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NMR and biochemical analysis of protein-protein interactions: insights on dopamine synthesis regulation and heterochromatin formation
Full text linkMolecular recognition between proteins plays a fundamental role in almost all biological processes. Therefore, the structural characterization of protein-protein interactions represents a fundamental tool to shed light on the molecular mechanisms of biological events. Moreover, it can elucidate the structural basis of the function for proteins without a detectable catalytic activity.
The regulation of dopamine synthesis is an example of the importance of protein-protein interactions in the modulation of a biological function.
L-Dopamine, one of the catecholaminergic neurotransmitters, has many functions in the brain, including regulation of behavior and cognition, motor activity, and sleep.
The enzyme responsible for the biosynthesis of its precursor L-DOPA, named Tyrosine Hydroxylase, is therefore tightly regulated by different mechanisms. One of the most important is the interaction with protein modulators. 14-3-3 proteins bind to the N-terminal regulatory domain of Tyrosine Hydroxylase, leading to the stabilization of the active conformer. Several published data suggest that also alpha-synuclein, a natively unfolded protein linked to the pathogenesis of Parkinson Disease, could play a role in dopamine synthesis, through the inhibition of Tyrosine Hydroxylase. Interestingly, experimental evidence exists also for the interaction between 14-3-3 protein and alpha-synuclein: the binding has been supposed to have functional significance in the network regulation of dopamine synthesis.
The first part of this research is focused on the characterization of the interactions among these three key protein modulators of dopamine biosynthesis using a combined biochemical and spectroscopic approach.
The work can be devided into different levels. Molecular biology, biochemical and spectroscopic methods were used to obtain and characterise each protein sample separately. Subsequently, we analysed the interaction between each pair of protein partners using both biochemical and biophysical tools, in particular NMR spectroscopy.
Electrophoretic and chromatographic results revealed that alpha-synuclein and 14-3-3 proteins fail to form a stable complex in solution, while NMR experiments suggest a transient interaction involving only the first N-terminal residues of monomeric alpha-synuclein. However, a strong indication of interaction comes from aggregation experiments followed by fluorescence polarization, where 14-3-3 proteins were demonstrated to slow down the alpha-synuclein aggregation rate. Taken together, these results suggest that 14-3-3 proteins may bind to the alpha-synuclein oligomeric species, thus opening a possible chaperon function of 14-3-3 proteins to prevent alpha-synuclein aggregation and fibrillation.
On the other hand, both enzymatic and NMR experiments indicated that alha-synuclein does not inhibit Tyrosine Hydroxylase and there is no direct interaction between the two proteins in the experimental conditions tested.
Finally, we verified, using biochemical tools, the formation of a complex with high affinity between 14-3-3 proteins and Tyrosine Hydroxylase phosphorylated in Serine 19, and carried out preliminary crystallization screenings of the complex.
Moreover, this research includes the NMR structural investigation of a second example of protein-protein interaction, involved in a different biological function. We analysed the molecular recognition mechanism between Heterochromatin protein 1 (Hp1beta) and a twenty-one residues peptide that mimics the histone 3 N-terminal tail of the nucleosome. The Hp1beta/histone 3 interaction leads to the packaging of the DNA in tertiary and quaternary structures, altogether named heterochromatin. As heterochromatin prevents access of the transcriptional machinery to the DNA, its formation represents a regulatory mechanism to inhibit gene expression in specific regions of the genome.
As necessary premise to study this interaction by NMR, we assigned, using heteronuclear 3D experiments, the 1H,15N and 13C backbone chemical shifts of Hp1beta protein. At present, we completed the assignment of the resonances from the domain that mediates the interaction with histone 3, called chromo-domain. The assignment of the signals from the other two parts of the molecule, named hinge region and chromo-shadow domain, is in progress. We then characterised the tumbling of the different parts of Hp1beta protein by cross-correlation experiments. Residual dipolar couplings were used to compare the structure of the chromo-domain domain in the full-length protein with that published for the domain produced as deleted mutant.
