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Functional and Structural Studies of AKR1B15 and AKR1B16: Two Novel Additions to Human and Mouse Aldo-Keto Reductase Superfamily

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Functional and Structural Studies of AKR1B15 and AKR1B16: Two Novel Additions to Human and Mouse Aldo-Keto Reductase Superfamily ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en Functional and structural studies of AKR1B15 and AKR1B16: Two novel additions to human and mouse aldo-keto reductase superfamily Mem`oriapresentada per JOAN GIMENEZ´ DEJOZ per optar al Grau de Doctor en Bioqu´ımicai Biologia Molecular Treball realitzat al departament de Bioqu´ımicai Biologia Molecular de la Universitat Aut`onomade Barcelona, sota la direcci´odels Doctors SERGIO PORTE´ ORDUNA, JAUME FARRES´ VICEN´ i XAVIER PARES´ CASASAMPERA Sergio Port´eOrduna Jaume Farr´esVic´en Xavier Par´esCasasampera Joan Gim´enezDejoz Bellaterra, 17 de juny de 2016 2 Agra¨ıments En primer lloc vull agrair als caps del grup, el Dr. Jaume Farr´esi el Dr. Xavier Par´esl'oportunitat de posar un peu a la ci`encia,iniciar-me en la carrera cient´ıfica,els seus bons consells i l’obtenci´o final d'aquesta tesi. Tamb´evoldria agrair especialment a tots els ADHs amb qui he compartit laboratori, sense vosaltres aix`ono hauria estat possible. Gr`aciesper aguantar tants dies a les fosques! Al Sergio-せんせい, per tot el que m'has ensenyat durant aquests anys, la bona guia, direcci´o, les inacabables correccions, per ensenyar-me a ser cr´ıticamb la meva pr`opiafeina i intentar sempre ser rigor´osi auto exigent tant amb els experiments com amb la redacci´oi preparaci´ode les figures (repassar cada figura mil cops buscant defectes abans de donar-la per bona ja s'ha convertit en costum) i tots i cada un dels inacabables \Podries..."que tant hem trobat a faltar en la `epoca final. Raquel, gr`aciesper tantes i tantes estones de feina compartida amb el llum apagat a l'HPLC (la meva companya del costat fosc), l'inigualable moment del canvi de la bombona de nitrogen, mai m'he rigut tant al lab com aquell dia xD, per aguantar la meva m´usicai no posar-me sempre la teva, tota la xocolata i caf`escompartits i tot el suport d'aquest ´ultimany en el que hem estat m´essols per aqu´ı. Per ser una persona tan incansable i pel teu bon humor i optimisme. Anims,` que encara que no ho vegis ara mateix s´eque far`asuna gran tesi! (nom´est'hi has de posar) Al Julio, per la seva const`anciai treball, els infortunis de l'HPLC i les seves les reparacions m`agiques de l'HPLC (nom´est'estima a tu aquest trasto!) i ensenyar-me a bussejar. El crack de l'Isidro, tu si que tens pasta per ser un bon cient´ıfic! Estic segur que te'n sortir`asde tot el que et proposis. Gr`aciesper totes les millores inform`atiquesi trucs que m'has ensenyat! A la Cristina, pel teu somriure i car`acter que alegraven el laboratori, per la estrella P feta amb post-its decorant el laboratori, per fer les comandes quan a la pissarra ja no hi cabia ninguna m´es,a tots ens hauria agradat que haguessis pogut continuar al lab amb nosaltres per`osegur que all`aon vagis t’anir`a b´e. Xavi-せS1い el primer en ensenyar-me el m´ondel laboratori, la recerca i les AKRs. Jo vaig comen¸car just quan tu acabaves, per`ot'estic agra¨ıtper tots els protocols ensenyats, i a mirar a ojimetro la OD dels cultius. Espero haver estat un relleu a l’al¸cada,encara que ha estat dif´ıcil aix`o. I tamb´ea tots els padawans, m`asters,gent de pr`actiques, m`astersi lacais que han passat pel lab durant aquets anys, especialment els \mariquites" de l'Alvaro` i el Javi per les cervesetes compartides, la bona feina i encara m´esbon humor. La nostra amiga hongaresa Esther, l'Alba i 3 la seva manera curiosa de fer, l'Agrin, i tamb´ea en Roger, la Marina, la Natasha i l'Ignacio. Tamb´evull agrair a tota la gent del departament amb qui m´esho menys he compartit temps i feina a la torre. A tota la gent de la planta 3, les meves disculpes per apagar les llums del passad´ıs i sala de cultius! El grup germ`adels llevats, amb el Biosca i la Chari, gr`aciesper tots els consells i el bon humor, sempre disposats a ajudar en el que calgu`es.Als kinasics Fani i Jordi, especialment pels consells amb els westerns (el protocol que utilitzem ´esbasicament Made in kinasics) i les estones a la sala de cultius. No voldria deixar-me ning´udel departament amb qui he viscut bones