PARylation Activity Research Articles

Quantification of PARP Activity in Human Tissues: Ex Vivo Assays in Blood Cells and Immunohistochemistry in Human Biopsies.

Poly(ADP-ribosyl)ation of proteins is a posttranslational modification mediated by poly(ADP-ribose) polymerases (PARPs) that use NAD+ as substrate to form the negatively charged polymer of poly(ADP-ribose) (PAR). After DNA damage, PARP-1 is responsible for approximately 90% of the total cellular PARylation activity. Numerous studies showed activation of PARP-1 in various conditions associated with oxidative and nitrosative stress, such as ischemia-reperfusion injury, diabetes mellitus, and inflammation, and also proved the beneficial effects of PARP inhibitors. Several pharmacological inhibitors of PARP moved toward clinical testing for a variety of indications, including cardioprotection and malignant tumors, and in late 2014, olaparib became the first PARP inhibitor approved for human use for the therapy of ovarian cancer. These advances necessitate the detection of PARP activation in human tissues. In the present chapter, we review specific methods used to detect PARP activation in human circulating leukocytes and in human tissue sections.

Tankyrase inhibition aggravates kidney injury in the absence of CD2AP.

Inappropriate activation of the Wnt/β-catenin pathway has been indicated in podocyte dysfunction and injury, and shown to contribute to the development and progression of nephropathy. Tankyrases, multifunctional poly(ADP-ribose) polymerase (PARP) superfamily members with features of both signaling and cytoskeletal proteins, antagonize Wnt/β-catenin signaling. We found that tankyrases interact with CD2-associated protein (CD2AP), a protein essential for kidney ultrafiltration as CD2AP-knockout (CD2AP−/−) mice die of kidney failure at the age of 6–7 weeks. We further observed that tankyrase-mediated total poly-(ADP-ribosyl)ation (PARylation), a post-translational modification implicated in kidney injury, was increased in mouse kidneys and cultured podocytes in the absence of CD2AP. The data revealed increased activity of β-catenin, and upregulation of lymphoid enhancer factor 1 (LEF1) (mediator of Wnt/β-catenin pathway) and fibronectin (downstream target of Wnt/β-catenin) in CD2AP−/− podocytes. Total PARylation and active β-catenin were reduced in CD2AP−/− podocytes by tankyrase inhibitor XAV939 treatment. However, instead of ameliorating podocyte injury, XAV939 further upregulated LEF1, failed to downregulate fibronectin and induced plasminogen activator inhibitor-1 (PAI-1) that associates with podocyte injury. In zebrafish, administration of XAV939 to CD2AP-depleted larvae aggravated kidney injury and increased mortality. Collectively, the data reveal sustained activation of the Wnt/β-catenin pathway in CD2AP−/− podocytes, contributing to podocyte injury. However, we observed that inhibition of the PARylation activity of tankyrases in the absence of CD2AP was deleterious to kidney function. This indicates that balance of the PARylation activity of tankyrases, maintained by CD2AP, is essential for normal kidney function. Furthermore, the data reveal that careful contemplation is required when targeting Wnt/β-catenin pathway to treat proteinuric kidney diseases associated with impaired CD2AP.

Involvement of PARP1 in the regulation of alternative splicing.

Specialized chromatin structures such as nucleosomes with specific histone modifications decorate exons in eukaryotic genomes, suggesting a functional connection between chromatin organization and the regulation of pre-mRNA splicing. Through profiling the functional location of Poly (ADP) ribose polymerase, we observed that it is associated with the nucleosomes at exon/intron boundaries of specific genes, suggestive of a role for this enzyme in alternative splicing. Poly (ADP) ribose polymerase has previously been implicated in the PARylation of splicing factors as well as regulation of the histone modification H3K4me3, a mark critical for co-transcriptional splicing. In light of these studies, we hypothesized that interaction of the chromatin-modifying factor, Poly (ADP) ribose polymerase with nucleosomal structures at exon–intron boundaries, might regulate pre-mRNA splicing. Using genome-wide approaches validated by gene-specific assays, we show that depletion of PARP1 or inhibition of its PARylation activity results in changes in alternative splicing of a specific subset of genes. Furthermore, we observed that PARP1 bound to RNA, splicing factors and chromatin, suggesting that Poly (ADP) ribose polymerase serves as a gene regulatory hub to facilitate co-transcriptional splicing. These studies add another function to the multi-functional protein, Poly (ADP) ribose polymerase, and provide a platform for further investigation of this protein’s function in organizing chromatin during gene regulatory processes.

Poly(ADP-ribose) polymerase 1 regulates nuclear reprogramming and promotes iPSC generation without c-Myc

Poly(ADP-ribose) polymerase 1 (Parp1) catalyzes poly(ADP-ribosylation) (PARylation) and induces replication networks involved in multiple nuclear events. Using mass spectrometry and Western blotting, Parp1 and PARylation activity were intensively detected in induced pluripotent stem cells (iPSCs) and embryonic stem cells, but they were lower in mouse embryonic fibroblasts (MEFs) and differentiated cells. We show that knockdown of Parp1 and pharmacological inhibition of PARylation both reduced the efficiency of iPSC generation induced by Oct4/Sox2/Klf4/c-Myc. Furthermore, Parp1 is able to replace Klf4 or c-Myc to enhance the efficiency of iPSC generation. In addition, mouse iPSCs generated from Oct4/Sox2/Parp1-overexpressing MEFs formed chimeric offspring. Notably, the endogenous Parp1 and PARylation activity was enhanced by overexpression of c-Myc and repressed by c-Myc knockdown. A chromatin immunoprecipitation assay revealed a direct interaction of c-Myc with the Parp1 promoter. PAR-resin pulldown, followed by proteomic analysis, demonstrated high levels of PARylated Chd1L, DNA ligase III, SSrp1, Xrcc-6/Ku70, and Parp2 in pluripotent cells, which decreased during the differentiation process. These data show that the activation of Parp1, partly regulated by endogenous c-Myc, effectively promotes iPSC production and helps to maintain a pluripotent state by posttranslationally modulating protein PARylation.