Endoplasmic reticulum quality control regulates the fate of transthyretin variants in the cell

Abstract

The secretion of transthyretin (TTR) variants contributes to the pathogenesis of amyloidosis because they form aggregates in the extracellular environment. However, the mechanism of how TTR variants pass the quality control system in the endoplasmic reticulum (ER) has not yet been elucidated. We investigated here the mechanism of how TTR passes ER monitoring. Monomeric mutation introduced in TTRs (M-TTRs) resulted in the ER retention of amyloidogenic M-TTRs but not non-amyloidogenic M-TTRs. Retention of amyloidogenic M-TTRs induced the unfolded protein response and upregulated the expression of ER chaperones BiP and glucose-regulated protein (GRP) 94. Additionally, we showed that the ER-retained amyloidogenic M-TTRs are subject to ER-associated degradation. On the other hand, the amyloidogenic TTR variants and non-amyloidogenic M-TTRs were secreted normally. These findings suggest that unlike for wild-type TTR, the ER quality control system may differentially regulate the fate of the TTR variants and their monomeric counterparts.

Figure 1

Secretion pattern of wild type and variant TTRs. (A, B) Wild-type TTR, non-amyloidogenic TTRs (A) or amyloidogenic TTRs (B) were transiently transfected in CHO-K1 cells. Medium samples were non-reduced and non-boiled (left panels), whereas the cell lysate samples were reduced and boiled (right panels) before loading. Samples were analyzed by Western blotting using anti-human TTR or anti-calnexin (CNX) antibody. The arrowheads indicate the TTR monomer (M), dimer (D) and tetramer (T). ^* indicates wild-type TTR protein purified from human serum.

Figure 2

Amyloidogenic monomeric TTRs (M-TTRs) are not secreted efficiently from the cells. (A, B) Wild-type M-TTR, non-amyloidogenic M-TTRs (A) or amyloidogenic M-TTRs (B) were transiently transfected in CHO-K1 cells. Medium samples and the cell lysate samples were reduced and boiled before loading. Samples were analyzed by Western blotting using anti-human TTR or anti-calnexin (CNX) antibody. The arrowheads indicate the TTR monomer (M). ^*Indicates wild-type TTR protein purified from human serum.

Figure 3ab

D18G TTR and amyloidogenic monomeric TTRs (M-TTRs) are retained in the ER. HeLa cells were transfected with TTRs (A, B) or M-TTRs (C, D). After 48 h, cells were fixed, permeabilized and immunostained with anti-human TTR and anti-KDEL (A, C) or anti-GM130 (B, D) antibodies and visualized with Alexa Fluor546-conjugated and Alexa Fluor488-conjugated secondary antibodies, respectively. Red fluorescence in the left subpanels represents TTRs, green fluorescence in the middle subpanels are KDEL (A, C) or GM130 (B, D), and right panels represent the overlay (merge). TTR overlap with organelle markers is indicated by the yellow fluorescence. Scale bars, 20 μm.

Figure 3cd

Continued.

Figure 4

Retention of D18G TTR and amyloidogenic monomeric TTRs (M-TTRs) upregulates ER chaperone, BiP and GRP94. (A, E) HeLa cells were transfected with empty vector (E.V.) or the indicated M-TTR constructs. Cell lysates were recovered 48 h post-transfection, boiled and subjected to SDS–PAGE under reduced condition. Proteins were probed with anti-human TTR, anti-KDEL (BiP and GRP94), anti-PDI and anti-actin antibodies. As positive control for ER chaperones induction, cells transfected with empty vector were treated for 16 h with 2 μg/ml tunicamycin (Tm) 32 h post-transfection (Figure 4E, lane 2). (B–D and F–H) Densitometric quantifications were performed on BiP, GRP94 and PDI Western blots of HeLa cells. (B–D) Bars shown are the average expression of ER chaperones in cells transfected with non-amyloidogenic and amyloidogenic M-TTRs. (F–H) Bars shown are the average of three independent experiments for the expression ratio of ER chaperones in cells transfected with the indicated constructs. Values represent the mean±s.e. (^**P<0.01, ^***P<0.001)

Figure 5abc

Amyloidogenic M-TTRs are degraded by proteasome. (A–E) HeLa cells were transfected with the indicated TTR constructs. After 42 h, cells were treated with 500 μM cycloheximide and the proteasome inhibitor, MG132 (30 μM) or DMSO. Cell lysate and media were collected immediately (0 h) or 2, 6 h after the addition of cycloheximide. Densitometric quantifications were performed on the intracellular TTR and secreted TTR from HeLa cells. Intracellular TTR expression at different time points was calculated as relative expression: relative expression (%)=100 × (intracellular TTR at given time t/intracellular TTR at 0 h). Secreted TTR was calculated as follows: (secretion level at given time t/secretion level at 0 h). White circles, CHX and black circles, CHX+MG132. (F) The % relative expression level of intracellular M-TTRs at 6 h after cycloheximide treatment in HeLa cells untreated or treated with 30 μM MG132. (G) The secretion ratio of wild-type or non-amyloidogenic M-TTRs at 6 h after cycloheximide treatment in HeLa cells untreated or treated with 30 μM MG132. Conditions in (F) and (G) are identical to those described above. All experiments were performed in triplicate. Values represent the mean±s.e. (^*P<0.05, ^**P<0.01, ^***P<0.001).

Figure 5defg

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Figure 6

Effect of thyroxine (T₄) and DF on the level of TTR monomers in the cell media. (A, B) Wild-type TTR or variant TTRs were transiently transfected in CHO-K1 cells. Cells were maintained for 24 h in serum-free DMEM with or without 1 μM T₄ or 1 μM DF. Medium samples were analyzed by Western blotting using anti-human TTR antibody. Western blots of secreted TTR monomer were quantified by densitometric scanning. Ratio of 1 μM T₄ or DF monomer to 0 μM T₄ or DF monomer in the media was calculated as follows: T₄ (DF) (1 μM)/T₄ (DF) (0 μM) monomer ratio (%)=100 × (non-reduced, non-boiled monomer at 1 μM T₄ or DF)/(non-reduced, non-boiled monomer at 0 μM T₄ or DF). Bars shown are representative of three independent experiments. Values represent the mean±s.e.