74 research outputs found
In Silico Analysis of Nanoplastics’ and β-amyloid Fibrils’ Interactions
Get PDFPlastic pollution has become a global environmental threat, which leads to an increasing concern over the consequences of plastic exposition on global health. Plastic nanoparticles have been shown to influence the folding of proteins and influence the formation of aberrant amyloid proteins, therefore potentially triggering the development of systemic and local amyloidosis. This work aims to study the interaction between nanoplastics and β-amyloid fibrils to better understand the potential role of nanoplastics in the outbreak of neurodegenerative disorders. Using microsecond-long coarse-grained molecular dynamics simulations, we investigated the interactions between neutral and charged nanoparticles made of the most common plastic materials (i.e., polyethylene, polypropylene, and polystyrene) and β-amyloid fibrils. We observe that the occurrence of contacts, region of amyloid fibril involved, and specific amino acids mediating the interaction depend on the type and charge of the nanoparticles
Heptad stereotypy, S/Q layering, and remote origin of the SARS-CoV-2 fusion core
Get PDF: The fusion of the SARS-CoV-2 virus with cells, a key event in the pathogenesis of Covid-19, depends on the assembly of a six-helix fusion core (FC) formed by portions of the spike protein heptad repeats (HRs) 1 and 2. Despite the critical role in regulating infectivity, its distinctive features, origin, and evolution are scarcely understood. Thus, we undertook a structure-guided positional and compositional analysis of the SARS-CoV-2 FC, in comparison with FCs of related viruses, tracing its origin and ongoing evolution. We found that clustered amino acid substitutions within HR1, distinguishing SARS-CoV-2 from SARS-CoV-1, enhance local heptad stereotypy and increase sharply the FC serine-to-glutamine (S/Q) ratio, determining a neat alternate layering of S-rich and Q-rich subdomains along the post-fusion structure. Strikingly, SARS-CoV-2 ranks among viruses with the highest FC S/Q ratio, together with highly syncytiogenic respiratory pathogens (RSV, NDV), whereas MERS-Cov, HIV, and Ebola viruses display low ratios, and this feature reflects onto S/Q segregation and H-bonding patterns. Our evolutionary analyses revealed that the SARS-CoV-2 FC occurs in other SARS-CoV-1-like Sarbecoviruses identified since 2005 in Hong Kong and adjacent regions, tracing its origin to >50 years ago with a recombination-driven spread. Finally, current mutational trends show that the FC is varying especially in the FC1 evolutionary hotspot. These findings establish a novel analytical framework illuminating the sequence/structure evolution of the SARS-CoV-2 FC, tracing its long history within Sarbecoviruses, and may help rationalize the evolution of the fusion machinery in emerging pathogens and the design of novel therapeutic fusion inhibitors
Colocalization and Interaction Study of Neuronal JNK3, JIP1, and β-Arrestin2 Together with PSD95
No full textc-Jun N-terminal kinases (JNKs) are stress-activated serine/threonine protein kinases belonging to the mitogen-activated protein kinase (MAPK) family. Among them, JNK3 is selectively expressed in the central nervous system, cardiac smooth muscle, and testis. In addition, it is the most responsive JNK isoform to stress stimuli in the brain, and it is involved in synaptic dysfunction, an essential step in neurodegenerative processes. JNK3 pathway is organized in a cascade of amplification in which signal transduction occurs by stepwise, highly controlled phosphorylation. Since different MAPKs share common upstream activators, pathway specificity is guaranteed by scaffold proteins such as JIP1 and β-arrestin2. To better elucidate the physiological mechanisms regulating JNK3 in neurons, and how these interactions may be involved in synaptic (dys)function, we used (i) super-resolution microscopy to demonstrate the colocalization among JNK3–PSD95–JIP1 and JNK3–PSD95–β-arrestin2 in cultured hippocampal neurons, and (ii) co-immunoprecipitation techniques to show that the two scaffold proteins and JNK3 can be found interacting together with PSD95. The protein-protein interactions that govern the formation of these two complexes, JNK3–PSD95–JIP1 and JNK3–PSD95–β-arrestin2, may be used as targets to interfere with their downstream synaptic events
Super-resolution study of PIAS SUMO E3-ligases in hippocampal and cortical neurons
