A matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) analysis of proteins released from isolated liver mitochondria treated with recombinant truncated Bid

Abstract

A crucial event in the process of apoptosis is caspasedependent generation of truncated Bid (tBid), inducing release of cytochrome c. In an in vitro reconstitution system we combined purified recombinant tBid with isolated liver mitochondria and identified the released proteins using a proteomic matrix-assisted laser desorption ionization postsource decay (MALDI-PSD) approach. In order to meet physiological conditions, the concentration of tBid was chosen such that it was unable to induce cytochrome c release in mitochondriaderivedfrom liver-specificBcl-2-transgenicmice. Several mitochondrial proteins were identified to be released in a tBid-dependent way, among which cytochrome c, DIABLO/ Smac, adenylate kinase 2, acyl-CoA-binding protein, endonuclease G, polypyrimidine tract-binding protein, a type-I RNA helicase, a WD-40 repeat-containing protein and the serine protease Omi. Western blotting confirmed the absence of adenylate kinase 3, a matrix mitochondrial protein. These results demonstrate that a physiologically relevant concentration of tBid is sufficient to induce release of particular intermembrane mitochondrial proteins belonging to a broad molecular-mass range.

Apoptosis-inducing factor (AIF): caspase-independent after all

“…7 Accordingly, in mammalian cell-free systems, the addition of recombinant t-Bid or Bax protein to isolated mitochondria can lead to the release of cytochrome c without that AIF (and endonuclease-G) would be released. 26,27 Although this is at odds with other reports showing that recombinant Bax and tBid can release AIF (and endonuclease-G) from isolated mitochondria, 18,28,29 it suggests that in some particular circumstances, proapoptotic protein of the Bcl-2 family can mediate the selective release of cytochrome c from mitochondria, which simultaneously retain the caspase-independent death effectors AIF and endonuclease-G. Accordingly, it has been found in several cellular models of apoptosis that cytochrome c can be released before AIF translocates to the nucleus, at least within the limits of immunofluorescence detection, 26,27,30 and that the release of cytochrome c (but not that of AIF) occurs in a caspase-independent manner (Table 1a).…”

“…Thus, addition of recombinant caspases to purified mitochondria can trigger the release of cytochrome c, [15][16][17] and caspase-2-or caspase-8-activated Bid (truncated Bid) can act on mitochondria to trigger the release of cytochrome c and AIF. 17,18 When cytochrome c is microinjected into cells, it can cause AIF release from mitochondria (as well as the translocation of endogenous cytochrome c), and this release depends on the activation of caspases, meaning that it is suppressed by the addition of Z-VAD.fmk or by the deletion of the Apaf-1 gene. 5,6,19,20 Thus, the primary activation of caspases may occur upstream of the release of AIF.…”

The serine protease Omi/HtrA2 is released from mitochondria during apoptosis. Omi interacts with caspase-inhibitor XIAP and induces enhanced caspase activity

“…14 One of the proteins identified by mass spectrometry and PSD analysis 18 was the serine protease Omi (Figure 1). Although Omi has a theoretical molecular weight of 49 kDa, the protein that is identified in mitochondria has a size of 37 kDa as the N-terminal presequence is probably cleaved off after mitochondrial transport producing mature Omi ( Figure 1D).…”

“…7,10 ± 12 After triggering of death receptors such as TNF-R1 and Fas, procaspase-8 is activated resulting in Bid cleavage generating truncated Bid (tBid) that translocates to mitochondria releasing apoptogenic proteins such as cytochrome c and DIABLO/Smac. 13 Studying the mitochondrial release of such proteins during tBid-treatment of isolated mitochondria, 14 we identified the serine protease Omi as a potential new player in this complex interaction between caspases, inhibitors and regulatory proteins. Omi, also named HtrA2, was identified as the mammalian homologue of the bacterial HtrA endoprotease and is highly conserved from bacteria to mammalians.…”

Early work on the role of mitochondria in apoptosis, an interview with Guido Kroemer

“…Today, it is known that mitochondria can release dozens of different potentially apoptogenic proteins, with multiple distinct biochemical activities. 23,24 Moreover, many different proteins regulate the permeability of mitochondrial membranes. In addition to the members of the Bcl-2 family, the first to be recognized to regulate mitochondrial permeability, 25 proteins like ANT, 26 VDAC, 27 transcription factors such as p53, 28 as well as lipid pores 29 may contribute to the apoptotic permeabilization of mitochondrial membranes.…”