Intracellular Signaling Processes Research Articles

Monitoring the Reversibility of GPCR Signaling by Combining Photochromic Ligands with Label-free Impedance Analysis.

G protein-coupled cell surface receptors (GPCR) trigger complex intracellular signaling cascades upon agonist binding. Classic pharmacological assays provide information about binding affinities, activation or blockade at different stages of the signaling cascade, but real time dynamics and reversibility of these processes remain often disguised. We show that combining photochromic NPY receptor ligands, which can be toggled in their receptor activation ability by irradiation with light of different wavelengths, with whole cell label-free impedance assays allows observing the cell response to receptor activation and its reversibility over time. The concept demonstrated on NPY receptors may be well applicable to many other GPCRs providing a deeper insight into the time course of intracellular signaling processes.

Association Between LMP-1 and p16 Expression with Prognostic Factor of Nasopharyngeal Carcinoma

Background: LMP-1 and p16 expression are potential biomarkers for nasopharyngeal prognostic factors. EBV interferes with intracellular signaling processes related to cell cycle regulation and LMP-1, inactivating p16 expression and increasing cell proliferation. LMP-1 has been also reported to remove the promoter of p16 via methylation and inactivation of the p16 gene leads to uncontrolled proliferation of cell tumors.Objective: To analyze the association between LMP-1 and p16 expression with prognostic factors of nasopharyngeal carcinoma.Methods: This is a cross-sectional design analyzed that LMP-1 and p16 expression based on immunostaining of nasopharyngeal carcinoma. The patient clinical data used as prognostic factors are histopathological type, quality of life, TNM, and tumor stage. Statistical analysis used chi-square and spearman correlation.Results: Of the 61 patients, 49 were non-keratinizing carcinoma, undifferentiated type (80,3%). About 54 (88.5%) of the 61 samples are LMP-1 positive, 47 of which were non-keratinizing carcinoma. We figured out the significant association between the p16 expression with lymph node involvement (p=0.026) and staging (p = 0.196) in nasopharyngeal carcinoma. No correlation between LMP and p16 expression (p=0,134) (r=-0,194).Conclusion: p16 overexpression is associated with lymph node involvement and advanced stage in nasopharyngeal carcinoma

The overexpressed transcription factor RGPR-p117 suppresses the proliferation of normal rat kidney proximal tubular epithelial NRK-52E cells: Involvement of diverse signaling pathways

AimsRGPR-p117 was originally discovered as a novel transcription factor, which specifically binds to a nuclear factor I (NFI) consensus motif TTGGC(N)6CC in the promoter region of the regucalcin gene. RGPR-p117 is also called as Lztr2 and SEC16B. The role of RGPR-p117 in cell regulation is poorly understood. This study was undertaken to determine whether the overexpression of RGPR-p117 impacts the proliferation of normal rat kidney proximal tubular epithelial NRK-52E cells in vitro. Main methodsThe NRK-52E wild-type cells and RGPR-p117-overexpressing NRK-52E cells were cultured in DMEM containing fetal bovine serum. Key findingsThe overexpression of RGPR-p117 repressed colony formation and proliferation of NRK-52E cells. Interestingly, RGPR-p117 overexpression blocked cell proliferation promoted by culturing with Bay K 8644, a calcium-entry agonist, and phorbol 12-myristate 13-acetate, an activator of protein kinase C. The depressive effects of RGPR-p117 overexpression on cell proliferation were not occurred by culturing with various inhibitors of cell cycle and intracellular signaling processes. RGPR-p117 overexpression increased the translocation of RGPR-p117 into the nucleus of NRK-52E cells. Mechanistically, RGPR-p117 overexpression diminished the levels of Ras, PI3 kinase, Akt, mitogen-activated protein kinase, and mTOR, while it raised the levels of p53, Rb, p21, and regucalcin. Furthermore, RGPR-p117 overexpression protected cell death caused by apoptosis-inducing factors, suggesting that the suppressive effects of RGPR-p117 on cell growth are independent of cell death. SignificanceThe present study demonstrates that the overexpressed transcription factor RGPR-p117 suppresses cell proliferation via targeting diverse signaling processes, suggesting a role of RGPR-p117 in cell regulation.

Circadian rhythm of lipid metabolism.

