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Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis
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Pre-amyloid oligomers of the proteotoxic RepA-WH1 prionoid assemble at the bacterial nucleoid

María Moreno-Del Álamo1, Susana Moreno-Díaz de la Espina1, M Elena Fernández-Tresguerres1

  • 1Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas - CSIC, Madrid E28040, Spain.

Scientific Reports
|October 2, 2015
PubMed

Insights

Researchers identified a novel monoclonal antibody, B3h7, that targets specific amyloid oligomers of the RepA-WH1 protein. This antibody helps reveal how amyloid proteinopathies form in bacteria, offering insights into disease mechanisms.

Area of Science:

  • Microbiology
  • Biochemistry
  • Structural Biology

Background:

  • The N-terminal WH1 domain of plasmid DNA replication initiator RepA forms amyloid fibers upon binding to dsDNA.
  • RepA-WH1 expression in Escherichia coli induces a synthetic amyloid proteinopathy with prion-like characteristics, including transmissibility and distinct strains.
  • Mammalian prion diseases share features with this bacterial model, highlighting its significance.

Purpose of the Study:

  • To characterize the amyloidogenic process of RepA-WH1.
  • To identify specific intermediates in RepA-WH1 amyloid formation.
  • To investigate the cellular location of RepA-WH1 oligomerization.

Main Methods:

  • Generation and characterization of a monoclonal antibody (B3h7) specific for RepA-WH1 oligomers.
  • In vitro studies of RepA-WH1 fiber assembly using B3h7.
  • Immuno-electron microscopy on thin sections of E. coli expressing RepA-WH1.

Main Results:

  • B3h7 specifically recognizes RepA-WH1 oligomers but not mature amyloid fibers.
  • B3h7 inhibits in vitro assembly of RepA-WH1 fibers, identifying oligomers as on-pathway intermediates.
  • Immuno-electron microscopy localizes RepA-WH1 oligomers to the bacterial nucleoid, suggesting DNA-initiated assembly.

Conclusions:

  • Oligomeric RepA-WH1 species are crucial, on-pathway intermediates in amyloid formation.
  • The bacterial nucleoid serves as the site for the initial nucleation and oligomerization of RepA-WH1 amyloid precursors.
  • Amyloid fiber growth likely transitions from the nucleoid to the cytoplasm.

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