PARP1-DNA co-condensation drives DNA repair site assembly to prevent disjunction of broken DNA ends.

DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that the synapsis of broken DNA is mediated by the DSB sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, we reconstitute functional DSB sites and show that DSB sites form through co-condensation of PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep DNA ends together and become enzymatically active for PAR synthesis. PARylation promotes release of PARP1 from DNA ends and the recruitment of effectors, such as Fused in Sarcoma, which stabilizes broken DNA ends against separation, revealing a finely orchestrated order of events that primes broken DNA for repair. We provide a comprehensive model for the hierarchical assembly of DSB condensates to explain DNA end synapsis and the recruitment of effector proteins for DNA damage repair.

Poly(ADP-ribosyl)ation enhances nucleosome dynamics and organizes DNA damage repair components within biomolecular condensates.

Nucleosomes, the basic structural units of chromatin, hinder recruitment and activity of various DNA repair proteins, necessitating modifications that enhance DNA accessibility. Poly(ADP-ribosyl)ation (PARylation) of proteins near damage sites is an essential initiation step in several DNA-repair pathways; however, its effects on nucleosome structural dynamics and organization are unclear. Using NMR, cryoelectron microscopy (cryo-EM), and biochemical assays, we show that PARylation enhances motions of the histone H3 tail and DNA, leaving the configuration of the core intact while also stimulating nuclease digestion and ligation of nicked nucleosomal DNA by LIG3. PARylation disrupted interactions between nucleosomes, preventing self-association. Addition of LIG3 and XRCC1 to PARylated nucleosomes generated condensates that selectively partition DNA repair-associated proteins in a PAR- and phosphorylation-dependent manner in vitro. Our results establish that PARylation influences nucleosomes across different length scales, extending from the atom-level motions of histone tails to the mesoscale formation of condensates with selective compositions.

PARP1 UFMylation ensures the stability of stalled replication forks.

The S-phase checkpoint involving CHK1 is essential for fork stability in response to fork stalling. PARP1 acts as a sensor of replication stress and is required for CHK1 activation. However, it is unclear how the activity of PARP1 is regulated. Here, we found that UFMylation is required for the efficient activation of CHK1 by UFMylating PARP1 at K548 during replication stress. Inactivation of UFL1, the E3 enzyme essential for UFMylation, delayed CHK1 activation and inhibits nascent DNA degradation during replication blockage as seen in PARP1-deficient cells. An in vitro study indicated that PARP1 is UFMylated at K548, which enhances its catalytic activity. Correspondingly, a PARP1 UFMylation-deficient mutant (K548R) and pathogenic mutant (F553L) compromised CHK1 activation, the restart of stalled replication forks following replication blockage, and chromosome stability. Defective PARP1 UFMylation also resulted in excessive nascent DNA degradation at stalled replication forks. Finally, we observed that PARP1 UFMylation-deficient knock-in mice exhibited increased sensitivity to replication stress caused by anticancer treatments. Thus, we demonstrate that PARP1 UFMylation promotes CHK1 activation and replication fork stability during replication stress, thus safeguarding genome integrity.

Histone ADP-ribosylation promotes resistance to PARP inhibitors by facilitating PARP1 release from DNA lesions.

Poly(ADP-ribose) polymerase 1 (PARP1) has emerged as a central target for cancer therapies due to the ability of PARP inhibitors to specifically kill tumors deficient for DNA repair by homologous recombination. Upon DNA damage, PARP1 quickly binds to DNA breaks and triggers ADP-ribosylation signaling. ADP-ribosylation is important for the recruitment of various factors to sites of damage, as well as for the timely dissociation of PARP1 from DNA breaks. Indeed, PARP1 becomes trapped at DNA breaks in the presence of PARP inhibitors, a mechanism underlying the cytotoxitiy of these inhibitors. Therefore, any cellular process influencing trapping is thought to impact PARP inhibitor efficiency, potentially leading to acquired resistance in patients treated with these drugs. There are numerous ADP-ribosylation targets after DNA damage, including PARP1 itself as well as histones. While recent findings reported that the automodification of PARP1 promotes its release from the DNA lesions, the potential impact of other ADP-ribosylated proteins on this process remains unknown. Here, we demonstrate that histone ADP-ribosylation is also crucial for the timely dissipation of PARP1 from the lesions, thus contributing to cellular resistance to PARP inhibitors. Considering the crosstalk between ADP-ribosylation and other histone marks, our findings open interesting perspectives for the development of more efficient PARP inhibitor-driven cancer therapies.