Lipids comprise a diverse group of metabolites that are indispensable as energy storage molecules, cellular membrane components and mediators of inter- and intra-cellular signaling processes. Lipid homeostasis plays a crucial role in maintaining metabolic health in mammals including human beings. A growing body of evidence suggests that the circadian clock system ensures temporal orchestration of lipid homeostasis, and that perturbation of such diurnal regulation leads to the development of metabolic disorders comprising obesity and type 2 diabetes. In view of the emerging role of circadian regulation in maintaining lipid homeostasis, in this review, we summarize the current knowledge on lipid metabolic pathways controlled by the mammalian circadian system. Furthermore, we review the emerging connection between the development of human metabolic diseases and changes in lipid metabolites that belong to major classes of lipids. Finally, we highlight the mechanisms underlying circadian organization of lipid metabolic rhythms upon the physiological situation, and the consequences of circadian clock dysfunction for dysregulation of lipid metabolism.

Accurate, high-coverage assignment of in vivo protein kinases to phosphosites from in vitro phosphoproteomic specificity data.

Phosphoproteomic experiments routinely observe thousands of phosphorylation sites. To understand the intracellular signaling processes that generated this data, one or more causal protein kinases must be assigned to each phosphosite. However, limited knowledge of kinase specificity typically restricts assignments to a small subset of a kinome. Starting from a statistical model of a high-throughput, in vitro kinase-substrate assay, I have developed an approach to high-coverage, multi-label kinase-substrate assignment called IV-KAPhE (“In vivo-Kinase Assignment for Phosphorylation Evidence”). Tested on human data, IV-KAPhE outperforms other methods of similar scope. Such computational methods generally predict a densely connected kinase-substrate network, with most sites targeted by multiple kinases, pointing either to unaccounted-for biochemical constraints or significant cross-talk and signaling redundancy. I show that such predictions can potentially identify biased kinase-site misannotations within families of closely related kinase isozymes and they provide a robust basis for kinase activity analysis.

A Tandem Activity‐Based Sensing and Labeling Strategy Enables Imaging of Transcellular Hydrogen Peroxide Signaling

Hydrogen Peroxide (H2O2) is one of many reactive oxygen species (ROS) that plays vital roles in intracellular signaling and stress processes, but its transient nature has made deciphering its roles in transcellular signaling a significant challenge. We have designed boronate caged small‐molecule probes bearing a fluoromethylene pendant group that serves as a latent quinone methide source that can undergo a tandem activity‐based sensing and biomolecule labeling reaction cascade to produce a spatially localized fluorescence signal upon exposure to H2O2. This strategy enables us to capture and record spatial information on ROS fluxes and has enabled us to move beyond studying intracellular H2O2 signaling to identifying and mapping cell‐to‐cell H2O2 communication in a microglia‐neuron co‐culture cell model, where selective activation of microglia for ROS production increases H2O2 in nearby neurons.

Lipid metabolism in T cell signaling and function.

T cells orchestrate adaptive immunity against pathogens and other immune challenges, but their dysfunction can also mediate the pathogenesis of cancer and autoimmunity. Metabolic adaptation in response to immunological and microenvironmental signals contributes to T cell function and fate decision. Lipid metabolism has emerged as a key regulator of T cell responses, with selective lipid metabolites serving as metabolic rheostats to integrate environmental cues and interplay with intracellular signaling processes. Here, we discuss how extracellular, de novo synthesized and membrane lipids orchestrate T cell biology. We also describe the roles of lipids as regulators of intracellular signaling at the levels of transcriptional, epigenetic and post-translational regulation in T cells. Finally, we summarize therapeutic targeting of lipid metabolism and signaling, and conclude with a discussion of important future directions. Understanding the molecular and functional interplay between lipid metabolism and T cell biology will ultimately inform therapeutic intervention for human disease.

