Benedict, M. A., Hu, Y., Inohara, N. & Nunez, G. Expression and functional analysis of Apaf-1 isoforms. Extra Wd-40 repeat is required for cytochrome c binding and regulated activation of procaspase-9. J. Biol. Chem. 275, 8461?8468
Apaf-1 is an important apoptotic signaling molecule that can activate procaspase-9 in a cytochrome c/dATP-dependent fashion. Alternative splicing can create an NH(2)-terminal 11-amino acid insert between the caspase recruitment domain and ATPase domains or an additional COOH-terminal WD-40 repeat. Recently, several Apaf-1 isoforms have been identified in tumor cell lines, but their expression in tissues and ability to activate procaspase-9 remain poorly characterized. We performed analysis of normal tissue mRNAs to examine the relative expression of the Apaf-1 forms and identified Apaf-1XL, containing both the NH(2)-terminal and COOH-terminal inserts, as the major RNA form expressed in all tissues tested. We also identified another expressed isoform, Apaf-1LN, containing the NH(2)-terminal insert, but lacking the additional WD-40 repeat. Functional analysis of all identified Apaf-1 isoforms demonstrated that only those with the additional WD-40 repeat activated procaspase 9 in vitro in response to cytochrome c and dATP, while the NH(2)-terminal insert was not required for this activity. Consistent with this result, in vitro binding assays demonstrated that the additional WD-40 repeat was also required for binding of cytochrome c, subsequent Apaf-1 self-association, binding to procaspase-9, and formation of active Apaf-1 oligomers. These experiments demonstrate the expression of multiple Apaf-1 isoforms and show that only those containing the additional WD-40 repeat bind and activate procaspase-9 in response to cytochrome c and dATP.
"Activation of the apoptosome apparatus, a stress-induced cell death-signaling platform, in the cytoplasm is an essential step for pro-caspase-9 activation. The initiation step for the apoptosome apparatus assembly is the binding of cytochrome-c to the functional Apaf-1 splice variants, Apaf-1LX and/or Apaf-1LC in the presence of dATP/ATP . Once activated in apoptosomes, the apoptosome-associated caspase-9 cleaves and activates the down-stream effector pro-caspase-3 and -7 [44-46]. "
[Show abstract][Hide abstract] ABSTRACT: Minnelide, a pro-drug of triptolide, has recently emerged as a potent anticancer agent. The precise mechanisms of its cytotoxic effects remain unclear.
Cell viability was studied using CCK8 assay. Cell proliferation was measured real-time on cultured cells using Electric Cell Substrate Impedence Sensing (ECIS). Apoptosis was assayed by Caspase activity on cultured lung cancer cells and TUNEL staining on tissue sections. Expression of pro-survival and anti-apoptotic genes (HSP70, BIRC5, BIRC4, BIRC2, UACA, APAF-1) was estimated by qRTPCR. Effect of Minnelide on proliferative cells in the tissue was estimated by Ki-67 staining of animal tissue sections.
In this study, we investigated in vitro and in vivo antitumor effects of triptolide/Minnelide in non-small cell lung carcinoma (NSCLC). Triptolide/Minnelide exhibited anti-proliferative effects and induced apoptosis in NSCLC cell lines and NSCLC mouse models. Triptolide/Minnelide significantly down-regulated the expression of pro-survival and anti-apoptotic genes (HSP70, BIRC5, BIRC4, BIRC2, UACA) and up-regulated pro-apoptotic APAF-1 gene, in part, via attenuating the NF-κB signaling activity.
In conclusion, our results provide supporting mechanistic evidence for Minnelide as a potential in NSCLC.
PLoS ONE 10/2013; 8(10):e77411. DOI:10.1371/journal.pone.0077411 · 3.23 Impact Factor
"The large family of WD40 repeat-containing proteins can be engaged in a broad range of cellular events, such as signal transduction (Parent et al., 2008; Smith et al., 1999), RNA processing complexes (Mitsuzawa et al., 2001), transcriptional regulation (Pickles et al., 2002), cytoskeleton assembly and mitotic spindle formation (Welch et al., 1997), vesicle formation and trafficking (Fath et al., 2007), cell division control (Yu, 2007) and programmed cell death (Benedict et al., 2000). The basic common function of WD40 repeat-containing proteins is to coordinate multi-protein complex assembly, where the repeating units serve as a scaffold for protein interactions. "
[Show abstract][Hide abstract] ABSTRACT: We identified the WD-repeat-containing protein, WDR36, as an interacting partner of the β isoform of thromboxane A(2) receptor (TPβ) by yeast two-hybrid screening. We demonstrated that WDR36 directly interacts with the C-terminus and the first intracellular loop of TPβ by in vitro GST-pulldown assays. The interaction in a cellular context was observed by co-immunoprecipitation, which was positively affected by TPβ stimulation. TPβ-WDR36 colocalization was detected by confocal microscopy at the plasma membrane in non-stimulated HEK293 cells but the complex translocated to intracellular vesicles following receptor stimulation. Coexpression of WDR36 and its siRNA-mediated knockdown, respectively, increased and inhibited TPβ-induced Gαq signalling. Interestingly, WDR36 co-immunoprecipitated with Gαq, and promoted TPβ-Gαq interaction. WDR36 also associated with phospholipase Cβ (PLCβ) and increased the interaction between Gαq and PLCβ, but prevented sequestration of activated Gαq by GRK2. In addition, the presence of TPβ in PLCβ immunoprecipitates was augmented by expression of WDR36. Finally, disease-associated variants of WDR36 affected its ability to modulate Gαq-mediated signalling by TPβ. We report that WDR36 acts as a new scaffold protein tethering a G-protein-coupled receptor, Gαq and PLCβ in a signalling complex.
"Apaf-1, procaspase-9, and procaspase-3 were cloned into pFastbacH, a modified version of pFastbac with a C-terminal 6 histidine tag, expressed in insect cells (Sf9) and purified on Nickel agarose as previously described , , , . "
[Show abstract][Hide abstract] 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.
PLoS ONE 01/2011; 6(1):e16379. DOI:10.1371/journal.pone.0016379 · 3.23 Impact Factor
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