Stéphane Marchal

Characterization of the Pressure-induced Intermediate and Unfolded State of Red-shifted Green Fluorescent Protein—A Static and Kinetic FTIR, UV/VIS and Fluorescence Spectroscopy Study

The green fluorescence proteins (GFP) are widely used as reporters in molecular and cell biology. For their use it in high-pressure microbiology and biotechnology studies, their structural properties, thermodynamic parameters and stability diagrams have to be known. We investigated the pressure stability of the red-shifted green fluorescent protein (rsGFP) using Fourier-transform infrared spectroscopy, fluorescence and UV/Vis spectroscopy. We found that rsGFP does not unfold up to approximately 9kbar at room temperature. Its unique three-dimensional structure is held responsible for the high-pressure stability. At higher temperatures, its secondary structure collapses below 9kbar (e.g. the denaturation pressure at 58 degrees C is 7.8kbar). The analysis of the IR data shows that the pressure-denatured state contains more disordered structures at the expense of a decrease of intramolecular beta-sheets. As indicated by the large volume change of DeltaV degrees (u) approximately -250(+/-50)mlmol(-1) at 58 degrees C, this highly cooperative transition can be interpreted as a collapse of the beta-can structure of rsGFP. For comparison, the temperature-induced unfolding of rsGFP has also been studied. At high temperature (T(m)=78 degrees C), the unfolding resulted in the formation of an aggregated state. Contrary to the pressure-induced unfolding, the temperature-induced unfolding and aggregation of GFP is irreversible. From the FT-IR data, a tentative p,T-stability diagram for the secondary structure collapse of GFP has been obtained. Furthermore, changes in fluorescence and absorptivity were found which are not correlated to the secondary structural changes. The fluorescence and UV/Vis data indicate smaller conformational changes in the chromophore region at much lower pressures ( approximately 4kbar) which are probably accompanied by the penetration of water into the beta-can structure. In order to investigate also the kinetics of this initial step, pressure-jump relaxation experiments were carried out. The partial activation volumes observed indicate that the conformational changes in the chromophore region when passing the transition state are indeed rather small, thus leading to a comparably small volume change of -20 ml mol(-1) only. The use of the chromophore absorption and fluorescence band of rsGFP in using GFP as reporter for gene expression and other microbiological studies under high pressure conditions is thus limited to pressures of about 4kbar, which still exceeds the pressure range relevant for studies in vivo in micro-organisms, including piezophilic bacteria from deep-sea environments.

Time-course and regional analyses of the physiopathological changes induced after cerebral injection of an amyloid β fragment in rats.

Alzheimers disease (AD) is a neurodegenerative pathology characterized by the presence of senile plaques and neurofibrillary tangles, accompanied by synaptic and neuronal loss. The major component of senile plaques is an amyloid β protein (Aβ) formed by pathological processing of the Aβ precursor protein. We assessed the time-course and regional effects of a single intracerebroventricular injection of aggregated Aβ fragment 25-35 (Aβ(25-35)) in rats. Using a combined biochemical, behavioral, and morphological approach, we analyzed the peptide effects after 1, 2, and 3 weeks in the hippocampus, cortex, amygdala, and hypothalamus. The scrambled Aβ(25-35) peptide was used as negative control. The aggregated forms of Aβ peptides were first characterized using electron microscopy, infrared spectroscopy, and Congo Red staining. Intracerebroventricular injection of Aβ(25-35) decreased body weight, induced short- and long-term memory impairments, increased endocrine stress, cerebral oxidative and cellular stress, neuroinflammation, and neuroprotective reactions, and modified endogenous amyloid processing, with specific time-course and regional responses. Moreover, Aβ(25-35), the presence of which was shown in the different brain structures and over 3 weeks, provoked a rapid glial activation, acetylcholine homeostasis perturbation, and hippocampal morphological alterations. In conclusion, the acute intracerebroventricular Aβ(25-35) injection induced substantial central modifications in rats, highly reminiscent of the human physiopathology, that could contribute to physiological and cognitive deficits observed in AD.

