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Title: Identification and roles of cell substrates of intracellular mono-ADP-ribosylation
Author: Grimaldi, Giovanna
Awarding Body: Open University
Current Institution: Open University
Date of Award: 2011
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Mono-ADP-ribosylation is a reversible post-translational modification that is catalysed by either bacterial pathogenic toxins or endogenous cellular ADP- ribosyltransferases (ARTs), and that can modulate the functions of target proteins. This latter aspect has only recently emerged as an important mechanism in mammalian cells for the control of a vast array of cellular processes. Some of the members of the poly-ADP-ribosyl-polymerase (PARP) family are among the mammalian enzymes that can catalyse mono-ADP-ribosylation. Here, based on biochemical data, I provide evidence that the P ARP family member P ARP 12 is a mono-ART (mART) that can modify acidic amino acid residues. Using an affinity purification method based on a protein module that recognises ADP-ribosylated proteins (the macro domain), together with liquid chromatography coupled to tandem mass spectrometry, I have identified 25 putative PARP12 substrates that are involved in different cellular processes. The identification of this range of substrates suggests that P ARP 12 is involved in multiple cellular functions, ranging from regulation of small-GTPases to the control of newly synthesized RNA. To determine its biological role, I analysed its intracellular localisation. Data reported here show that P ARPl2 is a mART that is localised tothe Golgi complex, where it can use the NAD+ pool present on this organelle to modify Golgi-localised proteins. In line with these findings, among the PARP12 substrates, I identified Rab1A, a small GTPase that controls both . transport from the endoplasmic reticulum to the Golgi- complex, and the structure of the Golgi itself My data suggest a role for PARP12 in maintenance of Golgi structure, potentially through ADP-ribosylation of RablA. A further aspect I have analysed is the ADP-ribosylation of the dual function protein C-terrninal binding protein 11 BFA- dependent ADP-ribosylated substrate (CtBP1IBARS) as a mechanism of action of the toxin brefeldin A (BFA). BFA-induced ADP-ribosylation of CtBP1IBARS occurs through a novel mechanism that comprises two steps: (i) synthesis of a BF A-ADP- ribose conjugate (BAC); and (ii) covalent binding of the BAC into the NAD+ -binding pocket of CtBPIIBARS. Here, I demonstrate that this reaction requires the involvement of ADP-ribosyl-cyc1ases, such as CD38. Importantly, this modification abolishes CtBPIIBARS fissioning activity, while it increases CtBPIIBARS co- repressor activity, in a promoter-dependent context. As with sumoylation and phosphorylation, ADP-ribosylation thus represents a new mechanism for the regulation of the functions ofCtBPlIBARS.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available