Umami taste receptor functions as an amino acid sensor via Gαs subunit in N1E-115 neuroblastoma cells

The sensing of the nutritional level of the body fluid is pivotal for maintaining homeostasis in animals. However, it is not yet understood how the cells detect nutritional levels. In the present study, we examined the function of umami taste receptor, which has a dimeric protein structure composed of Tas1r1 and Tas1r3, as amino acid sensor in the cells. We found that deprivation of amino acids induced neurite outgrowth in N1E-115 cells. The neurite outgrowth was inhibited by almost all of the amino acids tested. To investigate the involvement of the umami taste receptor, siRNA against each of Tas1r1 or Tas1r3 was administered, resulting in suppression of the inhibitory effects of amino acids on neurite outgrowth. In addition, inosine 5'-monophosphate, which potentiates the response to amino acids in the taste cells, enhanced the inhibitory effect of glutamine on neurite outgrowth. These results suggest that Tas1r1 + 3 functions as an amino acid sensor in N1E-115 cells. Because glutamine increased intracellular cAMP concentration, we investigated the involvement of the Gαs subunit of the heterotrimeric G protein in signal transduction. The treatments to inhibit the Gαs subunit significantly suppressed the increase of intracellular cAMP concentration induced by glutamine and the inhibitory effect of amino acids on neurite outgrowth. In addition, the reagents for increasing intracellular cAMP concentration inhibited neurite outgrowth induced by deprivation of amino acids. We concluded that Tas1r1 + 3 functions as an amino acid sensor and activates the intracellular signaling pathway through the Gαs subunit in N1E-115 cells.     

Nucleotide-dependent displacement and dynamics of the α-1 helix in kinesin revealed by site-directed spin labeling EPR

In kinesin X-ray crystal structures, the N-terminal region of the α-1 helix is adjacent to the adenine ring of the bound nucleotide, while the C-terminal region of the helix is near the neck-linker (NL). Here, we monitor the displacement of the α-1 helix within a kinesin monomer bound to microtubules (MTs) in the presence or absence of nucleotides using site-directed spin labeling EPR. Kinesin was doubly spin-labeled at the α-1 and α-2 helices, and the resulting EPR spectrum showed dipolar broadening. The inter-helix distance distribution showed that 20% of the spins have a peak characteristic of 1.4–1.7 nm separation, which is similar to what is predicted from the X-ray crystal structure, albeit 80% were beyond the sensitivity limit (>2.5 nm) of the method. Upon MT binding, the fraction of kinesin exhibiting an inter-helix distance of 1.4–1.7 nm in the presence of AMPPNP (a non-hydrolysable ATP analog) and ADP was 20% and 25%, respectively. In the absence of nucleotide, this fraction increased to 40–50%. These nucleotide-induced changes in the fraction of kinesin undergoing displacement of the α-1 helix were found to be related to the fraction in which the NL undocked from the motor core. It is therefore suggested that a shift in the α-1 helix conformational equilibrium occurs upon nucleotide binding and release, and this shift controls NL docking onto the motor core.     

Rotational diffusion of the α(2a) adrenergic receptor revealed by FlAsH labeling in living cells

The fluorescein arsenical hairpin binder (FlAsH) shows much promise to determine the relative orientations of protein regions and structures even in living cells and in the plasma membrane. In this study, we characterized FlAsH's photophysical properties by steady-state anisotropy and time-resolved single photon counting for further applications with G-protein coupled receptors. We find that FlAsH has a relatively high initial anisotropy of 0.31 ± 0.01 and a three-component fluorescence lifetime with an average of 4.1 ± 0.1 ns. We characterized the FlAsH fluorophore orientation in the α(2A) adrenergic receptor revealing rigid orientations of FlAsH in the membrane plane for rotational correlation times of ～50 ns in living cells. To elucidate the fluorophore-membrane orientation and rotational correlation time, an anisotropy treatment similar to that of another researcher (Axelrod, D. 1979. Biophys. J. 26:557-573) was developed. The rotational correlation times were observed to increase by up to 16 ns after agonist addition. The rotational correlation time also allowed for a comparison to the theoretical relationship between translational and rotational diffusion (originally proposed by Saffman, P. G., and M. Delbrück. 1975. Proc. Natl. Acad. Sci. USA. 72:3111-3113) and revealed a discrepancy of a factor between 10 and 100.     

