Identification of glutamine synthetase as a contryphan-Bt binding protein by His-tag pull-down

Background: Contryphan-Bt is a D-tryptophan-containing disulfide-constrained decapeptide recently isolated from the venom of Conus betulinus. The molecular targets of contryphans are controversial, and the identification of its interacting proteins may be of great importance. Methods: His-tag pull-down assays were performed to investigate intracellular binding proteins of contryphan-Bt from rat brain lysate. Bt-Acp-[His]6, a contryphan-Bt derivative containing hexahistidine tag, was synthesized and used as the bait. As a control, Acp-[His]6 was used to exclude nonspecific bindings. Results: Glutamine synthetase was identified as a potential contryphan-Bt binding protein by pull-down assays and subsequent LC-MS/MS. The binding of contryphan-Bt to glutamine synthetase was confirmed and determined using microscale thermophoresis, with a Kd of 74.02 ± 2.8 μM. The binding did not affect glutamine synthetase activity, suggesting that the interaction site was distinct from the catalytic center. Conclusions: Glutamine synthetase was identified as a novel contryphan-Bt binding protein. This is the first report in which the conopeptide binds to an intracellular protein.

Determination of intracellular protein–ligand binding affinity by competition binding in-cell NMR

Structure-based drug development suffers from high attrition rates due to the poor activity of lead compounds in cellular and animal models caused by low cell penetrance, off-target binding or changes in the conformation of the target protein in the cellular environment. The latter two effects cause a change in the apparent binding affinity of the compound, which is indirectly assessed by cellular activity assays. To date, direct measurement of the intracellular binding affinity remains a challenging task. In this work, in-cell NMR spectroscopy was applied to measure intracellular dissociation constants in the nanomolar range by means of protein-observed competition binding experiments. Competition binding curves relative to a reference compound could be retrieved either from a series of independent cell samples or from a single real-time NMR bioreactor run. The method was validated using a set of sulfonamide-based inhibitors of human carbonic anhydrase II with known activity in the subnanomolar to submicromolar range. The intracellular affinities were similar to those obtained in vitro, indicating that these compounds selectively bind to the intracellular target. In principle, the approach can be applied to any soluble intracellular target that gives rise to measurable chemical shift changes upon ligand binding.

Conformations of voltage-sensing domain III differentially define NaV channel closed- and open-state inactivation

Voltage-gated Na+ (NaV) channels underlie the initiation and propagation of action potentials (APs). Rapid inactivation after NaV channel opening, known as open-state inactivation, plays a critical role in limiting the AP duration. However, NaV channel inactivation can also occur before opening, namely closed-state inactivation, to tune the cellular excitability. The voltage-sensing domain (VSD) within repeat IV (VSD-IV) of the pseudotetrameric NaV channel α-subunit is known to be a critical regulator of NaV channel inactivation. Yet, the two processes of open- and closed-state inactivation predominate at different voltage ranges and feature distinct kinetics. How inactivation occurs over these different ranges to give rise to the complexity of NaV channel dynamics is unclear. Past functional studies and recent cryo-electron microscopy structures, however, reveal significant inactivation regulation from other NaV channel components. In this Hypothesis paper, we propose that the VSD of NaV repeat III (VSD-III), together with VSD-IV, orchestrates the inactivation-state occupancy of NaV channels by modulating the affinity of the intracellular binding site of the IFMT motif on the III-IV linker. We review and outline substantial evidence that VSD-III activates in two distinct steps, with the intermediate and fully activated conformation regulating closed- and open-state inactivation state occupancy by altering the formation and affinity of the IFMT crevice. A role of VSD-III in determining inactivation-state occupancy and recovery from inactivation suggests a regulatory mechanism for the state-dependent block by small-molecule anti-arrhythmic and anesthetic therapies.

