Deena M. Leslie Pedrioli

The mono-ADP-ribosylhydrolase MacroD1 has been recently reported to localize to mitochondria exclusively. However, the extent and means by which MacroD1 regulates metabolic homeostasis remains unclear. Here we show that the absence of MacroD1 in mice decreased mitochondrial load and negatively impacted muscle function, reducing maximal exercise capacity. Knockdown of MacroD1 in C2C12 myoblast cells amplified the production of reactive oxygen species which ultimately resulted in increased mitochondrial fission. Proteomic and metabolomic profiling showed that loss of MacroD1 re-routed metabolite flux from glucose to the pentose-phosphate cycle instead of the tricarboxylic acid cycle to support the production of antioxidants, including glutathione and NADPH. This resulted in increased glucose uptake and dependency both in vitro and in vivo. Hence, our research establishes MacroD1 as a regulator of metabolic homeostasis, which ensures the coordination of cellular carbohydrate flux and optimal mitochondrial function.

Several ADP-ribosyltransferases are upregulated during viral infections and are crucial for the cellular immune response. While interferon-induced PARP14 ADP-ribosylates various substrates, viruses such as SARS-CoV-2 counteract this by reversing ADP-ribosylation. The exact mechanism of PARP14’s antiviral activity and the targets of viral macrodomains remain unknown. Here, we observe that PARP14 mono-ADP-ribosylates the selective autophagy adaptor SQSTM1/p62 at cysteine residues 113, 289/90, and 331 following interferon treatment. This correlates with the ADP-ribosylation of cytoplasmic p62 foci that colocalize with ubiquitin and PARP14 but not with LC3, thereby distinguishing them from classical autophagosomes. Moreover, the SARS-CoV-2 macrodomain effectively prevented this p62 modification, suggesting an antiviral function for this ADP-ribosylated target. Furthermore, our results indicate that TRIM21 prevents the autophagic degradation of ADP-ribosylated p62, suggesting that the identified p62 foci may have autophagy-independent roles. This study contributes to our understanding of the molecular dynamics involved in host-virus interactions and highlights the potential role of ADP-ribosylation in the regulation of innate immunity.

Pluripotency is established in E4.5 preimplantation epiblast. Embryonic stem cells (ESCs) represent the immortalization of pluripotency, however, their gene expression signature only partially resembles that of developmental ground-state. Induced PRAMEL7 expression, a protein highly expressed in the ICM but lowly expressed in ESCs, reprograms developmentally advanced ESC+serum into ground-state pluripotency by inducing a gene expression signature close to developmental ground-state. However, how PRAMEL7 reprograms gene expression remains elusive. Here we show that PRAMEL7 associates with Cullin2 (CUL2) and this interaction is required to establish ground-state gene expression. PRAMEL7 recruits CUL2 to chromatin and targets regulators of repressive chromatin, including the NuRD complex, for proteasomal degradation. PRAMEL7 antagonizes NuRD-mediated repression of genes implicated in pluripotency by decreasing NuRD stability and promoter association in a CUL2-dependent manner. Our data link proteasome degradation pathways to ground-state gene expression, offering insights to generate in vitro models to reproduce the in vivo ground-state pluripotency.

Metastatic melanoma is either intrinsically resistant or rapidly acquires resistance to targeted therapy treatments, such as MAPK inhibitors (MAPKi). A leading cause of resistance to targeted therapy is a dynamic transition of melanoma cells from a proliferative to a highly invasive state, a phenomenon called phenotype switching. Mechanisms regulating phenotype switching represent potential targets for improving treatment of patients with melanoma. Using a drug screen targeting chromatin regulators in patient-derived three-dimensional MAPKi-resistant melanoma cell cultures, we discovered that PARP inhibitors (PARPi) restore sensitivity to MAPKis, independent of DNA damage repair pathways. Integrated transcriptomic, proteomic, and epigenomic analyses demonstrated that PARPis induce lysosomal autophagic cell death, accompanied by enhanced mitochondrial lipid metabolism that ultimately increases antigen presentation and sensitivity to T-cell cytotoxicity. Moreover, transcriptomic and epigenetic rearrangements induced by PARP inhibition reversed epithelial–mesenchymal transition-like phenotype switching, which redirected melanoma cells toward a proliferative and MAPKi-sensitive state. The combination of PARP and MAPKis synergistically induced cancer cell death both in vitro and in vivo in patient-derived xenograft models. Therefore, this study provides a scientific rationale for treating patients with melanoma with PARPis in combination with MAPKis to abrogate acquired therapy resistance.Significance:PARP inhibitors can overcome resistance to MAPK inhibitors by activating autophagic cell death and reversing phenotype switching, suggesting that this synergistic combination could help improve the prognosis of patients with melanoma.

