Type 2 transglutaminase is involved in the autophagy-dependent clearance of ubiquitinated proteins

Abstract

Eukaryotic cells are equipped with an efficient quality control system to selectively eliminate misfolded and damaged proteins, and organelles. Abnormal polypeptides that escape from proteasome-dependent degradation and aggregate in the cytosol can be transported via microtubules to inclusion bodies called 'aggresomes', where misfolded proteins are confined and degraded by autophagy. Here, we show that Type 2 transglutaminase (TG2) knockout mice display impaired autophagy and accumulate ubiquitinated protein aggregates upon starvation. Furthermore, p62-dependent peroxisome degradation is also impaired in the absence of TG2. We also demonstrate that, under cellular stressful conditions, TG2 physically interacts with p62 and they are localized in cytosolic protein aggregates, which are then recruited into autophagosomes, where TG2 is degraded. Interestingly, the enzyme's crosslinking activity is activated during autophagy and its inhibition leads to the accumulation of ubiquitinated proteins. Taken together, these data indicate that the TG2 transamidating activity has an important role in the assembly of protein aggregates, as well as in the clearance of damaged organelles by macroautophagy.

Figure 1

Effect of starvation on liver and heart autophagy in TG2^+/+ GFP-LC3 or TG2^−/− GFP-LC3 mice. (a and b) Liver samples from TG2^+/+ GFP-LC3 transgenic mice before (a) and after 48 h of starvation (b) were analyzed by fluorescence microscopy. (c and d) Liver samples from TG2^−/− GFP-LC3 transgenic mice before (c) and after 48 h of starvation (d) were analyzed by fluorescence microscopy. Scale bar, 8 μm. (e) Quantitative analysis of the formation of GFP-LC3 dots following autophagy-induction. The number of dots was counted in five independent fields of three independent mice. Results are expressed as the mean±S.D. Statistical analysis was performed by analysis of variance (^*P<0.05). (f and g) Heart samples from TG2^+/+ GFP-LC3 transgenic mice before (f) and after 48 h of starvation (g) were analyzed by fluorescence microscopy. (h and i) Heart samples from TG2^−/− GFP-LC3 transgenic mice before (h) and after 48 h of starvation (i) were analyzed by fluorescence microscopy. Scale bar, 3 μm. (j) Quantitative analysis of the formation of GFP-LC3 dots following autophagy-induction. The number of dots was counted in five independent fields of three independent mice. Results are expressed as the mean±S.D. Statistical analysis was performed by analysis of variance (^*P<0.05, ^**P<0.001, ^***P<0.0001)

Figure 2

TG2 is degraded by autophagy. (a, upper panel) Immunoblot analysis of TG2 in liver tissues from TG2^+/+ and TG2^−/− mice, before and after 24 and 48 h of starvation. Actin was used as loading control. (a, lower panel) Densitometric analysis of blots (quantification of TG2 bands normalized to actin levels). (b, upper panel) Immunoblot analysis of TG2 in liver tissues from TG2^+/+ mice, before and after 24 and 48 h of starvation in the presence or not of CQ. For the autophagy inhibition the CQ was administered in the drinking water during the whole starvation time. Actin was used as loading control for immunoblot. (b, lower panel) Densitometric analysis of blots (quantification of TG2 bands normalized to actin levels). TG2 expression is reduced following autophagy induction and is rescued from degradation when the lysosomal activity is blocked by CQ. (c, upper panel) Western blot analysis of TG2 in 2fTGH cells subjected to different treatments to induce autophagy (see Materials and Methods). Actin was used as loading control for immunoblot. (c, lower panel) Densitometric analysis of blots (quantification of TG2 bands normalized to actin levels)

Figure 3

TG2 colocalizes with LC3 in the autophagosomes. (a and b) Immunofluorescence microscopy analysis of 2fTGH-GFP-LC3 cells undergoing autophagy. Cells expressing GFP-LC3 protein were cultured in complete medium (a) or subjected to starvation (b) in EBSS for 16 h, stained with an anti-TG2 antibody and analyzed by microscopy. Green dots represent autophagosomes, red dots TG2 protein, and yellow dots sites of overlap of GFP-LC3 with TG2. Scale bar, 6 μm. (c and d) Immuno-gold analysis in human 2fTGH cells double-labeled against TG2 (15-nm gold particles) and p62 (5-nm gold particles). The colloidal gold particles, indicating the presence of TG2 (arrows) and p62 (arrowheads), were detected in typical autophagosome-like structures. Original magnification: × 85 000

Figure 4

TG2 interacts with autophagy-related cargo proteins. (a) Western blot analysis of TG2, p62 and VCP proteins in human 2fTGH cells subjected to immunoprecipitation for TG2. After 4 h of treatment as indicated, cells were lysed and proteins were immunoprecipitated using anti-TG2 antibody (see Materials and Methods). Immuno- and co-immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. WC, whole cell lysate, was used as protein control. (b) Western blot analysis of TG2, p62 and VCP proteins in HEK293^TG2 cells stably transfected with TG2 protein, subjected to immunoprecipitation for TG2. After 2 h of treatment as indicated, cells were lysed and proteins were immunoprecipitated using anti-TG2 antibody. Immuno- and co-immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. WC, whole cell lysate, was used as protein control. (c) Representative blot of the TG-catalyzed incorporation of 5-(biotinamido)pentylamine into proteins in 2fTGH cell line. Cells were labelled with 5-(biotinamido)pentylamine and treated as indicated for 4 h. After separation by SDS-PAGE, biotinylated proteins were revealed with HRP-conjugated streptavidin. p53 and p27 proteins were used as a control for monitoring proteasome inhibition, and actin was used as loading control

