Biology subjects

Pagotto, A.

Publications and source records attributed to Pagotto, A..

3 recordsLinked to original sources

bioRxiv2025

Systematic structural and functional analysis of metal-binding sites in native proteomes

Metal-binding proteins (MBPs) are widespread and critical for many biological processes. Proteomics-based approaches have identified MBPs in various systems but typically cannot determine the location of metal-binding sites or shed light on the functional roles of metals. Thus, a systems-wide map of metal binding sites and an understanding of the consequences of metal binding for protein function are still lacking. Here, we used limited proteolysis-coupled mass spectrometry (LiP-MS) combined with chelator treatment to globally map metal-protein interactions and binding sites in native protein lysates. We applied the pipeline, named Metal-LiP, to Escherichia coli, identified 981 peptides on 297 proteins that exhibited dose responsiveness to chelator treatment, and quantified the relative metal-binding affinity. These proteins are strongly enriched for known or predicted MBPs, based on our novel compiled ground truth dataset of previously annotated (n=1013) and predicted (n=850) MBPs. Further, chelator-responsive peptides mapped at or near annotated metal-binding sites in 3D protein structures, showing that Metal-LiP can identify metal-binding sites, and provided novel experimental evidence for previously predicted sites. Notably, Metal-LiP identified 225 novel candidate MBPs and we validated five of these novel MBPs (GatZ, AhpC, GrxB, MsrC, TrhO) using inductively coupled plasma-mass spectrometry to detect metal binding in purified proteins. Finally, we used orthogonal approaches to probe the broad functional consequences of metal binding. Using a combination of Metal-LiP, thermal proteome profiling, size-exclusion chromatography coupled to mass spectrometry, and structural analyses, we identified cases where metal binding affects protein stability, modulates the assembly states of protein complexes, or is likely to affect enzymatic activity. We thereby propose functional roles for metal binding in 98 of our Metal-LiP hits and in 225 proteins in total. In summary, we describe an approach to identify bona fide MBPs with binding site-level resolution across the proteome. We have combined it with other methods to identify 424 candidate MBPs in the E coli proteome, to map known and novel candidate metal-binding sites for 297 of these proteins, and to suggest functional roles of metal binding.

systems biology↗

bioRxiv2024

The Main protease (Mpro) from SARS-CoV-2 triggers plasma clotting in vitro by activating coagulation factors VII and FXII

Although the connection between COVID-19 and coagulopathy has been clear since the early days of SARS-CoV-2 pandemic, the underlying molecular mechanisms remain unclear. Available data support that the burst of cytokines and bradykinin, observed in some COVID-19 patients, sustains systemic inflammation and the hypercoagulant state, thus increasing thrombotic risk. Here we show that the SARS-CoV-2 main protease (Mpro) can play a direct role in the activation of the coagulation cascade. Adding Mpro to human plasma from healthy donors increased clotting probability by 2.5-fold. The results of enzymatic assays and degradomics analysis indicate that Mpro triggers plasma clotting by proteolytically activating coagulation factors zymogens VII and XII at their physiological activation sites, i.e. Arg152-Ile153 bond for FVII and Arg353-Val354 bond for FXII, where FVII and FXII are strategically positioned at the very beginning of the extrinsic or intrinsic pathways of blood coagulation. These findings are not compatible with the substrate specificity of the protease known so far, displaying a prevalence for a Gln-residue in P1 and a hydrophobic amino acid in P2 position. This apparent discrepancy was resolved by High Throughput Protease Screen assay, unveiling an extended, time-dependent, secondary specificity of Mpro for Arg-X bonds, which was further confirmed by Hydrogen-Deuterium Exchange Mass spectrometry analysis of Arg-containing inhibitors binding to Mpro and by enzymatic assays showing that the protease can cleave peptide substrates containing Arg in P1. Overall, integrating biochemical, proteomics and structural biology experiments, we unveil a novel mechanism linking SARS-CoV-2 infection to thrombotic complications in COVID-19.

biochemistry↗

bioRxiv2021

Human -Synuclein Inhibits Platelets Aggregation in vitro by Interfering with the -Thrombin/Protease-Activated Receptor 1 Functional Axis

-Synuclein (Syn) is a small (140 amino acids) disordered, acidic (pI: 4.7) protein, highly conserved in vertebrates and implicated in the pathogenesis of Parkinsons disease (PD), a neurodegenerative disease characterized by the deposition of Syn amyloid fibrils in dopaminergic neurons. Beyond the central nervous system, significant expression of Syn has also been measured in the blood (~1 M), where platelets are the main cellular hosts of Syn. Although the pathological implication of Syn in PD is widely accepted, the physiological role of blood Syn is still elusive. Starting from the notion that platelets are either the major cellular reservoir of Syn in the blood and, concomitantly, act as key players in hemostasis, being activated also by -thrombin (T) via cleavage of protease-activated receptors (PARs), we decided to investigate the possibility that Syn could modulate platelet activation by interfering with the T-PAR functional axis. Using multiple electrode aggregometry, i.e. a fast and specific platelet-function-testing method, as well as steady-state fluorescence spectroscopy, surface plasmon resonance, and fluorescence microscopy, we show here that monomeric Syn functions as a negative regulator of T-mediated platelets activation. Syn acts either directly, via competitive inhibition of PAR1 activation by T and TRAP6 agonist, and indirectly, by scavenging T on the platelet plasma membrane. A simple electrostatic model of Syn platelet antiaggregating effect is proposed and the possible role of the protein at the interplay of amyloidosis and thrombosis is discussed.

biochemistry↗