Tom70 mediates Sendai virus-induced apoptosis on mitochondria.

UNLABELLED: Virus infection triggers immediate innate immune responses. Apoptosis represents another effective means to restrict virus invasion, besides robust expression of host cytokines and chemokines. IRF3 was recently demonstrated to be indispensable for Sendai virus (SeV)-induced apoptosis, but the underlying mechanism is not fully understood. Here we report that a dynamic protein complex, Tom70/Hsp90/IRF3/Bax, mediates SeV-induced apoptosis. The cytosolic proapoptotic protein Bax interacts specifically with IRF3 upon virus infection. The mitochondrial outer membrane protein Tom70 recruits IRF3 to mitochondria via Hsp90. Consequently, the relocation of Bax onto mitochondria induces the leakage of cytochrome c into the cytosol and initiates the corresponding apoptosis. Interestingly, IKK-i is essential for this apoptosis, whereas TBK1 is dispensable. Collectively, our study characterizes a novel protein complex that is important for SeV-induced apoptosis. IMPORTANCE: Apoptosis is an effective means of sacrificing virus-infected cells and restraining the spread of virus. In this study, we demonstrate that IRF3 associates with Bax upon virus infection. Tom70 recruits this protein complex to the mitochondrial outer membrane through Hsp90, which thus induces the release of cytochrome c into the cytosol, initiating virus-induced apoptosis. Interestingly, IKK-i plays an essential role in this activation. This study uncovers a novel mechanism of SeV-induced apoptosis.

Studies on the temperature effect on bacteriorhodopsin of purple and blue membrane by fluorescence and absorption spectroscopy.

Fluorescence and absorption spectra were used to study the temperature effect on the conformation of bacteriorhodopsin (bR) in the blue and purple membranes (termed as bRb and bRp respectively). The maximum emission wavelengths of tryptophan fluorescence in both proteins at room temperature are 340 nm, and the fluorescence quantum yield of bRb is about 1.4 fold higher than that of bRp. As temperature increases, the tryptophan fluorescence of bRb decreases, while the tryptophan fluorescence of bRp increases. The binding study of extrinsic fluorescent probe bis-ANS indicated that the probe can bind only to bRb, but not to bRp. These results suggest that significant structural difference existed between bRb and bRp. It was also found that both kinds of bR are highly thermal stable. The maximum wavelength of the protein fluorescence emission only shifted from 340 nm to 346 nm at 100 degrees C. More interestingly, as temperature increased, the characteristic absorption peak of bRb at 605 nm decreased and a new absorption peak at 380 nm formed. The transition occurred at a narrow temperature range (65 degrees C-70 degrees C). These facts indicated that an intermediate can be induced by high temperature. This phenomenon has not been reported before.

Six specific lysine residues are crucial in maintaining the structure and function of soluble manganese stabilizing protein.

When manganese stabilizing protein (MSP) was treated with 0.5 mM N-succinimidyl propionate (NSP), the rebinding ability and oxygen-releasing capabilities of the modified MSP were not altered, in spite of changes of MSP surface Lys residues. Furthermore, far-ultraviolet circular dichroism and intrinsic fluorescence spectra analysis revealed that 0.5 mM NSP-modified MSP retained most of its native secondary and tertiary structure. Mapping of the sites of NSP modification by Staphylococcus V(8) protease digestion of the modified protein, as well as analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry, indicated that seven Lys residues were modified. The results suggested that these residues are not absolutely essential to the structure and function of MSP. However, when the NSP concentration was increased to 4 mM, the modified MSP was unable to bind photosystem II and completely lost its reactivating capability. Both far-ultraviolet circular dichroism and intrinsic fluorescence spectra analysis revealed a clear conformational change in MSP after 4 mM NSP treatment, suggesting that some Lys residues are involved in maintaining the structure and function of MSP. Analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated that another six Lys residues, namely Lys20, Lys101, Lys196, Lys207, Lys130 (or Lys137) and Lys66 (or Lys76), were modified by 4 mM NSP. Therefore, these six Lys residues are crucial in maintaining the structure and function of soluble MSP.

Folding studies of two hydrostatic pressure sensitive proteins.

High hydrostatic pressure combined with various spectroscopies is a powerful technique to study protein folding. An ideal model system for protein folding studies should have the following characteristics. (1) The protein should be sensitive to pressure, so that the protein can be unfolded under mild pressure. (2) The folding process of the protein should be easily modulated by several chemical or physical factors. (3) The folding process should be easily monitored by some spectroscopic parameters. Here, we summarized the pressure induced folding studies of two proteins isolated from spinach photosystem II, namely the 23-kDa and the 33-kDa protein. They have all the characteristics mention above and might be an ideal model protein system for pressure studies.

