Chemokine G-protein coupled receptors are validated drug targets for many diseases, including cancer, neurological, and inflammatory disorders. Despite much time and effort spent on therapeutic ...development, very few chemokine receptor antagonists are approved for clinical use. Among potential reasons for the slow progress in developing chemokine receptor inhibitors, antagonist tolerance, a progressive reduction in drug efficacy after repeated administration, is likely to play a key role. The mechanisms leading to antagonist tolerance remain poorly understood. In many cases, antagonist tolerance is accompanied by increased receptor concentration on the cell surface after prolonged exposure to chemokine receptor antagonists. This points to a possible role of altered receptor internalization and presentation on the cell surface, as has been shown for agonist (primarily opioid) tolerance. In addition, examples of antagonist tolerance in the context of other G-protein coupled receptors suggest the involvement of noncanonical signal transduction in opposing the effects of the antagonists. In this review, we summarize the available progress and challenges in therapeutic development of chemokine receptor antagonists, describe the available knowledge about antagonist tolerance, and propose new avenues for future investigation of this important phenomenon. Furthermore, we highlight the modern methodologies that have the potential to reveal novel mechanisms leading to antagonist tolerance and to propel the field forward by advancing the development of potent "tolerance-free" antagonists of chemokine receptors.
KRAS forms transient dimers and higher-order multimers (nanoclusters) on the plasma membrane, which drive MAPK signaling and cell proliferation. KRAS is a frequently mutated oncogene, and while it is ...well known that the most prevalent mutation, G12D, impairs GTP hydrolysis, thereby increasing KRAS activation, G12D has also been shown to enhance nanoclustering. Elucidating structures of dynamic KRAS assemblies on a membrane has been challenging, thus we have refined our NMR approach that uses nanodiscs to study KRAS associated with membranes. We incorporated paramagnetic relaxation enhancement (PRE) titrations and interface mutagenesis, which revealed that, in addition to the symmetric 'α-α' dimerization interface shared with wild-type KRAS, the G12D mutant also self-associates through an asymmetric 'α-β' interface. The 'α-β' association is dependent on the presence of phosphatidylserine lipids, consistent with previous reports that this lipid promotes KRAS self-assembly on the plasma membrane in cells. Experiments using engineered mutants to spoil each interface, together with PRE probes attached to the membrane or free in solvent, suggest that dimerization through the primary 'α-α' interface releases β interfaces from the membrane promoting formation of the secondary 'α-β' interaction, potentially initiating nanoclustering. In addition, the small molecule BI-2852 binds at a β-β interface, stabilizing a new dimer configuration that outcompetes native dimerization and blocks the effector-binding site. Our data indicate that KRAS self-association involves a delicately balanced conformational equilibrium between transient states, which is sensitive to disease-associated mutation and small molecule inhibitors. The methods developed here are applicable to biologically important transient interactions involving other membrane-associated proteins.
Studies of membrane-dependent dimerization of KRAS on nanodiscs using paramagnetic NMR titrations and mutagenesis revealed a novel asymmetric 'α-β' interface that provides a potential mechanism for the enhanced assembly of KRAS-G12D nanoclusters.
RIP1 and RIP3, cell death mediators, form fibrous amyloids. How RIP1/RIP3 amyloidal oligomers assemble functional necrosomes and control cell death is largely unknown. Here we use super-resolution ...microscopy to directly visualize cellular necrosomes as mosaics of RIP1 and RIP3 oligomers. The small (initial) mosaic complexes are round, and the large mosaics are in a rod shape. RIP3 oligomers with sizes of tetramer or above are the domains in mosaics that allow MLKL, recruited by phosphorylated RIP3, to oligomerize for necroptosis. Unexpectedly, RIP1 autophosphorylation not only controls the ordered oligomerization of RIP1 but also is required for RIP1-initiated RIP3 homo-oligomerization in correct organization, which is indispensable for the formation of functional rod-shaped mosaics. Similarly, apoptosis initiated by enzymatically defective RIP3 requires the formation of rod-shaped mosaics of RIP3 and RIP1 oligomers. The revealing of nanoscale architecture of necrosomes here innovates our understanding of the structural and organizational basis of this signalling hub in cell death.
Abnormal deposition of misprocessed and aggregated proteins is a common final pathway of most neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized by the extraneuronal ...deposition of the amyloid β (Aβ) protein in the form of plaques and the intraneuronal aggregation of the microtubule-associated protein tau in the form of filaments. Based on the biochemically diverse range of pathological tau proteins, a number of approaches have been proposed to develop new potential therapeutics. Here we discuss some of the most promising ones: inhibition of tau phosphorylation, proteolysis and aggregation, promotion of intra- and extracellular tau clearance, and stabilization of microtubules. We also emphasize the need to achieve a full understanding of the biological roles and post-translational modifications of normal tau, as well as the molecular events responsible for selective neuronal vulnerability to tau pathology and its propagation. It is concluded that answering key questions on the relationship between Aβ and tau pathology should lead to a better understanding of the nature of secondary tauopathies, especially AD, and open new therapeutic targets and strategies.
The heat-activated persulfate oxidation of bisphenol A (BPA), a representative endocrine disrupting compound, was investigated with respect to the effect of several process variables on degradation ...rates. The activation temperature appears to be the single most important parameter, i.e. a temperature increase from 40 to 70 °C results in an 80-fold rate increase. Regarding initial BPA concentration, the reaction follows a pseudo-first order rate expression, where the kinetic constant decreases from 11.5 10−2 to 3.5 10−2 min−1 when BPA concentration increases from 110 to 440 μg/L. Reactions in actual water matrices, such as bottled water and secondary treated wastewater, are slower than in pure water since various organic/inorganic water constituents compete with BPA for being oxidized by the reactive oxidizing species; this was confirmed with experiments in pure water spiked with humic acid or bicarbonate. Interestingly though, the presence of chloride seems to promote BPA degradation. Furthermore, degradation is favored at near-neutral pH and increased sodium persulfate (SPS) concentrations.
