[Show abstract][Hide abstract] ABSTRACT: The crystal structure of 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDO8PS) from Helicobacter pylori (HpKDO8PS) was determined alone and within various complexes, revealing an extra helix (HE) that is absent in the structures of KDO8PS from other organisms. In contrast to the metal coordination of the KDO8PS enzyme from Aquifex aeolicus, HpKDO8PS is specifically coordinated with Cd(2+) or Zn(2+) ions, and isothermal titration calorimetry (ITC) and differential scanning fluorimetry (DSF) revealed that Cd(2+) thermally stabilizes the protein structure more efficiently than Zn(2+). In the substrate-bound structure, water molecules play a key role in fixing residues in the proper configuration to achieve a compact structure. Using the structures of HpKDO8PS and API [arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) bisubstrate inhibitor], we generated 21 compounds showing potential HpKDO8PS-binding properties via in silico virtual screening. The capacity of three, avicularin, hyperin, and MC181, to bind to HpKDO8PS was confirmed through saturation transfer difference (STD) experiments, and we identified their specific ligand binding modes by combining competition experiments and docking simulation analysis. Hyperin was confirmed to bind to the A5P binding site, primarily via hydrophilic interaction, whereas MC181 bound to both the PEP and A5P binding sites through hydrophilic and hydrophobic interactions. These results were consistent with the epitope mapping by STD. Our results are expected to provide clues for the development of HpKDO8PS inhibitors.
No preview · Article · Jan 2016 · European Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: The Alba superfamily proteins have been regarded as a conserved group of proteins in archaea and eukarya, which have shown to be important in nucleic acid binding, chromatic organization and gene regulation. These proteins often belong to the N-acetyltransferase (NAT) category (N(α)-acetyltransferases or N(ε)-acetyltransferases) and undergo post-translational modifications. Here, we report the crystal structure of Alba from Thermoplasma volcanium (Tv Alba) at 2.4 Å resolution. The acetylation of Tv Alba was monitored and the N-terminal of Tv Alba has been shown to interact with acetyl coenzyme A (Ac-CoA). The chemical shift perturbation experiments of Tv Alba were performed in the presence of Ac-CoA and/or Tv Ard1, another T. volcanium protein that treats Tv Alba as a substrate. To examine the DNA binding capabilities of Tv Alba alone and in the presence of Ac-CoA and/or Tv Ard1, EMSA experiments were carried out. It is shown that although Tv Alba binds to Ac-CoA, the acetylation of Tv Alba is not related with its binding to dsDNA, and the involvement of the N-terminus in Ac-CoA binding demonstrates that Tv Alba belongs to the N(α)-acetyltransferase family.
No preview · Article · Nov 2015 · Archives of Biochemistry and Biophysics
[Show abstract][Hide abstract] ABSTRACT: Phosphoinositide-dependent protein kinase 1 (PDK1) plays a key role in the phosphoinositide 3-kinase (PI3K)-PDK1-Akt pathway that induces cell survival and cardiovascular protections through anti-apoptosis, vasodilation, anti-inflammation, and anti-oxidative stress activities. Although several reports have proposed the negative role of PDK1 in Toll-like receptor 4 (TLR4) signaling, the molecular mechanism is still unknown. Here we show that PDK1 inhibits tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) ubiquitination by interrupting the complex between transforming growth factor beta-activated kinase 1 (TAK1) and TAK1 binding protein 2 (TAB2), which negatively regulates TAK1 activity. The overexpression of PDK1 in 293/TLR4 cells resulted in suppressions of nuclear factor kappa B (NF-κB) activation and production of proinflammatory cytokines including interleukin (IL)-6 and TNF-α in response to lipopolysaccharide stimulation. Conversely, THP-1 human monocytes transiently cultured in low glucose medium displayed down-regulated PDK1 expression, and significantly enhanced TLR4-mediated signaling for the activation of NF-κB, demonstrating a negative role of PDK1. Biochemical studies revealed that PDK1 significantly interacted with TAK1, resulting in the inhibition of the association of TAB2 with TAK1, which led to the attenuation of TRAF6 ubiquitination. Moreover, PDK1-knockdown THP-1 cells displayed enhancement of downstream signals, activation of NF-κB, and increased production of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α, which potentially led to the up-regulation of NF-κB-dependent genes in response to TLR4 stimulation. Collectively, the results demonstrate that PDK1 inhibits the formation of the TAK1-TAB2-TRAF6 complex and leads to the inhibition of TRAF6 ubiquitination, which negatively regulates the TLR4-mediated signaling for NF-κB activation.