Get PDFThe SUMOylation machinery is a regulator of neuronal activity and synaptic plasticity. It is composed of SUMO isoforms and specialized enzymes named E1, E2 and E3 SUMO ligases. Recent studies have highlighted how SUMO isoforms and E2 enzymes localize with synaptic markers to support previous functional studies but less information is available on E3 ligases. PIAS proteins - belonging to the protein inhibitor of activated STAT (PIAS) SUMO E3-ligase family - are the best-characterized SUMO E3-ligases and have been linked to the formation of spatial memory in rodents. Whether however they exert their function co-localizing with synaptic markers is still unclear. In this study, we applied for the first time structured illumination microscopy (SIM) to PIAS ligases to investigate the co-localization of PIAS1 and PIAS3 with synaptic markers in hippocampal and cortical murine neurons. The results indicate partial co-localization of PIAS1 and PIAS3 with synaptic markers in hippocampal neurons and much rarer occurrence in cortical neurons. This is in line with previous super-resolution reports describing the co-localization with synaptic markers of other components of the SUMOylation machinery
Patient-derived C-terminal mutation of FANCI causes protein mislocalization and reveals putative EDGE motif function in DNA repair
No full textFanconi anemia (FA) is a rare familial genome instability syndrome caused by mutations in FA genes that results in defective DNAcrosslink repair. Activation of the FA pathway requires the FA core ubiquitin ligase complex-dependent monoubiquitination of 2 interacting FA proteins, FANCI and FANCD2. Although loss of either FANCI or FANCD2 is known to prevent monoubiquitination of its respective partner, it is unclear whether FANCI has any additional domains that may be important in promoting DNA repair, independent of its monoubiquitination. Here, we focus on an FA-I patient-derived FANCI mutant protein, R1299X (deletion of 30 residues from its C-terminus), to characterize important structural region(s) in FANCI that is required to activate the FA pathway. We show that, within this short 30 amino acid stretch contains 2 separable functional signatures, a nuclear localization signal and a putative EDGE motif, that is critical for the ability of FANCI to properly monoubiquitinate FANCD2 and promote DNA crosslink resistance. Our study enable us to conclude that, although proper nuclear localization of FANCI is crucial for robust FANCD2 monoubiquitination, the putative FANCI EDGE motif is important for DNA crosslink repair
Neuronal Localization of SENP Proteins with Super Resolution Microscopy
Get PDFSUMOylation of proteins plays a key role in modulating neuronal function. For this reason, the balance between protein SUMOylation and deSUMOylation requires fine regulation to guarantee the homeostasis of neural tissue. While extensive research has been carried out on the localization and function of small ubiquitin-related modifier (SUMO) variants in neurons, less attention has been paid to the SUMO-specific isopeptidases that constitute the human SUMO-specific isopeptidase (SENP)/Ubiquitin-Specific Protease (ULP) cysteine protease family (SENP1-3 and SENP5-7). Here, for the first time, we studied the localization of SENP1, SENP6, and SENP7 in cultured hippocampal primary neurons at a super resolution detail level, with structured illumination microscopy (SIM). We found that the deSUMOylases partially colocalize with pre-and post-synaptic markers such as synaptophysin and drebrin. Thus, further confirming the presence with synaptic markers of the negative regulators of the SUMOylation machinery
Telework and employee empowerment: effective practices amid the Covid-19 pandemic
Get PDFThe purpose of this paper is to provide a guideline of critical success practices for companies looking to empower their remote workforce during the Covid-19 pandemic. The research will explore current literature around Telework and Employee Empowerment, and provide suggestions in three key areas: Ageism, Work-life balance, and Goal Internalization. Using a qualitative approach this paper adds to the limited literature investigating the correlation between the two subjects and provide points for further research
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The Role of Smoking in Cocaine Addiction
