Marinieve Montero | Université de Montréal (original) (raw)

Papers by Marinieve Montero

The author has further granted permission to Simon Fraser University to keep or make a digital co... more The author has further granted permission to Simon Fraser University to keep or make a digital copy for use in its circulating collection (currently available to the public at the "Institutional Repository" link of the SFU Library website <www.lib.sfu.ca> at: <http://ir.lib.sfu.ca/handle/1892/112>) and, without changing the content, to translate the thesis/project or extended essays, if technically possible, to any medium or format for the purpose of preservation of the digital work. The author has further agreed that permission for multiple copying of this work for scholarly purposes may be granted by either the author or the Dean of Graduate Studies. It is understood thatcopying or publication of this work for financial gain shall not be allowed without the author's written permission. Permission for public performance, or limited permission for private scholarly use, of any multimedia materials forming part of this work, may have been granted by the author. This information may be found on the separately catalogued multimedia material and in the signed Partial Copyright Licence. While licensing SFU to permit the above uses, the author retains copyright in the thesis, project or extended essays, including the right to change the work for subsequent purposes, including editing and publishing the work in whole or in part, and licensing other parties, as the author may desire.

Senescence is a stable arrest of cell proliferation in which the cells remain viable and metaboli... more Senescence is a stable arrest of cell proliferation in which the cells remain viable and metabolically active but display a constitutive activation of the DNA damage response and of the tumor suppressors p53 and RB. The senescent phenotype can be induced by multiple stresses including short telomeres and oncogenes. We have shown that senescence involves the ERK-dependent degradation of selective proteins involved in cell cycle progression and tumorigenesis. We called this process senescence associated protein degradation (SAPD) and it involves many nucleolar proteins that play a role in ribosome biogenesis. Using tritium pulse labelling we found a strong decrease of rRNA synthesis in senescent cells. In addition, qPCR with primers flanking known rRNA processing sites demonstrated defects in the processing of rRNA in senescent cells. Knocking down some of the nucleolar proteins degraded in senescence was sufficient to trigger the process indicating that a decrease in ribosome biogenesis is causal to cellular senescence. Senescence induced by depletion of ribosome biogenesis factors was characterized by accumulation of p53 and in some cases rescued by inhibition of p53 using shRNAs or the viral oncoprotein E6. We will discuss how reduced ribosome biogenesis impact protein biosynthesis in senescent cells. Citation Format: Frédéric Lessard, Véronique Bourdeau, Sebastian Igelmann, Xavier Deschênes-Simard, Marinieve Montero, Gerardo Ferbeyre. Ribosome biogenesis is reduced by oncogenic stress in normal cells and is sufficient to trigger cellular senescence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1270. doi:10.1158/1538-7445.AM2015-1270

Senescence is a stable arrest of cell proliferation in which the cells remain viable and metaboli... more Senescence is a stable arrest of cell proliferation in which the cells remain viable and metabolically active but display a constitutive activation of the DNA damage response and of the tumor suppressors p53 and RB. The senescent phenotype can be induced by multiple stresses including short telomeres and oncogenes. We have shown that senescence, involves the ERK-dependent degradation of selective proteins involved in cell cycle progression and tumorigenesis. We call this process senescence associated protein degradation (SAPD) and it involves many nucleolar proteins that play a role in ribosome biogenesis. Using tritium pulse labelling we found a strong decrease of rRNA synthesis in senescent cells indicating that the degradation of nucleolar proteins is functionally relevant. Because we know how exactly the human 47S precursor rRNA is processed, it was possible to design primers on both sides of some processing sites and study their maturation by QPCR. In this way we showed defects in the processing of rRNA in senescent cells. Knocking down some of the nucleolar proteins degraded in senescence was sufficient to trigger the process indicating that a decrease in ribosome biogenesis is causal to cellular senescence. Mechanistically, the degradation of nucleolar proteins during senescence involves the ubiquitin-proteasome system suggesting that E3 ligases link the oncogenic stress that trigger senescence to nucleolar proteins degradation. We will discuss ongoing efforts to identify these enzymes. Citation Format: Frédéric Lessard, Véronique Bourdeau, Xavier Deschênes-Simard, Sebastian Igelmann, Marinieve Montero, Gerardo Ferbeyre. Senescence as a result of impaired ribosome biogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2246. doi:10.1158/1538-7445.AM2014-2246

