ERK1/2 signalling and protein ubiquitylation in the control of apoptosis

Programmed cell death, or apoptosis, is critical for normal developmental processes that involve cell turnover including embryogenesis and development and function of the immune system. It is preceded by classical changes in cell morphology, driven by biochemical changes including caspase activation. Apoptosis is deregulated in multiple human diseases, with suppression of apoptosis being critical for carcinogenesis. As such, proteins that regulate apoptosis are tightly regulated by cell fate signalling pathways. The ERK1/2 signalling pathway is a key regulator of cell intrinsic apoptosis, in part through regulation of the pro-apoptotic protein ‘BCL2-interacting mediator of cell death’ (BIM). BIM is phosphorylated by ERK1/2 and this serves to drive its K48-linked polyubiquitylation and proteasome-dependent degradation, thereby promoting cell survival. βTrCP, an F-box protein that acts in a larger SCF complex, is one of several E3 ligases that have been proposed to polyubiquitinate BIM. This study demonstrated that of the major isoforms of BIM only BIMEL interacts with βTrCP. ERK1/2-driven phosphorylation of BIMEL is essential for this interaction, leading to BIMEL destabilisation and degradation. As a consequence, tumour cells that are addicted to ERK1/2 signalling undergo BIM-dependent cell death in response to MEK1/2 inhibitors when combined with BH3-mimetics such as ABT263, small molecules that inhibit pro-survival proteins of the apoptotic pathway. The RSK1/2 protein kinases, immediate downstream targets of ERK1/2, have also been implicated in the destabilisation of BIMEL. Specifically phosphorylation of BIMEL by RSK1/2 is proposed to be required for βTrCP binding. This study revealed that whilst the putative RSK1/2 phosphorylation sites in BIMEL may be required for βTrCP binding, inhibition of RSK activity by three distinct RSK inhibitors does not block BIMEL:βTrCP binding or BIMEL turnover. Furthermore, tumour cells that are addicted to ERK1/2 signalling for survival are not addicted to RSK activity, arguing against a role for RSK in the regulation of BIMEL. This suggests that ERK1/2 and an as yet unidentified kinase cooperate to drive BIMEL degradation. Deubiquitylating enzymes (DUBs) remove ubiquitin from target proteins. In the context of BIMEL, DUB activity might oppose E3 ligases and thus cause its accumulation. Until recently the DUB for BIM was unknown however, USP27x has now been suggested. Follow-up validation of the reported interaction between BIMEL and USP27x was challenging but loss of BIMEL polyubiquitination was observed following overexpression of USP27x, suggesting that USP27x may serve as a DUB for BIM. Numerous DUBs control cellular processes that are dysregulated in cancer, including proliferation and apoptosis, making them attractive therapeutic targets. Recent interest in the DUB USP30 has increased as it has been shown to inhibit parkin-mediated mitophagy, with defective mitophagy being linked to Parkinson’s disease. USP30 has also been suggested to play a role in apoptosis and its depletion was shown to sensitise cells to BH3 mimetics. These findings suggest that USP30 depletion or inhibition could provide a means for inducing tumour cell death. Indeed, combining a novel USP30 inhibitor (MTX32), provided by Mission Therapeutics, with the BH3 mimetic, ABT-263, induced apoptotic tumour cell death that required BAX and caspase activation. However, this was not replicated by a more selective USP30 inhibitor (MTX48), suggesting that the observed apoptotic cell death reflected the off-target effects of MTX32 rather than specific inhibition of USP30. Finally, an RNAi screen, targeting 94 DUBs, and 8 sentrin/SUMO-specific proteases (SENPs), in the human genome, was performed to identify DUBs that modulate cell death induced by MEK1/2 or mTOR inhibition. As such, inhibitors of identified ‘hit’ DUBs might be suitable as a combinatorial therapy with MEK1/2 or mTOR inhibitors in the treatment of cancer. The RNAi screen identified several DUBs, that, when knocked down, sensitised HCT116 cells to MEK1/2 inhibitor treatment and enhanced MEK1/2 inhibitor induced cell death in a BAX-dependent fashion. This work is discussed in the context of the role of ERK1/2 signalling as a pro-survival pathway, its specific role in BIM regulation, the potential for co-targeting DUBs and the ERK1/2 pathway to inhibit the growth of ERK1/2 addicted tumour cells and suggestions for future work are outlined.iCase studentship - partially funded by Mission Therpeautic

Glyphosate in waters and soils from genetically modified canola cultivation in Parkes, NSW, Australia

Investigations were conducted of farmland from the Parkes region of New South Wales, Australia, cultivated with genetically modified canola, involving the determination of glyphosate (N-(phosphonomethyl)glycine) concentrations in water and soils, and its sorption. The soils are classified as loam under the USDA system (clay 13.8-15.8%, silt 39-43%, sand 41.2-47.2%). Firstly, a low-cost fluorometric method was developed for the analysis of glyphosate in waters and soils, calibrated against analytical standards and spectrophotometric and enzyme-linked immunosorbent assay (ELISA) methods. Soil and water samples were then collected using the NEPM sampling protocol into glass containers, chilled and analysed within two weeks. The samples were collected in multiple episodes, taking account of glyphosate and pesticide crop applications. The soil and water physical and chemical properties were characterised, and glyphosate levels were determined. Field concentrations of glyphosate ranged between 0.01 - 0.067 mg/L in water and 0.10 - 0.575 mg/kg in soil. The aqueous levels lie below Australian and international drinking water guidelines, but reach a Canadian freshwater guideline. Glyphosate levels varied with time of application and rainfall events. Glyphosate sorption isotherms were also constructed by batch tests on several soils, and were fitted with Freundlich and Langmuir isotherms. Desorption tests indicated 25% to 58% of soil glyphosate is extractable by 0.1M KH2PO4

Pramlintide but Not Liraglutide Suppresses Meal-Stimulated Glucagon Responses in Type 1 Diabetes

Postprandial hyperglycemia remains a challenge in type 1 diabetes (T1D) due, in part, to dysregulated increases in plasma glucagon levels after meals