Defective Molecular Timer in the Absence of Nucleotides Leads to Inefficient Caspase Activation

Abstract

In the intrinsic death pathway, cytochrome C (CC) released from mitochondria to the cytosol triggers Apaf-1 apoptosome formation and subsequent caspase activation. This process can be recapitulated using recombinant Apaf-1 and CC in the presence of nucleotides ATP or dATP [(d)ATP] or using fresh cytosol and CC without the need of exogenous nucleotides. Surprisingly, we found that stored cytosols failed to support CC-initiated caspase activation. Storage of cytosols at different temperatures led to the loss of all (deoxy)nucleotides including (d)ATP. Addition of (d)ATP to such stored cytosols partially restored CC-initiated caspase activation. Nevertheless, CC could not induce complete caspase-9/3 activation in stored cytosols, even with the addition of (d)ATP, despite robust Apaf-1 oligomerization. The Apaf-1 apoptosome, which functions as a proteolytic-based molecular timer appeared to be defective as auto-processing of recruited procaspase-9 was inhibited. Far Western analysis revealed that procaspase-9 directly interacted with Apaf-1 and this interaction was reduced in the presence of physiological levels of ATP. Co-incubation of recombinant Apaf-1 and procaspase-9 prior to CC and ATP addition inhibited CC-induced caspase activity. These findings suggest that in the absence of nucleotide such as ATP, direct association of procaspase-9 with Apaf-1 leads to defective molecular timer, and thus, inhibits apoptosome-mediated caspase activation. Altogether, our results provide novel insight on nucleotide regulation of apoptosome.

Resveratrol depletes mitochondrial DNA and inhibition of autophagy enhances resveratrol-induced caspase activation

“…Preparation of the whole cell lysate, isolation and purification of mitochondrial and cytosolic fractions, Western blotting were performed as described previously (Chandra et al, 2007; Zhang et al, 2011). …”

“…DEVDase, LEHDase, and IETDase activities were determined as described previously (Gogada et al, 2011b; Zhang et al, 2011). …”

Mechanism of neem limonoids-induced cell death in cancer: Role of oxidative phosphorylation

“…Preparation of whole cell lysates, mitochondrial and cytosolic fractions, and Western blotting were performed as mentioned previously [27, 28]. For whole cell lysates preparation, cells were lysed in NP-40 buffer (50 mM HEPES-KOH, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT) and a mixture of protease inhibitors (1 mM PMSF, 1% aprotinin, 1 mM leupeptin, 1 mg/ml pepstatin A and 1 mg/ml chymostatin).…”

“…Samples were loaded on SDS polyacrylamide gel for Western blotting. After protein transfer, the membrane was probed/reprobed with various primary and corresponding secondary antibodies followed by immunodetection using ECL as previously described [27, 28]. …”

“…DEVDase, LEHDase and IETDase activities were measured as described previously [27, 28]. Briefly, proteins were added to the caspase reaction mixture containing 30 μM fluorogenic peptide substrates, DEVD-AFC (for caspase-3) or LEHD-AFC (for caspase-9) or IETD-AFC (for caspase-8), 50 mM HEPES, pH 7.4, 10% glycerol, 0.1% CHAPS, 100 mM NaC1, 1 mM EDTA, and 10 mM DTT, in a total volume of 100 μl and incubated at 37°C for 90 min.…”