The transport of class I major histocompatibility complex antigens is determined by sequences in the α1 and α2 protein domains

The transport of human-mouse hybrid class I histocompatibility antigens has been studied in a mutant human cell line, 174 × CEM.T2 (T2). T2, a somatic cell hybrid of human B- and T-lymphoblastoid cell lines (B-LCL and T-LCL, respectively), synthesizes HLA-A2 and HLA-B5 glycoproteins, but expresses only low levels of A2 and undetectable levels of B5 at the cell surface. We have previously shown that the products of human class I genes introduced into T2 by transfection behave like the endogenous HLA-B5 glycoproteins, while the products of mouse class I alleles similarly introduced are transported normally to the cell surface. We have now determined that the surface expression of class I glycoproteins in T2 depends on the origin of the ₁ and ₂ domains. Human (HLA-B7) and mouse (H-2D ^p ) hybrid class I genes, encoding the leader, ₁, and ₂ sequences of one species fused to the ₃, transmembrane, and cytoplasmic domains of the other, were transfected into T2. Normal surface expression of the hybrid class I molecule was observed in T2 only when the leader, ₁, and ₂-encoding exons were derived from the mouse gene. The reciprocal construct, encoding human leader, ₁, and ₂ domains fused to the mouse ₃, transmembrane, and cytoplasmic regions, resulted in biosynthesis of a hybrid glycoprotein which was not transported to the cell surface. The products of both constructs were expressed normally in control cells. The effects of glycosylation on class I antigen transport were also studied using mutant class I constructs with altered glycosylation sites. Two mutant B7 genes encoding either an extra glycosylation site at position 176 or no glycosylation sites were transfected into T2. These mutant products were expressed at the cell surface in control cells, but were synthesized and not surface-expressed in T2. These data demonstrate that the HLA/H-2 transport dichotomy in T2 is a function of the origin of the ₁ and/or ₂ domains of the class I glycoprotein, and is not a reflection of glycosylation differences between the human and mouse molecules. Offprint requests to: P. Cresswell.     

Backbone dynamics of an alamethicin in methanol and aqueous detergent solution determined by heteronuclear 1H−15N NMR spectroscopy

Summary The ¹⁵N relaxation rates of the -aminoisobutyric acid (Aib)-rich peptide alamethicin dissolved in methanol at 27°C and 5°C, and dissolved in aqueous sodium dodecylsulfate (SDS) at 27°C, were measured using inverse-detected one-and two-dimensional ¹H–¹⁵N NMR spectroscopy. Measurements of ¹⁵N longitudinal (R_N(N_z)) and transverse (R_N(N_x,y)) relaxation rates and the {¹H} ¹⁵N nuclear Overhauser enhancement (NOE) at 11.7 Tesla were used to calculate (quasi-) spectral density values at 0, 50, and 450 MHz for the peptide in methanol and in SDS. Spectral density mapping at 0, 50, 450, 500, and 550 MHz was done using additional measurements of the ¹H–¹⁵N lingitudinal two-spin order, R_NH(2H _infZ ^supN N_Z), two-spin antiphase coherence, R_NH(2H _infN ^supZ N_x,y), and the proton longitudinal relaxation rate, R_H(H _infN ^supZ ), for the peptide dissolved in methanol only. The spectral density of motions was also modeled using the three-parameter Lipari-Szabo function. The overall rotational correlation times were determined to be 1.1, 2.5, and 5.7 ns for alamethicin in methanol at 27°C and 5°C, and in SDS at 27°C, respectively. From the rotational correlation time determined in SDS the number of detergent molecules associated with the peptide was estimated to be about 40. The average order parameter was about 0.7 and the internal correlation times were about 70 ps for the majority of backbone amide ¹⁵N sites of alamethicin in methanol and in SDS. The relaxation data, spectral densities, and order parameters suggest that the peptide N-H vectors of alamethicin are not as highly constrained as the core regions of folded globular proteins. However, the peptide backbone is clearly not as mobile as the most unconstrained regions of folded proteins, such as those found in the frayed C-and N-termini of some proteins, or in randomcoil peptides. The data also suggest significant mobility at both ends of the peptide dissolved in methanol. In SDS the mobility in the middle and at the ends of the peptide is reduced. The implications of the results with respect to the sterically hindered Aib residues and the biological activities of the peptide are discussed.To whom correspondence should be addressed.     

