Activated microglia mediate synapse loss and short-term memory deficits in a mouse model of transthyretin-related oculoleptomeningeal amyloidosis.

Oculoleptomeningeal amyloidosis (OA) is a fatal and untreatable hereditary disease characterized by the accumulation of transthyretin (TTR) amyloid within the central nervous system. The mechanisms underlying the pathogenesis of OA, and in particular how amyloid triggers neuronal damage, are still unknown. Here, we show that amyloid fibrils formed by a mutant form of TTR, A25T, activate microglia, leading to the secretion of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and nitric oxide. Further, we found that A25T amyloid fibrils induce the activation of Akt, culminating in the translocation of NFκB to the nucleus of microglia. While A25T fibrils were not directly toxic to neurons, the exposure of neuronal cultures to media conditioned by fibril-activated microglia caused synapse loss that culminated in extensive neuronal death via apoptosis. Finally, intracerebroventricular (i.c.v.) injection of A25T fibrils caused microgliosis, increased brain TNF-α and IL-6 levels and cognitive deficits in mice, which could be prevented by minocycline treatment. These results indicate that A25T fibrils act as pro-inflammatory agents in OA, activating microglia and causing neuronal damage.

Highlights of the 3rd International Conference on High Pressure Bioscience and Biotechnology.

The 3rd International Conference on High Pressure Bioscience and Biotechnology was held in the city of Rio de Janeiro from September 27 to September 30, 2004. The meeting, promoted by the International Association of High Pressure Bioscience and Biotechnology (IAHPBB), congregated top scientists and researchers from all over the world. In common, they shared the use of hydrostatic pressure for research, technical development, or industrial applications. The meeting consisted of invited lectures, contributed papers and a well-attended poster session. Very exciting discussions were held inside and outside the sessions, and the goals of discussing state-of-the-art data and establishing working collaborations and co-operations were fully attained.

Highlights of the 3rd International Conference on High Pressure Bioscience and Biotechnology

The 3rd International Conference on High Pressure Bioscience and Biotechnology was held in the city of Rio de Janeiro from September 27 to September 30, 2004. The meeting, promoted by the International Association of High Pressure Bioscience and Biotechnology (IAHPBB), congregated top scientists and researchers from all over the world. In common, they shared the use of hydrostatic pressure for research, technical development, or industrial applications. The meeting consisted of invited lectures, contributed papers and a well-attended poster session. Very exciting discussions were held inside and outside the sessions, and the goals of discussing state-of-the-art data and establishing working collaborations and co-operations were fully attained.

DNA converts cellular prion protein into the beta-sheet conformation and inhibits prion peptide aggregation.

The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which in most cases undergoes aggregation. In an organism infected with PrP(Sc), PrP(C) is converted into the beta-sheet form, generating more PrP(Sc). We find that sequence-specific DNA binding to recombinant murine prion protein (mPrP-(23-231)) converts it from an alpha-helical conformation (cellular isoform) into a soluble, beta-sheet isoform similar to that found in the fibrillar state. The recombinant murine prion protein and prion domains bind with high affinity to DNA sequences. Several double-stranded DNA sequences in molar excess above 2:1 (pH 4.0) or 0.5:1 (pH 5.0) completely inhibit aggregation of prion peptides, as measured by light scattering, fluorescence, and circular dichroism spectroscopy. However, at a high concentration, fibers (or peptide aggregates) can rescue the peptide bound to the DNA, converting it to the aggregating form. Our results indicate that a macromolecular complex of prion-DNA may act as an intermediate for the formation of the growing fiber. We propose that host nucleic acid may modulate the delicate balance between the cellular and the misfolded conformations by reducing the protein mobility and by making the protein-protein interactions more likely. In our model, the infectious material would act as a seed to rescue the protein bound to nucleic acid. Accordingly, DNA would act on the one hand as a guardian of the Sc conformation, preventing its propagation, but on the other hand may catalyze Sc conversion and aggregation if a threshold level is exceeded.

