Anne-Cecile Duc - Academia.edu (original) (raw)

Papers by Anne-Cecile Duc

HAL (Le Centre pour la Communication Scientifique Directe), Sep 21, 2021

Ovarian cancer (OC) is the most deadly gynaecological cancer and the 8 th leading cause of cancer... more Ovarian cancer (OC) is the most deadly gynaecological cancer and the 8 th leading cause of cancer-related deaths in women. Unfortunately, screening programs and early-stage diagnostic methods are not available, so late diagnosis remains a major contributor to the poor prognosis. Recently, aptamers have emerged as useful tools in cancer diagnostics. Aptamers are short single-stranded DNA or RNA oligonucleotides that bind with high specificity to diverse targets, including cancer biomarker proteins. Aptamers are developed in vitro using different variations of Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Herein, we describe the advances in the development of high-specificity DNA and RNA aptamers for detection for OC protein biomarkers. Here, we present a discussion on their diagnostic application in the detection of two clinically relevant ovarian cancer biomarkers: Cancer Antigen 125 (CA125) and Human Epididymis protein 4 (HE4). Ten promising candidate aptamers (seven for CA125 and three for HE4) have been identified. The stability in human serum and target detection in clinically relevant concentration range demonstrates the utility of aptamers as molecular probes in OC biomarker tests. Aptamer-based diagnostic platforms, such as biosensors and clinical assays could enable easy and rapid protein detection, with potential for earlier diagnosis of OC that could be implemented in clinical practice in the future.

ABSTRACT Towards the development of new anti-infective compounds, two approaches were followed in... more ABSTRACT Towards the development of new anti-infective compounds, two approaches were followed in this thesis work, with one being the development of a new class of compounds to an existing target, and the second one focusing on the interaction of an existing class of drugs to a new target.^ The research first focused on interaction of the peptide HPVHHYQ with an A-site rRNA hairpin model. A single binding site was identified at the internal bulge of the RNA, with an average dissociation constant of 17 μM. The roles of the amino-acid side chains in the peptide•RNA interaction were studied by alanine scanning, and showed that enhancement of the affinity could be observed upon P2A and Y2A mutations; whereas H1A and Q7A were unfavorable for binding. Similar results were obtained for the A-site rRNA hairpin and the 30 S ribosome. The parent peptide HPVHHYQ retained flexibility in its binding, such that it was able to bind to three A-site rRNA mutant hairpins in a 1:1 fashion, at the asymmetric bulge. These results are significant with respect to the development os new ligands towards potentially resistant bacterial mutants.^ The last part of the research focused on the interaction of aminoglycosides with a helix 69 rRNA hairpin model. The binding affinities and stochiometries of the interactions with the modified as well as the unmodified RNA were investigated, yielding Kds ranging from 0.5 to 14 μM. The preferred binding observed to the modified construct ober unmodified RNA was correlated to a long-range conformational change leading to base flipping of nucleotide A1913. Lower pH and lack of modifications lessen the affinity of the aminoglycosides for the target RNA, suggesting that a flipped-out conformation of 1913 is a driving force for the binding to helix 69.^ Overall, this thesis work explored new ligand•RNA interactions, with the long-term objective to develop new antibiotic compounds. Studies with mutant RNAs will assist with identifying compounds that will have greater efficacy against drug-resistance bacterial strains.^

Critical reviews in immunology, 2015

Ribosomal proteins have long been known to serve critical roles in facilitating the biogenesis of... more Ribosomal proteins have long been known to serve critical roles in facilitating the biogenesis of the ribosome and its ability to synthesize proteins. However, evidence is emerging that suggests ribosomal proteins are also capable of performing tissue-restricted, regulatory functions that impact normal development and pathological conditions, including cancer. The challenge in studying such regulatory functions is that elimination of many ribosomal proteins also disrupts ribosome biogenesis and/or function. Thus, it is difficult to determine whether developmental abnormalities resulting from ablation of a ribosomal protein result from loss of core ribosome functions or from loss of the regulatory function of the ribosomal protein. Rpl22, a ribosomal protein component of the large 60S subunit, provides insight into this conundrum; Rpl22 is dispensable for both ribosome biogenesis and protein synthesis yet its ablation causes tissue-restricted disruptions in development. Here we revie...

