Greg Crowther, UW Dept. of Medicine October 7, 2009

Study guide for research assistants

Read "Toxoplasma gondii cathepsin L is the primary target of the invasion-inhibitory compound morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl" (E. T. Larson et al.,Journal of Biological Chemistry284: 26839-50, 2009). The full text of this paper can be accessed onlineby following the links from this 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, pleaseanswer the "Questions for lab notebook" in yournotebook; these won't be given a letter grade but will be checked!

General background

As we have discussed previously, identifying the specific protein targets of cell-active compounds can be a complicated and difficult process. For example, the fact that a given antimalaria compound inhibits a certain purified Plasmodium enzyme does not prove that the compound kills the parasite by acting on this particular enzyme. Showing that resistance to the compound arises via mutation or amplification of the suspected target is one way to strengthen the case for a compound-target linkage; the assigned paper shows some alternative ways of pinning a compoundto a specific target. In this case, the compound is morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS) and the target is the cysteine protease cathepsin L from Toxoplasma gondii(TgCPL).

T. gondii, the protozoan parasite that causes toxoplasmosis, is commonly studied for two main reasons. First, as noted in the second paragraph of the Introduction, it is extremely threatening to immunocompromised individuals such as AIDS patients. Second, it is a model organism for understanding the Plasmodium parasites, since it is phylogenetically related to Plasmodium (both belong to phylum Apicomplexa) but is easier than Plasmodium to manipulate genetically.

Abstract

• First sentence: Proteolysis means lysis of proteins. You knew that, right?

• LHVS impairs the release of proteins from micronemes. Micronemes, according to Wikipedia, are "specialized secretory organelles important for gliding motility and host cell invasion."

• Once T. gondii is inside a host cell, TgCPL is found in multiple cytoplasmic "puncta" --i.e., several discrete locations.

• Note that the structural model for inhibition of TgCPL by LHVS is based on a crystal structure of the inactive precursor of the enzyme (with the propeptide still attached). The significance of this will become clear later.

Introduction

• First paragraph: The fact that cathepsins are "cysteine proteases" does not mean that the substrate proteins are cleaved at cysteine residues; rather, it means that the proteases have a key cysteine in the active site that is essential for catalysis. Similarly, serine proteases have a serine in the active site, threonine proteases have a threonine in the active site, etc.

• Third paragraph: Reference #10 showed that TgCPL has a low pH optimum. This is a clue to the intracellular location of the enzyme; i.e., it probably resides in an acidic organelle. While Larson et al. do not definitively nail down TgCPL's location, they say in the Discussion (top right of p. 26848) that TgCPL resides in "an endocytic organelle possibly related to a lysosome," which would be consistent with the low pH optimum.

Experimental Procedures

• Cloning, Protein Expression, Purification, Refolding, and Autoactivation: First, note the mention of the "prodomain" and the "mature domain." Before this enzyme can act upon other proteins, it must cleave itself, releasing its prodomain (propeptide). This is called autoactivation. Second, note that a six-histidine (His6) tag was added to TgCPL, as is usually done with our Plasmodium proteins. This allows the protein to be purified with immobilized metal affinity chromatography (IMAC). A column containing immobilized metal ions such as Ni2+is used to attract the protein's six consecutive histidine residues so that the protein of interest is separated from other proteins. Third, note all of the processing that was done on the protein once it was isolated from the E. coli cells. This is because the "raw" protein that was gathered was both denatured and inactive (i.e., in the "proform"), and the researchers wanted to study the properly folded, activated protein.

• Activity-based Profiling: In short, this allowed the researchers to prove that LHVS binds to TgCPL, as shown in Figures 1 and 3. They used a modified LHVS called BO-LHVS (see Figure 1A), whose fluorescent label makes it easy to track.

• Targeted disruption of TgCPL: This section explains how the TgCPL gene was knocked out. A TgCPL knockout strain serves asan important control because, if you think LHVS exerts its effects via its interaction with TgCPL, those effects should disappear when TgCPL is missing.

• You may skim the remaining sections (Immunoblotting, Immunoprecipitation, and Fluorescence Microscopy; Protein Crystallization; Data Collection and Structure Determination; Modeling of the LHVS Inhibitor into the TgCPL Active Site), which are beyond the scope of this assignment.

Results

• Figure 1 is about location, location, location. Where's the TgCPL? Where's the (BO-)LHVS? Are they in the same place? Figure1B is a protein gel (with weights in kilodaltons listed on the left side), with bands of fluorescence showing that the BO-LHVS binds to recombinant TgCPL (a 30-kilodalton protein) but not denatured (heat-inactivated,or HI) TgCPL. Figure 1C shows that similar results are obtained with "native" TgCPL (produced naturally by T. gondii cells). Figure 1D is the subject of a question below. Figure 1E shows that BO-LHVS appears in most of the same locations as TgCPL in live cells. (The authors say that, since BO-LHVS only binds to activated TgCPL, the spots of TgCPL that don't coincide with BO-LHVS signal might indicate the "proform" enzyme that has not yet activated itself.)

• Figure 2 is about knocking out (deleting) TgCPL. Most panels of this figure simply show in various ways that TgCPL is not expressed in the knockout strains (RHΔcpl and Ku80Δcpl). Note also, though, that, according to Figure 2D, the knockout strains are able to invade host cells! Does this call into question TgCPL's suitability as a drug target? The second paragraph of the Discussion has furtherrelevant information.

• X-ray Crystal Structure of TgCPL and Its Propeptide Reveals the Canonical Catalytic Triad and Active Site Cleftand Modeling the Binding Mode of LHVS: You should read these sections, but you do not need to master them or their associated figures (4 and 5). A brief summary is as follows. The researchers wanted to crystallize the mature, active TgCPL with LHVS bound (so that they could see how it fits into the TgCPL active site), but they were unable to get the cleaved propeptide out of the active site, and LHVS was unable to displace it, so they wound up with a crystal structure of the TgCPL with its propeptide still bound. They then made a model of how the LHVS is likely to bind based on comparisons of their crystal structure with structures of similar proteins containing molecules similar to LHVS. The final sentence of the results concludes, "The binding mode predicted by this model strongly suggests that a covalent complex between TgCPL and LHVS is favorable and that, similar to other cathepsin L proteases, LHVS will inhibit TgCPL activity."

Discussion

• The first paragraph clearly summarizes several of the paper's main points. Read it carefully.

• The second-to-last paragraph explains why the authors think the structural model of LHVS inhibition of TgCPL is valid even though it is based on the structure of an inactive form of the enzyme.

Questions for lab notebook

1. The second paragraph of the Introduction says, "T. gondii is an obligate intracellular organism that uses an actin-myosin-based motility system to actively invade nucleated host cells." How does this make it unlike Plasmodium?

2. Why do you think that a truncated version of the TgCPL gene was expressed (as described in the left column of p. 26840)?

3. The top right paragraph of p. 26840 states, "The efficiency of maturation was monitored using SDS-PAGE." Explain.

4. What is the point of Figure 1D? Hint: is it possible that BO-LHVS acts differently than LHVS?

5. What is the main difference between Figures 2B and 2C?

6. What is the point of Figure 3B?

7. Based on this paper – especially the information in the right column of p. 26848 – what role is TgCPL likely to play in T. gondii cells?

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