Cellular Signal Transduction Processes Research Articles

Direct modulation of G protein-gated inwardly rectifying potassium (GIRK) channels.

Ion channels play a pivotal role in regulating cellular excitability and signal transduction processes. Among the various ion channels, G-protein-coupled inwardly rectifying potassium (GIRK) channels serve as key mediators of neurotransmission and cellular responses to extracellular signals. GIRK channels are members of the larger family of inwardly-rectifying potassium (Kir) channels. Typically, GIRK channels are activated via the direct binding of G-protein βγ subunits upon the activation of G-protein-coupled receptors (GPCRs). GIRK channel activation requires the presence of the lipid signaling molecule, phosphatidylinositol 4,5-bisphosphate (PIP2). GIRK channels are also modulated by endogenous proteins and other molecules, including RGS proteins, cholesterol, and SNX27 as well as exogenous compounds, such as alcohol. In the last decade or so, several groups have developed novel drugs and small molecules, such as ML297, GAT1508 and GiGA1, that activate GIRK channels in a G-protein independent manner. Here, we aim to provide a comprehensive overview focusing on the direct modulation of GIRK channels by G-proteins, PIP2, cholesterol, and novel modulatory compounds. These studies offer valuable insights into the underlying molecular mechanisms of channel function, and have potential implications for both basic research and therapeutic development.

Hardware Development for Plant Cultivation Allowing In Situ Fluorescence Analysis of Calcium Fluxes in Plant Roots Under Microgravity and Ground-Control Conditions.

Maintaining an optimal leaf and stem orientation to yield a maximum photosynthetic output is accomplished by terrestrial plants using sophisticated mechanisms to balance their orientation relative to the Earth's gravity vector and the direction of sunlight. Knowledge of the signal transduction chains of both gravity and light perception and how they influence each other is essential for understanding plant development on Earth and plant cultivation in space environments. However, in situ analyses of cellular signal transduction processes in weightlessness, such as live cell imaging of signaling molecules using confocal fluorescence microscopy, require an adapted experimental setup that meets the special requirements of a microgravity environment. In addition, investigations under prolonged microgravity conditions require extensive resources, are rarely accessible, and do not allow for immediate sample preparation for the actual microscopic analysis. Therefore, supply concepts are needed that ensure both the viability of the contained plants over a longer period of time and an unhindered microscopic analysis in microgravity. Here, we present a customized supply unit specifically designed to study gravity-induced Ca2+ mobilization in roots of Arabidopsis thaliana. The unit can be employed for ground-based experiments, in parabolic flights, on sounding rockets, and probably also aboard the International Space Station.

Nanomedicine-Enabled Chemical Regulation of Reactive X Species for Versatile Disease Treatments.

Reactive X species (RXS), encompassing elements such as O, N, C, S, Se, Cl, Br, I, and H, play vital roles in cell biology and physiological function, impacting cellular signal transduction, metabolic regulation, and disease processes. The redox unbalance of RXS is firmly implicated in an assortment of physiological and pathological disorders, including cancer, diabetes, cardiovascular disease, and neurodegenerative diseases. However, the intricate nature and multifactorial dependence of RXS pose challenges in comprehending and precisely modulating their biological behavior. Nanomaterials with distinct characteristics and biofunctions offer promising avenues for generating or scavenging RXS to maintain redox homeostasis and advance disease therapy. This minireview provides a tutorial summary of the relevant chemistry and specific mechanisms governing different RXS, focusing on cellular metabolic regulation, stress responses, and the role of nanomedicine in RXS generation and elimination. The challenges associated with chemically regulating RXS for diverse disease treatments are further discussed along with the future prospects, aiming to facilitate the clinical translation of RXS-based nanomedicine and open new avenues for improved therapeutic interventions.

Nanomedicine‐Enabled Chemical Regulation of Reactive X Species for Versatile Disease Treatments

AbstractReactive X species (RXS), encompassing elements such as O, N, C, S, Se, Cl, Br, I, and H, play vital roles in cell biology and physiological function, impacting cellular signal transduction, metabolic regulation, and disease processes. The redox unbalance of RXS is firmly implicated in an assortment of physiological and pathological disorders, including cancer, diabetes, cardiovascular disease, and neurodegenerative diseases. However, the intricate nature and multifactorial dependence of RXS pose challenges in comprehending and precisely modulating their biological behavior. Nanomaterials with distinct characteristics and biofunctions offer promising avenues for generating or scavenging RXS to maintain redox homeostasis and advance disease therapy. This minireview provides a tutorial summary of the relevant chemistry and specific mechanisms governing different RXS, focusing on cellular metabolic regulation, stress responses, and the role of nanomedicine in RXS generation and elimination. The challenges associated with chemically regulating RXS for diverse disease treatments are further discussed along with the future prospects, aiming to facilitate the clinical translation of RXS‐based nanomedicine and open new avenues for improved therapeutic interventions.