Immunotechnology, Feb 1, 1996

Backgroutd Experimental evidence suggests that neutralizing antibodies could constitute an import... more Backgroutd Experimental evidence suggests that neutralizing antibodies could constitute an important factor in the control of AIDS progression and that the V3 loop of gpl20 constitutes the main target for such purposes. We have previously developed two neutralizing murine monoclonal antibodies (Mabs) against the V3 region of the HIV-l MN strain. Objecrives: To characterize those Mabs in terms of fine specificity and DNA sequence of their V regions and to study if Fab fragments retain their neutralizing potential in vitro. Study design: A set of 1Zmer alanine substituted peptides were employed for epitope mapping using two ELBA procedures: (1) indirect, with each peptide bound to polystyrene plates, and (2) competitive, with co-incubation of peptides and Mabs in solution. The V regions of both Mabs were PCR amplified from cDNA and their nucleotide sequences were determined. Finally, Fab fragments of Mab 1OFlO were generated and their neutralizing capacity against the MN isolated was assessed. Resulfs: We first restricted the minimal length of the epitopes recognized by 2C4 and 1OFlO to the 12-mer peptide KRIHIGPGRAFY. The core of the epitopes recognized by Mabs 2C4 and 1OFlO were IHIGP-R and IHIG-R, respectively. While substitution of proline in position 7 completely abolished the binding of 2C4, it only reduced that of 1OFlO by 50%. Finally, Fab fragments of Mab 1OFlO were still able to neutralize the HIV-l MN strain in vitro. Conclusion: This subtle distinction in the tine mapping of the epitope recognized by Mabs 2C4 and 1OFlO should correspond to three amino acid differences that we found in the heavy chain V-regions. The Fab fragments of Mab 1OFlO retained the neutralizing capacity. This indicates that HIV neutralization by anti V3 Mabs is an Fc independent process.

American Journal of Physiology-gastrointestinal and Liver Physiology, Aug 1, 2018

Paneth cells are a key subset of secretory epithelial cells found at the base of small intestinal... more Paneth cells are a key subset of secretory epithelial cells found at the base of small intestinal crypts. Unlike intestinal goblet cells, which secrete the mucin Muc2, Paneth cells are best known for producing an array of antimicrobial factors. We unexpectedly identified Muc2 staining localized around Paneth cell granules. Electron microscopy (EM) confirmed an electron lucent halo around these granules, which was lost in Paneth cells from Muc2-deficient (Ϫ/Ϫ) mice. EM and immunostaining for lysozyme revealed that Muc2 Ϫ/Ϫ Paneth cells contained larger, more densely packed granules within their cytoplasm, and we detected defects in the transcription of key antimicrobial genes in the ileal tissues of Muc2 Ϫ/Ϫ mice. Enteroids derived from the small intestine of wild-type and Muc2 Ϫ/Ϫ mice revealed phenotypic differences in Paneth cells similar to those seen in vivo. Moreover, lysozyme-containing granule release from Muc2 Ϫ/Ϫ enteroid Paneth cells was shown to be impaired. Surprisingly, Paneth cells within human ileal and duodenal tissues were found to be Muc2 negative. Thus Muc2 plays an important role in murine Paneth cells, suggesting links in function with goblet cells; however human Paneth cells lack Muc2, highlighting that caution should be applied when linking murine to human Paneth cell functions. NEW & NOTEWORTHY We demonstrate for the first time that murine Paneth cell granules possess a halo comprised of the mucin Muc2. The presence of Muc2 exerts an impact on Paneth cell granule size and number and facilitates the release and dispersal of antimicrobials into the mucus layer. Interestingly, despite the importance of Muc2 in murine Paneth cell function, our analysis of Muc2 in human intestinal tissues revealed no trace of Muc2 expression by human Paneth cells.

Proceedings of the National Academy of Sciences of the United States of America, Sep 27, 2010

Journal of Molecular Biology, Jul 13, 2007

The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclo... more The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary HIV-1 isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N-terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 Å resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against

