepG2) cells. Our results demonstrated that oleate-induced HepG2 cells treated with ethyl acetate (EA) extract greatly decreased intracellular and secreted total triglyceride (TG) and total cholesterol (TC) compared with other extracts. Further investigation of cellular expression of selected apolipoproteins also revealed that oleate-induced HepG2 cells treated with the EA extract best attenuated the apolipoprotein (Apo) profile by downregulating ApoB-100 and ApoE while upregulating ApoA1. Because both ApoB-100 and ApoE are key components of low-density lipoprotein (LDL) and very LDL (VLDL), which are recognized as "bad cholesterol," this result indicates that treatment with the EA extract inhibited LDL and VLDL production in oleate-induced HepG2 cells. On the other hand, increasing ApoA1 evidence shows antiatherogenic benefits to increasing ApoA1, the key component of high-density lipoprotein (HDL), particularly in relation to its role in promoting reverse cholesterol transport and preventing LDL oxidation; this indicates that the EA extract upregulated the production of HDL ("good cholesterol"). Hence, the EA extract is a good source of lipid-ameliorating agents in the management of dyslipidemia.
- Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features. [Journal Article]
- FJFEBS J 2018 Feb 12
- Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probabl...
Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probably, in part, due to their complex oligomeric nature and a whole spectrum of active site conformations related to interactions with different ligands. Here we report an extensive structural characterization of the apo forms of hexameric PNP from Helicobacter pylori (HpPNP), as well as its complexes with phosphate (Pi) and an inhibitor, formycin A (FA), together with kinetic, binding, docking and molecular dynamics studies. X-ray structures show previously unseen distributions of open and closed active sites. Microscale thermophoresis results indicate that a two-site model describes Pibinding, while a three-site model is needed to characterize FA binding, irrespective of Pipresence. The latter may be related to the newly found non-standard mode of FA binding. The ternary complex of the enzyme with Piand FA shows, however, that Pibinding stabilizes the standard mode of FA binding. Surprisingly, HpPNP has low affinity towards the natural substrate adenosine. Molecular dynamics simulations show that Pimoves out of most active sites, in accordance with its weak binding. Conformational changes between non-standard and standard binding modes of nucleoside are observed during the simulations. Altogether, these findings show some unique features of HpPNP and provide new insights into the functioning of the active sites, with implications for understanding the complex mechanism of catalysis of this enzyme. This article is protected by copyright. All rights reserved.
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- Engineered exosome-mediated delivery of functionally active miR-26a and its enhanced suppression effect in HepG2 cells. [Journal Article]
- IJInt J Nanomedicine 2018; 13:585-599
- CONCLUSIONS: Our gene delivery strategy can be adapted to treat a broad spectrum of cancers by expressing proteins on the surface of miRNA-loaded exosomes that recognize specific biomarkers on the tumor cell.