The neuronal apoptosis inhibitory protein is a direct inhibitor of caspases 3 and 7 - PubMed (original) (raw)
The neuronal apoptosis inhibitory protein is a direct inhibitor of caspases 3 and 7
Johannes K X Maier et al. J Neurosci. 2002.
Abstract
The neuronal apoptosis inhibitory protein (NAIP) was identified as a candidate gene for the inherited neurodegenerative disorder spinal muscular atrophy. NAIP is the founding member of a human protein family that is characterized by highly conserved N-terminal motifs called baculovirus inhibitor of apoptosis repeats (BIR). Five members of the human family of inhibitor of apoptosis proteins including NAIP have been shown to be antiapoptotic in various systems. To date, a mechanism for the antiapoptotic effect of NAIP has not been elucidated. To investigate NAIP function, we found cytoprotection of NAIP-expressing primary cortical neurons treated to undergo caspase-3-dependent apoptosis. The additional treatment of these neurons with the pancaspase inhibitor boc-aspartyl(OMe)-fluoromethylketone did not result in increased survival. Similar cytoprotective effects were obtained using HeLa cells transiently transfected with a NAIP N-terminal construct and treated to undergo a caspase-3-dependent cell death. To examine whether NAIP inhibits caspases directly, recombinant N-terminal NAIP protein containing BIR domains was overexpressed, purified, and tested for caspase inhibition potential. Our results demonstrate that inhibition of caspases is selective and restricted to the effector group of caspases, with K(i) values as low as approximately 14 nm for caspase-3 and approximately 45 nm for caspase-7. Additional investigations with NAIP fragments containing either one or two NAIP BIRs revealed that the second BIR and to a lesser extent the third BIR alone are sufficient to mediate full caspase inhibition.
Figures
Fig. 1.
Cytoprotection of Ad-NAIP in primary cortical neurons treated to undergo caspase-3-dependent cell death. The neurons were infected with Ad-lacZ, Ad-NAIP, or Ad-XIAP at the MOI indicated at the bottom. After 3 d of culture, the cells were treated with camptothecin alone (10 μ
m
) or with addition of the pancaspase inhibitor BAF (100 μ
m
). Control samples were kept untreated. A neuronal survival assay (MTT) was assessed 24 hr later. Data are expressed as number of surviving cells treated with camptothecin alone or with BAF versus nontreated cells (taken as 100%), and are the mean ± SEM of six independent experiments. *p < 0.05; **p < 0.01; Student's t test.
Fig. 2.
A, Illustration of the NAIP deletion constructs generated for caspase inhibition and cell death assays. B, Amino acid sequence alignment of BIR domains of human IAP family members known to have an antiapoptotic effect. The alignment was generated with the program CLUSTALW 1.8 (Thompson et al., 1994) with default settings (
). The_black_ regions in highlight amino acids conserved in BIR motifs across species as determined by Uren et al. (1998); the gray regions indicate identity with NAIP BIR2 domain. The sequences of NAIP BIR2 deletion constructs (B2A, B2B, B2C) are illustrated above the NAIP BIR2 sequence. C, Phylogenetic tree diagram (rooted) of the BIR sequences aligned in_B_ generated with the program DRAWGRAM (Inference Package, version 3.5; Department of Genetics, University of Washington, Seattle, WA) using NAIP B2 as the leader sequence.
Fig. 3.
Cytoprotection assay of apoptotic HeLa cells transfected with IAP constructs. HeLa cells were transiently cotransfected with the pEGFP-N1 vector and either pcDNA3.0-myc vector alone (control) or pcDNA3.0-myc containing B123xt, XIAP, or Survivin (Surv) and treated with the topoisomerase II inhibitor etoposide for 8 hr (control samples were kept untreated). Seventy-two hours after treatment, cell death was assessed in GFP-positive cells by flow cytometry. Results (mean ± SEM) of four independently performed experiments are shown. Cell survival was calculated in an adaptation of the method of Scaffidi et al. (1998). *p < 0.05; **p < 0.01; ***p < 0.001; Student's _t_test.
Fig. 4.
Caspase inhibition assay, group I and III caspases. A, Inhibition of caspase-1, a representative member of the group I caspases. The tetrapeptide Ac-YVAD-AMC (10 μ
m
) served as the substrate. Enzyme concentration was held constant at 1 n
m
. The peptide aldehyde_Ac-YVAD-CHO_ was used as control for enzyme inhibition at an initial concentration of 10 μ
m
. Continuous readings (λex, 380 nm; λem, 460 nm) were performed for 30 min at room temperature. The IC50 of the control inhibitor_Ac-YVAD-CHO_ was 74 n
m
. B, Inhibition of caspase-8, a representative member of group III caspases. The assay was performed as described above with a substrate concentration (Ac-DEVD-AMC) of 10 μ
m
and an enzyme concentration of 2.25 n
m
. The peptide aldehyde_Ac-DEVD-CHO_ was applied as inhibition control, inhibiting the enzyme with an IC50 of 18 n
m
.
Fig. 5.
Caspase inhibition assay, group II caspases.A, Representative illustration of caspase-7 inhibition. The IC50 of NAIP (B123xt) was 101 n
m
in this experiment; XIAP was 99 n
m;
and_Ac-DEVD-CHO_ was 39 n
m
. The enzyme concentration in this experiment was 1.4 n
m
.B, Representative experiment of caspase-3 inhibition by NAIP (B123xt). The assay was performed as described above using caspase-3 at 50 p
m
. The IC50 values obtained from this experiment were 23 n
m
for_XIAP_, 28 n
m
for NAIP (B123xt), and 4 n
m
for the control inhibitor_Ac-DEVD-CHO_.
Fig. 6.
Caspase-3 inhibition assay, NAIP BIR deletion proteins. A, Caspase-3 inhibition assays of NAIP BIR deletion constructs using NAIP proteins with two BIR domains. The NAIP_B12_ protein does not inhibit caspase-3,B23, with an IC50 of 614 n
m
, ∼20-fold lower than XIAP, with an IC50 of 32 n
m
. B, Typical experiment of caspase-3 inhibition by NAIP proteins containing a single BIR domain. The experimental procedure is described above and revealed IC50values for B3 of 348 n
m
and for_B2_ of 17 n
m
; values of control inhibitors are as in A.
Fig. 7.
Cytoprotection assay of apoptotic HeLa cells transfected with NAIP BIR deletion constructs. Description and procedure are as described in Figure 3. Surv, Survivin.
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