Human Serum Albumin: from molecular aspects to biotechnological applications

This PhD project aims to investigate the role of human serum albumin (HSA) in the context of innate immunity, specifically investigating the molecular determinants of the interaction between HSA and bacterial toxins and viral proteins. Furthermore, a further aim is to lay the foundation for the development of biosensors based on HSA and its unique binding capabilities. HSA is a globular protein composed of a single chain, structured into three homologous domains (I, II, and III), each of which contains two subdomains (A and B). As the most abundant protein in plasma, HSA plays a critical role as a transporter, a key regulator of plasma oncotic pressure, and the primary antioxidant protein. Additionally, HSA binds a wide range of drugs, affecting their pharmacodynamic and pharmacokinetic properties. Recent research has highlighted a new protective function of HSA in defending against pathogenic infections. HSA recognizes molecules from microorganisms, such as toxins and growth signals, neutralizing their toxic effects and inhibiting pathogen growth. Our research group has shown that HSA plays a significant role in the innate immune response, particularly during Clostridioides difficile (C. difficile) and Streptococcus pyogenes (S. pyogenes) infections. In this context, HSA functions as a self-defense mechanism by binding both TcdA and TcdB toxins, inducing conformational changes that lead to their autoproteolysis and inactivation, and neutralizing cytotoxic and hemolytic effect of SLO toxin. In addition, HSA has shown to neutralize cytotoxic effect of candidalysin toxin produced by Candida Albicans. Specifically, HSA interacts with TcdA, TcdB, SLO and candidalysin toxins through the domain II (DII), an unconventional binding site likely evolved to target pathogens. Here, an efficient protocol to produce the DII in bacteria has been assessed. Subsequently, the capability of the recombinant DII to recognize TcdA, TcdB and SLO has been reported. In detail, the capability of the DII to recognize these toxins has been assessed through ELISA. DII has shown to recognize these toxins with high affinity, with KD values comparable to those obtained for the interaction of HSA with these toxins (i.e., KD values of 26,5 nM, 21,4 nM e 12,2 nM for DII binding to TcdA, TcdB and SLO, respectively, and 46,45 nM, 7,1 nM e 2,5 nM for HSA binding to TcdA, TcdB and SLO, respectively). Furthermore, SPR results indicate that TcdA exhibit strong affinity for the DII, with KD value of 31 (± 8) nM. Additionally, preliminary bioinformatics data indicate that α-hemolysin, a toxin produced by Staphylococcus aureus, could be recognized by HSA, and specifically by the DII. 5 Consequently, we also evaluated if HSA and the recombinant DII bind α-hemolysin. Surprisingly, despite promising molecular docking analysis, neither HSA or DII has shown to be able to recognize a-hemolysin or to protect red blood cells (RBCs) from the hemolytic effects of the toxin. A further aim of this PhD thesis was to assess the molecular basis behind the clinical correlation between hypoalbuminemia and COVID-19 severity. COVID-19, caused by SARS- CoV-2, infects cells through the interaction between the virus spike protein and the ACE2 receptor expressed on host cells membrane. This interaction disrupts the renin-angiotensin system (RAS) and contributes to disease progression. With the aim of dissecting the role of HSA in SARS-CoV-2 infection, we analyze the capability of albumin to interact with spike. Obtained results indicated that HSA binds spike protein (KD = 2.2 µM), through an aminoacidic region of spike protein (i.e., the receptor binding motif (RBM)) that is also involved in the interaction with ACE2. This suggests that HSA may inhibit SARS-CoV-2 entry into host cells by competing with ACE2 for the binding to spike. Obtained data also show that HSA can reduce the replication rate of SARS-CoV-2 in VERO E6 cells, further supporting the idea that HSA limits viral infection. Additionally, we demonstrated that HSA modulates the expression of various components of the renin-angiotensin system (RAS). The study demonstrated that HSA upregulates the expression of ACE2, AT2, and MAS receptors in various human cell lines (i.e., A549 lung carcinoma cells and Hep-G2 liver cancer cells), and in VERO E6 cells infected with SARS-CoV-2. This HSA-mediated regulation of the RAS system counteracts the RAS imbalance