Pressure provides new insights into protein folding, dynamics and structure.

Hydrostatic pressure is a powerful tool for studying protein folding, and the dynamics and structure of folding intermediates. Recently, pressure techniques have opened two important fronts to aid our understanding of how polypeptides fold into highly structured conformations. The first advance is the stabilization of folding intermediates, making it possible to characterize their structures and dynamics by different methodologies. Kinetic studies under pressure constitute the second advance, promising detailed appraisal and understanding of protein folding landscapes. The combination of these two approaches enables dissection of the roles of packing and cavities in folding, and in assembly of multimolecular structures such as protein-DNA complexes and viruses. The study of aggregates and amyloids, derived from partially folded intermediates at the junction between productive and off-pathway folding, have also been studied, promising better understanding of diseases associated with protein misfolding.

The metastable state of nucleocapsids of enveloped viruses as probed by high hydrostatic pressure.

Enveloped viruses fuse their membranes with cellular membranes to transfer their genomes into cells at the beginning of infection. What is not clear, however, is the role of the envelope (lipid bilayer and glycoproteins) in the stability of the viral particle. To address this question, we compared the stability between enveloped and nucleocapsid particles of the alphavirus Mayaro using hydrostatic pressure and urea. The effects were monitored by intrinsic fluorescence, light scattering, and binding of fluorescent dyes, including bis(8-anilinonaphthalene-1-sulfonate) and ethidium bromide. Pressure caused a drastic dissociation of the nucleocapsids as determined by tryptophan fluorescence, light scattering, and gel filtration chromatography. Pressure-induced dissociation of the nucleocapsids was poorly reversible. In contrast, when the envelope was present, pressure effects were much less marked and were highly reversible. Binding of ethidium bromide occurred when nucleocapsids were dissociated under pressure, indicating exposure of the nucleic acid, whereas enveloped particles underwent no changes. Overall, our results demonstrate that removal of the envelope with the glycoproteins leads the particle to a metastable state and, during infection, may serve as the trigger for disassembly and delivery of the genome. The envelope acts as a "Trojan horse," gaining entry into the host cell to allow release of a metastable nucleocapsid prone to disassembly.

DNA tightens the dimeric DNA-binding domain of human papillomavirus E2 protein without changes in volume.

The recognition of palindromic specific DNA sequences by the human papillomavirus (HPV) E2 proteins is responsible for regulation of virus transcription. The dimeric E2 DNA-binding domain of HPV-16 (E2c) dissociates into a partially folded state under high hydrostatic pressure. We show here that pressure-induced monomers of E2c are highly structured, as evidenced by NMR hydrogen-deuterium exchange measurements. On binding to both specific and nonspecific DNA, E2c becomes stable against pressure. Competitive binding studies using fluorescence polarization of fluorescein-labeled DNA demonstrate the reversibility of the specific binding. To assess the thermodynamic parameters for the linkage between protein dissociation and DNA binding, urea denaturation curves were obtained at different pressures in the presence of specific and nonspecific DNA sequences. The change in free energy on denaturation fell linearly with increase in pressure for both protein-DNA complexes, and the measured volume change was similar to that obtained for E2c alone. The data show that the free energy of dissociation increases when E2c binds to a nonspecific DNA sequence but increases even more when the protein binds to the specific DNA sequence. Thus, specific complexes are tighter but do not entail variation in the volume change. The thermodynamic data indicate that DNA-bound E2c dissociates into monomers bound to DNA. The existence of monomeric units of E2c bound to DNA may have implications for the formation of DNA loops, as an additional target for viral and host factors binding to the loosely associated dimer of the N-terminal module of the E2 protein.

The preaggregated state of an amyloidogenic protein: hydrostatic pressure converts native transthyretin into the amyloidogenic state.