Ultrafast structural changes direct the first molecular events of vision

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.

Photo-control of small GTPase Ras GTPase cycle using photochromic molecule.

The small G protein Ras is a central regulator of cellular signal transduction processes, functioning as a molecular switch. Structure and regulatory mechanisms of Ras have been well studied at the molecular level. In this study, we have tried to photocontrol of Ras function using photochromic molecule as a regulatory molecular device. The two methods utilizing azobenzene derivatives as photochromic molecule were employed. The first one is that incorporating bifunctional azobenzene derivatives into the inhibitory peptides and photocontrol Ras function with the photoswitching inhibitory peptide. The second one is to incorporate azobenzene derivatives directly into the functional site of GEF. Recently, a novel peptide inhibitor of Ras (KRpep-2d) was reported to inhibit the guanine nucleotide exchange of Ras with GEF potently. We prepared the KRpep-2d modified with azobenzene derivative (ABDM, DIAAB, BSBCA22) to photocontrol of Ras GTPase activity. The GAP and GEF dependent GTPase of Ras was measured in the presence of peptide inhibitor upon UV and visible light irradiations. We also examined the photocontrol of Ras GTPase by incorporation of azobenzene into the functional site of GEF. The GEF mutant S732C, S881C, H905C, H911C and K679C were prepared and modified with phenylazophenyl maleimide (PAM), Amino azobenzene maleimide (AABM), Sulfonate azobenzene maleimide (SABM). These mutants modified with PAM did not show significant change of GTPase activity accompanied by cis-trans isomerization. Activity of these mutants decrease lower than wild type, but mutant(K679C) does not decrease.

Small GTPase Ras fused with light responsive protein exhibits photo-reversible regulation of GTPase activity.

The small G protein Ras is a central regulator of cellular signal transduction processes, functioning as a molecular switch. Switch mechanisms utilizing conformational changes in nucleotide-binding motifs have been well studied at the molecular level. Previously, we have demonstrated that H-Ras GTPase cycle activity is photoreversibly regulated by photochromic H-Ras, which is modified with 4-chloroacetoamido-4-sulfo-azobenzene (CASAB). However, the efficiency of photoregulation was not sufficient to utilize as a photo-switching. In addition, direct chemical modification to H-Ras carries risk of inactivating Ras function and chemical modification of H-Ras is hard to apply in vivo experiment. In this study, we tried to photoregulate H-Ras activity using photochromic protein; Dronpa or Aureochrome1. We designed H-Ras fusion Dronpa (HRas-Dronpa) or Aureochrome1 (HRas-Aureo1) and the recombinant proteins were prepared by E. coli expression system. HRas-Dronpa showed photo-isomerization upon 366-nm and 500-nm light irradiations. However, multimer formation accompanied by photoisomerization was not observed. On the other hand, HRas-Aureo1 also showed photo-isomerization accompanied by blue light irradiation and in the dark, and HRas-Aureo1 exhibited dimerization by light irradiation. Moreover, GTPase activity of HRas-Dronpa was changed accompanied by isomerization. GTPase activity of HRas-Aureo1 in the dark was approximately 50% of that under blue light irradiation. It has been suggested that light responsive protein is applicable to photo switching of enzymatic activity for small GTPase.

Genetic Variation in Schizothorax kozlovi Nikolsky in the Upper Reaches of the Chinese Yangtze River Based on Genotyping for Simplified Genome Sequencing.

