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Biomedical Studies of Human Adenosine Deaminase Acting on Transfer RNA and Related Therapeutic Strategies

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Biomedical Studies of Human Adenosine Deaminase Acting on Transfer RNA and Related Therapeutic Strategies Biomedical studies of human adenosine deaminase acting on transfer RNA and related therapeutic strategies Helena Roura Frigolé ADVERTIMENT. La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX (www.tdx.cat) i a través del Dipòsit Digital de la UB (diposit.ub.edu) ha estat autoritzada pels titulars dels drets de propietat intel·lectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposició des d’un lloc aliè al servei TDX ni al Dipòsit Digital de la UB. No s’autoritza la presentació del seu contingut en una finestra o marc aliè a TDX o al Dipòsit Digital de la UB (framing). Aquesta reserva de drets afecta tant al resum de presentació de la tesi com als seus continguts. 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On having consulted this thesis you’re accepting the following use conditions: Spreading this thesis by the TDX (www.tdx.cat) service and by the UB Digital Repository (diposit.ub.edu) has been authorized by the titular of the intellectual property rights only for private uses placed in investigation and teaching activities. Reproduction with lucrative aims is not authorized nor its spreading and availability from a site foreign to the TDX service or to the UB Digital Repository. Introducing its content in a window or frame foreign to the TDX service or to the UB Digital Repository is not authorized (framing). Those rights affect to the presentation summary of the thesis as well as to its contents. In the using or citation of parts of the thesis it’s obliged to indicate the name of the author. Biomedical studies of human adenosine deaminase acting on transfer RNA and related therapeutic strategies Helena Roura Frigolé UNIVERSITAT DE BARCELONA FACULTAT DE FARMÀCIA I CIÈNCIES DE L’ALIMENTACIÓ Programa de doctorat en Biotecnologia INSTITUTE FOR RESEARCH IN BIOMEDICINE Biomedical studies of human adenosine deaminase acting on transfer RNA and related therapeutic strategies Memòria presentada per Helena Roura Frigolé per optar al títol de doctor per la Universitat de Barcelona Lluís Ribas de Pouplana Helena Roura Frigolé Elena Escubedo Rafa Thesis director PhD candidate Tutor Helena Roura Frigolé 2018 Our greatest weakness lies in giving up. The most certain way to succeed is always to try just one more time. Thomas A. Edison Biomedical studies of human adenosine deaminase Index acting on transfer RNA and related therapeutic strategies Summary 1 Resum 3 Abbreviations 5 Introduction 9 Nucleic acids 11 Proteins 13 Transcription 16 Genetic code 18 Translation 19 Transfer RNAs 22 tRNA-derived fragments 26 tRFs and disease 28 tRNA modifications 30 Inosine 34 and ADAT 33 ADAT substrates 36 ADAT and codon usage 38 V128M mutation in ADAT3 is linked to intellectual disability 41 Additional inosines in RNA 43 Analysis of post-transcriptional RNA modifications 44 Objectives 47 Methods 51 Results 63 Development of an in vitro activity assay to monitor ADAT-mediated deamination 65 Restriction fragment length polymorphism (RFLP) method 65 Fluorescence-based assay 70 Biomedical studies of human adenosine deaminase Index acting on transfer RNA and related therapeutic strategies New insights into substrate recognition and regulation of human ADAT 75 ADAT substrates do not share a common signature sequence 75 Unfolded tRNAs are not ADAT substrates 77 Short tRNA variants are poor ADAT substrates 78 The substrate recognition mode of ADAT varies between different tRNAs 80 ADAT substrate recognition might involve the acceptor stem of some tRNAs 81 It is the whole tRNA architecture rather than a compilation of local structural features the main determinant for ADAT recognition 83 Some of the most abundant tRNA-derived fragments (tRFs) in human cells can modulate ADAT activity 84 Study of ADAT structure and kinetics and effect of a mutation linked to intellectual disability 87 Human ADAT structure and effect of V128M mutation 87 Characterization of the enzymatic activity of human ADAT and effect of V128M mutation 93 Analysis of the catalytic activity of ADAT2 homodimers 97 Small molecules targeting ADAT 100 Deaminase inhibitors as potential ADAT inhibitors 101 Virtual screening of small molecules targeting ADAT 106 In vitro analysis of the virtual screening hits 108 Virtual screening of small molecules targeting ADAT 110 Discussion 113 Conclusions 125 Supplementary material 129 Acknowledgments 