Publications by authors named "Aurélien Fouillen"

13 Publications

  • Page 1 of 1

A hydride transfer complex reprograms NAD metabolism and bypasses senescence.

Mol Cell 2021 09;81(18):3848-3865.e19

CRCHUM, 900 Saint-Denis St, Montréal, QC H2X 0A9, Canada; Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3C 3J7, Canada. Electronic address:

Metabolic rewiring and redox balance play pivotal roles in cancer. Cellular senescence is a barrier for tumorigenesis circumvented in cancer cells by poorly understood mechanisms. We report a multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP. This hydride transfer complex (HTC) is assembled by malate dehydrogenase 1, malic enzyme 1, and cytosolic pyruvate carboxylase. HTC is found in phase-separated bodies in the cytosol of cancer or hypoxic cells and can be assembled in vitro with recombinant proteins. HTC is repressed in senescent cells but induced by p53 inactivation. HTC enzymes are highly expressed in mouse and human prostate cancer models, and their inactivation triggers senescence. Exogenous expression of HTC is sufficient to bypass senescence, rescue cells from complex I inhibitors, and cooperate with oncogenic RAS to transform primary cells. Altogether, we provide evidence for a new multi-enzymatic complex that reprograms metabolism and overcomes cellular senescence.
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http://dx.doi.org/10.1016/j.molcel.2021.08.028DOI Listing
September 2021

A proline rich protein from the gingival seal around teeth exhibits antimicrobial properties against Porphyromonas gingivalis.

Sci Rep 2021 01 27;11(1):2353. Epub 2021 Jan 27.

Laboratory for the Study of Calcified Tissues and Biomaterials, Faculty of Dental Medicine, Université de Montréal, Montreal, QC, Canada.

The gingival seal around teeth prevents bacteria from destroying the tooth-supporting tissues and disseminating throughout the body. Porphyromonas gingivalis, a major periodontopathogen, degrades components of the specialized extracellular matrix that mediates attachment of the gingiva to the tooth. Of these, secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1) protein has a distinctive resistance to degradation, suggesting that it may offer resistance to bacterial attack. In silico analysis of its amino acid sequence was used to explore its molecular characteristics and to predict its two- and three-dimensional structure. SCPPPQ1 exhibits similarities with both proline-rich and cationic antimicrobial proteins, suggesting a putative antimicrobial potential. A combination of imaging approaches showed that incubation with 20 μM of purified SCPPPQ1 decrease bacterial number (p < 0.01). Fluorescence intensity decreased by 70% following a 2 h incubation of Porphyromonas gingivalis with the protein. Electron microscopy analyses revealed that SCPPPQ1 induced bacterial membrane disruption and breaches. While SCPPPQ1 has no effect on mammalian cells, our results suggest that it is bactericidal to Porphyromonas gingivalis, and that this protein, normally present in the gingival seal, may be exploited to maintain a healthy seal and prevent systemic dissemination of bacteria.
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http://dx.doi.org/10.1038/s41598-021-81791-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840901PMC
January 2021

New Insights Into Sulfur Metabolism Through Coupled Gene Expression, Solution Chemistry, Microscopy, and Spectroscopy Analyses.

Front Microbiol 2020 13;11:411. Epub 2020 Mar 13.

School of Geography and Earth Science, Faculty of Science, McMaster University, Hamilton, ON, Canada.

Here, we experimentally expand understanding of the reactions and enzymes involved in ATCC 19377 S and metabolism by developing models that integrate gene expression analyzed by RNA-Seq, solution sulfur speciation, electron microscopy and spectroscopy. The metabolism model involves the conversion of to , S and mediated by the sulfur oxidase complex (Sox), tetrathionate hydrolase (TetH), sulfide quinone reductase (Sqr), and heterodisulfate reductase (Hdr) proteins. These same proteins, with the addition of rhodanese (Rhd), were identified to convert S to , and polythionates in the S metabolism model. Our combined results shed light onto the important role specifically of TetH in metabolism. Also, we show that activity of Hdr proteins rather than Sdo are likely associated with S oxidation. Finally, our data suggest that formation of intracellular is a critical step in S metabolism, and that recycling of internally generated occurs, through comproportionating reactions that result in . Electron microscopy and spectroscopy confirmed intracellular production and storage of S during growth on both S and substrates.
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http://dx.doi.org/10.3389/fmicb.2020.00411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082400PMC
March 2020

Nanoporosity Stimulates Cell Spreading and Focal Adhesion Formation in Cells with Mutated Paxillin.

