Aggregation prone regions and gatekeeping residues in protein sequences

Most proteins sequences contain one or several short aggregation prone regions (APR) that can nucleate protein aggregation. Under normal conditions these APRs are protected from aggregation by protein interactions or because they are buried in the hydrophobic core of native protein domains. However, mutation, physiological stress or age-related disregulation of protein homeostasis increases the probability that aggregation-nucleating regions become solvent exposed. Aggregation then results from the self-assembly of APRs into -structured agglomerates that vary from small soluble oligomeric assemblies to large insoluble inclusions containing thousands of molecules. The functional effects of APR-driven aggregation are diverse and protein-specific leading to distinct disease phenotypes ranging from neurodegeneration to cancer. On a cellular and physiological level both wild type loss-of-function as well as aggregation-dependent gain-of-function effects have been shown to contribute to disease. Several molecular mechanism have been proposed to contribute to gain-of-function activity of protein aggregates includingcellular membrane disregulation, saturation of the protein quality control machinery or the ability of aggregates to engage non-native interactions with proteins and nucleic acids. These different mechanisms will all to some extent contribute to gain-of-function as in essence they all contribute to the rewiring ofthe cellular interactome by aggregation-specific interactions resulting for instance in the pronounced neurotoxicity of TDP43 aggregates by the sequestration of RNA molecules or the promotion of cell proliferation by the entrapment of homologous tumor suppressor proteins in p53 aggregates in cancer. In this review we discuss the mechanism of APR driven aggregation and how APRscontribute to modifying the cellular interactome by recruiting both misfolded as well as active proteins thereby inhibiting or activating specific cellular functions. Finally, we discuss the ubiquity of APRs in protein sequences and how selective pressure shaped protein sequences to minimizeAPR aggregation.status: publishe

WALTZ-DB: a benchmark database of amyloidogenic hexapeptides

Accurate prediction of amyloid-forming amino acid sequences remains an important challenge. We here present an online database that provides open access to the largest set of experimentally characterized amyloid forming hexapeptides. To this end, we expanded our previous set of 280 hexapeptides used to develop the Waltz algorithm with 89 peptides from literature review and by systematic experimental characterisation of the aggregation of 720 hexapeptides by transmission electron microscopy, dye binding and Fourier transform infrared spectroscopy. This brings the total number of experimentally characterized hexapeptides in the WALTZ-DB database to 1089, of which 244 are annotated as positive for amyloid formation. Availability and implementation: The WALTZ-DB database is freely available without any registration requirement at http://waltzdb.switchlab.org.status: publishe

WALTZ-DB: A benchmark database of amyloidogenic hexapeptides

Summary: Accurate prediction of amyloid-forming amino acid sequences remains an important challenge. We here present an online database that provides open access to the largest set of experimentally characterized amyloid forming hexapeptides. To this end, we expanded our previous set of 280 hexapeptides used to develop the Waltz algorithm with 89 peptides from literature review and by systematic experimental characterisation of the aggregation of 720 hexapeptides by transmission electron microscopy, dye binding and Fourier transform infrared spectroscopy. This brings the total number of experimentally characterized hexapeptides in the WALTZ-DB database to 1089, of which 244 are annotated as positive for amyloid formation

Aggregation gatekeepers modulate protein homeostasis of aggregating sequences and affect bacterial fitness