Simple SummarySchizothorax kozlovi Nikolsky is a unique cold−water fish in the upper reaches of the Yangtze River in China and has high economic value. In our study, genetic diversity and population structure analyses were performed on seven wild populations in the upper reaches of the Yangtze River by GBS. The above results indicate that the populations of S. kozlovi have different degrees of tolerance and selection pressure in response to temperature and altitude. The Wujiang population was genetically differentiated from the Jinsha River and Yalong River populations. The Wujiang intrapopulation has greater genetic diversity and differentiation than the Jinsha River and Yalong River populations, which demonstrates that the Jinsha and Yalong populations require more attention and resources for their protection. The results of this study will increase our understanding of the diversity of S. kozlovi in the upper reaches of the Yangtze River and provide a basis for the conservation and utilization of wild resources.Schizothorax kozlovi Nikolsky is a unique cold−water fish in the upper reaches of the Yangtze River in China and has high economic value. In our study, genetic diversity and population structure analyses were performed on seven wild populations (originating from the Jinsha River, Yalong River, and Wujiang River) in the upper reaches of the Yangtze River by genotyping by sequencing (GBS). The results indicated that a total of 303,970 single−nucleotide polymorphisms (SNPs) were identified from the seven wild populations. Lower genetic diversity was exhibited among the intrapopulations of the three tributaries, and the Wujiang River population had significant genetic differentiation when compared to the Jinsha River and Yalong River populations. Furthermore, the selected SNPs were enriched in cellular processes, environmental adaptation, signal transduction, and related metabolic processes between the Wujiang population and the other two populations. The above results indicate that the populations of S. kozlovi have different degrees of tolerance and selection pressure in response to temperature and altitude. The Wujiang intrapopulation has greater genetic diversity and differentiation than the Jinsha River and Yalong River populations, which demonstrates that the Jinsha and Yalong populations require more attention and resources for their protection. The results of this study will increase our understanding of the diversity of S. kozlovi in the upper reaches of the Yangtze River and provide a basis for the conservation and utilization of wild resources.

Phosphorylation of plant virus proteins: Analysis methods and biological functions.

Phosphorylation is one of the most extensively investigated post-translational modifications that orchestrate a variety of cellular signal transduction processes. The phosphorylation of virus-encoded proteins plays an important regulatory role in the infection cycle of such viruses in plants. In recent years, molecular mechanisms underlying the phosphorylation of plant viral proteins have been widely studied. Based on recent publications, our study summarizes the phosphorylation analyses of plant viral proteins and categorizes their effects on biological functions according to the viral life cycle. This review provides a theoretical basis for elucidating the molecular mechanisms of viral infection. Furthermore, it deepens our understanding of the biological functions of phosphorylation in the interactions between plants and viruses.

The activity of the serotonergic 5-HT1A receptor is modulated by voltage and sodium levels

G protein–coupled receptors are known to play a key role in many cellular signal transduction processes, including those mediating serotonergic signaling in the nervous system. Several factors have been shown to regulate the activity of these receptors, including membrane potential and the concentration of sodium ions. Whether voltage and sodium regulate the activity of serotonergic receptors is unknown. Here, we used Xenopus oocytes as an expression system to examine the effects of voltage and of sodium ions on the potency of one subtype of serotonin (5-hydroxytryptamine [5-HT]) receptor, the 5-HT1A receptor. We found that the potency of 5-HT in activating the receptor is voltage dependent and that it is higher at resting potential than under depolarized conditions. Furthermore, we found that removal of extracellular Na+ resulted in a decrease of 5-HT potency toward the 5-HT1A receptor and that a conserved aspartate in transmembrane domain 2 is crucial for this effect. Our results suggest that this allosteric effect of Na+ does not underlie the voltage dependence of this receptor. We propose that the characterization of modulatory factors that regulate this receptor may contribute to our future understanding of various physiological functions mediated by serotonergic transmission.

Photocontrol of small GTPase Ras GTPase cycle using photochromic molecule

The small G protein Ras is a central regulator of cellular signal transduction processes, functioning as a molecular switch. Structure and regulatory mechanism of Ras have been well studied at the molecular level. In this study, we have tried to photocontrol of Ras function using photochromic molecule as a regulatory molecular device. The two methods utilizing azobenzene derivatives as photochromic molecule were employed. The first one is that incorporating bifunctional azobenzene derivatives into the inhibitory peptides and photocontrol Ras function with the photoswitching inhibitory peptide.

Photoregulation of small G-protein RAS by the modification at HVR using azobenzene derivatives

Ras is one of the Small G proteins functioning as a molecular switch, is a central regulator of cellular signal transduction processes leading to transcription, cell cycle progression and differentiation by cycling between the inactive GDP-bound and the active GTP-bound states using GAP and GEF regulators. The structure and functional mechanism of Ras have been well studied and considered as a GTP driven nanomachine which control cellular function. Hyper Variable Region (HVR) is one of the functional parts of the G proteins in which modification induce multimerization and interaction with plasma membrane of Ras.

