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991.
Daisuke Suzuki Keiichi Inoue Makoto Sakai Hideki Kandori Yuki Sudo 《Journal of molecular biology》2009,392(1):48-4922
Microbial organisms utilize light not only as energy sources but also as signals by which rhodopsins (containing retinal as a chromophore) work as photoreceptors. Sensory rhodopsin I (SRI) is a dual photoreceptor that regulates both negative and positive phototaxis in microbial organisms, such as the archaeon Halobacterium salinarum and the eubacterium Salinibacter ruber. These organisms live in highly halophilic environments, suggesting the possibility of the effects of salts on the function of SRI. However, such effects remain unclear because SRI proteins from H. salinarum (HsSRI) are unstable in dilute salt solutions. Recently, we characterized a new SRI protein (SrSRI) that is stable even in the absence of salts, thus allowing us to investigate the effects of salts on the photochemical properties of SRI. In this study, we report that the absorption maximum of SrSRI is shifted from 542 to 556 nm in a Cl−-dependent manner with a Km of 307 ± 56 mM, showing that Cl−-binding sites exist in SRI. The bathochromic shift was caused not only by NaCl but also by other salts (NaI, NaBr, and NaNO3), implying that I−, Br−, and NO3− can also bind to SrSRI. In addition, the photochemical properties during the photocycle are also affected by chloride ion binding. Mutagenesis studies strongly suggested that a conserved residue, His131, is involved in the Cl−-binding site. In light of these results, we discuss the effects of the Cl− binding to SRI and the roles of Cl− binding in its function. 相似文献
992.
Mesenchymal stromal cells (MSCs) have a critical role in cancer progression and metastasis. Despite extensive studies of the physiological responses in cancer cells, the molecular mechanisms regulating gene expression in MSCs by cancer cells remain undefined. Here we demonstrate that CC chemokine ligand 5 (CCL5) expression was increased in MSCs co-cultured with pancreatic cancer cells (PCCs), and this activation was dependent on extracellular insulin-like growth factor (IGF-I). Moreover, CCL5 induction in MSCs was required for the activation of IGF-I pathway in PCCs. These results reveal a link between the IGF-I pathway in PCCs and CCL5 pathway in MSCs through the interaction of those cells. 相似文献
993.
Kimiko Tatsumi Shoji Tsuji Hideki Miwa Mutsuo Nuriya Kazunari Kaneko Masato Yasui 《FEBS letters》2009,583(12):2077-2082
The neurogenic gene Drosophilabig brain (bib) has a high sequence homology to aquaporin-4. However, its cellular functions in Drosophila neurogenesis have remained elusive. Here we investigated cell adhesion, and the ion and water permeability of Bib. The adhesive function was examined by a cell aggregation assay using L cells. Bib-transfected L cells formed aggregated clusters, while control-L cells remained as a single cell suspension. Ion permeation was not confirmed in L cells stably expressing Bib. When expressed in COS7 cells, Bib exhibited limited water permeability. This newly found cell adhesive function of Bib may be important for Drosophila neurogenesis. 相似文献
994.
Hideki Watanabe Hiroyuki Matsumaru Ayako Ooishi YanWen Feng Takayuki Odahara Kyoko Suto Shinya Honda 《The Journal of biological chemistry》2009,284(18):12373-12383
Protein-protein interaction in response to environmental conditions enables
sophisticated biological and biotechnological processes. Aiming toward the
rational design of a pH-sensitive protein-protein interaction, we engineered
pH-sensitive mutants of streptococcal protein G B1, a binder to the IgG
constant region. We systematically introduced histidine residues into the
binding interface to cause electrostatic repulsion on the basis of a rigid
body model. Exquisite pH sensitivity of this interaction was confirmed by
surface plasmon resonance and affinity chromatography employing a clinically
used human IgG. The pH-sensitive mechanism of the interaction was analyzed and
evaluated from kinetic, thermodynamic, and structural viewpoints.
