Conversion of the Native N-Terminal Domain of TDP-43 into a Monomeric Alternative Fold with Lower Aggregation Propensity.

TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and ß-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were compared with and without pre-incubation with SB3-10, but at the identical final SB3-10 concentration, a slower aggregation was found in the former case, despite the reversible attainment of the native conformation in both cases. This was attributed to protein monomerization and oligomeric seeds disruption by the conditions promoting the alternative conformation. Overall, the results show a high plasticity of TDP-43 NTD and identify strategies to monomerise TDP-43 NTD for methodological and biomedical applications.

CUBAN, a Case Study of Selective Binding: Structural Details of the Discrimination between Ubiquitin and NEDD8.

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.

Thymosin α1 Interacts with Hyaluronic Acid Electrostatically by Its Terminal Sequence LKEKK.

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.

Thymosin α1 Interacts with Exposed Phosphatidylserine in Membrane Models and in Cells and Uses Serum Albumin as a Carrier.

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.

New studies about the insertion mechanism of Thymosin α1 in negative regions of model membranes as starting point of the bioactivity.

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.

Structure of the cyclic peptide [W8S]contryphan Vn: effect of the tryptophan/serine substitution on trans-cis proline isomerization.

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.

Thymosin α1 interacts with Galectin-1 modulating the ß-galactosides affinity and inducing alteration in the biological activity.

The study of mechanism of action of Thymosin alpha 1 (Tα1) and the basis of the pleiotropic effect in health and disease, is one of the main focus of our ongoing research. Tα1 is a thymic peptide that demonstrates a peculiar ability to restore homeostasis in different physiological and pathological conditions (i.e., infections, cancer, immunodeficiency, vaccination, and aging) acting as multitasking protein depending on the host state of inflammation or immune dysfunction. However, few are the information about mechanisms of action mediated by specific Tα1-target protein interaction that could explain its pleiotropic effect. We investigated the interaction of Tα1 with Galectin-1 (Gal-1), a protein belonging to an oligosaccharide binding protein family involved in a variety of biological and pathological processes, including immunoregulation, infections, cancer progression and aggressiveness. Using molecular and cellular methodological approaches, we demonstrated the interaction between these two proteins. Tα1 specifically inhibited the hemagglutination activity of Gal-1, the Gal-1 dependent in vitro formation of endothelial cell tubular structures, and the migration of cancer cells in wound healing assay. Physico-chemical methods revealed the details of the molecular interaction of Tα1 with Gal-1. Hence, the study allowed the identification of the not known until now specific interaction between Tα1 and Gal-1, and unraveled a novel mechanism of action of Tα1 that could support understanding of its pleiotropic activity.

Calcium Binds to Transthyretin with Low Affinity.

The plasma protein transthyretin (TTR), a transporter for thyroid hormones and retinol in plasma and cerebrospinal fluid, is responsible for the second most common type of systemic (ATTR) amyloidosis either in its wild type form or as a result of destabilizing genetic mutations that increase its aggregation propensity. The association between free calcium ions (Ca2+) and TTR is still debated, although recent work seems to suggest that calcium induces structural destabilization of TTR and promotes its aggregation at non-physiological low pH in vitro. We apply high-resolution NMR spectroscopy to investigate calcium binding to TTR showing the formation of labile interactions, which leave the native structure of TTR substantially unaltered. The effect of calcium binding on TTR-enhanced aggregation is also assessed at physiological pH through the mechano-enzymatic mechanism. Our results indicate that, even if the binding is weak, about 7% of TTR is likely to be Ca2+-bound in vivo and therefore more aggregation prone as we have shown that this interaction is able to increase the protein susceptibility to the proteolytic cleavage that leads to aggregation at physiological pH. These events, even if involving a minority of circulating TTR, may be relevant for ATTR, a pathology that takes several decades to develop.

Stability Evaluation and Degradation Studies of DAC® Hyaluronic-Polylactide Based Hydrogel by DOSY NMR Spectroscopy.

The stability and the degradation of polymers in physiological conditions are very important issues in biomedical applications. The copolymer of hyaluronic acid and poly-D,L-lactic acid (made available in a product called DAC®) produces a hydrogel which retains the hydrophobic character of the poly-D,L-lactide sidechains and the hydrophilic character of a hyaluronic acid backbone. This hydrogel is a suitable device for the coating of orthopedic implants with structured surfaces. In fact, this gel creates a temporary barrier to bacterial adhesion by inhibiting colonization, thus preventing the formation of the biofilm and the onset of an infection. Reabsorbed in about 72 h after the implant, this hydrogel does not hinder bone growth processes. In the need to assess stability and degradation of both the hyaluronan backbone and of the polylactic chains along time and temperature, we identified NMR spectroscopy as a privileged technique for the characterization of the released species, and we applied diffusion-ordered NMR spectroscopy (DOSY-NMR) for the investigation of molecular weight dispersion. Our diffusion studies of DAC® in physiological conditions provided a full understanding of the product degradation by overcoming the limitations observed in applying classical chromatography approaches by gel permeation UV.

CoCUN, a Novel Ubiquitin Binding Domain Identified in N4BP1.

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 15N isotopically labeled 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 coupling of ubiquitin conjugation to endoplasmic-reticulum (ER) degradation (CUE) domain family, where an invariant proline, usually following a phenylalanine, is required for ubiquitin binding. Interestingly, this novel UBD, which is not evolutionary related to CUE domains, shares a 40% identity and 47% similarity with cullin binding domain associating with NEDD8 (CUBAN), a protein module that also recognizes the ubiquitin-like NEDD8. Based on these features, we dubbed the region spanning the C-terminal 50 residues of N4BP1 the CoCUN domain, for Cousin of CUBAN. By performing circular dichroism and 15N NMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the CoCUN domain lacks the NEDD8 binding properties observed in CUBAN. We also show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both CUBAN and CoCUN are poly-ubiquitinated in cells. The structural and the functional characterization of this novel UBD can contribute to a deeper understanding of the molecular mechanisms governing N4BP1 function, providing at the same time a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved.

Erratum: Nepravishta, R., et al. CoCUN, a Novel Ubiquitin Binding Domain Identified in N4BP1. Biomolecules 2019, 9, 284.

The Biomolecules Editorial Offices wishes to make the following erratum to this paper [...].

Selectivity of the CUBAN domain in the recognition of ubiquitin and NEDD8.

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.

Potential mechanism of thymosin-α1-membrane interactions leading to pleiotropy: experimental evidence and hypotheses.

INTRODUCTION: 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. AREAS COVERED: 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. EXPERT OPINION: 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 ß4 was outlined.

The mechanisms of humic substances self-assembly with biological molecules: The case study of the prion protein.

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.

Thymosin α1 inserts N terminus into model membranes assuming a helical conformation.

OBJECTIVE: 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. METHODS: The folding and interaction of Tα1 with sodium dodecyl sulfate micelles was monitored by NMR and CD spectroscopy techniques. RESULTS: 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. CONCLUSIONS: 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.