The most common mechanism by which proteins aggregate consists in the assembly of short hydrophobic primary sequence segments into extended -structured agglomerates. A significant enrichment of charged residues is observed at the flank of these aggregation-prone sequence segments, suggesting selective pressure against aggregation. These so-called aggregation gatekeepers act by increasing the intrinsic solubility of aggregating sequences in vitro, but it has been suggested that they could also facilitate chaperone interactions. Here, we address whether aggregation gatekeepers affect bacterial fitness. In Escherichia coli MC4100 we overexpressed GFP fusions with an aggregation-prone segment of sigma 32 (further termed sigma 32) flanked by gatekeeper and non-gatekeeper residues and measured pairwise competitive growth. We found that the identity of flanking residues had significant effect on bacterial growth. Overexpression of sigma 32 flanked by its natural gatekeepers displayed the greatest competitive fitness, followed by other combinations of gatekeepers, while absence of gatekeepers strongly affects bacterial fitness. Further analysis showed the diversity of effects of gatekeepers on the proteostasis of sigma 32 including synthesis and degradation rates, in vivo aggregation propensity and chaperone response. Our results suggest that gatekeeper residues affect bacterial fitness not only by modulating the intrinsic aggregation propensity of proteins but also by the manner in which they affect the processing of sigma 32GFP by the protein quality control machinery of the cell. In view of these observations, we hypothesize that variation at gatekeeper positions offers a flexible selective strategy to modulate the proteostatic regulation of proteins to the match intrinsic aggregation propensities of proteins with required expression levels.status: publishe

Structural hot spots for the solubility of globular proteins

Natural selection shapes protein solubility to physiological requirements and recombinant applications that require higher protein concentrations are often problematic. This raises the question whether the solubility of natural protein sequences can be improved. We here show an anti-correlation between the number of aggregation prone regions (APRs) in a protein sequence and its solubility, suggesting that mutational suppression of APRs provides a simple strategy to increase protein solubility. We show that mutations at specific positions within a protein structure can act as APR suppressors without affecting protein stability. These hot spots for protein solubility are both structure and sequence dependent but can be computationally predicted. We demonstrate this by reducing the aggregation of human α-galactosidase and protective antigen of Bacillus anthracis through mutation. Our results indicate that many proteins possess hot spots allowing to adapt protein solubility independently of structure and function.status: publishe

Exposure of a cryptic Hsp70 binding site determines the cytotoxicity of the ALS-associated SOD1-mutant A4V

The accumulation of toxic protein aggregates is thought to play a key role in a range of degenerative pathologies, but it remains unclear why aggregation of polypeptides into non-native assemblies is toxic and why cellular clearance pathways offer ineffective protection. We here study the A4V mutant of SOD1, which forms toxic aggregates in motor neurons of patients with familial amyotrophic lateral sclerosis (ALS). A comparison of the location of aggregation prone regions (APRs) and Hsp70 binding sites in the denatured state of SOD1 reveals that ALS-associated mutations promote exposure of the APRs more than the strongest Hsc/Hsp70 binding site that we could detect. Mutations designed to increase the exposure of this Hsp70 interaction site in the denatured state promote aggregation but also display an increased interaction with Hsp70 chaperones. Depending on the cell type, in vitro this resulted in cellular inclusion body formation or increased clearance, accompanied with a suppression of cytotoxicity. The latter was also observed in a zebrafish model in vivo. Our results suggest that the uncontrolled accumulation of toxic SOD1A4V aggregates results from insufficient detection by the cellular surveillance network.status: publishe

Exposure of a cryptic Hsp70 binding site determines the cytotoxicity of the ALS-associated SOD1-mutant A4V

The accumulation of toxic protein aggregates is thought to play a key role in a range of degenerative pathologies, but it remains unclear why aggregation of polypeptides into non-native assemblies is toxic and why cellular clearance pathways offer ineffective protection. We here study the A4V mutant of SOD1, which forms toxic aggregates in motor neurons of patients with familial amyotrophic lateral sclerosis (ALS). A comparison of the location of aggregation prone regions (APRs) and Hsp70 binding sites in the denatured state of SOD1 reveals that ALS-associated mutations promote exposure of the APRs more than the strongest Hsc/Hsp70 binding site that we could detect. Mutations designed to increase the exposure of this Hsp70 interaction site in the denatured state promote aggregation but also display an increased interaction with Hsp70 chaperones. Depending on the cell type, in vitro this resulted in cellular inclusion body formation or increased clearance, accompanied with a suppression of cytotoxicity. The latter was also observed in a zebrafish model in vivo. Our results suggest that the uncontrolled accumulation of toxic SOD1A4V aggregates results from insufficient detection by the cellular surveillance network