Fig 1.
Identification of E2 and E3 activity in T. spiralis SP.
A. Streptavidin-HRP blot of in vitro ubiquitination reactions including 1) human E1 (UBE1A), E2 (UBE2L3), E3 (parkin), and Ub-biotin, 2) in the absence of the E2, 3) SP substituted for the human E2, 4) SP substituted for the human E3, 5) SP substituted for the human E1, 6) SP substituted for both the human E2 and E3, 7) SP and Ub-biotin alone. B. T.sp SP were separated by SDS-PAGE and visualized by silver-staining. 50 μg of SP were de-glycosylated by PNGase treatment and analyzed by LC/MS/MS (10 μg of glycosylated proteins were analyzed for comparison). Peptides matching a T. spiralis E2, TsUBE2L3, were identified from the boxed section of the gel.
Fig 2.
Verification of expression, secretion, activity and localization of TsUBE2L3.
A. T.sp SP, HEK 293T cell lysate and T. spiralis muscle larvae lysate were separated by SDS-PAGE and immuno-blotted with anti-HsUBE2L3 and anti-tubulin antibodies. B. Auto-ubiquitination of parkin with Ub-biotin was probed by streptavidin-HRP blot. Reactions from left to right: lane 1: human E1 (UBE1A), E3 (parkin) and Ub-biotin only (no E2). Reactions in lanes 2–4 included human E1, parkin and Ub-biotin, with the following E2 substitution: lane 2: T.sp SP after resin-bound BSA depletion (BSA), lane 3: T.sp SP after resin-bound anti-tubulin depletion (α-tubulin), lane 4: T.sp SP after resin-bound α-HsUBE2L3 depletion (α-HsUBE2L3). In lanes 5–7 the elution fractions from the depletions shown in lanes 2–4 were used to substitute E2. C. The pixel intensity of lanes 2–7 of the depletion assay (B) was analysed using an ImageJ gel analysis plugin. For each of the three samples tested in the assay (BSA, α-tubulin and α-HsUBE2L3) the sum of the pixel intensity of the depletion plus elution lanes was taken as 100% (intensity of lane 2 + 5 for BSA and so on). The relative percentages of the depletion and elution lanes were then calculated (intensity of lane 2/(2+5) x 100) and plotted. D. A TsUBE2L3-specific antibody was made and its specificity was assessed by immuno-blot against T.sp L1 lysate, T.sp SP and rat (R.n) skeletal muscle tissue (SMT) lysate. The arrow indicates the expected size of TsUBE2L3. E. T. spiralis infected rat skeletal muscle tissue (R.n SMT) was sectioned and analyzed by immuno-histofluorescence. A single T.sp L1 inside the nurse cell (n), surrounded by a collagen capsule (c) is displayed. Tissue was probed with anti-TsUBE2L3 antibodies (Alexa-568, red) and nuclei were stained using DAPI (blue). Brightfield (BF)/DAPI/anti-TsUBE2L3 and DAPI/anti-TsUBE2L3 merged images are displayed. As a control, infected R.nSMT was probed with PBS and DAPI only. Arrows indicate stacks of stichocyte cells in the stichosome. F. Annotated diagram of T. spiralis L1 morphology. Image adapted and reprinted from Trichinosis in Man and Animals by S.E. Gould under a CC BY license, with permission from Charles C. Thomas Publisher LTD, original copyright 1970.
Fig 3.
Development of expression system for TsUBE2L3 analysis in C2C12 myotubes.
A. Wild-type C2C12 undifferentiated myoblasts and terminally differentiated myotubes were probed by immuno-fluorescence (IFA) with anti-tubulin antibodies (Alexa-488, green) and nuclei were stained with DAPI (blue). B. Schematic of the pLVX expression construct containing the coding sequence for TsUBE2L3-HA, showing the mechanism of induction by doxycycline (DOX). C. Myotubes transduced with empty vector pLVX or TsUBE2L3-HA pLVX and induced with DOX for 24 h were probed by IFA with anti-HA antibodies (Alexa-568, red); nuclei were stained with DAPI (blue). D. Empty vector and TsUBE2L3-HA cell lysates were reacted with myogenic differentiation markers, myogenin and myosin heavy chain II (MHC), and anti-HA antibodies, at indicated time points after 24 h DOX induction and analyzed by immuno-blot. The same samples were probed with anti-tubulin as a loading control.
