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SARS-CoV-2 non-structural protein 3 (Nsp3) contains a macrodomain that is essential for virus replication and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the removal of ADP-ribose modifications catalysed by host poly(ADP-ribose) polymerases (PARPs). Here, we show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this can be used to screen for cell-active macrodomain inhibitors without the requirement for BSL-3 facilities. This IFN-induced ADP-ribosylation is dependent on the PARP9/DTX3L heterodimer, but surprisingly the expression of Nsp3 macrodomain or PARP9/DTX3L deletion do not impair STAT1 phosphorylation or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyse this end product of IFN signalling, and not to suppress the IFN response itself.

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Cell context is key for cell phenotype. Using physiologically relevant models of laminin-rich ECM (lrECM) induction of mammary epithelial cell quiescence and differentiation, we have provided a landscape of the status of key molecular players involved in the proliferation/quiescence decision. Repression of some positive regulators of the cell cycle, such as cyclins and CDKs, occurred already at the mRNA level, whereas negative regulators of the cell cycle, such as Pten and p27, were upregulated only at the protein level. Interestingly, cell cycle arrest occurred despite the active status of Fak, Src and PI3k, because their downstream proliferative signalling pathways were repressed, suggesting the existence of a disconnecting node between upstream and downstream proliferative signalling in quiescent cells. Pten fulfils this role. Inhibition of Pten increased proliferation and restored Akt/mTORC1/2 and Mapk signalling in cells exposed to lrECM. In mice, Pten levels were positively correlated to the basement membrane thickness in the developing mammary epithelia, and Pten localized to the apicolateral membrane of luminal cells both in in ducts and near the nascent lumen in terminal end bud, characteristics consistent with a role for Pten in inducing and sustaining quiescence and tissue architecture. Accordingly, in 3D acininogenesis models, Pten was required for the onset and maintenance of quiescence, cell polarity and lumen assembly. The notion that lrECM-triggered differentiation involves a signalling circuitry with many layers of regulation provides an explanation for the resilience of quiescence within a growth-suppressive microenvironment, and that perturbations in master regulators, such as Pten, could disrupt the quiescent phenotype.

Aims/hypothesis: Efficient mitochondrial oxidative phosphorylation is essential for pancreatic beta cell responses to nutrient levels. Consequently, the evaluation of mitochondrial oxygen consumption and ATP production is important to investigate essential aspects of pancreatic islet pathophysiology. Currently, most studies use cell lines instead of primary islets due to difficulties in measuring primary islet respiration, which requires specific equipment and consumables that are expensive, complicated to use, and poorly reproducible. The aim of this study is to establish a robust and practical method to assess primary islet metabolic fluxes using Extracellular Flux Technology and standard commercial consumables. Methods: Pancreatic islets were isolated from 8 to 12-week-old mice and rats, and submitted to a dispersion protocol using trypsin. Dispersed islets were adhered overnight to pre-coated standard Seahorse microplates, and oxygen consumption rates were evaluated using a Seahorse Extracellular Flux Analyzer. We also validated the functionality of dispersed islets by analyzing glucose-stimulated insulin secretion (GSIS) and calcium (Ca2+) influx in response to different modulators by fluorescence microscopy. Results: We provide a detailed protocol with all steps necessary to optimize islet isolation and dispersion, in order to achieve a high yield of functional islets and perform metabolic flux analysis. With this method, which requires only a few islets per replicate, both rat and mouse islets present robust basal respiration and proper response to mitochondrial modulators (oligomycin, CCCP, antimycin and rotenone) and glucose addition. Both oligomycin and CCCP concentrations were titrated. Our method was also validated by other functional assays, which show these cells present conserved Ca2+ influx and insulin secretion in response to glucose. Conclusions/interpretation: We established a practical and robust method to assess ex vivo islet metabolic fluxes and oxidative phosphorylation. Our findings cover an important gap in primary islet physiology studies, providing a valuable tool we hope is useful to uncover basic beta cell metabolic mechanisms, as well as for translational investigations, such as pharmacological candidate discovery and islet transplantation protocols.