[1H,15N] HSQC experiments were obtained titrating Hp1beta with the histone 3 derived peptide. The results of the chemical shift perturbation analysis allowed us to map the residues of Hp1beta protein that are responsible for the interaction with the histone 3 N-terminal tail.Il riconoscimento molecolare tra proteine riveste un ruolo fondamentale nella gran parte dei processi biologici. Pertanto, la caratterizzazione strutturale delle interazioni proteina-proteina rappresenta una chiave di lettura fondamentale per far luce sui meccanismi molecolari alla base dei processi biologici. Inoltre tale caratterizzazione può chiarire le basi strutturali della funzione di quelle proteine che mancano di attività catalitica rilevabile.
La regolazione della sintesi di dopamina è un esempio dell'importanza delle interazioni proteina-proteina nella modulazione delle funzioni biologiche. La dopamina, uno dei neurotrasmettitori catecolamminergici, regola molte funzioni del sistema nervoso, tra cui quelle legate al comportamento ed ai processi cognitivi, all’attività motoria e al sonno. L'enzima responsabile della sintesi del suo precursore L-DOPA, è la Tirosina Idrossilasi. Data l’importanza della sua attività catalitica, l’enzima è finemente regolato da molteplici meccanismi. Uno dei più importanti è basato sull’interazione con modulatori proteici.
Le proteine 14-3-3 legano il dominio regolatore ammino-terminale della Tirosina Idrossilasi, stabilizzandone la conformazione cataliticamente attiva. Diversi dati pubblicati in letteratura sostengono che anche l’alfa-sinucleina, una proteina “natively unfolded” correlata all’insorgenza del morbo di Parkinson, potrebbe avere una funzione nella sintesi della dopamina, attraverso l'inibizione della Tirosina Idrossilasi. Di particolare interesse sono le evidenze sperimentali che dimostrano l'interazione tra alfa-sinucleina e proteine 14-3-3, che potrebbe giocare un ruolo importante nella complessa rete di regolazione della sintesi della dopamina.
La prima parte di questa ricerca è focalizzata sulla caratterizzazione delle interazioni fra i tre modulatori proteici della sintesi della dopamina, attraverso l’utilizzo combinato di tecniche biochimiche e spettroscopiche.
Il lavoro si articola in più livelli. Metodi di biologia molecolare, biochimica e spettroscopia sono stati impiegati per produrre e caratterizzare i campioni di proteine per i successivi studi. Abbiamo poi analizzato l'interazione di ciascuna coppia di proteine, utilizzando sia tecniche di biochimica e di biofisica, in particolare spettroscopia di risonanza magnetica nucleare (NMR). Dai risultati elettroforetici e cromatografici è emerso che alfa-sinucleina e proteine 14-3-3 non formano un complesso stabile in soluzione, mentre esperimenti NMR suggeriscono che vi sia una interazione debole che coinvolge solo i primi residui della regione ammino-terminale dell' alfa-sinucleina. Tuttavia, esperimenti di aggregazione seguendo il segnale di polarizzazione di fluorescenza, portano ad una forte indicazione dell’interazione tra le due proteine, dimostrando che le proteine 14-3-3 sono in grado di rallentare la velocità di aggregazione dell’alfa-sinucleina. Complessivamente i risultati suggeriscono che le proteine 14-3-3 potrebbero legarsi alle specie oligomeriche dell' alfa-sinucleina, aprendo quindi la strada ad una possibile funzione di “chaperon” nell’ inibizione del processo di aggregazione e fibrillazione dell’ alfa-sinucleina.
Invece, sia gli studi di attività enzimatica che NMR, indicano che l’ alfa-sinucleina non è un inibitore della Tirosina Idrossilasi, e che non c’è interazione diretta tra le due proteine nelle condizioni sperimentali utilizzate.
