PARP10 Mediates Mono-ADP-Ribosylation of Aurora-A Regulating G2/M Transition of the Cell Cycle.

Intracellular mono-ADP-ribosyltransferases (mono-ARTs) catalyze the covalent attachment of a single ADP-ribose molecule to protein substrates, thus regulating their functions. PARP10 is a soluble mono-ART involved in the modulation of intracellular signaling, metabolism and apoptosis. PARP10 also participates in the regulation of the G1- and S-phase of the cell cycle. However, the role of this enzyme in G2/M progression is not defined. In this study, we found that genetic ablation, protein depletion and pharmacological inhibition of PARP10 cause a delay in the G2/M transition of the cell cycle. Moreover, we found that the mitotic kinase Aurora-A, a previously identified PARP10 substrate, is actively mono-ADP-ribosylated (MARylated) during G2/M transition in a PARP10-dependent manner. Notably, we showed that PARP10-mediated MARylation of Aurora-A enhances the activity of the kinase in vitro. Consistent with an impairment in the endogenous activity of Aurora-A, cells lacking PARP10 show a decreased localization of the kinase on the centrosomes and mitotic spindle during G2/M progression. Taken together, our data provide the first evidence of a direct role played by PARP10 in the progression of G2 and mitosis, an event that is strictly correlated to the endogenous MARylation of Aurora-A, thus proposing a novel mechanism for the modulation of Aurora-A kinase activity.

PKD-dependent PARP12-catalyzed mono-ADP-ribosylation of Golgin-97 is required for E-cadherin transport from Golgi to plasma membrane.

Adenosine diphosphate (ADP)-ribosylation is a posttranslational modification involved in key regulatory events catalyzed by ADP-ribosyltransferases (ARTs). Substrate identification and localization of the mono-ADP-ribosyltransferase PARP12 at the trans-Golgi network (TGN) hinted at the involvement of ARTs in intracellular traffic. We find that Golgin-97, a TGN protein required for the formation and transport of a specific class of basolateral cargoes (e.g., E-cadherin and vesicular stomatitis virus G protein [VSVG]), is a PARP12 substrate. PARP12 targets an acidic cluster in the Golgin-97 coiled-coil domain essential for function. Its mutation or PARP12 depletion, delays E-cadherin and VSVG export and leads to a defect in carrier fission, hence in transport, with consequent accumulation of cargoes in a trans-Golgi/Rab11-positive intermediate compartment. In contrast, PARP12 does not control the Golgin-245-dependent traffic of cargoes such as tumor necrosis factor alpha (TNFα). Thus, the transport of different basolateral proteins to the plasma membrane is differentially regulated by Golgin-97 mono-ADP-ribosylation by PARP12. This identifies a selective regulatory mechanism acting on the transport of Golgin-97- vs. Golgin-245-dependent cargoes. Of note, PARP12 enzymatic activity, and consequently Golgin-97 mono-ADP-ribosylation, depends on the activation of protein kinase D (PKD) at the TGN during traffic. PARP12 is directly phosphorylated by PKD, and this is essential to stimulate PARP12 catalytic activity. PARP12 is therefore a component of the PKD-driven regulatory cascade that selectively controls a major branch of the basolateral transport pathway. We propose that through this mechanism, PARP12 contributes to the maintenance of E-cadherin-mediated cell polarity and cell-cell junctions.

ADP-ribosyltransferases, an update on function and nomenclature.

ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD+ onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.

Targeting ADP-ribosylation as an antimicrobial strategy.

ADP-ribosylation (ADPr) is an ancient reversible modification of cellular macromolecules controlling major biological processes as diverse as DNA damage repair, transcriptional regulation, intracellular transport, immune and stress responses, cell survival and proliferation. Furthermore, enzymatic reactions of ADPr are central in the pathogenesis of many human diseases, including infectious conditions. By providing a review of ADPr signalling in bacterial systems, we highlight the relevance of this chemical modification in the pathogenesis of human diseases depending on host-pathogen interactions. The post-antibiotic era has raised the need to find alternative approaches to antibiotic administration, as major pathogens becoming resistant to antibiotics. An in-depth understanding of ADPr reactions provides the rationale for designing novel antimicrobial strategies for treatment of infectious diseases. In addition, the understanding of mechanisms of ADPr by bacterial virulence factors offers important hints to improve our knowledge on cellular processes regulated by eukaryotic homologous enzymes, which are often involved in the pathogenesis of human diseases.