Microbiology and Molecular Biology Reviews, Jun 1, 2008

Journal of Molecular Biology, Jun 1, 2007

PLOS ONE, Mar 30, 2011

Background: Antibodies (Abs) produced during HIV-1 infection rarely neutralize a broad range of v... more Background: Antibodies (Abs) produced during HIV-1 infection rarely neutralize a broad range of viral isolates; only eight broadly-neutralizing (bNt) monoclonal (M)Abs have been isolated. Yet, to be effective, an HIV-1 vaccine may have to elicit the essential features of these MAbs. The V genes of all of these bNt MAbs are highly somatically mutated, and the V H genes of five of them encode a long ($20 aa) third complementarity-determining region (CDR-H3). This led us to question whether long CDR-H3s and high levels of somatic mutation (SM) are a preferred feature of anti-HIV bNt MAbs, or if other adaptive immune responses elicit them in general. Methodology and Principal Findings: We assembled a V H-gene sequence database from over 700 human MAbs of known antigen specificity isolated from chronic (viral) infections (ChI), acute (bacterial and viral) infections (AcI), and systemic autoimmune diseases (SAD), and compared their CDR-H3 length, number of SMs and germline V H-gene usage. We found that anti-HIV Abs, regardless of their neutralization breadth, tended to have long CDR-H3s and high numbers of SMs. However, these features were also common among Abs associated with other chronic viral infections. In contrast, Abs from acute viral infections (but not bacterial infections) tended to have relatively short CDR-H3s and a low number of SMs, whereas SAD Abs were generally intermediate in CDR-H3 length and number of SMs. Analysis of V H gene usage showed that ChI Abs also tended to favor distal germline V H-genes (particularly V H 1-69), especially in Abs bearing long CDR-H3s. Conclusions and Significance: The striking difference between the Abs produced during chronic vs. acute viral infection suggests that Abs bearing long CDR-H3s, high levels of SM and V H 1-69 gene usage may be preferentially selected during persistent infection.

Anti-inflammatory & anti-allergy agents in medicinal chemistry, Sep 1, 2009

Inflammatory bowel diseases (IBD) are thought to occur because of impaired mucosal integrity that... more Inflammatory bowel diseases (IBD) are thought to occur because of impaired mucosal integrity that allows enteric bacteria to leak out of the intestine, triggering maladaptive intestinal inflammation. While the exact pathogenesis of IBD is unclear, studies have recently demonstrated that bacterial activation of innate receptors not only causes inflammation, but may also play an important role in modulating intestinal epithelial barrier function, as well as intestinal epithelial cell proliferation and repair. These mucosal homeostatic mechanisms are essential in limiting as well as repairing mucosal damage. Therefore bacterial activation of the innate immune system can have both inflammatory as well as protective healing roles in the intestine. Strikingly, these findings suggest that dysregulation of these processes could be responsible for both the barrier dysfunction as well as the heightened inflammatory tone that characterize IBD. In this review we explore the current state of knowledge underlying this novel role for innate immunity in the gastrointestinal tract, and discuss the strengths and weaknesses of the chemical and infectious models used in these studies. In addition, we discuss preliminary evidence that exaggerated microbial activation of the innate immune system may cause the fibrotic responses that develop in some patients with IBD.

Nature Cell Biology, Jun 25, 2018

ellular senescence opposes neoplastic transformation by preventing the proliferation of cells tha... more ellular senescence opposes neoplastic transformation by preventing the proliferation of cells that have experienced oncogenic stimuli 1. Senescence is efficient as a tumour suppressor mechanism as long as it prevents cell cycle progression. Some benign lesions contain senescent cells that remain out of the cell cycle 2,3. Senescent cells also stimulate their own clearance by the immune system, thereby strengthening the defenses against tumourigenesis 4,5. However, the process is not infallible and lesions containing senescent cells can progress into malignant cancers 6,7. To escape from senescence, cells need to increase protein biosynthesis and, more importantly, the machinery to do so. Indeed, ribosome biogenesis is upregulated in cancer cells 8,9. Senescent cells exhibit reduced ribosome biogenesis and this has been explained by a delay in ribosomal RNA (rRNA) processing eventually leading to the accumulation of the ribosomal proteins L5 (uL18) and L11 (uL5), which form a complex with the 5S rRNA and disable the E3 ligase MDM2 (or HDM2), thus activating p53 10. Conversely, drugs that reduce ribosome biogenesis can trigger senescence and are considered very promising anticancer therapeutics 11-13. Cellular senescence can be induced in cells in the absence of p53 14 through a pathway that involves several members of the cyclin-dependent kinase inhibitor (CKI) family, such as p16INK4a (p16) and p15INK4b (or CDKN2B), and the retinoblastoma (Rb1 or Rb) tumour suppressor 15,16. Rb controls cell cycle progression by repressing E2F activity, and in senescent cells, by promoting the formation of heterochromatin at E2F target promoters 3,17. In proliferating cells or cells that escape from senescence, Rb is inhibited by cyclin-dependent kinases such as CDK2, CDK4 and CDK6 18 .