Infine, mediante tecniche biochimiche, abbiamo verificato che le proteine 14-3-3 e Tirosina Idrossilasi fosforilata sulla serina 19, formano un complesso ad alta affinità. Abbiamo quindi purificato il complesso su larga scala, ed effettuato lo screening preliminare di cristallizzazione.
La seconda parte del lavoro di ricerca si occupa dell’ indagine strutturale, mediante NMR, di un secondo esempio di interazione proteina-proteina che è alla base di una differente funzione biologica. Abbiamo analizzato il meccanismo di riconoscimento molecolare tra “Heterochromatin protein 1” (Hp1beta) ed un peptide di ventuno residui che mima la regione ammino-terminale della proteina istonica, appartenente al complesso proteico nucleosoma. L’interazione tra Hp1beta e l’istone III porta all’impacchettamento del DNA in strutture terziarie e quaternarie, definite complessivamente eterocromatina. Dal momento che l’eterocromatina impedisce l'accesso dell’apparato di trascrizione al DNA, la sua formazione rappresenta un meccanismo di regolazione per inibire l'espressione genica in specifiche regioni del genoma.
Il requisito fondamentale per studiare questa interazione mediante NMR, è l’assegnazione, utilizzando esperimenti tridimensionali eteronucleari, delle risonanze di 1H, 15N e 13C della catena polipeptidica della proteina Hp1beta. Ad oggi abbiamo completato l’assegnazione dei segnali relativi al dominio che media l’interazione con la proteina istonica III, definito “chromo-domain”. L'assegnazione dei segnali relativi alle altre due regioni della molecola, definiti “hinge-region” e “chromo-shadow domain”, è tuttora in corso. Abbiamo poi caratterizzato le proprietà di “tumbling” delle diverse regioni della proteina Hp1beta, mediante particolari esperimenti di rilassamento di magnetizzazione, definiti di “cross-correlation”. I valori di “residual dipolar coupling” sono stati utilizzati per confrontare la struttura del “chromo-domain” compreso nella proteina intera, con la struttura pubblicata dello stesso dominio prodotto come mutante di delezione.
Tramite esperimenti di titolazione per risonanza magnetica nucleare abbiamo infine individuato i residui amminoacidici della proteina Hp1beta che sono coinvolti nel riconoscimento molecolare del peptide. I risultati dell’analisi delle differenze nei valori di “chemical shift”, ha consentito di mappare le regioni della proteina Hp1beta che sono coinvolte nell'interazione con la parte ammino-terminale dell'istone III
Solution NMR insights into dynamic supramolecular assemblies of disordered amyloidogenic proteins
No full textThe extraordinary flexibility and structural heterogeneity of intrinsically disordered proteins (IDP) make them functionally versatile molecules. We have now begun to better understand their fundamental role in biology, however many aspects of their behaviour remain difficult to grasp experimentally. This is especially true for the intermolecular interactions which lead to the formation of transient or highly dynamic supramolecular self-assemblies, such as oligomers, aggregation intermediates and biomolecular condensates. Both the emerging functions and pathogenicity of these structures have stimulated great efforts to develop methodologies capable of providing useful insights. Significant progress in solution NMR spectroscopy has made this technique one of the most powerful to describe structural and dynamic features of IDPs within such assemblies at atomic resolution. Here, we review the most recent works that have illuminated key aspects of IDP assemblies and contributed significant advancements towards our understanding of the complex conformational landscape of prototypical disease-associated proteins. We also include a primer on some of the fundamental and innovative NMR methods being used in the discussed studies
Untangling the complexity and impact of tau protein ubiquitination
No full textThe microtubule-associated protein tau is an intrinsically disordered protein highly expressed in neuronal axons. Inhealthy neurons, tau regulates microtubule dynamics and neurite outgrowth. However, pathological conditions cantrigger aberrant tau aggregation into insoluble filaments, a hallmark of neurodegenerative disorders known astauopathies. Tau undergoes diverse posttranslational modifications (PTMs), suggesting complex regulation andpotentially varied functions. Among PTMs, the role and mechanisms of ubiquitination in physiology and disease haveremained enigmatic. The past three decades have witnessed the emergence of key studies on tau protein ubiquitination.In this concept, we discuss how these investigations have begun to shed light on the ubiquitination patterns ofphysiological and pathological tau, the responsible enzymatic machinery, and the influence of ubiquitination on tauaggregation. We also provide an overview of the semi-synthetic methods that have enabled in vitro investigations ofconformational transitions of tau induced by ubiquitin modification. Finally, we discuss future perspectives in the fieldnecessary to elucidate the molecular mechanisms of tau ubiquitination and clearance
