Changes in protein expression in two cholangiocarcinoma cell lines undergoing formation of multicellular tumor spheroids In vitro

Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and re- sistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutami- nase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The aris- ing protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conver- sion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the car- bohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen

Band 3 Erythrocyte Membrane Protein Acts as Redox Stress Sensor Leading to Its Phosphorylation by p (72) Syk

In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i) in intact erythrocytes, at 2 mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii) the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii) disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv) protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis

Band 3 Erythrocyte Membrane Protein Acts as Redox Stress Sensor Leading to Its Phosphorylation by p (72) Syk

In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i) in intact erythrocytes, at 2\u2009mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii) the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii) disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv) protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis

Alpha-Synuclein and polyunsaturated fatty acids molecular characterization of the interaction and implication in protein aggregation

The project of my PhD Thesis focuses on the general problem of the protein folding and misfolding in line with the research conducted in the laboratory of Protein Chemistry at CRIBI, where the work was mainly conducted. My research activity can be divided in two parts. In the first year of the PhD course I studied the effect of pH in protein fibrillogenesis using a peptide model. During the second and the third years, my research was focused into the molecular interaction between alpha-synuclein and fatty acids and its implications in alpha-synuclein aggregation. Thus, this PhD Thesis is composed of a minor part concerning the analysis of the aggregative properties of the peptide model apoMb1-29 (Chapter 1 and 2) and of a major part dealing with the characterization of the interaction of alpha-synuclein and fatty acids (Chapter 3 and 4). Several human diseases, defined also misfolding disease, result from the failure of protein folding of the involved proteins. An increasing number of human diseases, such as Alzheimer’s and Parkinson’s diseases (PD), have been linked to protein aggregation and the aberrant accumulation of protein deposits in different tissues and organs. These pathological deposits are characterized by the presence of highly organized fibrillar aggregates called amyloid fibrils. Amyloid is a non-covalent polymer of extended, intermolecularly hydrogen bonded betha-sheets that laterally self-assemble to yield twisted fibers. Since amyloid fibrils are formed from disease-associated as well as from disease unrelated proteins and peptides under appropriate conditions, there is the belief that the ability to form fibrils is a generic property of the polypeptide chain (Chiti and Dobson, 2006). However, the propensity to aggregate and the stability of the mature fibrils depends on the amino acid sequence, so intrinsic determinants, such as net charge, hydrophobicity, the presence of aromatic residues and betha-sheet propensity, have important roles in amyloidogenicity of polypeptides (Pawar et al., 2005). In order to investigate the role of the net charge in the aggregation process of unfolded proteins and to analyze the importance of electrostatic interaction in the stability of the resulting fibrils, the aggregation properties of a peptide model derived from the N-terminal region of apomyoglobin were analyzed under different pH conditions. The N-terminal fragment 1-29 of horse heart apomyoglobin (apoMb1-29) is highly prone to form amyloid-like fibrils at low pH. Fibrillogenesis at pH 2.0 occurs following a nucleation-dependent growth mechanism, as evidenced by the thioflavin T (ThT) assay. Transmission electron microscopy (TEM) confirms the presence of regular amyloid-like fibrils and far-UV circular dichroism (CD) spectra indicate the acquisition of a high content of betha-sheet structure. Using peptides deriving from the proteolysis of apoMb1–29, we identified the region 7-16 as the most amyloidogenic, indeed it contains in terms of hydrophobicity, betha-sheet propensity and low net