AKT-dependent nuclear localization of EPRS1 activates PARP1 in breast cancer cells.

Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser473-AKT+. EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors.

Decrotonylation of cGAS K254 prompts homologous recombination repair by blocking its DNA binding and releasing PARP1.

Cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, also exhibits nuclear genomic localization and is involved in DNA damage signaling. In this study, we investigated the impact of cGAS crotonylation on the regulation of the DNA damage response, particularly homologous recombination repair, following exposure to ionizing radiation (IR). Lysine 254 of cGAS is constitutively crotonylated by the CREB-binding protein; however, IR-induced DNA damage triggers sirtuin 3 (SIRT3)-mediated decrotonylation. Lysine 254 decrotonylation decreased the DNA-binding affinity of cGAS and inhibited its interaction with PARP1, promoting homologous recombination repair. Moreover, SIRT3 suppression led to homologous recombination repair inhibition and markedly sensitized cancer cells to IR and DNA-damaging chemicals, highlighting SIRT3 as a potential target for cancer therapy. Overall, this study revealed the crucial role of cGAS crotonylation in the DNA damage response. Furthermore, we propose that modulating cGAS and SIRT3 activities could be potential strategies for cancer therapy.

An E3 ubiquitin ligase localization screen uncovers DTX2 as a novel ADP-ribosylation-dependent regulator of DNA double-strand break repair.

DNA double-strand breaks (DSBs) elicit an elaborate response to signal damage and trigger repair via two major pathways: nonhomologous end-joining (NHEJ), which functions throughout the interphase, and homologous recombination (HR), restricted to S/G2 phases. The DNA damage response relies, on post-translational modifications of nuclear factors to coordinate the mending of breaks. Ubiquitylation of histones and chromatin-associated factors regulates DSB repair and numerous E3 ubiquitin ligases are involved in this process. Despite significant progress, our understanding of ubiquitin-mediated DNA damage response regulation remains incomplete. Here, we have performed a localization screen to identify RING/U-box E3 ligases involved in genome maintenance. Our approach uncovered 7 novel E3 ligases that are recruited to microirradiation stripes, suggesting potential roles in DNA damage signaling and repair. Among these factors, the DELTEX family E3 ligase DTX2 is rapidly mobilized to lesions in a poly ADP-ribosylation-dependent manner. DTX2 is recruited and retained at DSBs via its WWE and DELTEX conserved C-terminal domains. In cells, both domains are required for optimal binding to mono and poly ADP-ribosylated proteins with WWEs playing a prominent role in this process. Supporting its involvement in DSB repair, DTX2 depletion decreases HR efficiency and moderately enhances NHEJ. Furthermore, DTX2 depletion impeded BRCA1 foci formation and increased 53BP1 accumulation at DSBs, suggesting a fine-tuning role for this E3 ligase in repair pathway choice. Finally, DTX2 depletion sensitized cancer cells to X-rays and PARP inhibition and these susceptibilities could be rescued by DTX2 reexpression. Altogether, our work identifies DTX2 as a novel ADP-ribosylation-dependent regulator of HR-mediated DSB repair.

Pseudorabies virus usurps non-muscle myosin heavy chain IIA to dampen viral DNA recognition by cGAS for antagonism of host antiviral innate immunity.

