Paolo Mereghetti

Intrinsically disordered proteins (IDPs) and regions constitute a significant fraction of the proteome. Unlike globular proteins, IDPs exist as dynamic conformational ensembles, allowing functional versatility while challenging conventional structural methods, necessitating an integrated, multi-method approach. Here, we systematically compare IDPs and globular proteins using Fourier Transform Infrared (FTIR) and circular dichroism (CD) spectroscopies to investigate not simply the presence of disorder but also its structural organization. By analyzing the second derivatives of the Amide I′ absorption spectra, we identified a distinct spectral fingerprint of IDPs. Detailed analysis reveals that the spectral features encode distinct modes of structural organization within conformationally heterogeneous ensembles. Notably, the integration of second-derivative FTIR (SD-IR) and CD spectroscopies uncovers conformational states that are not captured by conventional techniques devoted to secondary structure characterization. Specifically, we identified conditions in which proteins retain a conformational order despite exhibiting random coil CD signatures (“ordered random coils”) as well as states displaying canonical α-helical CD features while maintaining substantial structural heterogeneity (“disordered α-helices”). Molecular dynamics simulations support these assignments, revealing the structural basis underlying the spectroscopic findings. Finally, we demonstrate the efficacy of SD-IR spectroscopy for validating putative IDPs, exemplified by the silkworm protein sericin. Overall, this study establishes second-derivative analysis of the Amide I′ band not merely as a diagnostic marker of disorder but also as a powerful approach to resolve distinct organizational states within conformationally heterogeneous protein ensembles.

Macrophages are among the first immune cells involved in the initiation of the inflammatory response to protect the host from pathogens. THP-1 derived macrophages (TDM) are used as a model to study the pro-inflammatory effects of lipopolysaccharide (LPS) exposure. Intact TDM cells were analysed by Fourier transform infrared (FTIR) microspectroscopy, supported by multivariate analysis, to obtain a snapshot of the molecular events sparked by LPS stimulation in macrophage-like cells. This spectroscopic analysis enabled the untargeted identification of the most significant spectral components affected by the treatment, ascribable mainly to lipid, protein, and sulfated sugar bands, thus stressing the fundamental role of these classes of molecules in inflammation and in immune response. Our study, therefore, shows that FTIR microspectroscopy enabled the identification of spectroscopic markers of LPS stimulation and has the potential to become a tool to assess those global biochemical changes related to inflammatory and anti-inflammatory stimuli of synthetic and natural immunomodulators different from LPS.

Biofluid analysis by optical spectroscopy techniques is attracting considerable interest due to its potential to revolutionize diagnostics and precision medicine, particularly for neurodegenerative diseases. However, the lack of effective biomarkers combined with the unaccomplished identification of convenient biofluids has drastically hampered optical advancements in clinical diagnosis and monitoring of neurodegenerative disorders. Here, we show that vibrational spectroscopy applied to human tears opens a new route, offering a non-invasive, label-free identification of a devastating disease such as amyotrophic lateral sclerosis (ALS). Our proposed approach has been validated using two widespread techniques, namely, Fourier transform infrared (FTIR) and Raman microspectroscopies. In conjunction with multivariate analysis, this vibrational approach made it possible to discriminate between tears from ALS patients and healthy controls (HCs) with high specificity (∼97% and ∼100% for FTIR and Raman spectroscopy, respectively) and sensitivity (∼88% and ∼100% for FTIR and Raman spectroscopy, respectively). Additionally, the investigation of tears allowed us to disclose ALS spectroscopic markers related to protein and lipid alterations, as well as to a reduction of the phenylalanine level, in comparison with HCs. Our findings show that vibrational spectroscopy is a new potential ALS diagnostic approach and indicate that tears are a reliable and non-invasive source of ALS biomarkers.

Amyloid aggregation of human ataxin-3 (ATX3) is responsible for spinocerebellar ataxia type 3, which belongs to the class of polyglutamine neurodegenerative disorders. It is widely accepted that the formation of toxic oligomeric species is primarily involved in the onset of the disease. For this reason, to understand the mechanisms underlying toxicity, we expressed both a physiological (ATX3-Q24) and a pathological ATX3 variant (ATX3-Q55) in a simplified cellular model, Escherichia coli. It has been observed that ATX3-Q55 expression induces a higher reduction of the cell growth compared to ATX3-Q24, due to the bacteriostatic effect of the toxic oligomeric species. Furthermore, the Fourier transform infrared microspectroscopy investigation, supported by multivariate analysis, made it possible to monitor protein aggregation and the induced cell perturbations in intact cells. In particular, it has been found that the toxic oligomeric species associated with the expression of ATX3-Q55 are responsible for the main spectral changes, ascribable mainly to the cell envelope modifications. A structural alteration of the membrane detected through electron microscopy analysis in the strain expressing the pathological form supports the spectroscopic results.

Deposition of misfolded proteins as extracellular amyloid aggregates is the pathological hallmark of systemic amyloidoses. Subcutaneous fat acquired by fine needle aspiration is the preferred screening tissue in suspected patients. In this study we employed Fourier transform infrared (FTIR) spectroscopy in attenuated total reflection (ATR) to investigate human abdominal fat aspirates with the aim to detect disease-related changes in the molecular structure and composition of the tissue and to exploit the potentiality of the method to discriminate between amyloid-positive and negative samples. The absorption and second derivative spectra of Congo Red (CR) positive and CR-negative specimens were analyzed by three multivariate methods in four spectral regions. The proposed ATR-FTIR method is label-free, rapid, relatively inexpensive and requires minimal sample preparation. We found that the ATR-FTIR approach can differentiate fat aspirates containing amyloid deposits from control specimens with high sensitivity and specificity, both of 100 [89-100]%. Noteworthy, the wavenumbers most important for discrimination indicate that changes both in the protein conformation and in resident lipids are intrinsic features of affected subcutaneous fat in comparison with the CR-negative controls. In this proof of concept study, we show that this approach could be useful for assessing tissue amyloid aggregates and for acquiring novel knowledge on the molecular bases of the disease.