charge, all the determinants that favor the aggregation. In conclusion, the modulation of the net charge of apoMb1-29 and its sub-fragments by change of pH is of utmost importance for fibril formation. Moreover, we demonstrated that the electrostatic interaction, in apoMb1-29 system, is the force that primarily stabilizes the betha-sheet structure of the mature fibrils. Indeed, ThT assay, TEM and CD highlight fast and complete disaggregation of the fibrils, if the pH of a suspension of mature fibrils is increased to neutral values. In the second part of my PhD project, I investigated the molecular details that regulate the interaction between alpha-synuclein (alpha-syn) and fatty acids (FAs), analyzing the conformational features of the protein bound to FAs and the physical state of the lipids. Moreover, the aggregation process FA-mediated was analyzed in order provides insights into the implication of lipids in amyloid formation in vivo. Human alpha-syn is a 140 amino acid natively unfolded protein of still unknown function. It is highly expressed in the central nervous system and enriched in the presynaptic nerve terminals. alpha-Syn is characterized by 7 repetitive amino acid sequences (KTKEGV) in the N-terminal portion, by a central hydrophobic region (non-amyloid component, NAC) and by acidic stretches in the C-terminal tail. Mutations or overexpression of the human alpha-syn gene cause early-onset autosomal dominant Parkinson’s disease (PD). alpha-Syn is the major component of Lewy bodies, the cytoplasmic proteinaceous aggregates pathognomonic for PD (Spillantini et al., 1998). The mechanism by which an abnormality in structure or expression of alpha-syn causes PD has not been elucidated. Despite the evidence for a key role of alpha-syn in the onset of PD, there is very little information about its physiological function in the brain. Among several hypotheses, the role of alpha-syn is also associated to FAs. alpha-Syn seems to interact with unsatured and polyunsatured fatty acids (PUFAs), but it is not known if this interaction involves free FA molecules (Sharon et al., 2001), or aggregate states of FAs (micelles, vesicles, oil droplets) (Broersen et al., 2006; Lücke et al., 2006). Furthermore, this interaction promotes the oligomerization of alpha-syn. alpha-Syn forms multimers in vitro upon exposure to vesicles containing certain PUFA acyl groups and this process occurs at physiological concentration (Perrin et al., 2001). Moreover, since exposure of neuronal cell lines to PUFA increases alpha-syn oligomer levels, the in vivo interaction of alpha-syn with PUFAs seems to promote the formation of soluble oligomers that precede the aggregates associated with neurodegeneration (Sharon et al., 2003). First, a systematic study on the conformational transitions of alpha-syn in the presence of several fatty acids was conducted. Since the number of unsaturations and the length of the acyl chain have been shown to deeply affect the aggregate state of fatty acid (monomer, micelle, vesicle and oil droplet) and consequently, the interaction with the protein, the analysis was conducted using several fatty acids: palmitic acid (saturated), oleic acid (unsaturated), and docosahexaenoic acid (DHA, polyunsaturated). In particular, the last one is an essential omega-3 fatty acid, abundant in brain. DHA levels have been shown to be elevated in those brains areas containing alpha-syn inclusions in PD patients (Sharon et al., 2003). The FAs effects on alpha-syn structure were analyzed by far-UV circular dichroism and by proteolytic mapping. The protein is unfolded in the absence of FAs or in the presence of palmitic acid. Instead, upon binding to oleic acid (OA) and DHA, alpha-syn acquires alpha-helical conformation in a simple two-state transition. In the presence of DHA, alpha-syn is quite resistant to proteolysis by proteinase K and trypsin. We reported that the segment 70-90 in the NAC region is more susceptible to proteolytic attack than the N-terminal region. Probably, This region is flexible and sufficiently protruded to be protease-sensitive even if the analysis of CD spectra in the far-UV demonstrates that this region has an alpha-helix conformation and the NMR experiment indicates that only the C-terminal ~ 40 residues continue to be unfolded and mobile in the presence of DHA. Furthermore, we observed that alpha-syn strongly affects the self-association process of DHA. The physical state of the lipid in the presence of the protein was analyzed by turbidity measurements, dynamic light scattering (DLS), pyrene fluorescence analysis and transmission electron microscopy (TEM). At pH 7.4, DHA assembles in oil droplets with a large size distribution (Namani et al., 2007). alpha-Syn disrupts these lipid aggregates, stabilizing a new product of DHA self-assembly. These species are formed at lower concentrations range and they have a regular shape, a smaller diameter and a reduced hydrophobic volume. Truncated forms of alpha-syn corresponding to different parts of its polypeptide chain (syn1-99, syn1-52, syn57-102, and syn108-140) were also used to extend the knowledge on the role of different protein regions in the interaction with the lipid. CD data suggest that there is an important role of the repeats in the alpha-helix transition and thereby in the interaction with DHA. The C-terminal region, at variance, seems to modulate the portion of alpha-syn buried into the lipid compartment. Moreover, with the exception of syn 108-140, all the polypeptides affect the self-assembly process of DHA. We can hypothesize that the N-terminal region of alpha-syn has a crucial role even in the regulation of DHA aggregation process. Finally, a general consideration concerns the ability of DHA and probably of other long chain PUFAs to induce oligomerization and fibrillation of alpha-syn (Perrin et al., 2001; Sharon et al., 2003; Broersen et al., 2006). The molecular effect of DHA on aggregation process of alpha-syn was analyzed by CD, native gel electrophoresis, Thioflavin T assay and TEM observation. The presence of DHA, in a molar ratio [DHA]/[alpha-syn] of 10, promotes aggregation and fibrils formation of alpha-syn. On the contrary, in the presence of saturating conditions of DHA, only oligomeric species are formed. DHA exerts a direct effect on protein structure, stabilizing an amyloidogenic conformation and generates an environment that can promote protein aggregation. Sharon R., Goldberg M.S., Bar-Josef I., Betensky R.A., Shen J., Selkoe D.J. (2001). Alpha-Synuclein occurs in lipid-rich high molecular weight complexes, binds fatty acids, and shows homology to the fatty acid-binding proteins. Proc. Natl. Acad. Sci. U S A. 98(16), 9110?9115. Broersen K., van den Brink D., Fraser G., Goedert M., Davletov B. (2006). Alpha-synuclein adopts an alpha-helical conformation in the presence of polyunsaturated fatty acids to hinder micelle formation. Biochem. 45(51), 15610?15616. Chiti, F. and Dobson, C. M. (2006). Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem., 75, 333?366. Lücke C., Gantz D. L., Klimtchuk E., Hamilton J. A. (2006). Interactions between fatty acids and alpha-synuclein. J. Lipid Res. 47, 1714?1724. Namani T., Ishikawa T., Morigaki K., Walde P. (2007). Vesicles from docosahexaenoic acid. Colloids and Surfaces B: Biointerfaces. 54, 118?123. Pawar, A. P., DuBay, K. F., Zurdo, J., Chiti, F., Vendruscolo, M. & Dobson, C. M. (2005). Prediction of “aggregation-prone” and “aggregation-susceptible” regions in proteins associated with neurodegenerative disease. J. Mol. Biol. 350, 379?392. Perrin R.J., Woods W.S., Clayton D.F., George J.M. (2001). Exposure to long chain polyunsaturated fatty acids triggers rapid multimerization of synucleins. J. Biol. Chem. 276(45), 41958?41962. Sharon R., Bar-Joseph I., Frosch M.P., Walsh D.M., Hamilton J.A., Selkoe D.J. (2003). The formation of highly soluble oligomers of alpha-synuclein is regulated by fatty acids and enhanced in Parkinson's disease. Neuron. 37(4), 583?595. Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M. & Goedert, M. (1998). alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies. Proc. Natl. Acad. Sci. U S A, 95, 6469–6473.Il progetto della mia Tesi di dottorato riguarda il problema del folding di proteine ed il loro misfolding, in linea con la ricerca condotta nel laboratorio di Chimica delle Proteine dove è stato principalmente svolto lo studio. La ricerca svolta può essere divisa in due parti. Durante il primo anno di dottorato è stato studiato l’effetto del pH nella fibrillogenesi di proteine, mediante l’analisi delle caratteristiche di un peptide modello. Nel secondo e terzo anno di dottorato, è stata analizzato il complesso formato da alpha-sinucleina umana ed acidi grassi e le implicazioni di questa interazione nel processo di aggregazione della proteina. Di conseguenza, la Tesi è composta da una prima parte riguardante lo studio delle proprietà di aggregazione del peptide apoMb1-29 (Capitolo 1, 2) e di una seconda parte dedicata alla caratterizzazione dell’interazione di alpha-sinucleina con acidi grassi (Capitolo 3, 4). Molte