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‣ An expanded view of bacterial DNA replication

Noirot-Gros, Marie-Françoise; Dervyn, Etienne; Wu, Ling Juan; Mervelet, Peggy; Errington, Jeffery; Ehrlich, S. Dusko; Noirot, Philippe
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 11/06/2002 Português
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A protein-interaction network centered on the replication machinery of Bacillus subtilis was generated by genome-wide two-hybrid screens and systematic specificity assays. The network consists of 91 specific interactions linking 69 proteins. Over one fourth of the interactions take place between homologues of proteins known to interact in other organisms, indicating the high biological significance of the other interactions we report. These interactions provide insights on the relations of DNA replication with recombination and repair, membrane-bound protein complexes, and signaling pathways. They also lead to the biological role of unknown proteins, as illustrated for the highly conserved YabA, which is shown here to act in initiation control. Thus, our interaction map provides a valuable tool for the discovery of aspects of bacterial DNA replication.

‣ Paracrine in vivo inhibitory effects of hepatitis B virus X protein (HBx) on liver cell proliferation: An alternative mechanism of HBx-related pathogenesis

Tralhao, J. Guilherme; Roudier, Jean; Morosan, Serban; Giannini, Carlo; Tu, Hong; Goulenok, Cyril; Carnot, Françoise; Zavala, Flora; Joulin, Virginie; Kremsdorf, Dina; Bréchot, Christian
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 14/05/2002 Português
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451.23344%
The role of the hepatitis B virus X protein (HBx) in the pathogenesis of hepatitis B virus (HBV) infection remains unclear. HBx exhibits pleiotropic biological effects, whose in vivo relevance is a matter for debate. In the present report, we have used a combination of HBx-expressing transgenic mice and liver cell transplantation to investigate the in vivo impact of HBx expression on liver cell proliferation and viability in a regenerative context. We show that moderate HBx expression inhibits liver regeneration after partial hepatectomy in HBx-expressing transgenic mice. We also demonstrate that the transplantation of HBx-expressing liver cells, isolated from HBx transgenic mice, is sufficient to inhibit overall recipient liver regeneration after partial hepatectomy. Moreover, the injection of serum samples drawn from HBx-expressing transgenic mice mimicked the inhibitory effect of HBx on liver regeneration. Finally, the incubation of primary rat hepatocytes with the supernatant of HBx-expressing liver cells inhibits cellular DNA synthesis. Taken together, our results demonstrate a paracrine inhibitory effect of HBx on liver cell proliferation and lead us to propose HBV as one of the few viruses implicated in human cancer which act...

‣ Identification of a protein secretory pathway for the secretion of heat-labile enterotoxin by an enterotoxigenic strain of Escherichia coli

Tauschek, Marija; Gorrell, Rebecca J.; Strugnell, Richard A.; Robins-Browne, Roy M.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 14/05/2002 Português
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451.23344%
Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen that causes cholera-like diarrhea in humans and animals. ETEC secretes a heat-labile enterotoxin (LT), which resembles cholera toxin, but the actual mechanism of LT secretion is presently unknown. We have identified a previously unrecognized type II protein secretion pathway in the prototypic human ETEC strain, H10407 (serotype O78:H11). The genes for this pathway are absent from E. coli K-12, although examination of the K-12 genome suggests that it probably once possessed them. The secretory pathway bears significant homology at the amino acid level to the type II protein secretory pathway required by Vibrio cholerae for the secretion of cholera toxin. With this in mind, we determined whether the homologous pathway of E. coli H10407 played a role in the secretion of LT. To this end, we inactivated the pathway by inserting a kanamycin-resistance gene into one of the genes (gspD) of the type II secretion pathway by homologous recombination. LT secretion by E. coli H10407 and the gspD mutant was assayed by enzyme immunoassay, and its biological activity was assessed by using Y-1 adrenal cells. This investigation showed that the protein secretory pathway is functional and necessary for the secretion of LT by ETEC. Our findings have revealed the mechanism for the secretion of LT by ETEC...

‣ Nanocrystal targeting in vivo

Åkerman, Maria E.; Chan, Warren C. W.; Laakkonen, Pirjo; Bhatia, Sangeeta N.; Ruoslahti, Erkki
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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451.23344%
Inorganic nanostructures that interface with biological systems have recently attracted widespread interest in biology and medicine. Nanoparticles are thought to have potential as novel intravascular probes for both diagnostic (e.g., imaging) and therapeutic purposes (e.g., drug delivery). Critical issues for successful nanoparticle delivery include the ability to target specific tissues and cell types and escape from the biological particulate filter known as the reticuloendothelial system. We set out to explore the feasibility of in vivo targeting by using semiconductor quantum dots (qdots). Qdots are small (<10 nm) inorganic nanocrystals that possess unique luminescent properties; their fluorescence emission is stable and tuned by varying the particle size or composition. We show that ZnS-capped CdSe qdots coated with a lung-targeting peptide accumulate in the lungs of mice after i.v. injection, whereas two other peptides specifically direct qdots to blood vessels or lymphatic vessels in tumors. We also show that adding polyethylene glycol to the qdot coating prevents nonselective accumulation of qdots in reticuloendothelial tissues. These results encourage the construction of more complex nanostructures with capabilities such as disease sensing and drug delivery.

