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Thymosins have been extracted, characterized, and identified from Thymus. The Thymosins are hormones whose therapeuric applications have seen a recent increase. The action of Thymosin α1 is based on the stimulation of the immune response with a large number of results in a variety of pathologies. The absence of a specific receptor prompted us to investigate the direct interaction with membranes, particularly those exposing phosphatidylserine thus contributing to assess the Thymosin α1's pleiotropy.The interaction with membranes has been studied with a number of models indicating that Thymosin α1 interacts preferentially with negative regions of the membrane (SDS mixed with dodecylphosphocholine) or, better, with vesicles of dipalmitoylphosphatidylcholine with exposed phosphatidylserine.The study of the role of the membrane in the mechanism of action of Thymosin α1 indicated that probably the first interaction occurs at the membrane level with recognition of negative surface due to the phosphatidylserine exposure. Upon assuming a conformation, with two helices with a disordered tract in between, it diffuses on the membrane surface by lateral diffusion. Then the interaction with membrane receptor(s) causes a membrane complex to be formed, with an activation of a signalling cascade. This can be considered the basis of its pleiotropy. Differences in structuration mechamism of Thymosin β4 was outlined.

The newly identified CUBAN (Cullin binding domain associating with NEDD8) domain recognizes both ubiquitin and the ubiquitin-like NEDD8. Despite the high similarity between the two molecules, CUBAN shows a clear preference for NEDD8, free and conjugated to cullins. We previously characterized the domain structure, both alone and in complex with NEDD8. The results here reported are addressed to investigate the determinants that drive the selective binding of CUBAN towards NEDD8 and ubiquitin. The 15N HSQC NMR perturbation pattern of the labeled CUBAN domain, when combined with either NEDD8 or ubiquitin, shows a clear involvement of hydrophobic residues that characterize the early stages of these interactions. After a slow conformational selection step, hydrophobic and then neutral and polar interactions take place, which drive the correct orientation of the CUBAN domain, leading to differences in the recognition scheme of NEDD8 and ubiquitin. As a result, a cascade of induced fit steps seems to determine the structural preference shown for NEDD8 and therefore the basis of the selectivity of the CUBAN domain. Finally, molecular dynamics analysis was performed to determine by fluctuations the internal flexibility of the CUBAN/NEDD8 complex. We consider that our results, based on a structural investigation mainly focused on the early stages of the recognition, provide a fruitful opportunity to report the different behavior of the same protein with two highly similar binding partners.

During a penal trial, it was requested to our laboratory to contribute the public minister prosecutor to study with a solid-state technique the effect of the time on a photographic negative and, eventually, to determine if the date of a certain photographic development can be located in a particular time range. It was important the determination of the time when a picture has been developed. Particularly, it was asked to define an estimate, more precisely possible, of the time elapsed from when the picture was taken assuming that it was close to the time of the processing of the negative of the photograph. The 13C cross polarization magic angle spinning nuclear magnetic resonance spectroscopy is known as a technique that is able to characterize the crystallinity of a sample. Among many materials, cellulose matrix is one whose crystallinity is more influenced on time. Assuming that after the wet development of the negative, the transformation of the crystallinity decreases upon time toward the amorphous state, it was possible, with a suitable reference scale, to obtain the approximate date of the development of the picture as required in the trial. This was possible by the preparation of a crystallinity reference scale obtained by photographic negatives developed at precise intervals along time.

Thymosin α1 is a peptidic hormone with pleiotropic activity and is used in the therapy of several diseases. It is unstructured in water solution and interacts with negative regions of vesicles by assuming two tracts of helical conformation with a structural break between them. This study reports on Thymosin α1's interaction with mixed phospholipids phosphatidylcholine and phosphatidylserine, the negative component of the membranes, by ¹H and natural abundance ¹⁵N nuclear magnetic resonance (NMR). The results indicate that interaction occurs when the membrane is negatively charged by exposing phosphatidylserine. Moreover, the direct interaction of Thymosin α1 with K562 cells with an overexposure of phosphatidylserine as a consequence of resveratrol-induced apoptosis was conducted. Thymosin α1's interaction with human serum albumin was also investigated by NMR spectroscopy. Steady-state saturation transfer, transfer nuclear Overhauser effect spectroscopy, and diffusion-ordered spectroscopy methodologies all reveal that the C-terminal region of Thymosin α1 is involved in the interaction with serum albumin. These results may shed more light on Thymosin α1's mechanism of action by its insertion in negative regions of membranes due to the exposure of phosphatidylserine. Once Thymosin α1's N-terminus has been inserted into the membrane, the rest may interact with nearby proteins and/or receptors acting as effectors and causing a biological signaling cascade, thus exerting thymosin α1's pleiotropy.