Histidine-mediated electrostatic repulsion resulted in significant loss of
exothermic heat of the binding that decreased the affinity only at acidic
conditions, thereby improving the pH sensitivity. The reduced binding energy
was partly recovered by “enthalpy-entropy compensation.” Crystal
structures of the designed mutants confirmed the validity of the rigid body
model on which the effective electrostatic repulsion was based. Moreover, our
data suggested that the entropy gain involved exclusion of water molecules
solvated in a space formed by the introduced histidine and adjacent tryptophan
residue. Our findings concerning the mechanism of histidine-introduced
interactions will provide a guideline for the rational design of pH-sensitive
protein-protein recognition.Molecular interactions govern a number of biological processes, including
metabolism, signal transduction, and immunoreaction. A better understanding of
the molecular basis for these interactions is crucial for a complete
elucidation of biological phenomena and redesign of interactions for drug
discovery and industrial biotechnology applications. Interactions between
biomolecules are generally characterized by their affinity, specificity, and
environmental responsiveness, such as sensitivity to pH. Such pH-dependent
ligand binding enables biological processes to function in an “on and
off” manner in response to environmental conditions, resulting in
sophisticated systems of regulation (e.g. pheromone production
(1,
2), immune systems
(3-5),
and mechanisms of virus survival
(6)).From an industrial perspective, pH sensitivity is advantageous to various
fields, such as drug delivery systems for medications
(7), biosensing techniques
(8,
9), and affinity chromatography
(10,
11). Although structure-based
protein design is a promising technique for improving molecular function
(12-15),
it is yet difficult to specifically modulate pH sensitivity of a
protein-protein interaction without an associated loss of inherent function
and/or structural stability. Some naturally occurring proteins undergo
substantial conformational change by pH shift, thereby achieving pH-dependent
binding for small molecules (2,
4,
16,
17). However, artificial
design of an equivalent mechanism involving conformational change is highly
problematic. Indeed, proteins have multiple degrees of freedom and consist of
a large number of atoms. Therefore, given that the resulting protein must
maintain both its innate binding ability and structural stability, the system
appears too complicated for rational design. By contrast to the method based
on conformational change, a rigid body-based model (i.e. introduction
of electrostatic repulsion or attraction into a binding interface between
rigid protein domains) could be a more promising approach for pH switching.
Naturally occurring proteins with pH sensitivity generally conserve histidine
residues
(18-21),
which function as a pH switch at slightly acidic conditions (pH ∼6.5) near
the pKa of the histidine side chain. In the presence of a
histidine residue at a binding interface, dissociation under acidic conditions
would be driven by electrostatic repulsion between rigid domains without
conformational change (Fig. 1).
This mechanism is rather simple and applicable to protein engineering
(22,
23). However, to our
knowledge, it still remains unclear how systematic design should be carried
out and, in particular, how histidine-mediated electrostatic repulsion
influences protein-protein interactions. Indeed, very little experimental data
are available for the molecular basis of histidine-introduced protein
binders.Open in a separate windowFIGURE 1.A schematic model for introduction of histidine-mediated electrostatic
repulsion into the binding interface between protein G (GB) and
Fc. Protein G residues positioned closely to basic side chains (depicted
as B) on Fc were systematically identified by distance calculations
and mutated into histidine to cause electrostatic repulsion under acidic
conditions. The inset shows an example of candidate positions for the
mutation.To better understand the design methodology for a pH-sensitive
protein-protein interaction, we generated a number of pH-sensitive
streptococcal protein G B1
(24) mutants by rationally
introducing histidine residues onto the binding surface. Protein G, a
bacterial Fc (fragment of crystallizable region) receptor to the constant
region of IgG, has been used as an affinity chromatography binder for antibody
immobilization and purification. Protein G has an acidic pH optimum for
binding relative to another bacterial Fc receptor, Staphylococcus
aureus protein A. The harsh elution conditions are likely to induce
acidic conformational changes in antibodies
(25,
26) during the purification
procedure, causing aggregation that is problematic for pharmaceutical
applications. The usefulness of the histidine-mediated electrostatic repulsion
for antibody purification was examined by constructing affinity chromatography
columns. Using the designed mutants, we analyzed the molecular basis of the
histidine-mediated interaction from a kinetic, thermodynamic, and structural
perspective. The observed data revealed functional and structural consequences
for the introduction of histidine residues. Analysis of our results provides a
guideline for the design of pH-dependent protein-protein interactions. 相似文献
995.
