Trafficking of an acylated cytosolic protein: newly synthesized p56(lck) travels to the plasma membrane via the exocytic pathway

The Src-related tyrosine kinase p56(lck) (Lck) is primarily expressed in T lymphocytes where it localizes to the cytosolic side of the plasma membrane and associates with the T cell coreceptors CD4 and CD8. As a model for acylated proteins, we studied how this localization of Lck is achieved. We followed newly synthesized Lck by pulse-chase analysis and found that membrane association of Lck starts soon after synthesis, but is not complete until at least 30-45 min later. Membrane-binding kinetics are similar in CD4/CD8-positive and CD4/CD8-negative cells. In CD4-positive T cells, the interaction with CD4 rapidly follows membrane association of Lck. Studying the route via which Lck travels from its site of synthesis to the plasma membrane, we found that: CD4 associates with Lck within 10 min of synthesis, long before CD4 has reached the plasma membrane; Lck associates with intracellular CD4 early after synthesis and with cell surface CD4 at later times; and transport of CD4-bound Lck to the plasma membrane is inhibited by Brefeldin A. These data indicate that the initial association of newly synthesized Lck with CD4, and therefore with membranes, occurs on intracellular membranes of the exocytic pathway. From this location Lck is transported to the plasma membrane.     

Ultrastruktur des Neurohaemalorgans im Nervus corporis allati II vonAcheta domesticus

Summary InAcheta domesticus the proximal part of the nervus corporis allati II (Nca II) is differentiated as a neurohemal organ und consists of several hundred fiber profiles. The neurosecretory region is confined to the peripheral layer and contains axons with different vesicular inclusions. The liberation of neurohormones is accomplished by exocytosis and the formation of synaptoids. Structures resembling synaptic ribbons were observed in contact with axons, glial profiles and interstitial stroma. The central area of the nerve contains only non-neurosecretory axons of various sizes. Connection with the subesophageal ganglion is attained by only 9 large axons of the central region.Zusammenfassung BeiAcheta domesticus ist der proximale Abschnitt des Nervus corporis allati II (Nca II) als Neurohaemalorgan mit mehreren hundert Faserprofilen ausgebildet. Die neurosekretorische Region ist auf die Peripherie des Nerven begrenzt und enthält Fasern mit unterschiedlichen vesikulären Einschlüssen. Die Freisetzung der Neurohormone erfolgt exocytotisch und durch Bildung von Synaptoiden. Es werden stiftchenförmige synapsenähnliche Kontaktstellen mit Axonen, Gliaprofilen und dem interzellulären Stroma beschrieben. Der zentrale Teil des Nerven führt nicht-neurosekretorische Axone unterschiedlichen Durchmessers, von denen lediglich 9 große Fasern die Verbindung mit dem Unterschlundganglion herstellen.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

A hypopharyngeal gland protein of the worker honeybee Apis mellifera L. enhances proliferation of primary-cultured rat hepatocytes and suppresses apoptosis in the absence of serum

The worker honeybees of Apis mellifera L. age-dependently change from nurse bees which take care of their brood to forager bees which collect for nectar and convert it into honey during their lifespan of 30-40 days. A 56-kDa protein (p56kP-4) was shown to be present only in the nurse-bee hypopharyngeal gland. Here, we investigated the physiological effects of p56kP-4 on primary-cultured rat hepatocytes in the absence of serum. The p56kP-4 recombinant protein expressed in Escherichia coli significantly stimulated hepatocyte DNA synthesis and protected cells from apoptosis induced by serum deprivation. It also activated mitogen-activated protein kinase, as well as protein kinase B, which is a key regulator of cell survival. These findings suggest that p56kP-4 has a growth factor-like action on primary-cultured rat hepatocytes.     

Structure-activity studies on different modifications of nociceptin/orphanin FQ: identification of highly potent agonists and antagonists of its receptor

