Transmembrane Signaling Pathways Research Articles

Is cholesterol both the lock and key to abnormal transmembrane signals in Autism Spectrum Disorder?

Disturbances in cholesterol homeostasis have been associated with ASD. Lipid rafts are central in many transmembrane signaling pathways (including mTOR) and changes in raft cholesterol content affect their order function. Cholesterol levels are controlled by several mechanisms, including endoplasmic reticulum associated degradation (ERAD) of the rate limiting HMGCoA reductase. A new approach to increase cholesterol via temporary ERAD blockade using a benign bacterial toxin-derived competitor for the ERAD translocon is suggested.A new lock and key model for cholesterol/lipid raft dependent signaling is proposed in which the rafts provide both the afferent and efferent 'tumblers' across the membrane to allow 'lock and key' receptor transmembrane signals.

Circularized fluorescent nanodiscs for probing protein–lipid interactions

Protein–lipid interactions are vital for numerous transmembrane signaling pathways. However, simple tools to characterize these interactions remain scarce and are much needed to advance our understanding of signal transduction across lipid bilayers. To tackle this challenge, we herein engineer nanodisc as a robust fluorescent sensor for reporting membrane biochemical reactions. We circularize nanodiscs via split GFP and thereby create an intensity-based fluorescent sensor (isenND) for detecting membrane binding and remodeling events. We show that isenND responds robustly and specifically to the action of a diverse array of membrane-interacting proteins and peptides, ranging from synaptotagmin and synuclein involved in neurotransmission to viral fusion peptides of HIV-1 and SARS-CoV-2. Together, isenND can serve as a versatile biochemical reagent useful for basic and translational research of membrane biology.

The Efficient Regeneration of Corneal Nerves via Tunable Transmembrane Signaling Channels Using a Transparent Graphene-Based Corneal Stimulation Electrode.

The efficient regeneration of corneal nerves is of limited success in the field of ophthalmology. This work reports the use of a non-invasive electrical stimulation technique that uses a transparent graphene-based corneal stimulation electrode and that can achieve efficient regeneration of corneal nerves. The corneal stimulation electrode is prepared using electroactive nitrogen-containing conducting polymers such as polyaniline functionalized graphene (PAG). This composite can carry a high capacitive current. It can be used to tune transmembrane signaling pathways including calcium channels and the MAPK signaling pathway. Tuning can lead to the efficient regeneration of corneal damaged nerves after the surgery of laser in-situ keratomileusis (LASIK). The composite and its application reported have the potential to provide a new way to treat nerve-related injuries.

针刺对5-HT7R跨膜信号通路的影响与其改善PCPA失眠中枢5-HT障碍机制的关联性探讨

针刺对5-HT7R跨膜信号通路的影响与其改善PCPA失眠中枢5-HT障碍机制的关联性探讨

SUN-030 Expression Profiles of Short RNAs in Mouse Serum Employing Endocrine Organs as Controls

Over 90% of the human genome expresses many different forms of non-protein-coding (nc) RNAs in addition to mRNAs. They are categorized into short and long ncRNAs. Although their overall functions and biological importance are still under investigation, they appear to be essential for supporting complex human activities mainly by regulating genome transcription and/or protein translation. It is known that some short RNAs are identified in several body fluids of humans, including serum, urine and saliva. Biological and/or pathological roles of serum micro (mi) RNAs have recently been reported, whereas little is known about other short non-coding (snc) RNAs circulating in serum. Here we present a comprehensive analysis on the short RNAs present in mouse serum using the RNA-sequencing by employing two endocrine organs (adrenal gland and ovary) and liver as controls. We found that serum has numerous short RNA reads and most of them (~90%) are unable to be mapped to the reference genome. Among the mapped reads, short RNAs expressed from the intergenic area (intergenic sncRNAs) represent the majority (97.7%) of short RNAs appeared in mouse serum. These intergenic sncRNAs are mapped to the intergenic area located closely to the nearby protein-coding genes (~20 kb distance), indicating their potential regulatory functions on the transcription of these coding genes. Most of the serum intergenic sncRNAs are distinct from those identified in the three control organs, whereas the intergenic sncRNAs found in the control organs highly overlap with each other. Among known sncRNAs, fragments of ribosomal (r) RNAs (72.0%), transfer (t) RNAs (18.1%), piwi-interacting (pi) RNAs (4.01%) and micro (mi) RNAs (3.96%) are most abundant, whereas small nuclear (sn) RNAs (1.46%) and small nucleolar (sno) RNAs (0.44%) are found as minor fractions. Very few short RNAs derived from mRNAs or long non-coding RNAs are also identified in mouse serum. Interestingly, many of the mRNA fragments identified in serum are derived from the genes associated with the transmembrane signaling pathways in the Ingenuity® Pathway Analysis, suggesting easy liberation of these mRNA fragments from respective cells possibly due to their local translation as reported in neuronal cells. Taken together, our findings indicate that sncRNAs, particularly intergenic sncRNAs, rRNA fragments and tRNAs, are abundant in mouse serum. These results suggest in humans that they may have important biological actions and/or potential clinical implications as valuable clinical markers/therapeutic targets in addition to miRNAs.

