Multi-histidinic fragments binding biological metals: an NMR study

Cap43 protein can be considered as a “stress protein” since it is involved in a number of noxious events inside the cell, like hypoxia, cancerous states and metastasis suppression, and stress response just to quote some. Another interesting feature is that its expression is triggered by the rise in concentration of some metals, amongst which nickel gives the highest response. We have examined the whole sequence of this protein in the search of a suitable site for metal binding, finding a remarkable aspect that prompted us to deepen our investigation. In fact, Cap43 presents in its C-terminal region a mono-histidinic decapeptide whose sequence is repeated consecutively three times (TRSRSHTSEGTRSRSHTSEG- TRSRSHTSEG). The occurrence of such a repeated motif containing a histidine residue reminded us of neurodegenerative deseases and prions, where an octapeptide fragment bearing an histidine residue is repeated four times, and proved to be very active in binding Cu(II) ions, so that the tetra-repeat sequence is able to bind up to four metal ions. Another peptide, the β-amyloid fibril, contains a multi-histidinic sequence which seems involved in metal coordination within the process of aggregation. It is thus possible that also Cap43 could have a similar behaviour toward metals. We have used the 10-amino acid monohistidinic basic fragment (TRSRSHTSEG) and its two- and three-repeats to investigate their coordination mode towards metal ions such as Cu(II), Ni(II) and Zn(II). Multidimensional and multinuclear NMR techniques have been employed to uncover the details of metal coordination at different pH values and metal-to-ligand molar ratios. The data collected in our experiments allowed us to calculate structural models for the metal complexes, both at low and high pH-values.</br

A Social Internet of Things Smart City Solution for Traffic and Pollution Monitoring in Cagliari

In the last years, the smart city (SC) paradigm has been deeply studied to support sustainable mobility and to improve human living conditions. In this context, a new SC based on the Social Internet of Things paradigm is presented in this article. Starting from the tracking of all vehicles (that is, private and public) and pedestrians, integrated with air quality measurements (that is, in real time by mobile and fixed sensors), the system aims to improve the viability of the city, both for pedestrian and vehicular users. A monitoring network based on sensors and devices hosted on board in local public transport allows real-time monitoring of the most sensitive areas both from traffic congestion and from an environmental point of view. The proposed solution is equipped with an appropriate intelligence that takes into account instantaneous speed, type of traffic, and instantaneous pollution data, allowing to evaluate the congestion and pollution condition in a specific moment. Moreover, specific tools support the decisions of public administration facilitating the identification of the most appropriate actions for the implementation of effective policies relating to mobility. All collected data are elaborated in real time to improve traffic viability suggesting new directions and information to citizens to better organize how to live in the city

A Social IoT-Based Solution for Real-Time Forest Fire Detection

Conservation of the natural ecosystem is a hot topic that is receiving increasing attention not only from the scientific community, but from the entire world population. Forests and woodlands are major contributors to climate change mitigation, able to absorb significant amounts of carbon dioxide. This paper proposes a novel real-time fire monitoring and detection system based on Digital Mobile Radio (DMR) nodes and a Social Internet of Things (SIoT) platform on which fire detection decision making algorithms have been implemented. The results obtained by employing a K-Nearest Neighbors (KNN) algorithm and a Recurrent Neural Network (RNN) show the ability to detect the slightest variation in the observed parameters, determining the direction and speed of fire propagation with an accuracy of more than 98%

Structural and Functional Characterization of a New Double Variant Haemoglobin (HbG-Philadelphia/Duarte α268Asn→Lysβ262Ala→Pro)

We report the first case of cosegregation of two haemoglobins (Hbs): HbG-Philadelphia [α68(E17)Asn → Lys] and HbDuarte [β62(E6)Ala → Pro]. The proband is a young patient heterozygous also for β°-thalassaemia. We detected exclusively two haemoglobin variants: HbDuarte and HbG-Philadelphia/Duarte. Functional study of the new double variant HbG-Philadelphia/Duarte exhibited an increase in oxygen affinity, with a slight decrease of cooperativity and Bohr effect. This functional behaviour is attributed to β62Ala → Pro instead of α68Asn → Lys substitution. Indeed, HbG-Philadelphia isolated in our laboratory from blood cells donor carrier for this variant is not affected by any functional modification, whereas purified Hb Duarte showed functional properties very similar to the double variant. NMR and MD simulation studies confirmed that the presence of Pro instead of Ala at the β62 position produces displacement of the E helix and modifications of the tertiary structure. The substitution α68(E17)Asn → Lys does not cause significant structural and dynamical modifications of the protein. A possible structure-based rational of substitution effects is suggested