Positive charges on lysine residues of the extrinsic 18 kDa protein are important to its electrostatic interaction with spinach photosystem II membranes.

To determine the contribution of charged amino acids to binding with the photosystem II complex (PSII), the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N-succinimidyl propionate (NSP) or glycine methyl ester (GME) in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.

An oligonucleotide microarray for microRNA expression analysis based on labeling RNA with quantum dot and nanogold probe.

MicroRNAs (miRNAs) play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. They have diverse expression patterns and might regulate various developmental and physiological processes. Profiling miRNA expression is very helpful for studying biological functions of miRNAs. We report a novel miRNA profiling microarray, in which miRNAs were directly labeled at the 3' terminus with biotin and hybridized with complementary oligo-DNA probes immobilized on glass slides, and subsequently detected by measuring fluorescence of quantum dots labeled with streptavidin bound to miRNAs through streptavidin-biotin interaction. The detection limit of this microarray for miRNA was approximately 0.4 fmol, and the detection dynamic range spanned about 2 orders of magnitude. We made a model microarray to profile 11 miRNAs from leaf and root of rice (Oryza sativa L. ssp. indica) seedlings. The analysis results of the miRNAs had a good reproducibility and were consistent with the northern blot result. To avoid using high-cost detection equipment, colorimetric detection, a method based on nanogold probe coupled with silver enhancement, was also successfully introduced into miRNA profiling microarray detection.

Pressure equilibrium and jump study on unfolding of 23-kDa protein from spinach photosystem II.

Pressure-induced unfolding of 23-kDa protein from spinach photosystem II has been systematically investigated at various experimental conditions. Thermodynamic equilibrium studies indicate that the protein is very sensitive to pressure. At 20 degrees C and pH 5.5, 23-kDa protein shows a reversible two-state unfolding transition under pressure with a midpoint near 160 MPa, which is much lower than most natural proteins studied to date. The free energy (DeltaG(u)) and volume change (DeltaV(u)) for the unfolding are 5.9 kcal/mol and -160 ml/mol, respectively. It was found that NaCl and sucrose significantly stabilize the protein from unfolding and the stabilization is associated not only with an increase in DeltaG(u) but also with a decrease in DeltaV(u). The pressure-jump studies of 23-kDa protein reveal a negative activation volume for unfolding (-66.2 ml/mol) and a positive activation volume for refolding (84.1 ml/mol), indicating that, in terms of system volume, the protein transition state lies between the folded and unfolded states. Examination of the temperature effect on the unfolding kinetics indicates that the thermal expansibility of the transition state and the unfolded state of 23-kDa protein are closer to each other and they are larger than that of the native state. The diverse pressure-refolding pathways of 23-kDa protein in some conditions were revealed in pressure-jump kinetics.

Detection of tumor marker CA125 in ovarian carcinoma using quantum dots.

Semiconductor quantum dots (QDs) offer several advantages over organic dyes in fluorescence-imaging applications, such as higher quantum yield, exceptional photostability, and a narrow, tunable, and symmetric emission spectrum. To explore whether QDs could specifically and effectively label tumor markers and be used in immunohistochemistry as a novel type of fluorescent probe, we used quantum dots with maximum emission wavelength 605 nm (QD605) to detect the ovarian carcinoma marker CA125 in specimens of different types (fixed cells, tissue sections, and xenograft piece). Additionally, we compared the photostability of QD signals with that of a conventional organic dye, FITC. All labeling signals of QDs were found to be more specific and brighter than those of FITC. Moreover, the QDs exhibited exceptional photostability during continuous illumination for 1 h by a high-intensity laser (Ar laser power 100 mW) at 488 nm, while the FITC signals faded very quickly and became undetectable after 24 min of illumination. These results indicate that QD-based probes can offer substantial advantages over existing fluorophores in many applications, and can be used effectively in immunohistochemistry as a novel class of fluorescent probes.

Colorimetric detection of protein microarrays based on nanogold probe coupled with silver enhancement.

This work presents a method for analyzing protein microarrays using a colorimetric nanogold probe coupled with silver enhancement (gold-silver detection). In this method, the gold nanoparticles were introduced to the microarray by the specific binding of the gold-conjugated antibodies or streptavidins and then coupled with silver enhancement to produce black image of microarray spots, which can be easily detected with a commercial CCD camera. The method showed high detection sensitivity (1 pg of IgG immobilized on slides or 2.75 ng/ml IgG in solution) and a good linear correlation between the signal intensity and the logarithm of the sample concentration. The examination of this method in analyzing a demonstrational ToRCH antigen microarray developed in our lab showed an identical result as in the fluorescent method. These results suggest the colorimetric gold-silver detection method has potential applications in proteomics research and clinical diagnosis.

[Thermodynamic and kinetic analysis of unfolding of P23k protein isolated from spinach photosystem II].

