Search

Protein modification by ubiquitin and ubiquitin-like proteins is one of the most complex and intensely studied mechanisms of posttranslational protein regulation in eukaryotes. Conjugation of the 76-amino-acid protein ubiquitin is first and foremost a signal for targeting proteins to the proteasome

Thin layer chromatography (TLC) was used to determine the free pool amino acid content of four larval stages (rediae, cercariae, encysted metacercariae, and excysted metacercariae) of the medically important digenetic trematode, Echinostoma caproni. These larval stages were obtained from experimentally infected Biomphalaria glabrata snails. Larvae were pooled, extracted in ethanol, and their free pool amino acids separated using four types of layers with different separation mechanisms. Zones were detected with ninhydrin spray and quantified by densitometry. Qualitative analysis revealed the presence of valine, leucine, lysine, histidine, and alanine in rediae; histidine in cercariae; histidine and alanine in encysted metacercariae; and histidine, alanine, and leucine in excysted metacercariae. Quantitative analysis showed that rediae contained 0.76 ± 0.20 ng of lysine per organism and excysted metacercariae contained 0.16 ± 0.05 ng of leucine per organism.

SummaryThe Plasmodium proteasome has been suggested to be a potential antimalarial drug target; however, toxicity of inhibitors has prevented validation of this enzyme in vivo. We report a screen of a library of 670 analogs of the recent US Food and Drug Administration-approved inhibitor, carfilzomib, to identify compounds that selectively kill parasites. We identified one compound, PR3, that has significant parasite killing activity in vitro but dramatically reduced toxicity in host cells. We found that this parasite-specific toxicity is not due to selective targeting of the Plasmodium proteasome over the host proteasome, but instead is due to a lack of activity against one of the human proteasome subunits. Subsequently, we used PR3 to significantly reduce parasite load in Plasmodium berghei infected mice without host toxicity, thus validating the proteasome as a viable antimalarial drug target.

We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, self-cleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD), an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP(6)), a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His(6)-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP(6) to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s) and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms.

Rediae of the trematode Echinostoma trivolvis, from naturally infected Helisoma trivolvis snails, form a black pigment while inside the snail host. Here we examine the black pigment to show that the insolubility characteristics in detergent and weak base solution are identical to Plasmodium falciparum hemozoin. Laser desorption mass spectrometry of the purified pigment demonstrates the presence of heme. Examination of purified pigment under polarized light microscopy illuminates ordered birefringent crystals. Field emission in lens scanning electron microscopy reveals irregular ovoid crystals of 200–300 nm in diameter. The purified pigment crystals seeded extension of monomeric heme onto the crystal which by Fourier Transform Infrared analysis is β-hematin. Rediae of a second echinostome parasite, Echinostoma caproni, from experimentally infected Biomphalaria glabrata, do not produce measurable or recoverable heme crystals. These observations are consistent with heme crystal formation by a hematophagous parasite within a non-vertebrate intermediate host.

Thin-layer chromatography was used to analyze the free-pool amino acids of the digestive gland-gonad complex (DGG) of Biomphalaria glabrata infected with Echinostoma caproni and uninfected (control) snails. Qualitative analysis revealed the presence of histidine, lysine, serine, alanine, valine, and isoleucine or leucine in all samples. Quantitative analysis of lysine and valine gave mean weight percentages of 0.00699 +/- 0.0022 and 0.00174 +/- 0.00056, respectively, in the DGG of uninfected snails, and 0.00504 +/- 0.0014 and 0.00254 +/- 0.00033, respectively, in the DGG of infected snails. The differences in values between infected and uninfected snails were not statistically significant (Student's t-test, P0.05).

Thin layer chromatography (TLC) was used to analyze the amino acids in worm incubates isotonic and hypotonic to the intestinal habitat of adult Echinostoma caproni and to analyze the free pool amino acids of these trematodes after incubation. Qualitative analysis revealed the presence of histidine, lysine, alanine, and proline in all samples of incubate and worm tissue. Quantification of histidine and lysine by TLC with densitometry gave mean concentrations of 24.1 micro g histidine/g worm per ml incubate in Locke's solution and 195.0 micro g lysine/g worm per ml incubate in deioinized (DI) water. Quantification of histidine and lysine in the worm tissue gave mean weight percents of 0.0587 and 0.0263, respectively, in worms incubated in Locke's solution and 0.0229 and 0.0163, respectively, for worms incubated in DI water. Our findings suggest that E. caproni adults may leak amino acids for osmoregulation in hypotonic environments.

Summary Sentrin specific proteases (SENPs) are responsible for activating and deconjugating SUMO (Small Ubiquitin-like MOdifier) from target proteins. It remains difficult to study this posttranslational modification due to the lack of reagents that can be used to block the removal of SUMO from substrates. Here, we describe the identification of small molecule SENP inhibitors and active site probes containing aza-epoxide and acyloxymethyl ketone (AOMK) reactive groups. Both classes of compounds are effective inhibitors of hSENPs 1, 2, 5, and 7 while only the AOMKs efficiently inhibit hSENP6. Unlike previous reported peptide vinyl sulfones, these compounds covalently labeled the active site cysteine of multiple recombinantly expressed SENP proteases and the AOMK probe showed selective labeling of these SENPs when added to complex protein mixtures. The AOMK compounds therefore represent promising new reagents to study the process of SUMO deconjugation.