Subnuclear localization and mitotic phosphorylation of HIRA, the human homologue of Saccharomyces cerevisiae transcriptional regulators Hir1p/Hir2p.

The HIRA gene encodes a nuclear protein with histone-binding properties that have been conserved from yeast to humans. Hir1p and Hir2p, the two HIRA homologues in Saccharomyces cerevisiae, are transcriptional co-repressors whose action resides at the chromatin level and occurs in a cell-cycle-regulated fashion. In mammals, HIRA is an essential gene early during development, possibly through the control of specific gene-transcription programmes, but its exact function remains to be deciphered. Here we report on the subnuclear distribution and cell-cycle behaviour of the HIRA protein. Using both biochemical and immunofluorescence techniques, a minor fraction of HIRA was found tightly associated with the nuclear matrix, the material that remains after nuclease treatment and high-salt extraction. However, most HIRA molecules proved extractable. In non-synchronized cell populations, extraction from chromatin necessitated 300 mM NaCl whereas 150 mM was sufficient in mitotic cells. Immunofluorescence staining and microscopic examination of mitotic cells revealed HIRA as excluded from condensed chromosomes, confirming a lack of association with chromatin during mitosis. Western-blot analysis indicated that HIRA molecules were hyper-phosphorylated at this point in the cell cycle. Metabolic labelling and pulse-chase experiments characterized HIRA as a stable protein with a half-life of approx. 12 h. The mitotic phosphorylation of HIRA could provide the dividing cell with a way to retarget HIRA-containing multi-protein complexes to different chromatin regions in daughter compared with parental cells.

The Brucella suis homologue of the Agrobacterium tumefaciens chromosomal virulence operon chvE is essential for sugar utilization but not for survival in macrophages.

Brucella strains possess an operon encoding type IV secretion machinery very similar to that coded by the Agrobacterium tumefaciens virB operon. Here we describe cloning of the Brucella suis homologue of the chvE-gguA-gguB operon of A. tumefaciens and characterize the sugar binding protein ChvE (78% identity), which in A. tumefaciens is involved in virulence gene expression. B. suis chvE is upstream of the putative sugar transporter-encoding genes gguA and gguB, also present in A. tumefaciens, but not adjacent to that of a LysR-type transcription regulator. Although results of Southern hybridization experiments suggested that the gene is present in all Brucella strains, the ChvE protein was detected only in B. suis and Brucella canis with A. tumefaciens ChvE-specific antisera, suggesting that chvE genes are differently expressed in different Brucella species. Analysis of cell growth of B. suis and of its chvE or gguA mutants in different media revealed that ChvE exhibited a sugar specificity similar to that of its A. tumefaciens homologue and that both ChvE and GguA were necessary for utilization of these sugars. Murine or human macrophage infections with B. suis chvE and gguA mutants resulted in multiplication similar to that of the wild-type strain, suggesting that virB expression was unaffected. These data indicate that the ChvE and GguA homologous proteins of B. suis are essential for the utilization of certain sugars but are not necessary for survival and replication inside macrophages.

Yersinia enterocolitica promotes deactivation of macrophage mitogen-activated protein kinases extracellular signal-regulated kinase-1/2, p38, and c-Jun NH2-terminal kinase. Correlation with its inhibitory effect on tumor necrosis factor-alpha production.

The enteropathogenic bacterium Yersinia enterocolitica counteracts host defense mechanisms by interfering with eukaryotic signal transduction pathways. In this study, we investigated the mechanism by which Y. enterocolitica prevents macrophage tumor necrosis factor-alpha (TNFalpha) production. Murine J774A.1 macrophages responded to Y. enterocolitica infection by rapid activation of mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK). However, after initial activation, the virulent Y. enterocolitica strain harboring the Y. enterocolitica virulence plasmid caused a substantial decrease in ERK1/2 and p38 tyrosine phosphorylation. Simultaneously, the virulent Y. enterocolitica strain gradually suppressed phosphorylation of the transcription factors Elk-1, activating transcription factor 2 (ATF2), and c-Jun, indicating time-dependent inhibition of ERK1/2, p38, and JNK kinase activities, respectively. Analysis of different Y. enterocolitica mutants revealed that (i) MAPK inactivation parallels the inhibition of TNFalpha release, (ii) the suppressor effect on TNFalpha production, which originates from the lack of TNFalpha mRNA, is distinct from the ability of Y. enterocolitica to resist phagocytosis and to prevent the oxidative burst, (iii) the tyrosine phosphatase YopH, encoded by the Y. enterocolitica virulence plasmid, is not involved in the decrease of ERK1/2 and p38 tyrosine phosphorylation or in the cytokine suppressive effect. Altogether, these results indicate that Y. enterocolitica possesses one or more virulence proteins that suppress TNFalpha production by inhibiting ERK1/2, p38, and JNK kinase activities.