malattie umane, definite anche misfolding diseases, derivano da una non corretta strutturazione delle proteine coinvolte. Un numero sempre maggiore di malattie, come il morbo di Alzheimer e di Parkinson, è correlato al fenomeno dell’aggregazione proteica e all’accumulo anomalo di depositi proteici in diversi tessuti e organi. Questi depositi patologici sono formati da aggregati proteici fibrillari, chiamati fibrille amiloidi. L’amiloide è un polimero proteico non-covalente, stabilizzato da struttura di tipo beta, in cui i diversi betha-strands sono lateralmente associati e formano aggregati fibrillari. Poiché anche proteine e peptidi non direttamente coinvolti in patologie sono in grado di formare fibrille amiloidi in appropriate condizioni, si ritiene che la capacità di formare fibrille sia una proprietà generica delle backbone polipeptidico (Chiti and Dobson, 2006). Comunque, la tendenza ad aggregare e la stabilità delle fibrille dipende dalla sequenza aminoacidica, quindi determinanti intrinseci, come la carica netta, l’idrofobicità, la presenza di residui aromatici e la propensione a formare struttura beta, hanno un ruolo determinante nell’amiloidogenicità di una catena polipeptidica (Pawar et al., 2005). Per comprendere l’importanza della carica netta di una proteina nel suo processo di aggregazione e per analizzare gli effetti dell’interazione elettrostatica nella stabilità delle risultanti fibrille, le proprietà di aggregazione di un peptide, corrispondente al frammento 1-29 di apomioglobina da cuore di cavallo (apoMb1-29), sono state studiate in differenti condizioni di pH. Questo peptide forma velocemente fibrille amiloidi a pH acidi. Il processo a pH 2.0 segue un meccanismo di crescita nucleazione-dipendente, come determinato dall’analisi fluorimetrica mediante Tioflavina T (ThT). Osservazioni mediante microscopia elettronica (TEM) confermano la presenza di fibrille e misure di dicroismo circolare (CD) indicano l’acquisizione di un alto contenuto di struttura secondaria di tipo beta. Mediante l’uso di peptidi derivanti dalla proteolisi di apoMb1-29, è stata poi identificata la regione 7-16 come la più amiloidogenica, infatti, ha un alto grado di idrofobicità, propensione a formare beta-sheet e bassa carica netta. In conclusione, la modulazione della carica netta dei peptidi analizzati, derivante da un cambiamento del pH, è il fattore che primariamente regola formazione di aggregati fibrillari. Inoltre, è stato dimostrato che interazioni di tipo elettrostatico hanno un ruolo determinante anche nel stabilizzare la struttura beta di fibrille mature. Infatti, ThT, TEM e CD hanno evidenziato una veloce e completa disaggregazione delle fibrille, se il pH della sospensione viene portato a valori più basici. Nella seconda parte del mio progetto di dottorato, ho studiato i dettagli molecolari che regolano l’interazione tra alpha-sinucleina (alpha-syn) e acidi grassi, analizzando sia le caratteristiche conformazionali della proteina acquisite in presenza dell’acido grasso, sia lo stato fisico dello stesso lipide. Inoltre, è stato studiato il processo di aggregazione di alpha-syn mediato da acidi grassi, allo scopo di comprendere l’implicazione dei lipidi nella formazione amiloide in vivo. ?-Sinucleina è una proteina solubile di 140 aminoacidi, natively unfolded con funzione sconosciuta. Essa è altamente espressa nel sistema nervoso centrale ed è abbondante nei terminali presinaptici dei neuroni. Questa proteina è caratterizzata dalla presenza di sette ripetizioni imperfette di sequenza aminoacidica (KTKEGV) nella regione N-terminale, da una regione idrofobica centrale (NAC, non-amyloid component) e da una coda C-terminale che presenta numerosi residui acidi. La sovraespressione di ?-syn e mutazioni nel suo gene sono associati a forme precoci della sindrome di Parkinson. Inoltre, alpha-syn è il componente principale dei corpi di Lewy, accumuli citoplasmatici caratteristici del morbo di Parkinson (Spillantini et al., 1998). Il meccanismo con cui un cambiamento nella struttura e nell’espressione della proteina possa portare allo sviluppo della malattia non è ancora stato chiarito. Nonostante l’evidenza di un ruolo chiave nella patogenesi, ci sono ancora poche informazioni sulla funzione fisiologica di alpha-syn a livello neuronale. Tra le varie ipotesi, la funzione di alpha-syn è stata associata anche ad acidi grassi. alpha-Syn sembra essere in grado di interagire con acidi grassi insaturi e polinsaturi, ma non è ancora chiaro se l’interazione coinvolga molecole libere (Sharon et al., 2001), o