Ubiquitin binding domains (UBDs) are modular elements that bind non-covalently to ubiquitin and act as downstream effectors and amplifiers of the ubiquitination signal. With few exceptions, UBDs recognize the hydrophobic path centered on Ile44 (Leu-8, Ile-44, Val-70). Nevertheless, a variety of different orientations, which can be attributed to specific contacts between each UBD and surface residues surrounding the hydrophobic patch, specify how each class of UBD recognizes ubiquitin. Here, we describe the structure of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis and NMR spectroscopy of the 15N isotopically labelled protein, we demonstrate that a Phe-Pro motif in N4BP1 recognizes the canonical hydrophobic patch of ubiquitin. This recognition mode resembles the molecular mechanism evolved in the CUE (Coupling of ubiquitin conjugation to ER degradation) domain family, where an invariant proline, usually following a phenylalanine, is required for binding to ubiquitin. Interestingly, the UBD of N4BP1 is evolutionary related to CUBAN (Cullin binding domain associating with NEDD8) (40% identity and 47% similarity), a protein module that also recognizes the ubiquitin-like NEDD8, which is the closest relative of ubiquitin (58% identity and 80% similarity). By performing circular dichroism and 15NNMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the UBD of N4BP1 lacks the NEDD8 binding properties observed in CUBAN and it recognizes the Ile44-centered patch of ubiquitin through a dedicated binding site, which share some of the features observed in the CUE domain family. Moreover, we show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both the CUBAN and the UBD of N4BP1 are poly-ubiquitinated in cells. This modification is dependent on the presence of a functional domain. We believe that the structural and functional characterization of this novel UBD will allow a deeper understanding of the molecular mechanisms governing N4BP1 function, while at the same time providing a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved.

Ubiquitin binding domains (UBDs) are modular elements that bind non-covalently to ubiquitin and act as downstream effectors and amplifiers of the ubiquitination signal. With few exceptions, UBDs recognize the hydrophobic path centered on Ile44 (Leu-8, Ile-44, Val-70). Nevertheless, a variety of different orientations, which can be attributed to specific contacts between each UBD and surface residues surrounding the hydrophobic patch, specify how each class of UBD recognizes ubiquitin. Here, we describe the structure of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis and NMR spectroscopy of the 15N isotopically labelled protein, we demonstrate that a Phe-Pro motif in N4BP1 recognizes the canonical hydrophobic patch of ubiquitin. This recognition mode resembles the molecular mechanism evolved in the CUE (Coupling of ubiquitin conjugation to ER degradation) domain family, where an invariant proline, usually following a phenylalanine, is required for binding to ubiquitin. Interestingly, the UBD of N4BP1 is evolutionary related to CUBAN (Cullin binding domain associating with NEDD8) (40% identity and 47% similarity), a protein module that also recognizes the ubiquitin-like NEDD8, which is the closest relative of ubiquitin (58% identity and 80% similarity). By performing circular dichroism and 15NNMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the UBD of N4BP1 lacks the NEDD8 binding properties observed in CUBAN and it recognizes the Ile44-centered patch of ubiquitin through a dedicated binding site, which share some of the features observed in the CUE domain family. Moreover, we show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both the CUBAN and the UBD of N4BP1 are poly-ubiquitinated in cells. This modification is dependent on the presence of a functional domain. We believe that the structural and functional characterization of this novel UBD will allow a deeper understanding of the molecular mechanisms governing N4BP1 function, while at the same time providing a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved.

Among the members of the ubiquitin-like (Ubl) protein family, neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8) is the closest in sequence to ubiquitin (57% identity). The two modification mechanisms and their functions, however, are highly distinct and the two Ubls are not interchangeable. A complex network of interactions between modifying enzymes and adaptors, most of which are specific while others are promiscuous, ensures selectivity. Many domains that bind the ubiquitin hydrophobic patch also bind NEDD8 while no domain that specifically binds NEDD8 has yet been described. Here, we report an unbiased selection of domains that bind ubiquitin and/or NEDD8 and we characterize their specificity/promiscuity. Many ubiquitin-binding domains bind ubiquitin preferentially and, to a lesser extent, NEDD8. In a few cases, the affinity of these domains for NEDD8 can be increased by substituting the alanine at position 72 with arginine, as in ubiquitin. We have also identified a unique domain, mapping to the carboxyl end of the protein KHNYN, which has a stark preference for NEDD8. Given its ability to bind neddylated cullins, we have named this domain CUBAN (Cullin-Binding domain Associating with NEDD8). We present here the solution structure of the CUBAN domain both in the isolated form and in complex with NEDD8. The results contribute to the understanding of the discrimination mechanism between ubiquitin and the Ubl. They also provide new insights on the biological role of a ill-defined protein, whose function is hitherto only predicted.