Dual effects of α-synuclein on neurotoxicity induced by low dosage of rotenone are dependent on exposure time in dopaminergic neuroblastoma cells

This study was designed to investigate the effects of α-synuclein on toxicity induced by long-term exposure to relatively low concentrations of rotenone.Compared with the control groups,the inhibition of cell viability which overexpressed α-synuclein(SH-SY5Y-Syn) improved after 1 and 2 weeks of rotenone treatment.The complex I activity was greater and the mitochondrial membrane swelling intensity was reduced after 1 and 2 weeks of treatment,which indicated that α-synuclein,at least in part,resists the rotenone-induced oxidative stress.The results indicate that α-synuclein has a dual effect on toxicity of rotenone according to exposure time in human SH-SY5Y cells.     

The thiol proteinase inhibitor E-64-d ameliorates amyloid-β-induced reduction of sAPPα secretion by reversing ceramide-induced protein kinase C down-regulation in SH-SY5Y neuroblastoma cells

In Alzheimer’s disease (AD), enhancing α-secretase processing of amyloid precursor protein (APP) is an important pathway to decrease neurotoxic amyloid β (Aβ) secretion. The α-secretase is reported to be regulated by protein kinase C (PKC) and various endogenous proteins or cell surface receptors. In this report, we first examined whether Aβ reduces α-secretase activity, and showed that Aβ peptide 1–40 (0.001 and 0.01 μM) reduced the secretion of soluble amyloid precursor protein α (sAPPα) in carbachol-stimulated SH-SY5Y neuroblastoma cells. E-64-d (3 μM), which is a potent calpain inhibitor that prevents PKC degradation, ameliorated the Aβ-induced reduction of sAPPα secretion. In addition, we observed that Aβ significantly enhanced ceramide production by activating neutral sphingomyelinase. The cell-permeable ceramide analog, C₂-ceramide (1 μg/mL), also reduced sAPPα secretion, and in addition, E-64-d eliminated the observed decrease of sAPPα secretion. C₂-ceramide induced down-regulation of PKC-α, -β₁, and -β₂ isozymes in SH-SY5Y cells. These findings suggest that ceramide may play an important role in sAPPα processing by modulating PKC activity.     

Biosynthesis of a blood group H1 antigen by α1,2-fucosyltransferase in PC12 cells

We have examined the expression of GDP-fucose: glycosphingolipid fucosyltransferase activity in PC12 cells and PC12 sublines in relation to the neuronal differentiation induced by nerve growth factor (NGF) or dexamethasone. Transfer of fucose to paragloboside (nLc₄Cer) yielded a product which was determined to be a blood group H₁ antigen (Fuc1-2Gal1-4GlcNAc1-3Gal1-4Glc-Cer) by gas chromatography/mass spectrometry analysis and enzymatic hydrolysis, suggesting that PC12 cells have an 1,2-fucosyltransferase. Lactosylceramide was also fucosylated at a reduced rate. When the differentiation of PC12 cells and PC12 subline cells, PC12D and MR31, was induced by exposure to either NGF or dexamethasone, the fucosyltransferase activity for nLc₄Cer was found to decrease in both cell lines, suggesting the association with cell differentiation. This is the first report of the presence of an 1,2-fucosyltransferase in cultured neuronal cell lines which catalyses thein vitro biosynthesis from nLc₄Cer of a type-2 chain glycosphingolipid having the blood group H₁ determinant. The disaccharides, -lactose andN-acetyllactosamine, were also fucosylated by PC12 cell enzyme, although the specificity for the carbohydrate structure was different from that for glycosphingolipids.Abbreviations Glc d-glucose - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Fuc l-fucose - Cer ceramide - nLc₄Cer neolactotetraosylceramide (paragloboside) - GDP guanosine diphosphate - CDP cytidine diphosphate - CTP cytidine triphosphate - NGF nerve growth factor - DX dexamethasone - GC/MS gas chromatography/mass spectrometry     

Retinoic acid-induced upregulation of the metalloendopeptidase nardilysin is accelerated by co-expression of the brain-specific protein p42(IP4) (centaurin α1; ADAP1) in neuroblastoma cells

