Aggregation Propensity of the Human Proteome

Formation of amyloid-like fibrils is involved in numerous human protein deposition diseases, but is also an intrinsic property of polypeptide chains in general. Progress achieved recently now allows the aggregation propensity of proteins to be analyzed over large scales. In this work we used a previously developed predictive algorithm to analyze the propensity of the 34,180 protein sequences of the human proteome to form amyloid-like fibrils. We show that long proteins have, on average, less intense aggregation peaks than short ones. Human proteins involved in protein deposition diseases do not differ extensively from the rest of the proteome, further demonstrating the generality of protein aggregation. We were also able to reproduce some of the results obtained with other algorithms, demonstrating that they do not depend on the type of computational tool employed. For example, proteins with different subcellular localizations were found to have different aggregation propensities, in relation to the various efficiencies of quality control mechanisms. Membrane proteins, intrinsically disordered proteins, and folded proteins were confirmed to have very different aggregation propensities, as a consequence of their different structures and cellular microenvironments. In addition, gatekeeper residues at strategic positions of the sequences were found to protect human proteins from aggregation. The results of these comparative analyses highlight the existence of intimate links between the propensity of proteins to form aggregates with β-structure and their biology. In particular, they emphasize the existence of a negative selection pressure that finely modulates protein sequences in order to adapt their aggregation propensity to their biological context

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

Iron-Catalyzed Reductive Amination of Aldehydes in Isopropyl Alcohol/Water Media as Hydrogen Sources

Reductive amination can be carried in i-PrOH/H2O as hydrogen sources using commercially available iron carbonyl complexes. Within an aqueous alkaline environment, a hydridocarboferrate is formed and its reducing potential is exploited for hydrogenation of the imine (or iminium ion) obtained in situ from aldehydes or ketones, and primary or secondary amines in almost equimolar ratio. This completely sustainable and hydrogen-free process proceeds at 100 °C using Fe3(CO)12as catalyst precursor under convectional heating while Fe2(CO)9gave better results when the reaction was carried out under MW dielectric heating. Both enolizable and non-enolizable aldehydes may be successfully employed in reactions with aliphatic and aromatic amines. (Figure presented.)

Folding and Aggregation Are Selectively Influenced by the Conformational Preferences of the α-Helices of Muscle Acylphosphatase

The native state of human muscle acylphosphatase (AcP) presents two alpha-helices. In this study we have investigated folding and aggregation of a number of protein variants having mutations aimed at changing the propensity of these helical regions. Equilibrium and kinetic measurements of folding indicate that only helix-2, spanning residues 55-67, is largely stabilized in the transition state for folding therefore playing a relevant role in this process. On the contrary, the aggregation rate appears to vary only for the variants in which the propensity of the region corresponding to helix-1, spanning residues 22-32, is changed. Mutations that stabilize the first helix slow down the aggregation process while those that destabilize it increase the aggregation rate. AcP variants with the first helix destabilized aggregate with rates increased to different extents depending on whether the introduced mutations also alter the propensity to form beta-sheet structure. The fact that the first alpha-helix is important for aggregation and the second helix is important for folding indicates that these processes are highly specific. This partitioning does not reflect the difference in intrinsic alpha-helical propensities of the two helices, because helix-1 is the one presenting the highest propensity. Both processes of folding and aggregation do not therefore initiate from regions that have simply secondary structure propensities favorable for such processes. The identification of the regions involved in aggregation and the understanding of the factors that promote such a process are of fundamental importance to elucidate the principles by which proteins have evolved and for successful protein design

Development of enzymatic activity during protein folding. Detection of a spectroscopically silent native-like intermediate of muscle acylphosphatase.

The recovery of enzymatic activity during the folding of muscle acylphosphatase and two single residue mutants (proline 54 to alanine and proline 71 to alanine) from 7 M urea has been monitored and compared with the development of intrinsic fluorescence emission. Fluorescence measurements reveal the presence in the wild-type protein of a major rapid refolding phase followed by a second low amplitude slow phase. The slow phase is absent in the fluorescence trace acquired with the proline 54 to alanine mutant, suggesting the involvement of this proline residue in the fluorescence-detected slow phase of the wild-type protein. The major kinetic phase is associated with a considerable recovery of enzymatic activity, indicating that a large fraction of molecules refolds with effective two-state behavior. The use of time-resolved enzymatic activity as a probe to follow the folding process reveals, however, the presence of another exponential slow phase arising from proline 71. This slow phase is not observable by utilizing optical probes, indicating that, unlike proline 54, the cis to trans isomerization of proline 71 can take place in an intermediate possessing a native-like fold. We suggest that, although spectroscopically silent and structurally insignificant, the cis-trans interconversion of proline residues in native-like intermediates may be crucial for the generation of enzymatic activity of functional enzymes

Drosophila melanogaster acylphosphatase: A common ancestor for acylphosphatase isoenzymes of vertebrate species

AbstractAn open reading frame encoding a putative acylphosphatase was found in Drosophila melanogaster. The corresponding gene product shows 40% identity and 22 additional amino acid residues at the C-terminus as compared to muscle- and common-type human acylphosphatases. Moreover, all the residues involved in the catalytic mechanism of vertebrate enzymes are conserved in the D. melanogaster acylphosphatase. The D. melanogaster protein and a deletion mutant, similar in length to vertebrate acylphosphatases, were produced by cloning the corresponding cDNA in Escherichia coli. The wild-type enzyme is a protein with a well-established three-dimensional fold and a markedly reduced conformational stability as compared to vertebrate isoenzymes. The specific activity of the enzyme is significantly lower than that found in vertebrate enzymes though the substrate binding capability is basically unaltered. The deletion of 22 residues does not cause a significant change in kcat, while affecting the apparent binding parameters. This work suggests that the genes encoding the vertebrate enzymes originate from an ancestor gene by duplication and subsequent evolution

Assessing the role of aromatic residues in the amyloid aggregation of human muscle acylphosphatase

Among the many parameters that have been proposed to promote amyloid fibril formation is the π-stacking of aromatic residues. We have studied the amyloid aggregation of several mutants of human muscle acylphosphatase in which an aromatic residue was substituted with a non-aromatic one. The aggregation rate was determined using the Thioflavin T test under conditions in which the variants populated initially an ensemble of partially unfolded conformations. Substitutions in aggregation-promoting fragments of the sequence result in a dramatically decreased aggregation rate of the protein, confirming the propensity of aromatic residues to promote this process. Nevertheless, a statistical analysis shows that the measured decrease of aggregation rate following mutation arises predominantly from a reduction of hydrophobicity and intrinsic β-sheet propensity. This suggests that aromatic residues favor aggregation because of these factors rather than for their aromaticity