Towards the development of new anti-infective compounds, two approaches were followed in this the... more Towards the development of new anti-infective compounds, two approaches were followed in this thesis work, with one being the development of a new class of compounds to an existing target, and the second one focusing on the interaction of an existing class of drugs to a new target.^ The research first focused on interaction of the peptide HPVHHYQ with an A-site rRNA hairpin model. A single binding site was identified at the internal bulge of the RNA, with an average dissociation constant of 17 μM. The roles of the amino-acid side chains in the peptide•RNA interaction were studied by alanine scanning, and showed that enhancement of the affinity could be observed upon P2A and Y2A mutations; whereas H1A and Q7A were unfavorable for binding. Similar results were obtained for the A-site rRNA hairpin and the 30 S ribosome. The parent peptide HPVHHYQ retained flexibility in its binding, such that it was able to bind to three A-site rRNA mutant hairpins in a 1:1 fashion, at the asymmetric b...

PLoS Genetics, 2013

Most yeast ribosomal protein genes are duplicated and their characterization has led to hypothese... more Most yeast ribosomal protein genes are duplicated and their characterization has led to hypotheses regarding the existence of specialized ribosomes with different subunit composition or specifically-tailored functions. In yeast, ribosomal protein genes are generally duplicated and evidence has emerged that paralogs might have specific roles. Unlike yeast, most mammalian ribosomal proteins are thought to be encoded by a single gene copy, raising the possibility that heterogenous populations of ribosomes are unique to yeast. Here, we examine the roles of the mammalian Rpl22, finding that Rpl22 2/2 mice have only subtle phenotypes with no significant translation defects. We find that in the Rpl22 2/2 mouse there is a compensatory increase in Rpl22-like1 (Rpl22l1) expression and incorporation into ribosomes. Consistent with the hypothesis that either ribosomal protein can support translation, knockdown of Rpl22l1 impairs growth of cells lacking Rpl22. Mechanistically, Rpl22 regulates Rpl22l1 directly by binding to an internal hairpin structure and repressing its expression. We propose that ribosome specificity may exist in mammals, providing evidence that one ribosomal protein can influence composition of the ribosome by regulating its own paralog.

Molecules, 2011

Ribosomal RNA is the catalytic portion of ribosomes, and undergoes a variety of conformational ch... more Ribosomal RNA is the catalytic portion of ribosomes, and undergoes a variety of conformational changes during translation. Structural changes in ribosomal RNA can be facilitated by the presence of modified nucleotides. Helix 31 of bacterial 16S ribosomal RNA harbors two modified nucleotides, m 2 G966 and m 5 C967, that are highly conserved among bacteria, though the degree and nature of the modifications in this region are different in eukaryotes. Contacts between helix 31 and the P-site tRNA, initiation factors, and ribosomal proteins highlight the importance of this region in translation. In this work, a heptapeptide M13 phage-display library was screened for ligands that target the wild-type, naturally modified bacterial helix 31. Several peptides, including TYLPWPA, CVRPFAL, TLWDLIP, FVRPFPL, ATPLWLK, and DIRTQRE, were found to be prevalent after several rounds of screening. Several of the peptides exhibited moderate affinity (in the high nM to low µM range) to modified helix 31 in biophysical assays, including surface OPEN ACCESS plasmon resonance (SPR), and were also shown to bind 30S ribosomal subunits. These peptides also inhibited protein synthesis in cell-free translation assays.

Developmental Cell, 2013

It remains controversial whether the highly homologous ribosomal protein (RP) paralogs found in l... more It remains controversial whether the highly homologous ribosomal protein (RP) paralogs found in lower eukaryotes have distinct functions and this has not been explored in vertebrates. Here we demonstrate that despite ubiquitous expression, the RP paralogs, Rpl22 and Rpl22-like1 (Rpl22l1) play essential, distinct, and antagonistic roles in hematopoietic development. Knockdown of Rpl22 in zebrafish embryos selectively blocks the development of T lineage progenitors after they have seeded the thymus. In contrast, knockdown of the Rpl22 paralog, Rpl22l1, impairs the emergence of hematopoietic stem cells (HSC) in the aorta-gonad-mesonephros by abrogating Smad1 expression and the consequent induction of essential transcriptional regulator, Runx1. Indeed, despite the ability of both paralogs to bind smad1 RNA, Rpl22 and Rpl22l1 have opposing effects on Smad1 expression. Accordingly, circumstances that tip the balance of these paralogs in favor of Rpl22 (e.g., Rpl22l1 knockdown or Rpl22 overexpression) result in repression of Smad1 and blockade of HSC emergence.

