Sumoylation and proteasomal activity determine the transactivation properties of the mineralocorticoid receptor.

MR is a hormone-activated transcription factor that carries a strong synergy inhibitory function at its N-terminus. Using this region as bait in a yeast two-hybrid screening, we isolated major components of the sumoylation pathway, including the SUMO-1-conjugating enzyme Ubc9, and SUMO-1 itself. We found that MR interacts with both, Ubc9 and SUMO-1 in mammalian cells, and that the receptor is sumoylated at four acceptor sites which are clustered within its AF-1 domain. We observed that MR can be poly-ubiquitinated and that proteasome activity is essential for MR-activated transcription. Disruption of the SUMO-1 attachment sites abolished MR sumoylation but interfered with neither the poly-ubiquitination of the receptor nor its transactivation potential on MMTV. However, the hormone-activated mutant displayed enhanced synergistic potential on a compound promoter and delayed mobility in the nucleus. FRAP analysis further showed that proteasome inhibition immobilizes a subpopulation of unliganded MR receptors in the nucleus, a phenomenon that is significantly attenuated in the presence of aldosterone. Interestingly, the ability of the hormone to counteract the immobilizing effect of MG132 requires the sumoylation-competent form of MR. Moreover, increasing exogenously SUMO-1 cellular levels resulted in a selective, dose-dependent inhibition of the activity of the sumoylation-deficient MR. This effect was observed only on a synergy-competent promoter, revealing a mode for negative regulation of synergy that might involve sumoylation of factors different from MR. The data suggest that the overall transcriptional activity of MR can be modulated by its sumoylation potential as well as the sumoylation level of MR-interacting proteins, and requires the continuous function of the proteasome.

PIASy, a nuclear matrix-associated SUMO E3 ligase, represses LEF1 activity by sequestration into nuclear bodies.

The Wnt-responsive transcription factor LEF1 can activate transcription in association with beta-catenin and repress transcription in association with Groucho. In search of additional regulatory mechanisms of LEF1 function, we identified the protein inhibitor of activated STAT, PIASy, as a novel interaction partner of LEF1. Coexpression of PIASy with LEF1 results in potent repression of LEF1 activity and in covalent modification of LEF1 with SUMO. PIASy markedly stimulates the sumoylation of LEF1 and multiple other proteins in vivo and functions as a SUMO E3 ligase for LEF1 in a reconstituted system in vitro. Moreover, PIASy binds to nuclear matrix-associated DNA sequences and targets LEF1 to nuclear bodies, suggesting that PIASy-mediated subnuclear sequestration accounts for the repression of LEF1 activity.

Ran GTPase cycle: oOne mechanism -- two functions.

The Ras-related GTPase Ran functions in nucleocytoplasmic transport by regulating interactions of transport receptors with transport cargo. Three recent studies suggest that Ran uses exactly the same mechanism to regulate spindle assembly during mitosis.

Rapid identification of Ralstonia eutropha strain CH34 using monoclonal antibodies.

Ralstonia eutropha strain CH34 (formerly Alcaligenes eutrophus CH34) is an aerobic Gram-negative and facultative chemolithotrophic bacteria with plasmid-bound resistance to heavy metals. The presence of Ralstonia eutropha strain CH34 is an indication of environmental heavy metals pollution. The major purpose of this work was to produce monoclonal antibodies (MAbs) against the metal transport outer membrane proteins. In this way, bacteria outer membranes, grown with or without iron, were purified. The electrophoretic pattern of the outer membrane revealed that, in iron starvation conditions, at least four proteins were overexpressed. These outer membranes were used to immunize mice to produce MAbs. About 200 hybridomas were tested by enzyme-linked immunoadsorbent assay (ELISA). Most of these hybridomas exhibited cross reactions with Escherichia coli and Klebsiella aerogenes. Two hybridomas, AE5/7 and AE5/9, produced MAbs that detected specifically Ralstonia eutropha strain CH34. Analysis by Western blotting showed that these MAbs recognized a protein with a molecular weight of about 41 kDa. Moreover, the presence of the two megaplasmids was required for the full expression of the 41-kDa protein, as demonstrated by screening of the derivatives strains by ELISA. These MAbs could be used for a specific and rapid detection of Ralstonia eutropha strain CH34, using direct immunological methods.

SUMO--nonclassical ubiquitin.