The mainly neuronally expressed protein p42^IP4 (centaurin α1; ADAP1), which interacts with the metalloendopeptidase nardilysin (NRD) was found to be localized in neuritic plaques in Alzheimer disease (AD) brains. NRD was shown to enhance the cleavage of the amyloid precursor protein (APP) by α-secretases, thereby increasing the release of neuroprotective sAPPα. We here investigated in vitro the biochemical interaction of p42^IP4 and NRD and studied the physiological interaction in SH-SY5Y cells. NRD is a member of the M16 family of metalloendopeptidases. Some members of this M16 family act bi-functionally, as protease and as non-enzymatic scaffold protein. Here, we show that p42^IP4 enhances the enzymatic activity of NRD 3-4 times. However, p42^IP4 is not a substrate for NRD. Furthermore, we report that differentiation of SH-SY5Y cells by stimulation with 10 μM retinoic acid (RA) results in upregulation of NRD protein levels, with a 6-fold rise after 15 days. NRD is expressed in the neurites of RA-stimulated SH-SY5Y cells, and localized in vesicular structures. Since p42^IP4 is not expressed in untreated SH-SY5Y cells, we could use this cell system as a model to find out, whether there is a functional interaction. Interestingly, SH-SY5Y cells, which we stably transfected with GFP-tagged-p42^IP4 showed an enhanced NRD protein expression already at an earlier time point after RA stimulation.     

Unique assignment of inter-subunit association in GABAA α1β3γ2 receptors determined by molecular modeling

Recent publications defined requirements for inter-subunit contacts in a benzodiazepine-sensitive GABA_A receptor (GABA_ARα1β3γ2). There is strong evidence that the heteropentameric receptor contains two α1, two β3, and one γ2 subunit. However, the available data do not distinguish two possibilities: When viewed clockwise from an extracellular viewpoint the subunits could be arranged in either γ2β3α1β3α1 or γ2α1β3α1β3 configurations. Here we use molecular modeling to thread the relevant GABA_AR subunit sequences onto a template of homopentameric subunits in the crystal structure of the acetylcholine binding protein (AChBP). The GABA_A sequences are known to have 15-18% identity with the acetylcholine binding protein and nearly all residues that are conserved within the nAChR family are present in AChBP. The correctly aligned GABA_A sequences were threaded onto the AChBP template in the γ2β3α1β3α1 or γ2α1β3α1β3  arrangements. Only the γ2α1β3α1β3 arrangement satisfied three known criteria: (1) α1 His¹⁰² binds at the γ2 subunit interface in proximity to γ2 residues Thr¹⁴², Phe⁷⁷, and Met¹³⁰; (2) α1 residues 80-100 bind near γ2 residues 91-104; and (3) α1 residues 58-67 bind near the β3 subunit interface. In addition to predicting the most likely inter-subunit arrangement, the model predicts which residues form the GABA and benzodiazepine binding sites.     

Neuroglobin upregulation induced by 17β-estradiol sequesters cytocrome c in the mitochondria preventing H2O2-induced apoptosis of neuroblastoma cells

The sex steroid hormone 17β-estradiol (E2) upregulates the levels of neuroglobin (NGB), a new neuroprotectant globin, to elicit its neuroprotective effect against H₂O₂-induced apoptosis. Several mechanisms could be proposed to justify the NGB involvement in E2 prevention of stress-induced apoptotic cell death. Here, we evaluate the ability of E2 to modulate the intracellular NGB localization and the NGB interaction with mitochondrial cytochrome c following the H₂O₂-induced toxicity. Present results demonstrate that NGB is expressed in the nuclei, mitochondria, and cytosol of human neuroblastoma SK-N-BE cells. E2, but not H₂O₂ treatment of SK-N-BE cells, reallocates NGB mainly at the mitochondria and contemporarily reduces the number of apoptotic nuclei and the levels of cleaved caspase-3. Remarkably, the E2 treatment strongly increases NGB–cytochrome c association into mitochondria and reduces the levels of cytochrome c into the cytosol of SK-N-BE cells. Although both estrogen receptors (ERα and ERβ) are expressed in the nucleus, mitochondria, and cytosol of SK-N-BE cells, this E2 effect specifically requires the mitochondrial ERβ activity. As a whole, these data demonstrate that the interception of the intrinsic apoptotic pathway into mitochondria (i.e., the prevention of cytochrome c release) is one of the pivotal mechanisms underlying E2-dependent NGB neuroprotection against H₂O₂ toxicity.     

