PBX1 (PreB-cell leukemia homeobox 1), PREP1 (PBX regulating protein 1), and MEIS1 (Myeloid ecotropic insertion site 1) are sub-families of TALE (Three Amino Acid Loop Extension) homeodomain transcription factors; they are essential in embryonic development and are also involved in adult physiological function and some pathological conditions. Both MEIS1 and PREP1 competitively heterodimerize with PBX1 to generate a complex able to translocate to the nucleus and act as a transcription factor binding DNA. These three TALE protein sub-families contain a DNA-binding homeodomain (HD) towards the C-terminus required for DNA binding; in addition, MEIS1 and PREP1 have similar high conserved sequences in the HR1 and HR2 regions at the N-terminal, involved in the interaction surface with PBX1. The heterodimers PREP1-PBX1 and MEIS1-PBX1 have opposite roles, depending on the transcriptional partner: PREP1 exerts a tumour-suppressive function and protects cells from DNA damage; in mouse and human cells in culture, Prep1 deficiency leads to genomic instability and conditions that favour tumorigenesis; MEIS1 acts as an oncogene, its overexpression induces generation of human and murine leukemias and tumors in general. MEIS1 requires dimerization with PBX1 to reach the nucleus; when MEIS1 is not in complex with PBX1 it is destabilized and degraded by the proteasome pathway. Therefore, we infected immortalized mouse embryonic fibroblast (in-MEFs) PREP1i/i cells with retroviral vectors encoding wild-type MEIS1 or two HR mutants unable to bind PBX1 and therefore translocate to the nucleus. Nanobodies that bind the regions involved in the binding of MEIS1-PBX1 are generated to avoid the complex formation or dissociate it when it is already formed; these were generated by immunizing two lamas with the MEIS1 (63-168) fragments and characterized in previous studies. The project aimed to engineer and test a nanobody that was able to inhibit the complex formation in vitro to make it able to cross the cellular membrane and perform its pharmacological activity. To verify the ability of MEIS1 to reach the nucleus when it binds PBX1, we first evaluated the formation of the MEIS1-PBX1 complex by immunofluorescence, comparing the cellular localization of wild-type MEIS1 with MEIS1 mutated in HR1 or HR2. In the presence of these mutations, MEIS1 cannot bind PBX1 and translocate to the nucleus; moreover, mutated MEIS1 has a shorter half-life than the wild-type. These results show the possibility of the HR1 and HR2 domains being an effective pharmacological target; therefore, we tested previously identified nanobodies to bind the HR1 and HR2 domains of MEIS1. In a previous study, the CA13279 nanobody was the most effective in preventing the binding interaction between MEIS1 and PBX1. Hence, the nanobody was engineered (NB79R) by modifying the surface charge to allow cell penetration. In vitro affinity studies result in the capacity of the NB79R to inhibit the formation of the MEIS1-PBX1 bond in vitro with good efficiency, close to that of the unmodified CA13279 nanobody. However, NB79R placed into immortalized mouse embryonic fibroblasts cannot prevent translocation of MEIS1 within the nucleus, probably due to its inability to cross cell membrane efficiently. We engineered a biological inhibitor of the MEIS1-PBX1 complex to make it able to cross the cell membrane and perform its pharmacological function in the cytoplasm. Although the engineering of the molecule was successful, further optimization of the molecule to make it more cell permeable is required.
PBX1 (PreB-cell leukemia homeobox 1), PREP1 (PBX regulating protein 1) e MEIS1 (Myeloid ecotropic insertion site 1) fanno parte della famiglia delle proteine TALE (Three Amino Acid Loop Extension), sono proteine essenziali nello sviluppo embrionale, ma anche coinvolte in funzioni fisiologiche adulte e alcune condizioni patologiche. MEIS1 e PREP1 competono per il legame con PBX1, quindi, per la formazione di un eterodimero MEIS1-PBX1 o PREP1-PBX1 in grado di traslocare nel nucleo e fungere da fattore trascrizionale legandosi al DNA. MEIS1 e PREP1 condividono sequenze altamente conservate quasi identiche: all’N-terminale le regioni HR1 e HR2, responsabili dell’interazione con PBX1, e nell’omeodominio (HD) richiesto per il legame al DNA. Fisiologicamente questi due fattori di trascrizione hanno una funzione opposta: PREP1 è un oncosoppressore, coinvolto nel controllo dell’attività trascrizionale di MEIS1 attraverso la competizione con esso per il legame con PBX1; è stato dimostrato che nelle colture cellulari umane e murine, la deficienza di Prep1 porta all’instabilità genomica e all’accumulo di danni al DNA, quindi, condizioni che favoriscono lo sviluppo di tumori; MEIS1 è un protooncogene, la cui sovraespressione determina un’attività tumorigenica, ma solo in assenza di PREP1 o se sono attivi altri oncogeni. Tuttavia, quando MEIS1 non è legato a PBX1 non può completare il suo ruolo oncogenico, in quanto non è in grado di raggiungere il nucleo. Lo scopo del progetto è stato di ingegnerizzare e testare una molecola biologica (“nanobody”), la quale era risultata in grado di inibire in vitro la formazione del complesso MEIS1-PBX1, per renderla in grado di attraversare la membrana cellulare per esplicare la propria azione farmacologica. Per verificare la capacità di MEIS1 di raggiungere il nucleo, una volta legato a PBX1, abbiamo innanzitutto valutato la formazione del complesso MEIS1-PBX1 attraverso immunofluorescenza, confrontando