The Molecular Basis of Functional Bacterial Amyloid Polymerization and Nucleation

Wang, Xuan; Hammer, Neal D.; Chapman, Matthew R.

doi:10.1074/jbc.m800466200

Cited by 117 publications

(163 citation statements)

References 28 publications

(52 reference statements)

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“…Preformed amyloid fibers can accelerate the polymerization of soluble subunits by a mechanism called seeding (18). Our previous results showed that R1 and R5 of CsgA are required for both CsgA-CsgB interactions and CsgACsgA interactions (12). Among five repeating units, only R1 and R5 polymerization can be accelerated by CsgB and CsgA (12).…”

Section: Resultsmentioning

confidence: 99%

“…Our previous results showed that R1 and R5 of CsgA are required for both CsgA-CsgB interactions and CsgACsgA interactions (12). Among five repeating units, only R1 and R5 polymerization can be accelerated by CsgB and CsgA (12). In vitro, peptides composed of repeating units of R1, R3, and R5 efficiently polymerize into ThT fluorescence-positive fibrous structures (19).…”

Section: Resultsmentioning

confidence: 99%

“…The repeats (R1, R2, R3, R4, and R5) share at least 30% identity at the amino acid level and are distinguished by the consensus sequence Ser-X 5 -Gln-X 4 -Asn-X 5 -Gln (11). We previously found that R1 and R5 direct CsgA to respond to CsgB-mediated nucleation and are critical for fiber elongation (12). Moreover, internally conserved Gln and Asn residues located in R1 and R5 of CsgA are required for CsgB nucleation and curli assembly (13).…”

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confidence: 99%

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Gatekeeper residues in the major curlin subunit modulate bacterial amyloid fiber biogenesis

Wang

Zhou

Ren

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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117

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Amyloid fibers are filamentous protein structures commonly associated with neurodegenerative diseases. Unlike disease-associated amyloids, which are the products of protein misfolding, Escherichia coli assemble membrane-anchored functional amyloid fibers called curli. Curli fibers are composed of two proteins, CsgA and CsgB. In vivo, the polymerization of the major curli subunit protein, CsgA, is dependent on CsgB-mediated nucleation. The amyloid core of CsgA features five imperfect repeats (R1-R5), and R1 and R5 govern CsgA responsiveness to CsgB nucleation and self-seeding by CsgA fibers. Here, the specificity of bacterial amyloid nucleation was probed, revealing that certain aspartic acid and glycine residues inhibit the intrinsic aggregation propensities and nucleation responsiveness of R2, R3, and R4. These residues function as "gatekeepers" to modulate CsgA polymerization efficiency and potential toxicity. A CsgA molecule lacking gatekeeper residues polymerized in vitro significantly faster than wild-type CsgA and polymerized in vivo in the absence of the nucleation machinery, resulting in mislocalized fibers. This uncontrolled polymerization was associated with cytotoxicity, suggesting that incorrectly regulated CsgA polymerization was detrimental to the cell.A myloids are ordered proteinaceous fibers commonly associated with mammalian neurodegenerative diseases and prion-based encephalopathies (1). Amyloid fibers have distinct biochemical and biophysical properties, such as remarkable resistance to chemical and thermal denaturation, and specific tinctorial properties when bound to Congo red and thioflavin T (ThT) (1). The molecular basis of neurodegenerative disease development induced by amyloid propagation remains elusive, partially because of the seemingly erratic and uncontrolled nature of amyloidogenesis. An emerging focus of amyloid biosynthesis has shown that amyloids can also be an integral part of physiology found in different organisms including bacteria, fungi, and mammals (2, 3). How nature coordinates functional amyloid propagation and reduces the associated cytotoxicity is poorly understood.Curli, a bacterially produced functional amyloid, is an important component of the extracellular matrix and is involved in bacterial community behaviors (4). Because of the amyloid properties of curli fibers (5, 6), the colonies of curli-producing Escherichia coli stain red when grown on Congo red indicator plates, which provides a convenient assay to monitor curli assembly in vivo (7). In E. coli, at least six proteins are dedicated to directing efficient curli formation. Curli fibers are composed of a major subunit CsgA and a minor subunit CsgB. CsgA remains unpolymerized until it encounters the surface-tethered nucleator CsgB, which initiates CsgA polymerization (8). CsgD is a transcriptional activator for the csgBA operon (4). CsgG, CsgE, and CsgF are nonfiber structural accessory proteins involved in secretion and stabilization of the fiber subunits and modulation of fiber assembly (6). CsgG is prop...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Gatekeeper residues in the major curlin subunit modulate bacterial amyloid fiber biogenesis

Wang

Zhou

Ren

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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117

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“…The first was an overlay assay using freshly purified CsgA or CsgA mutant proteins and cells expressing the CsgB nucleator protein. 21 In a CsgB-dependent manner, soluble wild-type CsgA was converted into an amyloid fiber within 1 minute of the overlay as evidenced by Congo red staining, or by TEM. 21 Various concentrations of CsgA, CsgA Q49A and CsgA N144A were overlaid on csgA cells (CsgB + ).…”

Section: Ala Scan Mutagenesis Of the Aromatic Residuesmentioning

confidence: 99%

“…21 In a CsgB-dependent manner, soluble wild-type CsgA was converted into an amyloid fiber within 1 minute of the overlay as evidenced by Congo red staining, or by TEM. 21 Various concentrations of CsgA, CsgA Q49A and CsgA N144A were overlaid on csgA cells (CsgB + ). At a concentration of 10 μM or higher, CsgA polymerized into an amyloid fiber in a CsgB-dependent fashion detected by Congo red staining ( Figure 5(a)).…”

Section: Ala Scan Mutagenesis Of the Aromatic Residuesmentioning

confidence: 99%

Sequence Determinants of Bacterial Amyloid Formation

Wang

Chapman

2008

Journal of Molecular Biology

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108

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SummaryAmyloids are proteinaceous fibers commonly associated with neurodegenerative diseases and prionbased encephalopathies. Many different polypeptides can form amyloid fibers, leading to the suggestion that amyloid is a primitive main-chain-dominated structure. A growing body of evidence suggests that amino acid side chains dramatically influence amyloid formation. The specific role fulfilled by side chains in amyloid formation, especially in vivo, remains poorly understood. Here, we determined the role of internally conserved polar and aromatic residues in promoting amyloidogenesis of the functional amyloid protein, CsgA. CsgA is the major protein component of curli fibers assembled by enteric bacteria such as Escherichia coli and Salmonella spp. In vivo CsgA polymerization into an amyloid fiber requires the CsgB nucleator protein. The CsgA amyloid core region is composed of five repeating units, defined by regularly spaced Ser, Gln and Asn residues. The results of a comprehensive alanine scan mutagenesis screen showed that Gln and Asn residues at positions 49, 54, 139 and 144 were critical for curli assembly. Alanine substitution of Q49 or N144 impeded the ability of CsgA to respond to CsgB-mediated heteronucleation, and the ability of CsgA to self-polymerize in vitro. However, CsgA proteins harboring these mutations were still seeded by preformed wild-type CsgA fibers in vitro. This suggests that CsgA-fibril-mediated seeding and CsgBmediated heteronucleation have distinguishable mechanisms. Remarkably, Gln residues at positions 49 and 139 could not be replaced by Asn residues without interfering with curli assembly, suggesting that the side chain requirements were especially stringent at these positions. This analysis demonstrates that bacterial amyloid formation is driven by specific side chain contacts and provides a clear illustration of the essential roles of specific side chains in promoting amyloid formation.

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