Large Proteins Have a Great Tendency to Aggregate but a Low Propensity to Form Amyloid Fibrils

et al. (2011) Large Proteins Have a Great Tendency to Aggregate but a Low Propensity to Form Amyloid Fibrils. PLoS ONE 6(1): e16075. doi:10.1371/journal.pone.0016075 Large Proteins Have a Great Tendency to Aggregate but a Low Propensity to Form Amyloid Fibrils Hassan Ramshini 0 Claudia Parrini 0 Annalisa Relini 0 Mariagioia Zampagni 0 Benedetta Mannini 0 Alessandra Pesce 0 Ali Akbar Saboury 0 Mohsen Nemat-Gorgani 0 Fabrizio Chiti 0 Vladimir N. Uversky, University of South Florida College of Medicine, United States of America 0 1 Dipartimento di Scienze Biochimiche, Universita` di Firenze , Florence , Italy , 2 Institute of Biochemistry and Biophysics, University of Tehran , Tehran, Iran, 3 Dipartimento di Fisica , Universita` di Genova , Genoa , Italy The assembly of soluble proteins into ordered fibrillar aggregates with cross-b structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively. The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger. We have then investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB) to form amyloid-like fibrils in vitro. This size is intermediate between the size distributions of amyloid and non-amyloid forming proteins. Aggregation was induced under conditions known to be most effective for amyloid formation by normally globular proteins: (i) low pH with salts, (ii) pH 5.5 with trifluoroethanol. In both situations YHKB aggregated very rapidly into species with significant b-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated a morphology distinct from typical amyloid fibrils. Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay. This analysis indicates that large proteins have a high tendency to form toxic aggregates, but low propensity to form regular amyloid in vivo and that such a behavior is intrinsically determined by the size of the protein, as suggested by the in vitro analysis of our sample protein. - Funding: This work was supported by grants from the Research Council of the University of Tehran, the Iranian National Science Foundation (INSF), the Italian Ministry of University, Research and Instruction (MIUR) and the European Community (EC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. A large family of human pathologies is associated with the conversion of peptides and proteins from their soluble functional forms into well-defined fibrillar aggregates, often called amyloid fibrils when they accumulate in the extracellular space [1]. Such diseases include neurodegenerative disorders, such as Alzheimers disease and spongiform encephalopathies, non neuropathic localized amyloidoses, such as type II diabetes and atrial amyloidosis and non neuropathic systemic amyloidoses, like light-chain amyloidosis and dialysis-related amyloidosis [1]. Images acquired by transmission electron microscopy show that amyloid fibrils are long, rigid, unbranched and usually consist of a number (typically 26) of protofilaments, each about 25 nm in diameter [2]. These protofilaments twist together to form rope-like fibrils that are typically 713 nm wide [2,3] or associate laterally to form long ribbons that are 25 nm thick and up to 30 nm wide [4,5]. The fibrils have the ability to bind specific dyes such as thioflavin T (ThT) and Congo red (CR) [6] and are characterized by an extended crossb structure, as revealed by X-ray fiber diffraction [3]. The peptides and proteins that form extracellular amyloid fibrils, or intracellular inclusions with recognized related morphological and structural characteristics, are generally small in size, often shorter than 250 residues [1]. Even proteins that have been converted into amyloid-like fibrils in vitro and have no link to human diseases are generally small, typically shorter than 150 residues [7,8]. The small percentage of large proteins recognized to form amyloid or amyloid-like fibrils is disproportionate to the fraction of such proteins in the human proteome, as more than 50% of natural human proteins are longer than 250 residues. The question thus arises as to why diseases associated with amyloid or amyloid-like deposits do not generally arise from large proteins. To address this issue we have carried out an extensive search in the literature of all proteins recognized to form deposits distinct from amyloid under pathological conditions and have compared the sizes of such proteins with those known to form amyloid deposits in disease. We will show that the size distributions of proteins forming amyloid and non-amyloid deposits in pathology are well separated and that proteins associated with non-amyloid deposits are remarkably longer. We have then investigated the aggregation process in vitro of a fairly large model protein, namely the 486-residue (55 kDa) protein hexokinase-B from the yeast Saccharomyces cerevisiae (YHKB). The size of this protein falls within the region of overlap of the size distributions of amyloid and non-amyloid forming proteins. In particular, we have determined the type of protein aggregates formed by such protein under two sets of conditions, both shown to be among the most effective in promoting amyloid fibril formation in vitro [923]. Using different biophysical techniques we will show that both conditions promote the very rapid formation of aggregates, characterized by significant b-sheet structure but only weak ability to bind ThT and CR and a morphology different from that of regular amyloid fibrils. Importantly, however, we will also show that both types of aggregates appear to be toxic to cultured cells. The results indicate that large proteins have a great tendency to aggregate into deleterious species but a poor propensity to form real amyloid fibrils and suggest that the difference in behavior between proteins forming amyloid structures and proteins producing non-amyloid deposits in pathology is primarily and intrinsically determined by the length of the polypeptide chain undergoing aggregation. Materials and Methods Materials 8-anilinonaphthalene-1-sulfonate (ANS), trifluoroethanol (TFE), trifluoroacetic acid (TFA), ThT, CR and a-cyclodextrin were purchased from Sigma-Aldrich (St. Louis, MO, USA). YHKB was also obtained from Sigma-Aldrich as a crystalline suspension in ammonium sulfate (code H6380). After r (...truncated)