Get PDFThe 2010 National Survey on Drug Use and Health reported that almost 37 million Americans,1 2 years of age or older, have tried cocaine in their lifetime, and 5.7 million have used it in the past year – making cocaine the second most commonly used drug of abuse in the United States. The reasons for its popularity are its immediate psychological effects. Snorted or injected cocaine induces self-confidence, euphoria and good feelings, magnifying the intensity of almost all normal pleasures. However, cocaine use also causes medical complications like respiratory failure, cardiovascular and gastrointestinal problems and paranoia. Moreover, after prolonged exposure, individuals become addicted to it, losing control over use of the drug and engaging in destructive behavior related to cocaine procurement. Cocaine addiction is a chronic relapsing condition with high risk of medical complications, cognitive impairment, financial ruin and social issues
Il disegno dello spazio commerciale urbano. Indirizzi progettuali e linee guida per il rilancio della Galleria del Toro a Milano
No full textLAUREA MAGISTRALEL'obiettivo della tesi è prefigurare un'ipotesi meta progettuale per il rilancio di un aggregato storico urbano di Milano: la Galleria del Toro. Gli argomenti trattati riguardano sia aspetti urbanistico-insediativi sia architettonico spaziali, variando la scala di indagine conoscitiva orientata a restituire un'articolazione di riflessioni metodologiche e progettuali fondate non solo sullo studio del singolo oggetto architettonico, ma del contesto urbano di prossimità inteso come valore aggiunto del progetto. Attraverso la restituzione di una serie di casi studio si sono definiti indirizzi progettuali per rilanciare un ambito problematico del centro urbano di Milano: la Galleria del Toro in Piazza San Babila
Effetto delle nanoplastiche sull'aggregazione di B-amiloidi : studi preliminari in silico ed in vitro
Get PDFLAUREA MAGISTRALEMicroplastiche (MP) e nanoplastiche (NP), termini ormai comuni nei mezzi di comunicazione, indicano particelle derivanti da oggetti di uso quotidiano prodotti con materiali plastici, come bottiglie e capi d'abbigliamento sintetici (es. pile), i quali, degradandosi, rilasciano nell'ambiente frammenti di diverse dimensioni. Precisamente, si fa riferimento alle microplastiche per indicare i frammenti di dimensione compresa tra 0,1 e 5000 mm e alle nanoplastiche quando i frammenti rientrano nel range da 1 a 100 nm.
Una volta disperse nell’ambiente, le particelle plastiche vengono ingerite dagli animali e assorbite dai vegetali entrando nella catena alimentare. Risulta importante quindi, indagare quali possano essere i meccanismi di interazione tra micro e nanoplastiche all’interno degli organismi biologici.
Molti studi in letteratura hanno analizzato le interazioni tra micro e nanoparticelle di plastica e le macromolecole quali lipidi e proteine. Per quanto riguarda le prime, in vari casi si è osservato come possano interagire con le membrane plasmatiche variandone la permeabilità e quindi selettività. Nel caso invece delle interazioni plastica-proteine, i vari studi hanno analizzato la formazione delle corone proteiche, osservando come le proteine avessero una predisposizione maggiore per l’aggregazione in presenza della plastica stessa.
Il presente lavoro di tesi ha come obiettivo lo studio, sia con approccio computazionale che sperimentale in vitro, delle interazioni che si instaurano tra diverse nanoparticelle di plastica e le proteine responsabili dell’Alzheimer. L’analisi è finalizzata alla comprensione del ruolo delle nanoplastiche nei processi di aggregazione dei peptidi amiloidi. Quest’ultimo rappresenta lo step iniziale nella formazione degli oligomeri, fisiologicamente tossici, che successivamente costituiscono le fibrille amiloidi.
Le analisi in silico sono state effettuate su sistemi costituiti da una particella di nanoplastica delle dimensioni di circa 5 nm in presenza di peptidi amiloidogenici. Per quanto riguarda i tipi di nanoplastiche si è considerato il polietilene (PE), il polistirene (PS), il polietilene tereftalato (PET) ed il polipropilene (PP). Per quanto riguarda le proteine amiloidogeniche si è considerato il singolo peptide Aβ1-42, sia nella sua interezza, sia come frammenti di diversa lunghezza (Aβ16-20, e Aβ25-42). In aggiunta ai singoli peptidi, sono state studiate forme pre-aggregate in oligomeri composti da 5 peptidi. Infine, le diverse forme del peptide Aβ1-42 state analizzate nel fenotipo wild type e nella forma mutata A2V.
