Prediction of tyrosine sulfation in seven-transmembrane peptide receptors

Posttranslational modification by tyrosine sulfation regulates many important protein-protein interactions and modulates the binding affinity and specificity of seven-transmembrane peptide receptors. We developed a log-odds position-specific-scoring-matrix (PSSM) to accurately predict tyrosine sulfation using 62 tyrosine sites known to be sulfated and 421 tyrosine sites known not to be sulfated. We predict that 49 tyrosines of 32 seven-transmembrane peptide receptors are sulfated. Although we did not incorporate characteristics of confirmed sulfation sites such as clustering and conservation across species into our PSSM, our predicted sites nevertheless exhibited these characteristics. The observed conservation suggests that there are strong evolutionary pressures to preserve selected biological activity of seven-transmembrane receptors. The predicted tyrosine sulfation sites predominantly occur in the extracellular tail and extracellular loop 2, regions consistent with their association with binding pockets of the receptor.     

Brief Report: Processing of a plant peptide hormone precursor facilitated by posttranslational tyrosine sulfation

Most peptide hormones and growth factors are matured from larger inactive precursor proteins by proteolytic processing and further posttranslational modification. Whether or how posttranslational modifications contribute to peptide bioactivity is still largely unknown. We address this question here for TWS1 (Twisted Seed 1), a peptide regulator of embryonic cuticle formation in Arabidopsis thaliana. Using synthetic peptides encompassing the N- and C-terminal processing sites and the recombinant TWS1 precursor as substrates, we show that the precursor is cleaved by the subtilase SBT1.8 at both the N and the C termini of TWS1. Recognition and correct processing at the N-terminal site depended on sulfation of an adjacent tyrosine residue. Arginine 302 of SBT1.8 was found to be required for sulfotyrosine binding and for accurate processing of the TWS1 precursor. The data reveal a critical role for posttranslational modification, here tyrosine sulfation of a plant peptide hormone precursor, in mediating processing specificity and peptide maturation.     

Species variation in the tyrosine sulfation of mammalian gastrins

The degree of tyrosine sulfation and the distribution between gastrin-17- and gastrin-34-like immunoreactivity (LI) were studied in the antra of ten mammalian species. Specific radioimmunoassays, gel-, and ion-exchange chromatography as well as enzymatic cleavage with trypsin and arylsulfatase were used. The percentage of sulfation varied from 24.4 +/- 4.2 (mean +/- SEM) in dogs to 80.1 +/- 2.6 in sheep, 46.8 +/- 3.3 in humans, 50.1 +/- 3.2 in cows, 55.9 +/- 2.3 in rats, 57.4 +/- 3.1 in pigs, 61.3 +/- 2.2 in guinea pigs, 64.1 +/- 4.7 in cats, 64.8 +/- 2.1 in mice and 68.2 +/- 2.8 in rabbits. Gastrin-34-LI in antral extracts could be converted to gastrin-17-LI by trypsin in all species. Five percent of antral gastrins eluted as gastrin-34-LI in all species. We conclude that while the ratio of gastrin-34-LI to gastrin-17-LI varies little in mammals, large differences occur in the degree of sulfation.     

酪氨酸羟化酶与生长相关蛋白43基因在心房颤动中的表达变化及其意义

目的了解酪氨酸羟化酶（TH）与生长相关蛋白43（GAP43）基因在心房颤动（房颤）形成与恢复过程中的表达变化及其意义。方法山羊24只,随机分为窦性心律组（窦律组）、房颤组、复律3个月组与复律6个月组（各6只）。开胸后于左心耳与左心房前壁连接处植入起搏电极导线,以（400±10）次／min连续起搏3个月,诱导房颤。复律组采用电复律法恢复窦性心律3～6个月。每周记录山羊体表心电图,分别采用Western blot与免疫组化方法检测TH与GAP43基因在窦律组、房颤组、复律3个月组与复律6个月组的蛋白水平变化。结果免疫组化检测结果显示房颤组左心房游离壁TH与GAP43基因蛋白水平较窦律组显著增高,差异有统计学意义[（22．50±16．47）个／mm^2vs（3．24±1．55）个／mm^2,（35．75±20．31）个／mm^2vs（4．83±3．97）个／mm^2,P〈0．01];复律3个月组,左心房游离壁TH与GAP43基因蛋白水平[（17．42±10．54）个／mm^2,（20．38±22．96）个／mm^2]较房颤组明显降低（P〈0．05）;复律6个月组,左心房游离壁TH与GAP43基因蛋白水平[（6．73±2．94）个／mm^2,（8．58±5．07）个／mm^2]较房颤组也明显降低（P〈0．01）。右心房游离壁TH与GAP43基因蛋白水平在房颤组、复律3个月组与复律6个月组变化趋势与左心房游离壁相似。Western blot检测结果与免疫组化结果一致。结论TH与GAP43基因表达变化在房颤形成与恢复过程中起着重要作用,可能是房颤治疗的潜在靶点。     

