11 research outputs found
Inflammation Induced by Lipopolysaccharide and Palmitic Acid Increases Cholesterol Accumulation via Enhancing Myeloid Differentiation Factor 88 Expression in HepG2 Cells
Recently, multiple studies have shown that chronic inflammation disturbs cholesterol homeostasis and promotes its accumulation in the liver. The underlying molecular mechanism remains to be revealed. The relationship between the toll-like receptor 4 (TLR4) inflammatory signaling pathway and cholesterol accumulation was investigated in HepG2 cells treated with lipopolysaccharide (LPS) or palmitic acid (PA) for different lengths of time. In addition, the effects of pretreatment with 20ÎĽmol/L ST2825 (MyD88 inhibitor) were also studied in LPS- or PA-treated HepG2 cells and myeloid differentiation factor 88 (MyD88)-overexpressing HEK293T cells. The intracellular total and free cholesterol levels were measured using a commercial kit and filipin staining, respectively. The expression levels of sterol regulatory element-binding protein-2 (SREBP-2) and components in the TLR4 signaling pathway were determined using Western blotting. The treatments with LPS for 12 h and with PA for 24 h significantly increased the contents of intracellular total and free cholesterol, as well as the expression levels of SREBP-2 and components in the TLR4 signaling pathway. The inhibition of MyD88 by ST2825 significantly decreased the cholesterol content and the expression levels of SREBP-2 and components of the TLR4/MyD88/NF-ÎşB pathway in HepG2 cells, as well as MyD88-overexpressing HEK293T cells. These results indicated that LPS and PA treatments increase SREBP-2-mediated cholesterol accumulation via the activation of the TLR4/MyD88/NF-ÎşB signaling pathway in HepG2 cells
What Contributes to Serotonin–Norepinephrine Reuptake Inhibitors’ Dual-Targeting Mechanism? The Key Role of Transmembrane Domain 6 in Human Serotonin and Norepinephrine Transporters Revealed by Molecular Dynamics Simulation
Dual
inhibition of serotonin and norepinephrine transporters (hSERT
and hNET) gives greatly improved efficacy and tolerability for treating
major depressive disorder (MDD) compared with selective reuptake inhibitors.
Pioneer studies provided valuable information on structure, function,
and pharmacology of drugs targeting both hSERT and hNET (serotonin–norepinephrine
reuptake inhibitors, SNRIs), and the differential binding mechanism
between SNRIs and selective inhibitors of 5-HT (SSRIs) or NE (sNRIs)
to their corresponding targets was expected to be able to facilitate
the discovery of a privileged drug-like scaffold with improved efficacy.
However, the dual-target mechanism of SNRIs was still elusive, and
the binding mode distinguishing SNRIs from SSRIs and sNRIs was also
unclear. Herein, an integrated computational strategy was adopted
to discover the binding mode shared by all FDA approved SNRIs. The
comparative analysis of binding free energy at the per-residue level
discovered that residues Phe335, Leu337, Gly338, and Val343 located
at the transmembrane domain 6 (TM6) of hSERT (the corresponding residues
Phe317, Leu319, Gly320, and Val325 in hNET) were the determinants
accounting for SNRIs’ dual-acting inhibition, while residues
lining TM3 and 8 (Ile172, Ser438, Thr439, and Leu443 in hSERT; Val148,
Ser419, Ser420, and Met424 in hNET) contributed less to the binding
of SNRIs than that of SSRIs and sNRIs. Based on these results, the
distances between an SNRI’s centroid and the centroids of its
two aromatic rings (measuring the depth of rings stretching into hydrophobic
pockets) were discovered as the key to the SNRIs’ dual-targeting
mechanism. This finding revealed SNRIs’ binding mechanism at
an atomistic level, which could be further utilized as structural
blueprints for the rational design of privileged drug-like scaffolds
treating MDD
Exploring the Binding Mechanism of Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators in Clinical Trials by Molecular Dynamics Simulations
Metabotropic
glutamate receptor 5 (mGlu<sub>5</sub>) plays a key role in synaptic
information storage and memory, which is a well-known target for a
variety of psychiatric and neurodegenerative disorders. In recent
years, the increasing efforts have been focused on the design of allosteric
modulators, and the negative allosteric modulators (NAMs) are the
front-runners. Recently, the architecture of the transmembrane (TM)
domain of mGlu<sub>5</sub> receptor has been determined by crystallographic
experiment. However, it has been not well understood how the pharmacophores
of NAMs accommodated into the allosteric binding site. In this study,
molecular dynamics (MD) simulations were performed on mGlu<sub>5</sub> receptor bound with NAMs in preclinical or clinical development
to shed light on this issue. In order to identify the key residues,
the binding free energies as well as per-residue contributions for
NAMs binding to mGlu<sub>5</sub> receptor were calculated. Subsequently,
the <i>in silico</i> site-directed mutagenesis of the key
residues was performed to verify the accuracy of simulation models.
As a result, the shared common features of the studied 5 clinically
important NAMs (mavoglurant, dipraglurant, basimglurant, STX107, and
fenobam) interacting with 11 residues in allosteric site were obtained.
This comprehensive study presented a better understanding of mGlu<sub>5</sub> receptor NAMs binding mechanism, which would be further used
as a useful framework to assess and discover novel lead scaffolds
for NAMs
Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/beta-catenin signaling in obstructed kidneys in vivo
Follistatin (FS)-like 1 (FSTL1) is a member of the FS-SPARC (secreted protein, acidic and rich in cysteine) family of secreted and extracellular matrix proteins. The functions of FSTL1 have been studied in heart and lung injury as well as in wound healing; however, the role of FSTL1 in the kidney is largely unknown. Here, we show using single-cell RNA-Seq that Fstl1 was enriched in stromal cells in obstructed mouse kidneys. In addition, immunofluorescence demonstrated that FSTL1 expression was induced in fibro-blasts during kidney fibrogenesis in mice and human patients. We demonstrate that FSTL1 overexpression increased renal fibrosis and activated the Wnt/beta-catenin signaling pathway, known to promote kidney fibrosis, but not the transforming growth factor beta (TGF-beta), Notch, Hedgehog, or Yes-associated protein (YAP) signaling pathways in obstructed mouse kidneys, whereas inhibition of FSTL1 lowered Wnt/beta-catenin signaling. Importantly, we show that FSTL1 interacted with Wnt ligands and the Frizzled (FZD) receptors but not the coreceptor lipoprotein receptor-related protein 6 (LRP6). Specifically, we found FSTL1 interacted with Wnt3a through its extracellular calcium-binding (EC) domain and von Willebrand factor type C-like (VWC) domain, and with FZD4 through its EC domain. Furthermore, we show that FSTL1 increased the association of Wnt3a with FZD4 and promoted Wnt/beta-catenin signaling and fibrogenesis. The EC domain interacting with both Wnt3a and FZD4 also enhanced Wnt3a signaling. Therefore, we conclude that FSTL1 is a novel extracellular enhancer of the Wnt/beta-catenin pathway