Protein misfolding and aggregation cause several diseases, by mechanisms that are poorly understood. The formation of amyloid aggregates is the hallmark of most of these diseases. Here, the properties and formation of amyloidogenic intermediates of transthyretin (TTR) were investigated by the use of hydrostatic pressure and spectroscopic techniques. Native TTR tetramers (T(4)) were denatured by high pressure into a conformation that exposes tryptophan residues to the aqueous environment. This conformation was able to bind the hydrophobic probe bis-(8-anilinonaphthalene-1-sulfonate), indicating persistence of elements of secondary and tertiary structure. Lowering the temperature facilitated the pressure-induced denaturation of TTR, which suggests an important role of entropy in stabilizing the native protein. Gel filtration chromatography showed that after a cycle of compression-decompression at 1 degrees C, the main species present was a tetramer, with a small population of monomers. This tetramer, designated T(4)*, had a non-native conformation: it bound more bis-(8-anilinonaphthalene-1-sulfonate) than native T(4), was less stable under pressure, and on decompression formed aggregates under mild acidic conditions (pH 5-5.6). Our data show that hydrostatic pressure converts native tetramers of TTR into an altered state that shares properties with a previously described amyloidogenic intermediate, and it may be an intermediate that lies on the aggregation pathway. This "preaggregated" state, which we call T(4)*, provides insight into the question of how a correctly folded protein may degenerate into the aggregation pathway in amyloidogenic diseases.

LexA repressor forms stable dimers in solution. The role of specific dna in tightening protein-protein interactions.

Cooperativity in the interactions among proteins subunits and DNA is crucial for DNA recognition. LexA repressor was originally thought to bind DNA as a monomer, with cooperativity leading to tighter binding of the second monomer. The main support for this model was a high value of the dissociation constant for the LexA dimer (micromolar range). Here we show that the protein is a dimer at nanomolar concentrations under different conditions. The reversible dissociation of LexA dimer was investigated by the effects of hydrostatic pressure or urea, using fluorescence emission and polarization to monitor the dissociation process. The dissociation constant lies in the picomolar range (lower than 20 pM). LexA monomers associate with an unusual large volume change (340 ml/mol), indicating the burial of a large surface area upon dimerization. Whereas nonspecific DNA has no stabilizing effect, specific DNA induces tightening of the dimer and a 750-fold decrease in the K(d). In contrast to the previous model, a tight dimer rather than a monomer is the functional repressor. Accordingly, the LexA dimer only loses its ability to recognize a specific DNA sequence by RecA-induced autoproteolysis. Our work provides insights into the linkage between protein-protein interactions, DNA recognition, and DNA repair.

Hydrostatic pressure rescues native protein from aggregates.

Misfolding and misassembly of proteins are major problems in the biotechnology industry, in biochemical research, and in human disease. Here we describe a novel approach for reversing aggregation and increasing refolding by application of hydrostatic pressure. Using P22 tailspike protein as a model system, intermediates along the aggregation pathway were identified and quantitated by size-exclusion high-performance liquid chromatography (HPLC). Tailspike aggregates were subjected to hydrostatic pressures of 2.4 kbar (35,000 psi). This treatment dissociated the tailspike aggregates and resulted in increased formation of native trimers once pressure was released. Tailspike trimers refolded at these pressures were fully active for formation of infectious viral particles. This technique can facilitate conversion of aggregates to native proteins without addition of chaotropic agents, changes in buffer, or large-scale dilution of reagents required for traditional refolding methods. Our results also indicate that one or more intermediates at the junction between the folding and aggregation pathways is pressure sensitive. This finding supports the hypothesis that specific determinants of recognition exist for protein aggregation, and that these determinants are similar to those involved in folding to the native state. An increased understanding of this specificity should lead to improved refolding methods.

Cavity defects in the procapsid of bacteriophage P22 and the mechanism of capsid maturation.