The Journal of Immunology

The limited success of vaccines targeting the MPER, a target of three broadly neutralizing (bNt) ... more The limited success of vaccines targeting the MPER, a target of three broadly neutralizing (bNt) monoclonal antibodies (MAbs), we hypothesize, reflects the difficulty of mimicking the neutralization-competent structure (NCS) of the MPER. We determined the contribution of the amino-acid sequence and the transmembrane domain (TM), to the antigenicity of the MPER in the context of the plasma membrane. DNA constructs encoding various gp41 ectodomain fragments, and the TM of either the platelet-derived growth factor receptor (PGDFR), or that of gp41, were produced and transiently expressed in COS-7 cells. Constructs expressing the MPER tethered to the gp41 TM followed by a 27-residue cytoplasmic tail fragment, MPER-TM1, produced optimal binding of MAbs 2F5, 4E10 and Z13e1. A series of 24 single amino-acid substitutions in the MPER-TM1 revealed critical binding residues for the three MAbs; similar substitutions were previously shown to ablate Ab-mediated viral neutralization. Neutralizati...

Journal of the International AIDS Society, 2012

Background: Many HIV databases and applications focus on a limited domain of HIV knowledge. Since... more Background: Many HIV databases and applications focus on a limited domain of HIV knowledge. Since even a ''simple'' organism like HIV represents a very complex system with many interacting elements, the fractured structure of existing databases and applications likely limits our ability to investigate and understand HIV. To facilitate research, therefore, we have built HIVToolbox, which integrates much of the knowledge about HIV proteins and presents the data in an interactive web application. HIVToolbox allows quick and convenient hypotheses generation, experiment interpretation, and potential new drug structure creation. Methods: HIVToolbox was built as a standard three-tier J2EE web application, consisting of 1) an underlying relational MySQL database, 2) a set of standard Java data access objects that pull data from the database, and 3) a set of dynamic web pages the user interacts with. HIV-1 data from external sources such as the Protein Data Bank, NCBI, Los Alamos, etc. was collected, curated, and stored in the HIVToolbox database. Additional data, such as homology and position statistics matrices, was generated from existing data. Since version 1, drug binding site and drug resistant mutation data has also been added. Results: HIVToolbox was used to create several new hypotheses about HIV-1 integrase, including predicting the location of a CK2 phosphorylation site, which was later confirmed by experiment. Track A Basic Science Figure 1. Protease with drug Amprenavir shown [Interactive HIV protein page].

Molecular Immunology, 2010

Peptide "mimics" (mimotopes) of linear protein epitopes and carbohydrate epitopes have been succe... more Peptide "mimics" (mimotopes) of linear protein epitopes and carbohydrate epitopes have been successfully used as immunogens to elicit cross-reactive antibodies against their cognate epitopes; however, immunogenic mimicry has been difficult to achieve for discontinuous protein epitopes. To explore this, we developed from phage-displayed peptide libraries optimized peptide mimics for three well-characterized discontinuous epitopes on hen egg lysozyme and horse cytochrome C. The peptides competed with their cognate antigens for antibody binding, displayed affinities in the nM range, and shared critical binding residues with their native epitopes. Yet, while immunogenic, none of the peptides elicited antibodies that cross-reacted with their cognate antigens. We analyzed the 3-D structure of the site within each discontinuous epitope that shared critical binding residues with its peptide mimic, and observed that in each case it formed a ridge-like patch on the epitope; in no case did it cover most or all of the epitope. Thus, the peptides' lack of immunogenic mimicry could be attributed to their inability to recapitulate the topological features of their cognate epitopes. Our results suggest that direct peptide immunizations are not a practical strategy for generating targeted antibody responses against discontinuous epitopes.

Journal of Innate Immunity, 2012

Paneth cell α-defensins are antimicrobial peptides involved in the control of the intestinal micr... more Paneth cell α-defensins are antimicrobial peptides involved in the control of the intestinal microbiota and immunological homeostasis. In mice, they are encoded by multiple, highly homologous genes (Defa). The transcriptional activity of ileal Defa genes was studied in response to pharmacological and genetic perturbations of the intestinal environment of C57BL/6 mice. Defa gene transcription was sensitive to oral antibiotic administration suggesting that commensal microbes regulate Defa expression. Ileal microbiota analysis showed that decreased transcription of Defa genes correlated with depletion of Lactobacillus. Defa expression was partially restored in vivo by lactobacillus administration to antibiotic-treated mice. Defa transcripts were less abundant in ex vivo, microbiota-free intestinal explants but recovered after explant exposure to UV-killed bacteria, Toll-like receptor (TLR)-2 or TLR4 agonists. Genetic deficiency of several TLRs or MyD88 led to dramatic drops in Defa tra...

