RESUMO
Glycosylation is a ubiquitous modification of proteins, necessitating approaches for its visualization and characterization. Bioorthogonally tagged monosaccharides have been instrumental to this end, offering a chemical view into the cell biology of glycans. Understanding the use of such monosaccharides by cellular biosynthetic pathways has expanded their applicability in cell biology, for instance through the strategy named Bio-Orthogonal Cell-specific TAgging of Glycoproteins (BOCTAG). Here, we show that the cellular use of two azide-tagged analogues of the monosaccharide N-acetylgalactosamine (GalNAzMe and GalNPrAz) can be promoted through expression of two biosynthetic enzymes. More precisely, cellular expression of the bacterial kinase NahK and the engineered human pyrophosphorylase AGX1F383A led to biosynthesis of the corresponding activated nucleotide-sugars and subsequent bioorthogonal tagging of the cellular glycoproteome. We explore the use of both sugars for BOCTAG, demonstrating the visualization of cell surface glycosylation tagged with GalNPrAz in a specific cell line in a co-culture system. Our work adds to the toolbox of glycoprotein analysis in biomedicine.
RESUMO
The F-box and WD repeat domain containing 7 (FBXW7) tumour suppressor gene encodes a substrate-recognition subunit of Skp, cullin, F-box (SCF)-containing complexes. The tumour-suppressive role of FBXW7 is ascribed to its ability to drive ubiquitination and degradation of oncoproteins. Despite this molecular understanding, therapeutic approaches that target defective FBXW7 have not been identified. Using genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens, focussed RNA-interference screens and whole and phospho-proteome mass spectrometry profiling in multiple FBXW7 wild-type and defective isogenic cell lines, we identified a number of FBXW7 synthetic lethal targets, including proteins involved in the response to replication fork stress and proteins involved in replication origin firing, such as cell division cycle 7-related protein kinase (CDC7) and its substrate, DNA replication complex GINS protein SLD5 (GINS4). The CDC7 synthetic lethal effect was confirmed using small-molecule inhibitors. Mechanistically, FBXW7/CDC7 synthetic lethality is dependent upon the replication factor telomere-associated protein RIF1 (RIF1), with RIF1 silencing reversing the FBXW7-selective effects of CDC7 inhibition. The delineation of FBXW7 synthetic lethal effects we describe here could serve as the starting point for subsequent drug discovery and/or development in this area.
Assuntos
Proteínas de Ciclo Celular , Neoplasias , Humanos , Proteína 7 com Repetições F-Box-WD/genética , Linhagem Celular Tumoral , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ubiquitinação , Interferência de RNA , Domínios Proteicos , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Cromossômicas não Histona/genéticaRESUMO
The PSMC3IP-MND1 heterodimer promotes meiotic D loop formation before DNA strand exchange. In genome-scale CRISPR-Cas9 mutagenesis and interference screens in mitotic cells, depletion of PSMC3IP or MND1 causes sensitivity to poly (ADP-Ribose) polymerase inhibitors (PARPi) used in cancer treatment. PSMC3IP or MND1 depletion also causes ionizing radiation sensitivity. These effects are independent of PSMC3IP/MND1's role in mitotic alternative lengthening of telomeres. PSMC3IP- or MND1-depleted cells accumulate toxic RAD51 foci in response to DNA damage, show impaired homology-directed DNA repair, and become PARPi sensitive, even in cells lacking both BRCA1 and TP53BP1. Epistasis between PSMC3IP-MND1 and BRCA1/BRCA2 defects suggest that abrogated D loop formation is the cause of PARPi sensitivity. Wild-type PSMC3IP reverses PARPi sensitivity, whereas a PSMC3IP p.Glu201del mutant associated with D loop defects and ovarian dysgenesis does not. These observations suggest that meiotic proteins such as MND1 and PSMC3IP have a greater role in mitotic DNA repair.
Assuntos
Antineoplásicos , Inibidores de Poli(ADP-Ribose) Polimerases , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Reparo do DNA , Dano ao DNA , Proteína BRCA1/genética , Reparo de DNA por Recombinação , Linhagem Celular TumoralRESUMO
To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.