SUMO (small ubiquitin-related modifier) is the best-characterized member of a growing family of ubiquitin-related proteins. It resembles ubiquitin in its structure, its ability to be ligated to other proteins, as well as in the mechanism of ligation. However, in contrast to ubiquitination-often the first step on a one-way road to protein degradation-SUMOlation does not seem to mark proteins for degradation. In fact, SUMO may even function as an antagonist of ubiquitin in the degradation of selected proteins. While most SUMO targets are still at large, available data provide compelling evidence for a role of SUMO in the regulation of protein-protein interactions and/or subcellular localization.

Two-way trafficking with Ran.

The small Ras-related GTPase Ran is directly involved in nuclear protein import and export. However, the question of how Ran functions in transport is highly controversial. Here, we suggest that Ran is important for the formation, vectorial movement and disassembly of many different classes of transport complexes that traverse the nuclear pore complex during import and export processes. Comparison of Ran with the translation elongation factor Ef-Tu raises the possibility that Ran might also be involved in a proofreading function related to the assembly of import complexes. Although aspects of this model are hypothetical and challenge some current dogma in the field, we believe that it can integrate most of the current data into a coherent picture of the import process.

Structure determination of the small ubiquitin-related modifier SUMO-1.

The recently discovered small ubiquitin-related modifier SUMO-1 belongs to the growing family of ubiquitin-related proteins involved in postranslational protein modification. Unlike ubiquitin, SUMO-1 does not appear to target proteins for degradation but seems to be involved in the modulation of protein-protein interactions. Independent studies demonstrate an essential function of SUMO-1 in the regulation of nucleo-cytoplasmic transport, and suggest a role in cell-cycle regulation and apoptosis. Here, we present the first three-dimensional structure of SUMO-1 solved by NMR. Although having only 18% amino acid sequence identity with ubiquitin, the overall structure closely resembles that of ubiquitin, featuring the betabetaalphabetabetaalphabeta fold of the ubiquitin protein family. In addition, the position of the two C-terminal Gly residues required for isopeptide bond formation is conserved between ubiquitin and SUMO-1. The most prominent feature of SUMO-1 is a long and highly flexible N terminus, which protrudes from the core of the protein and which is absent in ubiquitin. Furthermore, ubiquitin Lys48, required to generate ubiquitin polymers, is substituted in SUMO-1 by Gln69 at the same position, which provides an explanation of why SUMO-1 has not been observed to form polymers. Moreover, the hydrophobic core of SUMO-1 and ubiquitin is maintained by conserved hydrophobic residues, whereas the overall charge topology of SUMO-1 and ubiquitin differs significantly, suggesting specific modifying enzymes and target proteins for both proteins.

Modification of Ran GTPase-activating protein by the small ubiquitin-related modifier SUMO-1 requires Ubc9, an E2-type ubiquitin-conjugating enzyme homologue.

Covalent modification of the Ran GTPase-activating protein RanGAP1 with the ubiquitin-related protein SUMO-1 promotes its association with Nup358, a component of the cytoplasmic fibrils emanating from the nuclear pore complex (1,2). In Xenopus egg extracts, Nup358 can be found in a complex with Ubc9 (3), a structural homologue of the E2-type ubiquitin-conjugating enzymes (UBCs). Here we show that a subset of the human homologue of Ubc9 (HsUbc9) colocalizes with RanGAP1 at the nuclear envelope. HsUbc9 forms thiolester conjugates with recombinant SUMO-1, but not with recombinant ubiquitin, indicating that it is functionally distinct from E2-type UBCs. Finally, HsUbc9 is required for the modification of RanGAP1 by SUMO-1. These results suggest that HsUbc9 is a component of a novel enzymatic cascade that modifies RanGAP1, and possibly other substrates, with SUMO-1.

Molecular characterization of the SUMO-1 modification of RanGAP1 and its role in nuclear envelope association.

The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101- amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1-SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role.

RanGTP targets p97 to RanBP2, a filamentous protein localized at the cytoplasmic periphery of the nuclear pore complex.