Multimerization of small G-protein H-Ras induced by chemical modification at hyper variable region with caged compound.

The lipid-anchored small G protein Ras is a central regulator of cellular signal transduction processes, thereby functioning as a molecular switch. Ras forms a nanocluster on the plasma membrane by modifying lipids in the hypervariable region (HVR) at the C-terminus to exhibit physiological functions. In this study, we demonstrated that chemical modification of cysteine residues in HVR with caged compounds (instead of lipidation) induces multimerization of H-Ras. The sulfhydryl-reactive caged compound, 2-nitrobenzyl bromide, was stoichiometrically incorporated into the cysteine residue of HVR and induced the formation of the Ras multimer. Light irradiation induced the elimination of the 2-nitrobenzyl group, resulting in the conversion of the multimer to a monomer. Size-exclusion chromatography coupled with high-performance liquid chromatography and small-angle x-ray scattering analysis revealed that H-Ras forms a pentamer. Electron microscopic observation of the multimer showed a circular ring shape, which is consistent with the structure estimated from x-ray scattering. The shape of the multimer may reflect the physiological state of Ras. It was suggested that the multimerization and monomerization of H-Ras were controlled by modification with a caged compound in HVR under light irradiation.

AMICI: high-performance sensitivity analysis for large ordinary differential equation models.

SummaryOrdinary differential equation models facilitate the understanding of cellular signal transduction and other biological processes. However, for large and comprehensive models, the computational cost of simulating or calibrating can be limiting. AMICI is a modular toolbox implemented in C++/Python/MATLAB that provides efficient simulation and sensitivity analysis routines tailored for scalable, gradient-based parameter estimation and uncertainty quantification.Availabilityand implementationAMICI is published under the permissive BSD-3-Clause license with source code publicly available on https://github.com/AMICI-dev/AMICI. Citeable releases are archived on Zenodo.Supplementary information Supplementary data are available at Bioinformatics online.

Modulation of Insulin Receptor Activity Upon Plasma Membrane Sterol and Phospholipid Substitutions to Alter Lipid Domain Formation

Plasma membrane lipid organization into ordered (raft) and disordered domains has been thought to help cellular signal transduction processes by clustering membrane receptors in rafts to facilitate robust downstream signaling. Ordered (Lo) domains are enriched in sphingomyelin (SM) and cholesterol while disordered (Ld) domains are enriched in unsaturated lipids such as phosphatidyl choline (PC). Insulin receptor (IR) is a receptor tyrosine kinase that has reduced activity after cellular cholesterol depletion, and increased distribution in detergent resistant membranes, which can reflect localization in rafts, after activation.

Simultaneous Recording of Multiple Signaling Events by Frequency- and Spectrally-Tuned Multiplexing of Fluorescent Probes

Fluorescent probes that change their spectral properties upon binding to specific ligands, e.g., second messengers, or changes in the membrane potential (Vm) are invaluable tools to study cellular signal transduction. Here, we introduce a novel technique that facilitates simultaneous recording of multiple fluorescent probes: frequency- and spectrally-tuned multiplexing (FASTM). FASTM uses phase-sensitive signal detection, which renders various combinations of probes for Vm and ions accessible for multiplexing. Using stopped-flow fluorimetry, we show that FASTM allows for simultaneous recording of rapid changes in Ca2+, pH, Na+, and Vm with high sensitivity, millisecond time resolution, and minimal cross-talk. Moreover, FASTM is compatible with opto-chemical tools (e.g., caged compounds) used for manipulating signal transduction with light. Finally, FASTM is suited for multiplexing of genetically-encoded FRET biosensors and synthetic probes using single-cell fluorescence microscopy. Altogether, FASTM opens new experimental avenues for interrogating cellular signal transduction processes with high temporal resolution.

Sodium ions allosterically modulate the M2 muscarinic receptor

G protein coupled receptors (GPCRs) play a key role in the vast majority of cellular signal transduction processes. Previous experimental evidence has shown that sodium ion (Na+) allosterically modulate several class A GPCRs and theoretical studies suggested that the same also holds true for muscarinic receptors. Here we examined, using Xenopus oocytes as an expression system, the effect of Na+ on a prototypical GPCR, the M2 muscarinic receptor (M2R). We found that removal of extracellular Na+ resulted in a decrease in the potency of ACh toward the M2R and that a conserved aspartate in transmembrane domain 2 is crucial for this effect. We further show that this allosteric effect of Na+ does not underlie the voltage-dependence of this receptor.