No preview · Article · Sep 2015 · Cellular Signalling
[Show abstract][Hide abstract] ABSTRACT: ECSIT (evolutionarily conserved signaling intermediate in Toll pathways) is known as a multifunctional regulator in different
signals, including Toll-like receptors (TLRs), TGF-β, and BMP. Here, we report a new regulatory role of ECSIT in TLR4-mediated
signal. By LPS stimulation, ECSIT formed a high molecular endogenous complex including TAK1 and TRAF6, in which ECSIT interacted
with each protein and regulated TAK1 activity, leading to the activation of NF-κB. ECSIT-knockdown THP-1 (ECSITKD THP-1) cells exhibited severe impairments in NF-κB activity, cytokine production, and NF-κB-dependent gene expression, whereas
those were dramatically restored by reintroduction of wild type (WT) ECSIT gene. Interestingly, ECSIT mutants, which lack
a specific interacting domain for either TAK1 or TRAF6, could not restore these activities. Moreover, no significant changes
in both NF-κB activity and cytokine production induced by TLR4 could be seen in TAK1KD or TRAF6KD THP-1 cells transduced by WT ECSIT, strongly suggesting the essential requirement of TAK1-ECSIT-TRAF6 complex in TLR4 signaling.
Taken together, our data demonstrate that the ECSIT complex, including TAK1 and TRAF6, plays a pivotal role in TLR4-mediated
signals to activate NF-κB.
No preview · Article · Nov 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Recent evidence shows that evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) interacts with tumor necrosis factor receptor-associated factor 6 (TRAF6) ubiquitinated, and contributes to bactericidal activity during Toll-like receptor (TLR) signaling. Here, we first report a new regulatory role for ECSIT in TLR4 signaling. On TLR4 stimulation, endogenous ECSIT forms a molecular complex if p65/p50 NF- Κ: Bs. Our biochemical studies show that ECSIT specifically interacted with p65/p50 NF- Κ: Bs, which colocalized in the nucleus. Interestingly, these effects were critically dependent on ubiquitination of the ECSIT lysine (K) 372 residue. K372A mutant ECSIT did not interact with p65/p50 NF- Κ: Bs, and markedly attenuated nuclear colocalization. Additionally, ECSIT-knockdown THP-1 cells could not activate NF- Κ: B DNA-binding activities of p65 and p50, production of proinflammatory cytokines, and NF- Κ: B-dependent gene expression in response to TLR4 stimulation. However, these activities were markedly restored by expressing the wild-type ECSIT protein but not the K372A mutant ECSIT protein. These date strongly suggest that the ubiquitination of ECSIT might have the regulation of NF- Κ: B activity in TLR4 signaling.
Full-text · Article · Oct 2014 · Molecular Biology of the Cell
[Show abstract][Hide abstract] ABSTRACT: It is debatable whether AMP-activated protein kinase (AMPK) activation is involved in anti-apoptotic or pro-apoptotic signaling. AICAR treatment increases AMPK-α1 phosphorylation, decreases intracellular reactive oxygen species (ROS) levels, and significantly increases Annexin V-positive cells, DNA laddering, and caspase activity in human myeloid cell. AMPK activation is therefore implicated in apoptosis. However, AMPK-α1-knockdown THP-1 cells are more sensitive to apoptosis than control THP-1 cells are, suggesting that the apoptosis is AMPK-independent. Low doses of AICAR induce cell proliferation, whereas high doses of AICAR suppress cell proliferation. Moreover, these effects are significantly correlated with the downregulation of intracellular ROS, strongly suggesting that AICAR-induced apoptosis is critically associated with the inhibition of NADPH oxidase by AICAR. Collectively, our results demonstrate that in AICAR-induced apoptosis, intracellular ROS levels are far more relevant than AMPK activation.
