Genome-wide RNAi screening implicates the E3 ubiquitin ligase Sherpa in mediating innate immune signaling by Toll in Drosophila adults.” Sci Signal, 8, 400, Pp. ra107.Abstract. 2015. “
The Drosophila Toll pathway plays important roles in innate immune responses against Gram-positive bacteria and fungi. To identify previously uncharacterized components of this pathway, we performed comparative, ex vivo, genome-wide RNA interference screening. In four screens, we overexpressed the Toll adaptor protein dMyd88, the downstream kinase Pelle, or the nuclear factor κB (NF-κB) homolog Dif, or we knocked down Cactus, the Drosophila homolog of mammalian inhibitor of NF-κB. On the basis of these screens, we identified the E3 ubiquitin ligase Sherpa as being necessary for the activation of Toll signaling. A loss-of-function sherpa mutant fly exhibited compromised production of antimicrobial peptides and enhanced susceptibility to infection by Gram-positive bacteria. In cultured cells, Sherpa mediated ubiquitylation of dMyd88 and Sherpa itself, and Sherpa and Drosophila SUMO (small ubiquitin-like modifier) were required for the proper membrane localization of an adaptor complex containing dMyd88. These findings highlight a role for Sherpa in Drosophila host defense and suggest the SUMOylation-mediated regulation of dMyd88 functions in Toll innate immune signaling.
Ex vivo genome-wide RNAi screening of the Drosophila Toll signaling pathway elicited by a larva-derived tissue extract.” Biochem Biophys Res Commun, 467, 2, Pp. 400-6.Abstract. 2015. “
Damage-associated molecular patterns (DAMPs), so-called "danger signals," play important roles in host defense and pathophysiology in mammals and insects. In Drosophila, the Toll pathway confers damage responses during bacterial infection and improper cell-fate control. However, the intrinsic ligands and signaling mechanisms that potentiate innate immune responses remain unknown. Here, we demonstrate that a Drosophila larva-derived tissue extract strongly elicits Toll pathway activation via the Toll receptor. Using this extract, we performed ex vivo genome-wide RNAi screening in Drosophila cultured cells, and identified several signaling factors that are required for host defense and antimicrobial-peptide expression in Drosophila adults. These results suggest that our larva-derived tissue extract contains active ingredients that mediate Toll pathway activation, and the screening data will shed light on the mechanisms of damage-related Toll pathway signaling in Drosophila.
Molecular Interaction Search Tool (MIST): an integrated resource for mining gene and protein interaction data.” Nucleic Acids Res, 46, D1, Pp. D567-D574.Abstract. 11/16/2017. “
Model organism and human databases are rich with information about genetic and physical interactions. These data can be used to interpret and guide the analysis of results from new studies and develop new hypotheses. Here, we report the development of the Molecular Interaction Search Tool (MIST; http://fgrtools.hms.harvard.edu/MIST/). The MIST database integrates biological interaction data from yeast, nematode, fly, zebrafish, frog, rat and mouse model systems, as well as human. For individual or short gene lists, the MIST user interface can be used to identify interacting partners based on protein-protein and genetic interaction (GI) data from the species of interest as well as inferred interactions, known as interologs, and to view a corresponding network. The data, interologs and search tools at MIST are also useful for analyzing 'omics datasets. In addition to describing the integrated database, we also demonstrate how MIST can be used to identify an appropriate cut-off value that balances false positive and negative discovery, and present use-cases for additional types of analysis. Altogether, the MIST database and search tools support visualization and navigation of existing protein and GI data, as well as comparison of new and existing data.
Zinc Detoxification: A Functional Genomics and Transcriptomics Analysis in Drosophila melanogaster Cultured Cells.” G3 (Bethesda).Abstract. 12/9/2017. “
Cells require some metals, such as zinc and manganese, but excess levels of these metals can be toxic. As a result, cells have evolved complex mechanisms for maintaining metal homeostasis and surviving metal intoxication. Here, we present the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity, together with transcriptomics data for wildtype or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation. Altogether, we identified 47 genes for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment, and more than 1800 putative zinc-responsive genes. Analysis of the 'omics data points to the relevance of ion transporters, glutathione-related factors, and conserved disease-associated genes in zinc detoxification. Specific genes identified in the zinc screen include orthologs of human disease-associated genes CTNS, PTPRN (also known as IA-2), and ATP13A2 (also known as PARK9). We show that knockdown of red dog mine (rdog; CG11897), a candidate zinc detoxification gene encoding an ABCC-type transporter family protein related to yeast cadmium factor (YCF1), confers sensitivity to zinc intoxication in cultured cells and that rdog is transcriptionally up-regulated in response to zinc stress. As there are many links between the biology of zinc and other metals and human health, the 'omics datasets presented here provide a resource that will allow researchers to explore metal biology in the context of diverse health-relevant processes.
Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress.” Proc Natl Acad Sci U S A.Abstract. 12/11/2017. “
A systems-level understanding of cytokine-mediated, intertissue signaling is one of the keys to developing fundamental insight into the links between aging and inflammation. Here, we employed Drosophila, a routine model for analysis of cytokine signaling pathways in higher animals, to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca2+ signaling pathway mediates innate immune responses to GBP, we conducted a dsRNA library screen for genes that modulate Ca2+ mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. We discovered that the metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This we established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. We further report that organismal fitness pays a price for the utilization of Mthl10 to integrate all of these various homeostatic attributes of GBP: We found that elevated GBP expression reduces lifespan. Conversely, Mthl10 knockdown extended lifespan. We describe how our data offer opportunities for further molecular interrogation of yin and yang between homeostasis and longevity.
Accessing the Phenotype Gap: Enabling Systematic Investigation of Paralog Functional Complexity with CRISPR.” Dev Cell, 43, 1, Pp. 6-9.Abstract. 10/9/2017. “
Single-gene knockout experiments can fail to reveal function in the context of redundancy, which is frequently observed among duplicated genes (paralogs) with overlapping functions. We discuss the complexity associated with studying paralogs and outline how recent advances in CRISPR will help address the "phenotype gap" and impact biomedical research.