肝硬化时肝细胞胰岛素受体和酪氨酸蛋白激酶表达的定量研究时德仁东传凌陆立丛闻天周燕

目的探索肝硬化时肝组织细胞胰岛素受体(IR)和酪氨酸蛋白激酶(TPK)含量的变化规律.方法应用图象分析系统对12例血清HBV标志物阳性的肝炎后肝硬化患者肝组织石腊包埋切片对抗胰岛素受体和酪氨酸蛋白激酶抗体免疫组化标记物的定量测定.结果肝硬化糖耐量试验(GTT)异常组和GTT正常组IR含量分别为(41.44±6.47)AU和(43.36±4.78)AU,较对照组[(48.11±4.42)AU]显著减少(P＜0.01),肝硬化GTT异常组和GTT正常组IR含量无显著性差异(P＞0.05);TPK表达量肝硬化GTT异常组较GTT正常组显著性减少,分别为(41.31±5.55)AU和(51.16±5.83)AU.结论肝硬化时肝细胞IR减少;肝硬化时GTT异常与肝细胞胰岛素受体酪氨酸蛋白激酶活性减退有关.     

RimO, a MiaB-like enzyme, methylthiolates the universally conserved Asp88 residue of ribosomal protein S12 in Escherichia coli

Ribosomal protein S12 undergoes a unique posttranslational modification, methylthiolation of residue D88, in Escherichia coli and several other bacteria. Using mass spectrometry, we have identified the enzyme responsible for this modification in E. coli, the yliG gene product. This enzyme, which we propose be called RimO, is a radical-S-adenosylmethionine protein that bears strong sequence similarity to MiaB, which methylthiolates tRNA. We show that RimO and MiaB represent two of four subgroups of a larger, ancient family of likely methylthiotransferases, the other two of which are typified by Bacillus subtilis YqeV and Methanococcus jannaschii Mj0867, and we predict that RimO is unique among these subgroups in its modification of protein as opposed to tRNA. Despite this, RimO has not significantly diverged from the other three subgroups at the sequence level even within the C-terminal TRAM domain, which in the methyltransferase RumA is known to bind the RNA substrate and which we presume to be responsible for substrate binding and recognition in all four subgroups of methylthiotransferases. To our knowledge, RimO and MiaB represent the most extreme known case of resemblance between enzymes modifying protein and nucleic acid. The initial results presented here constitute a bioinformatics-driven prediction with preliminary experimental validation that should serve as the starting point for several interesting lines of further inquiry.     

The Molecular Nature of Cholecystokinin in Plasma: An in vivo Immunosorption Study in Rabbits

Rehfeld JF. The molecular nature of cholecystokinin in plasma. An in vivo immunosorption study in rabbits. Scand J Gastroenterol 1994;29:110-121.

