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- Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome (2019)
- Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.
- Contributions of ubiquitin- and PCNA-binding domains to the activity of Polymerase η in Saccharomyces cerevisiae (2007)
- Bypassing of DNA lesions by damage-tolerant DNA polymerases depends on the interaction of these enzymes with the monoubiquitylated form of the replicative clamp protein, PCNA. We have analyzed the contributions of ubiquitin and PCNA binding to damage bypass and damage-induced mutagenesis in Polymerase {eta} (encoded by RAD30) from the budding yeast Saccharomyces cerevisiae. We report here that a ubiquitin-binding domain provides enhanced affinity for the ubiquitylated form of PCNA and is essential for in vivo function of the polymerase, but only in conjunction with a basal affinity for the unmodified clamp, mediated by a conserved PCNA interaction motif. We show that enhancement of the interaction and function in damage tolerance does not depend on the ubiquitin attachment site within PCNA. Like its mammalian homolog, budding yeast Polymerase {eta} itself is ubiquitylated in a manner dependent on its ubiquitin-binding domain.
- BAG3 overexpression and cytoprotective autophagy mediate apoptosis resistance in chemoresistant breast cancer cells (2018)
- Target-specific treatment modalities are currently not available for triple-negative breast cancer (TNBC), and acquired chemotherapy resistance is a primary obstacle for the treatment of these tumors. Here we employed derivatives of BT-549 and MDA-MB-468 TNBC cell lines that were adapted to grow in the presence of either 5-Fluorouracil, Doxorubicin or Docetaxel in an aim to identify molecular pathways involved in the adaptation to drug-induced cell killing. All six drug-adapted BT-549 and MDA-MB-468 cell lines displayed cross resistance to chemotherapy and decreased apoptosis sensitivity. Expression of the anti-apoptotic co-chaperone BAG3 was notably enhanced in two thirds (4/6) of the six resistant lines simultaneously with higher expression of HSP70 in comparison to parental controls. Doxorubicin-resistant BT-549 (BT-549rDOX20) and 5-Fluorouracil-resistant MDA-MB-468 (MDA-MB-468r5-FU2000) cells were chosen for further analysis with the autophagy inhibitor Bafilomycin A1 and lentiviral depletion of ATG5, indicating that enhanced cytoprotective autophagy partially contributes to increased drug resistance and cell survival. Stable lentiviral BAG3 depletion was associated with a robust down-regulation of Mcl-1, Bcl-2 and Bcl-xL, restoration of drug-induced apoptosis and reduced cell adhesion in these cells, and these death-sensitizing effects could be mimicked with the BAG3/Hsp70 interaction inhibitor YM-1 and by KRIBB11, a selective transcriptional inhibitor of HSF-1. Furthermore, BAG3 depletion was able to revert the EMT-like transcriptional changes observed in BT-549rDOX20 and MDA-MB-468r5-FU2000 cells. In summary, genetic and pharmacological interference with BAG3 is capable to resensitize TNBC cells to treatment, underscoring its relevance for cell death resistance and as a target to overcome therapy resistance of breast cancer.
- Manatee invariants reveal functional pathways in signaling networks (2017)
- Background: Signal transduction pathways are important cellular processes to maintain the cell’s integrity. Their imbalance can cause severe pathologies. As signal transduction pathways feature complex regulations, they form intertwined networks. Mathematical models aim to capture their regulatory logic and allow an unbiased analysis of robustness and vulnerability of the signaling network. Pathway detection is yet a challenge for the analysis of signaling networks in the field of systems biology. A rigorous mathematical formalism is lacking to identify all possible signal flows in a network model. Results: In this paper, we introduce the concept of Manatee invariants for the analysis of signal transduction networks. We present an algorithm for the characterization of the combinatorial diversity of signal flows, e.g., from signal reception to cellular response. We demonstrate the concept for a small model of the TNFR1-mediated NF- κB signaling pathway. Manatee invariants reveal all possible signal flows in the network. Further, we show the application of Manatee invariants for in silico knockout experiments. Here, we illustrate the biological relevance of the concept. Conclusions: The proposed mathematical framework reveals the entire variety of signal flows in models of signaling systems, including cyclic regulations. Thereby, Manatee invariants allow for the analysis of robustness and vulnerability of signaling networks. The application to further analyses such as for in silico knockout was shown. The new framework of Manatee invariants contributes to an advanced examination of signaling systems.
- Diagnostic and clinical relevance of the autophago-lysosomal network in human gliomas (2016)
- Recently, the conserved intracellular digestion mechanism ‘autophagy’ has been considered to be involved in early tumorigenesis and its blockade proposed as an alternative treatment approach. However, there is an ongoing debate about whether blocking autophagy has positive or negative effects in tumor cells. Since there is only poor data about the clinico-pathological relevance of autophagy in gliomas in vivo, we first established a cell culture based platform for the in vivo detection of the autophago-lysosomal components. We then investigated key autophagosomal (LC3B, p62, BAG3, Beclin1) and lysosomal (CTSB, LAMP2) molecules in 350 gliomas using immunohistochemistry, immunofluorescence, immunoblotting and qPCR. Autophagy was induced pharmacologically or by altering oxygen and nutrient levels. Our results show that autophagy is enhanced in astrocytomas as compared to normal CNS tissue, but largely independent from the WHO grade and patient survival. A strong upregulation of LC3B, p62, LAMP2 and CTSB was detected in perinecrotic areas in glioblastomas suggesting micro-environmental changes as a driver of autophagy induction in gliomas. Furthermore, glucose restriction induced autophagy in a concentration-dependent manner while hypoxia or amino acid starvation had considerably lesser effects. Apoptosis and autophagy were separately induced in glioma cells both in vitro and in vivo. In conclusion, our findings indicate that autophagy in gliomas is rather driven by micro-environmental changes than by primary glioma-intrinsic features thus challenging the concept of exploitation of the autophago-lysosomal network (ALN) as a treatment approach in gliomas.