Reversible protein precipitation to ensure stability during encapsulation within PLGA microspheres.

Proteins were precipitated to ensure their stability upon subsequent encapsulation within PLGA microspheres. Spherical, nanosized protein particles were formed by the addition of a salt (sodium chloride) and a water-miscible organic solvent (glycofurol) to protein solutions. Various process parameters were modified to optimize the precipitation efficiency of four model proteins: lysozyme, alpha-chymotrypsin, peroxidase and beta-galactosidase. As monitored by enzymatic activity measurement of the rehydrated particles, conditions to obtain more than 95% of reversible precipitates were defined for each protein. The study of the structure of the rehydrated particles by absorbance spectroscopy, fluorescence spectroscopy and circular dichroism showed an absence of structural-perturbation after precipitation. Protein particles were then microencapsulated within PLGA microspheres using s/o/w technique. The average encapsulation yield was around 80% and no loss of protein activity occurred after the encapsulation step. Additionally, a lysozyme in vitro release study showed that all of the released lysozyme was biologically active. This method of protein precipitation is appropriate for the encapsulation in PLGA microspheres of various proteins without inactivation.

Alzheimer's Disease Related Markers, Cellular Toxicity and Behavioral Deficits Induced Six Weeks after Oligomeric Amyloid-β Peptide Injection in Rats

Alzheimer’s disease (AD) is a neurodegenerative pathology associated with aging characterized by the presence of senile plaques and neurofibrillary tangles that finally result in synaptic and neuronal loss. The major component of senile plaques is an amyloid-β protein (Aβ). Recently, we characterized the effects of a single intracerebroventricular (icv) injection of Aβ fragment (25–35) oligomers (oAβ25–35) for up to 3 weeks in rats and established a clear parallel with numerous relevant signs of AD. To clarify the long-term effects of oAβ25–35 and its potential role in the pathogenesis of AD, we determined its physiological, behavioral, biochemical and morphological impacts 6 weeks after injection in rats. oAβ25–35 was still present in the brain after 6 weeks. oAβ25–35 injection did not affect general activity and temperature rhythms after 6 weeks, but decreased body weight, induced short- and long-term memory impairments, increased corticosterone plasma levels, brain oxidative (lipid peroxidation), mitochondrial (caspase-9 levels) and reticulum stress (caspase-12 levels), astroglial and microglial activation. It provoked cholinergic neuron loss and decreased brain-derived neurotrophic factor levels. It induced cell loss in the hippocampic CA subdivisions and decreased hippocampic neurogenesis. Moreover, oAβ25–35 injection resulted in increased APP expression, Aβ1–42 generation, and increased Tau phosphorylation. In conclusion, this in vivo study evidenced that the soluble oligomeric forms of short fragments of Aβ, endogenously identified in AD patient brains, not only provoked long-lasting pathological alterations comparable to the human disease, but may also directly contribute to the progressive increase in amyloid load and Tau pathology, involved in the AD physiopathology.

Effect of various additives and polymers on lysozyme release from PLGA microspheres prepared by an s/o/w emulsion technique.

Incomplete protein release from PLGA-based microspheres due to protein interactions with the polymer is one of the main issues in the development of PLGA protein-loaded microspheres. In this study, a two-dimensional adsorption model was designed to rapidly assess the anti-adsorption effect of formulation components (additives, additives blended with the polymer or modified polymers). Lysozyme was chosen as a model protein because of its strong, non-specific adsorption on the PLGA surface. This study showed that PEGs, poloxamer 188 and BSA totally inhibited protein adsorption onto the PLGA37.5/25 layer. Similarly, it was emphasised that more hydrophilic polymers were less prone to protein adsorption. The correlation between this model and the in vitro release profile was made by microencapsulating lysozyme with a low loading in the presence of these excipients by a non-denaturing s/o/w encapsulation technique. The precipitation of lysozyme with the amphiphilic poloxamer 188 prior to encapsulation exhibited continuous release of active lysozyme over 3 weeks without any burst effect. To promote lysozyme release in the latter stage of release, a PLGA-PEG-PLGA tribloc copolymer was used; lysozyme was continuously released over 45 days in a biologically active form.