Malattie neurodegenerative e metalli: cosa lega la sindrome di Parkinson al manganese

E' noto come l'accumulo dei metalli nel cervello possa portare, o quantomeno contribuire, allo sviluppo di malattie neurodegenerative, attraverso meccanismi che solo ora cominciano a essere lentamente chiariti. La malattia di Parkinson, cosiddetta “idiopatica” in quanto non presenta alcuna causa apparente, è stata messa di recente in correlazione con l'esposizione o l'avvelenamento da manganese. Studi epidemiologici condotti da diverse università degli Stati Uniti hanno evidenziato come gli abitanti in zone urbane con alte concentrazioni di questo metallo avessero una probabilità di sviluppare il Parkinson quasi due volte più alta rispetto agli abitanti in zone meno inquinate, o inquinate da altri metalli (quali ad esempio il rame). Altri studi apparsi di recente in letteratura correlano l'esposizione al manganese con alcune modificazioni di un gene legato alla sinucleina, proteina presente con diverse funzioni in tutte le malattie neurodegenerative. Lo studio è stato effettuato su una proteina di un lievito la YPK9, al 58% simile e al 38% uguale all'analoga umana PARK9, la cui mutazione causa appunto lo sviluppo di una forma ereditaria di Parkinson. Silenziando il gene YPK9 nei lieviti si è notato che in assenza della relativa proteina questi mostravano disturbi nella crescita se sottoposti all'azione di diversi metalli, mentre in presenza del manganese la crescita era particolarmente ridotta. Veniva quindi dimostrata l'azione protettiva della YPK9 nei confronti dei cationi bivalenti, specialmente del manganese. Pare dunque possibile che una modifica sull'analogo umano, il PARK9, sia in grado di inficiare i normali meccanismi con cui il nostro organismo si protegge da ioni metallici dannosi, quali il manganese, e dando il via a una serie di processi che portano allo sviluppo della malattia neurodegenerativa. Abbiamo pertanto voluto verificare l'effettiva propensione di tale proteina a interagire con ioni Mn(II), selezionando sulla sequenza della YPK9 dei frammenti promettenti per il legame con il metallo e investigando la possibilità di una interazione efficace di questi frammenti con diversi cationi bivalenti, tra cui appunto il manganese, ma anche il calcio e lo zinco. I risultati preliminari, ottenuti attraverso alcune tecniche spettroscopiche quali l'NMR mono- e bidimensionale e l'EPR, verranno esposti in questa comunicazione

Interaction of divalent cations with Park9 protein fragments

Two peptide sequences from Park9 Parkinson’s disease (PD) gene, -P1D2E3K4H5E6L7- (1) and -F1C2G3D4G5A6N7D8C9G10- (2) have been tested for Mn(II), Zn(II) and Cu(II) binding. Park9 encoded protein can protect cells from manganese poisoning, which is an environmental risk factor for a Parkinson’s disease-like syndrome [1-4]. In fact, Park9 belongs to a family of ATP-ases involved in metal coordination and transportation; familial gene mutations may result in early development of PD. The chosen fragments are located from 1165 to 1171 and from 1184 to 1193 residues in the Park9 sequence, and are highly conserved in a number of organisms, going from yeasts to humans. Potentiometric, UV-vis experiments together with mono- and multidimensional NMR spectroscopy have been used to understand the details of metal binding sites at different pH values and at different ligand to metal molar ratios, showing that the three metals are able to effectively bind the examined peptides. From NMR measurements Mn(II) and Zn(II) coordination with peptide 1 involves imidazole Nε or Nδ of His5 and carboxyl γ-O of Asp2, Glu3 and Glu6 residues. Six donor atoms participate in Mn(II) binding, resulting in a distorted octahedral geometry, possibly involving bidentate interaction of carboxyl groups; four donor atoms participate in Zn(II) binding, resulting in a tetracoordinated geometry. Potentiometric data show that soluble, hydroxylated Zn(II) species are formed in the alkaline pH range. The formation of Cu(II) complexes with peptide 1 starts below pH: only mononuclear complexes have been potentiometrically detected also in the presence of excess of ligand. Imidazole nitrogen of His residues acts as first Cu(II) anchoring site; as pH is raised, ligand coordination proceeds with deprotonation and binding of neighbouring amide nitrogens of the peptidic backbone. UV-vis spectra agree that the main species at neutral pH is a {Nim, 2N-, O} complex, where the oxygen atom most likely belongs to an equatorially coordinated water molecule. Cu(II), Mn(II) and Zn(II) coordination involves the cysteine residues with peptide 2 and complex-formation invariably starts at lower pH with respect to ligand 1. Mn(II) accepts additional ligand bonds from D4 and D8 to complete the coordination sphere; the unoccupied sites may contain solvent molecules. When the ligand is in excess, both Zn(II) and Cu(II) ions form bis-complexes. The two metal ions behave in a very similar way and the stoichiometry of main species at physiological pH depends on the metal/ligand ratio: [ML]2- in equimolar solution or [MHL2]5- for the 1:2 ratio. Potentiometric data suggest for the former a {2S, 2O} and for the latter a {3S, 1O} coordination without any participation of amide nitrogens, as usually found for Cu(II)/peptide complexes