Galectin-3 promotes noncanonical inflammasome activation through intracellular binding to lipopolysaccharide glycans

Cytosolic lipopolysaccharides (LPSs) bind directly to caspase-4/5/11 through their lipid A moiety, inducing inflammatory caspase oligomerization and activation, which is identified as the noncanonical inflammasome pathway. Galectins, β-galactoside–binding proteins, bind to various gram-negative bacterial LPS, which display β-galactoside–containing polysaccharide chains. Galectins are mainly present intracellularly, but their interactions with cytosolic microbial glycans have not been investigated. We report that in cell-free systems, galectin-3 augments the LPS-induced assembly of caspase-4/11 oligomers, leading to increased caspase-4/11 activation. Its carboxyl-terminal carbohydrate-recognition domain is essential for this effect, and its N-terminal domain, which contributes to the self-association property of the protein, is also critical, suggesting that this promoting effect is dependent on the functional multivalency of galectin-3. Moreover, galectin-3 enhances intracellular LPS-induced caspase-4/11 oligomerization and activation, as well as gasdermin D cleavage in human embryonic kidney (HEK) 293T cells, and it additionally promotes interleukin-1β production and pyroptotic death in macrophages. Galectin-3 also promotes caspase-11 activation and gasdermin D cleavage in macrophages treated with outer membrane vesicles, which are known to be taken up by cells and release LPSs into the cytosol. Coimmunoprecipitation confirmed that galectin-3 associates with caspase-11 after intracellular delivery of LPSs. Immunofluorescence staining revealed colocalization of LPSs, galectin-3, and caspase-11 independent of host N-glycans. Thus, we conclude that galectin-3 amplifies caspase-4/11 oligomerization and activation through LPS glycan binding, resulting in more intense pyroptosis—a critical mechanism of host resistance against bacterial infection that may provide opportunities for new therapeutic interventions.

Nanobodies Right in the Middle: Intrabodies as Toolbox to Visualize and Modulate Antigens in the Living Cell

In biomedical research, there is an ongoing demand for new technologies to elucidate disease mechanisms and develop novel therapeutics. This requires comprehensive understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, post-translational modifications and dynamic interactions of cellular components. Traceable intracellular binding molecules provide new opportunities for real-time cellular diagnostics. Most prominently, intrabodies derived from antibody fragments of heavy-chain only antibodies of camelids (nanobodies) have emerged as highly versatile and attractive probes to study and manipulate antigens within the context of living cells. In this review, we provide an overview on the selection, delivery and usage of intrabodies to visualize and monitor cellular antigens in living cells and organisms. Additionally, we summarize recent advances in the development of intrabodies as cellular biosensors and their application to manipulate disease-related cellular processes. Finally, we highlight switchable intrabodies, which open entirely new possibilities for real-time cell-based diagnostics including live-cell imaging, target validation and generation of precisely controllable binding reagents for future therapeutic applications.

Modulation of Transient receptor potential melastatin 3 by protons through its intracellular binding sites

Transient receptor potential melastatin 3 channel (TRPM3) is a calcium-permeable nonselective cation channel that plays an important role in modulating glucose homeostasis in the pancreatic beta cells. However, how TRPM3 is regulated under physiological and pathological conditions is poorly understood. In this study, we found that both intracellular and extracellular protons block TRPM3 through its intracellular binding sites. We demonstrated that external protons indirectly block TRPM3, whereas internal protons inhibit TRPM3 directly with an inhibitory pH50 of 6.9 ± 0.11. We identified three titratable residues, D1059, D1062, and D1073, at the inner vestibule of the channel pore that contribute to pH sensitivity. The mutation of D1073Q reduces TRPM3 current intensity and pH sensitivity; Replacement of Asp 1073 by Gln 1073 changes the reduction of TRPM3 outward current by low external pH 5.5, from 62 ± 3 % in WT to 25 ± 6.0 % in D1073Q. These results indicate that D1073 is not only essential for intracellular pH sensitivity, but it is also crucial for TRPM3 channel gating. In addition, a single mutation of D1059 or D1062 enhances pH sensitivity. In summary, our findings provide a novel molecular determinant for pH regulation of TRPM3. The inhibition of TRPM3 by protons may indicate an endogenous mechanism governing TRPM3 gating and its physiological/ pathological functions.