caused by SARS-CoV-2, suggesting a protective effect against the progression of COVID-19. To further investigate the HSA protective roles in human plasma, the research activity conducted during this PhD project has been also focused on dissecting the role of HSA in the scavenging and delivery of heme. HSA binds free heme with high affinity, playing a crucial role in regulating heme metabolism, preventing toxicity, and potentially restricting heme availability to pathogens. Indeed, free heme, which contains ferrous iron (Fe(II)), can induce cellular damage by generating reactive oxygen species (ROS) through the Fenton reaction. Heme can disrupt cell membranes, trigger inflammation, oxidize LDLs, and impair vascular function by deactivating nitric oxide. HSA not only neutralizes heme-dependent harmful effects but also facilitates its transfer to hemopexin, which then transports it to the liver for safe degradation. This pathway is vital for protecting the body from oxidative stress, particularly in 6 conditions characterized by excessive red blood cell breakdown (e.g., severe hemolytic anemia, crush syndrome, and ischemia-reperfusion injuries). The above reported findings are particularly important considering the broader goal of this PhD thesis, that is the development of low-cost lab-on-chip HSA-based biosensors for the early detection of toxins and/or toxic metabolites in liquid biopsies. Given recent advancements in terahertz (THz) devices, including both sources and detectors, new possibilities have emerged in medicine and biomolecular research. Albumin-based biosensors show great potential for detecting a wide range of ligands such as drugs, hormones, metabolites, and pathogenic proteins. In detail, we created a protein-sensing platform by immobilizing albumin onto CMOS-compatible Ge-based THz plasmonic antennas using drop-cast biofunctionalization. To validate the biosensor, the platform was used to quantitatively measure the binding of heme. This measurement was performed using THz time-domain spectroscopy in dichroic transmission mode, achieving a sensitivity of approximately 200 GHz/mM for the HSA:hemin complex. These initial results demonstrate the potential of CMOS-compatible Ge- based THz plasmonic antennas as innovative sensors, capable of seamless integration with conventional electronics for data storage, processing, and communication within a unified system. The quantitative determination of heme is needed for everyday use and applications in developing countries, such as malaria control or point-of-care clinical applications. Moreover, due to HSA capacity to bind bacterial and fungal toxins, such biosensors could also aid in the early identification of toxemia in patients with infections. To further improve the sensing devices, additional immobilization strategies will be explored, aimed at achieving covalent binding of HSA to the antenna. Finally, with the aim to use zebrafish (Danio rerio) to further evaluate HSA role in the innate immunity, here we performed an in-depth analysis of zebrafish hematopoiesis, innate immune cell function, and interactions with various pathogens. Notably, zebrafish larvae depend solely on innate immunity in early development, as the adaptive immune system matures only 4–6 weeks post-fertilization. This window provides a unique opportunity to isolate and examine infection and inflammation mechanisms driven by the innate immune response without the confounding effects of adaptive immunity. The findings presented in this PhD thesis shed light on the role of HSA within the innate immune system. HSA appears to function as a soluble effector, capable of recognizing and neutralizing a range of pathogen-associated molecular patterns (PAMPs), such as toxins, envelope proteins, and nucleic acids. This function helps protect the body from infection and slows disease progression. The data also suggest that the DII of HSA serves as a unique binding 7 site evolved to interact with hydrophobic molecules and structures derived from microorganisms. This selective binding potential are of particular interest for the development of biosensors for the early detection of toxemia in liquid biopsy. Additionally, given HSA significant role in heme scavenging and delivery, a biosensor based on HSA could be valuable for detecting hemolytic events. Lastly, the zebrafish model has proven exceptionally useful for targeted studies of the innate immune response due to its unique developmental features.