Protease inhibition by Heterodera glycines cyst content: evidence for effects on the Meloidogyne incognita proteasome

in Nematology
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Proteases from Heterodera glycines and Meloidogyne incognita juveniles were inhibited by heat-stable content from H. glycines cysts (hHglCE), and by a polyphenol (EGCG) similar to a compound previously identified in Globodera cysts. General protease activities detected using the nematode peptide KSAYMRFa were inhibited by EGCG (IC50 1.19 mM, H. glycines; 0.34 mM, M. incognita) but not by hHglCE. However, hHglCE and EGCG each inhibited proteasome-associated chymotrypsin-like (CT-L) activity. EGCG IC50 values were 0.47 mM (H. glycines) and 0.15 mM (M. incognita). hHglCE IC50 values were 0.16 and 0.005 mM hHglCEeq μl−1 for H. glycines and M. incognita, respectively. Across all substrate-inhibitor combinations, M. incognita proteases were inhibited more robustly than those from H. glycines, particularly by hHglCE. In addition to CT-L protease, post-glutamate peptide hydrolysing (PGPH) and trypsin-like (T-L) proteasome proteases were detected in M. incognita, and each of these was also strongly inhibited by hHglCE. hHglCE inhibited CT-L, PGPH and T-L proteases within catalytic subunits from yeast (Saccharomyces cerevisiae) and human proteasomes. Proteasome inhibitors MG-132 and aclacinomycin A each inhibited M. incognita CT-L and PGPH activities by more than 80% at 20-100 μM, and hHglCE inhibited the same proteases by 70-80% at 0.04 hHglCEeq μl−1. hHglCE completely inhibited M. incognita T-L activity, but CT-L activity in native content from H. glycines cysts was not inhibited. Evidence that H. glycines cysts contain inhibitors of all proteases associated with the proteasome establishes the cyst as an important new target to explore for potential nematode control compounds. In addition, characterisation of protease activities from a core cellular metabolic component using M. incognita is novel for plant-parasitic nematodes.

Protease inhibition by Heterodera glycines cyst content: evidence for effects on the Meloidogyne incognita proteasome

in Nematology

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Figures

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    Inhibition of protease activities in preparations of infective juveniles of Heterodera glycines and Meloidogyne incognita by EGCG (A) and by HglCE (B). General protease (black symbols); CT-L protease (white symbols). Each data point represents the percent activity inhibition mean ± SEM for 3-9 independent reactions. A: Inhibition of H. glycines (black squares) and M. incognita (black circles) general protease, and H. glycines (white squares) and M. incognita (white circles) CT-L protease by EGCG; B: Inhibition of H. glycines (open squares) and M. incognita (open circles) CT-L protease by HglCE.

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    Effect of SDS on the detection of three proteasome-associated proteases in preparations of Meloidogyne incognita infective juveniles. Proteases detected were CT-L (black circles), PGPH (black squares), and T-L (black diamonds). Each data point represents the activity mean ± SEM for 4-9 independent reactions. For each substrate, means followed by different letters are significantly different (P<0.05; 1-way ANOVA).  = no activity.

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    Effect of heat-stable Heterodera glycines cyst content on the detection of three proteasome-associated proteases in preparations of Meloidogyne incognita infective juveniles. CT-L, PGPH, and T-L proteases were detected in the absence (white bars) or the presence (grey bars) of hHglCE. Data are expressed as the activity means ± SEM for a minimum of ten independent reactions, except for LLE and ARR with hHglCE where n=3. Between treatments and within substrate, means followed by different letters are significantly different. Across substrates and within treatments, means followed by different numbers are significantly different. (P<0.05, t-test;  = no activity). Inset: Inhibition of M. incognita CT-L protease by hHglCE in the presence of 0.04% SDS. Each data point is the percent inhibition mean ± SEM of three independent reactions, except for 0.0042 hHglCEeq μl−1 where n=2.

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    Effect of heat-stable Heterodera glycines cyst content on the detection of three proteasome-associated proteases in preparations of pure 20S catalytic subunits from Saccharomyces cerevisiae and Homo sapiens proteasomes. CT-L, PGPH and T-L proteases were detected in the absence (white bars) or presence (grey bars) of hHglCE. Data are activity means ± SEM for a minimum of six independent reactions, except for H. sapiens ARR where n=3, each for control and treated. Between treatments and within substrate, means followed by different letters are significantly different. Across substrates and within treatments, means followed by different numbers are significantly different. (P<0.05, t-test;  = no activity.) A: S. cerevisiae; Inset, LLE and ARR activities on an expanded scale; B: H. sapiens.

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