Biology subjects

Pozzi, N.

Publications and source records attributed to Pozzi, N..

6 recordsLinked to original sources

bioRxiv2025

POmAb, an antibody targeting open prothrombin, results in anticoagulation without excessive bleeding in mice

Anti-prothrombin antibodies are commonly found in patients with Antiphospholipid Syndrome (APS), yet their role in clinical manifestations remains unclear. We recently identified two classes of anti-prothrombin antibodies based on their ability to recognize closed and open forms of prothrombin. Type-I antibodies bind to the open form, while Type-II antibodies bind to both forms. POmAb is a prototypical Type-I antibody that specifically targets kringle-1 of prothrombin, maintaining it in an open state. In this study, we assess the effects of POmAb in mice using the cremaster arteriole laser-induced injury model. POmAb bound mouse prothrombin and decreased thrombin generation in mouse plasma. When administered intravenously shortly before the injury, POmAb quickly accumulated on the damaged vessel wall. This accumulation significantly reduced fibrin generation with a modest effect on platelet accumulation and without causing excessive bleeding. Results obtained with POmAb offer insights into the potential roles of the anti-prothrombin antibodies in APS. They also provide proof of concept for a new class of anticoagulants that, by specifically targeting open prothrombin, could mitigate thrombosis with reduced bleeding risk.

molecular biology↗

bioRxiv2024

Atomistic Characterization of Beta-2-Glycoprotein I Domain V Interaction with Anionic Membranes

BackgroundInteraction of beta-2-glycoprotein I ({beta}2GPI) with anionic membranes is crucial in antiphospholipid syndrome (APS), implicating the role of its membrane bind-ing domain, Domain V (DV). The mechanism of DV binding to anionic lipids is not fully understood. ObjectivesThis study aims to elucidate the mechanism by which DV of {beta}2GPI binds to anionic membranes. MethodsWe utilized molecular dynamics (MD) simulations to investigate the struc-tural basis of anionic lipid recognition by DV. To corroborate the membrane-binding mode identified in the HMMM simulations, we conducted additional simulations using a full mem-brane model. ResultsThe study identified critical regions in DV, namely the lysine-rich loop and the hydrophobic loop, essential for membrane association via electrostatic and hydrophobic interactions, respectively. A novel lysine pair contributing to membrane binding was also discovered, providing new insights into {beta}2GPIs membrane interaction. Simulations revealed two distinct binding modes of DV to the membrane, with mode 1 characterized by the insertion of the hydrophobic loop into the lipid bilayer, suggesting a dominant mechanism for membrane association. This interaction is pivotal for the pathogenesis of APS, as it facilitates the recognition of {beta}2GPI by antiphospholipid antibodies. ConclusionThe study advances our understanding of the molecular interactions be-tween {beta}2GPIs DV and anionic membranes, crucial for APS pathogenesis. It highlights the importance of specific regions in DV for membrane binding and reveals a predominant bind-ing mode. These findings have significant implications for APS diagnostics and therapeutics, offering a deeper insight into the molecular basis of the syndrome.

biophysics↗

bioRxiv2023

The PALB2 DNA binding domain is an intrinsically disordered recombinase.

The Partner and Localizer of BRCA2 (PALB2) is a scaffold protein that links BRCA1 with BRCA2 to initiate homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR efficiency in cells. The PALB2 DNA-binding domain (PALB2-DBD) supports strand exchange, a complex multistep reaction conducted by only a few proteins such as RecA-like recombinases and Rad52. Using bioinformatics analysis, small-angle X-ray scattering, circular dichroism, and electron paramagnetic spectroscopy, we determined that PALB2-DBD is an intrinsically disordered region (IDR) forming compact molten globule-like dimer. IDRs contribute to oligomerization synergistically with the coiled-coil interaction. Using confocal single-molecule FRET we demonstrated that PALB2-DBD compacts single-stranded DNA even in the absence of DNA secondary structures. The compaction is bimodal, oligomerization-dependent, and is driven by IDRs, suggesting a novel strand exchange mechanism. Intrinsically disordered proteins (IDPs) are prevalent in the human proteome. Novel DNA binding properties of PALB2-DBD and the complexity of strand exchange mechanism significantly expands the functional repertoire of IDPs. Multivalent interactions and bioinformatics analysis suggest that PALB2 function is likely to depend on formation of protein-nucleic acids condensates. Similar intrinsically disordered DBDs may use chaperone-like mechanism to aid formation and resolution of DNA and RNA multichain intermediates during DNA replication, repair and recombination.

biophysics↗

bioRxiv2022

Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit.

Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its regulation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing 8 key residues in the C-tail of RPL23 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to RPL23 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting a key residue in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

biochemistry↗

bioRxiv2020

A cysteine-based redox switch regulates millisecond dynamics of PDI and controls enzyme activity

Protein disulfide isomerase (PDI) is a ubiquitous redox-regulated enzyme that interacts with hundreds of client proteins intracellularly and extracellularly. It comprises two redox-sensitive domains, each hosting the conserved catalytic motif CxxC, two redox-insensitive protein-binding domains, and three linkers. Snapshots of oxidized and reduced PDI have been obtained by X-ray crystallography. Yet, how PDIs structure dynamically changes in response to the redox microenvironment and ligand binding remain unknown. Here, we used multiparameter confocal single-molecule Forster resonance energy transfer (smFRET) and multiple FRET pairs to track the movements of the two catalytic domains with high temporal resolution. Our studies document that, at equilibrium, PDI visits three structurally distinct conformational ensembles, two "open" (O1 and O2) and one "closed" (C). We show that the redox environment dictates the time spent in each ensemble and the rate at which they exchange. While oxidized PDI samples O1, O2 and C more evenly and in a slower fashion, reduced PDI predominantly populates O1 and O2, and exchanges between them more rapidly, on the sub-millisecond timescale. These findings were not expected based on crystallographic data. Using mutational analyses, we further demonstrate that the two active sites are structurally nonequivalent and that ligands targeting the active sites of reduced PDI shift the equilibrium towards closed conformations of the enzyme. This work introduces a new structural framework that challenges current views of PDI dynamics, helps rationalize the multifaced role of PDI in biology and may assist drug development.

biophysics↗

bioRxiv2020

X-ray and solution structures of human beta-2 glycoprotein I reveal a new mechanism of autoantibody recognition

Venous and arterial thromboses in patients suffering from the autoimmune disorder Antiphospholipid Syndrome (APS) are caused by the presence of antiphospholipid antibodies (aPL). Emerging evidence indicates that autoantibodies targeting the epitope R39-R43 in the N-terminal domain, Domain I (DI), of {beta}2-glycoprotein I ({beta}2GPI) are among the most pathogenic aPL in patients with APS. How such autoantibodies engage {beta}2GPI at the molecular level remains incompletely understood. Here, we have used X-ray crystallography, single-molecule FRET, and small-angle X-ray scattering to demonstrate that, in the free form, under physiological pH and salt concentrations, human recombinant {beta}2GPI adopts an elongated, flexible conformation in which DI is exposed to the solvent, thus available for autoantibody recognition. Consistent with this structural model, binding and mutagenesis studies revealed that the elongated form interacts with a pathogenic anti-DI antibody in solution, without the need of phospholipids. Furthermore, complex formation was affected neither by the neighboring domains, nor by the presence of the linkers, nor by the glycosylations. Since the pathogenic autoantibody requires residues R39 and R43 for optimal binding, these findings challenge longstanding postulates in the field envisioning {beta}2GPI adopting immunologic inert conformations featuring inaccessibility of the epitope R39-R43 in DI and support an alternative model whereby the preferential binding of anti-DI antibodies towards phospholipid-bound {beta}2GPI arises from the ability of the pre-existing elongated form to bind to the membranes and then oligomerize, processes that are likely to be supported by protein conformational changes. Interfering with these steps may limit the pathogenic effects of anti-DI antibodies in APS patients. SignificanceIn the autoimmune disorder called Antiphospholipid Syndrome (APS), the presence of autoantibodies targeting the plasma glycoprotein beta-2 glycoprotein I ({beta}2GPI) is associated with arterial and venous thrombosis as well as pregnancy complications. Understanding how {beta}2GPI becomes immunogenic and how autoantibodies in complex with {beta}2GPI cause the blood to clot remains a top priority in the field. By elucidating the structural architecture of {beta}2GPI free in solution, our studies challenge longstanding postulates in the field and shed new light on the pathogenic mechanisms of APS that may help the development of new diagnostics and therapeutic approaches.

biochemistry↗