Biology subjects

Acquasaliente, L.

Publications and source records attributed to Acquasaliente, L..

4 recordsLinked to original sources

bioRxiv2026

Structural conservation and expanded functionality of hyper-stable human serum albumin variants

Human serum albumin (hSA) is the most abundant protein in human plasma, and its pharmacological properties, such as long plasma half-life mediated by the neonatal Fc receptor (FcRn) and its ability to bind endogenous and exogenous molecules, make it attractive for biotechnological applications. Currently, most wild type (WT) SAs are derived from human or bovine serum or produced in yeast and mammalian cells. Although well established, these methods are costly, difficult to reproduce, and not environmentally sustainable. Building on a previous study to design highly mutated hSA sequences, we extend the validation through an in-depth analysis of three engineered hSA variants; hSA1, hSA2, and hSA3, containing 16, 25, or 73 amino acid substitutions, respectively. These variants were designed for enhanced solubility, stability, and expression in Escherichia coli. All three variants showed low- micromolar affinities for hFcRn at pH 5.5, and negligible binding at pH 7.4. In a human endothelial cell-based recycling assay (HERA), the engineered hSA variants were recycled by hFcRn to the same extent as hSA isolated from serum. Exploring the properties of canonical drug-binding sites, warfarin affinity was comparable to WT hSA, whereas ibuprofen binding differed. Complementary cytotoxicity assays on human macrophages confirmed negligible toxicity and biocompatibility. A cryo-electron microscopy structure of hSA3 revealed that, despite extensive engineering, the native heart-shape of hSA, folding of domains, and its open conformation were preserved. These findings validate the structural integrity and functional adaptability of engineered hSA variants, underscoring their potential as versatile, animal-free solutions for next-generation therapeutics and biotechnological applications.

biochemistry↗

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↗

bioRxiv2020

Mapping specificity, entropy, allosteric changes and substrates in blood proteases by a high-throughput protease screen

Proteases are among the largest protein families in eukaryotic phylae with more than 500 genetically encoded proteases in humans. By cleaving a wide range of target proteins, proteases are critical regulators of a vast number of biochemical processes including apoptosis and blood coagulation. Over the last 20 years, knowledge of proteases has been drastically expanded by the development of proteomic approaches to identify and quantify proteases and their substrates. In spite of their merits, some of these methods are laborious, not scalable or incompatible with native environments. Consequentially, a large number of proteases remain poorly characterized. Here, we introduce a simple proteomic method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter and their analysis in a mass spectrometer. The method is significantly faster, cheaper, technically less demanding, easily multiplexed and produces accurate protease fingerprints in near-native conditions. By using the blood cascade proteases as a case study we obtained protease substrate profiles of unprecedented depth that can be reliably used to map specificity, entropy and allosteric changes of the protease and to design fluorescent probes and predict physiological substrates. The native protease characterization method is comparable in performance, but largely exceeds the throughput of current alternatives.

biochemistry↗