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Journal ArticleDOI

Plasmodium palmitoylation machinery engineered in E. coli for high‐throughput screening of palmitoyl acyl‐transferase inhibitors

10 Jan 2019-FEBS Open Bio (John Wiley & Sons, Ltd)-Vol. 9, Iss: 2, pp 248-264
TL;DR: A novel strategy which involves engineering of Escherichia coli, a PTM‐null system, to enforce ectopic expression of palmitoyl acyl‐transferase in order to study Plasmodium‐specificPalmitoylation and screening of inhibitors is developed, suggesting that synthetic E. coli strains expressing PfDHHCs can enforce global palMIToylation in the E.ccoli proteome.
About: This article is published in FEBS Open Bio.The article was published on 2019-01-10 and is currently open access. It has received 12 citations till now. The article focuses on the topics: Palmitoylation & Escherichia coli.
Citations
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Journal ArticleDOI
TL;DR: Protein lipid modification involves the attachment of hydrophobic groups to proteins via ester, thioester, amide, or thioether linkages as discussed by the authors, and the role of click chemistry in elucidating specific biological features.
Abstract: Protein lipid modification involves the attachment of hydrophobic groups to proteins via ester, thioester, amide, or thioether linkages. In this review, the specific click chemical reactions that have been employed to study protein lipid modification and their use for specific labeling applications are first described. This is followed by an introduction to the different types of protein lipid modifications that occur in biology. Next, the roles of click chemistry in elucidating specific biological features including the identification of lipid-modified proteins, studies of their regulation, and their role in diseases are presented. A description of the use of protein-lipid modifying enzymes for specific labeling applications including protein immobilization, fluorescent labeling, nanostructure assembly, and the construction of protein-drug conjugates is presented next. Concluding remarks and future directions are presented in the final section.

42 citations

Journal ArticleDOI
Xu Wang1, Pengge Qian1, Huiting Cui1, Luming Yao1, Jing Yuan1 
TL;DR: This study identifies the first PAT–substrate pair in malaria parasites and uncovers a protein palmitoylation cascade regulating microtubule cytoskeleton.
Abstract: Morphogenesis of many protozoans depends on a polarized establishment of cytoskeletal structures. In malaria-causing parasites, this can be observed when a round zygote develops into an elongated motile ookinete within the mosquito stomach. This morphogenesis is mediated by the pellicle cytoskeletal structures, including the inner membrane complex (IMC) and the underlying subpellicular microtubules (SPMs). How the parasite maintains the IMC-SPM connection and establishes a dome-like structure of SPM to support cell elongation is unclear. Here, we show that palmitoylation of N-terminal cysteines of two IMC proteins (ISP1/ISP3) regulates the IMC localization of ISP1/ISP3 and zygote-to-ookinete differentiation. Palmitoylation of ISP1/ISP3 is catalyzed by an IMC-residing palmitoyl-S-acyl-transferase (PAT) DHHC2. Surprisingly, DHHC2 undergoes self-palmitoylation at C-terminal cysteines via its PAT activity, which controls DHHC2 localization in IMC after zygote formation. IMC-anchored ISP1 and ISP3 interact with microtubule component β-tubulin, serving as tethers to maintain the proper structure of SPM during zygote elongation. This study identifies the first PAT-substrate pair in malaria parasites and uncovers a protein palmitoylation cascade regulating microtubule cytoskeleton.

36 citations


Additional excerpts

  • ...Source data are available online for this figure. ª 2020 The Authors The EMBO Journal 39: e104168 | 2020 3 of 22 isp3::6HA, by tagging endogenous ISP1 with a triple V5 epitope (3V5) in the isp3::6HA parasite (Appendix Fig S1) and observed similar co-localization of ISP1/ISP3 at the periphery of zygote protrusion (Fig 2B)....

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  • ...18 of 22 The EMBO Journal 39: e104168 | 2020 ª 2020 The Authors...

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  • ...Indeed, the Cter75 fused with ª 2020 The Authors The EMBO Journal 39: e104168 | 2020 7 of 22 a BFP::Myc peptide could be palmitoylated by co-transfected WT DHHC2-HA in HEK293 cells, while the Cter75 containing C4A mutations (Cter75*) failed to be modified (Fig 5M)....

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  • ...We further generated a doubly tagged parasite (DTS), isp1::3V5/ 2 of 22 The EMBO Journal 39: e104168 | 2020 ª 2020 The Authors B C D E F in vitro 2 4 6 8 10 12 0 20 40 60 80 100 Time post fertilization (h) Δisp1/3 wildtype *** ****** P ar as ite o f s ta ge I an d II (% ) Δisp1/3 wildtype H J wildtype Δisp1/3 wild typ e ** 0 2 4 6 8 10 12 S al iv ar y gl an d sp or oz oi te s p er m os qu ito (× 10 3 ) Δis p1/ 3 wild typ e G wildtype Δisp1 Δisp3 Δisp1/3 Py/Pf.isp1 ISP3 (HA) Hoechst Py/Pf.isp3 6HA 4 5 6 7 O oc ys ts p er m os qu ito (D ay 7 ) 0 50 100 150 200 250 300 350 **** n=55 n=59 A Δis p3 wild typ e Δis p1 Δis p1/ 3 ** *** * ns C on ve rs io n ra te to m at ur e oo ki ne te (% ) 0 20 40 60 80 3V5 ISP1 (V5) Hoechst Pyisp1 Pfisp1 Pyisp3 Pfisp3 I Δis p1/ 3 0 20 40 60 80 C on ve rs io n ra te to m at ur e oo ki ne te (% ) *** *** *** ** wi ldt yp e Δis p1 /3 Δisp1/3 rescued with Py isp 1 Pf isp 1 Py isp 3 Pf isp 3 ** P ar as ite o f s ta ge I an d II (% ) 0 20 40 60 80 100 Δis p1/ 3 wild typ e in vivo 25 25 15 Δisp1/3 rescued with HA ISP3 V5 ISP1 P28 D TS Δ is p1 /3 P yi sp 1:...

