2 research outputs found
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Exploring G-Protein-Coupled Receptors Regulation, Specificity and Controllability of Exosomes Release in the Neuronal Cell Line SH-SY5Y
Parkinson's disease is a neurodegenerative disease characterized by the buildup of aggregated and spread of misfolded alpha-synuclein. How the misfolded alpha-synuclein contributing to the toxicity and death of neuronal cells has been the focal point of research. The spread of alpha-synuclein has been attributed to many mechanisms, one of which is via cell-derived vesicles called exosomes. This project aims to examine the controllability of exosome release. SH-SY5Y, MCF-7 and CHO-K1 cells were transfected with dopamine receptor 3-green fluorescent protein, G-protein receptor 143 or green fluorescent protein and treated with either dopamine or L-DOPA. Medium was harvested and subjected to ultracentrifugation and a silver stain and western blot were performed. There was no significant difference in the total protein in the exosome fraction lanes between the treatment groups or within them. Another aim was to test the specificity of exosomes. Exosomes isolated from SH-SY5Y or MCF-7 were labeled with Exo-Red dye and introduced to wells containing SH-SY5Y, MCF-7 and CHO-K1 cells at room temperature and -4C. At room temperature, exosomes were observed intercellular in all of the cell lines, however, they did not deliver their content. At -4C exosome uptake was halted and they remained on the surface of the cells. Exo-Red labeled SH-SY5Y exosomes were treated with proteinase K and were introduced to CHO-K1 cells at -4C and room temperature. CHO-K1 did not take up exosomes, suggesting exosomes contain one or more necessary proteins needed to interact with the cellular membrane to initiate internalization. CHO-K1 cells were treated with versene to examine the involvement of integrin proteins. Exo-Red labeled SH-SY5Y exosomes were trapped on the surface of CHO-K1 after versene treatment. Lastly, Exo-Red labeled SH-SY5Y exosomes were biotinylated and magnetically captured then introduced to SH-SY5Y and MCF-7 cells and a silver stain and a biotinylated blot were performed. MCF-7 bound more Exo-Red labeled SH-SY5Y exosomes
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Investigation Into the Role of BAP1 in DNA Damage and Repair
The DNA of living organisms is constantly experiencing wide range of DNA damaging agents that can result in altered DNA bases or more serious damage such as double-strand breaks (DSBs). Unrepaired DNA damage can lead to genome instability and it is implicated in disease process including cancer.TP53 binding-protein 1 (53BP1) is a key DSB repair protein that regulates the repair pathway choice1. It is implicated in PARP inhibitor resistance in BRCA1-dificient tumors2,3. In order to identify novel 53BP1 regulators, we performed a screen using an siRNA library against the human deubiquitinating (DUBs) enzymes with 53BP1 ionizing radiation induced foci as a read out. Two DUBs achieved a top negative score; BRCA1 Associated protein 1 (BAP1). BAP1 is an important tumor suppressor protein whose function is lost in more than 7% of all cancers4. Here we demonstrate that BAP1 stabilizes 53BP1 by preventing its ubiquitination and degradation by the proteasome. BAP1 associates with 53BP1 and it is required for 53BP1 ionizing radiation (IR)-induced foci (IRIF) formation, G2/M checkpoint and radiation sensitivity. Similar to 53BP1, BAP1 deficiency confers PARP inhibitor resistance in BRCA1-deficient cells, and it leads to impaired immunoglobulin class switch recombination.
Additionally, we discovered a ubiquitin ligase, UBE2O interaction with 53BP1 diminished after IR damage, and overexpression of UBE2O leads to diminished 53BP1 IRIF formation. We show evidence that BAP1 and UBE2O binds 53BP1 nuclear localization signal (NLS) basic stretch within its minimal focus forming region (FFR), and they regulate FFR ubiquitination and recruitment to the histone basic patch. Through further work, we discovered that an NLS binding family (Karyopherin (KPNA) family) interacts with and regulates 53BP1 recruitment.
Our findings shed light into important aspects of 53BP1 regulation through deubiquitination and provide mechanistic insights into the function of an important tumor suppressor BAP1