Identification of Aggregation-Prone and Gatekeeper Residues in Bacterial Amyloids Using Site-Directed Mutagenesis and Flow Cytometry

The TAR-DNA binding protein (TDP43) is a nuclear protein whose cytoplasmic inclusions are hallmarks of Amyotrophic Lateral Sclerosis (ALS). Acute stress in cells causes TDP43 mobilization to the cytoplasm and its aggregation through different routes. Although acute stress elicits a strong phenotype, is far from recapitulating the years-long aggregation process. We applied different chronic stress protocols and described TDP43 aggregation in a human neuroblastoma cell line by combining solubility assays, thioflavin-based microscopy and flow cytometry. This approach allowed us to detect, for the first time to our knowledge in vitro, the formation of 25 kDa C-terminal fragment of TDP43, a pathogenic hallmark of ALS. Our results indicate that chronic stress, compared to the more common acute stress paradigm, better recapitulates the cell biology of TDP43 proteinopathies. Moreover, we optimized a protocol for the detection of bona fide prions in living cells, suggesting that TDP43 may form amyloids as a stress response.

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Different Chronic Stress Paradigms Converge on Endogenous TDP43 Cleavage and Aggregation

Candelise

Caissutti

Zenuni

et al. 2023

Mol Neurobiol

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The Green Tea Polyphenol Epigallocatechin-Gallate (EGCG) Interferes with Microcin E492 Amyloid Formation

Aguilera,

Berríos-Pastén,

Veloso

et al. 2023

Molecules

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Microcin E492 (MccE492) is an antimicrobial peptide and proposed virulence factor produced by some Klebsiella pneumoniae strains, which, under certain conditions, form amyloid fibers, leading to the loss of its antibacterial activity. Although this protein has been characterized as a model functional amyloid, the secondary structure transitions behind its formation, and the possible effect of molecules that inhibit this process, have not been investigated. In this study, we examined the ability of the green tea flavonoid epigallocatechin gallate (EGCG) to interfere with MccE492 amyloid formation. Aggregation kinetics followed by thioflavin T binding were used to monitor amyloid formation in the presence or absence of EGCG. Additionally, synchrotron radiation circular dichroism (SRCD) and transmission electron microscopy (TEM) were used to study the secondary structure, thermal stability, and morphology of microcin E492 fibers. Our results showed that EGCG significantly inhibited the formation of the MccE492 amyloid, resulting in mainly amorphous aggregates and small oligomers. However, these aggregates retained part of the β-sheet SRCD signal and a high resistance to heat denaturation, suggesting that the aggregation process is sequestered or deviated at some stage but not completely prevented. Thus, EGCG is an interesting inhibitor of the amyloid formation of MccE492 and other bacterial amyloids.

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Identification of Aggregation-Prone and Gatekeeper Residues in Bacterial Amyloids Using Site-Directed Mutagenesis and Flow Cytometry

Cited by 2 publications

References 12 publications

Different Chronic Stress Paradigms Converge on Endogenous TDP43 Cleavage and Aggregation

Different Chronic Stress Paradigms Converge on Endogenous TDP43 Cleavage and Aggregation

The Green Tea Polyphenol Epigallocatechin-Gallate (EGCG) Interferes with Microcin E492 Amyloid Formation

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