The transferrin receptor (TfR) is a promising target in cancer therapy owing to its overexpression in most solid tumors and on the blood–brain barrier. Nanostructures chemically derivatized with transferrin are employed in TfR targeting but often lose their functionality upon injection in the bloodstream. As an alternative strategy, we rationally designed a peptide coating able to bind transferrin on suitable pockets not involved in binding to TfR or iron by using an iterative multiscale-modeling approach coupled with quantitative structure–activity and relationship (QSAR) analysis and evolutionary algorithms. We tested that selected sequences have low aspecific protein adsorption and high binding energy toward transferrin, and one of them is efficiently internalized in cells with a transferrin-dependent pathway. Furthermore, it promotes transferrin-mediated endocytosis of gold nanoparticles by modifying their protein corona and promoting oriented adsorption of transferrin. This strategy leads to highly effective nanostructures, potentially useful in diagnostic and therapeutic applications, which exploit (and do not suffer) the protein solvation for achieving a better targeting.

Hydrophobins, produced by filamentous fungi, are small amphipathic proteins whose biological functions rely on their unique surface-activity properties. Understanding the mechanistic details of the multimerization process is of primary importance to clarify the interfacial activity of hydrophobins. We used free energy calculations to study the role of a flexible β-hairpin in the multimerization process in hydrophobin II from Trichoderma reesei (HFBI). We characterized how the displacement of this β-hairpin controls the stability of the monomers/dimers/tetramers in solution. The regulation of the oligomerization equilibrium of HFBI will necessarily affect its interfacial properties, fundamental for its biological function and for technological applications. Moreover, we propose possible routes for the multimerization process of HFBI in solution. This is the first case where a mechanism by which a flexible loop flanking a rigid patch controls the protein-protein binding equilibrium, already known for proteins with charged binding hot-spots, is described within a hydrophobic patch. Fungi proliferate by creating a complex hyphal network growing within a wet environment. However, for most fungi to colonize new territories, they must produce spores carried by aerial hyphae and spread them into the air. Aerial structures need to overcome the surface tension of the surrounding water in order to grow into the air. This process requires hydrophobins, a remarkable class of self-associating fungal proteins which lower the surface tension at the air/water interface by creating a thin amphipathic layer. In solution they form multimers in equilibrium with the interfacial layer. Due to their unique surface-activity properties, hydrophobins have been used for a variety of biotechnical applications. We used enhanced sampling molecular dynamics simulations methods to study the multimerization process in solution of a hydrophobin from Trichoderma reesei (HFBI). We clarified the fundamental role of a small flexible region within the HFBI monomer involved in the formation of multimers. A flexible loop flanking a rigid interaction patch is able to fine-tune the interaction energy. This mechanism, already known for charged binding patches, is described here for hydrophobic hot-spots. This result is remarkably important in order to clarify the mechanism of arranging at the interface and enhancing hydrophobin-based technological applications.

Microbial lipids can represent a valuable alternative feedstock for biodiesel production in the context of a viable bio-based economy. This production can be driven by cultivating some oleaginous microorganisms on crude-glycerol, a 10 % (w/w) by-product produced during the transesterification process from oils into biodiesel. Despite attractive, the perspective is still economically unsustainable, mainly because impurities in crude glycerol can negatively affect microbial performances. In this view, the selection of the best cell factory, together with the development of a robust and effective production process are primary requirements. The present work compared crude versus pure glycerol as carbon sources for lipid production by three different oleaginous yeasts: Rhodosporidium toruloides (DSM 4444), Lipomyces starkeyi (DSM 70295) and Cryptococcus curvatus (DSM 70022). An efficient yet simple feeding strategy for avoiding the lag phase caused by growth on crude glycerol was developed, leading to high biomass and lipid production for all the tested yeasts. Flow-cytometry and fourier transform infrared (FTIR) microspectroscopy, supported by principal component analysis (PCA), were used as non-invasive and quick techniques to monitor, compare and analyze the lipid production over time. Gas chromatography (GC) analysis completed the quali-quantitative description. Under these operative conditions, the highest lipid content (up to 60.9 % wt/wt) was measured in R. toruloides, while L. starkeyi showed the fastest glycerol consumption rate (1.05 g L−1 h−1). Being productivity the most industrially relevant feature to be pursued, under the presented optimized conditions R. toruloides showed the best lipid productivity (0.13 and 0.15 g L−1 h−1 on pure and crude glycerol, respectively). Here we demonstrated that the development of an efficient feeding strategy is sufficient in preventing the inhibitory effect of crude glycerol, and robust enough to ensure high lipid accumulation by three different oleaginous yeasts. Single cell and in situ analyses allowed depicting and comparing the transition between growth and lipid accumulation occurring differently for the three different yeasts. These data provide novel information that can be exploited for screening the best cell factory, moving towards a sustainable microbial biodiesel production.

Brownian dynamics (BD) simulations can be used to study very large molecular systems, such as models of the intracellular environment, using atomic-detail structures. Such simulations require strategies to contain the computational costs, especially for the computation of interaction forces and energies. A common approach is to compute interaction forces between macromolecules by precomputing their interaction potentials on three-dimensional discretized grids. For long-range interactions, such as electrostatics, grid-based methods are subject to finite size errors. We describe here the implementation of a Debye-Hückel correction to the grid-based electrostatic potential used in the SDA BD simulation software that was applied to simulate solutions of bovine serum albumin and of hen egg white lysozyme.