PARPs and PAR as novel pharmacological targets for the treatment of stress granule-associated disorders.

Among the post-translational modifications, ADP-ribosylation has been for long time the least integrated in the scheme of the structural protein modifications affecting physiological functions. In spite of the original findings on bacterial-dependent ADP-ribosylation catalysed by toxins such as cholera and pertussis toxin, only with the discovery of the poly-ADP-ribosyl polymerase (PARP) family the field has finally expanded and the role of ADP-ribosylation has been recognised in both physiological and pathological processes, including cancer, infectious and neurodegenerative diseases. This is now a rapidly expanding field of investigation, centred on the role of the different PARPs and their substrates in various diseases, and on the potential of PARP inhibitors as novel pharmacological tools to be employed in relevant pathological context. In this review we analyse the role that members of the PARP family and poly-ADP-ribose (PAR; the product of PARP1 and PARP5a activity) play in the processes following the exposure of cells to different stresses. The cell response that arises following conditions such as heat, osmotic, oxidative stresses or viral infection relies on the formation of stress granules, which are transient cytoplasmic membrane-less structures, that include untranslated mRNA, specific proteins and PAR, this last one serving as the "collector" of all components (that bind to it in a non-covalent manner). The resulting phenotypes are cells in which translation, intracellular transport or pro-apoptotic pathways are reversibly inhibited, for the time the given stress holds. Interestingly, the formation of defective stress granules has been detected in diverse pathological conditions including neurological disorders and cancer. Analysing the molecular details of stress granule formation under these conditions offers a novel view on the pathogenesis of these diseases and, as a consequence, the possibility of identifying novel drug targets for their treatment.

ADP-ribosylation and intracellular traffic: an emerging role for PARP enzymes.

ADP-ribosylation is an ancient and reversible post-translational modification (PTM) of proteins, in which the ADP-ribose moiety is transferred from NAD+ to target proteins by members of poly-ADP-ribosyl polymerase (PARP) family. The 17 members of this family have been involved in a variety of cellular functions, where their regulatory roles are exerted through the modification of specific substrates, whose identification is crucial to fully define the contribution of this PTM. Evidence of the role of the PARPs is now available both in the context of physiological processes and of cell responses to stress or starvation. An emerging role of the PARPs is their control of intracellular transport, as it is the case for tankyrases/PARP5 and PARP12. Here, we discuss the evidence pointing at this novel aspect of PARPs-dependent cell regulation.

In Vitro Techniques for ADP-Ribosylated Substrate Identification.

ADP-ribosylation is a post-translational modification of proteins that has required the development of specific technical approaches for the full definition of its physiological roles and regulation. The identification of the enzymes and specific substrates of this reaction is an instrumental step toward these aims. Here we describe a method for the separation of ADP-ribosylated proteins based on the use of the ADP-ribose-binding macro domain of the thermophilic protein Af1521, coupled to mass spectrometry analysis for protein identification. This method foresees the coupling of the macro domain to resin, an affinity-based pull-down assay, coupled to a specificity step resulting from the clearing of cell lysates with a mutated macro domain unable to bind ADP-ribose. By this method both mono- and poly-ADP-ribosylated proteins have been identified.

PARP1-produced poly-ADP-ribose causes the PARP12 translocation to stress granules and impairment of Golgi complex functions.

Poly-ADP-ribose-polymerases (PARPs) 1 and 2 are nuclear enzymes that catalyze the poly-ADP-ribosylation of nuclear proteins transferring poly-ADP-ribose (PAR) polymers to specific residues. PARPs and PAR intervene in diverse functions, including DNA repair in the nucleus and stress granule assembly in the cytoplasm. Stress granules contribute to the regulation of translation by clustering and stabilizing mRNAs as well as several cytosolic PARPs and signaling proteins to modulate cell metabolism and survival. Our study is focused on one of these PARPs, PARP12, a Golgi-localized mono-ADP-ribosyltransferase that under stress challenge reversibly translocates from the Golgi complex to stress granules. PARP1 activation and release of nuclear PAR drive this translocation by direct PAR binding to the PARP12-WWE domain. Thus, PAR formation functionally links the activity of the nuclear and cytosolic PARPs during stress response, determining the release of PARP12 from the Golgi complex and the disassembly of the Golgi membranes, followed by a block in anterograde-membrane traffic. Notably, these functions can be rescued by reverting the stress condition (by drug wash-out). Altogether these data point at a novel, reversible nuclear signaling that senses stress to then act on cytosolic PARP12, which in turn converts the stress response into a reversible block in intracellular-membrane traffic.

ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells.

ZFP57 is necessary for maintaining repressive epigenetic modifications at Imprinting control regions (ICRs). In mouse embryonic stem cells (ESCs), ZFP57 binds ICRs (ICRBS) and many other loci (non-ICRBS). To address the role of ZFP57 on all its target sites, we performed high-throughput and multi-locus analyses of inbred and hybrid mouse ESC lines carrying different gene knockouts. By using an allele-specific RNA-seq approach, we demonstrate that ZFP57 loss results in derepression of the imprinted allele of multiple genes in the imprinted clusters. We also find marked epigenetic differences between ICRBS and non-ICRBS suggesting that different cis-acting regulatory functions are repressed by ZFP57 at these two classes of target loci. Overall, these data demonstrate that ZFP57 is pivotal to maintain the allele-specific epigenetic modifications of ICRs that in turn are necessary for maintaining the imprinted expression over long distances. At non-ICRBS, ZFP57 inactivation results in acquisition of epigenetic features that are characteristic of poised enhancers, suggesting that another function of ZFP57 in early embryogenesis is to repress cis-acting regulatory elements whose activity is not yet required.

ZFP57 recognizes multiple and closely spaced sequence motif variants to maintain repressive epigenetic marks in mouse embryonic stem cells.

Imprinting Control Regions (ICRs) need to maintain their parental allele-specific DNA methylation during early embryogenesis despite genome-wide demethylation and subsequent de novo methylation. ZFP57 and KAP1 are both required for maintaining the repressive DNA methylation and H3-lysine-9-trimethylation (H3K9me3) at ICRs. In vitro, ZFP57 binds a specific hexanucleotide motif that is enriched at its genomic binding sites. We now demonstrate in mouse embryonic stem cells (ESCs) that SNPs disrupting closely-spaced hexanucleotide motifs are associated with lack of ZFP57 binding and H3K9me3 enrichment. Through a transgenic approach in mouse ESCs, we further demonstrate that an ICR fragment containing three ZFP57 motif sequences recapitulates the original methylated or unmethylated status when integrated into the genome at an ectopic position. Mutation of Zfp57 or the hexanucleotide motifs led to loss of ZFP57 binding and DNA methylation of the transgene. Finally, we identified a sequence variant of the hexanucleotide motif that interacts with ZFP57 both in vivo and in vitro. The presence of multiple and closely located copies of ZFP57 motif variants emerges as a distinct characteristic that is required for the faithful maintenance of repressive epigenetic marks at ICRs and other ZFP57 binding sites.

From toxins to mammalian enzymes: the diversity of mono-ADP-ribosylation.

The ADP-ribosylation of proteins is a phylogenetically ancient mechanism that involves the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD⁺) to specific amino acids of target proteins post-translationally. In the first part of this review, we briefly describe ADP-ribosylation as the mechanism of action of toxins, while giving particular emphasis to a non-conventional ADP-ribosylation reaction that is mediated by the fungal toxin brefeldin A (BFA). This modification results in the loss of the membrane fission activity of the C-terminal binding protein (CtBP)1/ BFA-ADP-ribosylated substrate (BARS), thus blocking progression of cells into mitosis, with important implications for the design of new anticancer drugs. In addition, we summarize the most recent findings on mammalian, intracellular mono-ADP-ribosyl transferase enzymes, underlining the emerging functional roles in which they are involved, including immune responses, transcriptional regulation, stress responses, cell survival. The observation that several mono-ADP-ribosyl transferases, such as PARP-10, PARP-12, PARP-13, are involved in a range of physiological processes points at the multifunctional feature of these proteins.

High grade glioblastoma is associated with aberrant expression of ZFP57, a protein involved in gene imprinting, and of CPT1A and CPT1C that regulate fatty acid metabolism.

