RNA binding proteins (RBP) are a class of proteins which play a pivotal role in regulating gene expression. They bind to RNA to create a complex which has different roles in cell. In particular, their dysregulation leads to growth of neurodegenerative disease or cancer. In this contest, Embryonic Lethal Abnormal Vision (ELAV) or Hu protein class capture our attention. ELAV proteins are RBP which bind mRNA creating a stable complex whit it. The binding to mRNA occurs through three specific domain called RNA Recognition Motif (RRM) located on them and the recognition of a specific region on mRNA called ARE (adenine-uridine rich element). Within ELAV protein particular attention was given to HuR as it is involved in several pathologies (i.e. cancer etc.) when it is overexpressed. Consequently, HuR is considered the suitable candidate target for the development of new anticancer drugs. In order to modulate the interaction between HuR and mRNAs, several small molecules have been identified (natural or synthetic derived from an appropriate design) and tested by MedChemLab group under the supervision of Prof. Simona Collina. The basic idea is to identify molecules that can interfere with the HuR/RNA binding. This thesis is a part of this wide project, addressed to the identification and biological evaluation of new molecules. In particular, the purpose is the characterization, analysis of interaction and competition studies of a series of selected ligands with HuR using a potential tool called STD (Saturation Transfer Difference) NMR (Nuclear Magnetic Resonance). STD NMR is a technique based on the transfer of magnetization from the selectively irradiated protein to the ligand protons if they are in contact with the protein. It is used to study the interaction between small ligands and macromolecule. From this experiment it is possible to investigate if a ligand binds or not the target, and their affinity. STD experiments show the protons of the ligand interacting with the protein defining the so-called epitope. The protons strongly involved in ligand-protein interaction have the higher intensity in STD spectra. The final result is a mono-dimensional spectrum characterized by the presence of peaks of different intensity linked to their proximity/distance to protein binding site. Competition studies are important to verify the specificity of the binding site using a ligand known for its affinity with the protein, which acts as competitive antagonist towards the ligand that compete fort this site. In this thesis, four compounds (one natural and three synthetic) have been selected and after their characterization, their interaction with HuR through STD NMR have been studied. The epitope of the ligands has been mapped in order to observe which parts interact better and finally competition studies have been performed. Competition studies were performed by RNA decamer (UAUUAUUA), previously used for crystallization study. RNA is the natural ligand for HuR, but since has a low stability to environmental proteases and it is difficult to avoid its degradation due to the environmental proteases, to mimic the correct RNA sequence, we have synthetized (with the collaboration of Dr.ssa Silvia Cauteruccio, UNIMI) a fragment of PNA with the following sequence (TATTATTA), which is supposed to be a mimic of the RNA sequence and to bind HuR in the same binding site of mRNA. We have also analysed the PNA monomers, A-PNA and T-PNA. The results obtained in this thesis provide useful information about the binding site of HuR interested in the binding with the selected ligands. Moreover, these information can be useful for the design and identification of new compounds able to interfere the complex HuR-mRA
Le RNA binding proteins (RBPs) sono una classe di proteine che svolgono un ruolo importante nella regolazione dei processi post-trascrizionali. Tra queste, di particolare interesse sono le proteine ELAV (Embryonic Lethal Abnormal Vision) o proteine Hu, una sottoclasse delle RBPs che ha come target l’mRNA; attraverso il loro complesso con l’mRNA lo stabilizzano, regolano la sua degradazione e traduzione. HuR, il target scelto in questa tesi, fa parte della famiglia delle Hu proteins ed è in grado di legarsi agli mRNA attraverso una sua regione chiamata RNA Recognition Motif (RRM) in un sito presente sugli mRNA chiamato ARE (adenine-uridine rich element). Questa proteina è coinvolta in diversi processi fisiologici e il suo malfunzionamento può essere correlato a diverse malattie (quali ad esempio il cancro), in cui si osserva una sua sovraespressione e iperattivazione con conseguenze sui suoi mRNA target. Il mio lavoro di tesi fa parte di un progetto coordinato dalla Professoressa S. Collina che ha lo scopo di sintetizzare e testare delle piccole molecole che possano legarsi ad HuR e interferire perciò sulla formazione e stabilità del complesso HuR-mRNA. In dettaglio questo lavoro ha riguardato lo studio dell’interazione tra HuR e un set di molecole (sia naturali che di sintesi disegnate nell’ambito di questo progetto) tramite una tecnica innovativa chiamata STD (Saturation Transfer Difference) NMR (Nuclear Magnetic Resonance), e studi di competizione tra questi ligandi e frammenti di RNA o PNA (Peptide Nucleic Acid) che mimano i nucleotidi. L’STD NMR è una tecnica che si basa sul trasferimento di magnetizzazione dai protoni della proteina selettivamente irradiata ai protoni del ligando che sono in diretto contatto con essa. Lo scopo dell’STD è quello di andare ad osservare nel dettaglio i protoni del ligando per identificare se c’è