Induction of cystathionine gamma-lyase expression and metallothionein-1 S-sulfhydration alleviate cadmium-induced cell death in myoblast cells

Hydrogen sulfide (H2S), a multifunctional gasotransmitter, participates in a wide range of cellular signal transduction and pathophysiological processes. Cystathionine gamma-lyase (CSE) acts as a major H2S-generating enzyme in peripheral organs and tissues. As a cysteine-rich and heavy metal-binding protein, metallothionein-1 (MT-1) is known to protect cells from various environmental stresses. Here we demonstrated that exposure of cadmium (Cd) induced oxidative stress, depleted intracellular thiols, and stimulated apoptotic cell death in mouse myoblast cells. CSE expression and H2S production were significantly enhanced by Cd treatment. NaHS, a well-known H2S donor, at physiologically relevant concentration significantly alleviated Cd-induced damage in both myoblasts and mouse skeletal muscles. In contrast, down-regulation of CSE/H2S system deteriorated Cd-stimulated oxidative stress and cell death. Exposure of the cells to Cd lead to increased expressions of metal regulatory transcription factor 1 and MT-1, while siRNA-mediated MT-1 knockdown alleviated Cd-induced CSE expression and caused more oxidative stress and cell death. In addition, H2S post-translationally modified MT-1 by S-sulfhydration and stabilized zinc-protein complex. Taken together, these data suggest that CSE/H2S system would protect myoblasts and skeletal muscles from Cd-induced damage by S-sufhydrating MT-1.

Highly efficient catalytic scavenging of oxygen free radicals with graphene-encapsulated metal nanoshields

Normal levels of oxygen free radicals play an important role in cellular signal transduction, redox homeostasis, regulatory pathways, and metabolic processes. However, radiolysis of water induced by high-energy radiation can produce excessive amounts of exogenous oxygen free radicals, which cause severe oxidative damages to all cellular components, disrupt cellular structures and signaling pathways, and eventually lead to death. Herein, we show that hybrid nanoshields based on single-layer graphene encapsulating metal nanoparticles exhibit high catalytic activity in scavenging oxygen superoxide(· $${O_{\bar 2}}$$ ), hydroxyl (·OH), and hydroperoxyl (HO2·) free radicals via electron transfer between the single-layer graphene and the metal core, thus achieving biocatalytic scavenging both in vitro and in vivo. The levels of the superoxide enzyme, DNA, and reactive oxygen species measured in vivo clearly show that the nanoshields can efficiently eliminate harmful oxygen free radicals at the cellular level, both in organs and circulating blood. Moreover, the nanoshieldslead to an increase in the overall survival rate of gamma ray-irradiated mice to up to 90%, showing the great potential of these systems as protective agentsagainst ionizing radiation.

Adenylyl cyclase 1 as a major isoform to generate cAMP signaling for apoA-1-mediated cholesterol efflux pathway

HDL apoA-1-mediated cholesterol efflux pathway requires multiple cellular proteins and signal transduction processes, including adenylyl cyclase (AC)/cAMP signaling. Due to the existence of multiple transmembrane AC isoforms, it was not known how many AC isoforms are expressed and which ones are essential for cholesterol efflux in macrophage foam cells. These questions were investigated in THP-1 macrophages in this study. Quantitative RT-PCR detected mRNAs for all nine transmembrane AC isoforms, but only the mRNA and protein of the AC1 isoform were consistently upregulated by cholesterol loading and apoA-1. AC1 shRNA interference decreased AC1 mRNA and protein levels, resulting in reduction of apoA-1-mediated cAMP production and cholesterol efflux, while the intracellular cholesterol levels remained high. Confocal microscopy showed that apoA-1 promoted translocation of cholesterol and formation of cholesterol-apoA-1 complexes (protrusions) on the cholesterol-loaded macrophage surface. AC1 shRNA-interfered macrophages showed no translocation of cholesterol to the cell surface. AC1 shRNA interference also disrupted cellular localization of the intracellular cholesterol indicator protein adipophillin, and the expression as well as surface translocation of ABCA1. Together, our results show that AC1 is a major isoform for apoA-1-activated cAMP signaling to promote cholesterol transport and exocytosis to the surface of THP-1 macrophage foam cells.