Insights into antitrypanosomal drug mode-of-action from cytology-based profiling

Chemotherapy continues to have a major impact on reducing the burden of disease caused by trypanosomatids. Unfortunately though, the mode-of-action (MoA) of antitrypanosomal drugs typically remains unclear or only partially characterised. This is the case for four of five current drugs used to treat Human African Trypanosomiasis (HAT); eflornithine is a specific inhibitor of ornithine decarboxylase. Here, we used a panel of T. brucei cellular assays to probe the MoA of the current HAT drugs. The assays included DNA-staining followed by microscopy and quantitative image analysis, or flow cytometry; terminal dUTP nick end labelling to monitor mitochondrial (kinetoplast) DNA replication; antibody-based detection of sites of nuclear DNA damage; and fluorescent dye-staining of mitochondria or lysosomes. We found that melarsoprol inhibited mitosis; nifurtimox reduced mitochondrial protein abundance; pentamidine triggered progressive loss of kinetoplast DNA and disruption of mitochondrial membrane potential; and suramin inhibited cytokinesis. Thus, current antitrypanosomal drugs perturb distinct and specific cellular compartments, structures or cell cycle phases. Further exploiting the findings, we show that putative mitogen-activated protein-kinases contribute to the melarsoprol-induced mitotic defect, reminiscent of the mitotic arrest associated signalling cascade triggered by arsenicals in mammalian cells, used to treat leukaemia. Thus, cytology-based profiling can rapidly yield novel insight into antitrypanosomal drug MoA

Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation.

Four stress-sensing kinases phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) to activate the integrated stress response (ISR). In animals, the ISR is antagonised by selective eIF2α phosphatases comprising a catalytic protein phosphatase 1 (PP1) subunit in complex with a PPP1R15-type regulatory subunit. An unbiased search for additional conserved components of the PPP1R15-PP1 phosphatase identified monomeric G-actin. Like PP1, G-actin associated with the functional core of PPP1R15 family members and G-actin depletion, by the marine toxin jasplakinolide, destabilised the endogenous PPP1R15A-PP1 complex. The abundance of the ternary PPP1R15-PP1-G-actin complex was responsive to global changes in the polymeric status of actin, as was its eIF2α-directed phosphatase activity, while localised G-actin depletion at sites enriched for PPP1R15 enhanced eIF2α phosphorylation and the downstream ISR. G-actin's role as a stabilizer of the PPP1R15-containing holophosphatase provides a mechanism for integrating signals regulating actin dynamics with stresses that trigger the ISR.This work was funded by the Medical Research Council (UK) (MRC Ref G1002610) and a Wellcome Trust Strategic Award for core facilities to the Cambridge Institute for Medical Research (CIMR, Wellcome 100140). SJM holds a Senior Clinical Research Fellowship from the Medical Research Council (MRC Ref G1002610). DR is a Wellcome Trust Principal Research Fellow (Wellcome 084812/Z/08/Z). The June Hancock Mesothelioma Research Fund funded LED (JH09-2); the British Lung Foundation funded HJC (APHD11-4); CD is a member of the CIMR PhD programme funded by the Wellcome Trust; and VP holds a Diabetes UK Arthur and Sadie Pethybridge PhD Studentship.This is the final published version. It first appeared at http://elifesciences.org/content/4/e04872

The integrated stress response regulates BMP signalling through effects on translation.

BACKGROUND: Developmental pathways must be responsive to the environment. Phosphorylation of eIF2α enables a family of stress-sensing kinases to trigger the integrated stress response (ISR), which has pro-survival and developmental consequences. Bone morphogenetic proteins (BMPs) regulate multiple developmental processes in organisms from insects to mammals. RESULTS: Here we show in Drosophila that GCN2 antagonises BMP signalling through direct effects on translation and indirectly via the transcription factor crc (dATF4). Expression of a constitutively active GCN2 or loss of the eIF2α phosphatase dPPP1R15 impairs developmental BMP signalling in flies. In cells, inhibition of translation by GCN2 blocks downstream BMP signalling. Moreover, loss of d4E-BP, a target of crc, augments BMP signalling in vitro and rescues tissue development in vivo. CONCLUSION: These results identify a novel mechanism by which the ISR modulates BMP signalling during development

The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-α1-antitrypsin.

α1-Antitrypsin is a serine protease inhibitor produced in the liver that is responsible for the regulation of pulmonary inflammation. The commonest pathogenic gene mutation yields Z-α1-antitrypsin, which has a propensity to self-associate forming polymers that become trapped in inclusions of endoplasmic reticulum (ER). It is unclear whether these inclusions are connected to the main ER network in Z-α1-antitrypsin-expressing cells. Using live cell imaging, we found that despite inclusions containing an immobile matrix of polymeric α1-antitrypsin, small ER resident proteins can diffuse freely within them. Inclusions have many features to suggest they represent fragmented ER, and some are physically separated from the tubular ER network, yet we observed cargo to be transported between them in a cytosol-dependent fashion that is sensitive to N-ethylmaleimide and dependent on Sar1 and sec22B. We conclude that protein recycling occurs between ER inclusions despite their physical separation.-Dickens, J. A., Ordóñez, A., Chambers, J. E., Beckett, A. J., Patel, V., Malzer, E., Dominicus, C. S., Bradley, J., Peden, A. A., Prior, I. A., Lomas, D. A., Marciniak, S. J. The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-α1-antitrypsin.J.A.D. was funded by a Medical Research Council (MRC) Clinical Research Training Fellowship and a starter grant from the Academy of Medical Sciences; S.J.M. is a MRC Senior Clinical Research Fellow. D.A.L. is funded by the MRC and by the University College London Hospitals National Institute for Health Research (NIHR) Biomedical Research Centre (London, United Kingdom), and is an NIHR Senior Investigator. The work was also supported by the Alpha1 Foundation. The Cambridge Institute for Medical Research microscopy core facility is supported by a Wellcome Trust Strategic Award (100140) and a Wellcome Trust equipment grant (093026).This is the final version of the article. It first appeared from the Federation of American Societies for Experimental Biology via https://doi.org/10.1096/fj.201600430

The integrated stress response regulates BMP signalling through effects on translation.