Alphaherpesvirus pseudorabies virus (PRV) causes severe economic losses to the global pig industry and has garnered increasing attention due to its broad host range including humans. PRV has developed a variety of strategies to antagonize host antiviral innate immunity. However, the underlying mechanisms have not been fully elucidated. In our previous work, we demonstrated that non-muscle myosin heavy chain IIA (NMHC-IIA), a multifunctional cytoskeleton protein, attenuates innate immune responses triggered by RNA viruses. In the current study, we reported a previously unrecognized role of NMHC-IIA in counteracting PRV-induced cyclic GMP-AMP synthase (cGAS)-dependent type I interferon (IFN-I) production. Mechanistically, PRV infection led to an elevation of NMHC-IIA, strengthening the interaction between poly (ADP-ribose) polymerase 1 (PARP1) and cGAS. This interaction impeded cGAS recognition of PRV DNA and hindered downstream signaling activation. Conversely, inhibition of NMHC-IIA by Blebbistatin triggered innate immune responses and enhanced resistance to PRV proliferation both in vitro and in vivo. Taken together, our findings unveil that PRV utilizes NMHC-IIA to antagonize host antiviral immune responses via impairing DNA sensing by cGAS. This in-depth understanding of PRV immunosuppression not only provides insights for potential PRV treatment strategies but also highlights NMHC-IIA as a versatile immunosuppressive regulator usurped by both DNA and RNA viruses. Consequently, NMHC-IIA holds promise as a target for the development of broad-spectrum antiviral drugs.IMPORTANCECyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) axis plays a vital role in counteracting alphaherpesvirus infections. Alphaherpesviruses exploit various strategies for antagonizing cGAS-STING-mediated antiviral immune responses. However, limited examples of pseudorabies virus (PRV)-caused immunosuppression have been documented. Our findings reveal a novel role of non-muscle myosin heavy chain IIA (NMHC-IIA) in suppressing PRV-triggered innate immune responses to facilitate viral propagation both in vitro and in vivo. In detail, NMHC-IIA recruits poly (ADP-ribose) polymerase 1 (PARP1) to augment its interaction with cGAS, which impairs cGAS recognition of PRV DNA. Building on our previous demonstration of NMHC-IIA's immunosuppressive role during RNA virus infections, these findings indicate that NMHC-IIA acts as a broad-spectrum suppressor of host antiviral innate immunity in response to both DNA and RNA viruses. Therefore, NMHC-IIA will be a promising target for the development of comprehensive antiviral strategies.

Long noncoding RNA UCA1 promotes the chondrogenic differentiation of human bone marrow mesenchymal stem cells via regulating PARP1 ubiquitination.

Bone marrow mesenchymal stem cells (BMSCs) possess the potential to differentiate into cartilage cells. Long noncoding RNA (lncRNAs) urothelial carcinoma associated 1 (UCA1) has been confirmed to improve the chondrogenic differentiation of marrow mesenchymal stem cells (MSCs). Herein, we further investigated the effects and underlying mechanisms of these processes. The expression of UCA1 was positively associated with chondrogenic differentiation and the knockdown of UCA1 has been shown to attenuate the expression of chondrogenic markers. RNA pull-down assay and RNA immunoprecipitation showed that UCA1 could directly bind to PARP1 protein. UCA1 could improve PARP1 protein via facilitating USP9X-mediated PARP1 deubiquitination. Then these processes stimulated the NF-κB signaling pathway. In addition, PARP1 was declined in UCA1 knockdown cells, and silencing of PARP1 could diminish the increasing effects of UCA1 on the chondrogenic differentiation from MSCs and signaling pathway activation. Collectively, these outcomes suggest that UCA1 could act as a mediator of PARP1 protein ubiquitination and develop the chondrogenic differentiation of MSCs.

Poly (ADP-ribose) polymerase 1 promotes HuR/ELAVL1 cytoplasmic localization and inflammatory gene expression by regulating p38 MAPK activity.

Post-transcriptional regulation of cytokine/chemokine mRNA turnover is critical for immune processes and contributes to the mammalian cellular response to diverse inflammatory stimuli. The ubiquitous RNA-binding protein human antigen R (HuR) is an integral regulator of inflammation-associated mRNA fate. HuR function is regulated by various post-translational modifications that alter its subcellular localization and ability to stabilize target mRNAs. Both poly (ADP-ribose) polymerase 1 (PARP1) and p38 mitogen-activated protein kinases (MAPKs) have been reported to regulate the biological function of HuR, but their specific regulatory and crosstalk mechanisms remain unclear. In this study, we show that PARP1 acts via p38 to synergistically promote cytoplasmic accumulation of HuR and stabilization of inflammation-associated mRNAs in cells under inflammatory conditions. Specifically, p38 binds to auto-poly ADP-ribosylated (PARylated) PARP1 resulting in the covalent PARylation of p38 by PARP1, thereby promoting the retention and activity of p38 in the nucleus. In addition, PARylation of HuR facilitates the phosphorylation of HuR at the serine 197 site mediated by p38, which then increases the translocation of HuR to the cytoplasm, ultimately stabilizing the inflammation-associated mRNA expression at the post-transcriptional level.

