Techniques to Analyze sRNA Protein Cofactor Self-Assembly In Vitro

Partouche, David; Malabirade, Antoine; Bizien, Thomas; Vélez, Marisela; Marco, Sergio; Militello, Valeria; Sandt, Christophe; Wien, Frank; Arluison, Véronique

doi:10.1007/978-1-4939-7634-8_18

Cited by 4 publications

(6 citation statements)

References 36 publications

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“…A related question concerns the effect of DNA on amyloidogenesis of Hfq. Hfq-CTR self-assembly in vitro is quite a slow mechanism 33 , 34 , and cellular co-factors probably help to accelerate this process. Such an effect has already been demonstrated for lipids 41 .…”

Section: Resultsmentioning

confidence: 99%

Revised role for Hfq bacterial regulator on DNA topology

Malabirade

Partouche

Hamoui

et al. 2018

Sci Rep

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Hfq is a pleiotropic regulator that mediates several aspects of bacterial RNA metabolism. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, usually via its interaction with small regulatory RNA. Besides these RNA-related functions, Hfq has also been described as one of the nucleoid associated proteins shaping the bacterial chromosome. Therefore, Hfq appears as a versatile nucleic acid-binding protein, which functions are probably even more numerous than those initially suggested. For instance, E. coli Hfq, and more precisely its C-terminal region (CTR), has been shown to induce DNA compaction into a condensed form. In this paper, we establish that DNA induces Hfq-CTR amyloidogenesis, resulting in a change of DNA local conformation. Furthermore, we clarify the effect of Hfq on DNA topology. Our results evidence that, even if the protein has a strong propensity to compact DNA thanks to its amyloid region, it does not affect overall DNA topology. We confirm however that hfq gene disruption influences plasmid supercoiling in vivo, indicating that the effect on DNA topology in former reports was indirect. Most likely, this effect is related to small regulatory sRNA-Hfq-based regulation of another protein that influences DNA supercoiling, possibly a nucleoid associated protein such as H-NS or Dps. Finally, we hypothesise that this indirect effect on DNA topology explains, at least partially, the previously reported effect of Hfq on plasmid replication efficiency.

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Section: Resultsmentioning

confidence: 99%

Revised role for Hfq bacterial regulator on DNA topology

Malabirade

Partouche

Hamoui

et al. 2018

Sci Rep

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“…On average, self‐assembly of Hfq‐CTR into amyloid fibres occurs after a few weeks at 4°C. The process of self‐assembly was confirmed by FTIR spectroscopy and TEM (Partouche et al ., ).…”

Section: Materials and Technical Set‐upmentioning

confidence: 99%

“…To better characterise these fibrils, a broad range of techniques is currently available for the detection and visualisation of amyloid nanostructures. These include antibodies for amyloid identification in vivo or in vitro (Moreno-DelÁlamo et al, 2015), light microscopy using specific fluorescent dies such as Thioflavin T and S, infrared (IR) spectroscopy, synchrotron radiation circular dichroism, small angle X-ray scattering for their structural characterisation and molecular imaging at the nanoscale using either atomic force microscopy (AFM) or transmission electron microscopy (TEM) (Partouche et al, 2018). Alone, none of them is able to perform direct chemical characterisation of single protein fibrils.…”

Section: Introductionmentioning

confidence: 99%

“…Nevertheless, AFM measurement fails in its poor spatial (XY axis) resolution compared to TEM. A correlative analysis with TEM could solve the resolution issue and give a better structural and chemical description of the object (Partouche et al, 2018). The sample analysed herein is the c-terminal region (CTR) of a bacterial protein called Hfq.…”

Section: Introductionmentioning

confidence: 99%

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Correlative infrared nanospectroscopy and transmission electron microscopy to investigate nanometric amyloid fibrils: prospects and challenges

Partouche

Mathurin

Malabirade

et al. 2019

Journal of Microscopy

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Summary Propagation of structural information through conformational changes in host‐encoded amyloid proteins is at the root of many neurodegenerative disorders. Although important breakthroughs have been made in the field, fundamental issues like the 3D‐structures of the fibrils involved in some of those disorders are still to be elucidated. To better characterise those nanometric fibrils, a broad range of techniques is currently available. Nevertheless none of them is able to perform direct chemical characterisation of single protein fibrils. In this work, we propose to investigate the structure of the C‐terminal region of a bacterial protein called Hfq as a model amyloidogenic protein, using a correlative approach. The complementary techniques used are transmission electron microscopy and a newly developed infrared nanospectroscopy technique called AFM‐IR. We introduce and discuss the strategy that we have implemented as well as the protocol, challenges and difficulties encountered during this study to characterise amyloid assemblies at the nearly single‐molecule level. Lay Description Propagation of structural information through conformational changes in amyloid proteins is at the root of many neurodegenerative disorders. Amyloids are nanostructures originating from the aggregation of multiple copies of peptide or protein monomers that eventually form fibrils. Often described as being the cause for the development of various diseases, amyloid fibrils are of major significance in the public health domain. While important breakthroughs have been made in the field, fundamental issues like the 3D‐structures of the fibrils implied in some of those disorders are still to be elucidated. To better characterise these fibrils, a broad range of techniques is currently available for the detection and visualisation of amyloid nanostructures. Nevertheless none of them is able to perform direct chemical characterisation of single protein fibrils. In this work, we propose to investigate the structure of model amyloidogenic fibrils using a correlative approach. The complementary techniques used are transmission electron microscopy and a newly developed infrared nanospectroscopy technique called AFM‐IR that allows chemical characterisation at the nanometric scale. The strategy, protocol, challenges and difficulties encountered in this approach are introduced and discussed herein.

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“…Hfq forms organized nano-assemblies in vivo, associated with the membrane, whose cellular pattern resembles that of long-range bacterial cytoskeletal structures [24,25]. Strikingly, these nanostructures are observable only in the presence of Hfq CTR [24], an apparently intrinsically disordered fragment that spontaneously forms amyloid structures in vitro [24,28,29]. These amyloid structures form for both the full-length protein and the isolated CTR region [24,30].…”

Section: Introductionmentioning

confidence: 99%

In Situ Characterization of Hfq Bacterial Amyloid: A Fourier-Transform Infrared Spectroscopy Study

et al. 2019

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Hfq is a bacterial protein that regulates gene expression at the post-transcriptional level in Gram-negative bacteria. We have previously shown that Escherichia coli Hfq protein, and more precisely its C-terminal region (CTR), self-assembles into an amyloid-like structure in vitro. In the present work, we present evidence that Hfq unambiguously forms amyloid structures also in vivo. Taking into account the role of this protein in bacterial adaptation and virulence, our work opens possibilities to target Hfq amyloid self-assembly and cell location, with important potential to block bacterial adaptation and treat infections.

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Techniques to Analyze sRNA Protein Cofactor Self-Assembly In Vitro

Cited by 4 publications

References 36 publications

Revised role for Hfq bacterial regulator on DNA topology

Revised role for Hfq bacterial regulator on DNA topology

Correlative infrared nanospectroscopy and transmission electron microscopy to investigate nanometric amyloid fibrils: prospects and challenges

In Situ Characterization of Hfq Bacterial Amyloid: A Fourier-Transform Infrared Spectroscopy Study

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