PubMed Health⌕ Search

Biomedical subjects

Georgia Plakoutsi

Publications and source records attributed to Georgia Plakoutsi.

10 recordsLinked to original sources
PubMed2006

Stabilization of a native protein mediated by ligand binding inhibits amyloid formation independently of the aggregation pathway.

The acylphosphatases from Sulfolobus solfataricus and Drosophila melanogaster (Sso AcP and AcPDro2) were previously shown to form amyloid-like aggregates without the need to unfold initially. Inorganic phosphate (Pi), a competitive inhibitor binding specifically to the active site of these proteins, was found to stabilize, upon binding, the native state of AcPDro2 and to inhibit its conversion into amyloid-like fibrils. The inhibitory effect of Pi is suppressed only in a variant in which the Arg residue responsible for Pi binding is mutated. The study on Sso AcP shows that Pi retards both the formation of the initial nativelike oligomers and their subsequent conversion into protofibrils. Thus, stabilization of the native structure mediated by specific binding with small molecules can be an effective therapeutic strategy against protein deposition diseases that originate from initially folded proteins, independently of the structure of the protein, its aggregation pathway, and the particular aggregated species responsible for pathogenesis.

Acid Anhydride Hydrolases↗
PubMed2006

Structure, conformational stability, and enzymatic properties of acylphosphatase from the hyperthermophile Sulfolobus solfataricus.

The structure of AcP from the hyperthermophilic archaeon Sulfolobus solfataricus has been determined by (1)H-NMR spectroscopy and X-ray crystallography. Solution and crystal structures (1.27 A resolution, R-factor 13.7%) were obtained on the full-length protein and on an N-truncated form lacking the first 12 residues, respectively. The overall Sso AcP fold, starting at residue 13, displays the same betaalphabetabetaalphabeta topology previously described for other members of the AcP family from mesophilic sources. The unstructured N-terminal tail may be crucial for the unusual aggregation mechanism of Sso AcP previously reported. Sso AcP catalytic activity is reduced at room temperature but rises at its working temperature to values comparable to those displayed by its mesophilic counterparts at 25-37 degrees C. Such a reduced activity can result from protein rigidity and from the active site stiffening due the presence of a salt bridge between the C-terminal carboxylate and the active site arginine. Sso AcP is characterized by a melting temperature, Tm, of 100.8 degrees C and an unfolding free energy, DeltaG(U-F)H2O, at 28 degrees C and 81 degrees C of 48.7 and 20.6 kJ mol(-1), respectively. The kinetic and structural data indicate that mesophilic and hyperthermophilic AcP's display similar enzymatic activities and conformational stabilities at their working conditions. Structural analysis of the factor responsible for Sso AcP thermostability with respect to mesophilic AcP's revealed the importance of a ion pair network stabilizing particularly the beta-sheet and the loop connecting the fourth and fifth strands, together with increased density packing, loop shortening and a higher alpha-helical propensity.

Acid Anhydride Hydrolases↗
PubMed2006

Exploring the mechanism of formation of native-like and precursor amyloid oligomers for the native acylphosphatase from Sulfolobus solfataricus.

Over 40 human diseases are associated with the formation of well-defined proteinaceous fibrillar aggregates. Since the oligomers precursors to the fibrils are increasingly recognized to be the causative agents of such diseases, it is important to elucidate the mechanism of formation of these early species. The acylphosphatase from Sulfolobus solfataricus is an ideal system as it was found to form, under conditions in which it is initially native, two types of prefibrillar aggregates: (1) initial enzymatically active aggregates and (2) oligomers with characteristics reminiscent of amyloid protofibrils, with the latter originating from the structural reorganization of the initial assemblies. By studying a number of protein variants with a variety of biophysical techniques, we have identified the regions of the sequence and the driving forces that promote the first aggregation phase and show that the second phase consists in a cooperative conversion involving the entire globular fold.

Acid Anhydride Hydrolases↗
PubMed2006

Protein aggregation starting from the native globular state.

Amyloid formation by globular proteins that normally adopt a compact folded structure is generally induced in vitro under harsh conditions involving low pH, high temperature, high pressure, or in the presence of organic solvents. Under these conditions, folded proteins are generally unfolded, at least partially. The approach described here shows a rationale and two detailed examples as to how the mechanism of aggregation of a globular protein can be probed under conditions in which it is initially in its folded conformation, and hence relevant to a physiological environment.

Acid Anhydride Hydrolases↗
PubMed2006

Sequence and structural determinants of amyloid fibril formation.

Amyloid fibril formation is a process that represents an essential feature of the chemistry of proteins and plays a central role in human pathology and the biology of living organisms. In this Account, we shall describe some of the recent results on the sequence and structural determinants of protein aggregation. We shall describe the factors that govern aggregation of unfolded peptides and proteins. We shall then try to summarize the factors that pertain to the aggregation of partially structured states and will show that even fully folded states of proteins have an ability to aggregate into at least early oligomers with no need to undergo substantial conformational changes.

Amyloid↗
PubMed2005

Evidence for a mechanism of amyloid formation involving molecular reorganisation within native-like precursor aggregates.

