Biology subjects

Caillet-Saguy, C.

Publications and source records attributed to Caillet-Saguy, C..

8 recordsLinked to original sources

bioRxiv2025

HTRF-based identification of small molecules targeting SARS-CoV-2 E protein interaction with ZO-1 PDZ2

The SARS-CoV-2 E protein through its C-terminal PDZ-binding motif (PBM) interacts with several host PDZ-containing proteins, including the ZO-1 protein via its PDZ2 domain, thereby contributing to viral pathogenesis. Targeting this interaction represents a potential therapeutic strategy. In this study, we determined the X-ray structure of the E PBM peptide in complex with the ZO-1 PDZ2 domain at 1.7 [A] resolution. The structure revealed a domain-swapped dimer conformation of ZO-1 PDZ2, with the E PBM peptide conventionally bound within the PDZ domains canonical binding groove exhibiting key interactions characteristic of type II PBM-PDZ interactions. To identify potential inhibitors of the E PBM/ZO-1 PDZ2 interaction, we performed a HTRF screening using a protein-protein interaction-focused library of 1,000 compounds. This led to the identification of 36 hits that disrupted this interaction. Subsequent cytotoxicity and dose- response assays narrowed the selection to 14 promising compounds. Docking simulations showed that some compounds bind within or near the PBM-binding pocket, supporting a competitive mechanism of interaction inhibition, while others bind at a central interface between the two PDZ monomers, suggesting an inhibition of dimerization, which in turn prevents PBM binding. Thus, the ZO-1 PDZ2-E PBM interaction can be inhibited through both direct and indirect mechanisms. Finally, antiviral assays using a NanoLuciferase-expressing recombinant SARS-CoV-2 demonstrated that one compound, C19, significantly reduced viral replication, highlighting its potential as a candidate for further therapeutic development. HighlightsO_LICrystal structure reveals the binding determinant of E protein PBM to ZO-1 PDZ2 dimer C_LIO_LIHTRF screening identified 36 inhibitors of the E-ZO-1 protein interaction C_LIO_LIDocking revealed dual mechanisms: PBM groove binding and dimer interface disruption C_LIO_LICompound C19 reduced SARS-CoV-2 replication in a NanoLuc reporter assay C_LIO_LITargeting E-ZO-1 interaction may prevent barrier loss and systemic COVID-19 effects C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/665136v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1f900bcorg.highwire.dtl.DTLVardef@9f470aorg.highwire.dtl.DTLVardef@f0ec19org.highwire.dtl.DTLVardef@11c9115_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

The SARS-CoV-2 envelope PDZ Binding Motif acts as a virulence factor disrupting host's epithelial cell-cell junctions

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic, has significantly impacted global health, emphasizing the need to understand its pathogenicity and virulence mechanisms. SARS-CoV-2 disrupts the alveolar epithelial barrier and exacerbates airway inflammation, leading to acute respiratory failure, but the molecular details remain unclear. Additionally, SARS-CoV-2 infection causes neurological symptoms, potentially due to its weakly understood ability to cross the blood-brain barrier. The viral multifunctional Envelope (E) protein is crucial for its virulence, playing a key role in virus assembly, budding, and release. The E protein contains a PDZ-binding motif (PBM) that interacts with host PDZ domain-containing proteins, potentially affecting host signaling pathways and contributing to pathogenicity. This study focuses on the E protein PBM and its role in virulence, disrupting respiratory epithelial barriers and exacerbating airway inflammation. We generated recombinant mutant viruses lacking the PBM and conducted both in vitro and in vivo experiments to elucidate its impact on viral fitness, pathogenicity, and effects on the epithelial integrity. In vitro, the viral mutants showed delayed replication and reduced cytopathic effects. In vivo, experiments with hamsters revealed that PBM-deficient viruses caused less weight loss, lower viral loads, and reduced inflammation, indicating decreased pathogenicity. Histological analyses confirmed less airway damage in these hamsters compared to those infected with the wild-type virus. Additionally, PBM-deficient viruses had impaired interactions with tight junction proteins like ZO-1, a PDZ-containing protein, crucial for maintaining epithelial barrier integrity. Our findings also demonstrate that the PBM does not play a significant role in neuroinvasion during the acute phase of infection, as evidenced by comparable viral RNA loads across brain regions in infected hamsters, regardless of PBM presence. Histopathological and transcriptomic analyses further support this observation, suggesting that the PBM primarily affects specific epithelial barriers. Additionally, RNA-seq analysis on lung and brainstem from infected hamsters reveals that the PBM modulates inflammatory and immune responses, with a stronger impact in lung tissue than in the brainstem. PBM-deficient viruses induce lower levels of inflammation and cytokine expression, suggesting PBMs specific role in enhancing viral pathogenicity through the activation of pathways such as NF-{kappa}B and TNF. Thus, the E protein PBM plays a critical role in SARS-CoV-2s fitness, virulence, and pathogenicity, through the disruption of cell junctions and inflammation, underscoring its potential as a target for therapeutic interventions.