Extracellular Regucalcin Suppresses the Growth, Migration, Invasion, and Adhesion of Metastatic Human Prostate Cancer Cells

Introduction: Regucalcin plays a multifunctional role in the regulation of cellular function including metabolism, signaling process, and transcriptional activity in maintaining cell homeostasis. Downregulated expression or activity of regucalcin contributes to the development of malignancies in various types of human cancer. Survival of cancer patients, including metastatic prostate cancer, is prolonged with high expression of regucalcin in the tumor tissues. Methods: We elucidate whether extracellular regucalcin conquers the growth, migration, invasion, and adhesion of metastatic human prostate cancer PC-3 and DU-145 cells. Results: Extracellular regucalcin (0.1, 1, and 10 nM) of physiologic levels (1 nM at human serum) inhibited colony formation and growth of PC-3 and DU-145 cells, while it did not have an effect on cell death. Repressive effects of extracellular regucalcin on the proliferation were not exhibited by the presence of inhibitors of the cell cycle, intracellular signaling process, and transcriptional activity, suggesting that the signals of extracellular regucalcin are transmitted to block cell growth. Furthermore, extracellular regucalcin (0.1, 1, or 10 nM) inhibited migration, invasion, and adhesion of PC-3 and DU-145 cells. Mechanistically, extracellular regucalcin (10 nM) decreased the levels of various signaling proteins including Ras, posphatidylinositol-3 kinase, mitogen-activated protein kinase, mechanistic target of rapamycin, RSK-2, caveolin-1, and integrin β1 in PC-3 cells. Discussion and Conclusion: Thus, extracellular regucalcin may play a suppressive role in growth, migration, invasion, and adhesion, which are involved in the metastatic activity of human prostate cancer cells, via affecting diverse signaling processes. This study may provide a new strategy in preventing metastatic prostate cancer with exogenous regucalcin.

Analysis of the Glucose-Dependent Transcriptome in Murine Hypothalamic Cells.

Glucose provides vital energy for cells and contributes to gene expression. The hypothalamus is key for metabolic homeostasis, but effects of glucose on hypothalamic gene expression have not yet been investigated in detail. Thus, herein, we monitored the glucose-dependent transcriptome in murine hypothalamic mHypoA-2/10 cells by total RNA-seq analysis. A total of 831 genes were up- and 1390 genes were downregulated by at least 50%. Key genes involved in the cholesterol biosynthesis pathway were upregulated, and total cellular cholesterol levels were significantly increased by glucose. Analysis of single genes involved in fundamental cellular signaling processes also suggested a significant impact of glucose. Thus, we chose ≈100 genes involved in signaling and validated the effects of glucose on mRNA levels by qRT-PCR. We identified Gnai1–3, Adyc6, Irs1, Igfr1, Hras, and Elk3 as new glucose-dependent genes. In line with this, cAMP measurements revealed enhanced noradrenalin-induced cAMP levels, and reporter gene assays elevated activity of the insulin-like growth factor at higher glucose levels. Key data of our studies were confirmed in a second hypothalamic cell line. Thus, our findings link extra cellular glucose levels with hypothalamic lipid synthesis and pivotal intracellular signaling processes, which might be of particular interest in situations of continuously increased glucose levels.

Rab11 and Its Role in Neurodegenerative Diseases.

Vesicles mediate the trafficking of membranes/proteins in the endocytic and secretory pathways. These pathways are regulated by small GTPases of the Rab family. Rab proteins belong to the Ras superfamily of GTPases, which are significantly involved in various intracellular trafficking and signaling processes in the nervous system. Rab11 is known to play a key role especially in recycling many proteins, including receptors important for signal transduction and preservation of functional activities of nerve cells. Rab11 activity is controlled by GEFs (guanine exchange factors) and GAPs (GTPase activating proteins), which regulate its function through modulating GTP/GDP exchange and the intrinsic GTPase activity, respectively. Rab11 is involved in the transport of several growth factor molecules important for the development and repair of neurons. Overexpression of Rab11 has been shown to significantly enhance vesicle trafficking. On the other hand, a reduced expression of Rab11 was observed in several neurodegenerative diseases. Current evidence appears to support the notion that Rab11 and its cognate proteins may be potential targets for therapeutic intervention. In this review, we briefly discuss the function of Rab11 and its related interaction partners in intracellular pathways that may be involved in neurodegenerative processes.

Bioinformatic Analyses of Peroxiredoxins and RF-Prx: A Random Forest-Based Predictor and Classifier for Prxs.

