| 1. |
- Bednarska, Natalia G., et al.
(författare)
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Protein aggregation as an antibiotic design strategy
- 2016
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Ingår i: Molecular Microbiology. - : WILEY-BLACKWELL. - 0950-382X .- 1365-2958. ; 99:5, s. 849-865
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Tidskriftsartikel (refereegranskat)abstract
- Taking advantage of the xenobiotic nature of bacterial infections, we tested whether the cytotoxicity of protein aggregation can be targeted to bacterial pathogens without affecting their mammalian hosts. In particular, we examined if peptides encoding aggregation-prone sequence segments of bacterial proteins can display antimicrobial activity by initiating toxic protein aggregation in bacteria, but not in mammalian cells. Unbiased in vitro screening of aggregating peptide sequences from bacterial genomes lead to the identification of several peptides that are strongly bactericidal against methicillin-resistant Staphylococcus aureus. Upon parenteral administration in vivo, the peptides cured mice from bacterial sepsis without apparent toxic side effects as judged from histological and hematological evaluation. We found that the peptides enter and accumulate in the bacterial cytosol where they cause aggregation of bacterial polypeptides. Although the precise chain of events that leads to cell death remains to be elucidated, the ability to tap into aggregation-prone sequences of bacterial proteomes to elicit antimicrobial activity represents a rich and unexplored chemical space to be mined in search of novel therapeutic strategies to fight infectious diseases.
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| 2. |
- Claes, Filip, et al.
(författare)
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Exposure of a cryptic Hsp70 binding site determines the cytotoxicity of the ALS-associated SOD1-mutant A4V
- 2019
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 32:10, s. 443-457
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Tidskriftsartikel (refereegranskat)abstract
- 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 SOD1(A4V) aggregates results from insufficient detection by the cellular surveillance network.
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| 3. |
- Gallardo, Rodrigo, et al.
(författare)
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De novo design of a biologically active amyloid
- 2016
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Ingår i: Science. - : AMER ASSOC ADVANCEMENT SCIENCE. - 0036-8075 .- 1095-9203. ; 354:6313, s. 720-
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Tidskriftsartikel (refereegranskat)abstract
- Most human proteins possess amyloidogenic segments, but only about 30 are associated with amyloid-associated pathologies, and it remains unclear what determines amyloid toxicity. We designed vascin, a synthetic amyloid peptide, based on an amyloidogenic fragment of vascular endothelial growth factor receptor 2 (VEGFR2), a protein that is not associated to amyloidosis. Vascin recapitulates key biophysical and biochemical characteristics of natural amyloids, penetrates cells, and seeds the aggregation of VEGFR2 through direct interaction. We found that amyloid toxicity is observed only in cells that both express VEGFR2 and are dependent on VEGFR2 activity for survival. Thus, amyloid toxicity here appears to be both protein-specific and conditional-determined by VEGFR2 loss of function in a biological context in which target protein function is essential.
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| 4. |
- Ganesan, Ashok, et al.
(författare)
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Selectivity of Aggregation-Determining Interactions
- 2015
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:2, s. 236-247
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Tidskriftsartikel (refereegranskat)abstract
- Protein aggregation is sequence specific, favoring self-assembly over cross-seeding with non-homologous sequences. Still, as the majority of proteins in a proteome are aggregation prone, the high level of homogeneity of protein inclusions in vivo both during recombinant overexpression and in disease remains surprising. To investigate the selectivity of protein aggregation in a proteomic context, we here compared the selectivity of aggregation-determined interactions with antibody binding. To that purpose, we synthesized biotin-labeled peptides, corresponding to aggregation-determining sequences of the bacterial protein β-galactosidase and two human disease biomarkers: C-reactive protein and prostate-specific antigen. We analyzed the selectivity of their interactions in Escherichiacoli lysate, human serum and human seminal plasma, respectively, using a Western blot-like approach in which the aggregating peptides replace the conventional antibody. We observed specific peptide accumulation in the same bands detected by antibody staining. Combined spectroscopic and mutagenic studies confirmed accumulation resulted from binding of the peptide on the identical sequence of the immobilized target protein. Further, we analyzed the sequence redundancy of aggregating sequences and found that about 90% of them are unique within their proteome. As a result, the combined specificity and low sequence redundancy of aggregating sequences therefore contribute to the observed homogeneity of protein aggregation in vivo. This suggests that these intrinsic proteomic properties naturally compartmentalize aggregation events in sequence space. In the event of physiological stress, this might benefit the ability of cells to respond to proteostatic stress by allowing chaperones to focus on specific aggregation events rather than having to face systemic proteostatic failure.
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| 5. |
- Khodaparast, Ladan, et al.
(författare)
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Aggregating sequences that occur in many proteins constitute weak spots of bacterial proteostasis
- 2018
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Ingår i: Nature Communications. - : NATURE PUBLISHING GROUP. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Aggregation is a sequence-specific process, nucleated by short aggregation-prone regions (APRs) that can be exploited to induce aggregation of proteins containing the same APR. Here, we find that most APRs are unique within a proteome, but that a small minority of APRs occur in many proteins. When aggregation is nucleated in bacteria by such frequently occurring APRs, it leads to massive and lethal inclusion body formation containing a large number of proteins. Buildup of bacterial resistance against these peptides is slow. In addition, the approach is effective against drug-resistant clinical isolates of Escherichia coli and Acinetobacter baumannii, reducing bacterial load in a murine bladder infection model. Our results indicate that redundant APRs are weak points of bacterial protein homeostasis and that targeting these may be an attractive antibacterial strategy.
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