RanBP2, a protein containing FG repeat motifs and four binding sites for the guanosine triphosphatase Ran, is localized at the cytoplasmic periphery of the nuclear pore complex (NPC) and is believed to play a critical role in nuclear protein import. We purified RanBP2 from rat liver nuclear envelopes and examined its structural and biochemical properties. Electron microscopy showed that RanBP2 forms a flexible filamentous molecule with a length of approximately 36 nm, suggesting that it comprises a major portion of the cytoplasmic fibrils implicated in initial binding of import substrates to the NPC. Using in vitro assays, we characterized the ability of RanBP2 to bind p97, a cytosolic factor implicated in the association of the nuclear localization signal receptor with the NPC. We found that RanGTP promotes the binding of p97 to RanBP2, whereas it inhibits the binding of p97 to other FG repeat nucleoporins. These data suggest that RanGTP acts to specifically target p97 to RanBP2, where p97 may support the binding of an nuclear localization signal receptor/substrate complex to RanBP2 in an early step of nuclear import.

Plant polyketide synthases leading to stilbenoids have a domain catalyzing malonyl-CoA:CO2 exchange, malonyl-CoA decarboxylation, and covalent enzyme modification and a site for chain lengthening.

Stilbene synthases and the related bibenzyl synthases are plant polyketide synthases whose biological functions lie in the formation of antimicrobial phytoalexins. The formation of hydroxystilbenes from one molecule of acyl-CoA and three molecules of malonyl-CoA is catalyzed by a homodimeric 90 kDa protein and includes Claisen condensations and cleavage of a thioester followed by decarboxylation. Combining inhibitor studies, protein modifications, and site-directed mutagenesis, we were able to differentiate between the binding sites for malonyl-CoA and the regions responsible for the selection of the primer, p-coumaroyl-CoA or m-hydroxyphenylpropionyl-CoA, respectively. Mutations in the C-terminal part of the molecule or modification by photolabeling with p-azidocinnamoyl-CoA influence the overall reaction, the formation of hydroxystilbenes, but leave partial reactions, such as the malonyl-CoA:CO2 exchange and the malonyl-CoA-dependent modification of the enzyme, unaffected. Data obtained with several kinds of stilbene synthase and mutant forms suggest that the malonyl-CoA-dependent covalent modification takes place at a cysteine residue in the N-terminal part of the enzyme. Mutations in the C-terminal half of the enzyme molecule do not interfere with the malonyl-CoA-dependent reactions.

A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2.

We have found that the mammalian Ran GTPase-activating protein RanGAP1 is highly concentrated at the cytoplasmic periphery of the nuclear pore complex (NPC), where it associates with the 358-kDa Ran-GTP-binding protein RanBP2. This interaction requires the ATP-dependent posttranslational conjugation of RanGAP1 with SUMO-1 (for small ubiquitin-related modifier), a novel protein of 101 amino acids that contains low but significant homology to ubiquitin. SUMO-1 appears to represent the prototype for a novel family of ubiquitin-related protein modifiers. Inhibition of nuclear protein import resulting from antibodies directed at NPC-associated RanGAP1 cannot be overcome by soluble cytosolic RanGAP1, indicating that GTP hydrolysis by Ran at RanBP2 is required for nuclear protein import.

Increase of LBNP tolerance using elastic compression stockings.

BACKGROUND: Astronauts returning from spaceflight are affected by reduced orthostatic tolerance resulting from exposure to weightlessness. There are some countermeasures currently in use to improve cardiovascular performance of returning astronauts, while there are others that are being tested in flight and in ground-based investigations. This paper presents a study on the use of elastic compression stockings to reduce leg blood capacity (LBC) which is believed to be one of the determinants of orthostatic tolerance. METHODS: The data are from 6 healthy men with a mean age of 36 +/- 5 (SE) yr. Assessment of the effectiveness of stockings in improving orthostatic tolerance is based on a presyncopal-limited lower body negative pressure (LBNP) test, consisting of successive 3 min exposures to negative pressures of -20 hPa (-15 mmHg), -40 hPa (-30 mmHg), and decrements in steps of 10 hPa (7.5 mmHg) from then on until termination of the test. RESULTS: Results show an increase in the maximal level of LBNP tolerated (88 hPa or 66 mmHg for control vs. 108 hPa or 81 mmHg for stockings; p = 0.018) as well as in the cumulative stress index (CSI) (1122 hPa-min or 842 mmHg-min for control vs. 1734 hPa-min or 1300 mmHg-min for stockings; p = 0.029). CONCLUSIONS: The improvement of LBNP tolerance with elastic compression stockings coupled with their ease of use support the need for further experimental studies for evaluating their potential as a countermeasure for astronauts after return from spaceflight.

Cerebral blood flow velocity response induced by a 70-hPa Valsalva manoeuvre associated with normo- and hypergravity in humans.