pathology↗

bioRxiv2023

A short sequence in the tail of SARS-CoV-2 envelope protein controls accessibility of its PDZ Binding Motif to the cytoplasm.

The carboxy terminal tail of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope protein (E) contains a PDZ-binding motif (PBM) which is crucial for coronavirus pathogenicity. During SARS-CoV-2 infection, the viral E protein is expressed within the Golgi apparatus membrane of host cells with its PBM facing the cytoplasm. In this work we study the molecular mechanisms controlling the presentation of the PBM to host PDZ (PSD-95/Dlg/ZO-1) domain-containing proteins. We show that at the level of the Golgi apparatus, the PDZ-binding motif of the E protein is not detected by E C-terminal specific antibodies neither by PDZ domain-containing protein binding partner. Four alanine substitutions upstream of the PBM in the central region of the E protein tail is sufficient to generate immunodetection by anti-E antibodies and trigger robust recruitment of the PDZ domain-containing protein into the Golgi organelle. Overall, this work suggests that the presentation of the PBM to the cytoplasm is under conformational regulation mediated by the central region of the E protein tail and that PBM presentation probably does not occur at the surface of Golgi cisternae but likely at post-Golgi stages of the viral cycle.

cell biology↗

bioRxiv2023

Interactions of the Protein Tyrosine Phosphatase PTPN3 with Viral and Cellular Partners through its PDZ Domain: Insights into Structural Determinants and Phosphatase Activity

The human protein tyrosine phosphatase non-receptor type 3 (PTPN3) is a phosphatase containing a PDZ (PSD-95/Dlg/ZO-1) domain that has been found to play both tumor-suppressive and tumor-promoting roles in various cancers, despite limited knowledge of its cellular partners and signaling functions. Notably, the high-risk genital human papillomavirus (HPV) types 16 and 18 and the hepatitis B virus (HBV) target the PDZ domain of PTPN3 through PDZ-binding motifs (PBMs) in their E6 and HBc proteins respectively. This study focuses on the interactions between the PTPN3 PDZ domain (PTPN3-PDZ) and PBMs of viral and cellular protein partners. The solved X-ray structures of complexes between PTPN3-PDZ and PBMs of E6 of HPV18 and the tumor necrosis factor-alpha converting enzyme (TACE) reveal two novel interactions. We provide new insights into key structural determinants of PBM recognition by PTPN3 by screening the selectivity of PTPN3-PDZ recognition of PBMs, and by comparing the PDZome binding profiles of PTPN3-recognized PBMs and the interactome of PTPN3-PDZ. The PDZ domain of PTPN3 was known to auto-inhibit the proteins phosphatase activity. We discovered that the linker connecting the PDZ and phosphatase domains is involved in this inhibition, and that the binding of PBMs does not impact this catalytic regulation. Overall, the study sheds light on the interactions and structural determinants of PTPN3 with its cellular and viral partners, as well as on the inhibitory role of its PDZ domain on its phosphatase activity.

biophysics↗

bioRxiv2021

Interactions of SARS-CoV-2 protein E with cell junctions and polarity PDZ-containing proteins

