Biology subjects

Boeda, B.

Publications and source records attributed to Boeda, B..

6 recordsLinked to original sources

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

Specific subcellular localization drives the different functions of CDC42 isoforms during migration

The small G-protein CDC42 is an evolutionary conserved polarity protein and a key regulator of numerous polarized cell functions, including directed cell migration. In vertebrates, alternative splicing gives rise to two CDC42 proteins: the ubiquitously expressed isoform (CDC42u) and the brain isoform (CDC42b), whose specific roles are not fully elucidated. The two isoforms only differ in their carboxy-terminal sequence, which includes the CAAX motif essential for CDC42 interaction with membrane. Here we show that these divergent sequences do not directly affect the range of CDC42s potential binding partners, but indirectly influence CDC42-driven signaling by controlling the specific subcellular localization of the two isoforms. In astrocytes and neural precursors, which naturally express both variants, CDC42u is mainly cytosolic and associates with the leading-edge plasma membrane of migrating cells where it recruits the Par6-PKC{zeta} complex to fulfill its polarity function. In contrast, CDC42b mainly localizes to intracellular membrane compartments, where it interacts with N-WASP. CDC42b does not participate in cell polarization but embodies the major isoform regulating endocytosis. Both CDC42 isoforms act in concert by contributing their specific functions to promote chemotaxis of neural precursors, demonstrating that the expression pattern of the two isoforms is decisive for the tissue-specific behavior of cells.

cell biology↗

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↗

bioRxiv2018

Microtubule acetylation but not detyrosination promotes focal adhesion dynamics and cell migration

Microtubules play a crucial role in mesenchymal migration by controlling cell polarity and the turnover of cell adhesive structures on the extracellular matrix. The polarized functions of microtubules imply that microtubules are locally regulated. Here, we investigated the regulation and role of two major tubulin post-translational modifications, acetylation and detyrosination, which have been associated with stable microtubules. Using primary astrocytes in a wound healing assay, we show that these tubulin modifications are independently regulated during cell polarization and differently affect cell migration. In contrast to microtubule detyrosination, TAT1-mediated microtubule acetylation increases in the vicinity of focal adhesions and promotes cell migration. We further demonstrate that TAT1 increases focal adhesion turnover by promoting Rab6-positive vesicle fusion at focal adhesions. Our results highlight the specificity of microtubule post-translational modifications and bring new insight into the regulatory functions of tubulin acetylation.

cell biology↗

bioRxiv2018

Intermediate filaments control collective migration by restricting traction forces and sustaining cell-cell contacts

Mesenchymal cell migration relies on the coordinated regulation of the actin and microtubule networks which participate in polarised cell protrusion, adhesion and contraction. During collective migration, most of the traction forces are generated by the acto-myosin network linked to focal adhesions at the front of leader cells, which transmit these pulling forces to the followers. Here, using an in vitro wound healing assay to induce polarisation and collective directed migration of primary astrocytes, we show that the intermediate filament (IF) network composed of vimentin, GFAP and nestin contributes to directed collective movement by controlling the distribution of forces in the migrating cell monolayer. Together with the cytoskeletal linker plectin, these IFs control the organisation and dynamics of the acto-myosin network, promoting the actin-driven treadmilling of adherens junctions, thereby facilitating the polarisation of leader cells. Independently of their effect on adherens junctions, IFs influence the dynamics and localisation of focal adhesions and limit their mechanical coupling to the acto-myosin network. We thus conclude that IFs promote collective directed migration by restricting the generation of traction forces to the front of leader cells, preventing aberrant tractions in the followers and by contributing to the maintenance of lateral cell-cell interactions.

cell biology↗