Chemical genetic methodologies for identifying protein substrates of PARPs.

Poly-ADP-ribose-polymerases (PARPs) are a family of 17 enzymes that regulate a diverse range of cellular processes in mammalian cells. PARPs catalyze the transfer of ADP-ribose from NAD+ to target molecules, most prominently amino acids on protein substrates, in a process known as ADP-ribosylation. Identifying the direct protein substrates of individual PARP family members is an essential first step for elucidating the mechanism by which PARPs regulate a particular pathway in cells. Two distinct chemical genetic (CG) strategies have been developed for identifying the direct protein substrates of individual PARP family members. In this review, we discuss the design principles behind these two strategies and how target identification has provided novel insight into the cellular function of individual PARPs and PARP-mediated ADP-ribosylation.

HDAC activity is dispensable for repression of cell-cycle genes by DREAM and E2F:RB complexes.

Histone deacetylases (HDACs) play a crucial role in transcriptional regulation and are implicated in various diseases, including cancer. They are involved in histone tail deacetylation and canonically linked to transcriptional repression. Previous studies suggested that HDAC recruitment to cell-cycle gene promoters via the retinoblastoma (RB) protein or the DREAM complex through SIN3B is essential for G1/S and G2/M gene repression during cell-cycle arrest and exit. Here we investigate the interplay among DREAM, RB, SIN3 proteins, and HDACs in the context of cell-cycle gene repression. Knockout of SIN3B does not globally derepress cell-cycle genes in non-proliferating HCT116 and C2C12 cells. Loss of SIN3A/B moderately upregulates several cell-cycle genes in HCT116 cells but does so independently of DREAM/RB. HDAC inhibition does not induce general upregulation of RB/DREAM target genes in arrested transformed or non-transformed cells. Our findings suggest that E2F:RB and DREAM complexes can repress cell-cycle genes without relying on HDAC activity.

Rapid cell type-specific nascent proteome labeling in Drosophila.

Controlled protein synthesis is required to regulate gene expression and is often carried out in a cell type-specific manner. Protein synthesis is commonly measured by labeling the nascent proteome with amino acid analogs or isotope-containing amino acids. These methods have been difficult to implement in vivo as they require lengthy amino acid replacement procedures. O-propargyl-puromycin (OPP) is a puromycin analog that incorporates into nascent polypeptide chains. Through its terminal alkyne, OPP can be conjugated to a fluorophore-azide for directly visualizing nascent protein synthesis, or to a biotin-azide for capture and identification of newly-synthesized proteins. To achieve cell type-specific OPP incorporation, we developed phenylacetyl-OPP (PhAc-OPP), a puromycin analog harboring an enzyme-labile blocking group that can be removed by penicillin G acylase (PGA). Here, we show that cell type-specific PGA expression in Drosophila can be used to achieve OPP labeling of newly-synthesized proteins in targeted cell populations within the brain. Following a brief 2 hr incubation of intact brains with PhAc-OPP, we observe robust imaging and affinity purification of OPP-labeled nascent proteins in PGA-targeted cell populations. We apply this method to show a pronounced age-related decline in neuronal protein synthesis in the fly brain, demonstrating the capability of PhAc-OPP to quantitatively capture in vivo protein synthesis states. This method, which we call POPPi (PGA-dependent OPP incorporation), should be applicable for rapidly visualizing protein synthesis and identifying nascent proteins synthesized under diverse physiological and pathological conditions with cellular specificity in vivo.

HDAC activity is dispensable for repression of cell-cycle genes by DREAM and E2F:RB complexes.

Histone deacetylases (HDACs) are pivotal in transcriptional regulation, and their dysregulation has been associated with various diseases including cancer. One of the critical roles of HDAC-containing complexes is the deacetylation of histone tails, which is canonically linked to transcriptional repression. Previous research has indicated that HDACs are recruited to cell-cycle gene promoters through the RB protein or the DREAM complex via SIN3B and that HDAC activity is essential for repressing G1/S and G2/M cell-cycle genes during cell-cycle arrest and exit. In this study, we sought to explore the interdependence of DREAM, RB, SIN3 proteins, and HDACs in the context of cell-cycle gene repression. We found that genetic knockout of SIN3B did not lead to derepression of cell-cycle genes in non-proliferating HCT116 and C2C12 cells. A combined loss of SIN3A and SIN3B resulted in a moderate upregulation in mRNA expression of several cell-cycle genes in arrested HCT116 cells, however, these effects appeared to be independent of DREAM or RB. Furthermore, HDAC inhibition did not induce a general upregulation of RB and DREAM target gene expression in arrested transformed or non-transformed cells. Our findings provide evidence that E2F:RB and DREAM complexes can repress cell-cycle genes without reliance on HDAC activity.

Structurally distinct PARP7 inhibitors provide new insights into the function of PARP7 in regulating nucleic acid-sensing and IFN-ß signaling.

The mono-ADP-ribosyltransferase PARP7 has emerged as a key negative regulator of cytosolic NA-sensors of the innate immune system. We apply a rational design strategy for converting a pan-PARP inhibitor into a potent selective PARP7 inhibitor (KMR-206). Consistent with studies using the structurally distinct PARP7 inhibitor RBN-2397, co-treatment of mouse embryonic fibroblasts with KMR-206 and NA-sensor ligands synergistically induced the expression of the type I interferon, IFN-ß. In mouse colon carcinoma (CT-26) cells, KMR-206 alone induced IFN-ß. Both KMR-206 and RBN-2397 increased PARP7 protein levels in CT-26 cells, demonstrating that PARP7's catalytic activity regulates its own protein levels. Curiously, treatment with saturating doses of KMR-206 and RBN-2397 achieved different levels of PARP7 protein, which correlated with the magnitude of type I interferon gene expression. These latter results have important implications for the mechanism of action of PARP7 inhibitors and highlights the usefulness of having structurally distinct chemical probes for the same target.

Chemical genetics and proteome-wide site mapping reveal cysteine MARylation by PARP-7 on immune-relevant protein targets.

Poly(ADP-ribose) polymerase 7 (PARP-7) has emerged as a critically important member of a large enzyme family that catalyzes ADP-ribosylation in mammalian cells. PARP-7 is a critical regulator of the innate immune response. What remains unclear is the mechanism by which PARP-7 regulates this process, namely because the protein targets of PARP-7 mono-ADP-ribosylation (MARylation) are largely unknown. Here, we combine chemical genetics, proximity labeling, and proteome-wide amino acid ADP-ribosylation site profiling for identifying the direct targets and sites of PARP-7-mediated MARylation in a cellular context. We found that the inactive PARP family member, PARP-13-a critical regulator of the antiviral innate immune response-is a major target of PARP-7. PARP-13 is preferentially MARylated on cysteine residues in its RNA binding zinc finger domain. Proteome-wide ADP-ribosylation analysis reveals cysteine as a major MARylation acceptor of PARP-7. This study provides insight into PARP-7 targeting and MARylation site preference.

Peptide to Peptoid Substitutions Increase Cell Permeability in Cyclic Hexapeptides.

The effect of peptide-to-peptoid substitutions on the passive membrane permeability of an N-methylated cyclic hexapeptide is examined. In general, substitutions maintained permeability but increased conformational heterogeneity. Diversification with nonproteinogenic side chains increased permeability up to 3-fold. Additionally, the conformational impact of peptoid substitutions within a ß-turn are explored. Based on these results, the strategic incorporation of peptoid residues into cyclic peptides can maintain or improve cell permeability, while increasing access to diverse side-chain functionality.