Anti-G straining manoeuvres, derived from the Valsalva manoeuvre (VM), are physiological methods for protecting fighter pilots against positive accelerations (+Gz). The aim of this study was to investigate the effects of a standard VM on cerebral haemodynamics, in normo- and hypergravity. In six healthy male volunteers, we investigated the cerebral blood flow velocity response induced by a 10-s, 70-hPa (52.5 mmHg) VM, under normogravity, +2, +3 and +4 Gz acceleration plateaus. Mean blood flow velocity [formula: see text] in middle cerebral artery was monitored by transcranial Doppler velocimetry. In normogravity, no significant variation in [formula: see text] was observed at the onset of VM. After a maximal period of 1.2 s, while VM was sustained, [formula: see text] decreased significantly (P < 0.05). Following the end of the manoeuvre [formula: see text] did not change significantly. When the expiratory pressure had returned to the control value, [formula: see text] was transiently increased (P < 0.05) before returning to control values. During hypergravity, [formula: see text] was significantly decreased at +3 and +4 Gz (P < 0.05) before the onset of VM. While performing VM under +Gz, the main difference compared to the normogravity condition was a significant increase of [formula: see text] (P < 0.05) at the onset of the manoeuvre. Our findings would suggest that when performed under +Gz stress, a 70-hPa VM can transiently improve cerebral haemodynamics. However, when VM is sustained for more than 1.2 s it results in a lasting decrease of cerebral perfusion which may lower +Gz tolerance.

GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import.

Mediated import of proteins into the nucleus involves multiple cytosolic factors, including the small GTPase Ran. Whether Ran functions by interacting with other cytosolic proteins or components of the nuclear pore complex has been unclear. Furthermore, the precise transport step where Ran acts has not been determined. To address these questions, we have analyzed the binding interactions of Ran using permeabilized cells and isolated nuclear envelopes. By light and electron microscope immunolocalization, we have found that Ran accumulates specifically at the cytoplasmic surface of the nuclear pore complex when nuclear import in permeabilized cells is inhibited by nonhydrolyzable analogs of GTP. Ran associates with a peripheral pore complex region that is similar to the area where transport ligands accumulate by depletion of ATP, which arrests an early step of transport. Binding studies with isolated nuclear envelopes in the absence of added cytosol indicate that Ran-GTP directly interacts with a pore complex protein. Using blot overlay techniques, we detected a single prominent polypeptide of isolated nuclear envelopes that binds Ran-GTP. This corresponds to the 358-kD protein RanBP2, a Ran binding pore complex protein recently identified by two-hybrid screening. Thus, RanBP2 is likely to constitute the Ran-GTP-binding site detected at the cytoplasmic periphery of the pore complex. These data support a model in which initial ligand binding to the nuclear pore complex occurs at or near RanBP2, and that hydrolysis of GTP by Ran at this site serves to define commitment to the nuclear import pathway.

Mechanisms of nuclear protein import.

The past two years have seen a significant increase in our understanding of nuclear protein import. Five cytosolic import factors have been identified, two of which have been shown to directly interact with components of the nuclear pore complex. These findings enable refinement of previous models for steps in the nuclear import pathway, and provide a framework for future research.

RNA1 encodes a GTPase-activating protein specific for Gsp1p, the Ran/TC4 homologue of Saccharomyces cerevisiae.

Ran/TC4 is a ras-related GTP-binding protein predominantly located in the nucleus. Ran/TC4 is essential for nuclear transport and is involved in mitotic control. In Saccharomyces cerevisiae a gene highly homologous to Ran/TC4 has been identified and named GSP1. Like all ras-related GTP-binding proteins, Gsp1p undergoes cycles of GTP hydrolysis and GDP/GTP exchange. The switching between the two different nucleotide bound states regulates the function of these GTP-binding proteins. Here we identify the product of the yeast RNA1 gene as the GTPase-activating protein (GAP) of Gsp1p. RNA1 belongs to a group of genes which are conserved in a variety of different organisms. We have expressed and purified recombinant Gsp1p and Rna1p from Escherichia coli. The GTPase activity of Gsp1p is stimulated 10(7)-fold by Rna1p. In addition, we find that the previously identified human RanGAP1 and rna1p from Schizosaccharomyces pombe are also able to induce GTPase activity of Gsp1p. The GTP hydrolysis of Ran is induced by RanGAP1 and rna1p but not by Rna1p. Implications for the suggested functions of Ran/TC4/Gsp1p in nuclear transport and mitotic control are discussed.