Characterisation of the Trichinella spiralis deubiquitinating enzyme, TsUCH37, an evolutionarily conserved proteasome interaction partner

Abstract

Background: Trichinella spiralis is a zoonotic parasitic nematode that causes trichinellosis, a disease that has been identified on all continents except Antarctica. During chronic infection, T. spiralis larvae infect skeletal myofibres, severely disrupting their differentiation state.

Methodology and results: An activity-based probe, HA-Ub-VME, was used to identify deubiquitinating enzyme (DUB) activity in lysate of T. spiralis L1 larvae. Results were analysed by immuno-blot and immuno-precipitation, identifying a number of potential DUBs. Immuno-precipitated proteins were subjected to LC/MS/MS, yielding peptides with sequence homology to 5 conserved human DUBs: UCH-L5, UCH-L3, HAUSP, OTU 6B and Ataxin-3. The predicted gene encoding the putative UCH-L5 homologue, TsUCH37, was cloned and recombinant protein was expressed and purified. The deubiquitinating activity of this enzyme was verified by Ub-AMC assay. Co-precipitation of recombinant TsUCH37 showed that the protein associates with putative T. spiralis proteasome components, including the yeast Rpn13 homologue ADRM1. In addition, the UCH inhibitor LDN-57444 exhibited specific inhibition of recombinant TsUCH37 and reduced the viability of cultured L1 larvae.

Conclusions: This study reports the identification of the first T. spiralis DUB, a cysteine protease that is putatively orthologous to the human protein, hUCH-L5. Results suggest that the interaction of this protein with the proteasome has been conserved throughout evolution. We show potential for the use of inhibitor compounds to elucidate the role of UCH enzymes in T. spiralis infection and their investigation as therapeutic targets for trichinellosis.

Figure 1. Identification of deubiquitinating activity in T. spiralis L1 lysate.

(A) Lysate of T. spiralis L1 larvae was reacted with HA-Ub-VME, separated by 10% SDS-PAGE and analysed by immuno-blot using anti-HA-HRP. Reactions were carried out either in the absence or presence of NEM in order to determine cysteine specificity. Probe alone and lysate alone were included as controls (left hand lanes). Arrows indicate un-reacted probe or probe aggregates and molecular weights are shown in kDa. (B) Large-scale immuno-precipitation was carried out to isolate T. spiralis protein-HA-Ub-VME complexes on an anti-HA affinity matrix. Samples were separated by SDS-PAGE and 20 bands (numbered) were excised and analysed by tandem mass spectrometry. Arrows indicate the anti-HA antibody light chain (25 kDa) and un-reacted probe (10 kDa).

Figure 2. Protein alignment (MUSCLE) of the translated putative T. spiralis TsUCH37 sequence, EST gi157958881 and human UCH-L5 protein sequence.

The translated ORF sequence generated by AUGUSTUS (T.sp AUG) was aligned with the translated EST gi157958881 (T.sp EST) and human UCH-L5 (hUCH-L5) protein sequence (Drummond AJ et al, www.geneious.com). The full peptidase C12 domain (shaded box) and catalytic residues (asterisk) of hUCH-L5 are indicated. The TsUCH37 AUGUSTUS sequence displays 45% identity and 16% homology to hUCH-L5 and has a predicted molecular weight of 35.22 kDa. Conserved residues are highlighted where identical and homologous. Positions of the putative TsUCH37 start methionine, catalytic residues and the predicted end of the protein are numbered. The KEKE domain of the hUCH-L5 sequence and a potential KEKE domain of the putative TsUCH37 sequence have been outlined.

Figure 3. Expression, purification and verification of the deubiquitinating activity of recombinant TsUCH37.

(A) HIS-tagged recombinant TsUCH37 was expressed and purified yielding a protein of approximately 36 kDa when separated by SDS-PAGE. (B) Recombinant TsUCH37 (2 µM) was reacted with Ub-AMC. Ub-AMC hydrolysis was measured in RFU as fluorogenic AMC was released over time (minutes). All assays were carried out in triplicate. Points show the mean fluorescence with standard deviation (error bars are indicated). Deubiquitinating activity was fully inhibited by the pre-incubation of the protein with NEM. Plasmodium falciparum PfUCH-L3 (74 nM) was used as a positive control for deubiquitinating activity.

Figure 4. Identification of recombinant TsUCH37 associated T. spiralis proteins.

Ni-NTA bound TsUCH37 (with an N-terminal HIS-tag) was incubated with pre-cleared T. spiralis L1 larvae lysate. Associated proteins were isolated by co-precipitation and separated by SDS-PAGE. Samples containing native Ni-NTA resin incubated with pre-cleared T. spiralis lysate (middle lane) and Ni-NTA bound recombinant TsUCH37 incubated with lysis buffer (right lane) were included as controls. The gel was visualised using colloidal Coomassie staining and 19 (numbered) bands were excised and analysed by LC/MS/MS. Recombinant HIS-tagged TsUCH37 is indicated by an arrow and the location of peptides matching the putative T. spiralis ADRM1 protein sequence is marked with an asterisk. Molecular weights are shown in kDa.

Figure 5. LDN-57444 inhibition of recombinant TsUCH37 DUB activity and parasite viability.

Recombinant TsUCH37 was incubated with the UCH inhibitor LDN-57444. Panel A shows the cleavage of Ub from fluorogenic AMC by 12.5 nM TsUCH37 (measured as relative fluorescence units, RFU) after incubation with 50 µM, 100 µM and 500 µM of LDN-57444 solubilised in DMSO. Pre-incubation of TsUCH37 with NEM was included as a negative control and incubation with DMSO alone as a positive control. All assays were carried out in triplicate. Points show the mean fluorescence with standard deviation (error bars are indicated). (B), (C) and (D) The susceptibility of T. spiralis L1 larvae cultured in the presence of DMSO or 50 µM and 100 µM LDN-57444 after 24, 48 and 96 hours was measured as absorbance of formazan at 575 nm using the MTT assay. All assays were carried out in triplicate. The mean percentage of the DMSO control (100%) with 1 standard deviation is shown. Baseline absorbance was taken by MTT assay of heat-killed (HT) parasites. Statistically significant data are indicated with asterisks (*, P<0.05 versus parasites incubated with DMSO alone).