Eukaryotic 80S ribosomes of known structure are far more complex than their 70S bacterial counterparts. Those from Saccharomyces cerevisiae, Tetrahymena thermophila, and Triticum aestivum, for example, bear insertions of ribosomal RNA (rRNA) called expansion segments (ES) and additional ribosomal proteins. The ribosomes of the kinetoplastid Trypanosoma brucei, though, are especially fascinating: structurally and their other kinetoplastids’ ribosomes bear very large ESs, as well as smaller ESs, and protein extensions. Additionally, T. brucei ribosomes require novel protein factors for maturation, although they do not require several eukaryotic initiation factors or a recycling factor. As a species, T. brucei is fascinating not only in terms of structure, but also in terms of gene expression and even public health: the species is responsible for the incurable, terminal human African Trypanosomiasis (sleeping sickness); and during post-transcriptional regulation, a single common RNA segment called a splice leader is trans-spliced onto the 5′ ends of many of T. brucei’s mRNAs. The purpose of this splice event in translation is unknown. Here, we present a high-resolution structure of the T. brucei ribosome which contributes a great deal to addressing the above unknowns. We have employed map segmentation, homology modeling, ab initio rRNA modeling, and Molecular Dynamics Flexible Fitting (MDFF) to model the ribosome’s atomic structure. The positions and structures of the ribosome’s novel ESs and protein extensions were previously unknown, but our structure reveals the precise spatial contexts of these components. With this information in hand, we can begin to decipher T. brucei’s unusual translational requirements.
The paucity of selective agonists for TWIK-related acid-sensitive K+ 3 (TASK-3) channel, a member of two-pore domain K+ (K2P) channels, has contributed to our limited understanding of its biological functions. By targeting a druggable transmembrane cavity using a structure-based drug design approach, we discovered a biguanide compound, CHET3, as a highly selective allosteric activator for TASK-3-containing K2P channels, including TASK-3 homomers and TASK-3/TASK-1 heteromers. CHET3 displayed potent analgesic effects in vivo in a variety of acute and chronic pain models in rodents that could be abolished pharmacologically or by genetic ablation of TASK-3. We further found that TASK-3-containing channels anatomically define a unique population of small-sized, transient receptor potential cation channel subfamily M member 8 (TRPM8)-, transient receptor potential cation channel subfamily V member 1 (TRPV1)-, or tyrosine hydroxylase (TH)-positive nociceptive sensory neurons and functionally regulate their membrane excitability, supporting CHET3 analgesic effects in thermal hyperalgesia and mechanical allodynia under chronic pain. Overall, our proof-of-concept study reveals TASK-3-containing K2P channels as a druggable target for treating pain.
The absolute configuration of the title compound, C(12)H(15)NO(7)·0.5H(2)O, was assigned from the synthesis. There are two rhamnoside mol-ecules and one water mol-ecule in the asymmetric unit, displaying O-H⋯O hydrogen bonding. One of the nitro groups does not conjugate efficiently with the benzene ring.
Cys-loop ligand-gated ion channels assemble as pentameric proteins, and each monomer contributes two structural elements: an extracellular ligand-binding domain (LBD) and a transmembrane ion channel domain. Models of receptor activation include rotational movements of subunits leading to opening of the ion channel. We tested this idea using substituted cysteine accessibility to track conformational changes in the inner β sheet of the LBD. Using a nondesensitizing chick α7 background (L247T), we constructed 18 consecutive cysteine replacement mutants (Leu36 to Ile53) and tested each for expression of acetylcholine (ACh)-evoked currents and functional sensitivity to thiol modification. We measured rates of modification in the presence and absence of ACh to identify conformational changes associated with receptor activation. Resting modification rates of eight substituted cysteines in the β1 and β2 strands and the sequence between them (loop 2) varied over several orders of magnitude, suggesting substantial differences in the accessibility or electrostatic environment of individual side chains. These differences were in general agreement with structural models of the LBD. Eight of 18 cysteine replacements displayed ACh-dependent changes in modification rates, indicating a change in the accessibility or electrostatic environment of the introduced cysteine during activation. We were surprised that the effects of agonist exposure were difficult to reconcile with rotational models of activation. Acetylcholine reduced the modification rate of M40C but increased it at N52C despite the close physical proximity of these residues. Our results suggest that models that depend strictly on rigid-body rotation of the LBD may provide an incomplete description of receptor activation.
ABSTRACT Two-pore domain potassium (K2P) channels gate primarily within the selectivity filter, termed ‘C-type’ gating. Due to the lack of structural insights into the nonconductive (closed) state, ‘C-type’ gating mechanisms remain elusive. Here, molecular dynamics (MD) simulations on TREK-1, a K2P channel, revealed that M4 helix movements induce filter closing in a novel ‘deeper-down’ structure that represents a ‘C-type’ closed state. The ‘down’ structure does not represent the closed state as previously proposed and instead acts as an intermediate state in gating. The study identified the allosteric ‘seesaw’ mechanism of M4 helix movements in modulating filter closing. Finally, guided by this recognition of K2P gating mechanisms, MD simulations revealed that gain-of-function mutations and small-molecule activators activate TREK-1 by perturbing state transitions from open to closed states. Together, we reveal a ‘C-type’ closed state and provide mechanical insights into gating procedures and allosteric regulations for K2P channels.
The dysfunction of endothelial progenitor cells (EPCs) is closely associated with diabetic vascular complications. Both glucagonlike peptide-1 receptor (GLP-1R) and silent information regulator 1 (SIRT1) can control systemic glucose homeostasis and protect endothelial cells against hyperglycemia-induced oxidative stress. In this study, we mainly assessed the role played by SIRT1 and GLP-1R and their relationship in regulating the function of late EPCs under hyperglycemia stimulation. Human peripheral blood mononuclear cells (PBMCs) were cultured in EGM-2 medium and induced to differentiate into EPCs and 25 mM glucose was used to stimulate EPCs to obtain a hyperglycemia condition. Subsequently, the expression and location of GLP-1R and SIRT1 in EPCs were detected. After GLP-1R or SIRT1 knockdown, or the treatment by GLP-1R agonist and/or SIRT1 agonist/inhibitor, the effects of SIRT1 and GLP-1R and their relationship in regulating the function of late EPCs under hyperglycemia stimulation was studied by detecting the apoptosis, migration, adhesion and angiogenicity abilities of EPCs. Results demonstrated that, in high-glucose stimulated EPCs, the expression of GLP-1R and SIRT1 was down-regulated. The knockdown of either GLP-1R or SIRT1 could increase EPCs apoptosis and weaken the migration, adhesion and angiogenicity abilities of EPCs. In addition, the improvement effects of Exendin-4 or GLP-1R over-expression on EPCs dysfunction could be weakened to some degree under SIRT1 knockdown. In conclusion, both GLP-1R and SIRT1 expression played important roles in regulating EPCs dysfunction under hyperglycemia and the up-regulation of GLP-1R improved the dysfunction of late EPCs by regulating SIRT1 expression.
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