Quantitative proteomic analysis of Parkin substrates in Drosophila neurons.

Parkin (PARK2) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson's Disease (PD). In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1. PINK1 activates Parkin activity leading to ubiquitination of multiple proteins, which in turn promotes clearance of mitochondria by mitophagy. Many substrates have been identified using cell culture models in combination with depolarising drugs or proteasome inhibitors, but not in more physiological settings.Here we utilized the recently introduced BioUb strategy to isolate ubiquitinated proteins in flies. Following Parkin Wild-Type (WT) and Parkin Ligase dead (LD) expression we analysed by mass spectrometry and stringent bioinformatics analysis those proteins differentially ubiquitinated to provide the first survey of steady state Parkin substrates using an in vivo model. We further used an in vivo ubiquitination assay to validate one of those substrates in SH-SY5Y cells.We identified 35 proteins that are more prominently ubiquitinated following Parkin over-expression. These include several mitochondrial proteins and a number of endosomal trafficking regulators such as v-ATPase sub-units, Syx5/STX5, ALiX/PDCD6IP and Vps4. We also identified the retromer component, Vps35, another PD-associated gene that has recently been shown to interact genetically with parkin. Importantly, we validated Parkin-dependent ubiquitination of VPS35 in human neuroblastoma cells.Collectively our results provide new leads to the possible physiological functions of Parkin activity that are not overtly biased by acute mitochondrial depolarisation

Opposite feedback from mTORC1 to H-ras and K-ras4B downstream of SREBP1

As a major growth factor transducer, Ras is an upstream activator of mTORC1, which further integrates nutrient and energy inputs. To ensure a contextual coupling of cell division via Ras/MAPK-signalling and growth via mTORC1-signalling, feedback loops from one pathway back to the other are required. Here we describe a novel feedback from mTORC1, which oppositely affects oncogenic H-ras- and K-ras-signalling output, and as a consequence stemness properties of tumourigenic cells. Amino acid stimulation of mTORC1 increases the processed form of SREBP1, a major lipidome regulator. We show that modulation of the SREBP1 levels downstream of S6K1 has opposite effects on oncogenic H-ras and K-ras nanoscale membrane organisation, ensuing signalling output and promotion of mammospheres expressing these oncogenes. Our data suggest that modulation of phosphatidic acid, a major target of SREBP1 controlled lipid metabolism, is sufficient to affect H-ras and K-ras oppositely in the membrane. Thus mTORC1 activation increases H-ras-, but decreases K-ras-signalling output in cells transformed with the respective oncogene. Given the different impact of these two Ras isoforms on stemness, our results could have implications for stem cell biology and inhibition of cancer stem cells

Cancer stem cell drugs target K-ras signaling in a stemness context

Cancer stem cells (CSCs) are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. Salinomycin is the most established CSC inhibitor. However, its primary mechanistic target is still unclear, impeding the discovery of compounds with similar anti-CSC activity. Here, we show that salinomycin very specifically interferes with the activity of K-ras4B, but not H-ras, by disrupting its nanoscale membrane organization. We found that caveolae negatively regulate the sensitivity to this drug. On the basis of this novel mechanistic insight, we defined a K-ras-associated and stem cell-derived gene expression signature that predicts the drug response of cancer cells to salinomycin. Consistent with therapy resistance of CSC, 8% of tumor samples in the TCGA-database displayed our signature and were associated with a significantly higher mortality. Using our K-ras-specific screening platform, we identified several new candidate CSC drugs. Two of these, ophiobolin A and conglobatin A, possessed a similar or higher potency than salinomycin. Finally, we established that the most potent compound, ophiobolin A, exerts its K-ras4B-specific activity through inactivation of calmodulin. Our data suggest that specific interference with the K-ras4B/calmodulin interaction selectively inhibits CSC.Peer reviewe

PO-092 Inhibition of the mTORC1-pathway can feedback-activate H-RAS or K-RAS

ABSTRACTIntroductionPI3K/mTORC1- and Ras/MAPK-signalling pathways are aberrantly regulated in most cancers. Specific resistances to drugs targeting these pathways can emerge during tumour evolution. Preexisting, innate resistance mechanisms, such as stemming from feedback-loops, should ideally be known already during drug-target nomination. However, as the example of B-Raf-inhibitors that paradoxically activate MAPK-signalling has shown, feedback mechanisms may only become apparent at very late drug development stages.Material and methodsHEK cells expressing FRET-pairs of Ras proteins were used to study specific effects on Ras isoforms (nanoclustering). Breast cancer cells were grown in 2D for Western blotting of Ras and mTORC1-pathway proteins, or as spheres to analyse stemness traits.Results and discussionsHere, we describe two broad feedback loops from the mTor-pathway back to the nanoscopic membrane signalling complexes (nanocluster) of H-ras and K-ras4B (hereafter K-ras). Increased nanoclustering typically correlates with increased Ras output. The first, upstream loop leads to an inadvertent rapalog induced promotion of stemness traits and tumorigenicity in Ras transformed cells. This is due to an induction of the H-ras nanocluster scaffold galectin-1, when FKBP12 levels are low. Surprisingly we find that rapalogs do not only bind to but induce a loss of FKBP12 protein. Thus, rapalog treatment induces galectin-1, which stimulates H-ras signal output and stemness traits. Secondly, modulation of the activity in the mTORC1 pathway downstream of the major lipidome regulator SREBP1, oppositely regulates H-ras and K-ras nanoclustering. Thus, ablation of SREBP1 increases K-ras, but decreases H-ras nanoclustering and signal output. We show that altered levels of phosphatidic acid downstream of SREBP1 are sufficient for the opposite regulation of the two Ras isoforms.ConclusionThe described feedback loops may only become apparent in certain tumour settings. For example, tumour promotion during rapalog-treatment may only be relevant in H-ras mutant cancers, which make up a small portion of human cancers. In those cases, rapalog efficacy may be improved in combination with novel anti-galectin-1 drugs. Targeting the mTORC1 pathway downstream of SREBP1, may have opposite effects in H-ras and K-ras mutant cancers. Thus, care may have to be taken when targeting the mTORC1-pathway in a mutant Ras setting