Metastatic melanoma is either intrinsically resistant or rapidly acquires resistance to targeted therapy treatments, such as MAPK inhibitors (MAPKi). A leading cause of resistance to targeted therapy is a dynamic transition of melanoma cells from a proliferative to a highly invasive state, a phenomenon called phenotype switching. Mechanisms regulating phenotype switching represent potential targets for improving treatment of patients with melanoma. Using a drug screen targeting chromatin regulators in patient-derived three-dimensional MAPKi-resistant melanoma cell cultures, we discovered that PARP inhibitors (PARPi) restore sensitivity to MAPKis, independent of DNA damage repair pathways. Integrated transcriptomic, proteomic, and epigenomic analyses demonstrated that PARPis induce lysosomal autophagic cell death, accompanied by enhanced mitochondrial lipid metabolism that ultimately increases antigen presentation and sensitivity to T-cell cytotoxicity. Moreover, transcriptomic and epigenetic rearrangements induced by PARP inhibition reversed epithelial–mesenchymal transition-like phenotype switching, which redirected melanoma cells toward a proliferative and MAPKi-sensitive state. The combination of PARP and MAPKis synergistically induced cancer cell death both in vitro and in vivo in patient-derived xenograft models. Therefore, this study provides a scientific rationale for treating patients with melanoma with PARPis in combination with MAPKis to abrogate acquired therapy resistance.Significance:PARP inhibitors can overcome resistance to MAPK inhibitors by activating autophagic cell death and reversing phenotype switching, suggesting that this synergistic combination could help improve the prognosis of patients with melanoma.

Metastatic melanoma is either intrinsically resistant or rapidly acquires resistance to targeted therapy treatments, such as MAPK inhibitors (MAPKi). A leading cause of resistance to targeted therapy is a dynamic transition of melanoma cells from a proliferative to a highly invasive state, a phenomenon called phenotype switching. Mechanisms regulating phenotype switching represent potential targets for improving treatment of patients with melanoma. Using a drug screen targeting chromatin regulators in patient-derived three-dimensional MAPKi-resistant melanoma cell cultures, we discovered that PARP inhibitors (PARPi) restore sensitivity to MAPKis, independent of DNA damage repair pathways. Integrated transcriptomic, proteomic, and epigenomic analyses demonstrated that PARPis induce lysosomal autophagic cell death, accompanied by enhanced mitochondrial lipid metabolism that ultimately increases antigen presentation and sensitivity to T-cell cytotoxicity. Moreover, transcriptomic and epigenetic rearrangements induced by PARP inhibition reversed epithelial–mesenchymal transition-like phenotype switching, which redirected melanoma cells toward a proliferative and MAPKi-sensitive state. The combination of PARP and MAPKis synergistically induced cancer cell death both in vitro and in vivo in patient-derived xenograft models. Therefore, this study provides a scientific rationale for treating patients with melanoma with PARPis in combination with MAPKis to abrogate acquired therapy resistance. Significance: PARP inhibitors can overcome resistance to MAPK inhibitors by activating autophagic cell death and reversing phenotype switching, suggesting that this synergistic combination could help improve the prognosis of patients with melanoma.

Tumor necrosis factor (TNF) is a key component of the innate immune response. Upon binding to its receptor, TNFR1, it promotes production of other cytokines via a membrane-bound complex 1 or induces cell death via a cytosolic complex 2. To understand how TNF-induced cell death is regulated, we performed mass spectrometry of complex 2 and identified tankyrase-1 as a native component that, upon a death stimulus, mediates complex 2 poly–ADP-ribosylation (PARylation). PARylation promotes recruitment of the E3 ligase RNF146, resulting in proteasomal degradation of complex 2, thereby limiting cell death. Expression of the ADP-ribose–binding/hydrolyzing severe acute respiratory syndrome coronavirus 2 macrodomain sensitizes cells to TNF-induced death via abolishing complex 2 PARylation. This suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

Blood and plasma proteins are heavily investigated as biomarkers for different diseases. However, the post-translational modification states of these proteins are rarely analyzed since blood contains many enzymes that rapidly remove these modifications after sampling. In contrast to the well-described role of protein ADP-ribosylation in cells and organs, its role in blood remains mostly uncharacterized. Here, we discovered that plasma phosphodiesterases and/or ADP-ribosylhydrolases rapidly demodify in vitro ADP-ribosylated proteins. Thus, to identify the in vivo whole blood and plasma ADP-ribosylomes, we established a mass-spectrometry-based workflow that was applied to blood samples collected from LPS-treated pigs (Sus scrofa domesticus), which serves as a model for human systemic inflammatory response syndrome. These analyses identified 60 ADP-ribosylated proteins, 17 of which were ADP-ribosylated plasma proteins. This new protocol provides an important step forward for the rapidly developing field of ADP-ribosylation and defines the blood and plasma ADP-ribosylomes under both healthy and disease conditions.

Blood and plasma proteins are heavily investigated as biomarkers for different diseases. However, the post-translational modification states of these proteins are rarely analyzed since blood contains many enzymes that rapidly remove these modification after sampling. In contrast to the well-described role of protein ADP-ribosylation in cells and organs, its role in blood remains mostly uncharacterized. Here, we discovered that plasma phosphodiesterases and/or ADP-ribosylhydrolases rapidly demodify in vitro ADP-ribosylated proteins. Thus, to identify the in vivo whole blood and plasma ADP-ribosylomes, we established a novel mass-spectrometry based workflow that was applied to blood samples collected from LPS-treated pigs (Sus scrofa), which serves as a model for human systemic inflammatory response syndrome. These analyses identified 60 ADP-ribosylated proteins, 17 of which were ADP-ribosylated plasma proteins. This new protocol provides an important step forward for the rapidly developing field of ADP-ribosylation and defines the blood and plasma ADP-ribosylomes under both healthy and disease conditions.

Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions.