The ability to identify the site of a protein that can bind with high affinity to small, drug-like compounds has been an important goal in drug design. Sirtuin 2 (SIRT2), histone deacetylase protein ...family, plays a central role in the regulation of various pathways. Hence, identification of drug for SIRT2 has attracted great interest in the drug discovery community. To elucidate the molecular basis of the small molecules interactions to inhibit the SIRT2 function we employed the molecular docking, molecular dynamics simulations, and the molecular mechanism Poisson-Boltzmann/surface area (MM-PBSA) calculations. Five well know inhibitors such as suramin, mol-6, sirtinol, 67, and nf675 were selected to establish the nature of the binding mode of the inhibitors in the SIRT2 active site. The molecular docking and dynamics simulations results revealed that the hydrogen bonds between Arg97 and Gln167 are crucial to inhibit the function of SIRT2. In addition, the MM-PBSA calculations revealed that binding of inhibitors to SIRT2 is mainly driven by van der Waals/non-polar interactions. Although the five inhibitors are very different in structure, shape, and electrostatic potential, they are able to fit in the same binding pocket. These findings from this study provide insights to elucidate the binding pattern of SIRT2 inhibitors and help in the rational structure-based design of novel SIRT2 inhibitors with improved potency and better resistance profile.
Mitochondrial calcium uptake proteins 1 and 2 (MICU1 and MICU2) mediate mitochondrial Ca
influx via the mitochondrial calcium uniporter (MCU). Its molecular action for Ca
uptake is tightly controlled ...by the MICU1-MICU2 heterodimer, which comprises Ca
sensing proteins which act as gatekeepers at low Ca
or facilitators at high Ca
. However, the mechanism underlying the regulation of the Ca
gatekeeping threshold for mitochondrial Ca
uptake through the MCU by the MICU1-MICU2 heterodimer remains unclear. In this study, we determined the crystal structure of the apo form of the human MICU1-MICU2 heterodimer that functions as the MCU gatekeeper. MICU1 and MICU2 assemble in the face-to-face heterodimer with salt bridges and me-thio-nine knobs stabilizing the heterodimer in an apo state. Structural analysis suggests how the heterodimer sets a higher Ca
threshold than the MICU1 homodimer. The structure of the heterodimer in the apo state provides a framework for understanding the gatekeeping role of the MICU1-MICU2 heterodimer.
Buddleja officinalis
has been traditionally used in the supportive treatment of inflammatory and neuronal diseases in Korea and China. Although several reports have shown the anti-inflammatory ...effects of
Buddleja officinalis
, the anti-neuroinflammatory effect has remained unclear. In this study, we aimed to investigate the inhibitory effects of flower buds of
B. officinalis
Maximowicz water extract (BOWE) on LPS-induced inflammatory processes in BV-2 microglial cells. BOWE dose-dependently inhibited the production of nitric oxide as well as iNOS mRNA expression. Moreover, BOWE prevented IL-1β and IL-6 mRNA expression. However, BOWE had no effect on LPS-induced COX-2 or TNF-α mRNA expression. The extract also had no effect on LPS-stimulated p38 MAPK, JNK, and c-Jun phosphorylation, whereas ERK1/2 phosphorylation was strongly inhibited by BOWE. BOWE also inhibited the LPS-induced degradation of IκB-α, and LPS-induced phosphorylation of p65 NF-κB protein. These data indicate that BOWE inhibited the nitric oxide production and pro-inflammatory gene expression in BV-2 microglial cells, possibly through a negative regulation of the NF-κB and ERK1/2 pathways. Further identification of the direct target molecule(s) of BOWE is required to support its use as an anti-neuroinflammatory agent against the neurodegenerative disorders.
EF-hand proteins, which contain a Ca2+-binding EF-hand motif, are involved in regulating diverse cellular functions. Ca2+ binding induces conformational changes that modulate the activities of ...EF-hand proteins. Moreover, these proteins occasionally modify their activities by coordinating metals other than Ca2+, including Mg2+, Pb2+ and Zn2+, within their EF-hands. EFhd1 and EFhd2 are homologous EF-hand proteins with similar structures. Although separately localized within cells, both are actin-binding proteins that modulate F-actin rearrangement through Ca2+-independent actin-binding and Ca2+-dependent actin-bundling activity. Although Ca2+ is known to affect the activities of EFhd1 and EFhd2, it is not known whether their actin-related activities are affected by other metals. Here, the crystal structures of the EFhd1 and EFhd2 core domains coordinating Zn2+ ions within their EF-hands are reported. The presence of Zn2+ within EFhd1 and EFhd2 was confirmed by analyzing anomalous signals and the difference between anomalous signals using data collected at the peak positions as well as low-energy remote positions at the Zn K-edge. EFhd1 and EFhd2 were also found to exhibit Zn2+-independent actin-binding and Zn2+-dependent actin-bundling activity. This suggests the actin-related activities of EFhd1 and EFhd2 could be regulated by Zn2+ as well as Ca2+.
Background
In experimental models, bone marrow‐derived mesenchymal stem cells (BM‐MSCs) have the capacity to differentiate into hepatocytes and exhibit antifibrotic effects. However, there have been ...no studies in humans with alcoholic cirrhosis.
Aim
The aim of this study was to elucidate the antifibrotic effect of BM‐MSCs in patients with alcoholic cirrhosis, as a phase II clinical trial.
