Mitochondrial Dysfunction Induced by a Cytotoxic Adenine Dinucleotide Produced by ADP-ribosyl Cyclases from cADPR

ADP-ribosyl cyclases were previously shown to produce three new adenine dinucleotides, P1,P2 diadenosine 5′-diphosphate (Ap2A) and two isomers thereof (P18 and P24), from cyclic ADP-ribose (cADPR) and adenine (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 14509-14514). The Ap2A isomer P24, containing an unusual C1′-N3 N-glycosidic bond, is shown here to affect mitochondrial function through (i) opening of the permeability transition pore complex (and consequent proton gradient dissipation) and (ii) inhibition of Complex I of the respiratory chain. Whereas proton gradient dissipation is dependent upon the extracellular Ca2+ influx triggered by P24, the effect on oxygen consumption is Ca2+ independent. The proton gradient dissipation induces apoptosis in HeLa cells and thus appears to be responsible for the already described potent cytotoxic effect of P24 on several human cell types. The other products of ADP-ribosyl cyclase activity, Ap2A and cADPR, antagonize P24-induced proton gradient dissipation and cytotoxicity, suggesting that the relative concentration of P24, cADPR, and Ap2A in cyclase-positive cells may affect the balance between cell life and death.

Affinities of Mcl-1 and Bcl-XL for BH3-Only Proteins Define Distinct Survival Functions at the Outer Mitochondrial Membrane of Multiple Myeloma Cells.

The Bcl-2 family member Mcl-1 is a critical survival factor for MM cells. However, the functions of Mcl-1 that distinguish it from other anti-apoptotic factors remain unclear. We used fluorescence polarization (FP) to measure the affinity of purified Mcl-1 for amphipathic peptides derived from the BH3-domains of Bad, Bid, Bim, Bik and PUMA, and compared these results with data obtained with Bcl-XL. Functional assays were then performed with mitochondria isolated from 8226 MM cells or clones engineered to overexpress Mcl-1 or Bcl-XL. In contrast to Bcl-XL, Mcl-1 binds to BadBH3 and BikBH3 with very low affinity (>1 uM), confirmed by its inability to bind full-length Bad or Bik proteins. Mcl-1 exhibited high affinity for BimBH3 (90 ± 27 nM), and intermediate affinities for Bid and PUMA. Bcl-XL had intermediate affinities for each of the BH3-peptides tested (175–400 nM). Mitochondria from parental 8226 cells were very sensitive to BidBH3 and BimBH3 as assayed by cytochrome c (cyt c) release; BadBH3 and BikBH3 did not directly induce cyt c release. Mitochondria from 8226-Mcl-1 (which overexpress Mcl-1 5-fold) were very resistant to BidBH3 (even at 500 uM), suggesting that sequestration of BH3-peptide by Mcl-1 was not the sole mechanism of apoptosis inhibition. Paradoxically, Mcl-1-overexpressing mitochondria were more sensitive to BimBH3 (despite the higher affinity of Mcl-1 for Bim), and neither BadBH3 nor BikBH3 could potentiate BidBH3- or BimBH3-induced cyt c release from these mitochondria. In contrast, 8226-Bcl-XL mitochondria were more sensitive to BidBH3, were similarly resistant to BimBH3, and were sensitized by BadBH3 and BikBH3. Protein crosslinking of Mcl-1-overexpressing mitochondria after culture with BidBH3 indicated that peptide sequestration could account for resistance at low peptide concentrations (5–30 uM), but not at higher doses (≥100 uM) where a Bid-induced Bak conformational change occurred as confirmed by exposure of a trypsin-sensitive site. Despite this, formation of Bak multimers was inhibited. (Bax was not detected in 8226 mitochondria). BimBH3 (30 uM) induced detectable cyt c release and Bak dimerization in Mcl-1-overexpressing mitochondria, but 500 uM BimBH3 failed to enhance either effect, and most of the cyt c remained in the mitochondrial pellet. Taken together, these data demonstrate that Mcl-1 and Bcl-XL are not equivalent in their anti-apoptotic functions at the OMM of MM cells. Bad and Bik do not bind Mcl-1, and cannot displace other BH3-only peptides from Mcl-1, suggesting that Mcl-1 has evolved to specifically bind apoptosis-inducing BH3-only proteins in a resistant complex. Mcl-1 confers exceptional protection against BidBH3, and experiments are underway to confirm this with tBid protein. We propose a model in which low levels of BH3-peptide can be sequestered by Mcl-1, while at higher concentrations Mcl-1 binding capacity becomes saturated, allowing peptides to bind Bak and induce a conformational change. However pore formation is inhibited, perhaps by direct binding of Mcl-1 to Bak. To induce pore formation, BH3-peptides must dissociate the Mcl-1/Bak complex: BimBH3, which has a high affinity for Mcl-1 can perform this function, while BidBH3 cannot. This model is consistent with our measured affinity of Mcl-1 for BakBH3 as compared with its affinities for BimBH3 or BidBH3. We suggest that Mcl-1 prevents full cyt c release by preventing activation of the permeability transition pore complex.