Genetic Alterations and Tumor Mutation Burden of Poorly Differentiated Small Cell Euro-endocrine Carcinomas are Similar in Lung Lesions and Distant Metastatic Foci

Objective: Studying the genetic alterations of poorly differentiated small cell neuroendocrine carcinomas to improve the understanding of the biology of these aggressive cancers. Methods: Next generation sequencing was performed on the DNA extracted samples, using the Illumina HiSeq2000/4000 on 315 cancer related genes and tumor mutation burden was reported. Results: In 914 small cell lung cancer (SCLC) and 115 small cell of undefined primary (SCUP), there were similar and close rates of genetic alterations in lung lesions and distant metastatic foci in SCLC and SCUP. Also, the majority of tumors, both lung lesions and distant metastatic foci, did not carry a high tumor mutation burden. Multiple potentially targetable driver genes were identified. Despite common involvement of transmembrane signaling pathways and transcription machinery, other than TP53 and RB1, there was no considerable concurrent gene alteration. Conclusion: This study showed similar genetic alteration and tumor mutation burden in the lung lesions and in distant metastatic foci. TP53 and RB1 were the frequently altered concurrently.

Direct evidence for BBSome-associated intraflagellar transport reveals distinct properties of native mammalian cilia.

Cilia dysfunction underlies a class of human diseases with variable penetrance in different organ systems. Across eukaryotes, intraflagellar transport (IFT) facilitates cilia biogenesis and cargo trafficking, but our understanding of mammalian IFT is insufficient. Here we perform live analysis of cilia ultrastructure, composition and cargo transport in native mammalian tissue using olfactory sensory neurons. Proximal and distal axonemes of these neurons show no bias towards IFT kinesin-2 choice, and Kif17 homodimer is dispensable for distal segment IFT. We identify Bardet–Biedl syndrome proteins (BBSome) as bona fide constituents of IFT in olfactory sensory neurons, and show that they exist in 1:1 stoichiometry with IFT particles. Conversely, subpopulations of peripheral membrane proteins, as well as transmembrane olfactory signalling pathway components, are capable of IFT but with significantly less frequency and/or duration. Our results yield a model for IFT and cargo trafficking in native mammalian cilia and may explain the penetrance of specific ciliopathy phenotypes in olfactory neurons.

Role of PKCα−p38MAPK−Giα axis in peroxynitrite-mediated inhibition of β-adrenergic response in pulmonary artery smooth muscle cells

In the context of cross-talk between transmembrane signaling pathways, we studied the loci within the β-adrenergic receptor/G protein/adenyl cyclase system at which PKC exerts regulatory effects of peroxynitrite (ONOO−) on isoproterenol stimulated adenyl cyclase activity in pulmonary artery smooth muscle cells. Treatment of the cells with ONOO− stimulated PKC-α activity and that subsequently increased p38MAPK phosphorylation. Pretreatment with Go6976 (PKC-α inhibitor) and SB203580 (p38MAPK inhibitor) eliminated ONOO− caused inhibition on isoproterenol stimulated adenyl cyclase activity. Pretreatment with Go6976, but not SB203580, prevented ONOO− induced increase in PKC-α activity. Studies using genetic inhibitors of PKC-α (PKC-α siRNA) and p38MAPK (p38MAPK siRNA) also corroborated the findings obtained with their pharmacological inhibitors in eliminating the attenuation of ONOO− effect on isoproterenol stimulated adenyl cyclase activity. This inhibitory effect of ONOO− was found to be eliminated upon pretreatment of the cells with pertussis toxin thereby pointing to a Gi dependent mechanism. This hypothesis was reinforced by Giα phosphorylation as well as by the observation of the loss of the ability of Gpp(NH)p (a measure of Gi mediated response) to stimulate adenyl cyclase activity upon ONOO− treatment to the cells. We suggest the existence of a pertussis toxin sensitive G protein (Gi)-mediated mechanism in isoproterenol stimulated adenyl cyclase activity, which is regulated by PKCα-p38MAPK axis dependent phosphorylation of its α-subunit (Giα) in the pulmonary artery smooth muscle cells.