Applied Microbiology and Biotechnology, 1990

The production of human gamma interferon as intracellular inclusion bodies in Escherichia coli, w... more The production of human gamma interferon as intracellular inclusion bodies in Escherichia coli, which simplified the purification process, is described. An expression plasmid carrying lipoprotein and the tryptophane promoters in tandem was used. Preparation of highly pure interferon was achieved using high resolution chromatography after denaturation and renaturation steps. Structural characteristics of this protein were verified by mass spectrometric analysis. Additional control tests have shown the suitability of the final product for clinical purposes.

AIDS Research and Human Retroviruses, 1994

A gene encoding a multiepitope polypeptide (MEP) has been synthesized. It contains the informatio... more A gene encoding a multiepitope polypeptide (MEP) has been synthesized. It contains the information for (1) an 11-amino acid (aa) epitope from the Cl region of gpl20 of HIV-1 and (2) 3 epitopes of 15 amino acids each, from the central part of the V3 loop of isolates MN, SC, and WMJH. These four segments are linked by the short spacer peptide AGGGA. This gene was cloned in a plasmid vector and expressed in Escherichia coli as a fusion product with a 62-aa fragment of human IL-2. The recombinant protein TAB1 was purified by washed pellet procedures and reversed-phase HPLC. TAB1 was recognized in ELISAs by 25 of 27 sera from seropositive individuals. Mice were immunized and several hybridomas were obtained. Two of them secrete monoclonal antibodies that react with synthetic peptides from isolates MN, WMJI, WMJIII, and SC with an affinity constant in the range of 108 M_1. They also recognized peptides from isolates SF2 and WMJH, but at much lower affinity. The results obtained from peptide ELISAs indicate that the putative epitope recognized by these MAbs lies within the sequence IHIGPGRAFYT. Classic neutralization assays demonstrated that MAb 2C4 neutralizes 50% of the MN isolate at 0.6 (i-g/ml but fails to neutralize HIB and SF2 strains. The presence of antibodies directed against every one of the component peptides in the sera of rabbits immunized with TAB1 was also documented.

American Journal of Physiology-Gastrointestinal and Liver Physiology, 2012

Salmonella enterica serovar Typhimurium is a clinically important gram-negative, enteric bacteria... more Salmonella enterica serovar Typhimurium is a clinically important gram-negative, enteric bacterial pathogen that activates several Toll-like receptors (TLRs). While TLR signaling through the adaptor protein MyD88 has been shown to promote inflammation and host defense against the systemic spread of S. Typhimurium, curiously, its role in the host response against S. Typhimurium within the mammalian gastrointestinal (GI) tract is less clear. We therefore used the recently described Salmonella -induced enterocolitis and fibrosis model: wild-type (WT) and MyD88-deficient (MyD88−/−) mice pretreated with streptomycin and then orally infected with the Δ aroA vaccine strain of S. Typhimurium. Tissues were analyzed for bacterial colonization, inflammation, and epithelial damage, while fibrosis was assessed by collagen quantification and Masson's trichrome staining. WT and MyD88−/−mice carried similar intestinal pathogen burdens to postinfection day 21. Infection of WT mice led to acute m...

Senescence is a stable arrest of cell proliferation in which the cells remain viable and metaboli... more Senescence is a stable arrest of cell proliferation in which the cells remain viable and metabolically active but display a constitutive activation of the DNA damage response and of the tumor suppressors p53 and RB. The senescent phenotype can be induced by multiple stresses including short telomeres and oncogenes. We have shown that senescence, involves the ERK-dependent degradation of selective proteins involved in cell cycle progression and tumorigenesis. We call this process senescence associated protein degradation (SAPD) and it involves many nucleolar proteins that play a role in ribosome biogenesis. Using tritium pulse labelling we found a strong decrease of rRNA synthesis in senescent cells indicating that the degradation of nucleolar proteins is functionally relevant. Because we know how exactly the human 47S precursor rRNA is processed, it was possible to design primers on both sides of some processing sites and study their maturation by QPCR. In this way we showed defects in the processing of rRNA in senescent cells. Knocking down some of the nucleolar proteins degraded in senescence was sufficient to trigger the process indicating that a decrease in ribosome biogenesis is causal to cellular senescence. Mechanistically, the degradation of nucleolar proteins during senescence involves the ubiquitin-proteasome system suggesting that E3 ligases link the oncogenic stress that trigger senescence to nucleolar proteins degradation. We will discuss ongoing efforts to identify these enzymes. Citation Format: Frédéric Lessard, Véronique Bourdeau, Xavier Deschênes-Simard, Sebastian Igelmann, Marinieve Montero, Gerardo Ferbeyre. Senescence as a result of impaired ribosome biogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2246. doi:10.1158/1538-7445.AM2014-2246