143 References 147 Biomedical studies of human adenosine deaminase acting on transfer RNA and related therapeutic strategies Summary Adenosine deaminase acting on transfer RNA (ADAT) is a human heterodimeric enzyme that catalyzes the deamination of adenosine (A) to inosine (I) at the first position of the anticodon of transfer RNAs (tRNAs) (position 34, or wobble position); one of the few essential post-transcriptional modifications on tRNAs (1-5). Inosine 34 allows the recognition of three different nucleotides: cytidine, uridine and adenosine, at the third position of the codon, thus increasing the decoding capacity of tRNAs to more than one messenger RNA (mRNA) codon (adenosine 34 can in principle only pair with codons with uridine at the third position) (6, 7). This alters the tRNA pool available for each codon and it has been proved to align the correlation between codon usage and tRNA gene copy number (9). It has also been suggested to improve fidelity and efficiency of translation (9, 10), especially for mRNAs enriched in codons translated by modified tRNAs (11, 12). Monitoring ADAT-mediated deamination is crucial for the characterization of the enzyme in terms of activity, substrates, regulation, as well as for drug discovery purposes. However, this analysis is often challenging, laborious and lacks quantitativeness. We developed an in vitro deamination assay based on restriction fragment length polymorphism (RFLP) analyses to monitor ADAT activity in an efficient, cost-effective, and semiquantitative manner (13). To overcome a limitation of the method being the need of reverse transcription and amplification of the tRNA, we designed a direct method to quantify I34 formation in vitro using the first fluorescent analogs of nucleic acids that have been reported to undergo enzymatic deaminations (14-16). ADAT has been conserved over the evolution with the acquisition of multi-substrate specificity. Whereas its bacterial homolog TadA deaminates exclusively tRNAArg (2), the human enzyme deaminates eight different tRNAs (3, 17). However, the mechanisms that drove this evolution remain unknown. While the substrate recognition in TadA has been well studied, in the eukaryotic ADAT is poorly understood. Through in vitro enzymatic activity assays with different variants of tRNAArg and tRNAAla, we elucidated the most important features for efficient A34-to-I34 conversion and characterized the substrate recognition of the human enzyme. We also proposed a new potential mechanism of control of ADAT deamination activity by human tRNA-derived fragments, which provides new insights into the regulation of ADAT function and may open a door for the development of new strategies to modulate ADAT activity. A missense mutation (V128M) in one of the two subunits of the human ADAT enzyme causes intellectual disability and strabismus, but the molecular bases of the pathology are 1 Biomedical studies of human adenosine deaminase acting on transfer RNA and related therapeutic strategies unknown (18, 19). We characterized human ADAT in terms of kinetics and structure, and investigated the effect of the V128M mutation. We found that this substitution decreases ADAT deamination activity, and severely affects the stability of the quaternary structure of the enzyme. In this regard, we discovered small molecules with the ability to activate the enzyme, which could potentially recover the defective tRNA editing caused by the mutation. 2 Biomedical studies of human adenosine deaminase acting on transfer RNA and related therapeutic strategies Resum L’adenosina deaminasa específica per RNA de transferència (ADAT) és un enzim humà heterodimèric que catalitza la reacció de deaminació de l’adenosina (A) a inosina (I) a la primera posició de l’anticodó de RNAs de transferència (tRNAs) (també anomenada posició 34 o posició de balanceig); una de les poques modificacions post-transcripcionals essencials en tRNAs (1-5). La inosina 34 permet el reconeixement de tres nucleòtids diferents: citidina, uridina i adenosina, a la tercera posició del codó, augmentant per tant la capacitat de descodificació dels tRNAs a més d’un codó en l’RNA missatger (mRNA) (l’adenosina 34 en principi únicament pot aparellar-se amb uridina en la tercera posició) (6, 7). Això altera els nivells de tRNAs disponibles per cada codó i s’ha demostrat que alinea la correlació entre l’ús de codons i número de còpies gèniques de cada tRNA (9). També s’ha suggerit que millora la fidelitat i eficiència de la traducció (9, 10), especialment per mRNAs enriquits en codons traduïts per tRNA modificats (11, 12).
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