Heart Rate and Human Performance



Study guide for research assistants

Read "The malaria parasite cyclic GMP-dependent protein kinase plays a central role in blood-stage schizogony" (H. M. Taylor et al., Eukaryotic Cell 9: 37-45, 2010). The full text of this paper can be accessed from UW computers by following the links from this web page: .

Use the study guide below to help you understand the paper. You are welcome to discuss the paper with Greg and/or other people at any time. When you are satisfied with your overall understanding of the paper, please answer the "Questions for lab notebook" in your notebook; these won't be given a letter grade but will be checked!

General background

Your current research concerns several protein kinases thought to be important in various stages of the Plasmodium life cycle. This article offers a detailed look at one of these kinases, protein kinase G (PKG), and includes information on why it might be a good drug target.

To begin with, review the life cycle of the Plasmodium parasite, consulting the web as needed. Concentrate on gametogenesis and the intra-erythrocyte stages, as these are the focus of the PKG work. Then use the third paragraph of the Introduction to cement your understanding.

Title/Abstract

• Make sure you know what schizogony is!

• Some of the experiments are referred to as “erythrocyte invasion assays.” This is because the blood stages of the life cycle involve bursting out of erythrocytes (red blood cells) and invading new ones. The researchers determined whether compounds targeting PKG changed the parasites’ ability to pass through these stages.

• Note the sentence that begins, “To conclusively demonstrate…” In any experiment in which a compound is added to cells in order to inhibit a particular protein, a key issue is whether the compound also affects additional proteins. Here the issue was addressed effectively through the use of a mutant protein that is insensitive to the compound. When this mutant protein was present, the compound no longer stopped schizogony – strong evidence that the compound acts primarily via its effect on PKG.

Introduction

• First sentence: “Cyclic GMP (cGMP)-dependent protein kinases are the major intracellular mediators of cGMP signal transduction in eukaryotic cells.” First look up the chemical structure of cGMP, noting how it differs from GTP and ATP. Then consult the following web page (or a cellular/molecular biology textbook) for a simplified look at cGMP-mediated signal transduction: . The word “transduction” refers to the idea that an extracellular signal or message is transduced into some sort of intracellular response. The ultimate effects of the signal are not shown in the web page’s diagram, but result from phosphorylation of proteins by PKG, thus altering the function of these proteins.

• Later in the first paragraph, note the bit about the regulatory domain of PKG, which seems to fit into the substrate-binding site, preventing it from phosphorylating other proteins until cGMP changes the structure such that the substrate-binding site is opened up. cGMP is thus an allosteric effector of PKG.

• Second paragraph: the parasite PKGs have relatively a small “gatekeeper” residue in their ATP-binding site, so compound 1 has better access to this binding site in coccidian parasites than in mammalian cells.

• Note that the current study uses the same “trick” as a previous study: comparing the wild-type strain against a strain with a mutant (compound 1-insensitive) form of PKG.

Materials and Methods

• P. falciparum cultivation: Mutant strains generally contain antibiotic resistance genes so that they can easily be distinguished from the wild-type strain. That’s true of the T618Q mutant, which harbors a methotrexate resistance gene. The T618Q notation means that at amino acid position #618 in the protein, a threonine (T) was changed to a glutamine (Q).

• Erythrocyte invasion assays and statistical analysis: The researchers determined the fraction of parasite cells that were in particular stages such as the ring stage. This was done by inspecting cells under a microscope and counting them manually. There is potential for bias here – the researchers probably hoped that compound 1 would keep cells in the schizont stage – so the samples were “counted blind” (e.g., the researchers did not know which cells were exposed to compound 1 and which weren’t at the time of counting).

• IFA and Western blotting: you probably know what a Western blot is from your coursework. IFA is for detection and visualization of proteins inside cells (as opposed to proteins on gels, as in Western blotting). A primary antibody binds to the specific protein of interest, and a fluorescent secondary antibody binds to the primary antibody. This is called indirect immunofluorescence because the protein of interest and secondary antibody are not directly connected. (If the primary antibody is fluorescent, that is called direct immunofluorescence.)

Results

• Compound 1 prevents the progression of schizonts through to rings but does not interfere with host cell kinases required for invasion: Compounds like compounds 1-2 and staurosporine are generally dissolved in DMSO, and the DMSO itself could potentially affect PKG and/or other proteins – hence the need for DMSO-only controls.

• Schizonts treated with compound 1 for prolonged periods show aberrant morphology: Note that a bunch of proteins known to localize to different areas of the cell were examined to see whether compound 1 caused them to change their locations. (Rhoptries and micronemes are organelles specific to apicomplexan parasites.)

• PfPKG is central to schizogony: The T618Q mutant was generated by “allelic replacement.” That just means that the mutant gene took the place of the wild-type gene in the genome, as opposed to, say, providing an extra copy of the gene on a plasmid.

Discussion

• Go to and confirm that expression of PKG (gene ID PF14_0346) peaks in late schizonts. (In the “Expression” subsection of the gene page, look at the “Intraerythrocytic 3D7 (photolithographic oligo array)” dataset.)

• At the top right of page 43, consider the paragraph beginning, “To determine whether the inhibitors affect P. falciparum throughout the erythrocytic cycle…” Hypoxanthine is a purine used by the parasite to make purine nucleotides for nucleic acid synthesis, so researchers can measure the uptake of radioactive hypoxanthine by the cells to see how well they are growing.

Questions for lab notebook

1. This paper is a follow-up to a previous study of PKG conducted by the same lab (reference 23). What was the take-home message of that previous study?

2. Giemsa staining is used to mark the parasites and aid identification of their life-cycle stages. What is this stain and how does it work?

3. What is the key difference between the experiment reported in Fig. 1A and that reported in Fig. 1B?

4. What does Fig. 2 tell us that Fig. 1 doesn’t?

5. How do ring-stage parasites differ in appearance from schizonts? Feel free to include a sketch in your answer.

6. In Fig. 5C, which of the four panels is different from the others? Why?

7. This paper relies heavily on carefully selected images of cells. What reassurance (if any) do we have that these images are typical or representative of the experiments performed? What does this tell you about the nature of publishing scientific papers?

8. As discussed at the end of page 42, several cGMP-related genes have similar expression profiles. To what extent is this expected? Why?

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