The diagnosis of glioblastoma is still based on tumor histology, but emerging molecular diagnosis is becoming an important part of glioblastoma classification. Besides the well-known cell cycle-related circuitries that are associated with glioblastoma onset and development, new insights may be derived by looking at pathways involved in regulation of epigenetic phenomena and cellular metabolism, which may both be highly deregulated in cancer cells. We evaluated if in glioblastoma patients the high grade of malignancy could be associated with aberrant expression of some genes involved in regulation of epigenetic phenomena and lipid metabolism. We measured the mRNA levels of ZFP57, TRIM28, CPT1A, CPT1B, and CPT1C in a cohort of 80 patients divided in two groups: grade II and grade IV. We evidenced that high grade glioblastoma is associated with increased level of ZFP57, a protein involved in gene imprinting, and aberrant expression of CPT1A and CPT1C, regulators of fatty acid oxidation. Our study may pave the way to identify new markers that could be potentially useful for diagnosis and/or prognosis of glioblastoma.

Molecular mechanism and functional role of brefeldin A-mediated ADP-ribosylation of CtBP1/BARS.

ADP-ribosylation is a posttranslational modification that modulates the functions of many target proteins. We previously showed that the fungal toxin brefeldin A (BFA) induces the ADP-ribosylation of C-terminal-binding protein-1 short-form/BFA-ADP-ribosylation substrate (CtBP1-S/BARS), a bifunctional protein with roles in the nucleus as a transcription factor and in the cytosol as a regulator of membrane fission during intracellular trafficking and mitotic partitioning of the Golgi complex. Here, we report that ADP-ribosylation of CtBP1-S/BARS by BFA occurs via a nonconventional mechanism that comprises two steps: (i) synthesis of a BFA-ADP-ribose conjugate by the ADP-ribosyl cyclase CD38 and (ii) covalent binding of the BFA-ADP-ribose conjugate into the CtBP1-S/BARS NAD(+)-binding pocket. This results in the locking of CtBP1-S/BARS in a dimeric conformation, which prevents its binding to interactors known to be involved in membrane fission and, hence, in the inhibition of the fission machinery involved in mitotic Golgi partitioning. As this inhibition may lead to arrest of the cell cycle in G2, these findings provide a strategy for the design of pharmacological blockers of cell cycle in tumor cells that express high levels of CD38.

Genetic and epigenetic mutations affect the DNA binding capability of human ZFP57 in transient neonatal diabetes type 1.

In the mouse, ZFP57 contains three classical Cys2His2 zinc finger domains (ZF) and recognizes the methylated TGC(met)CGC target sequence using the first and the second ZFs. In this study, we demonstrate that the human ZFP57 (hZFP57) containing six Cys2His2 ZFs, binds the same methylated sequence through the third and the fourth ZFs, and identify the aminoacids critical for DNA interaction. In addition, we present evidences indicating that hZFP57 mutations and hypomethylation of the TNDM1 ICR both associated with Transient Neonatal Diabetes Mellitus type 1 result in loss of hZFP57 binding to the TNDM1 locus, likely causing PLAGL1 activation.

In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions.

The maintenance of H3K9 and DNA methylation at imprinting control regions (ICRs) during early embryogenesis is key to the regulation of imprinted genes. Here, we reveal that ZFP57, its cofactor KAP1, and associated effectors bind selectively to the H3K9me3-bearing, DNA-methylated allele of ICRs in ES cells. KAP1 deletion induces a loss of heterochromatin marks at ICRs, whereas deleting ZFP57 or DNMTs leads to ICR DNA demethylation. Accordingly, we find that ZFP57 and KAP1 associated with DNMTs and hemimethylated DNA-binding NP95. Finally, we identify the methylated TGCCGC hexanucleotide as the motif that is recognized by ZFP57 in all ICRs and in several tens of additional loci, several of which are at least ZFP57-dependently methylated in ES cells. These results significantly advance our understanding of imprinting and suggest a general mechanism for the protection of specific loci against the wave of DNA demethylation that affects the mammalian genome during early embryogenesis.

EphrinA1 activates a Src/focal adhesion kinase-mediated motility response leading to rho-dependent actino/myosin contractility.