ACS Appl Mater Interfaces 2020 Apr 19;12(13):14924-14932. Epub 2020 Mar 19.

Laboratory for the Study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dentistry, Université de Montréal, Montréal, Québec H3C3J7, Canada.

We have evaluated the response to nanotopography of CHO-K1 cells that express wild-type paxillin or paxillin with mutations at serine 273 that inhibit phosphorylation. Cells were grown on nanoporous and polished titanium surfaces. With all cell types, immunofluorescence showed that adhesion and spreading were minimally affected on the treated surface and that the actin filaments were more abundant and well-aligned. Scanning electron microscopy revealed changes in cell shape and abundant filopodia with lateral nanoprotrusions in response to nanoporosity. Gene expression of proteins associated with cellular adhesion and protrusions was significantly increased on the nanoporous surface regardless of the cell type. In particular, α-actinin, Rac1, Cdc42, and ITGα1 were upregulated in S273 cells with alanine substitutions, whereas FAK, Pxn, and Src were downregulated, leading to improved focal adhesion formation. These findings suggest that the surface nanoporosity can "compensate for" the genetic mutations that affect the biomechanical relationship of cells to surfaces.
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http://dx.doi.org/10.1021/acsami.0c01172DOI Listing
April 2020

Selective bacterial degradation of the extracellular matrix attaching the gingiva to the tooth.

Eur J Oral Sci 2019 08 22;127(4):313-322. Epub 2019 Jun 22.

Laboratory for the Study of Calcified Tissues and Biomaterials, Université de Montréal, Montréal, QC, Canada.

The junctional epithelium (JE) is a specialized portion of the gingiva that seals off the tooth-supporting tissues from the oral environment. This relationship is achieved via a unique adhesive extracellular matrix that is, in fact, a specialized basal lamina (sBL). Three unique proteins - amelotin (AMTN), odontogenic ameloblast-associated (ODAM), and secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1) - together with laminin-332 structure the supramolecular organization of this sBL and determine its adhesive capacity. Despite the constant challenge of the JE by the oral microbiome, little is known of the susceptibility of the sBL to bacterial degradation. Assays with trypsin-like proteases, as well as incubation with Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola, revealed that all constituents, except SCPPPQ1, were rapidly degraded. Porphyromonas gingivalis was also shown to alter the supramolecular network of reconstituted and native sBLs. These results provide evidence that proteolytic enzymes and selected gram-negative periodontopathogenic bacteria can attack this adhesive extracellular matrix, intimating that its degradation could contribute to progression of periodontal diseases.
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http://dx.doi.org/10.1111/eos.12623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771947PMC
August 2019

High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension.

BMC Biotechnol 2018 12 6;18(1):76. Epub 2018 Dec 6.

Département de Biochimie et Médecine Moléculaire, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, H3C 3J7, QC, Canada.

Background: Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as the ribonuclease III enzyme responsible for cleaving precursor miRNA substrates. Its enzymatic activity is highly regulated by protein factors, and this regulation can impact on the levels of miRNAs and modulate the behavior of a cell. To better understand the underlying mechanisms of regulation, detailed enzymatic and structural characterization of Dicer are needed. However, these types of studies generally require several milligrams of recombinant protein, and efficient preparation of such quantities of pure human Dicer remains a challenge. To prepare large quantities of human Dicer, we have optimized transfection in HEK293-6E cells grown in suspension and streamlined a purification procedure.

Results: Transfection conditions were first optimized to achieve expression levels between 10 and 18 mg of recombinant Dicer per liter of culture. A three-step purification protocol was then developed that yields 4-9 mg of purified Dicer per liter of culture in a single day. From SEC-MALS/RI analysis and negative stain TEM, we confirmed that the purified protein is monomerically pure ( ≥ 98%) and folds with the characteristic L-shape geometry. Using an electrophoretic mobility shift assay, a dissociation constant (K) of 5 nM was measured for Dicer binding to pre-let-7a-1, in agreement with previous reports. However, when probing the cleavage activity of Dicer for pre-let-7a-1, we measured k (7.2 ± 0.5 min) and K (1.2 ± 0.3 μM) values that are much higher than previously reported due to experimental conditions that better respect the steady-state assumption.