Peroxiredoxins (Prxs) are a protein superfamily, present in all organisms, that play a critical role in protecting cellular macromolecules from oxidative damage but also regulate intracellular and intercellular signaling processes involving redox-regulated proteins and pathways. Bioinformatic approaches using computational tools that focus on active site-proximal sequence fragments (known as active site signatures) and iterative clustering and searching methods (referred to as TuLIP and MISST) have recently enabled the recognition of over 38,000 peroxiredoxins, as well as their classification into six functionally relevant groups. With these data providing so many examples of Prxs in each class, machine learning approaches offer an opportunity to extract additional information about features characteristic of these protein groups.In this study, we developed a novel computational method named "RF-Prx" based on a random forest (RF) approach integrated with K-space amino acid pairs (KSAAP) to identify peroxiredoxins and classify them into one of six subgroups. Our process performed in a superior manner compared to other machine learning classifiers. Thus the RF approach integrated with K-space amino acid pairs enabled the detection of class-specific conserved sequences outside the known functional centers and with potential importance. For example, drugs designed to target Prx proteins would likely suffer from cross-reactivity among distinct Prxs if targeted to conserved active sites, but this may be avoidable if remote, class-specific regions could be targeted instead.

Homer signaling pathways as effective therapeutic targets for ischemic and traumatic brain injuries and retinal lesions.

Ischemic and traumatic insults to the central nervous system account for most serious acute and fatal brain injuries and are usually characterized by primary and secondary damage. Secondary damage presents the greatest challenge for medical staff; however, there are currently few effective therapeutic targets for secondary damage. Homer proteins are postsynaptic scaffolding proteins that have been implicated in ischemic and traumatic insults to the central nervous system. Homer signaling can exert either positive or negative effects during such insults, depending on the specific subtype of Homer protein. Homer 1b/c couples with other proteins to form postsynaptic densities, which form the basis of synaptic transmission, while Homer1a expression can be induced by harmful external factors. Homer 1c is used as a unique biomarker to reveal alterations in synaptic connectivity before and during the early stages of apoptosis in retinal ganglion cells, mediated or affected by extracellular or intracellular signaling or cytoskeletal processes. This review summarizes the structural features, related signaling pathways, and diverse roles of Homer proteins in physiological and pathological processes. Upregulating Homer1a or downregulating Homer1b/c may play a neuroprotective role in secondary brain injuries. Homer also plays an important role in the formation of photoreceptor synapses. These findings confirm the neuroprotective effects of Homer, and support the future design of therapeutic drug targets or gene therapies for ischemic and traumatic brain injuries and retinal disorders based on Homer proteins.

REPRODUCTIVE TOXICOLOGY: Environmental exposures, fetal testis development and function: phthalates and beyond.

Fetal development of the mammalian testis relies on a series of interrelated cellular processes: commitment of somatic progenitor cells to Sertoli and Leydig cell fate, migration of endothelial cells and Sertoli cells, differentiation of germ cells, deposition of the basement membrane, and establishment of cell-cell contacts, including Sertoli-Sertoli and Sertoli-germ cell contacts. These processes are orchestrated by intracellular, endocrine, and paracrine signaling processes. Because of this complexity, testis development can be disrupted by a variety of environmental toxicants. The toxicity of phthalic acid esters (phthalates) on the fetal testis has been the subject of extensive research for two decades, and phthalates have become an archetypal fetal testis toxicant. Phthalates disrupt the seminiferous cord formation and maturation, Sertoli cell function, biosynthesis of testosterone in Leydig cells, and impair germ cell survival and development, producing characteristic multinucleated germ cells. However, the mechanisms responsible for these effects are not fully understood. This review describes current knowledge of the adverse effects of phthalates on the fetal testis and their associated windows of sensitivity, and compares and contrasts the mechanisms by which toxicants of current interest, bisphenol A and its replacements, analgesics, and perfluorinated alkyl substances, alter testicular developmental processes. Working toward a better understanding of the molecular mechanisms responsible for phthalate toxicity will be critical for understanding the long-term impacts of environmental chemicals and pharmaceuticals on human reproductive health.