TLR9 activation induces immunosuppression and tumorigenesis via PARP1/PD-L1 signaling pathway in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer, and metastasis and immunosuppression are responsible for the poor prognosis of OSCC. Previous studies have shown that poly(ADP-ribose) polymerase (PARP)1 plays a key role in the pathogenesis of OSCC. Therefore, PARP1 may serve as an important research target for the potential treatment of OSCC. Here, we aimed to investigate the role of PARP1 in the tumorigenesis of OSCC and elucidate the key molecular mechanisms of its upstream and downstream regulation in vivo and in vitro. In human OSCC tissues and cells, Toll-like receptor (TLR)9 and PD-L1 were highly expressed and PARP1 was lowly expressed. Suppression of TLR9 remarkably repressed CAL27 and SCC9 cell proliferation, migration, and invasion. After coculture, we found that low expression of TLR9 inhibited PD-L1 expression and immune escape. In addition, TLR9 regulated PD-L1 expression through the PARP1/STAT3 pathway. PARP1 mediated the effects of TLR9 on OSCC cell proliferation, migration, and invasion and immune escape. Additionally, in vivo experiments further verified that TLR9 promoted tumor growth and immune escape by inhibiting PARP1. Collectively, TLR9 activation induced immunosuppression and tumorigenesis via PARP1/PD-L1 signaling pathway in OSCC, providing important insights for subsequent in-depth exploration of the mechanism of OSCC.NEW & NOTEWORTHY In this research, we took PARP1 as the key target to explore its regulatory effect on oral squamous cell carcinoma (OSCC). The key molecular mechanisms involved in its upstream and downstream regulation were elucidated in OSCC cell lines in vitro and tumor-bearing mice in vivo, combined with clinical OSCC tissues.

PARP1 in the intersection of different DNA repair pathways, memory formation, and sleep pressure in neurons.

Poly(ADP-ribose) polymerase-1 (PARP1) is a bottleneck that connects different DNA pathways during a DNA damage response. Interestingly, PARP1 has a dualist role in neurons, acting as a neuroprotector and inducer of cell death in distinct neurological diseases. Recent studies significantly expanded our knowledge of how PARP1 regulates repair pathways in neurons and uncovered new roles for PARP1 in promoting sleep to enhance DNA repair. Likewise, PARP1 is deeply associated with memory consolidation, implying that it has multiple layers of regulation in the neural tissue. In this review, we critically discuss PARP1 recent advances in neurons, focusing on its interplay with different DNA repair mechanisms, memory, and sleep. Provocative questions about how oxidative damage is accessed, and different hypotheses about the molecular mechanisms influenced by PARP1 in neurons are presented to expand the debate of future studies.

PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer.

BACKGROUND: Poly (ADP-ribose) polymerase inhibitor (PARPi) resistance poses a significant challenge in ovarian carcinoma (OC). While the role of DOT1L in cancer and chemoresistance is acknowledged, its specific role in PARPi resistance remains unclear. This study aims to elucidate the molecular mechanism of DOT1L in PARPi resistance in OC patients. METHODS: This study analyzed the expression of DOT1L in PARPi-resistant cell lines compared to sensitive ones and correlated it with clinical outcomes in OC patients. Comprehensive in vitro and in vivo functional experiments were conducted using cellular and mouse models. Molecular investigations, including RNA sequencing, chromatin immunoprecipitation (ChIP) and Cleavage Under Targets and Tagmentation (CUT&Tag) assays, were employed to unravel the molecular mechanisms of DOT1L-mediated PARPi resistance. RESULTS: Our investigation revealed a robust correlation between DOT1L expression and clinical PARPi resistance in non-BRCA mutated OC cells. Upregulated DOT1L expression in PARPi-resistant tissues was associated with diminished survival in OC patients. Mechanistically, we identified that PARP1 directly binds to the DOT1L gene promoter, promoting transcription independently of its enzyme activity. PARP1 trapping induced by PARPi treatment amplified this binding, enhancing DOT1L transcription and contributing to drug resistance. Sequencing analysis revealed that DOT1L plays a crucial role in the transcriptional regulation of PLCG2 and ABCB1 via H3K79me2. This established the PARP1-DOT1L-PLCG2/ABCB1 axis as a key contributor to PARPi resistance. Furthermore, we discovered that combining a DOT1L inhibitor with PARPi demonstrated a synergistic effect in both cell line-derived xenograft mouse models (CDXs) and patient-derived organoids (PDOs). CONCLUSIONS: Our results demonstrate that DOT1L is an independent prognostic marker for OC patients. The PARP1-DOT1L/H3K79me2-PLCG2/ABCB1 axis is identified as a pivotal contributor to PARPi resistance. Targeted inhibition of DOT1L emerges as a promising therapeutic strategy for enhancing PARPi treatment outcomes in OC patients.