The unfolding of 23kD (P23k) protein isolated from spinach photosystem II particle was studied by high pressure and fluorescence spectroscopy. The thermal equilibrium study indicated that the protein could be totally unfolded by 180 or 160 MPa at 20 degrees C and 3 degrees C, respectively. The standard free energy and standard volume change of the protein for unfolding at 20 degrees C is 23.45 kJ/mol and -150.3 ml/mol, respectively. Kinetics study indicated that at 20 degrees C the activation volume for unfolding, delta V(u)(++), was negative (-66.2 ml/mol), meanwhile the activation volume for folding, deltaV(f)(++), was positive (84.1 ml/mol). The rate constants for folding and unfolding (K(0f), K(0u)) were 1.87 s(-1) and 1.3x10(-4) s(-1), respectively, these results provide some clues to explain why the protein is so sensitive to pressure.

The assignment of the reactive sites of the double-headed arrowhead proteinase inhibitor A and B.

The arrowhead proteinase inhibitor A and B (APIA and APIB) are double-headed and multifunctional. Both their primary structure and cDNA sequence have been elucidated. To locate the possible reactive site residues Lys(44), Arg(76) and Arg(87) of APIB predicted according to the sequence comparison with other proteinase inhibitors, the above residues were substituted with Pro by site-directed mutagenesis respectively, and the mutated genes were expressed in the yeast secretion system. The mutant K(44)P-APIB displayed the same inhibitory activity as APIB does, while the mutants R(76)P-APIB and R(87)P-APIB could only inhibit one molecule, instead of two molecules of trypsin, indicating that Arg(76) and Arg87 but not Lys(44) are the two reactive sites of APIB. In order to further confirm this result, more mutants of APIB (K(44)P-R(76)P-APIB, K(44)P-R(87)P-APIB, R(76)P-R(87)P-APIB) were designed, in each mutant only one of the three possible reactive sites remained unchanged. Both the mutants K(44)P-R(76)P-APIB and K(44)P-R(87)P-APIB could only inhibit one molecule of trypsin, while R(76)P-R(87)P-APIB could no longer inhibit trypsin intensively, thus the residues Arg(76) and Arg(87) are definitely the reactive sites of APIB. Their K(i) were measured to be 0.39 nmol/L and 0.47 nmol/L, respectively. The mutant R(87)L-APIB lost about half activity towards trypsin but could inhibit one molecule of chymotrypsin as the wide type APIA did, indicating that Leu(87) was the reactive site towards trypsin but could inhibit one molecule of APIA for inhibiting chymotrypsin.

Conformation nearby Trp residues of APIA and APIB modulates the inhibitory specificity of the protease.

The relationship between the micro-environment of the two tryptophan residues and the inhibitory specificity of arrowhead protease inhibitors A and B (APIA and APIB) was studied by mutagenesis and fluorescence spectroscopy. The environment of the two Trp residues at positions 93 and 122 in APIB is more hydrophobic than in APIA. Study after substitution of Trp with Ala revealed that the environment of Trp(122) is more hydrophobic than that of Trp(93). Substitution of Leu(82) and Arg(87) in APIB with Ser and Leu respectively made the tryptophan fluorescence of APIB to be like that of APIA and the inhibitory specificity to be closer to APIA, indicating that the inhibitory specificity of the enzyme may be modulated by the conformation around the tryptophan residues.

[Application of quantum dot to life science].

As an eminent representation of nanotechnology, quantum dot (semiconductor nanocrystal) has caught great interests of scientists in physics, chemistry, material science, and biology extensively. Although its application to life science has just been explored, valuable progresses have been achieved in both biomacromolecule labeling and coding recently. The progress in quantum dot synthesis, its spectroscopic and photoelectronic properties, and its potential application to life science are reviewed.

Barotolerant E.coli Induced by High Hydrostatic Pressure.

The barotolerant E.coli TG1P, DH5alphaP, HB101P were obtained from TG1,DH5alpha, HB101 after treatment with high hydrostatic pressure, and the survival rates of the formers raised 2--3 magnitude, from 1.06x10(-6), 2.98x10(-4), 3.65x10(-3) to 8.31x10(-3), 3.40x10(-1), 1.69x10(-1),respectively. Comparison of partial proteomes of wild and barotolerant bacteria of TG1 and DH5alpha with two-dimensional electrophoresis showed that three proteins were obviously highly expressed in barotolerant bacteria. The N terminal sequence and molecular weight of one protein was shown to be V(L)EAGEFFMRA and 21 kD, with high identity to a known outmembrane protein in E.coli.

A Vaccine to Coxsackievirus Prepared by High Pressure.