Impact of three commercial feed formulations on farmed gilthead sea bream (Sparus aurata, L.) metabolism as inferred from liver and blood serum proteomics

Background: The zootechnical performance of three different commercial feeds and their impact on liver and serum proteins of gilthead sea bream (Sparus aurata, L.) were assessed in a 12 week feeding trial. The three feeds, named A, B, and C, were subjected to lipid and protein characterization by gas chromatography (GC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. Results: Feed B was higher in fish-derived lipids and proteins, while feeds C and A were higher in vegetable components, although the largest proportion of feed C proteins was represented by pig hemoglobin. According to biometric measurements, the feeds had significantly different impacts on fish growth, producing a higher average weight gain and a lower liver somatic index in feed B over feeds A and C, respectively. 2D DIGE/MS analysis of liver tissue and Ingenuity pathways analysis (IPA) highlighted differential changes in proteins involved in key metabolic pathways of liver, spanning carbohydrate, lipid, protein, and oxidative metabolism. In addition, serum proteomics revealed interesting changes in apolipoproteins, transferrin, warm temperature acclimation-related 65 kDa protein (Wap65), fibrinogen, F-type lectin, and alpha-1-antitrypsin. Conclusions: This study highlights the contribution of proteomics for understanding and improving the metabolic compatibility of feeds for marine aquaculture, and opens new perspectives for its monitoring with serological tests.Pubblicat

Coordination abilities of mono and multi-histidinic and glutamate peptide fragments towards manganese(II) and cobalt(II)

It is known that rich repeat domains in peptides can be of interest as the models for the study of molecular phenomena related to metal ion binding in proteins involved in neurodegenerative disorders. Imbalances in transition metal ions are assumed to contribute to the conversion of the multi-histidinic amyloid β-peptide (Aβ) from its soluble form to an amyloidogenic form, and to Aβ deposition. Of these ions, it has been reported that manganese binding to PrP is detrimental and causes a conformational change in the protein, suggesting that manganese binding could potentially play a role in prion disease progression in vivo. It appears that PrP is less stable on binding manganese and quickly converts to a misfolded form. The binding of manganese to PrP potentially results in the conversion of the protein to an abnormal isoform with properties reminiscent of PrPsc. In particular, although PrP can bind the same number of manganese atoms as of copper atoms, the resulting protein becomes proteinase resistant, forms fibrils and loses function.[1,2] Regarding cobalt, a novel low-affinity binding site for Co(II) was discovered between PrP residues 104 and 114, with residue His111 being the key amino acid for coordinating Co(II).[3] Thus, despite the interest in manganese and cobalt binding to PrP, a thorough analysis of the interaction of both metals with proteins related to brain pathies has not yet been reported. The (T1R2S3R4S5H6T7S8E9G10)3 fragment from Cap43 protein, which is induced by metal ions, is characterized by a decarepeat domain comprising three decapeptide units with one histidine and one glutamate residue in each repeat. Therefore the study of the interaction of the 30-aminoacid peptide from Cap43 protein with metal ions can contribute to the understanding of the crucial role of multi-imidazol and glutamate sites in the protein coordination processes and the possible role of divalent metal ions in the pathogenesis of prion disease and other related protein pathies.[4-8] Here we present our recent results on the Cobalt(II) and Manganese(II) complexes of terminally protected mono- and multi-histidine-glutamate peptides studied by combination of potentiometric measurements and spectroscopic techniques (NMR, UV-Vis and EPR). Metal complexation induces important structural changes with the C-terminal portion of the ligand, constraining it to leave its disordered conformation and promoting side chain orientation. Our results give rise to a molecular model of the induced structure for the peptides complexed with cobalt and manganese

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF-platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage

Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes

Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19