The aggregation of the alpha/beta protein acylphosphatase from Sulfolobus solfataricus has been studied under conditions in which the protein maintains a native-like, although destabilised, conformation and that therefore bear resemblance to a physiological medium. Static and dynamic light-scattering measurements indicate that under these conditions the protein aggregates rapidly, within two minutes. The initial aggregates are enzymatically active and have a secondary structure that is not yet characterized by the high content of cross-beta structure typical of amyloid, as inferred from Fourier transform infra-red and circular dichroism measurements. These species then convert slowly into enzymatically inactive aggregates that bind thioflavin T and Congo red, characteristic of amyloid structures, and contain extensive beta-sheet structure. Transmission electron microscopy reveals the presence in the latter aggregates of spherical species and thin, elongated protofibrils, both with diameters of 3-5 nm. Kinetic tests reveal that this process occurs without the need for dissolution and re-nucleation of the aggregates. Formation of thioflavin T-binding and beta-structured aggregates is substantially more rapid than unfolding of the native state, indicating that the initial aggregation process promotes formation of amyloid structures. Taken together, these findings suggest a mechanism of amyloid formation that may have physiological relevance and in which the amyloid structures result from reorganisation of the molecular interactions within the initially formed non-amyloid aggregates.

Acid Anhydride Hydrolases↗
PubMed2005

Amyloid formation from HypF-N under conditions in which the protein is initially in its native state.

Aggregation of the N-terminal domain of the Escherichia coli HypF (HypF-N) was investigated in mild denaturing conditions, generated by addition of 6-12% (v/v) trifluoroethanol (TFE). Atomic force microscopy indicates that under these conditions HypF-N converts into the same type of protofibrillar aggregates previously shown to be highly toxic to cultured cells. These convert subsequently, after some weeks, into well-defined fibrillar structures. The rate of protofibril formation, monitored by thioflavin T (ThT) fluorescence, depends strongly on the concentration of TFE. Prior to aggregation the protein has far-UV circular dichroism (CD) and intrinsic fluorescence spectra identical with those observed for the native protein in the absence of co-solvent; the quenching of the intrinsic tryptophan fluorescence by acrylamide and the ANS binding properties are also identical in the two cases. These findings indicate that HypF-N is capable of forming amyloid protofibrils and fibrils under conditions in which the protein is initially in a predominantly native-like conformation. The rate constants for folding and unfolding of HypF-N, determined in 10% TFE using the stopped-flow technique, indicate that a partially folded state is in rapid equilibrium with the native state and populated to ca 1%. A kinetic analysis reveals that aggregation results from molecules accessing such a partially folded state. The approach described here shows that it is possible to probe the mechanism of aggregation of a specific protein under conditions in which the protein is initially native and hence relevant to a physiological environment. In addition, the results indicate that toxic protofibrils can be formed from globular proteins under conditions that are only marginally destabilising and in which the large majority of molecules have the native fold. This conclusion emphasises the importance for cells to constantly combat the propensity for even the most stable of these proteins to aggregate.

Amyloid↗
PubMed2004

Monitoring the process of HypF fibrillization and liposome permeabilization by protofibrils.

Much information has appeared in the last few years on the low resolution structure of amyloid fibrils and on their non-fibrillar precursors formed by a number of proteins and peptides associated with amyloid diseases. The fine structure and the dynamics of the process leading misfolded molecules to aggregate into amyloid assemblies are far from being fully understood. Evidence has been provided in the last five years that protein aggregation and aggregate toxicity are rather generic processes, possibly affecting all polypeptide chains under suitable experimental conditions. This evidence extends the number of model proteins one can investigate to assess the molecular bases and general features of protein aggregation and aggregate toxicity. We have used tapping mode atomic force microscopy to investigate the morphological features of the pre-fibrillar aggregates and of the mature fibrils produced by the aggregation of the hydrogenase maturation factor HypF N-terminal domain (HypF-N), a protein not associated to any amyloid disease. We have also studied the aggregate-induced permeabilization of liposomes by fluorescence techniques. Our results show that HypF-N aggregation follows a hierarchical path whereby initial globules assemble into crescents; these generate large rings, which evolve into ribbons, further organizing into differently supercoiled fibrils. The early pre-fibrillar aggregates were shown to be able to permeabilize synthetic phospholipid membranes, thus showing that this disease-unrelated protein displays the same amyloidogenic behaviour found for the aggregates of most pathological proteins and peptides. These data complement previously reported findings, and support the idea that protein aggregation, aggregate structure and toxicity are generic properties of polypeptide chains.

Bacterial Proteins↗
PubMed2004

Aggregation of the Acylphosphatase from Sulfolobus solfataricus: the folded and partially unfolded states can both be precursors for amyloid formation.

Protein aggregation is associated with a number of human pathologies including Alzheimer's and Creutzfeldt-Jakob diseases and the systemic amyloidoses. In this study, we used the acylphosphatase from the hyperthermophilic Archaea Sulfolobus solfataricus (Sso AcP) to investigate the mechanism of aggregation under conditions in which the protein maintains a folded structure. In the presence of 15-25% (v/v) trifluoroethanol, Sso AcP was found to form aggregates able to bind specific dyes such as thioflavine T, Congo red, and 1-anilino-8-naphthalenesulfonic acid. The presence of aggregates was confirmed by circular dichroism and dynamic light scattering. Electron microscopy revealed the presence of small aggregates generally referred to as amyloid protofibrils. The monomeric form adopted by Sso AcP prior to aggregation under these conditions retained enzymatic activity; in addition, folding was remarkably faster than unfolding. These observations indicate that Sso AcP adopts a folded, although possibly distorted, conformation prior to aggregation. Most important, aggregation appeared to be 100-fold faster than unfolding under these conditions. Although aggregation of Sso AcP was faster at higher trifluoroethanol concentrations, in which the protein adopted a partially unfolded conformation, these findings suggest that the early events of amyloid fibril formation may involve an aggregation process consisting of the assembly of protein molecules in their folded state. This conclusion has a biological relevance as globular proteins normally spend most of their lifetime in folded structures.

Acid Anhydride Hydrolases↗