2C

Amino Acid Sequence

3D Structure (/structures/AF-P09874-F1-model_v6.cif)

Function

Poly-ADP-ribosyltransferase that mediates poly-ADP-ribosylation of proteins and plays a key role in DNA repair Mediates glutamate, aspartate, serine, histidine or tyrosine ADP-ribosylation of proteins: the ADP-D-ribosyl group of NAD+ is transferred to the acceptor carboxyl group of target residues and further ADP-ribosyl groups are transferred to the 2'-position of the terminal adenosine moiety, building up a polymer with an average chain length of 20-30 units. Serine ADP-ribosylation of proteins constitutes the primary form of ADP-ribosylation of proteins in response to DNA damage. Specificity for the different amino acids is conferred by interacting factors, such as HPF1 and NMNAT1. Following interaction with HPF1, catalyzes serine ADP-ribosylation of target proteins; HPF1 confers serine specificity by completing the PARP1 active site. Also catalyzes tyrosine ADP-ribosylation of target proteins following interaction with HPF1. Following interaction with NMNAT1, catalyzes glutamate and aspartate ADP-ribosylation of target proteins; NMNAT1 confers glutamate and aspartate specificity (By similarity). PARP1 initiates the repair of DNA breaks: recognizes and binds DNA breaks within chromatin and recruits HPF1, licensing serine ADP-ribosylation of target proteins, such as histones (H2BS6ADPr and H3S10ADPr), thereby promoting decompaction of chromatin and the recruitment of repair factors leading to the reparation of DNA strand breaks. HPF1 initiates serine ADP-ribosylation but restricts the polymerase activity of PARP1 in order to limit the length of poly-ADP-ribose chains. In addition to base excision repair (BER) pathway, also involved in double-strand breaks (DSBs) repair: together with TIMELESS, accumulates at DNA damage sites and promotes homologous recombination repair by mediating poly-ADP-ribosylation. Mediates the poly-ADP-ribosylation of a number of proteins, including itself, APLF, CHFR, RPA1 and NFAT5. In addition to proteins, also able to ADP-ribosylate DNA: catalyzes ADP-ribosylation of DNA strand break termini containing terminal phosphates and a 2'-OH group in single- and double-stranded DNA, respectively. Required for PARP9 and DTX3L recruitment to DNA damage sites. PARP1-dependent PARP9-DTX3L-mediated ubiquitination promotes the rapid and specific recruitment of 53BP1/TP53BP1, UIMC1/RAP80, and BRCA1 to DNA damage sites. PARP1-mediated DNA repair in neurons plays a role in sleep: senses DNA damage in neurons and promotes sleep, facilitating efficient DNA repair (By similarity). In addition to DNA repair, also involved in other processes, such as transcription regulation, programmed cell death, membrane repair, adipogenesis and innate immunity. Acts as a repressor of transcription: binds to nucleosomes and modulates chromatin structure in a manner similar to histone H1, thereby altering RNA polymerase II. Acts both as a positive and negative regulator of transcription elongation, depending on the context. Acts as a positive regulator of transcription elongation by mediating poly-ADP-ribosylation of NELFE, preventing RNA-binding activity of NELFE and relieving transcription pausing. Acts as a negative regulator of transcription elongation in response to DNA damage by catalyzing poly-ADP-ribosylation of CCNT1, disrupting the phase separation activity of CCNT1 and subsequent activation of CDK9. Involved in replication fork progression following interaction with CARM1: mediates poly-ADP-ribosylation at replication forks, slowing fork progression. Poly-ADP-ribose chains generated by PARP1 also play a role in poly-ADP-ribose-dependent cell death, a process named parthanatos (By similarity). Also acts as a negative regulator of the cGAS-STING pathway. Acts by mediating poly-ADP-ribosylation of CGAS: PARP1 translocates into the cytosol following phosphorylation by PRKDC and catalyzes poly-ADP-ribosylation and inactivation of CGAS. Acts as a negative regulator of adipogenesis: catalyzes poly-ADP-ribosylation of histone H2B on 'Glu-35' (H2BE35ADPr) following interaction with NMNAT1, inhibiting phosphorylation of H2B at 'Ser-36' (H2BS36ph), thereby blocking expression of pro-adipogenetic genes (By similarity). Involved in the synthesis of ATP in the nucleus, together with NMNAT1, PARG and NUDT5. Nuclear ATP generation is required for extensive chromatin remodeling events that are energy-consuming. Plays a role in sister chromatid cohesion by mediating ADP-ribosylation of RSMC during S phase which promotes the interaction between RSMC and CDCA5/sororin, leading to enhanced interaction of CDCA5/sororin with the cohesin complex and promotion of sister chromatid cohesion.

Mechanism

The unfolded protein response (UPR) is a quality-control mechanism triggered by the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen. Under ER stress, the chaperone BiP/GRP78 dissociates from three ER transmembrane sensors—IRE1α, PERK, and ATF6—activating three distinct UPR branches. Activated IRE1α splices XBP1 mRNA to generate XBP1s. PERK phosphorylates eIF2α, attenuating global translation while selectively inducing ATF4. ATF6 translocates to the Golgi apparatus and is cleaved to release its active nuclear form, ATF6(N). These transcription factors coordinately upregulate chaperones, folding enzymes, ER-associated degradation (ERAD), and autophagy to restore cellular proteostasis. However, if the stress persists or remains unresolved, prolonged UPR signaling triggers apoptosis through the CHOP and BAX/BAK-caspase pathways.

