17 results found
Anderson DP, Benns HJ, Tate EW, et al., 2020, CRISPR-TAPE: protein-centricCRISPRguide design for targeted proteome engineering, MOLECULAR SYSTEMS BIOLOGY, Vol: 16, ISSN: 1744-4292
Anderson DP, Benns HJ, Tate EW, et al., 2020, CRISPR-TAPE: protein-centric CRISPR guide design for targeted proteome engineering., Mol Syst Biol, Vol: 16
Rational molecular engineering of proteins with CRISPR-based approaches is challenged by the gene-centric nature of gRNA design tools. To address this, we have developed CRISPR-TAPE, a protein-centric gRNA design algorithm that allows users to target specific residues, or amino acid types within proteins. gRNA outputs can be customized to support maximal efficacy of homology-directed repair for engineering purposes, removing time-consuming post hoc curation, simplifying gRNA outputs and reducing CPU times.
Benns HJ, Tate EW, Child MA, 2018, Activity-Based Protein Profiling for the Study of Parasite Biology., Curr Top Microbiol Immunol, Vol: 420, Pages: 155-174, ISSN: 0070-217X
Parasites exist within most ecological niches, often transitioning through biologically and chemically complex host environments over the course of their parasitic life cycles. While the development of technologies for genetic engineering has revolutionised the field of functional genomics, parasites have historically been less amenable to such modification. In light of this, parasitologists have often been at the forefront of adopting new small-molecule technologies, repurposing drugs into biological tools and probes. Over the last decade, activity-based protein profiling (ABPP) has evolved into a powerful and versatile chemical proteomic platform for characterising the function of enzymes. Central to ABPP is the use of activity-based probes (ABPs), which covalently modify the active sites of enzyme classes ranging from serine hydrolases to glycosidases. The application of ABPP to cellular systems has contributed vastly to our knowledge on the fundamental biology of a diverse range of organisms and has facilitated the identification of potential drug targets in many pathogens. In this chapter, we provide a comprehensive review on the different forms of ABPP that have been successfully applied to parasite systems, and highlight key biological insights that have been enabled through their application.
Garland M, Schulze CJ, Foe IT, et al., 2018, Development of an activity-based probe for acyl-protein thioesterases, PLOS ONE, Vol: 13, ISSN: 1932-6203
Child MA, Garland M, Foe I, et al., 2017, Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1, mBio, Vol: 8, ISSN: 2150-7511
Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondii.IMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of the
Foe IT, Child MA, Majmudar JD, et al., 2015, Global Analysis of Palmitoylated Proteins in Toxoplasma gondii, CELL HOST & MICROBE, Vol: 18, Pages: 501-511, ISSN: 1931-3128
Bender KO, Garland M, Ferreyra JA, et al., 2015, A small-molecule antivirulence agent for treating Clostridium difficile infection, SCIENCE TRANSLATIONAL MEDICINE, Vol: 7, ISSN: 1946-6234
van der Linden WA, Segal E, Child MA, et al., 2015, Design and Synthesis of Activity-Based Probes and Inhibitors for Bleomycin Hydrolase, CHEMISTRY & BIOLOGY, Vol: 22, Pages: 995-1001, ISSN: 1074-5521
Treeck M, Sanders JL, Gaji RY, et al., 2014, The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis, PLOS PATHOGENS, Vol: 10, ISSN: 1553-7366
Child MA, 2013, Chemical biology approaches for the study of apicomplexan parasites, MOLECULAR AND BIOCHEMICAL PARASITOLOGY, Vol: 192, Pages: 1-9, ISSN: 0166-6851
Child MA, Hall CI, Beck JR, et al., 2013, Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion, NATURE CHEMICAL BIOLOGY, Vol: 9, Pages: 651-656, ISSN: 1552-4450
Child MA, Harris PK, Collins CR, et al., 2013, Molecular Determinants for Subcellular Trafficking of the Malarial Sheddase PfSUB2, TRAFFIC, Vol: 14, Pages: 1053-1064, ISSN: 1398-9219
Child MA, Hall CI, Beck JR, et al., 2013, Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion., Nat Chem Biol, Vol: 9, Pages: 651-656
Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.
Li H, Child MA, Bogyo M, 2012, Proteases as regulators of pathogenesis: Examples from the Apicomplexa, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, Vol: 1824, Pages: 177-185, ISSN: 1570-9639
Hall CI, Reese ML, Weerapana E, et al., 2011, Chemical genetic screen identifies Toxoplasma DJ-1 as a regulator of parasite secretion, attachment, and invasion, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 108, Pages: 10568-10573, ISSN: 0027-8424
Child MA, Epp C, Bujard H, et al., 2010, Regulated maturation of malaria merozoite surface protein-1 is essential for parasite growth, MOLECULAR MICROBIOLOGY, Vol: 78, Pages: 187-202, ISSN: 0950-382X
Koussis K, Withers-Martinez C, Yeoh S, et al., 2009, A multifunctional serine protease primes the malaria parasite for red blood cell invasion, EMBO JOURNAL, Vol: 28, Pages: 725-735, ISSN: 0261-4189
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