‣ Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry

Salomon, Arthur R.; Ficarro, Scott B.; Brill, Laurence M.; Brinker, Achim; Phung, Qui T.; Ericson, Christer; Sauer, Karsten; Brock, Ansgar; Horn, David M.; Schultz, Peter G.; Peters, Eric C.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
451.23344%
The reversible phosphorylation of tyrosine residues is an important mechanism for modulating biological processes such as cellular signaling, differentiation, and growth, and if deregulated, can result in various types of cancer. Therefore, an understanding of these dynamic cellular processes at the molecular level requires the ability to assess changes in the sites of tyrosine phosphorylation across numerous proteins simultaneously as well as over time. Here we describe a sensitive approach based on multidimensional liquid chromatography/mass spectrometry that enables the rapid identification of numerous sites of tyrosine phosphorylation on a number of different proteins from human whole cell lysates. We used this methodology to follow changes in tyrosine phosphorylation patterns that occur over time during either the activation of human T cells or the inhibition of the oncogenic BCR-ABL fusion product in chronic myelogenous leukemia cells in response to treatment with STI571 (Gleevec). Together, these experiments rapidly identified 64 unique sites of tyrosine phosphorylation on 32 different proteins. Half of these sites have been documented in the literature, validating the merits of our approach, whereas motif analysis suggests that a number of the undocumented sites are also potentially involved in biological pathways. This methodology should enable the rapid generation of new insights into signaling pathways as they occur in states of health and disease.

‣ Crystal structure of a photoactive yellow protein from a sensor histidine kinase: Conformational variability and signal transduction

Rajagopal, Sudarshan; Moffat, Keith
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
451.23344%
Photoactive yellow protein (E-PYP) is a blue light photoreceptor, implicated in a negative phototactic response in Ectothiorhodospira halophila, that also serves as a model for the Per–Arnt–Sim superfamily of signaling molecules. Because no biological signaling partner for E-PYP has been identified, it has not been possible to correlate any of its photocycle intermediates with a relevant signaling state. However, the PYP domain (Ppr-PYP) from the sensor histidine kinase Ppr in Rhodospirillum centenum, which regulates the catalytic activity of Ppr by blue light absorption, may allow such issues to be addressed. Here we report the crystal structure of Ppr-PYP at 2 Å resolution. This domain has the same absorption spectrum and similar photocycle kinetics as full length Ppr, but a blue-shifted absorbance and considerably slower photocycle than E-PYP. Although the overall fold of Ppr-PYP resembles that of E-PYP, a novel conformation of the β4–β5 loop results in inaccessibility of Met-100, thought to catalyze chromophore reisomerization, to the chromophore. This conformation also exposes a highly conserved molecular surface that could interact with downstream signaling partners. Other structural differences in the α3–α4 and β4–β5 loops are consistent with these regions playing significant roles in the control of photocycle dynamics and...

‣ Structural specificity of heparin binding in the fibroblast growth factor family of proteins

Raman, Rahul; Venkataraman, Ganesh; Ernst, Steffen; Sasisekharan, V.; Sasisekharan, Ram
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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451.23344%
Heparin and heparan sulfate glycosaminoglycans (HSGAGs) mediate a wide variety of complex biological processes by specifically binding proteins and modulating their biological activity. One of the best studied model systems for protein–HSGAG interactions is the fibroblast growth factor (FGF) family of molecules, and recent observations have demonstrated that the specificity of a given FGF ligand binding to its cognate receptor (FGFR) is mediated by distinct tissue-specific HSGAG sequences. Although it has been known that sulfate and carboxylate groups in the HSGAG chain play a key role by interacting with basic residues on the proteins, there is little understanding of how these ionic interactions provide the necessary specificity for protein binding. In this study, using all of the available crystal structures of different FGFs and FGF–HSGAG complexes, we show that in addition to the ionic interactions, optimal van der Waals contact between the HSGAG oligosaccharide and the protein is also very important in influencing the specificity of FGF–HSGAG interactions. Although the overall helical structure is maintained in the FGF-bound HSGAG compared with unbound HSGAG, we observe distinct changes in the backbone torsion angles of the oligosaccharide chain induced upon protein binding. These changes result in local deviations in the helical axis that provide optimal ionic and van der Waals contact with the protein. A specific conformation and topological arrangement of the HSGAG-binding loops of FGF...

‣ Mimicking natural evolution in vitro: An N-acetylneuraminate lyase mutant with an increased dihydrodipicolinate synthase activity

Joerger, Andreas C.; Mayer, Sebastian; Fersht, Alan R.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
451.23344%
N-acetylneuraminate lyase (NAL) and dihydrodipicolinate synthase (DHDPS) belong to the NAL subfamily of (β/α)8-barrels. They share a common catalytic step but catalyze reactions in different biological pathways. By rational design, we have introduced various mutations into the NAL scaffold from Escherichia coli to switch the activity toward DHDPS. These mutants were tested with respect to their catalytic properties in vivo and in vitro as well as their stability. One point mutation (L142R) was sufficient to create an enzyme that could complement a bacterial auxotroph lacking the gene for DHDPS as efficiently as DHDPS itself. In vitro, this mutant had an increased DHDPS activity of up to 19-fold as defined by the specificity constant kcat/KM for the new substrate l-aspartate-β-semialdehyde when compared with the residual activity of NAL wild-type, mainly because of an increased turnover rate. At the same time, mutant L142R maintained much of its original NAL activity. We have solved the crystal structure of mutant L142R at 1.8 Å resolution in complex with the inhibitor β-hydroxypyruvate. This structure reveals that the conformations of neighboring active site residues are left virtually unchanged by the mutation. The high flexibility of R142 may favor its role in assisting in catalysis. Perhaps...