Three-dimensional domain swapping in the folded and molten-globule states of cystatins, an amyloid-forming structural superfamily

The [URE3] phenotype in Saccharomyces cerevisiaepropagates by a prion mechanism, involving the aggregation of the normally soluble and highly helical protein Ure2. Previous data have shown that the protein spontaneously forms in vitro long, straight, insoluble fibrils at neutral pH that are similar to amyloids in that they bind Congo red and show green-yellow birefringence and have an increased resistance to proteolysis. These fibrils are not amyloids as they are devoid of a cross-␤ core. Here we further document the mechanism of assembly of Ure2p into fibrils. The critical concentration for Ure2p assembly is measured, and the minimal size of the nuclei that are the precursors of Ure2p fibrils is determined. Our data indicate that the assembly process is irreversible. As a consequence, the critical concentration is very low. By analyzing the elongation rates of preformed fibrils and combining the results with singlefiber imaging experiments of a variant Ure2p labeled by fluorescent dyes, we reveal the polarity of the fibrils and differences in the elongation rates at their ends. These results bring novel insight in the process of Ure2p assembly into fibrils and the mechanism of propagation of yeast prions.Creutzfeldt-Jacob disease, bovine spongiform encephalopathy, and sheep scrapie are transmissible spongiform encephalopathies. They are believed to be caused by a constitutive protein called prion protein (PrP) in an altered conformation (1). This rare form of the protein is thought to convert the normal form to the infectious conformation in a catalytic manner (for review, see Ref.2). The molecular events at the origin of propagation are not yet elucidated. In the lower eukaryotes Saccharomyces cerevisiae and Podospora anserina prion proteins have been identified taking advantage of traits they confer (3-7). These proteins are harmless for humans and constitute valuable model systems to characterize prion propagation.The [URE3] phenotype in S. cerevisiae is caused by a disorder in a signal transduction cascade that regulates nitrogen catabolism (8). Yeast cells exhibiting the [URE3] phenotype have the ability to take up ureidosuccinate, the structural analogue of allantoate, a poor nitrogen source in the presence of a good nitrogen source such as ammonium ions. This is believed to be because of the loss of the function of the protein determinant of [URE3], Ure2p, whose function is unknown although reported to be involved in yeast heavy metal and strong oxidant detoxification (9). Importantly, Ure2p [URE3] promotes the conversion of the newly synthesized active Ure2p into an inactive form. Thus, the Ure2p [URE3] state is heritable, independent of any nucleic acid (10).Native soluble Ure2p assembles in vitro into fibrils that exhibit the characteristics of amyloids in that they are about 20 nm wide and more than 1 m long, bind thioflavin T and Congo Red, show yellow-green birefringence in polarized light upon Congo Red binding, and exhibit an increased resistance to proteolysis (11-13). The assembly reaction fo...

Section: Discussionmentioning

confidence: 99%

Assembly of the Yeast Prion Ure2p into Protein Fibrils

Fay

Inoue

Bousset

et al. 2003

“…Observations that these regions of the sequence are solvent-exposed in the fibrils formed from this protein provide direct evidence that fibril formation does not originate from the assembly of protein molecules in their native conformation (7). Some other proteins such as cystatin C, which is associated with a hereditary form of cerebral amyloid angiopathy, and the cell cycle regulatory protein p13suc1 are suggested to fibrillize via domain swapping (8,9). Domain swapping is a mechanism in which part of the structure of each monomer replaces the corresponding structural elements of an identical monomer so as to form an oligomer where each subunit has a similar structure to the folded monomer (8,9).…”

mentioning

confidence: 94%

“…Some other proteins such as cystatin C, which is associated with a hereditary form of cerebral amyloid angiopathy, and the cell cycle regulatory protein p13suc1 are suggested to fibrillize via domain swapping (8,9). Domain swapping is a mechanism in which part of the structure of each monomer replaces the corresponding structural elements of an identical monomer so as to form an oligomer where each subunit has a similar structure to the folded monomer (8,9). Even in this case, aggregation is proposed to require a substantial unfolding of the folded monomer in order to facilitate exchange of structural elements (8,9).…”