PARP1 expression predicts PARP inhibitor sensitivity and correlates with metastatic potential and overall survival in melanoma.

Metastatic melanoma is still a difficult-to-treat cancer type owing to its frequent resistance mechanisms to targeted and immunotherapy. Therefore, we aimed to unravel novel therapeutic strategies for melanoma patients. Preclinical and clinical studies show that melanoma patients may benefit from a treatment with poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). In this study, we focus on PARP1 as a potential biomarker to predict the response of melanoma cells to PARPi therapy. We found that melanoma cells with high basal PARP1 expression exhibit significantly increased cell death after PARPi treatment owing to higher PARP1 trapping compared with melanoma cells with low PARP1 expression. In addition, we could demonstrate that PARP1 expression levels are low in nonmalignant skin cells, and metastatic melanomas show considerably higher PARP1 levels compared with primary melanomas. Most strikingly, we found that high PARP1 levels correlate with worse overall survival of late stage metastasized melanoma patients. In conclusion, we show that PARP1 might act as a biomarker to predict the response to PARPi therapy, and that in particular the late stage metastasized melanoma patients are especially sensitive to PARPi therapy owing to elevated PARP1 expression. Our data suggest that the PARPi cytotoxicity primarily will affect the high PARP1 expressing melanoma cells, rather than the low PARP1 expressing nonmalignant skin cells resulting in only low side effects.

A desthiobiotin labelled NAD+ analogue to uncover Poly(ADP-ribose) polymerase 1 protein targets.

ADP-ribosylation is a post-translational modification catalyzed by the enzyme family of polyadenosine diphosphate (ADP)-ribose) polymerases (PARPs). This enzymatic process involves the transfer of single or multiple ADP-ribose molecules onto proteins, utilizing nicotinamide adenine dinucleotide (NAD+ ) as a substrate. It, thus, plays a pivotal role in regulating various biological processes. Unveiling PARP-selective protein targets is crucial for a better understanding of their biological functions. Nonetheless, this task proves challenging due to overlapping targets shared among PARP family members. Therefore, we applied the "bump-and-hole" strategy to modify the nicotinamide binding site of PARP1 by introducing a hydrophobic pocket ("hole"). This PARP1-mutant binds an orthogonal NAD+ (Et-DTB-NAD+ ) containing an ethyl group ("bump") at the nicotinamide moiety. Furthermore, we added a desthiobiotin (DTB) tag directly to the adenosine moiety, enabling affinity enrichment of ADP-ribosylated proteins. Employing this approach, we successfully identified protein targets modified by PARP1 in cell lysate. This strategy expands the arsenal of chemically modified NAD+ analogs available for studying ADP-ribosylation, providing a powerful tool to study these critical post-translational modifications.

HBV DNA polymerase upregulates the transcription of PD-L1 and suppresses T cell activity in hepatocellular carcinoma.