Fig 4.
Co-immuno-precipitation (co-IP) and yeast-2-hybrid (Y2H) analyses of TsUBE2L3.
A. Transgenic C2C12 myotube lines (empty vector, eGFP-HA, TsUBE2L3-HA and mouse MmUBE2L3-HA) after 24 h DOX induction were probed by co-IP with anti-HA antibody. Co-IP elutions were silver-stained and all co-IP'd proteins were analyzed by LC/MS/MS. The same samples were reacted with anti-HA antibodies by immuno-blot (IB) as a control for transgene expression. B. Protein interaction network showing positive interactions between TsUBE2L3 and human E3 ligases as observed by yeast-2-hybrid (Y2H) and co-IP analyses. Key indicates if interactions were found by Y2H to be strong, weak or by co-IP.
Table 1.
Identification of TsUBE2L3 E3 interaction partners by co-IP and yeast-2-hybrid (Y2H) analyses.
Only interactions measured by Y2H as being strong are listed. Known ubiquitination substrates found by UbiScan analysis to be significantly and specifically upregulated (upreg.) or downregulated (downreg.) after expression of TsUBE2L3-HA are listed.
Fig 5.
Expression, purification and activity of 6His-TsUBE2L3 and 6His-ARIH2 in vitro.
A. Coommassie stain of 6His-TsUBE2L3 from un-induced (UI) and induced (I) E.coli cultures, nickel purification resin flow-through (FT1-2), wash (W) and elutions (E1-2). B and D. Streptavidin-HRP blots of in vitro parkin auto-ubiquitination reactions using human E1 (UBE1A), human Ub-biotin and either no E2, human E2 (HsUBE2L3) or 6His-TsUBE2L3 with (B) human parkin as the E3 or (C) human ARIH2 as the E3. C. Coomassie stain of 6His-HsARIH2ΔAri from un-induced (UI) E.coli cultures, inclusion body supernatants (S1-4) induced inclusion bodies (I) and refolded from inclusion bodies (Rf). E. Surface representation of ARIH2 (grey) and Ub (wheat) with HsARIH2 (green) and TsUBE2L3 (cyan) bound to the RING1 domain of ARIH2. Residue differences between the E2-E3 interfaces are shown as sticks (HsARIH2 in orange and TsUBE2L3 in magenta). Zinc ions are shown as blue spheres, and Val141 of ARIH2 is shown as sticks. F. Zoom of the ARIH2:E2:Ub interface.
Fig 6.
UbiScan analysis of TsUBE2L3 effect on the myotube ubiquitome.
A. Heatmap displaying proteins in which ubiquitinated peptides were found to be upregulated (blue) or downregulated (red) as measured by a fold change in relation to the empty vector C2C12 myotube cell line after 24 h expression of eGFP-HA (GFP), TsUBE2L3-HA (TsE2) or MmUBE2L3-HA (MmE2). Proteins listed showed a maximum % CV of 49.9 or less, a maximum intensity of 200,000 or more and a fold change in ubiquitination of 2.5 or more in response to TsUBE2L3-HA expression only. Where multiple ubiquitinated peptides were identified for the same protein (according to the protein description as assigned by Cell Signaling Technology), the mean fold change was calculated. Proteins were grouped according to annotated biological ontology. B. Myosin II/fast skeletal myosin (MYH2) (the protein with the largest significant fold change specifically in response to TsUBE2L3 expression) was analyzed by immuno-blot in the empty vector and TsUBE2L3 C2C12 myotube cell lines before and after 24 h of induction (DOX) using anti-myosin II antibodies and anti-vinculin as a loading control.