Alpha-Synuclein-Nanoparticle Interactions: Understanding, Controlling and Exploiting Conformational Plasticity
Full text linkAlpha-synuclein (alpha S) is an extensively studied protein due to its involvement in a group of neurodegenerative disorders, including Parkinson ' s disease, and its documented ability to undergo aberrant self-aggregation resulting in the formation of amyloid-like fibrils. In dilute solution, the protein is intrinsically disordered but can adopt multiple alternative conformations under given conditions, such as upon adsorption to nanoscale surfaces. The study of alpha S-nanoparticle interactions allows us to better understand the behavior of the protein and provides the basis for developing systems capable of mitigating the formation of toxic aggregates as well as for designing hybrid nanomaterials with novel functionalities for applications in various research areas. In this review, we summarize current progress on alpha S-nanoparticle interactions with an emphasis on the conformational plasticity of the biomolecule
Interaction of the intermembrane space domain of Tim23 protein with mitochondrial membranes.
Full text linkBackground: Tim23 mediates protein translocation into mitochondria. Results: Tim23 binds to mitochondria-like membranes through a hydrophobic anchor at its N terminus, with cardiolipin enhancing the interaction. Conclusion: The intermembrane space domain of Tim23 can interact with both inner and outer mitochondria-like membranes. Significance: Tim23 provides the central element for formation of the translocation contact. Tim23 mediates protein translocation into mitochondria. Although inserted into the inner membrane, the dynamic association of its intermembrane space (IMS) domain with the outer membrane promotes protein import. However, little is known about the molecular basis of this interaction. Here, we demonstrate that the IMS domain of Tim23 tightly associates with both inner and outer mitochondrial membrane-like membranes through a hydrophobic anchor at its N terminus. The structure of membrane-bound Tim23(IMS) is highly dynamic, allowing recognition of both the incoming presequence and other translocase components at the translocation contact. Cardiolipin enhances Tim23 membrane attachment, suggesting that cardiolipin can influence preprotein import
Alzheimer's disease-associated ubiquitin mutant Ubb+1: Properties of the carboxy-terminal domain and its influence on biomolecular interactions
No full textUbb+1, a ubiquitin (Ub) mutant protein originating from misreading of the Ub B gene, is found accumulated in brain tissues of Alzheimer's disease patients. The mutant attracts strong interest due to its possible participation in the molecular events leading to neurodegeneration. Ubb+1 is composed of the globular domain of Ub, linked to a 19-residue C-terminal peptide. Based on NMR relaxation and solvent accessibility measurements we obtained new insight into the molecular properties of Ubb+1. We further determined the thermal stability of Ubb+1 in the monomeric form, and in Lys48- and Lys63-linked dimers. Finally, we explored the influence of the C-terminal fragment on the interactions of Ubb+1 with an isolated UBA2 domain and with membrane mimics. Our data indicate that the C-terminal fragment of Ubb+1 is overall highly flexible, except for a short stretch which appears less solvent-exposed. While influencing the hydrodynamic properties of the globular domain, the fragment does not establish long-lived interactions with the globular domain. It results that the structure and stability of Ub are minimally perturbed by the peptide extension. However, binding to UBA2 and to membrane mimics are both affected, exemplifying possible changes in biomolecular recognition experienced by the disease-associated Ubb+1 compared to the wild-type protein
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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