Identification and Roles of Cell Substrates of Intracellular Mono-ADP-Ribosylation

Grimaldi, Giovanna (2012). Identification and Roles of Cell Substrates of Intracellular Mono-ADP-Ribosylation. PhD thesis The Open University.



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 PARP family member PARP12 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 PARP12 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 PARP12 is a mART that is localised to the 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 Rab1A. A further aspect I have analysed is the ADP-ribosylation of the dual function protein C-terminal binding protein 1/BFA- dependent ADP-ribosylated substrate (CtBP1/BARS) as a mechanism of action of the toxin brefeldin A (BFA). BFA-induced ADP-ribosylation of CtBP1/BARS 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 CtBP1/BARS. Here, I demonstrate that this reaction requires the involvement of ADP-ribosyl-cyclases, such as CD38. Importantly, this modification abolishes CtBPIIBARS fissioning activity, while it increases CtBP1/BARS 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 of CtBP1/BARS.

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