Le interazioni molecolari e macromolecolari ed i processi biofisici sono studiati con diverse tecniche in funzione del livello di dettaglio desiderato.
Tutte le simulazioni presentate in questo studio sono state eseguite con il software di modellazione molecolare NAMD e visualizzate con VMD.
Le plastiche sono state create attraverso repliche dello stesso monomero, realizzato con l’editor Avogadro, e tramite simulazioni in vuoto sono state compresse sino ad ottenere una sfera da 5 nm di diametro. Per le proteine con strutture già note in letteratura, si è utilizzato il Protein Data Bank (PDB), dal quale si è potuto scaricare il relativo file in formato .pdb, mentre gli oligomeri sono stati costruiti con il software Pymol. Per le diverse nanoparticelle, si è scelto di partire da numero diverso di catene a seconda della plastica scelta per ottenere polimeri di circa 7000 atomi. In particolare, il polietilene (PE) è stato realizzato a partire da 16 catene C72H146, per il polipropilene (PP) 36 catene C88H178, per il polistirene (PS) 16 catene C216H165, e per il polietilene tereftalato (PET) 25 catene C10H8O4.
Le simulazioni sono state fatte in condizioni al contorno periodiche e mantenendo costanti temperatura e pressione, precisamente ad una temperatura di 300 K e una pressione di 1 bar. Una volta prodotta la nanoplastica si verificata la sua stabilità in vuoto con una simulazione da 100 ps. Da questa è stato selezionato l’ultimo frame e usato come punto di partenza di ogni altra simulazione. I peptidi Aβ1-42, Aβ16-20, Aβ25-42 e la versione modificata A2V, sono stati implementati con Pymol. Infine, la fibrilla 5OQV è stata selezionata e scaricata dal PDB, dopo aver fatto una ricerca che prendeva in considerazione tutte le fibrille amiloidi presenti in letteratura. È stata selezionata la 5OQV in virtù del fatto che presenta una struttura maggiormente caratterizzata. Anche della fibrilla è stata studiata la versione che presentava la modifica A2V.
La durata delle simulazioni è variabile, con un tempo che va da pochi ns per le simulazioni più brevi a centinaia di ns per quelle più lunghe. Ogni nanoplastica è stata fatta interagire con tutte le diverse proteine e, per avere una migliore attendibilità, ogni sistema è stato ripetuto 5 volte. Per poter osservare un’eventuale differenza nel comportamento della proteina, sono state effettuate prove della sola proteina e della proteina in presenza della nanoplastica, in entrambi i casi in ambiente solvatato e ionizzato.
Per la caratterizzazione in vitro, svolta in parallelo a quella in silico, sono stati realizzati i peptidi A25-42 tramite il protocollo di sintesi peptidica standard. Successivamente sono state svolte analisi con il dicroismo circolare, con lo spettrofotometro e con l’AFM per studiare l’evoluzione della struttura secondaria della proteina in presenza di nanoparticelle di PS con dimensioni e carica diversa.
Nel presente lavoro di ricerca, sono stati ottenuti interessanti risultati sull’interazione delle proteine β amiloidi con diversi tipi di nanoplastiche.
Dall’analisi in silico è stato possibile stabilire che tra polistirene, polietilene tereftalato, polipropilene e polietilene, quest’ultima sia la nanoplastica che, instaurando il più alto numero di contatti/ns, determina una maggiore interazione con le componenti proteiche. Per il polistirene il discorso è analogo seppur presentando valori di Root-Mean-Square Deviation (RMSD) e numero di contatti leggermente inferiori. Per il polietilene tereftalato e il polipropilene si può affermare che entrambe hanno avuto un numero ancora inferiore di contatti, garantendo una maggiore stabilità nelle proteine durante l’analisi.
Inoltre, la struttura secondaria di tutte le proteine in versione oligomerica si è mantenuta costante per tutta la durata della simulazione ed è stato possibile identificare tre zone di contatto, costituite circa da 4/5 aminoacidi ciascuna.
Individuando gli amminoacidi di adesione tra nanoplastica e proteina è stato possibile definire il legame instaurato tra queste come interazione idrofobica.