An ABC transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas aeruginosa

Pseudomonas aeruginosa is capable of injecting protein toxins into other bacterial cells through one of its three type VI secretion systems (T6SSs). The activity of this T6SS is tightly regulated on the posttranslational level by phosphorylation-dependent and -independent pathways. The phosphorylation-dependent pathway consists of a Threonine kinase/phosphatase pair (PpkA/PppA) that acts on a forkhead domain-containing protein, Fha1, and a periplasmic protein, TagR, that positively regulates PpkA. In the present work, we biochemically and functionally characterize three additional proteins of the phosphorylation-dependent regulatory cascade that controls T6S activation: TagT, TagS and TagQ. We show that similar to TagR, these proteins act upstream of the PpkA/PppA checkpoint and influence phosphorylation of Fha1 and, apparatus assembly and effector export. Localization studies demonstrate that TagQ is an outer membrane lipoprotein and TagR is associated with the outer membrane. Consistent with their homology to lipoprotein outer membrane localization (Lol) components, TagT and TagS form a stable inner membrane complex with ATPase activity. However, we find that outer membrane association of T6SS lipoproteins TagQ and TssJ1, and TagR, is unaltered in a ΔtagTS background. Notably, we found that TagQ is indispensible for anchoring of TagR to the outer membrane fraction. As T6S-dependent fitness of P. aeruginosa requires TagT, S, R and Q, we conclude that these proteins likely participate in a trans-membrane signalling pathway that promotes H1-T6SS activity under optimal environmental conditions.

Alteration of Ceramide 1-O-Functionalization as a Promising Approach for Cancer Therapy

Sphingolipids, which are complex lipidic components of the cell membranes, lie in a key position to modulate the pathways of trans-membrane signaling and allow the cell to adapt to environmental stresses. In malignancies, reduced production of some sphingolipid species able to induce apoptosis such as ceramide and conversely, increased levels of some other metabolites involved in tumor progression and drug resistance of cancer cells, are often described. In this context, the discovery of new chemical entities able to specifically modify ceramide metabolism should offer novel pharmacological tools in cancer therapy. The review dedicates particular attention to the enzymes that modify ceramide at the C1-OH position generating other biologically important sphingolipids in cancer, such as sphingomyelin, ceramide-1-phosphate or glucosylceramide. Findings reported in the literature leading to the development of new chemical entities specifically designed to achieve the above goals have been collected and are discussed. The effects of enzyme inhibitors of sphingomyelin synthase, ceramide kinase and glucosylceramide synthase on cancer cell proliferation, sensitivity to chemotherapeutics, induction of apoptosis or growth of xenografts are presented.