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  • ...16 of 22 The EMBO Journal 39: e104168 | 2020 ª 2020 The Authors required to fully elucidate the mechanisms by which the IMC recruits the palmitoylated DHHC2....

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Journal ArticleDOI
TL;DR: It is shown that ionic imbalance caused by scaffold 7 induces autophagy that leads to onset of apoptosis in the parasite evident by the loss of mitochondrial membrane potential (ΔΨm) and DNA degradation.
Abstract: Natural products offer an abundant source of diverse novel scaffolds that inspires development of next generation anti-malarials. With this vision, a library of scaffolds inspired by natural biologically active alkaloids was synthesized from chiral bicyclic lactams with steps/scaffold ratio of 1.7:1. On evaluation of library of scaffolds for their growth inhibitory effect against malaria parasite we found one scaffold with IC50 in low micro molar range. It inhibited parasite growth via disruption of Na+ homeostasis. P-type ATPase, PfATP4 is responsible for maintaining parasite Na+ homeostasis and is a good target for anti-malarials. Molecular docking with our scaffold showed that it fits well in the binding pocket of PfATP4. Moreover, inhibition of Na+-dependent ATPase activity by our potent scaffold suggests that it targets parasite by inhibiting PfATP4, leading to ionic imbalance. However how ionic imbalance attributes to parasite's death is unclear. We show that ionic imbalance caused by scaffold 7 induces autophagy that leads to onset of apoptosis in the parasite evident by the loss of mitochondrial membrane potential (ΔΨm) and DNA degradation. Our study provides a novel strategy for drug discovery and an insight into the molecular mechanism of ionic imbalance mediated death in malaria parasite.

18 citations

Journal ArticleDOI
TL;DR: In this paper, metabolic labeling and click chemistry are employed as a fast transfection-independent method for the microscopic examination of protein S-palmitoylation, an important post-translational modification during the asexual intraerythrocytic replication of P. falciparum.
Abstract: The examination of the complex cell biology of the human malaria parasite Plasmodium falciparum usually relies on the time-consuming generation of transgenic parasites. Here, metabolic labeling and click chemistry are employed as a fast transfection-independent method for the microscopic examination of protein S-palmitoylation, an important post-translational modification during the asexual intraerythrocytic replication of P. falciparum. Applying various microscopy approaches such as confocal, single-molecule switching, and electron microscopy, differences in the extent of labeling within the different asexual developmental stages of P. falciparum and the host erythrocytes over time are observed.

9 citations

Posted ContentDOI
21 Jan 2020-bioRxiv
TL;DR: Reduced expression of Kell in palmitoylated protein pool of sickle-cell RBC ghost, with its diminished surface representation in these RBCs, proposed Kell, as one of the novel receptor-prototype for P. falciparum invasion.
Abstract: Squeezability of biconcave RBC raises a fundamental query, about, how it can restructure its bendable cytoskeleton for efficient micro-circulation. We report for the first time, the existence of dynamic palmitoylome in RBC composed of 118 palmitoylated proteins that reduced to 42 upon treatment with 2BP, a generic inhibitor of palmitoylation. In-depth analysis revealed that Semaphorin7A, CR1 and ABCB6, the known RBC receptors for P. falciparum were reduced to negligible in 2BP-treated RBCs, suggesting palmitoylation-dependent recruitment of parasite-specific receptors. Interestingly, Kell, a single disulphide-linked co-partner in Kell-Kx complex was undetected in 2BP-treated RBCs, while Kx remained intact. RBCs-blocked with anti-Kell antibody demonstrated signficant reduction in parasite invasion, thus suggesting it as a receptor proto-type for P. falciparum invasion. Finally, reduced expression of Kell in palmitoylated protein pool of sickle-cell RBC ghost, with its diminished surface representation in these RBCs, proposed Kell, as one of the novel receptor-prototype for P. falciparum invasion.

2 citations

References
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30,478 citations

Journal ArticleDOI
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19,213 citations

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TL;DR: AutoDock4 incorporates limited flexibility in the receptor and its utility in analysis of covalently bound ligands is reported, using both a grid‐based docking method and a modification of the flexible sidechain technique.
Abstract: We describe the testing and release of AutoDock4 and the accompanying graphical user interface AutoDockTools. AutoDock4 incorporates limited flexibility in the receptor. Several tests are reported here, including a redocking experiment with 188 diverse ligand-protein complexes and a cross-docking experiment using flexible sidechains in 87 HIV protease complexes. We also report its utility in analysis of covalently bound ligands, using both a grid-based docking method and a modification of the flexible sidechain technique.

15,616 citations