Description

To better understand the specificity and mechanism of HyT-induced PARP-1 degradation, we assessed changes in protein levels within the whole cell lysate of HyT-conjugate 2c-treated cells compared with olaparib treated cells. In total, 211 proteins were found to be differentially expressed (p = 0.01, Log2 FC ± 2; Fig. 6A). Of the 211 proteins identified, 103 were enriched in 2c-treated cells and 108 in olaparib-treated cells. Validation of PARP degradation under the conditions used in these proteomics experiments is shown in Fig. S9. Gene set enrichment analysis (GSEA) of the differentially ex-pressed proteins identified significantly upregulated (Fig. 6A–B, full list Fig. S10) gene/protein sets after treatment with 2c vs treatment with olaparib, including hallmarks of the “Unfolded protein response”, and biological processes including “Positive regulation of response to ER stress”, and “Intrinsic apoptotic signalling pathway in response to ER stress”. This included a large increase in levels of DDIT3 (CHOP) and XBP1 which are major regulators of ER stress/UPR, and DNAJC25 which is a Hsp40 protein and known ER chaperone [35]. To address the role of ER chaperones further, ES-2 cells were pre-treated with an Hsp90 inhibitor 17-AAG, which is known to upregulate Hsp70 and has previously been used to augment HyT-mediated degradation of Src-1 [28]. As a baseline, we used incubation conditions for 2c that did not induce substantial PARP-1 degradation. Pre-treating cells with 17-AAG before incubating with 2c under these conditions (Fig. 7), showed that 17-AAG promoted PARP-1 degradation by 2c, significantly decreasing PARP-1 protein levels. This indicates that ER chaperone Hsp70 can promote PARP-1 degradation, and these results provide further evidence for HyT-mediated activation of the UPR.

Mechanism

The autophagy-lysosomal pathway (ALP) is capable of degrading a broader range of substrates, including longer-lived and larger misfolded proteins and damaged organelles. ALP is classified into three subtypes: microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. In microautophagy, substrates are directly sequestered and degraded by lysosomes and late endosomes via membrane protrusion and invagination. In CMA, HSPA8/HSC70 recognizes specific substrates containing KFERQ-like sequences; these substrates subsequently bind to LAMP2A on the lysosomal surface and are delivered into lysosomes for degradation. In macroautophagy, substrates are encapsulated through the formation of double-membrane autophagosomes, which subsequently fuse with lysosomes to degrade the substrates.

Description

Using Western blot analysis, we found a significant increase in PARP-1 protein in cells pre-incubated with the autophagy inhibitor bafilomycin-A1 (BafA) prior to treatment with HyT-conjugate 2c, over cells treated with 2c alone (Fig. S11A–B). We further interconnected autophagy, UPR and ER stress and validated the proteomics results by probing the protein levels of lipidated LC3-II (typical autophagosome marker) and CHOP by Western blot, demonstrating that increases in both proteins were observed after2c treatment (Fig. S11C). Furthermore, a final assessment of the top hits from proteomics was carried out by RT-qPCR, determining that UPR/ER stress-related genes ATF4, DDIT3, DNAJB9, HSP90B1, and XBP1s (Fig. S12A), in addition to key UPR/autophagy players LC3A, LC3B and SQSTM1 (Fig. S12B), were all upregulated after treatment with 2c. This supports a mechanism whereby the HyT-olaparib conjugate 2c induces UPR/autophagy, thus facilitating the degradation of PARP-1.

Description

Using Western blot analysis of intracellular PARP-1 levels, HyT-olaparib conjugate 2c was shown to have a Dmax of 55 % and DC50 of 618 nM in ES-2 cells (Fig. 4). Although this activity is lower than reported for PROTAC-based degradation of PARP-1 [27], it is consistent with HyT-based degradation mechanisms of other proteins [1]. In addition, the release of potentially teratogenic cereblon ligands from UPS degraders through linker metabolism [29], means that activation of alternative degradation pathways by HyT remains an attractive therapeutic target even at these levels.

Description

Using Western blot analysis of intracellular PARP-1 levels, HyT-olaparib conjugate 2c was shown to have a Dmax of 55 % and DC50 of 618 nM in ES-2 cells (Fig. 4). Although this activity is lower than reported for PROTAC-based degradation of PARP-1 [27], it is consistent with HyT-based degradation mechanisms of other proteins [1]. In addition, the release of potentially teratogenic cereblon ligands from UPS degraders through linker metabolism [29], means that activation of alternative degradation pathways by HyT remains an attractive therapeutic target even at these levels.

Description

With the knowledge that 1a–1c and 2a–2c were comparatively potent HyT degraders of PARP-1, we next sought to determine whether PARP-1 degradation directly affected cell viability. ES-2 cells are commonly used in ovarian cancer research and are BRCA WT, allowing higher olaparib doses to be used for cellular Raman imaging. Using Alamar Blue as a readout, the viability of ES-2 cells was measured after 72 h incubation with olaparib, 1a–1c, 2a–2c and BADY ester control 3a (Fig. 5). Lead compound 2c effectively degrades intra cellular PARP-1 (DC50 = 0.618 μM, Dmax = 55 %) and has more potent antiproliferative activity than the parent compound olaparib (IC50 = 62 nM vs 11.52 μM for olaparib), whilst maintaining on-target PARP inhibitory activity.