Abstract
Caspases are universal effectors of apoptosis. The mitochondrial and death receptor pathways activate distinct apical caspases (caspase-9 and -8, respectively) that converge on the proteolytic activation of the downstream executioner caspase-3. Caspase-9 and -8 cleave procaspase-3 to produce a p24 processing intermediate (composed of its prodomain and large subunit), which then undergoes autoproteolytic cleavage to remove the prodomain from the active protease. Recently, several heat shock proteins have been shown to selectively inhibit the mitochondrial apoptotic pathway by disrupting the activation of caspase-9 downstream of cytochrome c release. We report here that the small heat shock protein alphaB-crystallin inhibits both the mitochondrial and death receptor pathways. In S-100 cytosolic extracts treated with cytochrome c/dATP or caspase-8, alphaB-crystallin inhibits the autoproteolytic maturation of the p24 partially processed caspase-3 intermediate. In contrast, neither the closely related small heat shock protein family member Hsp27 nor Hsp70 inhibited the maturation of the p24 intermediate. We also demonstrate that alphaB-crystallin co-immunoprecipitates with the p24 partially processed caspase-3 in vivo. Taken together, our results demonstrate that alphaB-crystallin is a novel negative regulator of apoptosis that acts distally in the conserved cell death machinery by inhibiting the autocatalytic maturation of caspase-3.
Highlights
The caspase family of cysteine proteases are critical effectors of apoptosis that selectively cleave key proteins at aspartate residues, thereby altering their function to promote cell death [1, 2]
Because the maturation of the p24 partially processed caspase-3 requires the autocatalytic removal of its prodomain (8 –12), these findings indicate that the principal mechanism by which ␣B-crystallin inhibits the cytochrome c-dependent activation of caspase-3 is by blocking its autoproteolytic maturation
Recent studies indicate that several heat shock proteins (HSPs) inhibit the mitochondrial apoptotic pathway by binding to components of the cell death apparatus and disrupting the assembly of the apoptosome
Summary
Cell Culture—Human MDA-MB-231 breast carcinoma cells were maintained in DMEM (Mediatech) supplemented with 10% fetal calf serum (FCS, Life Technologies). Transient and Stable Transfections—MDA-MB-231 and PC-3 cells were grown on glass coverslips and transiently transfected with 1 g of pcDNA3-FLAG plasmid containing human ␣B-crystallin or human Hsp, or 1 g of control vector (pEGFP-N1, CLONTECH) using LipofectAMINE Plus reagent (Life Technologies) according to the manufacturer’s instructions. MDA-MB-231 cells were transfected with 1 g of pcDNA3-FLAG vector or pcDNA3FLAG-␣B-crystallin and allowed to recover for 48 h; clones stably expressing these constructs were selected by growth in 800 g/ml G418 (Life Technologies) for 3 weeks. For stable transfections, pooled, vector-transfected cells or two clones stably expressing ␣B-crystallin were untreated or treated with 50 M etoposide for 65 h or 10 ng/ml TNF-␣ and 1 g/ml CHX for 36 h; cells were scored for apoptosis as above. Beads were washed four times in IP wash buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 100 mM NaF) and the immunoprecipated proteins were detected by immunoblotting with ␣B-crystallin mAb (StressGen Biotechnologies) or caspase-3 mAb as described [23]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
