Study of the Cellular Role of GRP78/BiP mono-ADP-ribosylation in UPR and Cancer

Fabrizio, Gaia (2018). Study of the Cellular Role of GRP78/BiP mono-ADP-ribosylation in UPR and Cancer. PhD thesis The Open University.



Mono-ADP-ribosylation is a reversible post-translational protein modification that modulates the function of proteins involved in different cellular processes, including signal transduction, protein transport, transcription, cell cycle regulation, DNA (deoxyribonucleic acid) repair and apoptosis. In mammals, mono-ADPribosylation is catalyzed by three different classes of enzymes: ARTCs, ARTDs, and members of the sirtuin family.

In the present study, hARTC1-mediated mono-ADP-ribosylation was investigated in terms of the cellular compartments involved, target(s) and roles. The collected results demonstrated that hARTC1 protein and enzymatic activity is mainly localized to the endoplasmic reticulum (ER), in contrast to other ARTCs, which are either typically GPI-anchored enzymes in the plasma membrane, or secreted enzymes. Previous studies in my laboratory demonstrated that a protein macro domain was useful for the study of APD-ribosylation. The data reported here indicate, for the first time, that the macro domain can be used for immunofluorescence, allowing visualization of ADP-ribosylated proteins in intact cells, and in far-Western Blotting, allowing the detection of specific ADPribosylated targets. These methodologies were employed to demonstrate that the ER-localized chaperone, GRP78/BiP, was a prime target of hARTC1. A doubly mutated hARTC1 mutant was designed, and used as a specific control for hARTC1 expression. The mutant enzyme localized to the ER, but did not catalyze GRP78/BiP ADP-ribosylation.

The demonstration that GRP78/BiP was mono-ADP-ribosylated by hARTC1 suggested that hARTC1 could be a key regulator of GRP78/BiP-mediated functions. Consistent with the key role of GRP78/BiP in the ER stress response, it was found that hARTC1 was activated during short-term cell treatment with ER stressors, resulting in acute GRP78/BiP ADP-ribosylation. However, the monoADP-ribosylation of the chaperone did not trigger an unfolded protein response. Recently, hARTC1 has been associated with cancer, suggesting a possible role in cell proliferation. In line with these findings, the results presented here demonstrated that hARTC1 over-expression inhibited cell proliferation.

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