Wnt transmembrane signaling and long‐term spatial memory

Transmembrane signaling mechanisms are critical for regulating the plasticity of neuronal connections underlying the establishment of long-lasting memory (e.g., Linden and Routtenberg (1989) Brain Res Rev 14:279-296; Sossin (1996) Trends Neurosci 19:215-218; Mayr and Montminy (2001) Nat Rev Mol Cell Biol 2:599-609; Chen et al. (2011) Nature 469:491-497). One signaling mechanism that has received surprisingly little attention in this regard is the well-known Wnt transmembrane signaling pathway even though this pathway in the adult plays a significant role, for example, in postsynaptic dendritic spine morphogenesis and presynaptic terminal neurotransmitter release (Inestrosa and Arenas (2010) Nat Rev Neurosci 11:77-86). The present report now provides the first evidence of Wnt signaling in spatial information storage processes. Importantly, this Wnt participation is specific and selective. Thus, spatial, but not cued, learning in a water maze selectively elevates the levels in hippocampus of Wnt 7 and Wnt 5a, but not the Wnt 3 isoform, indicating behavioral selectivity and isoform specificity. Wnt 7 elevation is subfield-specific: granule cells show an increase with no detectable change in CA3 neurons. Wnt 7 elevation is temporally specific: increased Wnt signaling is not observed during training, but is seen 7 days and, unexpectedly, 30 days later. If the Wnt elevation after learning is activity-dependent, then it may be possible to model this effect in primary hippocampal neurons in culture. Here, we evaluate the consequence of potassium or glutamate depolarization on Wnt signaling. This represents, to our knowledge, the first demonstration of an activation-dependent elevation of Wnt levels and surprisingly an increased number of Wnt-stained puncta in neurites suggestive of trafficking from the cell body to neuronal processes, probably dendrites. It is proposed that Wnt signaling pathways regulate long-term information storage in a behavioral-, cellular-, and isoform-specific manner.

Protein Footprinting in a Complex Milieu: Identifying the Interaction Surfaces of the Chemotaxis Adaptor Protein CheW

Characterizing protein–protein interactions in a biologically relevant context is important for understanding the mechanisms of signal transduction. Most signal transduction systems are membrane associated and consist of large multiprotein complexes that undergo rapid reorganization—circumstances that present challenges to traditional structure determination methods. To study protein–protein interactions in a biologically relevant complex milieu, we employed a protein footprinting strategy based on isotope-coded affinity tag (ICAT) reagents. ICAT reagents are valuable tools for proteomics. Here, we show their utility in an alternative application—they are ideal for protein footprinting in complex backgrounds because the affinity tag moiety allows for enrichment of alkylated species prior to analysis. We employed a water-soluble ICAT reagent to monitor cysteine accessibility and thereby to identify residues involved in two different protein–protein interactions in the Escherichia coli chemotaxis signaling system. The chemotaxis system is an archetypal transmembrane signaling pathway in which a complex protein superstructure underlies sophisticated sensory performance. The formation of this superstructure depends on the adaptor protein CheW, which mediates a functionally important bridging interaction between transmembrane receptors and histidine kinase. ICAT footprinting was used to map the surfaces of CheW that interact with the large multidomain histidine kinase CheA, as well as with the transmembrane chemoreceptor Tsr in native E. coli membranes. By leveraging the affinity tag, we successfully identified CheW surfaces responsible for CheA–Tsr interaction. The proximity of the CheA and Tsr binding sites on CheW suggests the formation of a composite CheW–Tsr surface for the recruitment of the signaling kinase to the chemoreceptor complex.

Over-accumulation of nuclear IGF-1 receptor in tumor cells requires elevated expression of the receptor and the SUMO-conjugating enzyme Ubc9

The insulin-like growth factor 1 receptor (IGF-1R) plays crucial roles in tumor cell growth and is overexpressed in many cancers. IGF-1R’s trans-membrane kinase signaling pathways have been well characterized. Very recently, we showed that SUMOylation mediates nuclear translocation of the IGF-1R, and that nuclear IGF-1R (nIGF-1R) binds to enhancer regions and activates transcription. We identified three lysine residues in the β-subunit of the receptor and that mutation of these blocks nuclear translocation and gene activation. Furthermore, accumulation of nIGF-1R was proven strongly dependent on the specific SUMO-conjugating enzyme Ubc9. Here we show that nIGF-1R originates solely from the cell membrane and that phosphorylation of the core tyrosine residues of the receptor kinase is crucial for nuclear accumulation. We also compared the levels of nIGF-1R, measured as nuclear/membrane ratios, in tumor and normal cells. We found that the breast cancer cell line MCF-7 has 13-fold higher amounts of nIGF-1R than breast epithelial cells (IME) which showed only a small amount of nIGF-1R. In comparison, the total expression of IGF-1R was only 3.7- higher in MCF-7. Comparison of several other tumor and normal cell lines showed similar tumor cell over-accumulation of nIGF-1R, exceeding the total receptor expression substantially. Ectopic overexpression (>10-fold) of the receptor increased nIGF-1R in IME cells but not to that high level as in wild type MCF-7. The levels of Ubc9 were higher in all tumor cell lines, compared to the normal cells, and this probably contributes to over-accumulation of nIGF-1R. Over-accumulation of nIGF-1R may contribute to deregulated gene expression and therewith play a pathophysiological role in cancer cells.

