Repozitorij Univerze v Novi Gorici

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Naslov:Structural and functional determinants of TDP-43 aggregation : Dissertation
Avtorji:Škaro, Sanja (Avtor)
Baralle, Marco (Mentor) Več o mentorju... Novo okno
Datoteke:.pdf Sanja_Skaro.pdf (6,84 MB)
 
Jezik:Angleški jezik
Vrsta gradiva:Doktorsko delo/naloga (mb31)
Tipologija:2.08 - Doktorska disertacija
Organizacija:FPŠ - Fakulteta za podiplomski študij
Opis:TDP-43 (TAR DNA-binding protein) is an hnRNP that was identified as the main component of the brain inclusions characteristically found in patients suffering of Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration. As an hnRNP protein, TDP-43 fulfills diverse roles in mRNA metabolism, localization and transport. Structurally, TDP-43 is composed of a well conserved N terminal domain (NTD), two RRM domains of which RRM1 is necessary for recognizing and binding to its target, UG rich RNA sequences, and the C-terminal domain (CTD) which is a Glycine rich domain. The CTD also contains a Q/N rich region that plays a key role in protein aggregation and interaction with another hnRNP proteins and polyglutamine repeats. This thesis focus on the structural determinants involved in the different TDP-43 interactions with itself and with other hnRNPs. Both the carboxyl and amino terminal domains are involved in these interactions. We have mapped the regions more relevant for the function of TDP-43 and for the aggregation process characteristic of the pathological pathway leading to neurodegeneration. We have started to further study the N-terminal domain. Previous results in our laboratory using a cellular aggregation model have shown that the N-terminal domain is also necessary for sequestering the endogenous TDP-43 into the aggregates. In particular, the intact NTD, specifically residues 1 to 77, have been shown to be needed to efficiently recruit TDP-43 monomers into these aggregates. We have extended our knowledge of NTD structure and function, by assessing the behavior of a series of proteins in which key structural features (α-helix and β-sheets) were modified and TDP-43 splicing function together with structure via NMR were analyzed. It was found that by disrupting protein secondary structure in the NTD (mutation in α-helix NTD-31V/R-32T/R) the capacity of the aggregates to sequester enough TDP-43 to induce loss of function was lost.In fact, this protein is also unable to recovery TDP-43 functionality when it is disrupted due to sequestration of the endogenous TDP-43 in add back experiments. Disturbing protein stability through substitution of residues in α-helix also affects its ability to form an active conformation. On the other hand, synthesis of hybrid peptides containing certain NTD and CTD segments was performed in order to see if they are capable to bind to the TDP-43 aggregates. However, it has been shown that these synthetic peptides have a greater ability to induce TDP-43 aggregation than to bind to them, probably due to specific functional characteristics of NTD and CTD segments used for their synthesis.The main focus of the thesis was on the C-terminal domain sequences involved in protein-protein interaction, misfolding and aggregation.A comparison of human, mouse, zebrafish, Annelida, flatworms and Drosophila showed a very strong conservation of the NTD and RRMs, but the C terminal regions of human and other TDP-43 orthologues are very different.I have studied Human and Drosophila melanogaster orthologues, because Drosophila orthologue contains different paralogs of TDP-43.Through a series of deletions and mutations it was shown that the shorter paralog of Drosophila TDP-43 (TBPH-RA) is more active than the longer one (TBPH-RC), and that this is due to a combination of two factors: 1. TBPH-RC by itself aggregates more than TBPH-RA, 2. The functionality of TBPH-RC is downregulated by intramolecular interactions in the C terminal domain. Apparently there is a cation-π interaction involving Tryptophan and Arginine in TBPH-RC that has a high relevance to the protein function and is lacking in the TBPH-RA.Overall this data has identified structural features essential for the proper function of TDP-43.In addition, we have also identified sequences that are critical in the pathological aggregation process of TDP-43 that lead to the characteristic brain inclusions in ALS and FTLD and to the loss of functionality
Ključne besede:TDP-43 structural determinants, hybrid peptides, protein-protein interactions, intramolecular interaction, cation-π interaction, Drosophila orthologues.
Leto izida:2019
Izvor:Nova Gorica
COBISS_ID:5426427 Povezava se odpre v novem oknu
URN:URN:SI:UNG:REP:Y8S7O2O4
Število ogledov:2139
Število prenosov:133
Metapodatki:XML RDF-CHPDL DC-XML DC-RDF
Področja:Gradivo ni uvrščeno v področja.
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Skupna ocena:(0 glasov)
Vaša ocena:Ocenjevanje je dovoljeno samo prijavljenim uporabnikom.