The modulation of radiation-induced cell death by genistein in K562 cells：Activation of thymidine kinase 1

Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML) cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization (SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression ofTK1 mRNA and TK 1 enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition, the expression of cell cycle-related proteins, cyclin A and cyclin B 1, provided the evidences of G I/S progression and G2-arrest, and their relationship with TKI in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.     

The solid-state catalytic isotope exchange of hydrogen in α-conotoxin G1 by the tritium spillover

Tritium-labeled α-conotoxin G1 with a molar radioactivity of 35 Ci/mmol and full biological activity (according to the binding to nicotinic acetylcholine receptor) was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE). The tritium distribution in the molecule of α-conotoxin G1 was revealed by³H NMR spectroscopy. Tritium was found in all amino acid residues except for the Asn4-Pro5-Ala6 fragment. The data on the comparative reactivity of C-H bonds, theab initio quantum-chemical calculation of the hydrogen exchange reaction, and the information on the spatial structures of α-conotoxin G1 in solution and in crystal state allowed us to establish that the reactivity of H atoms may be increased by their interaction with the electron donor O and N atoms at the transition state of the HSCIE reaction. A decrease in the rate of the HSCIE reaction could be caused by both a poor spatial accessibility of C-H bonds and a limited mobility of the peptide fragment containing these bonds.     

Volume regulatory decrease in UMR-106.01 cells is mediated by specific α1 subunits of L-type calcium channels

An early cellular response of osteoblasts to swelling is plasma membrane depolarization, accompanied by a transient increase in intracellular calcium ([Ca2+]i), which initiates regulatory volume decrease (RVD). The authors have previously demonstrated a hypotonically induced depolarization of the osteoblast plasma membrane, sufficient to open L-type Ca channels and mediate Ca2+ influx. Herein is described the initiation of RVD in UMR-106.01 cells, mediated by hypotonically induced [Ca2+]i transients resulting from the activation of specific isoforms of L-type Ca channels. The authors further demonstrate that substrate interaction determines which specific alpha1 Ca channel subunit isoform predominates and mediates Ca2+ entry and RVD. Swelling-induced [Ca2+]i transients, and RVD in cells grown on a type I collagen matrix, are inhibited by removal of Ca from extracellular solutions, dihydropyridines, and antisense oligodeoxynucleotides directed exclusively to the alpha1C isoform of the L-type Ca channel. Ca2+ transients and RVD in cells grown on untreated glass cover slips were inhibited by similar maneuvers, but only by antisense oligodeoxynucleotides directed to the alpha1S isoform of the L-type Ca channel. This represents the first molecular identification of the Ca channels that transduce the initiation signal for RVD by osteoblastic cells.     

Role of α5β1 and αvβ3 integrins in relation to adhesion and spreading dynamics of prostate cancer cells interacting with fibronectin under in vitro conditions

Interaction of cell integrins with the ECM (extracellular matrix) proteins is commonly assumed to be associated with cell dissemination and tumour metastases. Since these processes depend on the mechanism of cell-protein interaction, we have attempted to show the contribution of α5β1 and αvβ3 integrins of the prostate cancer PC-3 cells in in vitro interaction with FN (fibronectin) adsorbed on defined polystyrene surfaces. Cell adhesion, spreading and cytoskeleton organization were studied using antibodies against integrins or a GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) peptide. The results show that blocking the α5β1 integrin causes: (i) a decrease in the number of the adherent cells in the early phase of adhesion and (ii) a decrease in the dynamics of cell spreading and cell shape changes, and weaker reorganization of cytoskeletal proteins than in the control cells. Conversely, the blocking of the αvβ3 integrin: (i) causes no observable effect on the number of the adhered cells; however, (ii) causes an increase in the dynamics of cell spreading and cell shape changes, and stronger reorganization of cytoskeletal proteins than in the control cells. Interestingly, the blocking of integrins with a GRGDSP peptide strongly decreases the number of the adhered cells, and a complete inhibition of cell spreading. Our results strongly suggest that the α5β1 integrin plays the main role in the adhesion and spreading of PC-3 cells interacting with FN, whereas the αvβ3 integrin seems to regulate other receptors in the spreading process. Moreover, integrin-FN interaction through the RGD sequence evidently curbed the cell adhesion and spreading.