Conclusions: The expression and purification protocols described here provide high yields of monomerically pure and active human Dicer. Cleavage studies of a pre-let-7 substrate with this purified Dicer reveal higher k and K values than previously reported and support the current view that conformational changes are associated with substrate binding. Large quantities of highly pure Dicer will be valuable for future biochemical, biophysical and structural investigations of this key protein of the miRNA pathway.
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http://dx.doi.org/10.1186/s12896-018-0485-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282390PMC
December 2018

Interaction via the N terminus of the type IV secretion system (T4SS) protein VirB6 with VirB10 is required for VirB2 and VirB5 incorporation into T-pili and for T4SS function.

J Biol Chem 2018 08 5;293(35):13415-13426. Epub 2018 Jul 5.

From the Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Quebec H3C 3J7, Canada and

Many bacterial pathogens employ multicomponent protein complexes such as type IV secretion systems (T4SSs) to transfer virulence factors into host cells. Here we studied the interaction between two essential T4SS components: the very hydrophobic inner membrane protein VirB6, which may be a component of the translocation channel, and VirB10, which links the inner and outer bacterial membranes. To map the interaction site between these two T4SS components, we conducted alanine scanning and deleted six-amino acid stretches from the N-terminal periplasmic domain of VirB6 from Using the bacterial two-hybrid system to analyze the effects of these alterations on the VirB6-VirB10 interaction, we identified the amino acid regions 16-21 and 28-33 and Leu-18 in VirB6 as being required for this interaction. SDS-PAGE coupled with Western blotting of cell lysates and native PAGE of detergent-extracted membrane proteins revealed that the corresponding VirB6 residues in (Phe-20 and amino acids 18-23 and 30-35) modulate the stability of both VirB6 and VirB5. However, the results from immuno-EM and super-resolution microscopy suggested that these regions and residues are not required for membrane association or for polar localization of VirB6. The six-amino acid deletions in the N terminus of VirB6 abolished pilus formation and virulence of , and the corresponding deletions in the VirB6 homolog TraD from the plasmid pKM101-T4SS abrogated plasmid transfer. Our results indicate that specific residues of the VirB6 N-terminal domain are required for VirB6 stabilization, its interaction with VirB10, and the incorporation of VirB2 and VirB5 into T-pili.
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http://dx.doi.org/10.1074/jbc.RA118.002751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120205PMC
August 2018

VirB8 homolog TraE from plasmid pKM101 forms a hexameric ring structure and interacts with the VirB6 homolog TraD.

Proc Natl Acad Sci U S A 2018 06 21;115(23):5950-5955. Epub 2018 May 21.

Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, QC, Canada H3C 3J7;

Type IV secretion systems (T4SSs) are multiprotein assemblies that translocate macromolecules across the cell envelope of bacteria. X-ray crystallographic and electron microscopy (EM) analyses have increasingly provided structural information on individual T4SS components and on the entire complex. As of now, relatively little information has been available on the exact localization of the inner membrane-bound T4SS components, notably the mostly periplasmic VirB8 protein and the very hydrophobic VirB6 protein. We show here that the membrane-bound, full-length version of the VirB8 homolog TraE from the plasmid pKM101 secretion system forms a high-molecular-mass complex that is distinct from the previously characterized periplasmic portion of the protein that forms dimers. Full-length TraE was extracted from the membranes with detergents, and analysis by size-exclusion chromatography, cross-linking, and size exclusion chromatography (SEC) multiangle light scattering (MALS) shows that it forms a high-molecular-mass complex. EM and small-angle X-ray scattering (SAXS) analysis demonstrate that full-length TraE forms a hexameric complex with a central pore. We also overproduced and purified the VirB6 homolog TraD and show by cross-linking, SEC, and EM that it binds to TraE. Our results suggest that TraE and TraD interact at the substrate translocation pore of the secretion system.
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http://dx.doi.org/10.1073/pnas.1802501115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003364PMC
June 2018

TDP-43 regulates the alternative splicing of hnRNP A1 to yield an aggregation-prone variant in amyotrophic lateral sclerosis.

Brain 2018 05;141(5):1320-1333

Department of Neurosciences, Université de Montréal, Montréal, QC, Canada.

