The loss of lipid homeostasis can lead to lipid overload and is associated with a variety of disease states. However, little is known as to how the disruption of lipid regulation or lipid overload affects cell survival. In this study we investigated how excess diacylglycerol (DG), a cardinal metabolite suspected to mediate lipotoxicity, compromises the survival of yeast cells. We reveal that increased DG achieved by either genetic manipulation or pharmacological administration of 1,2-dioctanoyl-sn-glycerol (DOG) triggers necrotic cell death. The toxic effects of DG are linked to glucose metabolism and require a functional Rim101 signaling cascade involving the Rim21-dependent sensing complex and the activation of a calpain-like protease. The Rim101 cascade is an established pathway that triggers a transcriptional response to alkaline or lipid stress. We propose that the Rim101 pathway senses DG-induced lipid perturbation and conducts a signaling response that either facilitates cellular adaptation or triggers lipotoxic cell death. Using established models of lipotoxicity, i.e., high-fat diet in Drosophila and palmitic acid administration in cultured human endothelial cells, we present evidence that the core mechanism underlying this calpain-dependent lipotoxic cell death pathway is phylogenetically conserved.
BCR-ABL is the oncogenic fusion product of tyrosine kinase ABL1 and a highly frequent driver of acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (CML). The kinase activity of BCR-ABL is strongly elevated, however changes of substrate specificity in comparison to wild-type ABL1 kinase are less well characterized. Here, we heterologously expressed full-length BCR-ABL kinases in yeast. We exploited the proteome of living yeast as in vivo phospho-tyrosine substrate for assaying human kinases specificity. Phospho-proteomic analysis of ABL1 and BCR-ABL isoforms p190 and p210 yielded a high-confidence dataset of 1127 phospho-tyrosine sites on 821 yeast proteins. We used this data set to generate linear phosphorylation site motifs for ABL1 and the oncogenic ABL1 fusion proteins. The oncogenic kinases yielded a substantially different linear motif when compared to ABL1. Kinase set enrichment analysis with human pY-sites that have high linear motif scores well recalled BCR-ABL driven cancer cell lines from human phospho-proteome data sets.
The dynamic nature of the actin cytoskeleton controls many cellular processes and a loss of its plasticity has been linked to accelerated cell ageing. Cofilin is an actin-binding protein that controls actin dynamics and is linked to mitochondrial signalling pathways that control drug resistance and cell death. Here we show that cofilin driven stabilisation of actin dynamics leads to a loss in cell wall integrity, vacuole fragmentation and disruption of lipid homeostasis, lipid droplet accumulation and the promotion of necrosis. Our data suggest that these phenotypes are triggered by the inappropriate activation of Protein Kinase C (PKC) which leads to constitutive MAPK signalling. Cofilin-driven activation of the MAPK Slt2 requires the presence of the voltage dependent anion channel (VDAC), which resides in the mitochondrial outer membrane, referred to as Porin 1 (Por1) in yeast. This provides further evidence for a link between actin regulation and mitochondrial signalling. Porin has recently been implicated in lipid transport and we find that prevention of excessive lipid droplet accumulation, achieved by deleting the LRO1 and DGA1 genes, was sufficient to prevent Slt2 activation and restore cellular homeostasis in actin stabilised cells. These data suggest that the integrity of the actin cytoskeleton is essential to maintain the fidelity of MAPK signalling and that this in turn is crucial for the maintenance of lipid homeostasis and cell health in S. cerevisiae. Our data also suggest that chronic actin stabilisation leads to mitochondrial VDAC-dependent Slt2 activation and altered, pro-death, cell fate in yeast cells.
BCR-ABL is the oncogenic fusion product of tyrosine kinase ABL1 and a highly frequent driver of acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (CML). The kinase activity of BCR-ABL is strongly elevated; however, changes of substrate specificity in comparison to wild-type ABL1 kinase are less well characterized. Here, we heterologously expressed full-length BCR-ABL kinases in yeast. We exploited the proteome of living yeast as an in vivo phospho-tyrosine substrate for assaying human kinase specificity. Phospho-proteomic analysis of ABL1 and BCR-ABL isoforms p190 and p210 yielded a high-confidence data set of 1127 phospho-tyrosine sites on 821 yeast proteins. We used this data set to generate linear phosphorylation site motifs for ABL1 and the oncogenic ABL1 fusion proteins. The oncogenic kinases yielded a substantially different linear motif when compared to ABL1. Kinase set enrichment analysis with human pY-sites that have high linear motif scores well-recalled BCR-ABL driven cancer cell lines from human phospho-proteome data sets.
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