Bacteriophage P22 belongs to a family of double-stranded DNA viruses that share common morphogenetic features like DNA packaging into a procapsid precursor and maturation. Maturation involves cooperative expansion of the procapsid shell with concomitant lattice stabilization. The expansion is thought to be mediated by movement of two coat protein domains around a hinge. The metastable conformation of subunit within the procapsid lattice is considered to constitute a late folding intermediate. In order to understand the mechanism of expansion it is necessary to characterize the interactions stabilizing procapsid and mature capsid lattices, respectively. We employ pressure dissociation to compare subunit packing within the procapsid and expanded lattice. Procapsid shells contain larger cavities than the expanded shells, presumably due to polypeptide packing defects. These defects contribute to the metastable nature of the procapsid lattice and are cured during expansion. Improved packing contributes to the increased stability of the expanded shell. Comparison of two temperature-sensitive folding (tsf) mutants of coat protein (T294I and W48Q) with wild-type coat revealed that both mutations markedly destabilized the procapsid shell and yet had little effect on relative stability of the monomeric subunit. Thus, the regions affected by these packing defects constitute subunit interfaces of the procapsid shell. The larger activation volume of pressure dissociation observed for both T294I and W48Q indicates that the decreased stability of these particles is due to increase of cavity defects. These defects in the procapsid lattice are cured upon expansion suggesting that the intersubunit contacts affected by tsf mutations are absent or rearranged in the mature shell. The energetics of the in vitro expansion reaction also suggests that entropic stabilization contributes to the large free energy barrier for expansion.

Characterization of a partially folded monomer of the DNA-binding domain of human papillomavirus E2 protein obtained at high pressure.

The pressure-induced dissociation of the dimeric DNA binding domain of the E2 protein of human papillomavirus (E2-DBD) is a reversible process with a Kd of 5.6 x 10(-8) M at pH 5.5. The complete exposure of the intersubunit tryptophans to water, together with the concentration dependence of the pressure effect, is indicative of dissociation. Dissociation is accompanied by a decrease in volume of 76 ml/mol, which corresponds to an estimated increase in solvent-exposed area of 2775 A2. There is a decrease in fluorescence polarization of tryptophan overlapping the red shift of fluorescence emission, supporting the idea that dissociation of E2-DBD occurs in parallel with major changes in the tertiary structure. The dimer binds bis(8-anilinonaphthalene-1-sulfonate), and pressure reduces the binding by about 30%, in contrast with the almost complete loss of dye binding in the urea-unfolded state. These results strongly suggest the persistence of substantial residual structure in the high pressure state. Further unfolding of the high pressure state was produced by low concentrations of urea, as evidenced by the complete loss of bis(8-anilinonaphthalene-1-sulfonate) binding with less than 1 M urea. Following pressure dissociation, a partially folded state is also apparent from the distribution of excited state lifetimes of tryptophan. The combined data show that the tryptophans of the protein in the pressure-dissociated state are exposed long enough to undergo solvent relaxation, but the persistence of structure is evident from the observed internal quenching, which is absent in the completely unfolded state. The average rotational relaxation time (derived from polarization and lifetime data) of the pressure-induced monomer is shorter than the urea-denatured state, suggesting that the species obtained under pressure are more compact than that unfolded by urea.

Mimicry of the calcium-induced conformational state of troponin C by low temperature under pressure.

Calcium binding to the N-domain of troponin C initiates a series of conformational changes that lead to muscle contraction. Calcium binding provides the free energy for a hydrophobic region in the core of N-domain to assume a more open configuration. Fluorescence measurements on a tryptophan mutant (F29W) show that a similar conformational change occurs in the absence of Ca2+ when the temperature is lowered under pressure. The conformation induced by subzero temperatures binds the hydrophobic probe bis-aminonaphthalene sulfonate, and the tryptophan has the same fluorescence lifetime (7 ns) as in the Ca2+-bound form. The decrease in volume (delta V = -25.4 ml/mol) corresponds to an increase in surface area. Thermodynamic measurements suggest an enthalpy-driven conformational change that leads to an intermediate with an exposed N-domain core and a high affinity for Ca2+.

The use of hydrostatic pressure as a tool to study viruses and other macromolecular assemblages.