- Arsenic trioxide and (−)-gossypol synergistically target glioma stem-like cells via inhibition of hedgehog and notch signaling (2019)
- Glioblastoma is one of the deadliest malignancies and is virtually incurable. Accumulating evidence indicates that a small population of cells with a stem-like phenotype is the major culprit of tumor recurrence. Enhanced DNA repair capacity and expression of stemness marker genes are the main characteristics of these cells. Elimination of this population might delay or prevent tumor recurrence following radiochemotherapy. The aim of this study was to analyze whether interference with the Hedgehog signaling (Hh) pathway or combined Hh/Notch blockade using small-molecule inhibitors can efficiently target these cancer stem cells and sensitize them to therapy. Using tumor sphere lines and primary patient-derived glioma cultures we demonstrate that the Hh pathway inhibitor GANT61 (GANT) and the arsenic trioxide (ATO)-mediated Hh/Notch inhibition are capable to synergistically induce cell death in combination with the natural anticancer agent (−)-Gossypol (Gos). Only ATO in combination with Gos also strongly decreased stemness marker expression and prevented sphere formation and recovery. These synergistic effects were associated with distinct proteomic changes indicating diminished DNA repair and markedly reduced stemness. Finally, using an organotypic brain slice transplantation model, we show that combined ATO/Gos treatment elicits strong growth inhibition or even complete elimination of tumors. Collectively, our data show for the first time that ATO and Gos, two drugs that can be used in the clinic, represent a promising targeted therapy approach for the synergistic elimination of glioma stem-like cells.
- Sharpin prevents skin inflammation by inhibiting TNFR1-induced keratinocyte apoptosis (2014)
- Linear Ubiquitin chain Assembly Complex (LUBAC) is an E3 ligase complex that generates linear ubiquitin chains and is important for tumour necrosis factor (TNF) signaling activation. Mice lacking Sharpin, a critical subunit of LUBAC, spontaneously develop inflammatory lesions in the skin and other organs. Here we show that TNF receptor 1 (TNFR1)-associated death domain (TRADD)-dependent TNFR1 signaling in epidermal keratinocytes drives skin inflammation in Sharpin-deficient mice. Epidermis-restricted ablation of Fas-associated protein with death domain (FADD) combined with receptor-interacting protein kinase 3 (RIPK3) deficiency fully prevented skin inflammation, while single RIPK3 deficiency only delayed and partly ameliorated lesion development in Sharpin-deficient mice, showing that inflammation is primarily driven by TRADD- and FADD-dependent keratinocyte apoptosis while necroptosis plays a minor role. At the cellular level, Sharpin deficiency sensitized primary murine keratinocytes, human keratinocytes, and mouse embryonic fibroblasts to TNF-induced apoptosis. Depletion of FADD or TRADD in Sharpin-deficient HaCaT cells suppressed TNF-induced apoptosis, indicating the importance of FADD and TRADD in Sharpin-dependent anti-apoptosis signaling in keratinocytes.
- Ubiquitylation in immune disorders and cancer: from molecular mechanisms to therapeutic implications (2012)
- Conjugation of ubiquitin to proteins (ubiquitylation) has emerged to be one of the most crucial post-translational modifications controlling virtually all cellular processes. What was once regarded as a mere signal for protein degradation has turned out to be a major regulator of molecular signalling networks. Deregulation of ubiquitin signalling is closely associated with various human pathologies. Here, we summarize the current knowledge of ubiquitin signalling in immune deficiencies and cancer as well as the available therapeutic strategies targeting the ubiquitin system in combating these pathogenic conditions.
- IKKα controls ATG16L1 degradation to prevent ER stress during inflammation (2017)
- Inhibition of the IκB kinase complex (IKK) has been implicated in the therapy of several chronic inflammatory diseases including inflammatory bowel diseases. In this study, using mice with an inactivatable IKKα kinase (IkkαAA/AA), we show that loss of IKKα function markedly impairs epithelial regeneration in a model of acute colitis. Mechanistically, this is caused by compromised secretion of cytoprotective IL-18 from IKKα-mutant intestinal epithelial cells because of elevated caspase 12 activation during an enhanced unfolded protein response (UPR). Induction of the UPR is linked to decreased ATG16L1 stabilization in IkkαAA/AA mice. We demonstrate that both TNF-R and nucleotide-binding oligomerization domain stimulation promote ATG16L1 stabilization via IKKα-dependent phosphorylation of ATG16L1 at Ser278. Thus, we propose IKKα as a central mediator sensing both cytokine and microbial stimulation to suppress endoplasmic reticulum stress, thereby assuring antiinflammatory function during acute intestinal inflammation.