Figure 5

TG2 is present in cytosolic protein aggregates. (a and b) HEK293^TG2 cells were cultured under starved condition in presence of MG132 for 2 h. Cells were double-labelled against TG2 (15-nm gold particles), and against p62 (5 nm gold particles). Immuno-gold analysis shows that small cytosolic membrane-free electron-dense structures (arrowheads) are positively stained for TG2 while larger aggregates display simultaneous positivity for TG2 and p62 (arrows). Original magnification: × 85 000

Figure 6

Impaired in vivo pexophagy after ciprofibrate-induced peroxisomal proliferation in the liver of TG2^−/− mice. Ultrastructural catalase cytochemistry of TG2^+/+ (a) and TG2^−/− (b–d) mouse liver, after ciprofibrate treatment. Mice were given ciprofibrate-containing food for 10 days, then killed immediately (T0) or 7 days (T7) after suspension of treatment. Liver specimens were prepared as described in Materials and Methods and ultrathin sections were observed in a Zeiss EM 900 electron microscope. (a and b) Remarkable peroxisomal induction is observed in both TG2^+/+ and TG2^−/− hepatocytes, at T0. However, while autophagosomes with partially degraded material are readily detected in the TG2^+/+ samples (arrows), the knockout shows virtually no autophagic vacuoles. (c and d) Several autophagosomes are recognized in TG2^−/− samples at T7. These mostly contain indigested peroxisomes, with homogeneous catalase positivity (arrowheads). The higher magnification micrograph (d) shows the details of an autophagic vacuole (arrowhead), surrounding an intact peroxisome, in which the double membrane is clearly visible, catalase is cytochemically active and a typical peroxisomal core (white arrowhead) is present. N, nucleus; m, mitochondrion; p, peroxisome. Original magnification: (a and b) × 12 000; (c × 4400;) (d × 20 000.) (e and f) Densitometric analysis of western blotting data, obtained on liver extracts from TG2^+/+ and TG2^−/− mice before and after in vivo ciprofibrate administration, using the peroxisomal marker PMP70 (e) and the autophagic marker LC3 normalized to GADPH (f). LC3 levels are expressed as the ratio between LC3 II and LC3 I. Statistical analysis was performed by analysis of variance (^*P<0.05)

Figure 7

Effect of TG2 on ubiquitinated aggregates accumulation. (a–d) Immunofluorescence detection of ubiquitin in the liver of TG2^+/+ and TG2^−/− mice before (a and b) and after starvation (c and d). Scale bar, 8 μm. (e) Quantitative analysis of the formation of ubiquitin-positive aggregates in basal condition and after autophagy induction. The number of cells containing aggregates was counted in five independent fields of three independent mice. Results are expressed as the mean±S.D. Statistical analysis was performed by analysis of variance (^*P<0.05, ^**P<0.001). (f) Immunoblotting analysis of ubiquitinated proteins in HEK293 and HEK293^TG2 cell lines. Cells were incubated with or without MG132 for 2 h, and ubiquitin expression levels were assayed by immunoblotting with anti-ubiquitin antibody. Actin was used as loading control

Figure 8

Effect of TG2 transamidating activity on the clearance of ubiquitinated protein aggregates. (a) Western blot analysis of p62 and ubiquitinated proteins in HEK293, HEK293^TG2 and HEK293^C277S cell lines subjected to immunoprecipitation for TG2. Cells treated with MG132 for 2 h were lysed and proteins were immunoprecipitated using anti-TG2 antibody. Immuno- and co-immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. Arrows indicate ubiquitinated proteins present in TG2 interacting protein complexes. (b) Immunoblotting analysis of ubiquitinated proteins in HEK293, HEK293^TG2 and HEK293^C277S cell lines. Cells were incubated with MG132 for 2 h, and ubiquitin expression levels were assayed by immunoblotting with anti-ubiquitin antibody. Actin was used as loading control

Figure 9

Tentative scheme of TG2 action during autophagy. The accumulation of misfolded ubiquitinated proteins, sensed by the cell as a stress, leads to the activation of autophagy at ER level determining the release of free calcium ions in the cytoplasm (1). This event leads to the activation of TG2 transamidating activity associated with its 3D structural change from the inactive ‘closed conformation' to the active ‘opened conformation' (2). The activated enzyme may catalyze the misfolded protein crosslinking, leading to the formation of small aggregates (3) and subsequently large aggregates (4). The formation of these ubiquitinated inclusions is recognized via the UBA domains by p62 or other cargo proteins. These complexes finally interact with LC3II through the LIR domains present on the cargo proteins, leading to their uploading inside the pre-autophagic vesicles