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Microtubule associated proteins MAP7 and MAP7D1 promote 53BP1-mediated DNA double strand break repair in G1 cell cycle phase

ABSTRACT: In this project we have been looking for proteins binding to phosphorylated sites in DNA repair proteins. Previously, we and others have used peptide-pull down assays as a direct means of attributing specific binding functions to known sites phosphorylated by CK2 in DDR proteins. We have now applied the approach to identify proteins binding to four in vivo phosphorylated sites from DDR proteins RAD50 (Thr690), BRCA1 (Ser1336), MLH1 (Ser477) and XPC (Ser883, 884). These sites are conserved, reside within consensus sites for CK2, share similar sequence features, are mutated in cancer and/or their phosphorylation is known to affect (or is affected) by the DDR. We designed biotinylated 20 amino acid (aa) peptides encompassing the specific phosphorylated amino acids of BRCA1, RAD50, MLH1 and XPC that were either non-phosphorylated or phosphorylated. The biotinylated peptides were coupled to streptavidin beads and used to pull down proteins from HeLa nuclear extracts. Proteomic analysis revealed that all four sites bind proteins in a phospho-dependent manner. Each of the phosphorylated sites were binding various proteins including those implicated in DDR and cell cycle regulation as well as proteins involved in other cellular processes; some of these proteins have already known phospho-binding domains, while others do not. Interestingly, microtubule associated protein MAP7 featured prominently in all of the phosphorylated RAD50, BRCA1, MLH1 and XPC peptide pull-downs and MAP7 paralogue MAP7D1 also featured in the phosphorylated BRCA1 peptide pull-down. In the rest of the project we study the interactions and their function in DNA repair.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Vinothini Rajeeve

LAB HEAD: Professor Pedro R. Cutillas

PROVIDER: PXD033715 | Pride | 2023-03-01

REPOSITORIES: Pride

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Project description:PD-L1 Inhibitor Regulates the miR-33a-5p/PTEN Signaling Pathway and Can Be Targeted to Sensitize Glioblastomas to Radiation. Glioblastoma (GBM) is the most common and lethal brain tumor in adults. Ionizing radiation (IR) is a standard treatment for GBM patients and results in DNA damage. However, the clinical efficacy of IR is limited due to therapeutic resistance. The programmed death ligand 1 (PD-L1) blockade has a shown the potential to increase the efficacy of radiotherapy by inhibiting DNA damage and repair responses. The miR-33a-5p is an essential microRNA that promotes GBM growth and self-renewal. In this study, we investigated whether a PD-L1 inhibitor (a small molecule inhibitor) exerted radio-sensitive effects to impart an anti-tumor function in GBM cells by modulating miR-33a-5p. U87 MG cells and U251 cells were pretreated with PD-L1 inhibitor. The PD-L1 inhibitor-induced radio-sensitivity in these cells was assessed by assaying cellular apoptosis, clonogenic survival assays, and migration. TargetScan and luciferase assay showed that miR-33a-5p targeted the phosphatase and tensin homolog (PTEN) 3' untranslated region. The expression level of PTEN was measured by western blotting, and was also silenced using small interfering RNAs. The levels of DNA damage following radiation was measured by the presence of γ-H2AX foci, cell cycle, and the mRNA of the DNA damage-related genes, BRCA1, NBS1, RAD50, and MRE11. Our results demonstrated that the PD-L1 inhibitor significantly decreased the expression of the target gene, miR-33a-5p. In addition, pretreatment of U87 MG and U251 cells with the PD-L1 inhibitor increased radio-sensitivity, as indicated by increased apoptosis, while decreased survival and migration of GBM cells. Mir-33a-5p overexpression or silencing PTEN in U87 MG and U251 cells significantly attenuated PD-L1 radiosensitive effect. Additionally, PD-L1 inhibitor treatment suppressed the expression of the DNA damage response-related genes, BRCA1, NBS1, RAD50, and MRE11. Our results demonstrated a novel role for the PD-L1 inhibitor in inducing radio- sensitivity in GBM cells, where inhibiting miR-33a-5p, leading to PTEN activated, and inducing DNA damage was crucial for antitumor immunotherapies to treat GBM.

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Microtubule associated proteins MAP7 and MAP7D1 promote 53BP1-mediated DNA double strand break repair in G1 cell cycle phase

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