Project description:Restriction-modification (R-M) systems protect against phage infection by detecting and degrading invading foreign DNA. However, like many prokaryotic anti-phage defenses, R-M systems pose a significant risk of auto-immunity, exacerbated by the presence of hundreds to thousands of potential cleavage sites in the bacterial genome. Pseudomonas aeruginosa strains experience the temporary inactivation of restriction endonucleases (tiREN) upon growth at high temperatures, but the mechanisms and implications of this are unknown. Here, we report that P. aeruginosa Type I restriction endonuclease (HsdR) is degraded, and the methyltransferase (HsdMS) is partially degraded, by two Lon-like proteases when replicating above 41 °C. This post-translational regulation prevents self-DNA targeting and leads to partial genomic hypomethylation, as demonstrated by SMRT sequencing and eTAM-seq. Interestingly, upon return to 37 ºC, restriction activity and full genomic methylation do not fully recover for up to 60 bacterial generations. Our findings demonstrate that Type I R-M is tightly regulated post-translationally with a long memory effect that ensures genomic stability and mitigates auto-toxicity.
Project description:OMI (Uniprot ID O43464) is a pro-apoptotic protease localized to the mitochondria which was hypothesized to activate senescense following X-ray irradiation via cleavage of specific ligands. To elucidate cellular processes affected by OMI after irradiation, human lung cancer cell line was subjected to the following treatments: WT + X-ray irradiation WT - X-ray irradiation WT + OMI-inhibitor + X-ray irradiation WT + OMI-inhibitor - X-ray irradiation OMI-siRNA + X-ray irradiation OMI-siRNA - X-ray irradiation siRNA-empty vector (control) + X-ray irradiation siRNA-empty vector (control) - X-ray irradiation
Project description:We performed a deep mass-spec analysis to determine DAP5 proteomic signature on human embryonic stem cells (hESCs) by evaluating the steady state proteins level on DAP5-KD and NT control cells.
Project description:Mutations in Lissencephaly 1 (LIS1) result in various human brain developmental diseases, such as changes in brain structure, lissencephaly, and epilepsy. RNA-sequencing data from on-chip organoids derived from human embryonic stem cells (hESCs) revealed significant changes in the expression of extracellular matrix (ECM) – related genes in LIS1+/- samples. This project examined the biomechanical properties of LIS1+/- mutated and healthy hESCs-derived cortical organoids. A rheological test using the pipette aspiration technique revealed that LIS1+/- cortical organoids are stiffer than control organoids. The increased stiffness of the LIS1+/- cortices was proportional to an increased expression of the nuclear mechano-sensing protein, Lamin A, highlighting the adverse cellular and nuclear changes underlying the stiffening of LIS1+/- organoids. To delineate the ECM composition associated with the stiffening effect in LIS1+/-, healthy and mutated hippocampal and cortical organoids were examined at the protein, mRNA, and miRNA levels. Whole RNA sequencing showed altered expression of multiple collagen-related pathways, including the 'collagens containing ECM' and the 'Collagen trimer' pathways. Differential mRNA expression has inversely correlated with the expression of their targeting miRNAs in the LIS1+/- organoids. This inverse miRNA-mRNA expression was most pronounced in genes associated with the ECM-receptor signalling pathway. At the protein level, the mutated hippocampal organoids showed unilateral, substantial increased expression of collagens and proteins involved in collagen synthesis, modification, and remodelling. Following a collagenolytic treatment with the catalytic domain of the MMP9 enzyme, the stiffness levels in the LIS1+/- organoids were reduced to control values. Overall, this work provides new information about the role of LIS1 in controlling collagen expression and the abnormal mechanical properties associated with mutation to the LIS1 gene in the development of the human cortex and hippocampus.
Project description:LIS1 mutations are known to cause lissencephaly, a neurological disorder characterized by the absence of cerebral cortical convolutions. Here we show that brain organoids with LIS1 mutations exhibit increased expression of extracellular matrix (ECM) proteins, which are less organized. The mechanical properties of the mutant organoids demonstrated increased stiffness and steady-state stiffness. These changes are associated with dysregulated mRNA and microRNA. Short-term treatment of the mutant organoids, but not the control ones, with the catalytic subunit of MMP2, which proteolytically cleaves ECM, reduced stiffness. The changes were associated with changes in water diffusion measured by MRI (Magnetic Resonance Imaging) and gene expression. We generated a model incorporating the differences in the mutant's and control brain organoid composition and organization that matches and explains our findings. Our study provides insights into the importance of the composition and organization of the ECM during human brain development and the role of LIS1 in brain structure.
Project description:Syngap1 was detected in cultured human brain organoids by SP3 digestion, fractionation of the lysate, and LC-MS/MS analysis of the fractionated digest
Project description:Stochastic transition of cancer cells between drug-sensitive and drug-tolerant persister phenotypes has been proposed to play a key role in non-genetic resistance to therapy. Yet, we show here that cancer cells actually possess a highly stable inherited chance to persist (CTP) during therapy. This CTP is non-stochastic, determined pre-treatment, and has a unimodal distribution ranging from 0 to almost 100%. Importantly, CTP is drug-specific. We found that differential serine/threonine phosphorylation of the insulin receptor substrate 1 (IRS1) protein determines the CTP of lung and of head and neck cancer cells under EGFR inhibition, both in vitro and in vivo. Indeed, the first-in-class IRS1 inhibitor NT219 was highly synergistic with anti-EGFR therapy across multiple in vitro and in vivo models. Elucidation of drug-specific mechanisms that determine the degree and stability of cellular CTP may establish a framework for the elimination of cancer persisters, using novel rationally designed drug combinations.
Project description:Stochastic transition of cancer cells between drug-sensitive and drug-tolerant persister phenotypes has been proposed to play a key role in non-genetic resistance to therapy. Yet, we show here that cancer cells actually possess a highly stable inherited chance to persist (CTP) during therapy. This CTP is non-stochastic, determined pre-treatment, and has a unimodal distribution ranging from 0 to almost 100%. Importantly, CTP is drug-specific. We found that differential serine/threonine phosphorylation of the insulin receptor substrate 1 (IRS1) protein determines the CTP of lung and of head and neck cancer cells under EGFR inhibition, both in vitro and in vivo. Indeed, the first-in-class IRS1 inhibitor NT219 was highly synergistic with anti-EGFR therapy across multiple in vitro and in vivo models. Elucidation of drug-specific mechanisms that determine the degree and stability of cellular CTP may establish a framework for the elimination of cancer persisters, using novel rationally designed drug combinations.
Project description:Stochastic transition of cancer cells between drug-sensitive and drug-tolerant persister phenotypes has been proposed to play a key role in non-genetic resistance to therapy. Yet, we show here that cancer cells actually possess a highly stable inherited chance to persist (CTP) during therapy. This CTP is non-stochastic, determined pre-treatment, and has a unimodal distribution ranging from 0 to almost 100%. Importantly, CTP is drug-specific. We found that differential serine/threonine phosphorylation of the insulin receptor substrate 1 (IRS1) protein determines the CTP of lung and of head and neck cancer cells under EGFR inhibition, both in vitro and in vivo. Indeed, the first-in-class IRS1 inhibitor NT219 was highly synergistic with anti-EGFR therapy across multiple in vitro and in vivo models. Elucidation of drug-specific mechanisms that determine the degree and stability of cellular CTP may establish a framework for the elimination of cancer persisters, using novel rationally designed drug combinations.