experi`encies;Mohammed, Javi, Lu, Guillem, Salva, Magda, Santi, Laia, Jofre, Gisela, Andrea, Eva, Jordi, Valent´ın,Carla, Laura, Lucia, Irantzu, Inma, Alicia, Ana Paula, Edu... I a l'Helena per ser sempre tan alegre i positiva, pels consells donats, tooooooots els tampons i medis preparats, puntes posades i autoclavades, vialets netejats i per aguantar tamb´euns quants dies a les fosques. A l'Albert Suades, per les seves visites des de biof´ısica,els seus consells i el bon rollo. Iago, gran amic, ja company d'infortunis en el m`asteramb les puteines i company de pesars durant la tesi, estic segur que acabar`astrobant el que busques. Tenim pendents unes quantes idees de negoci per fer plegats, per quan el forn de pa/pastisseria a Tokyo o el nou producte revolucionari, la gayasa? Sento no haver-te visitat a Santander (potser en la defensa de la tesi?), per`ovas ser un magnific guia a Londres. Hem de fer una altre viatge a Jap´oplegats! ありがとうございます。 A les meves \Zorras", gr`aciesper les sessions frikis i les nits de vici, la gl`oriaobtinguda d'haver petat el Minecraft (tu no Andreu) per totes les birres gaudides en la vostra companyia, i les que ens queden! El businessman de l'Andreu, canviant el fosforito pel traje i ara el business casual, que arribar`aall`aon es proposi i tantes estones i aventures passades junts des de l'institut (i abans) (Mendoooosaaaaaaaaaaa). Marc, com han canviat les coses des de que et vaig tornar a con`eixera la granja... i mira't ara, casat amb l'Alba i amb un fill. Per sort tu segueixes sent el mateix, no canvi¨ıs mai! Moi, per obrir-me a un m´onencara m´esfriki, per ser com ets d'entusiasta, la nostra princesa friki del p`esol,gran persona i amic (Metaaaaaal GeeeaaAAAAAAAAAAAaaaar!! ). Hum- bert, per ser tan bona persona, bon amic, per estar sempre de bon humor, les lli¸consi morts al CoD (tot i haver perdut el mojo) i pel teu riure m`agic(Mmwwhhhoohhhhooohhooo, ya ves). Al David, per la teva amistat, i per totes les frikades, birres, i juergues passades, molta sort amb aquesta nova etapa que encetes! A tota la resta de la colla: l'Anna (p`adelteam!),la Mireia, l'Aza, el Vicente (moltes gr`aciesper aquesta gran portada!), el Sergi, l'Hugo, l'Alba, l'Alba i el Jordi, la Cris i el Ricard, l'Angel, el Carlos, l'Irene i el Mart´ı. Sou uns grans amics tots i m'alegro molt de ser part d'aquest magn´ıficgrup! A la Jenny, que tot i que est`asvoltant pel m´onsempre hi ets present. I per descomptat a la meva fam´ıliai especialment als meus pares, gr`aciesper haver-me aguantat tant de temps entendre ben b´efeia. Gr`aciesper confiar sempre en mi, aix`ono hauria estat possible sense vosaltres. 4 ~転3八起き。 \Fall seven times, stand up eight." Proverbi japon`es Functional and structural studies of AKR1B15 and AKR1B16: Two novel additions to human and mouse aldo-keto reductase superfamily Joan Gim´enezDejoz Abstract The aldo-keto reductase (AKR) superfamily comprises a large number of monomeric proteins with cytosolic localization and a molecular weight of about 36 kDa. AKRs have a common evo- lutionarily conserved protein fold which consists of a (α/β)8 barrel. Most AKRs act by reducing aldehydes and ketones to their respective alcohols in a NADPH-dependent reaction, with a broad spectrum of substrates ranging from simple sugars to potentially toxic aldehydes. This Thesis dissertation is a part of the structural and functional studies performed by our group on the characterization of AKRs, especially on their contribution in the biosynthesis pathway of retinoic acid (RA). Our group identified retinaldehyde reductase activity in the AKR family, and since then it has been involved in the deep functional and structural study of the human AKR proteins in relation to this important physiological role. We obtained the crystallographic structure of the highly efficient retinaldehyde reductase AKR1B10, a human enzyme overexpressed in cancer. A relationship was proposed between high retinaldehyde reductase activity and cancer development. The first part of the Thesis is based on the purification and characterization of a novel human AKR enzyme, AKR1B15, an enzyme sharing 92% sequence identity with AKR1B10. The recom- binant protein was expressed mostly in the insoluble fraction, showing limited activity when it was solubilized with detergents. We developed a novel approach in AKRs, using a coexpressing system with chaperones that was successful in increasing the amount of protein in the soluble fraction, in monomeric active form.
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