Experiments at an increased BPA concentration of 20 mg/L were performed to identify transformation by-products (TBPs), as well as assess the mineralization and toxicity of the treated samples. Liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) revealed the formation of eleven TBPs of BPA and plausible pathways including hydroxylation, oxidation, cleavage and oligomerization reactions are proposed. Mineralization occurs slower than BPA degradation, while the toxicity to marine bacteria Vibrio fischeri increases during the early stages of the reaction but it progressively decreases thereafter.
•A 30° temperature increase accelerates BPA degradation 80 times.•The presence of chloride may enhance BPA degradation.•Complex water matrices reduce kinetics.•Polymerization of BPA occurs at the early stages.
Fungal laccase has aroused great concern in rapidly removing estrogens because of its ability to accelerate humification and oligomerization. Here, the effect of two humic acids (HAs) on the reaction ...kinetics and products distribution of 17α-ethynylestradiol (EE2) in laccase-initiated humification and coupling was systematically elucidated. Laccase from Trametes versicolor exhibited over 98.3% removal rate for EE2 at pH 5.0 within 120 min, while HAs invariably restrained EE2 transformation by competing with target-substrate for the enzymatic catalytic center. EE2 removal followed pseudo-first-order kinetics, and the rate constant was decreased markedly with increasing concentration of two HAs (0–60 mg L−1). Additionally, laccase heightened the aromaticity and humification degrees (A250 nm/A365 nm ratio) of HAs probably due to the formation of new humic polymers such as (HA)m and/or (HA)m-(EE2)n (m and n represent the number of HA and EE2 units, respectively). Three major EE2 oligomers were identified, in accordance with a mechanism involving the phenoxy radical-driven polymerization to yield a wide variety of self-coupling products. Notably, HAs diminished the extent of EE2 self-coupling but aggrandized the cross-coupling between EE2 and HAs, and the inhibition degree of EE2 self-coupling increased with the concentration of HAs. One major reason is EE2 could be covalently incorporated into humic molecules to produce (HA)m-(EE2)n cross-coupling products via radical-caused C–C, C–O–C, and/or C–O–C bonds, thereby reducing EE2 self-oligomerization. These findings highlight that HAs play a vital role in the fungal laccase-induced humification and oligomerization of EE2, which obviously alter the geochemical fate and transport of EE2 in natural aquatic ecosystems.
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•EE2 was effectively eliminated by Trametes versicolor laccase at pH 5.0.•Humification and oligomerization of HAs with EE2 were accelerated by laccase.•Radical-induced step-polymerization was the primary EE2 transformation mechanism.•HAs decreased EE2 self-coupling but increased the cross-coupling of EE2 and HAs.•The natural water matrix constituents had no evident effect on removal of EE2.
Disproportionate activation of pattern recognition receptors plays a role in inflammatory bowel disease (IBD) pathophysiology. Diarrhea is a hallmark symptom of IBD, resulting at least in part from ...an electrolyte imbalance that may be caused by changes in potassium channel activity. We evaluated the impact of Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain 2 (NOD2) stimulation on potassium conductance of the basolateral membrane in human intestinal epithelial cells (IECs) and the role of potassium channels through electrophysiological assays under short-circuit current in Ussing chambers. TLRs and NOD2 were stimulated using specific agonists, and potassium channels were selectively blocked using triarylmethane-34 (TRAM-34), adenylyl-imidodiphosphate (AMP-PNP), and BaCl
. Potassium conductance of the basolateral membrane decreased upon activation of TLR2, TLR4, and TLR7 in T84 cells (means ± SE, -11.2 ± 4.5, -40.4 ± 7.2, and -19.4 ± 5.9, respectively) and in Caco-2 cells (-13.1 ± 5.7, -55.7 ± 7.4, and -29.1 ± 7.2, respectively). In contrast, activation of TLR5 and NOD2 increased basolateral potassium conductance, both in T84 cells (18.0 ± 4.1 and 18.4 ± 2.8, respectively) and in Caco-2 cells (21.2 ± 8.4 and 16.0 ± 3.6, respectively). TRAM-34 and AMP-PNP induced a decrease in basolateral potassium conductance upon TLR4 stimulation in both cell lines. Both K
3.1- and K
6-channels appear to be important mediators of this effect in IECs and could be potential targets for therapeutic agent development.
This study highlights that PRRs stimulation directly influences K
-channel conductance in IECs. TLR-2, -4, -7 stimulation decreased K
conductance, whereas TLR5 and NOD2 stimulation had the opposite effect, leading to an increase of it instead. This study reports for the first time that K
3.1- and K
6-channels play a role in K
transport pathways triggered by TLR4 stimulation. These findings suggest that K
3.1- and K
6-channels modulation may be a potential target for new therapeutic agents in IBD.
Genetically Encoded Oligomerization
Protein‐based optoelectronics is a novel field toward the development of eco‐friendly materials, replacing non‐sustainable elements while maintaining device ...figures‐of‐merit. However, such materials are not stable under device fabrication and operation conditions. In article number 2303993, Juan Pablo Fuenzalida‐Werner, Rubén D. Costa, and co‐workers report a genetically encoded macro‐oligomerization strategy that enables versatile, electrostatically controlled protein–protein interactions, ensuring the protein stability in non‐native environments and stressful conditions, significantly enhancing the performance of protein‐lighting devices.
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