la localizzazione cellulare di MEIS1 “wild-type” con MEIS1 mutato nei domini HR1 o HR2. In presenza di queste mutazioni, MEIS1 non è in grado di legare PBX1 e, quindi, di traslocare nel nucleo; inoltre, MEIS1 mutato presenta un’emivita ridotta rispetto al “wild-type”. Verificato pertanto che i domini HR1 e HR2 possono essere un efficace bersaglio farmacologico, abbiamo testato delle molecole biologiche (“nanobodies”) che erano state precedentemente selezionate per la loro capacità di legarsi ai domini HR1 e HR2 di MEIS1. In uno studio precedente, era stato individuato un nanobody (NB79) che mostrava un’alta affinità di legame per MEIS1, in grado di impedire in vitro la formazione del complesso MEIS1-PBX1. Il nanobody è stato, quindi, ingegnerizzato (NB79R) attraverso la modifica delle cariche superficiali, per renderlo in grado di attraversare la membrana cellulare. Mediante studi di affinità in vitro, il NB79R è risultato in grado di inibire la formazione del legame MEIS1-PBX1 in vitro, con un’efficienza paragonabile a quella del nanobody non modificato. Tuttavia, il NB79R inserito in fibroblasti embrionali di topo immortalizzati non è stato in grado di impedire la traslocazione di MEIS1 all’interno del nucleo, probabilmente, a causa della sua incapacità di attraversare in modo efficiente la membrana cellulare. Abbiamo ingegnerizzato un inibitore biologico del complesso MEIS1-PBX1 per renderlo in grado di attraversare la membrana cellulare e di espletare la propria funzione farmacologica nel citoplasma. Sebbene l’ingegnerizzazione della molecola sia riuscita con successo, è necessaria un’ulteriore ottimizzazione della molecola per renderla più permeabile alla membrana cellulare.
Studi sulla localizzazione intracellulare di Meis1 e caratterizzazione di un nanobody a superficie modificata contro Meis1
SERRICCHIO, ILARIA
2021/2022
Abstract
PBX1 (PreB-cell leukemia homeobox 1), PREP1 (PBX regulating protein 1), and MEIS1 (Myeloid ecotropic insertion site 1) are sub-families of TALE (Three Amino Acid Loop Extension) homeodomain transcription factors; they are essential in embryonic development and are also involved in adult physiological function and some pathological conditions. Both MEIS1 and PREP1 competitively heterodimerize with PBX1 to generate a complex able to translocate to the nucleus and act as a transcription factor binding DNA. These three TALE protein sub-families contain a DNA-binding homeodomain (HD) towards the C-terminus required for DNA binding; in addition, MEIS1 and PREP1 have similar high conserved sequences in the HR1 and HR2 regions at the N-terminal, involved in the interaction surface with PBX1. The heterodimers PREP1-PBX1 and MEIS1-PBX1 have opposite roles, depending on the transcriptional partner: PREP1 exerts a tumour-suppressive function and protects cells from DNA damage; in mouse and human cells in culture, Prep1 deficiency leads to genomic instability and conditions that favour tumorigenesis; MEIS1 acts as an oncogene, its overexpression induces generation of human and murine leukemias and tumors in general. MEIS1 requires dimerization with PBX1 to reach the nucleus; when MEIS1 is not in complex with PBX1 it is destabilized and degraded by the proteasome pathway. Therefore, we infected immortalized mouse embryonic fibroblast (in-MEFs) PREP1i/i cells with retroviral vectors encoding wild-type MEIS1 or two HR mutants unable to bind PBX1 and therefore translocate to the nucleus. Nanobodies that bind the regions involved in the binding of MEIS1-PBX1 are generated to avoid the complex formation or dissociate it when it is already formed; these were generated by immunizing two lamas with the MEIS1 (63-168) fragments and characterized in previous studies. The project aimed to engineer and test a nanobody that was able to inhibit the complex formation in vitro to make it able to cross the cellular membrane and perform its pharmacological activity. To verify the ability of MEIS1 to reach the nucleus when it binds PBX1, we first evaluated the formation of the MEIS1-PBX1 complex by immunofluorescence, comparing the cellular localization of wild-type MEIS1 with MEIS1 mutated in HR1 or HR2. In the presence of these mutations, MEIS1 cannot bind PBX1 and translocate to the nucleus; moreover, mutated MEIS1 has a shorter half-life than the wild-type. These results show the possibility of the HR1 and HR2 domains being an effective pharmacological target; therefore, we tested previously identified nanobodies to bind the HR1 and HR2 domains of MEIS1. In a previous study, the CA13279 nanobody was the most effective in preventing the binding interaction between MEIS1 and PBX1. Hence, the nanobody was engineered (NB79R) by modifying the surface charge to allow cell penetration. In vitro affinity studies result in the capacity of the NB79R to inhibit the formation of the MEIS1-PBX1 bond in vitro with good efficiency, close to that of the unmodified CA13279 nanobody. However, NB79R placed into immortalized mouse embryonic fibroblasts cannot prevent translocation of MEIS1 within the nucleus, probably due to its inability to cross cell membrane efficiently. We engineered a biological inhibitor of the MEIS1-PBX1 complex to make it able to cross the cell membrane and perform its pharmacological function in the cytoplasm. Although the engineering of the molecule was successful, further optimization of the molecule to make it more cell permeable is required.È 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/20.500.14239/14557