NAP-22 and MARCKS mRNA expression levels in spontaneously hypertensive rat kidneys: the effect of drinking water with different calcium contents

Objective. The aim of study was to assess the effect of the level of calcium intake with drinking water on NAP-22 and MARCKS mRNA expression in cortical and medullar kidney layers of spontaneously hypertensive rats.Design and methods. The study involved 90-day-old SHR (n = 8) and WKY (n = 8) strain rats of both sexes. We assessed tissue samples from cortical and medullar kidney layers. NAP-22 and MARCKS mRNA expression levels were determined by RT-PCR.Results. Sufficient drinking water calcium intake was associated with similar the expression of NAP-22 and MARCKS mRNA in kidneys of spontaneously hypertensive and normotensive rats. Consumption of drinking water with insufficient calcium content it decreases in both rat strains, being more evident in spontaneously hypertensive rats, especially in the medullar layer.Conclusions. Our results show that genetically determined impairments of calcium metabolism in cells of spontaneously-hypertensive rats (SHR line) and their effect on intracellular signaling processes are more evident with the reduced intake of exogenous calcium.

NAP-22 and MARCKS mRNA expression levels in spontaneously hypertensive rat kidneys: the effect of drinking water with different calcium contents

Objective. The aim of study was to assess the effect of the level of calcium intake with drinking water on NAP-22 and MARCKS mRNA expression in cortical and medullar kidney layers of spontaneously hypertensive rats.Design and methods. The study involved 90-day-old SHR (n = 8) and WKY (n = 8) strain rats of both sexes. We assessed tissue samples from cortical and medullar kidney layers. NAP-22 and MARCKS mRNA expression levels were determined by RT-PCR.Results. Sufficient drinking water calcium intake was associated with similar the expression of NAP-22 and MARCKS mRNA in kidneys of spontaneously hypertensive and normotensive rats. Consumption of drinking water with insufficient calcium content it decreases in both rat strains, being more evident in spontaneously hypertensive rats, especially in the medullar layer.Conclusions. Our results show that genetically determined impairments of calcium metabolism in cells of spontaneously-hypertensive rats (SHR line) and their effect on intracellular signaling processes are more evident with the reduced intake of exogenous calcium.

MUC1: Structure, Function, and Clinic Application in Epithelial Cancers.

The transmembrane glycoprotein mucin 1 (MUC1) is a mucin family member that has different functions in normal and cancer cells. Owing to its structural and biochemical properties, MUC1 can act as a lubricant, moisturizer, and physical barrier in normal cells. However, in cancer cells, MUC1 often undergoes aberrant glycosylation and overexpression. It is involved in cancer invasion, metastasis, angiogenesis, and apoptosis by virtue of its participation in intracellular signaling processes and the regulation of related biomolecules. This review introduces the biological structure and different roles of MUC1 in normal and cancer cells and the regulatory mechanisms governing these roles. It also evaluates current research progress and the clinical applications of MUC1 in cancer therapy based on its characteristics.

Increased expression of autophagy and Nrf2-dependent signaling pathway genes by new monophenolic antioxidants depends on their structure

Under certain conditions, both activators and inhibitors of Nrf2-dependent signaling and autophagy can serve as potential agents for the prevention, treatment, and maintenance therapy of cancer, as well as overcoming chemoresistance. We are developing a panel of structurally related synthetic monophenolic compounds, in addition to a direct antioxidant effect, having the ability to affect intracellular signaling pathways and processes, including the above mentioned. Aim of the study was to investigate the relationship between its structure and the ability to influence the expression of autophagy and Nrf2 pathway genes.Material and methods. Five original hydrophilic phenolic structurally related compounds were synthesized; the prototypical inductor of the Keap1/Nrf2/ARE system, monosubstituted phenol tert-butylhydro-quinone (tBHQ), served as a reference substance. We used cultures of murine Lewis lung carcinoma (LLC) and human breast adenocarcinoma MCF-7 cells, with the test compounds at a concentration of 20 gM cells were incubated for 24 hours. Changes in mRNA expression of Nrf2, Nqo1, LC3b, and Sqstm1 genes encoding Nrf2, NAD(P)H:quinone oxidoreductase-1, LC3B, p62/sequestosome-1, respectively, were determined by TaqMan real-time PCR.Results and discussion. Structurally related synthetic monophenols effectively induced the Keap1/Nrf2/ARE system in LLC and MCF-7 cells, increasing the expression of the Nrf2-driven Nqo1 gene, and ambiguously affected the transcription of genes of proteins mediating autophagy: the content of mRNA of the LC3B protein did not change, while the expression of mRNA of the p62 protein, which affects the operation of both systems (processes), is increased only by the action of ortho-monosubstituted phenol with an «active» sulfur atom in the para-propyl substituent TS-13 and its structural analogue TS-12 with a para-substituent shortened by one methylene. It can be assumed that the observed effect either indicates the ability of TS-13 and TS-12 both to induce the Keap1/Nrf2/ARE system and to stimulate autophagy, or reflects a compound-mediated mutually reinforcing cross-activation of these processes.

