Biology subjects

Paradela, A.

Publications and source records attributed to Paradela, A..

4 recordsLinked to original sources

bioRxiv2025

Aviadenovirus structure: a highly thermostable capsid in the absence of stabilizing proteins

High-resolution structural studies have mainly focused on two out of the six adenovirus genera: mastadenoviruses and atadenoviruses. Here we report the high-resolution structure of an aviadenovirus, the poultry pathogen fowl adenovirus serotype 4 (FAdV-C4). FAdV-C4 virions are highly thermostable, despite lacking minor coat and core proteins shown to stabilize the mast- and atadenovirus particles, and having no genus-specific cementing proteins. Unique structural features of the FAdV-C4 hexon include a large insertion at the trimer equatorial region, and a long N-terminal tail. Protein IIIa conformation is closer to atadenoviruses than to mastadenoviruses, while protein VIII diverges from all previously reported structures. We interpret these differences in light of adenovirus evolution. Finally, we discuss the possible role of core composition in determining capsid stability properties. These results enlarge our view on the structural diversity of adenoviruses, and provide useful information to counteract fowl pathogens or use non-human adenoviruses as vectors.

molecular biology↗

bioRxiv2024

Harnessing homeostatically active RhoC at cell junctions preserves human endothelial barrier function during inflammation

Rho GTPases are molecular targets of bacterial toxins that modulate their enzymatic activity. RhoA, RhoB and RhoC are almost identical and play critical roles in generating actomyosin-mediated contractile forces that cause endothelial hyperpermeability during inflammation. Searching for new treatments to modulate endothelial integrity, we demonstrate that the specific and simultaneous activation of these three Rho GTPases with a chimeric recombinant toxin does not induce cell contraction but enhances homeostatic endothelial barrier function, increases reticular adherens junctions and preserves the microvascular endothelium in response to pathological inflammatory challenges in vitro and in vivo. This pro-barrier effect is specifically mediated by RhoC, whose activity is increased by cell confluence. The uniqueness of RhoC relies on an arginine 188 within its hypervariable region that determines its junctional localization, high homeostatic activity, and barrier-protective function. Quantitative proteomics revealed that RhoC regulates the expression of myosin light chain proteins and junction-stabilizing actomyosin. Thus, harnessing the activity of RhoC represents a potential therapy for strengthening endothelial barriers during pathological inflammation.

cell biology↗

bioRxiv2023

ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin

Epithelial Intercellular Adhesion Molecule (ICAM)-1 is apically polarized, interacts with and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures (BCs). We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

cell biology↗

bioRxiv2021

An updated structural model of the A domain of the Pseudomonas putida XylR regulator exposes a distinct interplay with aromatic effectors

A revised model of the aromatic binding A domain of the {sigma}54-dependent regulator XylR of Pseudomonas putida mt-2 was produced based on the known 3D structures of homologous regulators PoxR, MopR, and DmpR. The resulting frame was instrumental for mapping the large number of mutations known to alter effector specificity, which were then reinterpreted under a dependable spatial reference. Some of these changes involved the predicted aromatic-binding pocket but others occurred in distant locations, including dimerization interfaces and putative zinc-binding site. The effector pocket was buried within the protein structure and accessible from the outside only through a narrow tunnel. The model was experimentally validated by treating the cells in vivo and the purified protein in vitro with benzyl bromide, which reacts with accessible nucleophilic residues on the protein surface. Proteomic analyses of the thereby tagged peptides confirmed the predicted in/out distribution of residues but also suggested that the fully-folded protein is not accessible by externally added effectors. The data thus suggested that XylR inducers assist the folding and/or the structuring of the A domain in an intramolecular non-repressive form rather than interacting dynamically with the aromatic partner once a fully structured protein is shaped. Originality-Significance StatementXylR is a transcriptional regulator of Pseudomonas putida strain mt-2 which activates the upper TOL pathway promoter Pu for catabolism of toluene and m-xylene upon binding of these aromatic effectors to its N-terminal A domain. While this feature has made XylR a popular platform for the development of whole-cell biosensors for aromatic compounds, the difficulty to crystallize the A domain --let alone the whole-length protein-- has made structural comprehension of the effector-regulator binding quite problematic. To overcome this impasse, we have combined homology-based structural predictions of the A domain of XylR with biochemical probing of exposed amino acids on the surface of the protein, both in vivo and in vitro. The results generally matched the effects of mutations known from previous genetic/phenotypic analyses of the protein. However, the data also suggested an intriguing mechanism of activation of XylR by effectors in which the inducer assists the shaping of the regulator in an active conformation rather than interacting a posteriori with an already formed protein invitro. This may in fact explain the longstanding failure to purify the protein in an effector-responsive form.

microbiology↗