zoom presentazione per Japanese society of biochemistry

Ruolo delle modificazioni post-traduzionali mediante ADP-ribosilazione di proteine nei regni animale, vegetale e batterico

 reazione ciclica reversibile: poli-ADP-ribosilazione e degradazione del polimero da parte della Poli-ADP-ribosio-glicoidrolasi (PARG) (disegno del Prof. Hisanori Suzuki)

Relazione tra precezione di un patogeno, attivazione della risposta immune e ADP-ribosilazione

importanza del Nicotinamide-adenina-dinucleotide (NAD) come substrato di varie reazioni biochimiche, sia per gli enzimi ADP-ribosilanti che per i recettori di membrana di tipo nucleotide-binding leucine-rich repeat (NLR) receptor:  many blast resistance (R) genes have been identified, which mainly encode NLRs and confer limited race-specific resistance to the fungal pathogen

 Annual Meeting of The Japanese Biochemical Society  held in Yokohama on September 14th to 16th.

symposium on "Poly(ADP-ribosylation) pathway and metabolism" 

ADP-ribosyltransferase (ARTs) enzymes attach mono-ADP-ribose (MAR) or poly ADP-ribose (PAR) to target proteins, using nicotinamide adenine dinucleotide (NAD+) as a substrate. This is referred as a "writer" activity. The MARylation and PARylation of proteins exerts key roles in various biological processes.

In addition to covalent PARylation of target proteins, the interaction of proteins with PAR in noncovalent manner triggers the formation of protein complexes (interactomes), participating to various biological processes. The recognition of PAR, MAR and iso-ADPr by binding proteins is referred as a "Reader" activity.

Finally, the degradation of PAR and MAR is performed by eraser enzymes, poly ADP ribose glycohydrolases, and ADP-ribosyl acceptor hydrolases, ARH1 and ARH3, in humans.

Sirtuins are NAD-dependent histone deacetylases (HDACs). Some sirtuin perform different enzymatic activities using NAD+ as a substrate: it is the case of sirtuins with ADP-ribosylating activity, producing MARylated proteins. This activity can be found in various organisms, as in vertebrates, lower eukaryotes, and plants. 

Bacteria produce virulence factors with ADP-ribosylating activity, important in human diseases (toxins) and plant immune response (effectors).

Bacteria possess, in addition to ART toxins, sirtuin enzymes (SirTs) with ART activity, that are involved in the control of lypoate: the SirTM operon (coding for a sirtuin and for a macro-domain) is flanked by two ORFs, the glycine cleavage system H-like (GcvH-L), coding for proteins involved in glycine detoxification, and lipoate-protein ligase homolog (LplA2), coding for an enzyme scavenging the lipoate cofactor. GCVH-L is ADP ribosylated when in the lipoylated form.

In viruses (flaviviruses, Coronaviruses), Macro-domain proteins bind to ADP-ribose and counteract ADP-ribosylation signals in host defence against viruses, altering the stress granule formation, and metabolism of ADP-ribose.

In plants, there are three PARP proteins and several PARP-domain proteins, such as radical-induced cell death 1 (RCD1) and Similar to RCD One (SRO) homologues. DAWDLE (DDL), a Forkhead associated (FHA) domain protein, binding to PAR and to phosphothreonine, is involved in plant immunity and in miRNA biogenesis: in vivo PARylation of DDL is important in plant immune response to pathogens.

Regarding ARTs in human genome, Parp9 forms heterodimers with Deltex homolog Dtx3L, a histone ubiquitin (Ub) E3 ligase: Dtx3L/Parp9 ADP-ribosylates the carboxyl group of Gly76 in ubiquitin, while E3 ligase adds  the carboxyl group of the C-terminal glycine 76 from ubiquitin to the epsilon-amino group of a lysine in the substrate. Parp9 ADP-ribosylation activity therefore restrains the E3 function of Dtx3L, while poly(ADP-ribose) binding to Parp9 increases E3 activity.

As for cytoplasmic PARP5a/b, tankyrases, synthesizing linear PAR, authors found Poly(ADP-Ribose)-dependent ubiquitination (PARdU).