Role of obestatin on growth hormone secretion: An in vitro approach

Obestatin, the ghrelin-associated peptide, showed to activate MAPK signaling with no effect on Akt nor cell proliferating activity in rat tumor somatotroph cells (growth cells, GC). A sequential analysis of the obestatin transmembrane signaling pathway indicated a route involving the consecutive activation of G i, PI3k, novel PKCε, and Src for ERK1/2 activation. Furthermore, obestatin treatment triggers growth hormone (GH) release in the first 30 min, being more acute at 15 min. At 1 h, obestatin treated cells showed the same levels in GH secretion than controls. Added to this functionality, obestatin was secreted by GC cells. Based on the capacity to stimulate GH release from somatotroph cells, obestatin may act directly in the pituitary through an autocrine/paracrine mechanism.

Obestatin stimulates Akt signalling in gastric cancer cells through β-arrestin-mediated epidermal growth factor receptor transactivation

Obestatin was identified as a gut peptide encoded by the ghrelin gene that interacts with the G protein-coupled receptor, GPR39. In this work, a sequential analysis of its transmembrane signalling pathway has been undertaken to characterize the intracellular mechanisms responsible for Akt activation. The results show that Akt activation requires the phosphorylation of T308 in the A-loop by the phosphoinositide-dependent kinase 1 (PDK1) and S473 within the HM by the mammalian target of rapamycin (mTOR) kinase complex 2 (mTORC2: Rictor, mLST8, mSin1, mTOR kinase) with participation neither of G(i)(/o)-protein nor Gbetagamma dimers. Obestatin induces the association of GPR39/beta-arrestin 1/Src signalling complex resulting in the transactivation of the epidermal growth factor receptor (EGFR) and downstream Akt signalling. Upon administration of obestatin, phosphorylation of mTOR (S2448) and p70S6K1 (T389) rise with a time course that parallels that of Akt activation. Based on the experimental data obtained, a signalling pathway involving a beta-arrestin 1 scaffolding complex and EGFR to activate Akt signalling is proposed.

Gastrointestinal stromal tumors - A model for understanding solid tumor biology and development of targeted therapies, or just another low-hanging fruit?

Gastrointestinal stromal tumors - A model for understanding solid tumor biology and development of targeted therapies, or just another low-hanging fruit?

Transmembrane signaling pathway mediates oxidized low-density lipoprotein-induced expression of plasminogen activator inhibitor-1 in vascular endothelial cells

Atherosclerotic cardiovascular disease is the number one cause of death for adults in Western society. Plasminogen activator inhibitor-1 (PAI-1), the major physiological inhibitor of plasminogen activators, has been implicated in both thrombogenesis and atherogenesis. Previous studies demonstrated that copper-oxidized low-density lipoprotein (C-oLDL) stimulated production of PAI-1 in vascular endothelial cells (EC). The present study examined the involvement of lectin-like oxidized LDL receptor-1 (LOX-1) and Ras/Raf-1/ERK1/2 pathway in the upregulation of PAI-1 in cultured EC induced by oxidized LDLs. The results demonstrated that C-oLDL or FeSO(4)-oxidized LDL (F-oLDL) increased the expression of PAI-1 or LOX-1 in human umbilical vein EC (HUVEC) or coronary artery EC (HCAEC). Treatment with C-oLDL significantly increased the levels of H-Ras mRNA, protein, and the translocation of H-Ras to membrane fraction in EC. LOX-1 blocking antibody, Ras farnesylation inhibitor (FTI-277), or small interference RNA against H-Ras significantly reduced C-oLDL or LDL-induced expression of H-Ras and PAI-1 in EC. Incubation with C-oLDL or F-oLDL increased the phosphorylation of Raf-1 and ERK1/2 in EC compared with LDL or vehicle. Treatment with Raf-1 inhibitor blocked Raf-1 phosphorylation and the elevation of PAI-1 mRNA level in EC induced by C-oLDL or LDL. Treatment with PD-98059, an ERK1/2 inhibitor, blocked C-oLDL or LDL-induced ERK1/2 phosphorylation or PAI-1 expression in EC. The results suggest that LOX-1, H-Ras, and Raf-1/ERK1/2 are implicated in PAI-1 expression induced by oxidized LDLs or LDL in cultured EC.