stati aggregati (micelle, vescicole, oil droplets) di acidi grassi (Broersen et al., 2006; Lücke et al., 2006). Questa interazione modula anche l’oligomerizzazione della proteina. Infatti, studi in vitro hanno evidenziato come alpha-Syn formi multimeri in seguito all’esposizione a vescicole formate da lipidi contenenti PUFA (Perrin et al., 2001). Inoltre, in linee cellulari neuronali trattate con PUFA è stato descritto un aumento della formazione di oligomeri di alpha-syn. Queste strutture potrebbero precedere la formazione di aggregati associati alla neurodegenerazione (Sharon et al., 2003). In questo lavoro di tesi è stato effettuato in primo luogo uno studio sistematico sulle transizioni conformazionali di alpha-syn in presenza di diversi acidi grassi. Dato che il numero di insaturazioni e la lunghezza della catena acilica hanno un importante effetto sullo stato aggregativo dell’acido grasso (monomero, micella, vescicola o oil droplet) e di conseguenza anche nell’interazione con la proteina, l’analisi è stata condotta usando acidi grassi con diverse caratteristiche: acido palmitico (saturo), acido oleico (monoinsaturo) e acido docosaesaenoico (DHA, polinsaturo). Quest’ultimo è un acido grasso omega-3 abbondante a livello delle membrane neuronali. E’ stato osservato che in aree del cervello di pazienti affetti da morbo di Parkinson contenenti inclusioni di alpha-syn, si registra un aumento nel livello di DHA. Gli effetti degli acidi grassi sulla struttura di alpha-syn sono stati analizzati mediante CD e mapping proteolitico. La proteina è unfolded in assenza degli acidi grassi e in presenza di acido palmitico, mentre in seguito al legame con acido oleico e DHA, acquisisce una conformazione alpha-elicoidale mediante una semplice transizione a due stadi. In presenza di DHA, alpha-syn è abbastanza resistente alla proteolisi con proteinasi K e tripsina e il segmento 70-90 contenuto nella regione NAC è maggiormente suscettibile all’attacco proteolitico rispetto alla regione N-terminale. Probabilmente, questo segmento è flessibile ed sufficientemente esposto all’azione proteolitica, nonostante l’analisi CD dimostri la presenza di struttura alpha-elica e gli esperimenti NMR indichino che solo 40 residui del C-terminale risultano essere destrutturati e mobili in presenza di DHA. Successivamente, abbiamo osservato che alpha-syn altera il processo di auto-associazione di DHA. Lo stato fisico del lipide in presenza di alpha-syn è stato analizzato mediante misure di torbidità, dynamic light scattering (DLS), TEM e studi di fluorescenza utilizzando il pirene come sonda. DHA forma oil droplets polidisperse a pH neutro (Namani et al., 2007). alpha-Syn disgrega questi aggregati lipidici, favorendo una diversa forma di auto associazione di DHA. In presenza di alpha-syn sono necessarie concentrazioni minori di acido grasso per ottenere questa specie, caratterizzata da forma più regolare, diametro inferiore e volume idrofobico ridotto. Forme tronche di alpha-syn corrispondenti a diverse parti della catena polipeptidica (syn1-99, syn1-52, syn57-102, syn108-140) sono state utilizzate per ulteriori studi sul ruolo che ciascuna regione ha nell’interazione con il lipide. Analisi CD evidenziano come le sequenze ripetute svolgano un’importante funzione nella transizione ad alpha-elica e, di conseguenza, nell’interazione con DHA. Invece, la regione C-terminale sembra modulare la porzione di proteina che si colloca nel compartimento lipidico. Questi peptidi sono stati utilizzati anche nello studio delle proprietà aggregative di DHA. Ad eccezione di syn108-140, tutti gli altri peptidi alterano il processo di auto-associazione di DHA. Possiamo, quindi, ipotizzare che la regione N-terminale svolge un ruolo cruciale anche nel regolare il processo aggregativo di DHA. Infine, in questo lavoro di tesi si discute l’abilità del DHA, e probabilmente di altri acidi grassi polinsaturi, di indurre la formazione di oligomeri e fibrille di alpha-syn (Perrin et al., 2001; Sharon et al., 2003; Broersen et al., 2006). Gli effetti molecolari di DHA sull’aggregazione di alpha-syn sono stati analizzati mediante CD, elettroforesi su gel nativo, ThT e TEM. La presenza di DHA in un rapporto molare [DHA]/[alpha-syn] di 10, promuove l’aggregazione e la formazione di fibrille della proteina. Al contrario, condizioni saturanti di DHA inducono la formazione di sole specie oligomeriche. DHA esercita un effetto diretto sulla struttura proteica, stabilizzandone una conformazione amiloidogenica, e crea un ambiente che promuove l’aggregazione proteica