The C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein E contains a PBM (PDZ binding motif) targeting PDZ (PSD-95/Dlg/ZO-1) domains identical to the PBM of SARS-CoV. The latter is involved in the pathogenicity of the virus. Recently, we identified ten human PDZ-containing proteins showing significant interactions with SARS-CoV-2 protein E PBM. We selected several of them involved in cellular junctions and cell polarity (TJP1, PARD3, MLLT4, LNX2) and MPP5/Pals1 previously shown to interact with SARS-CoV E PBM. Targeting cellular junctions and polarity components is a common strategy by viruses to hijack cell machinery to their advantage. In this study, we showed that these host PDZ domains TJP1, PARD3, MLLT4, LNX2 and MPP5/PALS1 interact in a PBM-dependent manner in vitro and colocalize with the full-length E protein in cellulo, sequestrating the PDZ domains to the Golgi compartment. We solved three crystal structures of complexes between human LNX2, MLLT4 and MPP5 PDZs and SARS-CoV-2 E PBM highlighting its binding preferences for several cellular targets. Finally, we showed different affinities for the PDZ domains with the original SARS-CoV-2 C-terminal sequence containing the PBM and the one of the beta variant that contains a mutation close to the PBM. The acquired mutations in E protein localized near the PBM might have important effects both on the structure and the ion-channel activity of the E protein and on the host machinery targeted by the variants during the infection.

biochemistry↗

bioRxiv2021

PDZ-containing proteins targeted by the ACE2 receptor

Angiotensin converting enzyme 2 (ACE2) is a main receptor for SARS-CoV-2 entry to the host cell. Indeed, the first step in viral entry is the binding of the viral trimeric spike protein to ACE2. Abundantly present in human epithelial cells of many organs, ACE2 is also expressed in the human brain. ACE2 is a type I membrane protein with an extracellular N-terminal peptidase domain and a C-terminal collectrin-like domain that ends with a single transmembrane helix and an intracellular 44-residues segment. This C-terminal segment contains a PDZ-binding motif (PBM) targeting protein interacting domains called PSD-95/Dlg/ZO-1 (PDZ). Here, we identified the human PDZ specificity profile of the ACE2 PBM using the high throughput holdup assay and measuring the binding intensities of the PBM of ACE2 against the full human PDZome. We discovered 14 human PDZ binders of ACE2 showing significant binding with dissociation constants values ranging from 3 to 81 M. NHERF, SHANK, and SNX27 proteins found in this study are involved in protein trafficking. The PDZ/PBM interactions with ACE2 could play a role on ACE2 internalization and recycling that could benefit for the virus entry. Interestingly, most of the ACE2 partners we identified are expressed in neuronal cells, such as SHANK and MAST families, and modifications of the interactions between ACE2 and these neuronal proteins may be involved in neurological symptoms of COVID-19.

biochemistry↗

bioRxiv2021

Host PDZ-containing proteins targeted by SARS-Cov-2

Small linear motif targeting protein interacting domains called PDZ have been identified at the C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins E, 3a, and N. Using a high-throughput approach of affinity-profiling against the full human PDZome, we identified sixteen human PDZ binders of SARS-CoV-2 proteins E, 3A and N showing significant interactions with dissociation constants values ranging from 3 M to 82 M. Six of them (TJP1, PTPN13, HTRA1, PARD3, MLLT4, LNX2) are also recognized by SARS-CoV while three (NHERF1, MAST2, RADIL) are specific to SARS-CoV-2 E protein. Most of these SARS-CoV-2 protein partners are involved in cellular junctions/polarity and could be also linked to evasion mechanisms of the immune responses during viral infection. Seven of the PDZ-containing proteins among binders of the SARS-CoV-2 proteins E, 3a or N affect significantly viral replication under knock-down gene expression in infected cells. This PDZ profiling identifying human proteins potentially targeted by SARS-CoV-2 can help to understand the multifactorial severity of COVID19 and to conceive effective anti-coronaviral agents for therapeutic purposes.

biochemistry↗

bioRxiv2020

Interactomic affinity profiling by holdup assay: acetylation and distal residues impact the PDZome-binding specificity of PTEN phosphatase

Protein domains often recognize short linear protein motifs composed of a core conserved consensus sequence surrounded by less critical, modulatory positions. Here we used an accurate experimental approach combining high-throughput holdup chromatographic assay and fluorescence polarization to measure quantitative binding affinity profiles of the PDZ domain-binding motif (PBM) of PTEN phosphatase towards the 266 known human PDZ domains. Inclusion of N-terminal flanking residues, acetylation or mutation of a lysine at a modulatory position significantly altered the PDZome-binding profile of the PTEN PBM. A specificity index is also introduced to quantify the specificity of a given PBM over the complete PDZome. Our results highlight the impact of modulatory residues and post-translational modifications on PBM interactomes and their specificity.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