A muscle-specific MuRF1-E2 network requires stabilization of MuRF1-E2 complexes by telethonin, a newly identified substrate

Abstract

Background: Muscle wasting is observed in the course of many diseases and also during physiological conditions (disuse, ageing). Skeletal muscle mass is largely controlled by the ubiquitin-proteasome system and thus by the ubiquitinating enzymes (E2s and E3s) that target substrates for subsequent degradation. MuRF1 is the only E3 ubiquitin ligase known to target contractile proteins (α-actin, myosins) during catabolic situations. However, MuRF1 depends on E2 ubiquitin-conjugating enzymes for ubiquitin chain formation on the substrates. MuRF1-E2 couples are therefore putative targets for preventing muscle wasting.

Methods: We focused on 14 E2 enzymes that are either expressed in skeletal muscle or up-regulated during atrophying conditions. In this work, we demonstrated that only highly sensitive and complementary interactomic approaches (surface plasmon resonance, yeast three-hybrid, and split green fluorescent protein) allowed the identification of MuRF1 E2 partners.

Results: Five E2 enzymes physically interacted with MuRF1, namely, E2E1, E2G1, E2J1, E2J2, and E2L3. Moreover, we demonstrated that MuRF1-E2E1 and MuRF1-E2J1 interactions are facilitated by telethonin, a newly identified MuRF1 substrate. We next showed that the five identified E2s functionally interacted with MuRF1 since, in contrast to the non-interacting E2D2, their co-expression in HEK293T cells with MuRF1 led to increased telethonin degradation. Finally, we showed that telethonin governed the affinity between MuRF1 and E2E1 or E2J1.

Conclusions: We report here the first MuRF1-E2s network, which may prove valuable for deciphering the precise mechanisms involved in the atrophying muscle programme and for proposing new therapeutical approaches.

Keywords: E3 ubiquitin ligase; Tcap; UBE2; muscle wasting; split-GFP; ubiquitin-conjugating enzyme.

Figure 1

Only surface plasmon resonance screen reveals MuRF1 interacting E2s (A) yeast two‐hybrid (Y2H) screen. Y2HGold strain containing MuRF1 was mated with Y187 strain expressing MuRF3, E2, or LT (Large‐T antigen). LT construct was used as negative control against MuRF1 to estimate MuRF1 potential background level. Colonies are considered positive when bigger than this background. Colonies were plated on selective medium [‐LTH + Aureo + 3‐AT] (Experimental section) and monitored during 21 days. Three independent transformation experiments were performed, and 11 to 32 colonies were analyzed for each E2. (B) MuRF1‐E2s interactions were screened by surface plasmon resonance (SPR), using a BIAcore T200 (GE Healthcare). GST‐MuRF1 and GST were covalently immobilized on CM5 chips. E2s diluted to 1 μM (or 0.5 μM for E2J2c) were injected in parallel onto GST‐MuRF1 and GST surfaces at 30 μL/min. GST surface was used as a reference to subtract non‐specific binding of E2 on GST and/or on the CM5 surface. Only subtracted sensorgrams are shown. Black box, injection/association phase; grey box, dissociation phase; RU, arbitrary response units.

Figure 2

Determination of the binding affinity constant (K_D) of E2L3‐MuRF1 and E2G1‐MuRF1 interactions MuRF1‐E2L3 (A, B) and MuRF1‐E2G1 (C, D, E) interactions was characterized using SPR analysis. (A) Sensorgram of a representative single cycle kinetics (SCK) experiment obtained by the sequential injection of serial dilutions of E2L3 (250 nM, 500 nM, 666 nM, 1 μM, and 2 μM) onto GST‐MuRF1 and GST control surfaces. Flow rate was 30 μL/min. Arrows denote sample injections. (B) Low residuals (<10% of the response, red line) indicated that the kinetics fitted well. Binding affinity constant (K_D) of E2L3 for MuRF1 was estimated to be ≈50 nM. (C) Sensorgram of a representative single cycle kinetics obtained by the injection of serial dilutions of E2G1 (750 nM, 1 μM, 1.5 μM, 2 μM, and 3 μM) onto the GST‐MuRF1 and GST control surfaces at 30 μL/min. Red curve, experimental data; black curve, calculated data when using the ‘heterogeneous analyte’ model. (D) Residuals. The kinetics fitted to this model (heterogeneous solution of E2G1 monomers and dimers) as seen by the low residuals of the fit. (E) E2G1 protein preparation contained monomeric and dimeric forms. The E2G1 recombinant protein produced was pure as shown by the Coomassie staining of the denaturating gel (left). E2G1 recombinant protein was submitted to size exclusion chromatography (hiload 16/600 Superdex 200; GE Healthcare), performed in native conditions (right). E2G1 protein elution pattern confirmed the presence of monomers (at 104.5 mL corresponding to ≈18 kDa) and dimers (at 91.5 mL, corresponding to ≈46 kDa).