Ubiquitin profiling in liver using a transgenic mouse with biotinylated ubiquitin

Ubiquitination is behind most cellular processes, with ubiquitin substrates being regulated variously according to the number of covalently conjugated ubiquitin molecules and type of chain formed. Here we report the first mammalian system for ubiquitin proteomics allowing direct validation of the MS-identified proteins. We created a transgenic mouse expressing biotinylated ubiquitin and demonstrate its use for the isolation of ubiquitinated proteins from liver and other tissues. The specificity and strength of the biotin-avidin interaction allow very stringent washes so only proteins conjugated to ubiquitin are isolated. In contrast to recently available antibody-based approaches, our strategy allows direct validation by immunoblotting, therefore revealing the type of ubiquitin chains (mono or poly) formed in vivo. We also identify the conjugating E2 enzymes that are ubiquitin-loaded in the mouse tissue. Furthermore, our strategy allows the identification of candidate cysteine-ubiquitinated proteins, providing a strategy to identify those in a proteomic scale. The novel in vivo system described here allows broad access to tissue-specific ubiquitomes and can be combined with established mouse disease models to investigate ubiquitin-dependent therapeutical approaches

Anatomical distribution and biochemical characterization of the novel RFamide peptide 26RFa in the human hypothalamus and spinal cord

International audience26RFa is a novel RFamide peptide originally isolated in the amphibian brain. The 26RFa precursor has been subsequently characterized in various mammalian species but, until now, the anatomical distribution and the molecular forms of 26RFa produced in the CNS of mammals, in particular in human, are unknown. In the present study, we have investigated the localization and the biochemical characteristics of 26RFa-like immunoreactivity (LI) in two regions of the human CNS--the hypothalamus and the spinal cord. Immunohistochemical labeling using specific antibodies against human 26RFa and in situ hybridization histochemistry revealed that in the human hypothalamus 26RFa-expressing neurons are located in the paraventricular and ventromedial nuclei. In the spinal cord, 26RFa-expressing neurons were observed in the dorsal and lateral horns. Characterization of 26RFa-related peptides showed that two distinct molecular forms of 26RFa are present in the human hypothalamus and spinal cord, i.e. 26RFa and an N-terminally elongated form of 43 amino acids designated 43RFa. These data provide the first evidence that 26RFa and 43RFa are actually produced in the human CNS. The distribution of 26RF-LI suggests that 26RFa and/or 43RFa may modulate feeding, sexual behavior and transmission of nociceptive stimuli

Multi-Omics Integration Highlights the Role of Ubiquitination in CCl4-Induced Liver Fibrosis

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in chronic liver disease. Ubiquitination is a post-translational modification that is crucial for a plethora of physiological processes. Even though the ubiquitin system has been implicated in several human diseases, the role of ubiquitination in liver fibrosis remains poorly understood. Here, multi-omics approaches were used to address this. Untargeted metabolomics showed that carbon tetrachloride (CCl4)-induced liver fibrosis promotes changes in the hepatic metabolome, specifically in glycerophospholipids and sphingolipids. Gene ontology analysis of public deposited gene array-based data and validation in our mouse model showed that the biological process “protein polyubiquitination” is enriched after CCl4-induced liver fibrosis. Finally, by using transgenic mice expressing biotinylated ubiquitin (bioUb mice), the ubiquitinated proteome was isolated and characterized by mass spectrometry in order to unravel the hepatic ubiquitinated proteome fingerprint in CCl4-induced liver fibrosis. Under these conditions, ubiquitination appears to be involved in the regulation of cell death and survival, cell function, lipid metabolism, and DNA repair. Finally, ubiquitination of proliferating cell nuclear antigen (PCNA) is induced during CCl4-induced liver fibrosis and associated with the DNA damage response (DDR). Overall, hepatic ubiquitome profiling can highlight new therapeutic targets for the clinical management of liver fibrosis

Multi-Omics Integration Highlights the Role of Ubiquitination in CCl4-Induced Liver Fibrosis

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in chronic liver disease. Ubiquitination is a post-translational modification that is crucial for a plethora of physiological processes. Even though the ubiquitin system has been implicated in several human diseases, the role of ubiquitination in liver fibrosis remains poorly understood. Here, multi-omics approaches were used to address this. Untargeted metabolomics showed that carbon tetrachloride (CCl4)-induced liver fibrosis promotes changes in the hepatic metabolome, specifically in glycerophospholipids and sphingolipids. Gene ontology analysis of public deposited gene array-based data and validation in our mouse model showed that the biological process “protein polyubiquitination” is enriched after CCl4-induced liver fibrosis. Finally, by using transgenic mice expressing biotinylated ubiquitin (bioUb mice), the ubiquitinated proteome was isolated and characterized by mass spectrometry in order to unravel the hepatic ubiquitinated proteome fingerprint in CCl4-induced liver fibrosis. Under these conditions, ubiquitination appears to be involved in the regulation of cell death and survival, cell function, lipid metabolism, and DNA repair. Finally, ubiquitination of proliferating cell nuclear antigen (PCNA) is induced during CCl4-induced liver fibrosis and associated with the DNA damage response (DDR). Overall, hepatic ubiquitome profiling can highlight new therapeutic targets for the clinical management of liver fibrosis