Background: Developmental pathways must be responsive to the environment. Phosphorylation of eIF2α enables a family of stress sensing kinases to trigger the integrated stress response (ISR), which has pro-survival and developmental consequences. Bone Morphogenetic Proteins (BMPs) regulate multiple developmental processes in organisms from insects to mammals. Results: Here we show in Drosophila that GCN2 antagonises BMP signaling through direct effects on translation and indirectly via the transcription factor crc (dATF4). Expression of a constitutively active GCN2 or loss of the eIF2α phosphatase dPPP1R15 impair developmental BMP signaling in flies. In cells, inhibition of translation by GCN2 blocks downstream BMP signaling. Moreover, loss of d4E-BP, a target of crc, augments BMP signaling in vitro and rescues tissue development in vivo. Conclusion: These results identify a novel mechanism by which the ISR modulates BMP signaling during development

A plasma membrane localized protein phosphatase in Toxoplasma gondii, PPM5C, regulates attachment to host cells

Abstract The propagation of Toxoplasma gondii is accomplished by repeated lytic cycles of parasite attachment to a host cell, invasion, replication within a parasitophorous vacuole, and egress from the cell. This lytic cycle is delicately regulated by calcium-dependent reversible phosphorylation of the molecular machinery that drives invasion and egress. While much progress has been made elucidating the protein kinases and substrates central to parasite propagation, little is known about the relevant protein phosphatases. In this study, we focused on the five protein phosphatases that are predicted to be membrane-associated either integrally or peripherally. We have determined that of these only PPM5C, a PP2C family member, localizes to the plasma membrane of Toxoplasma. Disruption of PPM5C results in a slow propagation phenotype in tissue culture. Interestingly, parasites lacking PPM5C divide and undergo egress at a normal rate, but have a deficiency in attaching to host cells. Both membrane localization and phosphatase activity are required for PPM5C’s role in attachment. Phosphoproteomic analysis show relatively few phosphorylation sites being affected by PPM5C deletion in extracellular parasites of which several are found on proteins involved in signaling cascades. This implies that PPM5C is part of a wider regulatory network important for attachment to host cells

New Concepts in Alpha-1 Antitrypsin Deficiency Disease Mechanisms.

Alpha-1 antitrypsin deficiency is predominantly caused by point mutations that alter the protein's folding. These mutations fall into two broad categories: those that destabilize the protein dramatically and lead to its post-translational degradation and those that affect protein structure more subtly to promote protein polymerization within the endoplasmic reticulum (ER). This distinction is important because it determines the cell's response to each mutant. The severely misfolded mutants trigger an unfolded protein response (UPR) that promotes improved protein folding but can kill the cell in the chronic setting. In contrast, mutations that permit polymer formation fail to activate the UPR but instead promote a nuclear factor-κB-mediated ER overload response. The ability of polymers to increase a cell's sensitivity to ER stress likely explains apparent inconsistencies in the alpha-1 antitrypsin-signaling literature that have linked polymers with the UPR. In this review we discuss the use of mutant serpins to dissect each signaling pathway

Additional file 1: of The integrated stress response regulates BMP signalling through effects on translation

Figure S1. Modulation of the ISR delays developmental delay and causes wing venation defects. (A) Phenotypes of animals expressing ppp1r15 RNAi under the control of a panel of tissue-selective drivers. (B) Representative photomicrographs (5× objective) of w 1118 ;esgGAL4 (esg) and esgGAL4 > UAS-ppp1r15 RNAi (esg > ppp1r15 RNAi) animals at 5 and 14 days after egg laying (AEL). Scale bar = 1 mm. (C) Representative photomicrographs of w1118;esgGAL4 (esg), esgGAL4 > UAS-ppp1r15 RNAi (esg > ppp1r15 RNAi), esgGAL4 > UAS-gcn2 RNAi (esg > gcn2 RNAi) and esgGAL4 > UAS-gcn2;UAS-ppp1r15 RNAi (esg > ppp1r15 RNAi;gcn2 RNAi) animals at 14 days AEL. (D) Quantification of indicated crosses at days 5 and 14 AEL. esgGAL4 > UAS-ppp1r15 RNAi (esg > ppp1r15 RNAi), esgGAL4 > UAS-dGCN2 RNAi (esg > gcn2 RNAi) and esgGAL4 > UAS-dPERK RNAi (esg > perk RNAi). n denotes number of animals counted. P values calculated using Χ2 statistic with Bonferroni correction for multiple comparisons. (E) Quantification of indicated crosses at days 5 and 14 AEL. enGAL4 > UAS-ppp1r15 RNAi (en > ppp1r15 RNAi), enGAL4 > UAS-gcn2 RNAi (en > gcn2 RNAi) and enGAL4 > UAS-perk RNAi (en > perk RNAi). n denotes number of animals counted. P values calculated using Χ 2 statistics with Bonferroni correction for multiple comparisons. (F) Representative photomicrographs of adult wings of the indicated genotypes. Scale bars = 250 μm. (PDF 1057 kb