Nociceptin/orphanin FQ (N/OFQ) and its receptor system modulate a variety of biological functions and further understandings of physiological and pathological roles of this system require new potent agonists and antagonists of its receptor. Two series of N/OFQ related analogues were synthesized to investigate the relationship of different modifications. We combined modifications including: (a) Phe⁴→(pF)Phe⁴; (b) Ala⁷, Ala¹¹→Aib⁷, Aib¹¹; (c) Leu¹⁴, Ala¹⁵→Arg¹⁴, Lys¹⁵. Compared with the first series, N-terminus of the second series was changed from Phe¹ to Nphe¹. All the analogues were amidated at C-terminus. These compounds were tested in binding studies on rat brain membranes and mouse vas deferens assay. Results indicated that the compounds of the first series showed higher affinity and potency than N/OFQ (pK_i = 9.33; pEC₅₀ = 7.50). In particular, [(pF)Phe⁴, Aib⁷, Aib¹¹, Arg¹⁴, Lys¹⁵] N/OFQ-NH₂ was found to be a highly potent agonist with pK_i = 10.78 in binding studies and pEC₅₀ = 9.37 in mouse vas deferens assay. The second series all competitively antagonized the effects of N/OFQ in mouse vas deferens assay. [Nphe¹, (pF)Phe⁴, Aib⁷, Aib¹¹, Arg¹⁴, Lys¹⁵] N/OFQ-NH₂ was the best antagonist with pA₂ = 8.39 and showed high binding affinity with pK_i = 9.99. Thus modifications which increase the potency of agonist have synergistic effect on biological activity and a replacement of N-terminus leads to shift of analogues from agonist to antagonist.     

Ca<Superscript>2+</Superscript>-induced permeability transition in human lymphoblastoid cell mitochondria from normal and Huntington’s disease individuals

Huntingtons disease (HD) is associated with expansion of polyglutamine tract in a protein named huntingtin (htt) that is expressed in virtually all body tissues. Thus mutated htt (HD-htt) might affect all organs, although clinical manifestations of HD are associated with selective loss of corticostriatal neurons of the brain. In this work we studied how HD-htt affects mitochondria in human peripheral blood cells. We compared various functions of mitochondria isolated from cultured lymphoblastoid cells derived from three HD patients with juvenile onset of the disease (HD-LBM) and three age-matched control (C-LBM) individuals. Respiratory parameters in different metabolic states, with succinate and glutamate plus malate were the same for all control and HD cell lines. State 4 membrane potential in HD-LBM was slightly lower than in C-LBM. The calcium retention capacity (CRC) of mitochondria was estimated using simultaneously several methods to register permeability transition (PT). We found that LBM do not undergo swelling upon Ca²⁺-induced PT, and do not increase CRC in the presence of ADP + oligomycin. Although each cell line had different CRC values, qualitatively PT was different in C-LBM and HD-LBM. With C-LBM cyclosporin A (CsA) increased CRC significantly, while with HD-LBM CsA was ineffective. In C-LBM depolarization of mitochondria and a large pore opening (PT) always occurred simultaneously. In HD-LBM depolarization occurred at 20–50% lower Ca²⁺ loads than PT. We suggest that HD-htt promotes low H⁺ conductance of the mitochondria by interacting with proteins at the contacts sites without directly promoting PT or hampering mitochondrial oxidative phosphorylation. (Mol Cell Biochem 269: 143–152, 2005)     

Plasma membrane oestrogen receptor mediates neuroprotection against beta-amyloid toxicity through activation of Raf-1/MEK/ERK cascade in septal-derived cholinergic SN56 cells

Rapid oestrogen neuroprotection against beta-amyloid peptide (Abeta)-induced toxicity, a main feature of Alzheimer's disease, may be partially initiated at the plasma membrane. However, the mechanism by which this oestrogen effect occurs is unknown. In a septal murine cell line (SN56), we observed that short exposures to either 17beta-oestradiol (E2) or membrane impermeant E2 bound to horseradish peroxidase (E-HRP) induced a biphasic stimulation of extracellular-signal regulated protein kinase (ERK1/2) phosphorylation, with peak inductions detected around 4-8 min in the early phase and a second maximum around 8 h after treatment. ERK1/2 phosphorylation was abolished by ERK1/2 kinase (MEK) inhibitors PD98059 and U0126. Interestingly, PD98059 was also shown to block rapid E2-related prevention of death in cells exposed to Abeta fragment 1-40 (Abeta1-40) for 24 h. In contrast, no neuroprotective effects were obtained when MEK inhibitor was used to selectively abolish the late phosphorylation phase. Furthermore, both ERK1/2 activation and E2-associated protection were blocked by an inhibitor of Raf-1 kinase. Raf-1 may be involved in these effects because oestrogen caused the rapid serine 338 (Ser338) phosphorylation of this protein. In addition, the oestrogen receptor (ER) antagonist ICI 182,780 was also observed to block ERK1/2 phosphorylation. We propose a novel mechanism in SN56 cells by which rapid effects of oestrogen leading to neuroprotection are signalled through Raf-1/MEK/ERK1/2 pathway, possibly by activation of a membrane-related ER.     