In the WNT signaling pathway, degradation of Axin1/2 is mediated by the E3 ligase RING finger protein 146 (RNF146), which is activated by binding of PAR. The substrates of PARP5a/b, such as Axin1/2, possess an amino acid motif defined as the “Tankyrase binding motif” (TBM), shown to be critical for PARdU. RNF146 and PARP5a/b are substrates of one another. Ubiquitin itself is ADP-ribosylated, which has been shown to block subsequent attachment of ubiquitin to target proteins. On the other hand, PARP5a/b ADP-ribosylates the proteasome regulator PI31, promoting the assembly of 26S proteasome.

PARP1 PARylation is required for RNF146-mediated ubiquitination of PARP1, and RNF146 PAR binding ability is required to decrease PARP1 protein levels. ADP-ribosylation-dependent ubiquitination mechanism of pathogenesis has been shown important in Legionella pneumophila. The SidE family of proteins (SdeA, SidE, SdeB and SdeC) ubiquitylates ER-associated Rab small GTPases and Rtn4, and is NAD+ dependent, to ADP-ribosylate and activate ubiquitin for subsequent conjugation. Once ADP-ribosylated ubiquitin is generated by the mART domain, the modified ubiquitin is utilized by a phosphodiesterase (PDE) domain within SidE proteins to complete the ubiquitylation of target substrates on serine.

Several molecular mechanisms of PARPs enzymes in inflammation- and metabolic-related diseases have been proposed.

targeting PARP has potential therapeutic applications in several diseases associated with inflammation and metabolism. PARPs also regulate the unfolded protein response (UPR) of the endoplasmic reticulum (ER). PARP16 (also known as ARTD15) is a tail-anchored ER protein, which is inserted into the ER membrane. It appears that both PARP16 itself and its catalytic activity are required for the ER stress responses by regulating the UPR signaling pathway. Stress granules contain RISC complexes processing microRNAs. On the other side, few microRNAs can regulate some PARP family member. In peripheral blood, neutrophils express miR-659, that targets among other proteins also PARP16.

ARTCs (Cholera toxin-like ARTs) are characterised by the R-S-E triad in the catalytic domain, typical of arginine specific ARTs, while Diphteria toxin-like ARTs (ARTD) have  histidine within the catalytic triad. hARTC1 protein localized to a tubular membrane network consistent with the ER, as they co-localized with typical ER marker proteins such as calnexin, calreticulin, PDI and GRP78/BiP, ARTC1 localises in the endoplasmic reticulum and, by modifying GRP78/BiP, functions in Endoplamsic Reticulum (ER) stress.

Various studies revealed the arginine-ADPr modification of soluble intracellular proteins (e.g. GAPDH, G protein beta/gamma subunit, Rabs, and tubulin), suggesting that mammalian ARTCs also regulates intracellular functions. Signals from the extracellular matrix, transduced by integrins, control cell growth, shape, migration and differentiation, and regulate the epithelial-mesenchymal transition (EMT). The selective expression of ARTC1 and integrin a7 in both cardiac and skeletal muscle, their similar developmental appearance, and the specific ADP-ribosylation, are consistent with the regulatory association between these two proteins. It has been suggested that mono-ADP-ribosylation of integrin a7 may modulate receptor signaling and could play a significant role in cell adhesion and signal transduction. A role of ARTC1 in tumor angiogenesis has also been suggested based on the demonstration that ART1 upregulates HIF-1a through the PI3K/ Akt signaling pathway, promote the expression of angiogenic factors such as VEGF and bFGF.

Finally, clinical trials are testing PARP1 inhibitors for DNA repair defective tumors, as in ovary and breast cancer, challenged with NAD-dependent PARP-1 inhibitors, such as olaparib, veliparib, and rucaparib. These compounds are prone to P-glycoprotein-mediated efflux and resistance induction. AZD2461 is an inhibitor less affected by this mechanism. Among the non-NAD-like inhibitors, for prostate cancer therapy, are histone-dependent 5F02 and analogs. 

It is expected further advancement in therapies able to target specific members of ARTs, or to address them to localise to the organ or cancer, delivering them in a specific manner (such as embedded in liposomes, and locally released due to pH changes).