Figure 3

E2E1, E2G1, E2J1, E2J2, and E2L3 interact with MuRF1 in the presence of telethonin (A) Telethonin does not interact with E2s. Y2H experiments were performed using telethonin as a bait to confirm that this protein cannot directly interact with the E2 enzymes used in this work. The empty vector and the vector containing the LT construct were used as negative controls against telethonin to estimate potential background level. Signals above ‘empty’ and ‘LT’ lanes were considered as positive. Colonies were plated on selective medium [‐LTH + Aureo + 3‐AT] (Experimental section) and monitored during 21 days. LT, Large‐T antigen; Tele, telethonin. (B) Telethonin expression level in yeast varies according to methionine (Met) concentration in the medium. BD‐Tele, fusion protein between the binding domain of GAL4 and telethonin; Tele, telethonin. (C) Densitometry analysis from the immunoblot presented in (B). (D) Yeast three‐hybrid (Y3H) experiments revealed E2E1, E2G1, E2J1, E2J2, and E2L3 as MuRF1 partners in the presence of telethonin. E2‐expressing yeasts were mated against strains expressing either MuRF1 alone or MuRF1 and telethonin. Colonies were plated on selective medium [‐LTH + Aureo + 3‐AT] containing 134 mM Met. Results were observed at day 6. Three to four independent transformation experiments were performed and 11 to 32 colonies were analyzed for each E2.

Figure 4

Telethonin favoured MuRF1‐E2E1 or MuRF1‐E2J1 interactions (A) dose‐dependent effect of telethonin on the growth of yeasts expressing MuRF1 and E2J1 or MuRF1 and E2E1. Yeasts expressing pBridge::MuRF1/telethonin were mated with yeasts expressing different E2s or MuRF3 (positive control) or LT (negative control). Y3H assays were carried out at different Met concentrations, that is, with different telethonin levels in yeast. Serial replica were performed by switching from low to high and high to low Met concentrations to avoid any bias due to the replica plating order. LT, Large‐T antigen; Tele, telethonin. (B) Yeast growth quantification from (A) is parallel to telethonin expression level (red curve). (C) Production of MuRF1/telethonin stable complexes. Immunoblots show the different steps of the production of crosslinked MuRF1/telethonin complexes that were thereafter bound on CM5 sensorchip for subsequent SPR experiments. IB, Immunoblot; L, lysat; W1, wash 1; El, eluted proteins; R, proteins remained on matrix; CL, cross linked proteins. (D, E, F) Telethonin stabilized MuRF1/E2E1 interaction. SPR experiments were performed using a single cycle kinetics method (SCK). Serial E2E1 solutions were injected in parallel over GST‐MuRF1 (D), GST‐MuRF1/telethonin complexes (E), and GST (reference) surfaces. Red curves, experimental data; black curve, calculated data for a fit using the ‘heterogeneous ligand model’. (F) Residual of the fit from (E).

Figure 5

Telethonin co‐localized with MuRF1/E2 complexes in mammalian cells (A) interaction and localization of MuRF1‐E2s complexes in HEK_GFP1‐9 cells. Green fluorescent signal only results from E2‐MuRF1 interactions. (B) E2D2, an E2 that did not interact with MuRF1 (Y2H, Y3H, SPR) did not generate green fluorescence signal. Representative confocal microscopy images of split‐GFP fluorescence (rGFP). + telethonin, additional co‐expression of a mCherry‐telethonin (red signal) fusion construct. GFPr fluorescence was visualized in the FITC channel (488 nm); DAPI nuclear labelling (cyan) and mCherry fluorescence (561 nm). Merge images indicate co‐localization (yellow) of telethonin with MuRF1‐E2 complexes. Signal was acquired over 18 h and represented the sum of all interaction events over this period. (B) Negative control with the non‐interacting E2D2. Scale bars: 10 μm.

Figure 6

Telethonin is degraded in presence of MuRF1 and its E2 partners (A) telethonin level is depressed in HEK293 cells co‐transfected with MuRF1, telethonin and an E2 interacting with MuRF1 but not with the negative control E2D2. Immunoblotting was performed on cell lysates against telethonin. IB, immunoblot; Load: membranes were stained using Blot‐FastStain dye (a portion of the membrane is shown). (B) Densitometric analysis was used to correct for uneven loading. *P < 0.05, n = 6; **P < 0.01, n = 6.