[Show abstract][Hide abstract] ABSTRACT: HP1492 is a NifU-like protein of Helicobacter pylori (H. pylori) and plays a role as a scaffold which transfer Fe-S cluster to Fe-S proteins like Ferredoxin. To understand how to bind to iron ion or iron-sulfur cluster, HP1492 was expressed and purified in Escherichia coli (E. coli). From the NMR measurement, we could carry out the sequence specific backbone resonance assignment of HP1492. Approximately 91% of all resonances could be assigned unambiguously. By analyzing results of CSI and TALOS from NMR data, we could predict the secondary structure of HP1492, which consists of three -helices and three -sheets. This study is an essential step towards the structural characterization of HP1492.
[Show abstract][Hide abstract] ABSTRACT: Transforming growth factor β (TGF-β)-activated kinase 1 (TAK1) is a key regulator in the signals transduced by proinflammatory
cytokines and Toll-like receptors (TLRs). The regulatory mechanism of TAK1 in response to various tissue types and stimuli
remains incompletely understood. Here, we show that ribosomal S6 kinase 1 (S6K1) negatively regulates TLR-mediated signals
by inhibiting TAK1 activity. S6K1 overexpression causes a marked reduction in NF-κB and AP-1 activity induced by stimulation
of TLR2 or TLR4. In contrast, S6K1−/− and S6K1 knockdown cells display enhanced production of inflammatory cytokines. Moreover, S6K1−/− mice exhibit decreased survival in response to challenge with lipopolysaccharide (LPS). We found that S6K1 inhibits TAK1
kinase activity by interfering with the interaction between TAK1 and TAB1, which is a key regulator protein for TAK1 catalytic
function. Upon stimulation with TLR ligands, S6K1 deficiency causes a marked increase in TAK1 kinase activity that in turn
induces a substantial enhancement of NF-κB-dependent gene expression, indicating that S6K1 is negatively involved in the TLR
signaling pathway by the inhibition of TAK1 activity. Our findings contribute to understanding the molecular pathogenesis
of the impaired immune responses seen in type 2 diabetes, where S6K1 plays a key role both in driving insulin resistance and
modulating TLR signaling.
Full-text · Article · Nov 2013 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Salt-inducible kinases (SIKs) are a family of related serine-threonine kinases and are involved in controlling various metabolisms such as liver glucose homeostasis, hepatic lipogenesis, steroidogenesis, and adipogenesis. Here we investigated the regulatory role of SIK proteins in Toll-like receptor 4 (TLR4)-mediated signaling. Overexpression of SIK1 and SIK3, but not SIK2, significantly inhibited nuclear factor-κB activity in response to lipopolysaccharide stimulation and affected the expression of proinflammatory cytokines. In contrast, both SIK1(KD) and SIK3(KD) Raw 264.7 cells exhibit dramatic elevations of nuclear factor-κB activation and activations of downstream signaling molecules, such as TGF-β-activated kinase 1, p38, and c-Jun N-terminal kinase, in response to TLR4 stimulation, indicating that SIK1 and SIK3 are negatively involved in the TLR4-mediated signaling. Through biochemical studies, we found that SIK1 and SIK3 interact with TGF-β-activated kinase 1-binding protein 2 (TAB2), and interrupt the functional complex of TAB2-TNF receptor-associated factor 6 (TRAF6). Interestingly, the molecular interruption is induced to suppress the ubiquitination of TRAF6 in response to TLR4 stimulation. These result suggest that SIK1 and SIK3 negatively regulate TLR4-mediated signaling through the interruption of TAB2-TRAF6 complex and thereby the inhibition of ubiquitination of TRAF6. The present findings can be useful for a better understanding of multilevel interactions between the metabolic and immune systems.
[Show abstract][Hide abstract] ABSTRACT: SAV0506 is an 87 residue hypothetical protein from Staphylococcus aureus strain Mu50 and also predicted to have similar function to ribosome associated heat shock protein, Hsp 15. Hsp15 is thought to be involved in the repair mechanism of erroneously produced 50S ribosome subunit. In this report, we present the sequence specific backbone resonance assignment of SAV0506. About 82.5% of all resonances could be assigned unambiguously. By analyzing deviations of the and chemical shift values, we could predict the secondary structure of SAV0506. This study is an essential step towards the structural characterization of SAV0506.