See Fratta and Isaacs (doi:10.1093/brain/awy091) for a scientific commentary on this article.The RNA binding proteins TDP-43 (encoded by TARDBP) and hnRNP A1 (HNRNPA1) are each mutated in certain amyotrophic lateral sclerosis cases and are often mislocalized in cytoplasmic aggregates within motor neurons of affected patients. Cytoplasmic inclusions of TDP-43, which are accompanied by a depletion of nuclear TDP-43, are observed in most amyotrophic lateral sclerosis cases and nearly half of frontotemporal dementia cases. Here, we report that TDP-43 binds HNRNPA1 pre-mRNA and modulates its splicing, and that depletion of nuclear TDP-43 results in increased inclusion of a cassette exon in the HNRNPA1 transcript, and consequently elevated protein levels of an isoform containing an elongated prion-like domain, referred to as hnRNP A1B. Combined in vivo and in vitro approaches demonstrated greater fibrillization propensity for hnRNP A1B, which drives protein aggregation and is toxic to cells. Moreover, amyotrophic lateral sclerosis patients with documented TDP-43 pathology showed neuronal hnRNP A1B cytoplasmic accumulation, indicating that TDP-43 mislocalization may contribute to neuronal vulnerability and loss via altered HNRNPA1 pre-mRNA splicing and function. Given that TDP-43 and hnRNP A1 each bind, and thus modulate, a third of the transcriptome, our data suggest a much broader disruption in RNA metabolism than previously considered.
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http://dx.doi.org/10.1093/brain/awy062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5917749PMC
May 2018

A nanoporous titanium surface promotes the maturation of focal adhesions and formation of filopodia with distinctive nanoscale protrusions by osteogenic cells.

Acta Biomater 2017 09 17;60:339-349. Epub 2017 Jul 17.

Laboratory for the Study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dentistry, Université de Montréal, C.P 6128 succursale Centre-Ville, Montréal, Québec H3C3J7, Canada; Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, C.P 6128 succursale Centre-Ville, Montréal, Québec H3C3J7, Canada. Electronic address:

While topography is a key determinant of the cellular response to biomaterials, the mechanisms implicated in the cell-surface interactions are complex and still not fully elucidated. In this context, we have examined the effect of nanoscale topography on the formation of filopodia, focal adhesions, and gene expression of proteins associated with cell adhesion and sensing. Commercially pure titanium discs were treated by oxidative nanopatterning with a solution of HSO/HO 50:50 (v/v). Scanning electron microscopy and atomic force microscopy characterizations showed that this facile chemical treatment efficiently creates a unique nanoporous surface with a root-mean-square roughness of 11.5nm and pore diameter of 20±5nm. Osteogenic cells were cultured on polished (control) and nanotextured discs for periods of 6, 24, and 72h. Immunofluorescence analysis revealed increases in the adhesion formation per cell area, focal adhesion length, and maturity on the nanoporous surface. Gene expression for various focal adhesion markers, including paxillin and talin, and different integrins (e.g. α1, β1, and α5) was also significantly increased. Scanning electron microscopy revealed the presence of more filopodia on cells grown on the nanoporous surface. These cell extensions displayed abundant and distinctive nanoscale lateral protrusions of 10-15nm diameter that molded the nanopore walls. Together the increase in the focal adhesions and abundance of filopodia and associated protrusions could contribute to strengthening the adhesive interaction of cells with the surface, and thereby, alter the nanoscale biomechanical relationships that trigger cellular cascades that regulate cell behavior.

Statement Of Significance: Oxidative patterning was exploited to create a unique three-dimensional network of nanopores on titanium surfaces. Our study illustrates how a facile chemical treatment can be advantageously used to modulate cellular behavior. The nanoscale lateral protrusions on filopodia elicited by this surface are novel adhesive structures. Altogether, the increases in focal adhesion, length, maturity, and filopodia with distinctive lateral protrusions could substantially increase the contact area and adhesion strength of cells, thereby promoting the activation of cellular signaling cascades that may explain the positive osteogenic outcomes previously achieved with this surface. Such physicochemical cueing offers a simple attractive alternative to the use of bioactive agents for guiding tissue repair/regeneration around implantable metals.
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http://dx.doi.org/10.1016/j.actbio.2017.07.022DOI Listing
September 2017

Interactions of AMTN, ODAM and SCPPPQ1 proteins of a specialized basal lamina that attaches epithelial cells to tooth mineral.

Sci Rep 2017 04 24;7:46683. Epub 2017 Apr 24.

Laboratory for the Study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dental medicine Université de Montréal, Montréal, Québec, Canada.