Per quanto concerne le analisi in vitro, si può affermare che il polistirene interagisce in maniera diversa con il peptide A25-42 in funzione di carica e dimensione. Si è visto infatti che a dimensioni inferiori, le nanoplastiche hanno dato dei valori più bassi in fluorescenza, indicando così una minore componente di aggregati proteici in forma amiloidogenica, se paragonati con quelli ottenuti con stesse in versione più grande. Inoltre, a parità di dimensione, le nanoplastiche neutre hanno dato dei segnali maggiori rispetto a quelle cariche. Infine, le nanoparticelle cariche positivamente generano dei valori più intensi rispetto a quelle negative di uguale grandezza.
Un ultimo risultato di rilievo è stato ottenuto grazie all’utilizzo della microscopia a forza atomica. Infatti, si è riscontrata una differenza tra i campioni che avevano in soluzione nanoplastiche e quelli dove invece non erano presenti. In particolare, si è notato come le nanoparticelle con il passare delle ore, fossero in grado in tutti i casi di rimuovere dalla superficie del substrato, il tappeto di fibre che al tempo zero era presente. Questo ha fatto sorgere due ipotesi relative al fatto che le nanoplastiche potessero essere in grado di adsorbire nella loro superficie le fibrille rimuovendole dalla soluzione, oppure, fossero capaci di sfaldarle riportandole quindi allo stadio precedente: quello oligomerico tossico.
In conclusione, si può affermare che le nanoplastiche presenti nell’ecosistema sono in grado, entrando all’interno degli organismi viventi, di interagire e modificare l’ambiente cellulare.
Come si è osservato in questo lavoro di tesi, tutte le nanoplastiche, anche se in maniera differente, sono in grado di influenzare le proteine studiate alterandone quindi la conformazione tridimensionale. Essendo le nanoplastiche non selettive, è ancora difficile prevedere e comprendere quali possano essere i peggiori danni che sono in grado di provocare. Il nostro studio si è concentrato sulla proteina principale responsabile dell’Alzheimer riscontrando che sicuramente è influenzabile dalle nanoparticelle.
Attualmente stiamo portando avanti delle simulazioni che hanno l’obbiettivo di comprendere se le nanoplastiche sono in grado di influenzare le fibrille amiloidi già formate, e nel caso, di sfaldarle riportandole allo stadio oligomerico.
Studi futuri possono focalizzarsi sulle interazioni prima tra nanoplastiche e neuroni, per poi passare a modelli animali della malattia di Alzheimer, e verificare come queste possano effettivamente essere correlate a malattie amiloidi, quali Alzheimer.Microplastics (MP) and nanoplastics (NP) are terms that now we can find in any media, which indicate small particles coming from daily plastic object such as, bottles and cloths made with synthetic fabrics (e.g. pile), that due to degradation, release into the environment fragments of different sizes. In particular, microplastic indicate bits between 0.1 and 5000mm, and nanoplastics when the fragments fall within the range of 1 to 100 nm.
Once dispersed in the environment, plastic particles are ingested by animals and absorbed by plants entering the food chain. It is therefore important to investigate the mechanisms of interaction between micro and nanoplastics within biological organisms.
Many studies in the literature have analyzed the interactions between micro and nanoparticles of plastic and macromolecules such as lipids and proteins. As regards the first, in several cases it has been observed how they can interact with plasma membranes varying the permeability and therefore selectivity. In the case of plastic-protein interactions, the various studies analyzed the formation of protein corona, observing how proteins had a greater predisposition for aggregation when the plastic particles were present.
Silico analyses were carried out in systems consisting 5 nm nanoplastic particles in the presence of amyloidogenic peptides. Polyethylene (PE), polystyrene (PS), polyethylene terephthalate (PET) and polypropylene (PP) were the types of nanoplastics considered. For amyloidogenic proteins, the single peptide Aβ1-42 was studied, both in its full length and as fragments of different dimensions (Aβ16-20, and Aβ25-42). In addition to individual peptides, pre-aggregate forms have been studied in oligomers composed of 5 peptides. Finally, the different forms of the peptide Aβ1-42 were analyzed in the wild type phenotype and in the mutated form A2V.