Norikiyo Ueno Carol L. Bratt Nilda E. Rodriguez Mary E. Wilson 《Cellular microbiology》2009,11(12):1827-1841
The obligate intracellular protozoan, Leishmania infantum chagasi (Lic) undergoes receptor-mediated phagocytosis by macrophages followed by a transient delay in phagolysosome maturation. We found differences in the pathway through which virulent Lic metacyclic promastigotes or avirulent logarithmic promastigotes are phagocytosed by human monocyte-derived macrophages (MDMs). Both logarithmic and metacyclic promastigotes entered MDMs through a compartment lined by the third complement receptor (CR3). In contrast, many logarithmic promastigotes entered through vacuoles lined by mannose receptors (MR) whereas most metacyclic promastigotes did not ( P < 0.005). CR3-positive vacuoles containing metacyclic promastigotes stained for caveolin-1 protein, suggesting CR3 localizes in caveolae during phagocytosis. Following entry, the kinetics of phagolysosomal maturation and intracellular survival also differed. Vacuoles containing metacyclic parasites did not accumulate lysosome-associated membrane protein-1 (LAMP-1) at early times after phagocytosis, whereas vacuoles with logarithmic promastigotes did. MDMs phagocytosed greater numbers of logarithmic than metacyclic promastigotes, yet metacyclics ultimately replicated intracellularly with greater efficiency. These data suggest that virulent metacyclic Leishmania promastigotes fail to ligate macrophage MR, and enter through a path that ultimately enhances intracellular survival. The relatively quiescent entry of virulent Leishmania spp. into macrophages may be accounted for by the ability of metacyclic promastigotes to selectively bypass deleterious entry pathways. 相似文献
996.
Jun Suzuki Katsumi Yamaguchi Masaki Kajikawa Kenji Ichiyanagi Noritaka Adachi Hideki Koyama Shunichi Takeda Norihiro Okada 《PLoS genetics》2009,5(4)
Long interspersed elements (LINEs) are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s) in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs—zebrafish ZfL2-2 and human L1—in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation. 相似文献
997.
Saneyoshi Ueno Yuriko Taguchi Nobuhiro Tomaru Yoshihiko Tsumura 《Conservation Genetics》2009,10(5):1477-1485
Fagus crenata Blume is widely distributed throughout Japanese cool-temperate deciduous broad-leaved forests, but there are two divergent
groups of populations in areas with contrasting winter climates separated by Japan’s Central Mountain Range. To facilitate
investigations of adaptive genetic differentiation of the species using potentially functional genes, we have collected Expressed
Sequence Tags and developed Simple Sequence Repeat markers using a cDNA library constructed from cambium and surrounding tissues.
In total, 270 primer pairs were designed, and 87 of the corresponding loci showed polymorphism in 16 individuals, with 2–21
alleles per locus and expected heterozygosities ranging from 0.06 to 0.97. EST-SSR markers developed in the present study
will be useful for genomic analyses of F. crenata populations. 相似文献
998.
Naoki Hida Muhammad Awais Masaki Takeuchi Naoto Ueno Mayuri Tashiro Chiyo Takagi Tanuja Singh Makoto Hayashi Yoshihiro Ohmiya Takeaki Ozawa 《PloS one》2009,4(6)
Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1–Smad4 and Smad2–Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects. 相似文献
999.
1000.
Shimamura T Shibata J Kurihara H Mita T Otsuki S Sagara T Hirai H Iwasawa Y 《Bioorganic & medicinal chemistry letters》2006,16(14):3751-3754
5-Pyrimidinyl-2-aminothiazole 1 was identified as an inhibitor of cyclin-dependent kinases (CDKs) by a screening of the Merck sample repository. The introduction of a methyl group at the C-5 or C-6 position on the pyrimidine ring, directed toward the gate keeper residue of CDK4 (Phe93), led to significant enhancement of selectivity for CDK4 over other CDKs. Compound 3 exhibited more than 300-fold selectivity for CDK4 over CDK1, 2, 5, 7, and 9. Subsequent improvements in aqueous solubility afforded compound 4, which is available for further in vivo studies and this compound inhibited pRb phosphorylation and BrdU incorporation in tumor models. 相似文献