Tyrosine phosphorylation and Ras activation is required for hydrogen peroxide-mediated Raf-1 kinase activation

Reactive oxygen species (ROS), such as hydrogen peroxide (H(2)O(2)), have been shown to play a significant role in regulating transmembrane signaling pathways to modulate cell proliferation and differentiation. Here we report findings that indicate that treatment of Sf9 cells expressing Raf-1 with H(2)O(2) results in significant and sustained activation of Raf-1 kinase. The activation of Raf-1 in response to H(2)O(2) treatment of Raf-expressing Sf9 cells was found to involve tyrosine phosphorylation, detected by immunoblotting with anti-phosphotyrosine antibody. The addition of tyrosine-specific phosphatase (PTP1B) to Raf-1 immunoprecipitated from Sf9 cells infected with Raf-1 after H(2)O(2) stimulation partially decreased the kinase activity of Raf-1. In a mammalian cell system, we also identified that the overexpression of a kinase-negative Raf-1 fragment (which acts as a dominant-negative inhibitor of Ras-Raf interaction) resulted in the inhibition of the H(2)O(2)-induced activation of Raf-1. Moreover, the blocking of the Ras function by the farnesyltransferase inhibitor, alpha-hydroxyfarnesylphosphonic acid, led to a 40% or greater reduction in Raf-1 kinase activity, suggesting that Ras is involved in the signaling pathway mediating the H(2)O(2) activation of Raf-1. Taken together, these results suggest that tyrosine phosphorylation and Ras activation are essential components of the mechanism by which H(2)O(2) activates Raf-1 kinase activity.

Physiology of the Prion Protein

Prion diseases are transmissible spongiform encephalopathies (TSEs), attributed to conformational conversion of the cellular prion protein (PrP(C)) into an abnormal conformer that accumulates in the brain. Understanding the pathogenesis of TSEs requires the identification of functional properties of PrP(C). Here we examine the physiological functions of PrP(C) at the systemic, cellular, and molecular level. Current data show that both the expression and the engagement of PrP(C) with a variety of ligands modulate the following: 1) functions of the nervous and immune systems, including memory and inflammatory reactions; 2) cell proliferation, differentiation, and sensitivity to programmed cell death both in the nervous and immune systems, as well as in various cell lines; 3) the activity of numerous signal transduction pathways, including cAMP/protein kinase A, mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt pathways, as well as soluble non-receptor tyrosine kinases; and 4) trafficking of PrP(C) both laterally among distinct plasma membrane domains, and along endocytic pathways, on top of continuous, rapid recycling. A unified view of these functional properties indicates that the prion protein is a dynamic cell surface platform for the assembly of signaling modules, based on which selective interactions with many ligands and transmembrane signaling pathways translate into wide-range consequences upon both physiology and behavior.

Stochastic Signal Processing and Transduction in Chemotactic Response of Eukaryotic Cells

Single-molecule imaging analysis of chemotactic response in eukaryotic cells has revealed a stochastic nature in the input signals and the signal transduction processes. This leads to a fundamental question about the signaling processes: how does the signaling system operate under stochastic fluctuations or noise? Here, we report a stochastic model of chemotactic signaling in which noise and signal propagation along the transmembrane signaling pathway by chemoattractant receptors can be analyzed quantitatively. The results obtained from this analysis reveal that the second-messenger-production reactions by the receptors generate noisy signals that contain intrinsic noise inherently generated at this reaction and extrinsic noise propagated from the ligand-receptor binding. Such intrinsic and extrinsic noise limits the directional sensing ability of chemotactic cells, which may explain the dependence of chemotactic accuracy on chemical gradients that has been observed experimentally. Our analysis also reveals regulatory mechanisms for signal improvement in the stochastically operating signaling system by analyzing how the SNR of chemotactic signals can be improved on or deteriorated by the stochastic properties of receptors and second-messenger molecules. Theoretical consideration of noisy signal transduction by chemotactic signaling systems can further be applied to signaling systems in general.