mentioning

confidence: 99%

“…Domain swapping is a mechanism in which part of the structure of each monomer replaces the corresponding structural elements of an identical monomer so as to form an oligomer where each subunit has a similar structure to the folded monomer (8,9). Even in this case, aggregation is proposed to require a substantial unfolding of the folded monomer in order to facilitate exchange of structural elements (8,9). The presence of structure in the fibrils formed from the amyloid ␤ peptide associated with Alzheimer's disease indicates that fibrils formed from peptides or proteins that are unstructured in their soluble form need to adopt a partially structured conformation either before or during assembly (10,11).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Aggregation of the Acylphosphatase from Sulfolobus solfataricus

Plakoutsi

Taddei

Stefani

et al. 2004

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.

“…Stefin B has a much higher tendency than stefin A to form dimers, higher oligomers, and amyloid fibrils, nevertheless, the stefin A domain-swapped dimer could be prepared under more extreme solution conditions and its structure was determined by NMR (22,23). The model for the mechanism of stefin B fibrillization involves off-pathway lower oligomer formation (probably involving domain-swapping, due to a high energetic barrier) and a larger nucleus in the order of 30 dimers, explaining both an unusual behavior at higher protein concentrations and a relatively long lag phase (21).…”

mentioning

confidence: 99%

Interaction between Oligomers of Stefin B and Amyloid-β in Vitro and in Cells

Škerget

Taler‐Verčič

Bavdek

et al. 2010

To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction between human stefin B (cystatin B) and amyloid-␤-(1-40) peptide (A␤). Using surface plasmon resonance and electrospray mass spectrometry we were able to show a direct interaction between the two proteins. As an interesting new fact, we show that stefin B binding to A␤ is oligomer specific. The dimers and tetramers of stefin B, which bind A␤, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit A␤ fibril formation. When expressed in cultured cells, stefin B co-localizes with A␤ intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the A␤ epitope. Thus, stefin B is another APP/A␤-binding protein in vitro and likely in cells.Neurodegenerative diseases present a huge burden in the developed world's aging population. They are all in one way or another connected to aberrant protein folding and aggregation of the proteins involved (1). Various protein conformational disorders of the central and peripheral nervous system are known, which often appear sporadically but also run in families. These are among others: Parkinson and Alzheimer diseases, dementia with Lewy bodies, vascular and fronto-temporal dementia, and amyotrophic lateral sclerosis.The A␤ peptide implicated in Alzheimer disease pathology is a cleavage product of the membrane A␤ precursor protein (APP).3 It is the main constituent of extracellular amyloid plaques, however, together with its oligomers, it also resides intracellularly (2). It has been shown that A␤ oligomers prepared in vitro and those extracted from living cells exert cytotoxicity and cause symptoms of reversible memory loss in animal models (3). Amyloid protein oligomers have special structural properties, which are reflected in a common antioligomer antibody (4). This antibody not only binds the oligomers against which it was raised but also binds chaperones and some other proteins involved in disaggregating protein aggregates in cells (5). A␤-binding proteins, the so called "amateur chaperones," were suggested to have a potential in Alzheimer disease therapy (6, 7).It has been shown before that human cystatin C is an A␤-binding protein (8). Cystatins are single chain proteins that inhibit cysteine cathepsins (9). Human stefin B (also known as cystatin B) is a member of subfamily A of cystatins, classified as family I25 in the MEROPS scheme (10). Stefin B, a protein of 98 amino acid residues and 1 Cys, is predominantly intracellular, whereas cystatin C, a protein of 120 residues and 2 disulfide bonds, is a secretory protein. Three-dimensional structures of stefins and cystatin C have been determined, among others, the solution structure of stefin A (11) and cystatin C (12, 13).Human cystatin C has been found as a constituent of senile plaques of Alzheimer disease patients (14) and stefins A and B have also been reported to localize to a...