Biopolymers, 2009

The use of bacteriophage T7 is presented as a peptide display platform to identify short binding ... more The use of bacteriophage T7 is presented as a peptide display platform to identify short binding sequences for PDZ domain proteins. Two different domains are examined, the 10th PDZ domain (PDZ10) of the multi-PDZ domain protein 1 (MUPP1) and the third PDZ domain (PDZ3) of postsynaptic density-95 (PSD-95) protein. Using the T7Select 415-1b construct, which displays 415 peptides per phage particle, a random heptapeptide and focused octapeptide libraries were constructed and subjected to iterative selection-enrichment cycles against surface-immobilized PDZ3 and PDZ10 proteins. The derived consensus sequences, together with those of high-frequency clones, were used as the basis for individual chemically synthesized peptides. Each peptide was subjected to isothermal titration calorimetry binding determinations against the corresponding PDZ domain under standard solution conditions. For MUPP1 PDZ10, binding analysis demonstrated that one of the heptapeptides, Ac-IGRISRV, displayed a two-fold improved affinity over the octapeptide derived from the carboxy terminus of the hc-Kit protein, which we had recently demonstrated as among the highest affinity ligands reported to date for that domain. In the case of PSD-95 PDZ3, peptides were found that possessed low-micromolar dissociation constants, as well as those that rediscovered the C-terminal sequence (KQTSV) of the protein CRIPT, a known natural binding protein of PDZ3. These successful examples of ligand discovery against two distinctly different PDZ domains demonstrate that the T7 phage platform could prove broadly applicable to the numerous other PDZ domains for which binding peptides are absent or of insufficient affinity.

Biochemistry, 2009

For almost five decades, antibiotics have been used successfully to control infectious diseases c... more For almost five decades, antibiotics have been used successfully to control infectious diseases caused by bacterial pathogens. More recently, however, two-thirds of bacterial pathogens exhibit resistance and are continually evolving new resistance mechanisms against almost every clinically used antibiotic. Novel efforts are required for the development of new drugs or drug leads to combat these infectious diseases. A number of antibiotics target the bacterial aminoacyl-tRNA site (A site) of 16S ribosomal RNA (rRNA). Mutations in the A-site region are known to cause antibiotic resistance. In this study, a bacterial (E. coli) A-site rRNA model was chosen as a target to screen for peptide binders. Two heptapeptides, HPVHHYQ and LPLTPLP, were selected through M13 phage display. Both peptides display selective binding to the A-site 16S rRNA with on-bead fluorescence assays. Dissociation constants (K d s) of the amidated peptide HPVHHYQ-NH 2 to various A-site RNA constructs were determined by using enzymatic footprinting, electrospray ionization mass spectrometry (ESI-MS), and isothermal titration calorimetry (ITC) under a variety of buffer and solution conditions. HPVHHYQ-NH 2 exhibits moderate affinity for the A-site RNA, with an average K d value of 16 μM. In addition, enzymatic footprinting assays and competition ESI-MS with a known A-site binder (paromomycin) revealed that peptide binding occurs near the asymmetric bulge at positions U1495 and G1494 and leads to increased exposure of residues A1492 and A1493.

Angewandte Chemie International Edition, 2012

Helix 69 (H69) of 23S ribosomal RNA serves as a unique model system to study the impact of modifi... more Helix 69 (H69) of 23S ribosomal RNA serves as a unique model system to study the impact of modified bases on RNA structure and function, and to screen potential antibiotics. H69 is located at the functionally important core domain of the bacterial ribosome, participates in key intersubunit bridge B2a interactions, and plays important roles in translation. This helix exists in multiple conformational states, and interacts with a number of translation factors at different stages of protein synthesis. Chemical probing analyses revealed that H69 undergoes structural rearrangements upon ribosome association, particularly at positions A1913 and A1918, with the various conformational states being influenced by solution conditions (e.g., concentration of Mg 2+ , pH value, and temperature) as well as pseudouridine (Y) modifications. Residue A1913 is proposed to be important for high-fidelity translation [2c, 4] and efficient termination. [2b] Moreover, flexibility of the H69 stem region may help to accommodate the twisting energy from rotation of the subunits. Since H69 is a highly dynamic RNA domain, altering or regulating these important conformational states with small molecules could be a promising way to disrupt bacterial ribosome translation. Development of a method to easily monitor these changes is therefore important in order to understand H69 dynamics in solution, as well as to discover H69-targeting ligands.