A specialized basal lamina (sBL) mediates adhesion of certain epithelial cells to the tooth. It is distinct because it does not contain collagens type IV and VII, is enriched in laminin-332, and includes three novel constituents called amelotin (AMTN), odontogenic ameloblast-associated (ODAM), and secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1). The objective of this study was to clarify the structural organization of the sBL. Fluorescence and immunogold labeling showed that the three proteins co-localize. Quantitative analysis of the relative position of gold particles on the sBL demonstrates that the distribution of ODAM is skewed towards the cell while that of AMTN and SCPPPQ1 tends towards the tooth surface. Bacterial two-hybrid analysis and co-immunoprecipitation, gel filtration of purified proteins and transmission electron and atomic force microscopies highlight the propensity of AMTN, ODAM, and SCPPPQ1 to interact with and among themselves and form supramolecular aggregates. These data suggest that AMTN, ODAM and SCPPPQ1 participate in structuring an extracellular matrix with the distinctive capacity of attaching epithelial cells to mineralized surfaces. This unique feature is particularly relevant for the adhesion of gingival epithelial cells to the tooth surface, which forms a protective seal that is the first line of defense against bacterial invasion.
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http://dx.doi.org/10.1038/srep46683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5402393PMC
April 2017

Cag-delta (Cag3) protein from the Helicobacter pylori 26695 cag type IV secretion system forms ring-like supramolecular assemblies.

FEMS Microbiol Lett 2017 01 15;364(1). Epub 2016 Dec 15.

Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montreal, QC H3C 3J7, Canada

Helicobacter pylori is an important cause of gastric pathologies and persistent infection can lead to stomach cancer. Virulent H. pylori strains encode a type IV secretion system responsible for translocation of the oncogenic CagA protein into cells of the gastric mucosa. Gene HP0522 encodes the essential component Cagδ (Cag3), and we show by gel filtration and cross-linking that purified Cagδ forms high molecular mass complexes. In contrast, its interaction partner CagT is mostly monomeric, but co-fractionates after gel filtration. Analysis by transmission electron microscopy revealed that purified Cagδ complexes can self-assemble ring-like structures. Cagδ-overexpressing Escherichia coli exhibits membrane-associated circular profiles in regions of the cell envelope with intense immunogold labelling with a Cagδ-specific antiserum. Our results suggest that Cagδ has the capacity to form macromolecular structures contributing to the assembly of the type IV secretion system.
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http://dx.doi.org/10.1093/femsle/fnw280DOI Listing
January 2017

Laminin γ2 knockout mice rescued with the human protein exhibit enamel maturation defects.

Matrix Biol 2016 May-Jul;52-54:207-218. Epub 2016 Mar 5.

Laboratory for the Study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dentistry, Université de Montréal, Montréal, Québec, Canada; Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Québec, Canada. Electronic address:

The epithelial ameloblasts are separated from the maturing enamel by an atypical basement membrane (BM) that is enriched in laminin 332 (LM-332). This heterotrimeric protein (α3, ß3 and γ2 chains) provides structural integrity to BMs and influences various epithelial cell processes including cell adhesion and differentiation. Mouse models that lack expression of individual LM-332 chains die shortly after birth. The lethal phenotype of laminin γ2 knockout mice can be rescued by human laminin γ2 (LAMC2) expressed using a doxycycline-inducible (Tet-on) cytokeratin 14 promoter-rtTA. These otherwise normal-looking rescued mice exhibit white spot lesions on incisors. We therefore investigated the effect of rescue with human LAMC2 on enamel maturation and structuring of the atypical BM. The maturation stage enamel organ in transgenic mice was severely altered as compared to wild type controls, a structured BM was no longer discernible, dystrophic matrix appeared in the maturing enamel layer, and there was residual enamel matrix late into the maturation stage. Microtomographic scans revealed excessive wear of occlusal surfaces on molars, chipping of enamel on incisor tips, and hypomineralization of the enamel layer. No structural alterations were observed at other epithelial sites, such as skin, palate and tongue. These results indicate that while this humanized mouse model is capable of rescue in various epithelial tissues, it is unable to sustain structuring of a proper BM at the interface between ameloblasts and maturing enamel. This failure may be related to the atypical composition of the BM in the maturation stage and reaffirms that the atypical BM is essential for enamel maturation.
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http://dx.doi.org/10.1016/j.matbio.2016.03.002DOI Listing
December 2017
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