Lamin A is one of the major splicing products of the LMNA gene, a gene involved in a group of rare diseases collectively known as laminopathies. Lamin A is a main constituent of the nuclear lamina, a protein meshwork located below the inner nuclear membrane. The nuclear lamina is involved in several functions, such as maintaining the nuclear shape, cooperating to mechanosignaling transduction and gene regulation. The role of lamin A in stress response is particularly intriguing, yet not fully elucidated, and involves lamin A post-translational processing. Mature lamin A is generated due to post-translational modifications of its precursor known as prelamin A. Four different molecular species are generated including non-farnesylated and farnesylated prelamin A forms. While prelamin A undergoes fast and efficient maturation under basal conditions, prelamin A accumulation occurs during ageing-related processes, as, for example, stress response. Further, LMNA gene mutations may affect prelamin A processing leading to the accumulation of lamin A precursors and causing progeroid laminopathies. The hypothesis behind the present research project is that different prelamin A forms modulate oxidative stress response in cells by differentially recruiting enzymes or other molecules involved in DNA damage repair (DDR), such as 53BP1. 53BP1 is an important nuclear protein with a pivotal role in the Non-Homologous End-Joining repair pathway (NHEJ). In physiological conditions, 53BP1 is diffused in the nucleoplasm, whereas, when DNA damage occurs, it localizes in foci at DNA break sites. Lamin A/C can bind 53BP1 under physiological conditions, helping its nuclear recruiting. We induced the accumulation of various prelamin A forms in human skin fibroblasts through in vitro treatments or DNA plasmid transfections. Cells were subjected to oxidative stress and the accumulation of prelamin A forms and the generation of 53BP1 foci were investigated. We further investigated the molecular interaction between mature lamin A and 53BP1 in cells that accumulated different prelamin A forms. As a whole, our results show that non-farnesylated and farnesylated prelamin A differently influence recruitment of 53BP1 to DNA damage foci. This finding helps to explain the role of lamin A in the stress response and has important implications for therapeutic options exploiting prelamin A inhibitors in laminopathic and non-laminopathic patients.
La lamina A è uno dei principali prodotti di splicing del gene LMNA, un gene coinvolto in un gruppo di malattie rare note come laminopatie. La lamina A è un costituente principale della lamina nucleare, un reticolo proteico situato al di sotto della membrana nucleare interna. La lamina nucleare è coinvolta in diverse funzioni, come mantenere la forma nucleare, cooperare alla trasduzione del mechanosignaling e alla regolazione genica. Il ruolo della lamina A nella risposta allo stress è particolarmente intrigante, ma non del tutto chiarito, e coinvolge l'elaborazione post-traduzionale della lamina A. La lamina A matura viene generata grazie a modifiche post-traduzionali del suo precursore, noto come prelamina A. Vengono generate quattro diverse molecole in questo processo, tra cui le forme di prelamina A non farnesilata e farnesilata. Mentre la prelamina A subisce una maturazione rapida ed efficiente in condizioni basali, l'accumulo di prelamina A si verifica durante i processi legati all'invecchiamento, come, ad esempio, la risposta allo stress. Inoltre, le mutazioni del gene LMNA possono influenzare il processing della prelamina A portando all'accumulo di precursori della lamina A e causando le laminopatie progeroidi. L'ipotesi alla base del presente progetto di ricerca è che diverse forme di prelamina A modulino la risposta allo stress ossidativo nelle cellule reclutando in modo differenziale enzimi o altre molecole coinvolte nella riparazione del danno al DNA (DDR), come 53BP1. 53BP1 è un'importante proteina nucleare con un ruolo fondamentale nel Non-Homologous End-Joining (NHEJ). In condizioni fisiologiche, 53BP1 è diffusa nel nucleoplasma, mentre, quando si verifica un danno al DNA, si localizza ai siti di danno al DNA. Lamin A/C può legare 53BP1 in condizioni fisiologiche, aiutando il suo recruiting all’interno del nucleo. Abbiamo indotto l'accumulo di varie forme di prelamina A nei fibroblasti cutanei umani attraverso trattamenti in vitro o trasfezioni di DNA plasmidici. Le cellule sono state sottoposte a stress ossidativo e abbiamo indagato l'accumulo di forme di prelamina A e la generazione di foci di 53BP1. Inoltre, abbiamo studiato l'interazione molecolare tra la lamina A matura e la 53BP1 nelle cellule che hanno accumulato diverse forme di prelamina A. Nel complesso, i nostri risultati mostrano che la prelamina A non farnesilata e farnesilata influenzano in modo diverso il reclutamento di 53BP1 nei foci di danno. Questa scoperta aiuta a spiegare il ruolo della lamina A nella risposta allo stress e ha importanti implicazioni per le opzioni terapeutiche che sfruttano gli inibitori della prelamina A nei pazienti laminopatici e non laminopatici.
Ruolo fisiologico e patologico della prelamina A nella risposta cellulare allo stress in fibroblasti cutanei umani
DI GIAMPIETRO, MARIA LETIZIA
2020/2021
Abstract
Lamin A is one of the major splicing products of the LMNA gene, a gene involved in a group of rare diseases collectively known as laminopathies. Lamin A is a main constituent of the nuclear lamina, a protein meshwork located below the inner nuclear membrane. The nuclear lamina is involved in several functions, such as maintaining the nuclear shape, cooperating to mechanosignaling transduction and gene regulation. The role of lamin A in stress response is particularly intriguing, yet not fully elucidated, and involves lamin A post-translational processing. Mature lamin A is generated due to post-translational modifications of its precursor known as prelamin A. Four different molecular species are generated including non-farnesylated and farnesylated prelamin A forms. While prelamin A undergoes fast and efficient maturation under basal conditions, prelamin A accumulation occurs during ageing-related processes, as, for example, stress response. Further, LMNA gene mutations may affect prelamin A processing leading to the accumulation of lamin A precursors and causing progeroid laminopathies. The hypothesis behind the present research project is that different prelamin A forms modulate oxidative stress response in cells by differentially recruiting enzymes or other molecules involved in DNA damage repair (DDR), such as 53BP1. 53BP1 is an important nuclear protein with a pivotal role in the Non-Homologous End-Joining repair pathway (NHEJ). In physiological conditions, 53BP1 is diffused in the nucleoplasm, whereas, when DNA damage occurs, it localizes in foci at DNA break sites. Lamin A/C can bind 53BP1 under physiological conditions, helping its nuclear recruiting. We induced the accumulation of various prelamin A forms in human skin fibroblasts through in vitro treatments or DNA plasmid transfections. Cells were subjected to oxidative stress and the accumulation of prelamin A forms and the generation of 53BP1 foci were investigated. We further investigated the molecular interaction between mature lamin A and 53BP1 in cells that accumulated different prelamin A forms. As a whole, our results show that non-farnesylated and farnesylated prelamin A differently influence recruitment of 53BP1 to DNA damage foci. This finding helps to explain the role of lamin A in the stress response and has important implications for therapeutic options exploiting prelamin A inhibitors in laminopathic and non-laminopathic patients.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/20.500.14239/13403