Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases

Abstract

The Fas/APO-1-receptor associated cysteine protease Mch5 (MACH/FLICE) is believed to be the enzyme responsible for activating a protease cascade after Fas-receptor ligation, leading to cell death. The Fas-apoptotic pathway is potently inhibited by the cowpox serpin CrmA, suggesting that Mch5 could be the target of this serpin. Bacterial expression of proMch5 generated a mature enzyme composed of two subunits, which are derived from the pre-cursor proenzyme by processing at Asp-227, Asp-233, Asp-391, and Asp-401. We demonstrate that recombinant Mch5 is able to process/activate all known ICE/Ced-3-like cysteine proteases and is potently inhibited by CrmA. This contrasts with the observation that Mch4, the second FADD-related cysteine protease that is also able to process/activate all known ICE/Ced-3-like cysteine proteases, is poorly inhibited by CrmA. These data suggest that Mch5 is the most upstream protease that receives the activation signal from the Fas-receptor to initiate the apoptotic protease cascade that leads to activation of ICE-like proteases (TX, ICE, and ICE-relIII), Ced-3-like proteases (CPP32, Mch2, Mch3, Mch4, and Mch6), and the ICH-1 protease. On the other hand, Mch4 could be a second upstream protease that is responsible for activation of the same protease cascade in CrmA-insensitive apoptotic pathways.

Figure 1

Subunit structure of mature Mch4 and Mch5. Truncated proMch5 (A) and proMch4 (C) lacking most of the FADD-like prodomain (see schematics B and D) were expressed in Escherichia coli, purified to homogeneity, analyzed by SDS/PAGE and Coomassie staining, and then microsequenced. (A) Lane 1, Total bacterial lysate; lane 2, Ni²⁺-affinity purified mature Mch5 enzyme. The N-terminal amino acid sequence of p11 is LSSPQTRYIPDEADFLLGMA, and the N-terminal amino acid sequences of p18 are SPREQDSESQTLDKVYQMKS and SESQTLD KVYQMKS. (B) A schematic diagram illustrating autoprocessing of proMch5. ProMch5 is autocatalytically processed in two steps to generate the mature p11/p18 enzyme complex. The first step involves processing at Asp-391 and Asp-401, which generates the p11 subunit and nonmature large subunit. This form is enzymatically active and can autoprocess the nonmature large subunit to the mature p18 species by cleavage at Asp-227 and Asp-233 to remove the FADD-like prodomain. (C) Lane 1, Ni²⁺-affinity purified mature Mch4 obtained after expression of truncated proMch4 (V220-I479); lane 2, Ni²⁺-affinity purified Mch4 obtained after expression of truncated proMch4 (I194-I479). The N-terminal amino acid sequence of p12 is ALNPEQAPTSLQDSIPAEAD, and the N-terminal amino acid sequence of p17 is VKTFLEALPRA. The N-terminal amino acid sequence of p23 is ASMTGGQQMGRDPIQIVTPP, which contains both the T7 tag (underlined) and the proMch4 sequence. (D) A schematic diagram illustrating autoprocessing of proMch4. Like proMch5, proMch4 undergoes autocatalytic processing in two steps to generate the mature p12/p17 enzyme complex. The first step involves processing at Asp-372, which generates the p12 subunit and nonmature large subunit (in this case p23). The nonmature large subunit is autocatalytically processed to the mature p17 species by cleavage at Asp-219 to remove the FADD-like prodomain. Hatched boxes at the N and C termini of truncated proMch4 or proMch5 represent the T7 and His₆ tags, respectively. Lanes M in A and C contain molecular weight markers.

Figure 2

Processing and enzymatic activity of wild-type and mutated proMch5. (A) In vitro processing of proMch5 by mature Mch5 enzyme. ³⁵S-labeled truncated wild-type (WT), Asp-391 mutant (M1), Asp-401 mutant (M2), or Asp-391/Asp-401 double mutant (M3) proMch5 was incubated with (+) or without (−) pure mature Mch5 (200 ng per reaction) for 2 h at 37°C. The reaction products were then analyzed by Tricine-SDS/PAGE and autoradiography. LS, large subunit. (B) Enzymatic activity of wild-type and mutant Mch5 enzymes. Pure wild-type (WT), Asp-391 mutant (391), Asp-401 mutant (401), or Asp-391/Asp-401 double mutant (391/401) Mch5 enzymes was incubated with the peptide substrate DEVD–7-amino-4-methylcoumarin (AMC) (50 μM final) for the indicated times at 37°C. The release of AMC was determined by spectrofluorometry and expressed in micromolar per microgram protein. (C) SDS/PAGE analysis of Ni²⁺-affinity purified wild-type and mutant Mch5 enzymes. Lanes 1–4 are wild-type, Asp-391 mutant, Asp-401 mutant, and Asp-391/Asp-401 double mutant Mch5 enzymes, respectively. The band seen below the p11 band in lane 3 is a contaminant bacterial protein seen occasionally in some batches of purified Mch5.

Figure 4

Processing of proASCPs by mature Mch4 or Mch5. ³⁵S-labeled proASCPs were incubated with (+) or without (−) purified equivalent amounts (200 ng per reaction) of Mch4 (A and C) or Mch5 (B and D) for 1 h at 37°C. The reaction products were then analyzed by Tricine-SDS/PAGE and autoradiography. (A and B) ICE-like (ICE30 and TX) and NEDD2-like (ICH1) proASCPs. (C and D) CED-3-like (CPP32, Mch2, Mch3, Mch4, Mch5, and Mch6) proASCPs. SS, small subunit.

Figure 3

Processing of proCPP32 and proMch3 by mature Mch5. ³⁵S-labeled wild-type (WT) or Asp-9 mutant (9D → A) proCPP32 (A) or proMch3 (B) were incubated with pure mature Mch5 (40 ng) for the indicated times at 37°C. The reaction products were then analyzed by Tricine-SDS/PAGE and autoradiography.

Figure 5

Effect of CrmA on the enzymatic activity of Mch4 and Mch5. Purified ³⁵S-labeled proCPP32 was incubated with equivalent amounts of pure mature Mch4 or Mch5 (20 ng per reaction) in the presence of increasing concentrations of CrmA for 45 min at 37°C. The reaction products were then analyzed by Tricine-SDS/PAGE, autoradiography, and densitometric scanning. The extent of cleavage was determined from the intensity of the cleavage products relative to the total input and expressed as percentage of cleavage.

Figure 6

A schematic diagram illustrating the molecular ordering of two potential apoptotic pathways. See text for details.