interazione tra questo e la proteina e per analizzare quali parti interagiscono con la proteina in soluzione. Il risultato finale è uno spettro monodimensionale caratterizzato dalla presenza di picchi dei protoni del ligando con diversa intensità. In particolare l’intensità dei picchi è correlata alla vicinanza/ lontananza dei protoni dal sito di legame della proteina. Poiché l’STD da solo indicazioni relative al ligando senza fornire informazioni circa il sito di binding della proteina, gli studi di competizione hanno lo scopo di identificare la specificità del sito di legame dei composti utilizzando come antagonisti delle molecole note per la loro affinità con la proteina in uno specifico sito di binding. In particolare in questa tesi è stato utilizzato un frammento di RNA (UAUUAUUA, di cui esiste la struttura cristallografica nel sito di bingi di HuR) e il corrispondente decamero PNA. Il lavoro è stato suddiviso in vari step che prevedono: 1. La caratterizzazione dei ligandi scelti, dei monomeri e del Decamero dei PNA, tramite tecniche NMR mono e bidimensionali (1H-NMR, COSY, TOCSY, HSQC, HMBC, NOESY). 2. Lo studio dell’interazione tra i suddetti composti e HuR tramite la tecnica dell’STD NMR con conseguente mappatura dell’epitopo 3. Studi di competizione di tre di questi composti in presenza di RNA e PNA tramite STD NMR. I risultati ottenuti hanno fornito informazioni utili per identificare la specificità dei composti in esame per il sito di binding che si vuole colpire e potranno essere usate anche per il design di nuovi composti che possano essere più affini a HuR e che possano interferire maggiormente nella formazione del complesso HuR-mRNA.
Uso dell'STD NMR per studi di interazione tra la proteina HuR e una serie di composti.
GRASSELLI, GAIA
2020/2021
Abstract
RNA binding proteins (RBP) are a class of proteins which play a pivotal role in regulating gene expression. They bind to RNA to create a complex which has different roles in cell. In particular, their dysregulation leads to growth of neurodegenerative disease or cancer. In this contest, Embryonic Lethal Abnormal Vision (ELAV) or Hu protein class capture our attention. ELAV proteins are RBP which bind mRNA creating a stable complex whit it. The binding to mRNA occurs through three specific domain called RNA Recognition Motif (RRM) located on them and the recognition of a specific region on mRNA called ARE (adenine-uridine rich element). Within ELAV protein particular attention was given to HuR as it is involved in several pathologies (i.e. cancer etc.) when it is overexpressed. Consequently, HuR is considered the suitable candidate target for the development of new anticancer drugs. In order to modulate the interaction between HuR and mRNAs, several small molecules have been identified (natural or synthetic derived from an appropriate design) and tested by MedChemLab group under the supervision of Prof. Simona Collina. The basic idea is to identify molecules that can interfere with the HuR/RNA binding. This thesis is a part of this wide project, addressed to the identification and biological evaluation of new molecules. In particular, the purpose is the characterization, analysis of interaction and competition studies of a series of selected ligands with HuR using a potential tool called STD (Saturation Transfer Difference) NMR (Nuclear Magnetic Resonance). STD NMR is a technique based on the transfer of magnetization from the selectively irradiated protein to the ligand protons if they are in contact with the protein. It is used to study the interaction between small ligands and macromolecule. From this experiment it is possible to investigate if a ligand binds or not the target, and their affinity. STD experiments show the protons of the ligand interacting with the protein defining the so-called epitope. The protons strongly involved in ligand-protein interaction have the higher intensity in STD spectra. The final result is a mono-dimensional spectrum characterized by the presence of peaks of different intensity linked to their proximity/distance to protein binding site. Competition studies are important to verify the specificity of the binding site using a ligand known for its affinity with the protein, which acts as competitive antagonist towards the ligand that compete fort this site. In this thesis, four compounds (one natural and three synthetic) have been selected and after their characterization, their interaction with HuR through STD NMR have been studied. The epitope of the ligands has been mapped in order to observe which parts interact better and finally competition studies have been performed. Competition studies were performed by RNA decamer (UAUUAUUA), previously used for crystallization study. RNA is the natural ligand for HuR, but since has a low stability to environmental proteases and it is difficult to avoid its degradation due to the environmental proteases, to mimic the correct RNA sequence, we have synthetized (with the collaboration of Dr.ssa Silvia Cauteruccio, UNIMI) a fragment of PNA with the following sequence (TATTATTA), which is supposed to be a mimic of the RNA sequence and to bind HuR in the same binding site of mRNA. We have also analysed the PNA monomers, A-PNA and T-PNA. The results obtained in this thesis provide useful information about the binding site of HuR interested in the binding with the selected ligands. Moreover, these information can be useful for the design and identification of new compounds able to interfere the complex HuR-mRAÈ consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/12984