Thymosin α1 (Tα1) is a peptide hormone whose therapeutic application has been approved in several diseases, but the description of a precise receptor for its therapeutic action still remains elusive and some knowledge of the mechanism of interaction with the cell membrane still needs to be clarified. This work is aimed at studying the folding and interaction of Tα1, which is completely unstructured in water solution, with model membranes.The folding and interaction of Tα1 with sodium dodecyl sulfate micelles was monitored by NMR and CD spectroscopy techniques.Tα1 assumes a helical conformation in the presence of sodium dodecyl sulfate micelles, showing a helical fold with a structural break around residues 9 and 14. These results were confirmed by circular dichroism and NMR spectroscopy. Moreover, by paramagnetic NMR relaxation it was found that Tα1 is inserted in the hydrophobic region of the micelles by the residues 1 - 5 of the N-terminal end. This result clarifies the modality of insertion that was not obtained in previous NMR studies in trifluoroethanol.These findings suggest that Tα1 folds on the membrane and, when inserted, may be able to interact with nearby proteins and/or receptors acting as an effector and causing a biological signaling cascade.

Thymosin α1 (Tα1), is a peptidic hormone, whose immune regulatory properties have been demonstrated both in vitro and in vivo and approved in different countries for treatment of several viral infections and cancers. Tα1 assumes a conformation in negative membranes upon insertion into the phosphatidylserine exposure as found in several pathologies and in apoptosis. These findings are in agreement with the pleiotropy of Tα1, which targets both normal and tumor cells, interacting with multiple cellular components, and have generated renewed interest in the topic. Hyaluronan (HA) occurs ubiquitously in the extracellular matrix and on cell surfaces and has been related to a variety of diseases, and developmental and physiological processes. Proteins binding HA, among them CD44 and the Receptor for HA-mediated motility (RHAMM) receptors, mediate its biological effects. NMR spectroscopy indicated preliminarily that an interaction of Tα1 with HA occurs specifically around lysine residues of the sequence LKEKK of Tα1 and is suggestive of a possible interference of Tα1 in the binding of HA with CD44 and RHAMM. Further studies are needed to deepen these observations because Tα1 is known to potentiate the T-cell immunity and anti-tumor effect. The binding inhibitory activity of Tα1 on HA-CD44 or HA-RHAMM interactions can suppress both T-cell reactivity and tumor progression.

The structural characterization of [W8S]contryphan Vn, an analogue of Contryphan Vn with tryptophan 8 substituted with a serine residue (W8S), was performed by NMR spectroscopy, molecular dynamics simulations and fluorescence spectroscopy. Contryphan Vn, a bioactive cyclic peptide from the venom of the cone snail Conus ventricosus, contains an S-S bridge between two cysteines and a D-tryptophan. Like other Contryphans, [W8S]contryphan Vn has proline 7 isomerized trans, while the proline 4 has nearly equivalent populations of cis and trans configurations. The thermodynamic and kinetic parameters of the trans-cis isomerization of proline 4 were measured. The isomers of [W8S]contryphan Vn with proline 4 in cis and trans show structural differences. The absence of the salt bridge between the same Asp2 and Lys6, present in Contryphan Vn, may be attributed to the lack of the hydrophobic side chain of Trp8 where it likely protects the electrostatic interactions. These results may contribute to identifying, in these cyclic peptides, the structural determinants of the mechanism of proline trans-cis isomerization, this being also an important step in protein folding.

Ubiquitin binding domains (UBDs) are modular elements that bind non-covalently to ubiquitin and act as downstream effectors and amplifiers of the ubiquitination signal. With few exceptions, UBDs recognize the hydrophobic path centered on Ile44, including residues Leu8, Ile44, His68, and Val70. A variety of different orientations, which can be attributed to specific contacts between each UBD and surface residues surrounding the hydrophobic patch, specify how each class of UBD specifically contacts ubiquitin. Here, we describe the structural model of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis, and nuclear magnetic resonance (NMR) spectroscopy of the

Humic substances (HS) are the largest constituent of soil organic matter and are considered as a key component of the terrestrial ecosystem. HS may facilitate the transport of organic and inorganic molecules, as well as the sorption interactions with environmentally relevant proteins such as prions. Prions enter the environment through shedding from live hosts, facilitating a sustained incidence of animal prion diseases such as Chronic Wasting Disease and scrapie in cervid and ovine populations, respectively. Changes in prion structure upon environmental exposure may be significant as they can affect prion infectivity and disease pathology. Despite its relevance, the mechanisms of prion interaction with HS are still not completely understood. The goal of this work is to advance a structural-level picture of the encapsulation of recombinant, non-infectious, prion protein (PrP) into different natural HS. We observed that PrP precipitation upon addition of HS is mainly driven by a mechanism of “salting-out” whereby PrP molecules are rapidly removed from the solution and aggregate in insoluble adducts with humic molecules. Importantly, this process does not alter the protein folding since insoluble PrP retains its α-helical content when in complex with HS. The observed ability of HS to promote PrP insolubilization without altering its secondary structure may have potential relevance in the context of “prion ecology”. These results suggest that soil organic matter interacts with prions possibly without altering the protein structures. This may facilitate prions preservation from biotic and abiotic degradation leading to their accumulation in the environment.

Thymosinα1 is a peptidic hormone with pleiotropic activity, which is used in the therapy of several diseases. It is unstructured in water solution and interacts with negative regions of micelles and vesicles assuming two tracts of helical conformation with a structural flexible break in between. The studies of the interaction of Thymosinα1 with micelles of mixed dipalmitoylphosphatidylcholine and sodium dodecylsulfate and vesicles with mixed dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylserine, the latter the negative component of the membranes, by (1)H and natural abundance (15)N NMR are herewith reported, reviewed, and discussed. The results indicate that the preferred interactions are those where the surface is negatively charged due to sodium dodecylsulfate or due to the presence of dipalmitoylphosphatidylserine exposed on the surface. In fact the unbalance of dipalmitoylphosphatidylserine on the cellular surface is an important phenomenon present in pathological conditions of cells. Moreover, the direct interaction of Thymosinα1 with K562 cells presenting an overexposure of phosphatidylserine as a consequence of resveratrol-induced apoptosis was carried out.

Inclusion of drugs in cyclodextrins (CDs) is a recognized tool for modifying several properties such as solubility, stability, bioavailability, and so on. The photoreactive behavior of the β-CD/ketoprofen (KP) complex upon UV exposure showed a significant increase in photodecarboxylation, whereas the secondary degradation products by hydroxylation of the benzophenone moiety were inhibited. The results may account for an improvement of KP photophysical properties upon inclusion, thus better fostering its topical use. To correlate the structural details of the inclusion with these results, an NMR spectroscopic study of KP upon inclusion in β-CD was performed. Effects of the magnetically anisotropic centers of KP, changing their orientations upon inclusion and giving chemical shift variations, were specifically correlated with the results of the molecular dynamic simulations and ab initio calculations. In the large variety of papers focusing on the structural analysis of β-CD complexes, this work represents one of the few examples in which a detailed analysis of these simultaneous upfield-downfield NMR shifts of the same aromatic molecule upon inclusion is reported. Interestingly, the results demonstrate that the observed upfield and downfield shifts upon inclusion are not related to any direct magnetic role of β-CD. The conformational change of KP upon the inclusion process consists of a slight reduction in the angle between the two phenyl rings and in a remarkable reduction in the mobility of the carboxyl group, the latter being one of the main contributions to the NMR resonance shifts. These structural details help in understanding the features of the inclusion complex and, eventually, the driving force for its formation.

Thymosin α1 is a peptidic hormone already used in the therapy of several diseases. Until now, the description of the precise receptor and mechanism for its action still remains elusive. The interaction of Thymosin α1, which is unstructured in water solution, has been recently studied in sodium dodecylsulphate micellar systems and it was reported that Thymosin α1 inserts in micelle assuming a conformation with two tracts of helix with a structural break in between. An investigation of its interaction both with micelles of dodecylphosphocholine alone and with mixed dodecylphosphocholine-sodium dodecylsulphate micelles is here reported. In these environments the results indicate that Thymosin α1 in phospholipidic membrane exposing choline polar heads interacts by aspecific modality and, oppositely, in the mixed dodecylphosphocholine-sodium dodecylsulphate micelles an insertion in the micellar hydrophobic region conformationally similar to that found in sodium dodecylsulphate micelles occurs. In presence of mixed micelles the insertion and structuration occur in preferred regions when the membrane models are negatively charged. From the point of view of the mechanism of action, insertion its N terminus in negative regions of membrane led to hypothesize that this process would be similar to a binding to phosphatidylserine exposed like in apoptotic cells. Thymosin α1 when inserted may interact with nearby proteins and/or receptors acting as effector and causing a biological signaling cascade. The recent attention to the phosphatidylserine exposure in cells may enforce the interest for these findings.

Nuclear Magnetic Resonance (NMR) methodologies offer a comprehensive characterization of foodstuff owing to the possibility to study a sample from different points of view including structural, compositional, functional, morphological etc. aspects. High resolution NMR spectroscopy applied to semi-solid food samples or to extracts in solution is used to determine the foodstuff composition. Here, some features of high resolution NMR methodologies related to food analysis such as quantitative analysis, chemometrics, and use of databases are included. Other NMR methodologies such as relaxometry and imaging described in this chapter give precious information regarding the morphology and texture of intact food samples.