The emerging three-dimensional structure of a receptor. The nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor is the neurotransmitter receptor with the most-characterized protein structure. The amino acid sequences of its five subunits have been elucidated by cDNA cloning and sequencing. Its shape and dimensions (approximately 12.5 nm x 8 nm) were deduced from electron-microscopy studies. Its subunits are arranged around a five-fold axis of pseudosymmetry in the order (clockwise) alpha H gamma alpha L delta beta. Its two agonist/competitive-antagonist-binding sites have been localized by photolabelling studies to a deep gorge between the subunits near the membrane surface. Its ion channel is formed by five membrane-spanning (M2) helices that are contributed by the five subunits. This finding has been generalized as the Helix M2 model for the superfamily of ligand-gated ion channels. The binding site for regulatory non-competitive antagonists has been localized by photolabelling and site-directed-mutagenesis studies within this ion channel. Therefore a three-dimensional image of the nicotinic acetylcholine receptor is emerging, the most prominent feature of which is an active site that combines the agonist/ competitive-antagonist-binding sites, the regulatory site and the ion channel within a relatively narrow space close to and within the bilayer membrane.

The handedness of the subunit arrangement of the nicotinic acetylcholine receptor from Torpedo californica.

Cross-linking an alpha-neurotoxin with a known three-dimensional structure and with photoactivatable groups in known positions to native membrane-bound acetylcholine receptor reveals its quaternary structure, including the handedness of its circular subunit arrangement. Photolabelling with alpha-neurotoxin carrying the photoactivatable group at position Lys46 is inhibited by the competitive antagonist (+)-tubocurarine in a biphasic manner, indicating that it reacts with both alpha-subunits that were shown to have different affinities for this antagonist [Neubig, R. R. & Cohen, J. B. (1979) Biochemistry 18, 5464-5475]. Lys46 is located on loop III of the neurotoxin. The other information necessary for the elucidation of the handedness was provided by the recent finding that the central loop of the toxin (loop II) is oriented towards the central pore of the receptor, securing the overall orientation of the bound toxin [Machold, J., Utkin, Y. N., Kirsch, D., Kaufmann, R., Tsetlin, V. & Hucho, F. (1995b) Proc. Natl Acad. Sci. USA 92, 7282-7286]. Looking at the receptor from the synaptic side of the postsynaptic membrane, it was concluded that the clockwise subunit arrangement is alpha H-gamma-alpha L-delta-beta (alpha H and alpha L are the alpha-subunits binding (+)-tubocurarine with high and low affinity, respectively). Its mirror image alpha alpha L-gamma-alpha H-beta-delta could thus be excluded.

Photolabeling reveals the proximity of the alpha-neurotoxin binding site to the M2 helix of the ion channel in the nicotinic acetylcholine receptor.

A photoactivatable derivative of neurotoxin II from Naja naja oxiana containing a 125I-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl label at Lys-25 forms a photo-induced cross-link with the delta subunit of the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR). The cross-linked radioactive receptor peptide was isolated by reverse-phase HPLC after tryptic digestion of the labeled delta subunit. The sequence of this peptide, delta-(260-277), and the position of the label at Ala-268 were established by matrix-assisted laser-desorption-ionization mass spectrometry based on the molecular mass and on post-source decay fragment analysis. With the known dimensions of the AChR molecule, of the photolabel, and of alpha-neurotoxin, finding the cross-link at delta Ala-268 (located in the upper part of the channel-forming transmembrane helix M2) means that the center of the alpha-neurotoxin binding site is situated at least approximately 40 A from the extracellular surface of the AChR, proximal to the channel axis.

Synthesis of nitrodiazirinyl derivatives of neurotoxin II from Naja naja oxiana and their interaction with the Torpedo californica nicotinic acetylcholine receptor.