Effects of high hydrostatic pressure on infectious and immune activity of coxsackievirus group B (CVB) was described. CVB was completely inactivated at 230 MPa pressure and -16 degrees. The inactivated viruses remained highly immunogenic and induced CVB-specific serum antibody at a concentration as low as 1:1 500. 67% mice vaccinated with the pressurized viruses survived the challenge of fatal dose of CVB. These results indicated that the pressurized CVB induced immune reactions to protect mice from the viruses, and would be a potential vaccine candidate for human myocarditis.

Comparison of the Catalytic Domains of Collagenase-1 and Stromelysin-1.

The catalytic domains of two matrix metalloproteinases--collagenase-1 and stromelysin-1 have been studied by means of fluorescence spectroscopy and high hydrostatic pressure. The hydrophobic fluorescence probe ANS could bind to stromelysin-1, with a dissociation constant of 26.3 &mgr;mol/L, but could not bind to collagenase-1, indicating that there exists a hydrophobic site on the surface of stromelysin-1. Further study suggests that the hydrophobic site may not be the catalytic site. The biological activity of catalytic domains of collagenase-1 and stromelysin-1 showed obvious difference under high pressure the activity of collagenase-1 increased with elevating pressure, with an activation volume of D18.9 ml/mol however, the activity of stromelysin-1 did not change under high pressure. The results indicate that there are some obvious differences between the catalytic domain conformations of these two enzymes, though the crystal analysis indicated that they were very similar as reported before.

High Immunogenicity of the Pressure-inactivated Virus.

Effects of high hydrostatic pressure on the activity of infectious bursal disease virus (IBDV) was described. The infectivity of IBDV decreased with the increasing of pressure and pressurizing time and may be completely lost, indicated by the tests with chicken embryo fibrablast cell and young chicken as models. How-ever, pressure-inactivated IBDV still remained high immunogenicity and might induce high levels of protecting serum antibody. Fluorescence spectroscopy showed obvious differences between the structures of native and pressurized IBDV, similar with the dissociation-reassociation of oligomeric proteins under high pressure. The changes of pressurized IBDV were also directly observed with electron microscopy. These results suggest that pressure-inactivated IBDV may be used as a vaccine.

An Emerging Field in Bioscience High Hydrostatic Pressure Study.

The goal of this mini review is to explain how hydrostatic pressure acts on bio-macromolecules(mainly on protein), giving some basic knowledge. Moreover, it will be shown that high pressure is a powerful tool both for the study of bio-macromolecules such as protein conformation, protein-protein (or protein-nucleic acid, or protein-ligands) interactions and the modulation of enzymatic activity etc. and for study of biotechnological application.

Effect of High Hydrostatic Pressure on Activity of Restriction Endonucleases.

The effect of high hydrostatic pressure on the activities of type II restriction enzymes HindIII and XbaI in digesting plasmid pSPORT1 was studied. The endonuclease activity of HindIII and XbaI at 37 degrees were gradually inhibited by increasing pressure and completely inhibited at 200 and 180 MPa, respectively. No obvious irreversible effect was observed for HindIII after suffering high pressure, while a considerable irreversible inactivation was observed for XbaI. The standard molar volume changes for HindIII and XbaI estimated from the inhibition of endonuclease activity at different pressures were 213 and 103 ml/mol, respectively. It was also concluded that pressurization did not change the substrate sequence specificity of both HindIII and XbaI.

Effect of Calcium Ion on Conformation of Horseradish Peroxidase Isoenzyme C.

Horseradish peroxidase isoenzyme C without ligand [apo-HRP(C)] was studied by fluorescence spectroscopy in this paper. It was observed: 1. there was a distinct Tyr fluorescence in the intrinsic fluorescence of apo-HRP(C), which was seldom observed in other "B" proteins; 2. low concentration denaturants (<2 mol/L urea or 0.2 mol/L GuHCl) could increase the fluorescence intensity of the Trp residue; however they did not change the spectrum characteristics; 3. further increase in the denaturant concentration exposed the Trp residue in HRP(C) to solvent, resulting in a little decrease in fluorescence intensity and a red shift in the spectrum; 4. the similar phenomena were also observed when EDTA, a chelator of calcium, was added to apo-HRP(C). It was also observed that, when the fluorescence of apo-HRP(C) had been affected by either EDTA or low concentration denaturant, it was not more affected by the other one, suggesting that the EDTA and low concentration denaturants might affect Trp fluorescent probe ANS to apo-HRP(C) indicated that the dissociation constants for ANS binding were 15.4 &mgr;mol/L and 7.3 &mgr;mol/L, in the absence and presence of calcium ion, respectively, implying that calcium ion could enhance the binding ability of ANS to the active site of apo-HRP(C), and this suggested that the effect of calcium ion in the enzyme activity might be related to its influence on the conformation of heme binding site in HRP(C).