Eph receptors and ephrin ligands are widely expressed in epithelial cells and mediate cell repulsive motility through heterotypic cell-cell interactions. Several Ephs, including EphA2, are greatly overexpressed in certain tumors, in correlation with poor prognosis and high vascularity in cancer tissues. The ability of several Eph receptors to regulate cell migration and invasion likely contribute to tumor progression and metastasis. We report here that in prostatic carcinoma cells ephrinA1 elicits a repulsive response that is executed through a Rho-dependent actino/myosin contractility activation, ultimately leading to retraction of the cell body. This appears to occur through assembly of an EphA2-associated complex involving the two kinases Src and focal adhesion kinase (FAK). EphrinA1-mediated repulsion leads to the selective phosphorylation of Tyr-576/577 of FAK, enhancing FAK kinase activity. The repulsive response elicited by ephrinA1 in prostatic carcinoma cells is mainly driven by a Rho-mediated phosphorylation of myosin light chain II, in which Src and FAK activation are required steps. Consequently, Src and FAK are upstream regulators of the overall response induced by ephrinA1/EphA2, instructing cells to retract the cell body and to move away, probably facilitating dissemination and tissue invasion of ephrin-sensitive carcinomas.

Redox regulation of ephrin/integrin cross-talk.

Interactions linking the Eph receptor tyrosine kinase and ephrin ligands transduce short-range repulsive signals regulating several motile biological processes including axon path-finding, angiogenesis and tumor growth. These ephrin-induced effects are believed to be mediated by alterations in actin dynamics and cytoskeleton reorganization. The members of the small Rho GTPase family elicit various effects on actin structures and are probably involved in Eph receptor-induced actin modulation. In particular, some ephrin ligands lead to a decrease in integrin-mediated cell adhesion and spread. Here we show that the ability of ephrinA1 to inhibit cell adhesion and spreading in prostatic carcinoma cells is strictly dependent on the decrease in the activity of the small GTPase Rac1. Given the recognized role of Rac-driven redox signaling for integrin function, reported to play an essential role in focal adhesion formation and in the overall organization of actin cytoskeleton, we investigated the possible involvement of oxidants in ephrinA1/EphA2 signaling. We now provide evidence that Reactive Oxygen Species are an integration point of the ephrinA1/integrin interplay. We identify redox circuitry in which the ephrinA1-mediated inhibition of Rac1 leads to a negative regulation of integrin redox signaling affecting the activity of the tyrosine phosphatase LMW-PTP. The enzyme in turn actively dephosphorylates its substrate p190RhoGAP, finally leading to RhoA activation. Altogether our data suggest a redox-based Rac-dependent upregulation of Rho activity, concurring with the inhibitory effect elicited by ephrinA1 on integrin-mediated adhesion strength.

Structure/function of KRAB repression domains: structural properties of KRAB modules inferred from hydrodynamic, circular dichroism, and FTIR spectroscopic analyses.

The abundant zinc finger proteins (ZFPs) sharing the KRAB motif, a potent transcription repression domain, direct the assembly on templates of multiprotein repression complexes. A pivotal step in this pathway is the assembly of a KRAB domain-directed complex with a primary corepressor, KAP1/KRIP-1/TIF1beta. The structure/function dependence of KRAB/TIF1beta protein-protein interaction and properties of the complex, therefore, play pivotal roles in diverse cellular processes depending on KRAB-ZFPs regulation. KRAB domains are functionally bipartite. The 42 amino acid-long KRAB-A module, indeed, is necessary and sufficient for transcriptional repression and for the interaction with the tripartite RBCC region of TIF1beta, while the KRAB-B motif seems to potentiate the assembly of the complex. The structural properties of KRAB-A and KRAB-AB domains from the human ZNF2 protein have been investigated by characterizing highly purified lone (A) and composite (AB) modules. Hydrodynamic and spectroscopic features, investigated by means of gel filtration, circular dichroism, and infrared spectroscopy, provide evidence that both KRAB-A and KRAB-AB domains present low compactness, structural disorder, residual secondary structure content, flexibility, and tendency to molecular aggregation. Comparative analysis among KRAB-A and KRAB-AB modules suggests that the presence of the -B module may influence the properties of lone KRAB-A by affecting the structural flexibility and stability of the conformers. The combined experimental data and the intrinsic features of KRAB-A and KRAB-AB primary structures indicate a potential role of specific subregions within the modules in driving structural flexibility, which is proposed to be of importance for their function.

Expression, purification and partial characterization of the Krüppel-associated box (KRAB) from the human ZNF2 protein.

The Krüppel-associated box (KRAB) domain is a potent transcription repression bipartite domain, shared by over 400 zinc finger proteins in humans, involved in the regulation of many functions. KRAB domains are both physically and functionally bipartite (A and B modules). The lone KRAB-A and composite KRAB-AB domains from the human ZNF2 protein were over-expressed as recombinant proteins in E. coli, isolated and purified to homogeneity to investigate their structure to function relationship.