Project description:DNA replication stress promotes cancer development and is associated with DNA synthesis beyond S-phase. Replication stress-induced DNA lesions and under-replicated DNA can persist into G2 and mitosis, where they undergo mitotic DNA synthesis (MiDAS). After cell division, inherited DNA lesions are protected by genome caretaker proteins and show signs of post-mitotic DNA synthesis (post-MiDAS) in G1. The genomic regions that undergo post-MiDAS, the cellular factors involved, and the implications for genome integrity remain poorly understood. Here, we provide evidence for post-MiDAS activity at centromeres and telomeres in cancer cells employing alternative lengthening of telomeres (ALT) and present data suggesting that a subset of these fragile genomic regions can cluster during post-MiDAS in G1. We further show that post-MiDAS is promoted by local SUMOylation and BLM recruitment into ALT-associated PML bodies (APBs) and involves DNA damage tolerance factors and proteins associated with ALT and MiDAS. Consistent with G1-specific regulation, complementary quantitative proteomics and targeted siRNA screening reveal that APC/C, together with its G1 co-activator CDH1 but independent of its mitotic co-activator CDC20, restrains excessive post-MiDAS in G1. Finally, we show that CDH1 depletion selectively impairs the survival of ALT-positive cancer cell lines, indicating that post-MiDAS might represent a potential ALT-specific vulnerability. This dataset contains RPA70 immunoprecipitation mass spectrometry data generated from chromatin-bound protein extracts of FACS-sorted G1-phase U-2 OS FUCCI cells to identify the interactome of RPA70 in G1.
Project description:DNA replication stress promotes cancer development and is associated with DNA synthesis beyond S-phase. Replication stress-induced DNA lesions and under-replicated DNA can persist into G2 and mitosis, where they undergo mitotic DNA synthesis (MiDAS). After cell division, inherited DNA lesions are protected by genome caretaker proteins and show signs of post-mitotic DNA synthesis (post-MiDAS) in G1. The genomic regions that undergo post-MiDAS, the cellular factors involved, and the implications for genome integrity remain poorly understood. Here, we provide evidence for post-MiDAS activity at centromeres and telomeres in cancer cells employing alternative lengthening of telomeres (ALT) and present data suggesting that a subset of these fragile genomic regions can cluster during post-MiDAS in G1. We further show that post-MiDAS is promoted by local SUMOylation and BLM recruitment into ALT-associated PML bodies (APBs) and involves DNA damage tolerance factors and proteins associated with ALT and MiDAS. Consistent with G1-specific regulation, complementary quantitative proteomics and targeted siRNA screening reveal that APC/C, together with its G1 co-activator CDH1 but independent of its mitotic co-activator CDC20, restrains excessive post-MiDAS in G1. Finally, we show that CDH1 depletion selectively impairs the survival of ALT-positive cancer cell lines, indicating that post-MiDAS might represent a potential ALT-specific vulnerability. This dataset contains RPA70 immunoprecipitation mass spectrometry data generated from whole-cell protein extracts of FACS-sorted G1-phase U-2 OS FUCCI cells to identify the interactome of RPA70 in G1.
Project description:Endometriosis shows significant lesion heterogeneity, but pathophysiological mechanisms leading to differences in clinical presentation are poorly understood. Here we utilized primary stromal cells from different types of endometriotic lesions to better understand the molecular mechanisms that lead to endometriosis heterogeneity. For this aim, endometrial stromal cells (ESCs) were obtained from eutopic endometrium (EU), endometrioma (OMA), superficial (SF) and deep (DE) endometriosis lesions and analyzed in vitro for their behavior regarding proliferation, migration, and contractility. Proteomics was used to explore the molecular mechanism underlying the observed changes.
Project description:Dysfunction in lysosomal membrane proteins and their associated complexes impairs the lysosome's essential role as a key signaling hub within the cell. This disruption underlies severe neurological disorders, such as lysosomal storage diseases and cancer. While direct visualization of the proteomic membrane environment of isolated lysosomes is crucial for advancing our understanding of its functions, it remains a significant challenge. To overcome this, we developed a method to enrich intact lysosomes using the essential lysosomal ion channel, transient receptor potential mucolipin 1. This enabled us to employ cryo electron tomography to reveal the heterogeneous molecular landscape of lysosomes. Notably, in concordance with quantitative mass spectrometry we identified protein densities on lysosomal membranes consistent with V-ATPase, Flotillin, clathrin-coated vesicles, mTORC1, HOPS, VPS13C, and dynein-dynactin. These findings demonstrate that our method offers a robust platform for advancing the structural and functional understanding of individual lysosomes, facilitating the visualization and resolution of endogenous protein complexes.
Project description:Mitofusin-1 (MFN1) and Mitofusin-2 (MFN2) are key players in mitochondrial fusion, endoplasmic reticulum (ER)-mitochondria yuxtaposition, and autophagy. However, the mechanisms by which these proteins participate in these processes remain poorly understood. To better understand their functions, we studied the interactomes of these two proteins. To this end, we used CRISPR/Cas9 technology to insert an HA-tag in the C-terminal domain of MFN1 and MFN2 and generated HeLa cell lines that endogenously expressed MFN1-HA or MFN2-HA, respectively. HA-pulldown followed by mass spectrometry identified potential interactors of MFN1 and MFN2. A substantial proportion of interactors were common for MFN1 and MFN2 and were regulated by nutrient deprivation. We validated novel ER and endosomal partners of MFN1 and/or MFN2 with a potential role in interorganelle communication. We characterized RAB5C as an endosomal modulator of mitochondrial dynamics through its interaction with MFN1 and SLC27A2 as a novel partner of MFN2 relevant in autophagy. Our findings reveal that MFN proteins participate in nutrient-modulated pathways involved in organelle communication.
Project description:To understand the transcriptomic, genomic and proteomics changes between HER2 overexpressingcells and those that have become resistant to lapatinib
Project description:A series of valdiazen chemical probes were developed and applied in affinity-based pulldown experiments to enrich potential valdiazen-binding proteins.
Project description:The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity, and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. The Estrogen Receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease. Applying interaction proteomics coupled to mass spectrometry (MS) to characterize nuclear factors cooperating with ERβ in gene regulation, we identify AGO2 as a novel partner of ERβ in human BC cells. ERβ-AGO2 association was confirmed in vitro and in vivo both in the nucleus and in cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association to a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ+ vs ERβ- cells, and before and after AGO2 knock-down in ERβ+ cells, reveals a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may able to control directly also mRNA translation efficiency and stability.These results demonstrate that AGO2 can act as a pleiotropic functional partner of ERβ, indicating that both factors are endowed with multiple roles in the control of key cellular functions
Project description:In the project “Dysregulated lipid metabolism and hypomyelination in postnatal peroxisome-deficient Pex2 knockout Zellweger mice” label-free mass spectrometry proteomics was used to study the proteome in different regions of the central nervous system of wild-type and peroxisome-deficient Pex2 knockout mice. Four sets of experiments were performed