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

A metal-binding site in the RTN1-C protein: new perspectives on the physiological role of a neuronal protein

Reticulon 1-C (RTN1-C) is an ER-associated neuronal protein characterized by horse-shoe-like topology with two transmembrane helices and the N- and C-terminal regions which are supposed in the cytosolic side of ER. The physiological role of this protein is not completely clarified, but several studies have suggested its involvement in the neuronal differentiation, membrane vesicle trafficking and induction of apoptosis. The C-terminal region of RTN1-C is characterized by the presence of a H4 histone consensus sequence that makes it able to interact with nucleic acids and HDAC enzymes both in vitro and in vivo. In the present study a potential metal ion binding motif (HxE/D) at the C-terminal of the RTN1-C has been identified and its capability to bind metals investigated by UV-vis, CD, multidimensional NMR spectroscopy and biological assays. The results suggest a possible implication of the metal ions in the mechanisms of formation of the recently observed RTNs multiprotein complexes contributing to understand the structure and function of this neuronal membrane protein, suggesting a possible effect of the metal binding property on its biological function

A Forensic Application of Solid-State 13C NMR Spectroscopy: the Date of a Photographic Development

Abstract 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

Reticulon RTN1-C(CT) peptide: a potential nuclease and inhibitor of histone deacetylase enzymes

RTN1-C protein is a membrane protein localized in the ER and expressed in the nervous system, and its biological role is not completely clarified. Our previous studies have shown that the C-terminal region of RTN1-C, corresponding to the fragment from residues 186 to 208, was able to bind the nucleic acids and to interact with histone deacetylase (HDAC) enzymes. In the present work the properties of the synthetic RTN1-C(CT) peptide corresponding to this region were studied with relation to its ability to bind the metal ions in its N-terminal region. RTN1-C(CT) peptide is characterized by the presence of high-affinity copper and nickel ion sites. The nuclease activity of the metal-peptide complex was observed due to the presence of an ATCUN-binding motif. Moreover, the effect of the Cu/Ni-RTN1-C(CT) complexes on the HDAC activity was investigated. The histone deacetylase inhibitors are a new class of antineoplastic agents currently being evaluated in clinical trials. Our data show that the acetylated form of the metal-peptide complex is able to inhibit the HDAC activity at micromolar concentrations. These results allow to propose the Cu/Ni-RTN1-C(CT) complexes as models for the design of antitumor agents

Reticulon RTN1-C(CT) peptide: a potential nuclease and inhibitor of histone deacetylase enzymes

RTN1-C protein is a membrane protein localized in the ER and expressed in the nervous system, and its biological role is not completely clarified. Our previous studies have shown that the C-terminal region of RTN1-C, corresponding to the fragment from residues 186 to 208, was able to bind the nucleic acids and to interact with histone deacetylase (HDAC) enzymes. In the present work the properties of the synthetic RTN1-C(CT) peptide corresponding to this region were studied with relation to its ability to bind the metal ions in its N-terminal region. RTN1-C(CT) peptide is characterized by the presence of high-affinity copper and nickel ion sites. The nuclease activity of the metal-peptide complex was observed due to the presence of an ATCUN-binding motif. Moreover, the effect of the Cu/Ni-RTN1-C(CT) complexes on the HDAC activity was investigated. The histone deacetylase inhibitors are a new class of antineoplastic agents currently being evaluated in clinical trials. Our data show that the acetylated form of the metal-peptide complex is able to inhibit the HDAC activity at micromolar concentrations. These results allow to propose the Cu/Ni-RTN1-C(CT) complexes as models for the design of antitumor agents

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.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