The nature of cholecystokinin (CCK) in rabbit plasma was examined by means of a novel in vivo immunosorption procedure. Cholecystokinin (CCK) antibodies in 11 rabbit antisera were denatured, and the released peptides characterized by size and reversed-phase chromatography. Five of six antisera specific for the COOH terminus of CCK contained substantial amounts of CCK-22- and CCK-8-like peptides and small amounts of CCK-33-like peptides (range, 120 to 1140nmol/l antiserum). In contrast, neither antisera for the NH₂-terminus and mid-sequence of porcine CCK-33 nor antisera against the glycine-extended COOH terminus released CCK peptides. Postprandial acidified plasma from non-immunized rabbits concentrated in vitro also contained mainly CCK-22- and -8-like peptides, whereas extracts of rabbit duodenum and jejunum in addition contained forms resembling CCK-58, -39, and/or -33. The results show that mainly small molecular forms of CCK circulate in rabbits, and that NH₂-terminal and mid-sequences of porcine and human CCK-33 differ from those of rabbit CCK-33. The results support the contention that plasma in most mammals contains small molecular forms of CCK.     

Endogenous tyrosine hydroxylase activity in the developing chick heart: a possible source of extraneuronal catecholamines

The source of catecholamines in the developing chick heart was investigated by using catecholamine assays and tyrosine hydroxylase assays on hearts from normal and chemically-sympathectomized chick embryos. A biochemical index of sympathetic nerve development in the heart was obtained by monitoring the ability of sympathetic nerves in the atria to take up [3H]-norepinephrine in vitro. Specific neuronal uptake of [3H]-norepinephrine in atria was first detected on incubation day 11 and increased throughout the incubation period. High performance liquid chromatography with electrochemical detection was used to measure the norepinephrine concentration and content of embryonic hearts. The cardiac norepinephrine concentration fluctuated throughout the incubation period but was particularly low (0.01 +/- 0.005 ng/mg wet wt) on incubation days 10 to 13, coincident with the arrival of sympathetic nerves in the heart. The highest norepinephrine concentration was measured on incubation day 7 (2.09 +/- 0.50 ng/mg wet wt) prior to the arrival of sympathetic nerves in the heart. Sympathetic nerve axotomy produced by chronic treatment with 6-hydroxydopamine reduced [3H]-norepinephrine uptake in atria and norepinephrine concentration in whole hearts on incubation day 20 to 33 and 47% of control, respectively. Tyrosine hydroxylase activity was detected in normal hearts on incubation day 7, 3 to 4 days before the heart is innervated by sympathetic nerves. Tyrosine hydroxylase activity persisted in the heart on incubation day 20, despite treatment with 6-hydroxydopamine on incubation days 13 to 19. The tyrosine hydroxylase activity in 6-hydroxydopamine lesioned hearts was not significantly different from saline-treated controls. This data indicates that tyrosine hydroxylase activity is present in the immature chick heart prior to the arrival of sympathetic innervation and following chemical sympathectomy; hence, an extraneuronal source of tyrosine hydroxylase, the rate limiting enzyme for catecholamine biosynthesis, exists in the embryonic chick heart.     

Pasteurella multocida toxin activation of heterotrimeric G proteins by deamidation

Pasteurella multocida toxin is a major virulence factor of Pasteurella multocida, which causes pasteurellosis in men and animals and atrophic rhinitis in rabbits and pigs. The ≈145 kDa protein toxin stimulates various signal transduction pathways by activating heterotrimeric G proteins of the Gα_q, Gα_i, and Gα_12/13 families by using an as yet unknown mechanism. Here, we show that Pasteurella multocida toxin deamidates glutamine-205 of Gα_i2 to glutamic acid. Therefore, the toxin inhibits the intrinsic GTPase activity of Gα_i and causes persistent activation of the G protein. A similar modification is also evident for Gα_q, but not for the closely related Gα₁₁, which is not a substrate of Pasteurella multocida toxin. Our data identify the α-subunits of heterotrimeric G proteins as the direct molecular target of Pasteurella multocida toxin and indicate that the toxin does not act like a protease, which was suggested from its thiol protease-like catalytic triad, but instead causes constitutive activation of G proteins by deamidase activity.     

肠易激综合征心理状态与去甲肾上腺素、酪氨酸羟化酶

肠易激综合征（IBS）为7种经典心身疾病之一,精神因素在发病中占据重要地位,80％以上的IBS患者症状加重与精神因素有明显关系。去甲肾上腺素（NE）能系统与情感、行为及意识状态有关,脑内NE功能异常可导致抑郁、焦虑类疾病并在某些应激状态下表现出来或恶化,肾上腺素能交感神经失调与部分IBS发病有关。酪氨酸羟化酶（TH）作为NE合成的限速酶,与抑郁症密切相关。研究脑-肠轴各层面中NE和TH的变化趋势有助于进一步了解IBS尤其是IBS伴焦虑、抑郁状态者的发病机制。     

流式细胞术分析慢性髓性白血病细胞酪氨酸磷酸化水平

目的：探讨慢性髓性白血病（CML）患者骨髓细胞酪氨酸磷酸化水平（p-Tyr），探讨甲磺酸伊马替尼（商品名：格列卫）治疗对p-Tyr水平的作用和影响。方法：对38例CML患者（其中16例接受格列卫治疗）及5例健康对照者的骨髓细胞进行细胞膜CD45和细胞质内PTyr（PY99）荧光标记，应用流式细胞术（FCM）分析不同病期CML各群细胞p-Tyr水平。结果：①未经甲磺酸伊马替尼治疗的慢性期（CP）、加速期（AP）患者骨髓各群细胞p-Tyr水平高于健康对照者，而CP与AP之间p-Tyr水平差异无统计学意义；②CP患者应用甲磺酸伊马替尼治疗〉12个月者的骨髓细胞p-Tyr水平低于用药〈12个月者；AP期用药≥40个月者的幼稚细胞群p-Tyr水平亦低于用药〈12个月者；③随CML病程延长，幼稚细胞群p-Tyr水平增高，但无明显影响；年龄对p-Tyr水平无明显影响。结论：①应用FCM测定细胞p-Tyr水平可作为快速、敏感的方法检测BCR-ABL（＋）细胞；②CML各期细胞的p-Tyr水平均高于正常对照；③甲磺酸伊马替尼对慢性期白血病细胞p-Tyr水平的降低程度随用药时间延长而增强。     

Palmitoylation regulates raft affinity for the majority of integral raft proteins

The physical basis for protein partitioning into lipid rafts remains an outstanding question in membrane biology that has previously been addressed only through indirect techniques involving differential solubilization by nonionic detergents. We have used giant plasma membrane vesicles, a plasma membrane model system that phase separates to include an ordered phase enriching for raft constituents, to measure the partitioning of the transmembrane linker for activation of T cells (LAT). LAT enrichment in the raft phase was dependent on palmitoylation at two juxtamembrane cysteines and could be enhanced by oligomerization. This palmitoylation requirement was also shown to regulate raft phase association for the majority of integral raft proteins. Because cysteine palmitoylation is the only lipid modification that has been shown to be reversibly regulated, our data suggest a role for palmitoylation as a dynamic raft targeting mechanism for transmembrane proteins.     

Effects of c-Src tyrosine kinase on ethanol sensitivity of recombinant NMDA receptors expressed in HEK 293 cells

N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that are important mediators of the actions of the excitatory amino acid neurotransmitter glutamate. Previous studies have shown that ethanol inhibits the function of both wild-type receptors found in neurons and recombinant NMDA receptors expressed in heterologous cells, such as oocytes and transfected mammalian cells. Although some studies have reported that certain subunit combinations display an enhanced sensitivity to ethanol, this effect is not observed in all experimental systems. This discrepancy may be due to varying levels of endogenous modulators, such as kinases, between different cell preparations. In this study, we investigated the effects of tyrosine phosphorylation on the ethanol sensitivity of NMDA receptor function using a recombinant cell system where levels of both NMDA subunits and protein kinases can be more carefully controlled. Human embryonic kidney (HEK 293) cells were transfected with different NMDA receptor subunits and a c-Src-green fluorescent protein (GFP) fusion protein that could be directly visualized in living cells. Agonist-stimulated calcium flux was measured in single cells using fura-2 video imaging. As expected, cells transfected with the NR1/NR2B subunits were more sensitive to inhibition by the NR2 selective antagonist ifenprodil than those transfected with NR1/ NR2A or NR1/NR2A/NR2B subunits. All receptor combinations were inhibited by ethanol (25 and 100 mM), with the NR1/NR2B combination being slightly more sensitive than NR1/NR2A or NR1/NR2A/NR2B. Control and NMDA-receptor transfected HEK 293 cells displayed a low degree of tyrosine phosphorylation as measured by immunofluorescence and Western immunoblotting using an antiphosphotyrosine antibody. Phosphorylation was markedly enhanced in cells transfected with the c-Src-GFP fusion protein. The sensitivity of NMDA receptors to either 25 or 100 mM ethanol, or 10 microM ifenprodil, was not significantly altered by co-transfection with c-Src-GFP. These results indicate that, although NMDA receptors can be a target of c-Src tyrosine kinase, tyrosine phosphorylation by this enzyme does not modulate the inhibitory effects of ethanol on NMDA-activated currents.     

Leptin promotes the tyrosine phosphorylation of SHC proteins and SHC association with GRB2

The identification and characterization of proteins that become tyrosine phosphorylated in response to growth factor stimulation is critical for furthering our understanding of the signal transduction pathways involved in the regulation of cell proliferation, differentiation as well as metabolic activities. In this report, we demonstrate for the first time, that leptin is able to induce the tyrosine phosphorylation of the SH₂ containing protein SHC. These studies have been carried out on a human embryonic cell line (HEK 293) transfected with the cDNA encoding for the long form of the leptin receptor and stably expressing the receptor itself. We also shown that upon tyrosine phosphorylation, SHC associated with the adaptor protein, Grb₂. The formation of this complex may directly link tyrosine phosphorylation events to Ras activation and may be a critical step in proliferation and/or differentiation of cells. In conclusion, these results indicate that leptin receptor, after binding the ligand, activates several pathways for signal transduction that might lead to mitogenic effect.     

RAS-converting enzyme 1-mediated endoproteolysis is required for trafficking of rod phosphodiesterase 6 to photoreceptor outer segments

Prenylation is the posttranslational modification of a carboxyl-terminal cysteine residue of proteins that terminate with a CAAX motif. Following prenylation, the last three amino acids are cleaved off by the endoprotease, RAS-converting enzyme 1 (RCE1), and the prenylcysteine residue is methylated. Although it is clear that prenylation increases membrane affinity of CAAX proteins, less is known about the importance of the postprenylation processing steps. RCE1 function has been studied in a variety of tissues but not in neuronal cells. To approach this issue, we generated mice lacking Rce1 in the retina. Retinal development proceeded normally in the absence of Rce1, but photoreceptor cells failed to respond to light and subsequently degenerated in a rapid fashion. In contrast, the inner nuclear and ganglion cell layers were unaffected. We found that the multimeric rod phosphodiesterase 6 (PDE6), a prenylated protein and RCE1 substrate, was unable to be transported to the outer segments in Rce1-deficient photoreceptor cells. PDE6 present in the inner segment of Rce1-deficient photoreceptor cells was assembled and functional. Synthesis and transport of transducin, and rhodopsin kinase 1 (GRK1), also prenylated substrates of RCE1, was unaffected by Rce1 deficiency. We conclude that RCE1 is essential for the intracellular trafficking of PDE6 and survival of photoreceptor cells.     

酪氨酸对高碘性小鼠甲状腺形态结构的影响

目的观察酪氨酸对高碘性小鼠甲状腺形态结构的影响。方法采用2×3析因设计,将碘酸钾加入饮水中,分为50、600μg/L2个水平。同时将酪氨酸加入饲料中,分为0、10%、20%3个水平,喂养6个月后,测定小鼠甲状腺质量,光镜和电镜下观察甲状腺形态结构的变化。结果与适碘组相比,高碘组小鼠甲状腺相对质量明显增加,而高碘酪氨酸组小鼠甲状腺相对质量恢复正常;光镜下甲状腺随着碘剂量增高,出现胶质潴留;电镜下甲状腺滤泡上皮细胞器减少。但是随着酪氨酸剂量增高,甲状腺滤泡中胶质潴留量明显减少,甲状腺肿大(甲肿)程度明显降低,甲状腺滤泡上皮细胞表现为功能活跃现象。结论高碘可以引起小鼠甲状腺胶质潴留性肿大,并引起甲状腺滤泡上皮细胞损伤,而酪氨酸对滤泡上皮细胞具有一定的保护作用,可以部分拮抗高碘引起的甲肿。     

A procedure for measuring the contributions of intracellular and extracellular tyrosine pools to the rate of myocardial protein synthesis

To determine the relative contribution of intracellular and extracellular amino acid sources as precursors for protein synthesis, rat left atria were incubated in l-[³H]tyrosine at medium tyrosine concentrations ranging from 0.05 to 2.5 mm. Under conditions where protein synthesis was shown to be constant, tyrosine incorporation rates calculated from either the intracellular or extracellular tyrosine concentration. This behavior is consistent with a model which postulates that amino acids inside and outside the cell simultaneously supply a common precursor pool. A method is presented for determining the contributions of the intracellular and extracellular pools to the total protein synthesis rate. Analysis of tyrosine incorporation data according to this procedure yields a total incorporation rate of 1.14 ± 0.18 (s.e.m.) nmol tyrosine/mg protein/h, of which 61 ± 15% is attributed to a mixed intracellular source and 39 ± 7% to amino acids taken directly from the extracellular medium. During a 3-h incubation of the isolated atria protein synthesis and degradation rates were equal, and the oxygen consumption and ATP and creatine phosphate levels were similar to those of the beating, perfused heart.     

Effects of metformin on insulin receptor tyrosine kinase activity in rat adipocytes

Summary The cellular mechanism(s) by which the biguanide, metformin, exerts its antihyperglycaemic effect was investigated. Rat adipocytes were either treated acutely (2 h) or maintained in a biochemically defined medium (20 h) in the presence or absence of metformin (1 x 10^–4 mol/1). Exposure to the drug resulted in a significant enhancement (p < 0.01) of hexose transport in both the absence (basal) and presence of insulin. Stimulation of transport was not explained by the increase in the basal state alone, since the incremental response to maximally effective concentrations of insulin was significantly enhancedp<0.025. Insulin-receptor tyrosine kinase activity was examined under the same experimental conditions. Activity of the kinase was unaltered as evaluated by phosphorylation of an artificial substrate and by phosphorylation of the receptor in situ. Furthermore, in this investigation neither insulin receptor number nor affinity was changed in adipose tissue treated with metformin. These studies indicate that metformin potentiates the effect of insulin on glucose transport at a site(s) beyond insulin receptor binding and phosphorylation.     

Calmodulin and a cyclic nucleotide-dependent protein kinase facilitate the prolactin-induced increase in tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons

Many aspects of tuberoinfundibular dopaminergic neuronal function are increased by elevated prolactin (PRL) levels, including the activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine. This study evaluated the roles of calmodulin, cyclic nucleotide-dependent protein kinase, and calcium/calmodulin-dependent protein kinase II in the PRL-induced increase in tyrosine hydroxylase activity. Ovariectomizerd rats were treated with haloperidol or ovine PRL (oPRL) for 20–30 h before the experiment, respectively. Treatment with haloperidol increased circulating PRL levels 8-fold and tyrosine hydroxylase activity in the stalk-median eminence 1.8-fold. Treatment with oPRL increased tyrosine hydroxylase activity 1.9-fold. W-7, a calmodulin antagonist, reversed both the haloperidol- and oPRL-induced increase in tyrosine hydroxylase activity to control levels. H-8, a cyclic nucleotide-dependent protein kinase inhibitor, also reversed the haloperidol induced increase in tyrosine hydroxylase activity. KN62, a selective calcium/calmodulin-dependent protein kinase II inhibitor, attenuated the haloperidol-induced increase in tyrosine hydroxylase activity, but KNO4, a structurally related control compound, had no effect. By contrast, the oPRL- and haloperidol-induced increases in tyrosine hydroxylase activity were not altered by KN93, a selective calcium/calmodulin-dependent protein kinase II inhibitor. These data indicate that calmodulin and a cyclic nucleotide-dependent protein kinase contribute to the PRL-induced increase in tyrosine hydroxylase activity, but the role of calcium/calmodulin-dependent protein kinase II is still unclear.