Structure-Function Perturbation and Dissociation of Tetrameric Urate Oxidase by High Hydrostatic Pressure

Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms.

Deciphering the structure, growth and assembly of amyloid-like fibrils using high-speed atomic force microscopy

Formation of fibrillar structures of proteins that deposit into aggregates has been suggested to play a key role in various neurodegenerative diseases. However mechanisms and dynamics of fibrillization remains to be elucidated. We have previously established that lithostathine, a protein overexpressed in the pre-clinical stages of Alzheimers disease and present in the pathognomonic lesions associated with this disease, form fibrillar aggregates after its N-terminal truncation. In this paper we visualized, using high-speed atomic force microscopy (HS-AFM), growth and assembly of lithostathine protofibrils under physiological conditions with a time resolution of one image/s. Real-time imaging highlighted a very high velocity of elongation. Formation of fibrils via protofibril lateral association and stacking was also monitored revealing a zipper-like mechanism of association. We also demonstrate that, like other amyloid ß peptides, two lithostathine protofibrils can associate to form helical fibrils. Another striking finding is the propensity of the end of a growing protofibril or fibril to associate with the edge of a second fibril, forming false branching point. Taken together this study provides new clues about fibrillization mechanism of amyloid proteins.

Lithostathine quadruple-helical filaments form proteinase K-resistant deposits in Creutzfeldt-Jakob disease.

Autocatalytic cleavage of lithostathine leads to the formation of quadruple-helical fibrils (QHF-litho) that are present in Alzheimers disease. Here we show that such fibrils also occur in Creutzfeldt-Jakob and Gerstmann-Sträussler-Scheinker diseases, where they form protease-K-resistant deposits and co-localize with amyloid plaques formed from prion protein. Lithostathine does not appear to change its native-like, globular structure during fibril formation. However, we obtained evidence that a cluster of six conserved tryptophans, positioned around a surface loop, could act as a mobile structural element that can be swapped between adjacent protein molecules, thereby enabling the formation of higher order fibril bundles. Despite their association with these clinical amyloid deposits, QHF-litho differ from typical amyloid fibrils in several ways, for example they produce a different infrared spectrum and cannot bind Congo Red, suggesting that they may not represent amyloid structures themselves. Instead, we suggest that lithostathine constitutes a novel component decorating disease-associated amyloid fibrils. Interestingly, [6,6′]bibenzothiazolyl-2,2′-diamine, an agent found previously to disrupt aggregates of huntingtin associated with Huntingtons disease, can dissociate lithostathine bundles into individual protofilaments. Disrupting QHF-litho fibrils could therefore represent a novel therapeutic strategy to combat clinical amyloidoses.

The fate of the prion protein in the prion/plasminogen complex

The cellular prion protein (PrP(c)) forms complexes with plasminogen. Here, we show that the PrP(c) in this complex is cleaved to yield fragments of PrP(c). The cleavage is accelerated by plasmin but does not appear to be dependent on it.

The powerful high pressure tool for protein conformational studies

The pressure behavior of proteins may be summarized as a the pressure-induced disordering of their structures. This thermodynamic parameter has effects on proteins that are similar but not identical to those induced by temperature, the other thermodynamic parameter. Of particular importance are the intermolecular interactions that follow partial protein unfolding and that give rise to the formation of fibrils. Because some proteins do not form fibrils under pressure, these observations can be related to the shape of the stability diagram. Weak interactions which are differently affected by hydrostatic pressure or temperature play a determinant role in protein stability. Pressure acts on the 2 degrees, 3 degrees and 4 degrees structures of proteins which are maintained by electrostatic and hydrophobic interactions and by hydrogen bonds. We present some typical examples of how pressure affects the tertiary structure of proteins (the case of prion proteins), induces unfolding (ataxin), is a convenient tool to study enzyme dissociation (enolase), and provides arguments to understand the role of the partial volume of an enzyme (butyrylcholinesterase). This approach may have important implications for the understanding of the basic mechanism of protein diseases and for the development of preventive and therapeutic measures.