Cleavage of Atg16L1 by the protease calpain

Calpains are a family of cysteine proteases which are activated by calcium.

Calpain-1 (μ-calpain) and calpain-2 (m-calpain) are the most common calpains and are ubiquitously expressed (reviewed in Goll 2003). In 2006, Yousefi et al. observed that Atg5 is cleaved by calpain to form a truncated 24 kDa fragment comprising residues 1-193. They found that this fragment translocates to the mitochondria and associates with Bcl-xL, which triggers apoptosis. It was our aim to crystallize the complex of Bcl-xL with the truncated form of Atg5.

Adi Bar Shalom, a lab rotation student, tried to express Atg5(1-193) on its own as well as co-expression with Bcl-XL(1-44, 85-196). Neither strategy yielded soluble protein.

Therefore, I tried to generate the cleavage product of Atg5 by digesting the Atg5~Atg16L1(1-231) complex with calpain-1. However, upon addition of calpain-1 (Calbiochem) to the complex, Atg5 remained intact while Atg16L1(1-231) was almost entirely cleaved, resulting in the formation of several fragments (Figure 3.19 panel A).

Upon addition of calpain-1, Atg16L1(1-231) was almost entirely digested within 7 hours at 4 °C and one hour at RT, while Atg5 remained intact. In contrast, without the addition of calpain-1, the complex was stable at 4 °C and at RT for up to a day (Figure 3.19 panel B).

Figure 3.19: Calpain-1 cleavage of the Atg5~Atg16L1(1-231) complex. 15% SDS-PAGE gels of time course comparing the stability of the Atg5~Atg16L1(1-231) complex (A) with and (B) without calpain-1 at 4 °C and RT. t= hr.

Calpain-2 (Calbiochem) was also tested. To investigate whether this cleavage was indeed due to calpains, several calpain inhibitors were added to the digestions: acetyl calpastatin(184-210) CS peptide (Anaspec) inhibits both of these calpains, whereas the peptides ALLN (Calbiochem) and ALLM (Sigma Aldrich) are specific for calpain-1 and calpain-2, respectively. As can be seen in Figure 3.20, both calpain-1 and calpain-2 cleaved Atg16L1(1-231) and this cleavage was blocked by the presence of ALLN, ALLM, or calpastatin. In contrast, Pefabloc SC (Roche), a serine protease inhibitor, did not inhibit cleavage. Together these results show that cleavage is indeed catalyzed by calpain.

To test if the N-terminal Atg5-binding domain of Atg16L1 is sufficient for calpain-1 cleavage, the Atg5~Atgcalpain-16Lcalpain-1(calpain-1-70) complex was also digested with calpain. Within calpain-15 min of addition of calpain-1, Atg16L1(1-70) was cleaved into smaller fragments while Atg5 remained intact (Figure 3.21). In the absence of calpain-1 or in the presence of ALLN, Atg16L1(1-70) remained intact even after incubation for 90 min at 37 °C.

Figure 3.20: Cleavage of the Atg5~Atg16L1(1-231) complex by calpain-1 and calpain-2. 15% SDS-PAGE gels of the Atg5~Atg16L1(1-231) complex after incubation in the absence or presence of calpain-1 (c-1) and calpain-2 (c-2) (at a ratio of 1:50 w/w) and specific inhibitors ALLN, ALLM, and calpastatin at 37 °C for 60 min.

Figure 3.21: Calpain-1 also specifically cleaves Atg16L1(1-70). 15% SDS-PAGE gels of time course of the Atg5~Atg16L1(1-70) complex after incubation at 37 °C in the absence or presence (at a ratio of 1:50 w/w) of calpain-1 and its specific inhibitor ALLN. t= min.

To characterize where calpain cleaved Atg16L1, the Atg5~Atg16L1(1-231) complex was loaded onto a HisTrap FF column after calpain digestion for 60 min at 37 °C (Figure 3.22). The Atg5-binding domain of Atg16L1 was intact since Atg5 co-eluted with an approximately 24 kDa Atg16L1 cleavage product. The band of Atg16L1 shown in the box in Fig. 3.22 was sent for N-terminal sequencing and the first four residues were determined to be G-S-H-M. These amino acid residues correspond to the thrombin cleavage site in the Atg16L1 construct. This indicates that calpain cleaved near the C-terminus of Atg16L1(1-231).

Figure 3.22: Cleaved Atg16L1 fragment co-elutes with Atg5. 15% SDS-PAGE gel after incubation of calpain-1 (1:50 w/w) at 37 °C for 60 min with Atg5~Atg16L1(1-231). The reaction was stopped with ALLN and loaded onto a HisTrap FF column. Several fragments of Atg16L1 co-eluted with Atg5. t= min. Box indicates fragment of Atg16L1 sent for N-terminal sequencing.

Atg16L1 might mask the calpain cleavage site of Atg5 when they form a complex.

To test this hypothesis, calpain-1 was added to Atg5 that was purified on its own. As seen in Figure 3.23 panel A, full-length Atg5 on its own does not seem to diminish upon incubation with calpain-1 for 60 min whereas Atg16L1(1-231) is effectively cleaved by calpain-1 in the presence of Atg5, which remains intact. Of note, there was always a constant band of ~24 kDa present in the Atg5 sample, even without addition of calpain.

The digestion was repeated again, and here the incubation time was doubled. It can be observed on a Western blot where an Atg5 antibody (rabbit anti-Apg5L polyclonal antibody, Proteintech Group Inc) was used for detection that a ~24 kDa band was present even in the absence of calpain and also when the calpain inhibitor ALLN was present (Figure 3.23 panel B). In addition, no band corresponding to an Atg5 cleavage product can be observed for the Atg5~Atg16L1(1-231) complex which was digested by calpain for two hours at 37 °C. These data demonstrate that Atg5, either on its own or in complex with Atg16L1, is not cleaved by calpain.

Figure 3.23: Calpain cleaves Atg16L1 but not Atg5. (A) 15% SDS-PAGE gel of calpain-1 (at a ratio of 1:50 w/w) cleavage of Atg5~Atg16L1(1-231) or Atg5 for 60 min at 37 °C. (B) Western blot using polyclonal anti-Apg5L antibody of Atg5 and Atg5~Atg16L1(1-231) after incubation with calpain-1 (at a ratio of 1:50 w/w) for 120 minutes at 37 °C. A 24 kDa fragment of Atg5 is observed even without addition of calpain (c-1) and is not enriched upon incubation with calpain. t=min t=hr, Pefa=pefabloc, cpst=calpastatin.