Methods
Twelve patients (11 males, 1 female) with baseline biopsy‐proven alcoholic cirrhosis who had been alcohol free for at least 6 months were enrolled. BM‐MSCs were isolated from each patient's BM and amplified for 1 month, and 5 × 107 cells were then injected twice, at weeks 4 and 8, through the hepatic artery. One patient was withdrawn because of ingestion of alcohol. Finally, 11 patients completed the follow‐up biopsy and laboratory tests at 12 weeks after the second injection. The primary outcome was improvement in the patients’ histological features.
Results
According to the Laennec fibrosis system, histological improvement was observed in 6 of 11 patients (54.5%). The Child‐Pugh score improved in ten patients (90.9%) and the levels of transforming growth factor‐β1, type 1 collagen and α‐smooth muscle actin significantly decreased (as assessed by real‐time reverse transcriptase polymerase chain reaction) after BM‐MSCs therapy (P < 0.05). No significant complications or side effects were observed during this study.
Conclusions
Bone marrow‐derived mesenchymal stem cells therapy in alcoholic cirrhosis induces a histological and quantitative improvement of hepatic fibrosis.
The RtcB protein has recently been identified as a 3′-phosphate RNA ligase that directly joins an RNA strand ending with a 2′,3′-cyclic phosphate to the 5′-hydroxyl group of another RNA strand in a ...GTP/Mn ²⁺-dependent reaction. Here, we report two crystal structures of Pyrococcus horikoshii RNA-splicing ligase RtcB in complex with Mn ²⁺ alone (RtcB/ Mn ²⁺) and together with a covalently bound GMP (RtcB-GMP/Mn ²⁺). The RtcB/ Mn ²⁺ structure (at 1.6 Å resolution) shows two Mn ²⁺ ions at the active site, and an array of sulfate ions nearby that indicate the binding sites of the RNA phosphate backbone. The structure of the RtcB-GMP/Mn ²⁺ complex (at 2.3 Å resolution) reveals the detailed geometry of guanylylation of histidine 404. The critical roles of the key residues involved in the binding of the two Mn ²⁺ ions, the four sulfates, and GMP are validated in extensive mutagenesis and biochemical experiments, which also provide a thorough characterization for the three steps of the RtcB ligation pathway: (i) guanylylation of the enzyme, (ii) guanylyl-transfer to the RNA substrate, and (iii) overall ligation. These results demonstrate that the enzyme’s substrate-induced GTP binding site and the putative reactive RNA ends are in the vicinity of the binuclear Mn ²⁺ active center, which provides detailed insight into how the enzyme-bound GMP is tansferred to the 3′-phosphate of the RNA substrate for activation and subsequent nucleophilic attack by the 5′-hydroxyl of the second RNA substrate, resulting in the ligated product and release of GMP.
•The crystal structure of GKAP homology domain 1 (GH1) was determined.•GKAP GH1 is a three-helix bundle connected by short flexible loops.•The predicted helix α4 associates weakly with the helix α3, ...suggesting dynamic nature of the GH1 domain.
Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domain of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.
The endoplasmic reticulum (ER) consists of the nuclear envelope and a connected peripheral network of tubules and interspersed sheets. The structure of ER tubules is generated and maintained by ...various proteins, including reticulons, DP1/Yop1p, atlastins, and lunapark. Reticulons and DP1/Yop1p stabilize the high membrane curvature of ER tubules, and atlastins mediate homotypic membrane fusion between ER tubules; however, the exact role of lunapark remains poorly characterized. Here, using isolated yeast ER microsomes and reconstituted proteoliposomes, we directly examined the function of the yeast lunapark Lnp1p for yeast atlastin Sey1p-mediated ER fusion and found that Lnp1p inhibits Sey1p-driven membrane fusion. Furthermore, by using a newly developed assay for monitoring trans-Sey1p complex assembly, a prerequisite for ER fusion, we found that assembly of trans-Sey1p complexes was increased by the deletion of LNP1 and decreased by the overexpression of Lnp1p, indicating that Lnp1p inhibits Sey1p-mediated fusion by interfering with assembly of trans-Sey1p complexes.
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•The yeast lunapark Lnp1p inhibits Sey1p-mediated ER fusion•Trans-Sey1p complexes can be preserved in the presence of GMP-PNP or GDP/AlF4−•The N-terminal region of Lnp1p prevents the formation of trans-Sey1p complexes
Biochemistry; Molecular biology; Cell biology
Abstract The increased permeability of the blood–retinal barrier is known to occur in patients with diabetes, and this defect contributes to retinal edema. This study aimed to determine the effects ...of silver nanoparticles (Ag-NPs) on advanced glycation end-products (AGEs)-induced endothelial cell permeability. Cultured porcine retinal endothelial cells (PRECs) were exposed to AGE-modified bovine serum albumin (AGE-BSA) and the endothelial cell permeability was detected by measuring the flux of RITC-dextran across the PREC monolayers. We found that AGE-BSA increased the dextran flux across a PREC monolayer and Ag-NPs blocked the solute flux induced by AGE-BSA. In order to understand the underlying signaling mechanism of Ag-NPs on the inhibitory effect of AGE-BSA-induced permeability, we demonstrated that Ag-NPs could inhibit the AGE-BSA-induced permeability via Src kinase pathway. AGE-BSA also increased the PREC permeability by stimulating the expression of intracellular adhesion molecule-1 (ICAM-1) and decreased the expression of occludin and ZO-1. Further, Ag-NPs inhibited the AGE-BSA-induced permeability by increased expression of tight junction proteins occludin and ZO-1, co-incident with an increase in barrier properties of endothelial monolayer. Together, our results indicate that Ag-NPs could possibly act as potent anti-permeability molecule by targeting the Src signaling pathway and tight junction proteins and it offers potential targets to inhibit the ocular related diseases.
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