Recent studies on the effect of pressure on macromolecular assemblages have provided new information on protein-protein and protein-nucleic acid interactions. New findings have recently emerged on the use of hydrostatic pressure to assess intermediate states in the assembly pathways of viruses, multimeric proteins and protein-nucleic acid complexes, addressing many questions of macromolecular recognition.

Role of entropic interactions in viral capsids: single amino acid substitutions in P22 bacteriophage coat protein resulting in loss of capsid stability.

Bacteriophage P22 is a double-stranded DNA containing phage. Its morphogenetic pathway requires the formation of a precursor procapsid that subsequently matures to the capsid. The stability of bacteriophage P22 coat protein in both monomeric and polymeric forms under hydrostatic pressure has been examined previously [Prevelige, P. E., King, J., & Silva, J. L. (1994) Biophys. J. 66, 1631-1641]. The monomeric protein is very unstable to pressure and undergoes denaturation at pressures below 1.5 kbar, whereas the procapsid shell is very stable to applied pressure and does not dissociate with pressure to 2.5 kbar. However, under applied pressure the procapsid shells are cold labile, suggesting they are entropically stabilized. We have analyzed the pressure stability of mutant procapsid shells having either of two single amino acid substitutions in the coat protein (G232D and W48Q) using light-scattering and fluorescence emission methods. While the wild-type shells were stable under 2.2 kbar of pressure at room temperature (22 degrees C), the G232D mutant shells showed time-dependent dissociation under these conditions. Decreasing the temperature to 1 degree C dramatically accelerated the dissociation of G232D mutant under applied pressure. On the other hand, the W48Q mutant shells could be dissociated easily by pressure at room temperature and displayed little dependence on temperature, suggesting a smaller entropic contribution to the stability of this mutant. The unpolymerized mutant subunits displayed a pressure stability similar to that of the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Cold denaturation of a repressor-operator complex: the role of entropy in protein-DNA recognition.

The mechanisms by which regulatory proteins recognize specific DNA sequences are not fully understood. Here we examine the basis for the stability of a protein-DNA complex, using hydrostatic pressure and low temperature. Pressure converts the DNA-binding Arc repressor protein from a native state to a denatured, molten-globule state. Our data show that the folding and dimerization of Arc repressor in the temperature range 0-20 degrees C are favored by a large positive entropy value, so that the reaction proceeds in spite of an unfavorable positive enthalpy. On binding operator DNA, Arc repressor becomes extremely stable against denaturation. However, the Arc repressor-operator DNA complex is cold-denatured at subzero temperatures under pressure, demonstrating that the favorable entropy increases greatly when Arc repressor binds tightly to its operator sequence but not a nonspecific sequence. We show how an increase in entropy may operate to provide the protein with a mechanism to distinguish between a specific and a nonspecific DNA sequence. It is postulated that the formation of the Arc-operator DNA complex is followed by an increase in apolar interactions and release of solvent which would explain its entropy-driven character, whereas this solvent would not be displaced in nonspecific complexes.

Effects of the Alkaloid Gramine on the Light-Harvesting, Energy Transfer, and Growth of Anabaena sp. (PCC 7119).

Long-term and short-term effects of gramine on cells of Anabaena sp. were studied. Culture death was observed after an initial growth in the presence of 0.5 mM gramine, and lower concentrations decreased both the specific growth rate and the growth yield. Cultures showed a reduction in the chlorophyll content as well as an increase in the level of accessory pigments, which were proportional to the alkaloid concentration. When cultures were excited with green light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the fluorescence spectra of the cells showed a shoulder at 685 nm related to the photosystem II (PSII) antennae emission. This band was reduced when gramine was present during the growth, suggesting that gramine suppresses the energy transfer between the phycobilisomes and PSII. At lethal concentrations for cellular growth, gramine suppressed immediately the photosynthetic oxygen production as well as the electron transport from H2O to p-benzoquinone. The influence of gramine on the PSII photochemical reactions was investigated by flash-induced fluorescence measurements, and the results suggest that the alkaloid could act as an electron donor to the PSII reaction center.