Biology subjects

Di Palma, M.

Publications and source records attributed to Di Palma, M..

4 recordsLinked to original sources

bioRxiv2026

Structural basis of ligand-selective transcriptional activation in the MerR-family antibiotic resistance regulator AlbA

Multidrug-resistant pathogens demand novel resistance-breaking strategies. The oligoarylamide albicidin and pyrrolobenzodiazepines (PBDs) are potent antibacterials with unrelated scaffolds, yet both are neutralized by albA gene products in many Gram-negatives: AlbA, an albicidin-responsive self-upregulating MerR-family factor, and the smaller AlbAS, its unique ligand binding domain (LBD) alone. How AlbA couples ligand sensing to transcriptional activation has remained elusive. Here, using an integrative multidisciplinary approach, we define this mechanism and show that albicidin and PBDs elicit different responses. Crystal structures reveal C8-linked PBDs bound to AlbAS at the N- and C-terminal subdomains (NTD, CTD) of the central tunnel in poses dictated by C8-tail chemistry. This plasticity sequesters diverse PBDs with nanomolar affinities. Cryo-EM shows AlbA as an autoinhibited dimer in which a reciprocal arm closes the NTD end of the partner's tunnel, restricting PBD binding to the CTD while the DNA binding domains (DBDs) are mobile. Albicidin binding is incompatible with this arrangement, whereas PBDs, as CTD plugs, do not perturb the native equilibrium between autoinhibited and promoter-competent states. We visualized the latter by cryo-EM in the RNAP-DNA-AlbA complex. By blocking albicidin-mediated enhancement in vitro, PBDs act as resistance-breaking partners for albicidin and a route to overcoming albA-dependent resistance to oligoarylamide antibiotics.

biochemistry↗

bioRxiv2025

Continuous Serial Electron Diffraction for High Quality Protein Structures

Determining macromolecular structures is crucial for understanding biological mechanisms and advancing drug discovery. Three-dimensional electron diffraction (3D ED), also known as microcrystal electron diffraction (MicroED) using continuous sample rotation has emerged as a powful method for solving structures from sub-micrometre-sized crystals. However, the resolution of MicroED data from protein crystals is often limited by radiation damage. Serial electron diffraction (SerialED) overcomes this limitation by merging single-shot diffraction patterns from thousands of crystals, but its widespread use has been hindered by the complexity and scarcity of equipment required for single shot data acquisition. Here, we introduce continuous SerialED (c-SerialED) - a simple, robust and widely accessible protocol. This approach collects diffraction data quickly and efficiently from all crystals within a given area, without prior crystal identification. We show that only using a standard cryo-EM instrument equipped with a simple widely available CMOS detector, c-SerialED greatly reduces radiation damage while improving the data quality. We demonstrate that c-SerialED enables determination of lysozyme structures at atomic resolution (0.83 [A]) and improves the data resolution of Dype Type Peroxidase Aa (DTPAa) crystals from 2.5 [A] (MicroED) to 1.3 [A]. Remarkably, the resulting structures are virtually free of radiation damage. The improved data quality and resolution allow visualization of radiation sensitive chemical features and protein-ligand interactions to state-of-the-art accuracy. By providing a convenient, fast, and damage-minimizing workflow on existing cryo-EM setups, c-SerialED significantly enhances the applicability of electron diffraction in structural biology. We anticipate our protocol will enable a wide range of studies requiring high-quality diffraction data from radiation-sensitive macromolecular crystals.

molecular biology↗

bioRxiv2025

Microtubule architecture connects AMOT stability to YAP/TAZmechanotransduction and Hippo signaling

Cellular mechanotransduction is a fundamental informational system by which cells read the structural features of their environment to control their own form and function. The YAP/TAZ transcriptional regulators are universal effectors of physical signals. Yet, the identity of proteins and subcellular structures serving as mechano-rheostats remains elusive. Here we demonstrate that perinuclear centrosome and microtubules architecture act functionally downstream of F-actin as cornerstones of cellular mechanotransduction. The mechanism revolves around the stability of AMOT proteins, that act as cytoplasmic inhibitory sinks for YAP/TAZ. Being degraded in mechano-activated cells and stabilized in mechanically-inhibited cells, AMOT serves as primary mechanical rheostat. In mechanically inhibited cells, microtubules form a cage-like network surrounding the nucleus, but, in mechanically activated cells, switch their architecture with formation of the centrosome from which microtubules sprout toward the cell periphery. In these conditions, AMOT proteins bound to the Dynein/Dynactin complex are subject to fast retrograde transport through the microtubular aster toward the pericentrosomal proteasome for quantitative and timely degradation. Restoring centrosomal condensation in mechanically inhibited cells by NLP1 overexpression is sufficient to restore mechanosignaling and YAP activation. AMOT proteins serves as universal integrator of distinct physical inputs from the ECM and the cytoskeleton, and their ablation renders cells invariably mechano-insensitive. Our findings also provide a unifying model that mechanistically merges mechanosignaling with the Hippo cascade. The current model by which YAP/TAZ are regulated by Hippo kinases is through direct YAP/TAZ phosphorylation. Our data instead show that, at least in the context of mechanotransduction, Hippo signaling inhibits YAP/TAZ largely indirectly, through LATS phosphorylation of AMOT averting it from its degradation route. We further show that Ras/RTK oncogenes hijack the AMOT degradation route to promote YAP/TAZ-mediated tumorigenesis. The findings imply the AMOT stabilization machinery as novel target for YAP/TAZ therapeutic modulation. In sum, our work reveals a previously unknown hierarchical coordination of distinct cytoskeletal and transport systems orchestrating mechanosignaling at the whole cell level.

cell biology↗

bioRxiv2023

Cysteine enrichment mediates co-option of uricase in reptilian skin and transition to uricotelism

Uric acid is the main means of nitrogen excretion in uricotelic vertebrates (birds and reptiles) and the end product of purine catabolism in humans and a few other mammals. While uricase is inactivated in mammals unable to degrade urate, the presence of orthologous genes without inactivating mutations in avian and reptilian genomes is unexplained. Here we show that the Gallus gallus gene we name cysteine-rich urate oxidase (CRUOX) encodes a functional protein representing a unique case of cysteine enrichment in the evolution of vertebrate orthologous genes. CRUOX retains the ability to catalyze urate oxidation to hydrogen peroxide and 5-hydroxyisourate (HIU), albeit with a 100-fold reduced efficiency. However, differently from all uricases hitherto characterized, it can also facilitate urate regeneration from HIU, a catalytic property which we propose depends on its enrichment in cysteine residues. X-ray structural analysis highlights differences in the active site compared to known orthologs and suggests a mechanism for cysteine-mediated self-aggregation under H2O2-oxidative conditions. Cysteine enrichment was concurrent with transition to uricotelism and a shift in gene expression from the liver to the skin where CRUOX is co-expressed with {beta}-keratins. Therefore, the loss of urate degradation in amniotes has followed opposite evolutionary trajectories: while uricase has been eliminated by pseudogenization in some mammals, it has been repurposed as a redox-sensitive enzyme in the reptilian skin.

evolutionary biology↗