Characterization of neutral sphingomyelinase activity and isoform expression in rodent skeletal muscle mitochondria

Skeletal muscle is composed of fiber types that differ in mitochondrial content, antioxidant capacity, and susceptibility to apoptosis. Ceramides have been linked to oxidative stress-mediated apoptotic intracellular signalling and the enzyme neutral sphingomyelinase (nSMase) is, in part, responsible for generating these ceramides through the hydrolysis of sphingomyelin. Despite the role of ceramides in mediating apoptosis, there is a gap in the literature regarding nSMase in skeletal muscle mitochondria. This study aimed to characterize total nSMase activity and individual isoform expression in isolated subsarcolemmal (SS) mitochondria from soleus, diaphragm, plantaris, and extensor digitorum longus (EDL). Total nSMase activity did not differ between muscle types. nSMase2 content was detectable in all muscles and higher in EDL, soleus, and plantaris compared to diaphragm whereas nSMase3 was undetectable in all muscles. Finally, total nSMase activity positively correlated to nSMase2 protein content in soleus but not the other muscles. These findings suggest that nSMase associated with SS mitochondria may play a role in intracellular signalling processes involving ceramides in skeletal muscle and nSMase2 may be the key isoform, specifically in slow twitch muscle like soleus. Further studies are needed to fully elucidate the specific contribution of nSMase, along with the role of the various isoforms and mitochondrial subpopulation in generating mitochondrial ceramides in skeletal muscle, and its potential effects on mediating apoptosis.

Altered Signal Transduction in the Immune Response to Influenza Virus and S. pneumoniae or S. aureus Co-Infections.

Influenza virus is a well-known respiratory pathogen, which still leads to many severe pulmonary infections in the human population every year. Morbidity and mortality rates are further increased if virus infection coincides with co-infections or superinfections caused by bacteria such as Streptococcus pneumoniae (S. pneumoniae) and Staphylococcus aureus (S. aureus). This enhanced pathogenicity is due to complex interactions between the different pathogens and the host and its immune system and is mainly governed by altered intracellular signaling processes. In this review, we summarize the recent findings regarding the innate and adaptive immune responses during co-infection with influenza virus and S. pneumoniae or S. aureus, describing the signaling pathways involved and how these interactions influence disease outcomes.

Characterization of voltage gated calcium channels in human sensory neurons

Voltage gated calcium channels (VGCCs) in sensory neurons, as with almost all excitable cells, are responsible for initiating a variety of cellular processes ranging from transmitter release to changes in gene expression. VGCCs are also a primary target for a number of drugs in current use to treat pain. Despite their importance, though, there has been no functional characterization of VGCCs in human sensory neurons. To address this knowledge gap, whole cell patch clamp was used to characterize high voltage activating (HVA) voltage-gated Ca2+ current in acutely dissociated human and rat dorsal root ganglion (DRG) neurons. The biophysical properties and relative proportions of pharmacologically-defined HVA current subtypes activated with square or action potential waveforms were assessed, as was the regulation of HVA currents downstream of µ-opioid receptor (MOR) activation. HVA currents in sensory neurons from humans and rats shared general features such as the voltage of activation and suppression following MOR activation. However, we did observe three significant species differences: (1) Ca2+ current density was significantly smaller in human sensory neurons, (2) the proportion of nifedipine-sensitive currents was greater and that of ω-conotoxin IVA-sensitive current was less in human sensory neurons compared to rat, and (3) a subpopulation of human neurons displayed relatively large constitutive inhibition of HVA currents relieved with a depolarizing pre-pulse. While not explored in the rat, there was a significant negative correlation between HVA current density and donor age. These results suggest that there may be far more extensive compartmentalization of intracellular signaling processes in human sensory neurons, and that it may be necessary to re-evaluate the current subtypes targeted for therapeutic interventions to treat pain. R01DK107966,R01AR063772, UG3TR003090.