The role of tryptophan in protein fibrillogenesis: relevance of Trp7 and Trp14 to the amyloidogenic properties of myoglobin

In order to understand the role of tryptophan in the mechanisms of fibrils formation, the ability of a series of analogs of the residue 7-18 span of myoglobin to form amyloid-like fibrils was investigated. Alternatively one or both tryptophans were substituted with alanine and leucine, to determine the contribution of hydrophobicity and aromaticity. The scale of aggregation propensity of the peptides determined indicates that tryptophan is crucial for the amyloidogenic process. Since the rare tryptophan residue is generally engaged in structural roles in proteins, or when exposed serves as binding sites, we surmise that its exposure in the amyloidogenic fragments allows for intermolecular clustering with residues from other molecules leading to the formation of amyloid aggregates

Molecular Insights into the Interaction between \u3b1-Synuclein and Docosahexaenoic Acid

\u3b1-Synuclein (\u3b1-syn) is a 140-residue protein of unknown function, involved in several neurodegenerative disorders, such as Parkinson's disease. Recently, the possible interaction between \u3b1-syn and polyunsaturated fatty acids has attracted a strong interest. Indeed, lipids are able to trigger the multimerization of the protein in vitro and in cultured cells. Docosahexaenoic acid (DHA) is one of the main fatty acids (FAs) in cerebral gray matter and is dynamically released following phospholipid hydrolysis. Moreover, it has been found in high levels in brain areas containing \u3b1-syn inclusions in patients affected by Parkinson's disease. Debated and unsolved questions regard the nature of the molecular interaction between \u3b1-syn and DHA and the effect exerted by the protein on the aggregated state of the FA. Here, we show that \u3b1-syn is able to strongly interact with DHA and that a mutual effect on the structure of the protein and on the physical state of the lipid derives from this interaction. \u3b1-Syn acquires an \u3b1-helical conformation in a simple two-state transition. The binding of the protein to the FA leads to a reduction of the size of the spontaneously formed aggregated species of DHA as well as of the critical aggregate concentration of the lipid. Specifically, biophysical methods and electron microscopy observations indicated that the FA forms oil droplets in the presence of \u3b1-syn. Limited proteolysis experiments showed that, when the protein is bound to the FA oil droplets, it is initially cleaved in the 89\u2013102 region, suggesting that this chain segment is sufficiently flexible or unfolded to be protease-sensitive. Subsequent proteolytic events produce fragments corresponding to the first 70\u201380 residues that remain structured and show high affinity for the lipid. The fact that a region of the polypeptide chain remains accessible to proteases, when interacting with the lipid, suggests that this region could be involved in other interactions, justifying the ambivalent propensity of \u3b1-syn towards folding or aggregation in the presence of FAs

The Oleic Acid Complexes of Proteolytic Fragments of Alpha-lactalbumin Display Apoptotic Activity

The complexes formed by partially folded human and bovine alpha-lactalbumin with oleic acid (OA) have been reported to display selective apoptotic activity against tumor cells. These complexes were named human (HAMLET) or bovine (BAMLET) alpha-lactalbumin made lethal to tumor cells. Here, we analyzed the OA complexes formed by fragments of bovine alpha-lactalbumin obtained by limited proteolysis of the protein. Specifically, the fragments investigated were 53-103 and the two-chain fragment species 1-40/53-123 and 1-40/104-123, these last being the N-terminal fragment 1-40 covalently linked via disulfide bridges to the C-terminal fragment 53-123 or 104-123. The OA complexes were obtained by mixing the fatty acid and the fragments in solution (10-fold and 15-fold molar excess of OA over protein fragment) or by chromatography of the fragments loaded onto an OA-conditioned anion exchange column and salt-induced elution of the OA complexes. Upon binding to OA, all fragments acquire an enhanced content of alpha-helical secondary structure. All OA complexes of the fragment species showed apoptotic activity for Jurkat tumor cells comparable to that displayed by the OA complex of the intact protein. We conclude that the entire sequence of the protein is not required to form an apoptotic OA complex, and we suggest that the apoptotic activity of a protein-OA complex does not imply specific binding of the protein