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Sekundarni jezik

Jezik:Slovenski jezik
Naslov:Strukturne in funkcionalne determinante agregacije TDP-43 : Disertacija
Opis:DNA vezavni transaktivacijski protein TDP-43 spada med heterogene jedrne ribonukleoproteinske delce (ang. hnRNPs).Izkazalo se je,da je TDP-43 glavna komponenta inkluzijskih telesc,ki jih običajno najdemo pri pacientih z amiotrofno lateralno sklerozo in frontotemporalno lobarno degeneracijo.Kot hnRNP ima TDP-43 različne vloge pri metabolizmu,lokalizaciji in transportu mRNA.Strukturno je sestavljen iz dobro ohranjene N-terminalne domene,dveh RNA-prepozavnih motivov ki sta potrebna za prepoznavanje in vezavo na UG bogata zaporedja RNK,in iz C-terminalne domene,bogate z glicinom.C-terminalna domena vsebuje tudi poliglutaminske ponovitve in Q/N bogato regijo,ki igrajo pomembno vlogo pri agregaciji samega TDP-43,interakcijah z drugimi hnRNP proteini in poliglutaminskimi ponovitvami.Pričujoče delo se osredotoča na strukturne determinante,ki sodelujejo pri interakcijah TDP-43 s samim seboj in z drugimi hnRNP proteini.V te interakcije sta vključeni tako C- kot tudi N-terminalna domena.V sklopu te raziskave smo mapirali regije,ki so relevantne za normalno elovanje TDP-43 kot tudi pri agregacijskih procesih značilnih za patološko kaskado,ki vodi v nevrodegeneracijo.V prvem delu smo se osredotočili na N-terminalno domeno.Predhodni rezultati,ki smo jih v našem laboratoriju pridobili s pomočjo modela celične agregacije,kažejo na to,da tudi N-terminalna domena sodeluje pri sekvestraciji endogenega TDP-43 v agregate. Da se monomeri TDP-43 lahko povežejo in agregirajo,je potrebno celotno zaporedje N-terminalne domene,natančneje aminokislinska ostanka 1 in 77.Z namenom da bi podrobneje preiskali vlogo N-terminalne domene v povezavi s strukturo proteina kot tudi z njegovo funkcijo,smo pripravili serijo proteinov z modifikacijami v glavnih strukturnih motivih (α-heliksih in β-ploskvah),katerih funkcijo in strukturo smo analizirali s pomočjo tehnike NMR.Odkrili smo, da z zrušenjem sekundarne strukture N-terminalne domene (mutacija v α-heliksu NTD-31V/R-32T/R),TDP-43 agregati sekvestrirajo manj endogenega TDP-43 kot sicer ter da prisotnost mutiranega proteina ne povzroči zadostne agregacije endogenega TDP-43,da bi to motilo njegovo normalno delovanje v celici.Poleg tega z ekspresijo mutiranega TDP-43 ne uspemo nadomestiti funkcije endogenega TDP-43,ko je normalno delovanje le-tega onemogočeno zaradi sekvestracije v citoplazmi.Manjša stabilnost proteina,do katere privede zamenjava aminokislinskih ostankov v α-heliksu,vpliva na sposobnost proteina za tvorbo aktivne konformacije.Da bi razumeli,kateri segmenti TDP-43 so sposobni vezave v agregate,smo sintetizirali hibridne peptide,ki vsebujejo določene segmente N- in C-terminalnih regij.Rezultati so pokazali,da imajo ti peptidi večjo tendenco,da spodbudijo agregacijo TDP-43,in ne toliko da bi se sami vezali v agregate.To je najverjetneje posledica specifičnih funkcionalnih karakteristik segmentov N- in C- terminalnih domen,ki smo jih uporabili pri sintezi.Glavni fokus te naloge so bila zaporedja C-terminalne domene,ki so vključena v interakcije med proteini,nepravilno zvijanje in njegovo agregacijo.Primerjava aminokislinskega zaporedja pri človeku,miši,navadni cebrici,ploskem črvu in vinski mušici je pokazala visoko ohranjenost N-terminalne domene,hkrati pa velike razlike v zaporedju C-terminalnega konca.Študija se osredotoča na človeške ortologe in ortologe vinske mušice,saj ortolog vinske mušice vključuje tri izoforme proteina TDP-43.Skozi serijo delecij in mutacij smo pokazali,da je krajša izoforma TDP-43 pri vinski mušici (TBPH-RA) aktivnejša kot daljša (TBPH-RC).Razlog za to tiči v kombinaciji dveh faktorjev;TBPH-RP je bolj dovzeten za agregacijo,poleg tega pa funkcionalnost TBPH-RC negativno regulirajo intramolekularne interakcije C-terminalne domene.Kot kaže,obstaja kationska-π interakcija med triptofanom in argininom v C-terminalni domeni TBPH-RC,ki je pomembna pri sami funkciji.Le-ta ni prisotna pri TBPH-RA izoformi.V sklopu naloge smo okarakterizirali strukturne lastnosti TDP-43...
Ključne besede:strukturne determinante TDP-43, hibridni peptidi, interakcije protein-protein, intramolekularna interakcija, kationsko-π interakcija, Drosophila orthologues.


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