Five singly modified nitrodiazirine derivatives of neurotoxin II (NT-II) from Naja naja oxiana were obtained after NT-II reaction with N-hydroxysuccinimide ester of (2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3yl]phenoxy)acet ic acid followed by chromatographic separation of the products. To localize the label positions, each derivative was first UV-irradiated and then subjected to reduction, carboxymethylation, and trypsinolysis. Tryptic digests were separated by reversed phase-HPLC, the labeled peptides being identified by mass spectrometry. The derivatives containing the photolabel at the position Lys 25, Lys 26, Lys 44, or Lys 46 were [125I]iodinated by the chloramine T procedure. Each iodinated derivative was found to form photoinduced cross-links with the membrane-bound nicotinic acetylcholine receptor (AChR) from Torpedo californica. The pattern of labeling the receptor's alpha, beta, gamma, or delta subunits was dependent on the photolabel position in the NT-II molecule and differed from that obtained earlier with an analogous series of p-azidobenzoyl derivatives of NT-II. The results obtained indicate that (i) different sides of the neurotoxin molecule are involved in the AChR binding, and (ii) fragments of the different AChR subunits are located close together at the neurotoxin-binding sites.

A new class of photoactivatable and cleavable derivatives of neurotoxin II from Naja naja oxiana. Synthesis, characterisation, and application for affinity labelling of the nicotinic acetylcholine receptor from Torpedo californica.

A new series of photoactivatable and cleavable derivatives of neurotoxin II from the cobra Naja naja oxiana is investigated which can be used for mapping the surface topology of the nicotinic acetylcholine receptor from Torpedo electric tissue. The preparation and characterisation of five toxin derivatives, each with a radioactive 125I-azidosalicylamidoethyl-1,3'-dithiopropyl group in a defined position within the primary structure, are described. The photoinduced cross-linking reaction of the toxin derivatives with membrane-bound receptor is investigated. The photoactivatable group located at position K25 reacts almost exclusively with the delta subunit of the receptor, whereas the K15 derivative reacts with the alpha and beta subunits. The other derivatives did not react with the receptor to any significant extent. It is shown that, with respect to the receptor subunits, the cross-linking pattern depends on the length and chemical nature of the cross-linking group.

Mapping the functional topography of a receptor.

Photoactivatable derivatives of the alpha-neurotoxin II from Naja naja oxiana are useful tools for investigating the three dimensional architecture of the extra-membrane part of the nicotinic acetylcholine receptor from the electric tissue of Torpedo californica. Three derivatives, carrying an azidobenzoyl group in position Lys-15, Lys-26, and Lys-46, respectively, are shown to react differently within the receptor's quaternary structure. Especially the Lys-26 and Lys-46 derivatives can be used for differentiating between the two nonequivalent alpha-subunits. The Lys-26 derivative is applied for probing the receptor subunits next to the alpha-subunit: the gamma-subunit is shown to be located next to the alpha-subunit binding d-tubocurarine with high affinity. The delta-subunit is the neighbor of the low affinity alpha-subunit. We radioiodinated the toxin derivatives and localized the 125I at the His-31 residue of the toxin. Very little label was found in position Tyr-24, the only tyrosine residue of the toxin, or in position His-4, the only other histidine residue. This result is important for the cleavage experiments necessary in attempts to identify the receptor sequence which reacted with the photolabel.

Investigation of ligand-binding sites of the acetylcholine receptor using photoactivatable derivatives of neurotoxin II from Naja naja oxiana.

Several photoaffinity derivatives of neurotoxin II from the venom of the central Asian cobra Naja naja oxiana have been prepared. After reaction of the 125I-labeled derivatives with the nicotinic acetylcholine receptor from electric organ, the alpha-subunit of the nAChR is almost exclusively labeled by the derivative carrying the photoactivatable group in position Lys46. In contrast to this, a reactive group at Lys26 predominantly labels the gamma- and delta-subunits, while the alpha- and beta-subunits incorporate much less radioactivity. Competition experiments with d-tubocurarine show that the gamma-subunit is labeled when this derivative occupies the high affinity d-tubocurarine-binding site, while the delta-subunit is labeled by the toxin bound at the low-affinity d-tubocurarine site. A model is discussed for the orientation of different loops of the toxin molecules in the binding site for agonists and competitive antagonists.