GPR80/99, proposed to be the P2Y<Subscript>15</Subscript> receptor activated by adenosine and AMP,is not a P2Y receptor

The orphan receptor GPR80 (also called GPR99) was recently reported to be the P2Y₁₅ receptor activated by AMP and adenosine and coupled to increases in cyclic AMP accumulation and intracellular Ca²⁺ mobilization (Inbe et al. J Biol Chem 2004; 279: 19790–9). However, the cell line (HEK293) used to carry out those studies endogenously expresses A_2A and A_2B adenosine receptors as well as multiple P2Y receptors, which complicates the analysis of a potential P2Y receptor. To determine unambiguously whether GPR80 is a P2Y receptor subtype, HA-tagged GPR80 was either stably expressed in CHO cells or transiently expressed in COS-7 and HEK293 cells, and cell surface expression was verified by radioimmunoassay (RIA). COS-7 cells overexpressing GPR80 showed a consistent twofold increase in basal inositol phosphate accumulation. However, neither adenosine nor AMP was capable of promoting accumulation of either cyclic AMP or inositol phosphates in any of the three GPR80-expressing cells. A recent paper (He et al. Nature 2004; 429: 188–93) reported that GPR80 is a Gq-coupled receptor activated by the citric acid cycle intermediate, -ketoglutarate. Consistent with this report, -ketoglutarate promoted inositol phosphate accumulation in CHO and HEK293 cells expressing GPR80, and pretreatment of GPR80-expressing COS-7 cells with glutamate dehydrogenase, which converts -ketoglutarate to glutamate, decreased basal levels of inositol phosphates. Taken together, these data demonstrate that GPR80 is not activated by adenosine, AMP or other nucleotides, but instead is activated by -ketoglutarate. Therefore, GPR80 is not a new member of the P2Y receptor family.     

Determination of the solution structure of the SH3 domain of human p56 Lck tyrosine kinase

Summary The solution structure of the SH3 domain of human p56 Lck tyrosine kinase (Lck-SH3) has been determined by multidimensional heteronuclear NMR spectroscopy. The structure was calculated from a total of 935 experimental restraints comprising 785 distance restraints derived from 1017 assigned NOE cross peaks and 150 dihedral angle restraints derived from 160 vicinal coupling constants. A novel combination of the constant-time ¹H–¹³C NMR correlation experiment recorded with various delays of the constant-time refocusing delays and a fractionally ¹³C-labelled sample was exploited for the stereo-specific assignment of prochiral methyl groups. Additionally, 28 restraints of 14 identified hydrogen bonds were included. A family of 25 conformers was selected to characterize the solution structure. The average root-mean-square deviations of the backbone atoms (N, C, C, O) among the 25 conformers is 0.42 Å for residues 7 to 63. The N- and C-terminal residues, 1 to 6 and 64 to 81, are disordered, while the well-converged residues 7 to 63 correspond to the conserved sequences of other SH3 domains. The topology of the SH3 structure comprises five anti-parallel -strands arranged to form two perpendicular -sheets, which are concave and twisted in the middle part. The overall secondary structure and the backbone conformation of the core -strands are almost identical to the X-ray structure of the fragment containing the SH2-SH3 domains of p56 Lck [Eck et al. (1994) Nature, 368, 764–769]. The X-ray structure of the SH3 domain in the tandem SH2-SH3 fragment is spatially included within the ensemble of the 25 NMR conformers, except for the segment of residues 14 to 18, which makes intermolecular contacts with an adjacent SH2 molecule and the phosphopeptide ligand in the crystal lattice. Local structural differences from other known SH3 domains are also observed, the most prominent of which is the absence in Lck-SH3 of the two additional short -strands in the regions Ser¹⁵ to Glu¹⁷ and Gly²⁵ to Glu²⁷ flanking the so-called RT-Src loop. This loop (residues Glu¹⁷ to Leu²⁴), together with the n-Src loop (residues Gln³⁷ to Ser⁴⁶) confines the ligand interaction site which is formed by a shallow patch of hydrophobic amino acids (His¹⁴, Tyr¹⁶, Trp⁴¹, Phe⁵⁴ and Phe⁵⁹). Both loops are flexible and belong to the most mobile regions of the protein, which is assessed by the heteronuclear ¹⁵N,¹H-NOE values characterizing the degree of internal backbone motions. The aromatic residues of the ligand binding site are arranged such that they form three pockets for interactions with the polyproline ligand.Abbreviations CT constant time - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - SH2 Src homology domain 2 - SH3 Src homology domain 3     

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose