High-level expression, purification, and characterization of recombinant wheat xylanase inhibitor TAXI-I secreted by the yeast Pichia pastoris

Triticum aestivum xylanase inhibitor I (TAXI-I) is a wheat protein that inhibits microbial xylanases belonging to glycoside hydrolase family 11. In the present study, recombinant TAXI-I (rTAXI-I) was successfully produced by the methylotrophic yeast Pichia pastoris at high expression levels (approximately 75 mg/L). The rTAXI-I protein was purified from the P. pastoris culture medium using cation exchange and gel filtration chromatographic steps. rTAXI-I has an iso-electric point of at least 9.3 and a mass spectrometry molecular mass of 42,013 Da indicative of one N-linked glycosylation. The recombinant protein fold was confirmed by circular dichroism spectroscopy. Xylanase inhibition by rTAXI-I was optimal at 20-30 degrees C and at pH 5.0. rTAXI-I still showed xylanase inhibition activity at 30 degrees C after a 40 min pre-incubation step at temperatures between 4 and 70 degrees C and after 2 h pre-incubation at room temperature at a pH ranging from 3.0 to 12.0, respectively. All tested glycoside hydrolase family 11 xylanases were inhibited by rTAXI-I whereas those belonging to family 10 were not. Specific inhibition activities against family 11 Aspergillus niger and Bacillus subtilis xylanases were 3570 and 2940IU/mg protein, respectively. The obtained biochemical characteristics of rTAXI-I produced by P. pastoris (no proteolytical cleft) were similar to those of natural TAXI-I (mixture of proteolytically processed and non-processed forms) and non-glycosylated rTAXI-I expressed in Escherichia coli. The present results show that xylanase inhibition activity of TAXI-I is only affected to a limited degree by its glycosylation or proteolytic processing.     

The human polycomb group EED protein interacts with the integrase of human immunodeficiency virus type 1

Human EED, a member of the superfamily of WD-40 repeat proteins and of the Polycomb group proteins, has been identified as a cellular partner of the human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein (R. Peytavi et al., J. Biol. Chem. 274:1635-1645, 1999). In the present study, EED was found to interact with HIV-1 integrase (IN) both in vitro and in vivo in yeast. In vitro, data from mutagenesis studies, pull-down assays, and phage biopanning suggested that EED-binding site(s) are located in the C-terminal domain of IN, between residues 212 and 264. In EED, two putative discrete IN-binding sites were mapped to its N-terminal moiety, at a distance from the MA-binding site, but EED-IN interaction also required the integrity of the EED last two WD repeats. EED showed an apparent positive effect on IN-mediated DNA integration reaction in vitro, in a dose-dependent manner. In situ analysis by immunoelectron microscopy (IEM) of cellular distribution of IN and EED in HIV-1-infected cells (HeLa CD4(+) cells or MT4 lymphoid cells) showed that IN and EED colocalized in the nucleus and near nuclear pores, with maximum colocalization events occurring at 6 h postinfection (p.i.). Triple colocalizations of IN, EED, and MA were also observed in the nucleoplasm of infected cells at 6 h p.i., suggesting the ocurrence of multiprotein complexes involving these three proteins at early steps of the HIV-1 virus life cycle. Such IEM patterns were not observed with a noninfectious, envelope deletion mutant of HIV-1.     

RLIP,an effector of the Ral GTPases,is a platform for Cdk1 to phosphorylate epsin during the switch off of endocytosis in mitosis

The Ral signaling pathway is critically involved in Ras-dependent oncogenesis. One of its key actors, RLIP/RalBP1, which participates in receptor endocytosis during interphase, is also involved in mitotic processes when endocytosis is switched off. During mitosis, RLIP76 is located on the duplicated centrosomes and is required for their proper separation and movement to the poles. We have looked for actors that associate with RLIP during mitosis. We show here that RLIP/RalBP1 interacts with an active p34cdc2.cyclinB1 (cdk1) enzyme and that this interaction is crucial for the mitotic phosphorylation of Epsin that, once phosphorylated, is no longer competent for endocytosis. We show also that this latter phosphorylation is dependent on Ral signaling. We propose that RLIP/RalBP1 is used as a platform by the mitotic cdk1 to facilitate the phosphorylation of Epsin, which makes Epsin incompetent for endocytosis during mitosis, when endocytosis is switched off.     

A single surface tryptophan in the chitin-binding domain from Bacillus circulans chitinase A1 plays a pivotal role in binding chitin and can be modified to create an elutable affinity tag

Site-directed mutagenesis was carried out to investigate the roles of a number of highly conserved residues of the chitin-binding domain (ChBD) of Bacillus circulans chitinase A1 (ChiA1) in the binding of chitin. Analysis of single alanine replacement mutants showed that mutation of an exposed tryptophan residue (Trp(687)) impaired the binding to chitin, while mutation of other highly conserved residues, most carrying aromatic or hydrophobic side chains, did not significantly affect the binding activity. Interestingly, replacement of Trp(687) with phenylalanine significantly reduced chitin-binding activity at lower salt concentrations (0-1 M NaCl) but allowed strong binding to chitin at 2 M NaCl. Since Trp(687) is conserved among the ChBDs belonging to the bacterial ChiA1 subfamily, the data presented suggest a general mechanism in which this exposed tryptophan, which is located in the cleft formed between two beta-sheets as revealed by the solution structure [J. Biol. Chem. 275 (2000) 13654], makes a major contribution to ligand binding presumably through hydrophobic interactions. Furthermore, modulation of the chitin-binding activity by the conserved amino acid replacement (W687F) and a shift in the ionic strength of buffer has led to the development of an elutable affinity tag for single column purification of recombinant proteins.     

Involvement of a novel fast inward sodium current in the invasion capacity of a breast cancer cell line

This work reports the finding of a unique fast inward sodium current (I(Na)) in MDA-MB-231 cells which is missing in MDA-MB-468 cells and in MCF-7 cells. This current is high-voltage-activated and displays a window current at the membrane potential of MDA-MB-231 cells. This current is blocked by high concentrations of tetrodotoxin (TTX). In MDA-MB-231 cells, which are the most invasive cells among the three cell lines tested, proliferation and migration were not sensitive to TTX while invasion was reduced by approximately 30%. These experiments suggest that I(Na) is involved in the invasion process, probably through its participation to the regulation of the intracellular sodium homeostasis.     

A mixed strategy in the annual endemic Aster laurentianus (Asteraceae)--a stress-tolerant, yet opportunistic species

Several environmental factors influence the distribution of plants in coastal salt marshes. Substrate salinity is among the major factors preventing several species from establishing near the water line. However, interspecific competition for light and nutrients is often significant in determining the upper limit of plants along the salt marsh gradient. In this study, we tested the effects of substrate salinity and light and nutrient availability on the performance of the annual Aster laurentianus (Asteraceae), an endangered species of eastern Canadian salt marshes. This species is typically found in a narrow band along the shores of shallow lagoons, cornered between the high water line and the dense, herbaceous community of the upper marsh. Low light availability was the most significant factor limiting plant performance. Salinity had little effect on A. laurentianus as, unexpectedly, did nutrient availability. Yet plants were able to absorb nutrients when these were made more available. Luxury consumption, the uptake of excess nutrients, may make sense for this annual plant because the habitat in which it grows is subject to frequent disturbances (e.g., sand accretion and salinity pulses) that may kill canopy species and release suppressed A. laurentianus individuals. These results suggest that interspecific competition for light may play a significant role in restraining A. laurentianus from the upper part of salt marshes. Luxury consumption may help the species to opportunistically take advantage of release from taller species, particularly towards the upper edge of the salt marsh gradient.     

Human immunodeficiency virus-1 Tat protein interacts with distinct proteasomal alpha and beta subunits

The human immunodeficiency virus-1 (HIV-1) Tat protein was previously reported to compete the association of PA28 regulator with the alpha rings of the 20S proteasome and to inhibit its peptidase activity. However, the distinct interaction sites within the proteasome complex remained to be determined. Here we show that HIV-1 Tat binds to alpha4 and alpha7, six beta subunits of the constitutive 20S proteasome and the interferon-gamma-inducible subunits beta2i and beta5i. A Tat-proteasome interaction can also be demonstrated in vivo and leads to inhibition of proteasomal activity. This indicates that Tat can modulate or interfere with cellular proteasome function by specific interaction with distinct proteasomal subunits.     

Mapping of synergistic components of weakly interacting protein-protein motifs using arrays of paired peptides

Protein-protein recognition usually involves multiple interactions among different motifs that are scattered over protein surfaces. To identify such weak interactions, we have developed a novel double peptide synthesis (DS) method. This method allows us to map protein-protein interactions that involve two linear dis- continuous components from a polypeptide by the use of spatially addressable synergistic pairs of synthetic peptides. The DS procedure is based on the "SPOT" membrane-bound peptide synthesis technique, but to synthesize a mixture of two peptides, it uses both Fmoc (N-(9-fluorenyl)methoxycarbonyl))-alanine and Alloc-alanine at the first cycle. This allows their selective deprotection by either piperidine or tributyltin/palladium treatment, respectively. Using SPOT DS, we confirmed as a proof of principle that Elk-1 Ser(383) phosphorylation by ERK-2 kinase is stimulated by the presence of the Elk-1-docking domain. SPOT DS can also be used to dissect protein-protein motifs that define phosphatase substrate affinity. Using this technique, we identified three new regions in the insulin receptor that stimulate the dephosphorylation of the receptor by protein-tyrosine phosphatase (PTP) 1B and presumably increase the selectivity of PTP for this substrate. These data demonstrate that the SPOT DS technique allows the identification of non-linear weakly interacting protein motifs, which are an important determinant of protein kinase and phosphatase substrate specificity and of protein-protein interactions in general.     

The anti-HIV pentameric pseudopeptide HB-19 binds the C-terminal end of nucleolin and prevents anchorage of virus particles in the plasma membrane of target cells

The multivalent pseudopeptide HB-19 that binds the cell-surface-expressed nucleolin is a potent inhibitor of human immunodeficiency virus (HIV) infection by blocking virus particle attachment and thus anchorage in the plasma membrane. We show that cross-linking of surface-bound HB-19A (like HB-19 but with a modified template) results in aggregation of HB-19A with surface nucleolin. Consistent with its specific action, HB-19A binding to different types of cells reaches saturation at concentrations that have been reported to result in inhibition of HIV infection. By using Chinese hamster ovary mutant cell lines, we confirm that the binding of HB-19A to surface nucleolin is independent of heparan and chondroitin sulfate proteoglycans. In vitro generated full-length nucleolin was found to bind HB-19A, whereas the N-terminal part containing the acidic amino acid stretches of nucleolin did not. The use of various deletion constructs of the C-terminal part of nucleolin then permitted the identification of the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif, RGG, as the domain that binds HB-19A. Finally, a synthetic peptide corresponding to the last C-terminal 63 amino acids was able to inhibit HIV infection at the stage of HIV attachment to cells, thus suggesting that this domain could be functional in the HIV anchorage process.     

Revision of<Emphasis Type="Italic">Cheliderpeton vranyi</Emphasis><Emphasis Type="SmallCaps">Fritsch</Emphasis>, 1877 (Amphibia,Temnospondyli) from the Lower Permian of Bohemia (Czech Republic)

The temnospondylCheliderpeton vranyi Fritsch, 1877 from the Lower Permian Ruprechtice horizon (Rotliegend) of the Intrasudetic Basin (Bohemia, Czech Republic) is redescribed. Many features of the skeleton permit a new understanding of the type species and consequently of the genus. Diagnostic characters are the narrow and round-tipped snout, straight to convex outline of the skull roof, narrow and long otic notch, posteriorly expanded quadratojugal, and the relatively wide and short rhombic interclavicle. The ilium with a short, expanded dorsal branch and the missing contact of nasal/maxilla are features shared with the related Upper PermianIntasuchus from Russia and the Eryopidae.Actinodon germanicus is a junior synonym ofCheliderpeton vranyi.