Molecular and macromolecular interactions and biophysical processes are studied with different techniques according to the desired detail’s level.
All simulations presented in this study were performed with the molecular modeling software NAMD and displayed with VMD.
The plastics were created through repetitions of the same monomer, made with the editor Avogadro, and through simulations in vacuum were compressed to obtain the desired a sphere of 5 nm in diameter. Protein Data Bank (PDB) was used for the search for beta amyloid proteins whose structures are known in the literature, from which it was possible to download the relevant file in .pdb format, while oligomers were built with Pymol software. For the different nanoparticles, it was chosen to start from a different number of chains depending on the plastic selected, but always getting a system composed of about 7000 atoms. In particular, the polyethylene (PE) was made from 16 C72H146 chains, for the polypropylene (PP) 36 C88H178 chains, for the polystyrene (PS) 16 C216H165 chains, and for the polypropylene terephthalate (PET) 25 C10H8O4 chains.
The duration of the simulations is variable, with a length ranging from a few ns for the shorter simulations to hundreds of ns for the longer ones. Each nanoplastic has been made to interact with all the different proteins, and to get a better reliability, each system has been replicated 5 times. In order to observe possible difference in protein behavior, tests were carried out both in presence and absence of nanoplastics, and in a solvated and ionized environment.
For the in vitro characterization, carried out in parallel with the in silico one, Aβ 25-42 peptides have been realized through the standard protocol of peptide synthesis. Subsequently, analyses were carried out with circular dichroism, spectrophotometer and AFM to study the evolution of the secondary structure of the protein in the presence of nanoparticles of PS with different size and charge.
In this work, interesting results have been obtained on the interaction of β amyloid proteins with different types of nanoplastics.
From the in silico analysis it has been possible to establish that, between polystyrene, polyethylene terephthalate, polypropylene and polyethylene, the last nanoplastic is the one that establishing the highest number of contacts/ns, determining the greatest interaction with the protein components. Polystyrene has a similar behavior, even though it has slightly lower number of contacts and RMSD values. For polyethylene terephthalate and polypropylene, both had an even lower number of contacts, ensuring greater stability in proteins during the analysis.
In addition, the secondary structure of all the proteins in the oligomeric version, remained constant throughout the simulation and it has been possible to identify three contact zones, consisting of about 4/5 amino acids each.
By identifying the amino acids of adhesion between nanoplastic and protein it has been possible to define the bond established between these as hydrophobic interactions.
As regards in vitro analyses, it has been observed that polystyrene interacts differently with the peptide A25-42 as a function of charge and size. In fact, it has been seen that at smaller sizes, nanoplastics have given lower values in fluorescence, when compared with those obtained with the same in the larger version. In addition, neutral nanoplastics have given higher signals than charged nanoplastics with same size. Finally, positively charged nanoparticles generate more intense values than negative ones of equal size.
A final important result was obtained thanks to the use of the Atomic Force Microscopy. In fact, there was a difference between the samples that had nanoplastic in solution and those where there were not. It was noted that nanoparticles, as the hours passed, were able in all cases to remove from the surface of the substrate, the layer of fibers that at zero time was present.
This gave rise to two hypotheses about the fact that nanoplastics could be able to adsorb fibrils in their surface by removing them from the solution or were able to flake them back then to the previous stage: the toxic oligomeric one.
In conclusion, it can be said that the nanoplastics present in the ecosystem are able, entering within living organisms, to interact and modify the cellular environment.
As has been observed in this thesis work that all the nanoplastics analyzed, even if in a different way, are able to influence the proteins, thus altering their function. As nanoplastics are non-selective, it is still difficult to predict and understand what is the worst damage they can cause. Our study focused on the main protein responsible for Alzheimer and found that it is susceptible to nanoparticles
Currently we are carrying out simulations that have the aim to understand if the nanoplastics can influence the amyloid fibrils already formed, and in the case, to bring them back to the oligomeric stage.
Further studies may first focus on the interactions between nanoplastics and neurons, then move on to animal models, and finally verify how nanoplastics can be related to amyloid diseases, such as Alzheimer’s and tauopathies
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