BACKGROUND: In HBV-associated HCC, T cells often exhibit a state of functional exhaustion, which prevents the immune response from rejecting the tumor and allows HCC to progress. Moreover, polymerase-specific T cells exhibit more severe T-cell exhaustion compared to core-specific T cells. However, whether HBV DNA polymerase drives HBV-specific CD8+ T cell exhaustion in HBV-related HCC remains unclear. METHODS: We constructed a Huh7 cell line stably expressing HA-HBV-DNA-Pol and applied co-culture systems to clarify its effect on immune cell function. We also examined how HBV-DNA-Pol modulated PD-L1 expression in HCC cells. In addition, HBV-DNA-Pol transgenic mice were used to elucidate the underlying mechanism of HBV-DNA-Pol/PD-L1 axis-induced T cell exhaustion. RESULTS: Biochemical analysis showed that Huh7 cells overexpressing HBV-DNA-Pol inhibited the proliferation, activation, and cytokine secretion of Jurkat cells and that this effect was dependent on their direct contact. A similar inhibitory effect was observed in an HCC mouse model. PD-L1 was brought to our attention during screening. Our results showed that the overexpression of HBV-DNA-Pol upregulated PD-L1 mRNA and protein expression. PD-L1 antibody blockade reversed the inhibitory effect of Huh7 cells overexpressing HBV-DNA-Pol on Jurkat cells. Mechanistically, HBV-DNA-Pol interacts with PARP1, thereby inhibiting the nuclear translocation of PARP1 and further upregulating PD-L1 expression. CONCLUSIONS: Our findings suggest that HBV-DNA-Pol can act as a regulator of PD-L1 in HCC, thereby directing anti-cancer immune evasion, which further provides a new idea for the clinical treatment of liver cancer.

Unlocking cardioprotection: iPSC exosomes deliver Nec-1 to target PARP1/AIFM1 axis, alleviating HF oxidative stress and mitochondrial dysfunction.

BACKGROUND: Heart failure (HF) is characterized by oxidative stress and mitochondrial dysfunction. This study investigates the therapeutic potential of Necrostatin-1 (Nec-1) delivered through exosomes derived from induced pluripotent stem cells (iPSCs) to address these pathologies in HF. METHODS: An HF rat model was established, and comprehensive assessments were performed using echocardiography, hemodynamics, and ventricular mass index measurements. iPSCs were used to isolate exosomes, loaded with Nec-1, and characterized for efficient delivery into cardiomyocytes. The interaction between Nec-1-loaded exosomes (Nec-1-Exos), poly (ADP-ribose) polymerase 1 (PARP1), and apoptosis-inducing factor mitochondria-associated 1 (AIFM1) was explored. Gain-of-function experiments assessed changes in cardiomyocyte parameters, and histological analyses were conducted on myocardial tissues. RESULTS: Cardiomyocytes successfully internalized Nec-1-loaded exosomes, leading to downregulation of PARP1, inhibition of AIFM1 nuclear translocation, increased ATP and superoxide dismutase levels, reduced reactive oxygen species and malonaldehyde levels, and restored mitochondrial membrane potential. Histological examinations confirmed the modulation of the PARP1/AIFM1 axis by Nec-1, mitigating HF. CONCLUSIONS: iPSC-derived exosomes carrying Nec-1 attenuate oxidative stress and mitochondrial dysfunction in HF by targeting the PARP1/AIFM1 axis. This study proposes a promising therapeutic strategy for HF management and highlights the potential of exosome-mediated drug delivery.

PARP-1, EpCAM, and FRα as potential targets for intraoperative detection and delineation of endometriosis: a quantitative tissue expression analysis.

BACKGROUND: Endometriosis is a gynecological disease characterized by the presence of endometrial tissue in abnormal locations, leading to severe symptoms, inflammation, pain, organ dysfunction, and infertility. Surgical removal of endometriosis lesions is crucial for improving pain and fertility outcomes, with the goal of complete lesion removal. This study aimed to analyze the location and expression patterns of poly (ADP-ribose) polymerase 1 (PARP-1), epithelial cell adhesion molecule (EpCAM), and folate receptor alpha (FRα) in endometriosis lesions and evaluate their potential for targeted imaging. METHODS: Gene expression analysis was performed using the Turku endometriosis database (EndometDB). By immunohistochemistry, we investigated the presence and distribution of PARP-1, EpCAM, and FRα in endometriosis foci and adjacent tissue. We also applied an ad hoc platform for the analysis of images to perform a quantitative immunolocalization analysis. Double immunofluorescence analysis was carried out for PARP-1 and EpCAM, as well as for PARP-1 and FRα, to explore the expression of these combined markers within endometriosis foci and their potential simultaneous utilization in surgical treatment. RESULTS: Gene expression analysis revealed that PARP-1, EpCAM, and FOLR1 (FRα gene) are more highly expressed in endometriotic lesions than in the peritoneum, which served as the control tissue. The results of the immunohistochemical study revealed a significant increase in the expression levels of all three biomarkers inside the endometriosis foci compared to the adjacent tissues. Additionally, the double immunofluorescence analysis consistently demonstrated the presence of PARP-1 in the nucleus and the expression of EpCAM and FRα in the cell membrane and cytoplasm. CONCLUSION: Overall, these three markers demonstrate significant potential for effective imaging of endometriosis. In particular, the results emphasize the importance of PARP-1 expression as a possible indicator for distinguishing endometriotic lesions from adjacent tissue. PARP-1, as a potential biomarker for endometriosis, offers promising avenues for further investigation in terms of both pathophysiology and diagnostic-therapeutic approaches.

Differences between long- and short-wavelength light-induced retinal damage and the role of PARP-1 in retinal injury induced by blue light.

Photobiomodulation (PBM) therapy uses light of different wavelengths to treat various retinal degeneration diseases, but the potential damage to the retina caused by long-term light irradiation is still unclear. This study were designed to detect the difference between long- and short-wavelength light (650-nm red light and 450-nm blue light, 2.55 mW/cm2, reference intensity in PBM)-induced injury. In addition, a comparative study was conducted to investigate the differences in retinal light damage induced by different irradiation protocols (short periods of repeated irradiation and a long period of constant irradiation). Furthermore, the protective role of PARP-1 inhibition on the molecular mechanism of blue light-induced injury was confirmed by a gene knockdown technique or a specific inhibitor through in vitro and in vivo experiments. The results showed that the susceptibility to retinal damage caused by irradiation with long- and short-wavelength light is different. Shorter wavelength lights, such as blue light, induce more severe retinal damage, while the retina exhibits better resistance to longer wavelength lights, such as red light. In addition, repeated irradiation for short periods induces less retinal damage than constant exposure over a long period. PARP-1 plays a critical role in the molecular mechanism of blue light-induced damage in photoreceptors and retina, and inhibiting PARP-1 can significantly protect the retina against blue light damage. This study lays an experimental foundation for assessing the safety of phototherapy products and for developing target drugs to protect the retina from light damage.

The involvement of Sting in exacerbating acute lung injury in sepsis via the PARP-1/NLRP3 signaling pathway.

BACKGROUND: Interferon gene stimulator (Sting) is an indispensable adaptor protein that plays a crucial role in acute lung injury (ALI) induced by sepsis, and the PARP-1/NLRP3 signaling pathway may be an integral component of the inflammatory response mediated by Sting. However, the regulatory role of Sting in the PARP-1/NLRP3 pathway in ALI remains insufficiently elucidated. METHODS: Using lipopolysaccharide (LPS) to induce ALI in C57BL/6 mice and HUVEC cells, an in vivo and in vitro model was established. In vivo, Sting agonists and inhibitors were administered, while in vitro, Sting was knocked down using siRNA. ELISA was employed to quantify the levels of IL-1ß, IL-6, and TNF-α. TUNEL staining was conducted to assess cellular apoptosis, while co-immunoprecipitation was utilized to investigate the interaction between Sting and NLRP3. Expression levels of Sting, NLRP3, PARP-1, among others, were assessed via Western blotting and RT-qPCR. Lung HE staining and lung wet/dry ratio were evaluated in the in vivo mouse model. To validate the role of the PARP-1/NLRP3 signaling pathway, PARP-1 inhibitors were employed both in vivo and in vitro. RESULTS: In vitro experiments revealed that the Sting agonist group exacerbated LPS-induced pulmonary pathological damage, pulmonary edema, inflammatory response (increased levels of IL-6, TNF-α, and IL-1ß), and cellular injury, whereas the Sting inhibitor group significantly ameliorated the aforementioned injuries, with further improvement observed in the combination therapy of Sting inhibitor and PARP-1 inhibitor. Western blotting and RT-qPCR results demonstrated significant suppression of ICAM-1, VCAM-1, NLRP3, and PARP-1 expression in the Sting inhibitor group, with this reduction further enhanced in the Sting inhibitor + PARP-1 inhibitor treatment group, exhibiting opposite outcomes to the agonist. Furthermore, in vitro experiments using HUVEC cell lines validated these findings. CONCLUSIONS: Our study provides new insights into the roles of Sting and the PARP-1/NLRP3 signaling pathway in inflammatory responses, offering novel targets for the development of therapeutic interventions against inflammatory reactions.