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, 289 (32), 22183-95

Anamorsin, a Novel caspase-3 Substrate in Neurodegeneration

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Anamorsin, a Novel caspase-3 Substrate in Neurodegeneration

Nuri Yun et al. J Biol Chem.

Abstract

Activated caspases play a central role in the execution of apoptosis by cleaving endogenous substrates. Here, we developed a high throughput screening method to identify novel substrates for caspase-3 in a neuronal cell line. Critical steps in our strategy consist of two-dimensional electrophoresis-based protein separation and in vitro caspase-3 incubation of immobilized proteins to sort out direct substrates. Among 46 putative substrates identified in MN9D neuronal cells, we further evaluated whether caspase-3-mediated cleavage of anamorsin, a recently recognized cell death-defying factor in hematopoiesis, is a general feature of apoptosis. In vitro and cell-based cleavage assays indicated that anamorsin was specifically cleaved by caspase-3 but not by other caspases, generating 25- and 10-kDa fragments. Thus, in apoptosis of neuronal and non-neuronal cells induced by various stimuli including staurosporine, etoposide, or 6-hydroxydopamine, the cleavage of anamorsin was found to be blocked in the presence of caspase inhibitor. Among four tetrapeptide consensus DXXD motifs existing in anamorsin, we mapped a specific cleavage site for caspase-3 at DSVD(209)↓L. Intriguingly, the 25-kDa cleaved fragment of anamorsin was also detected in post-mortem brains of Alzheimer and Parkinson disease patients. Although the RNA interference-mediated knockdown of anamorsin rendered neuronal cells more vulnerable to staurosporine treatment, reintroduction of full-length anamorsin into an anamorsin knock-out stromal cell line made cells resistant to staurosporine-induced caspase activation, indicating the antiapoptotic function of anamorsin. Taken together, our approach seems to be effective to identify novel substrates for caspases and has the potential to provide meaningful insights into newly identified substrates involved in neurodegenerative processes.

Keywords: Apoptosis; Caspase; Neurodegeneration; Neurotoxin; Proteomics.

Figures

FIGURE 1.
FIGURE 1.
A schematic view of the novel caspase-3 substrate screening method. After the IEF step, the strips were incubated in a caspase-3 activation buffer with or without an empirically predetermined amount of the recombinant human caspase-3 (Cas-3). Subsequently, SDS-PAGE was performed, and the resulting gels were stained with Coomassie Brilliant Blue G-250. The separated protein spots were analyzed by using ProteomWeaver software system. Assuming that the cleaved forms of the putative caspase-3 substrates (indicated by blue arrows) appear below the uncleaved full-length forms (indicated by yellow arrows) on the merged gel, all of the spots indicated by arrows were subjected to MALDI-TOF mass spectrometry for identification.
FIGURE 2.
FIGURE 2.
Representative gel images. Total cellular lysates (1.5 mg) were processed for protein separation by two-dimensional electrophoresis. Incubation with buffer only (A) and 25 μg of recombinant human caspase-3 in caspase-3 activation buffer (B) was done after the IEF step on Immobiline DryStrips (pI 4–7 linear). Representative gels demonstrate typical protein profiles of untreated and caspase-3-incubated samples. Arrows indicate protein spots that were altered significantly as summarized in Table 1.
FIGURE 3.
FIGURE 3.
Comparative close-up view of the identified caspase-3 substrates. After MN9D cells were solubilized in 1× sample buffer, the resulting cellular lysates (1.5 mg) were absorbed into Immobiline DryStrips (pI 4–7 linear). Following the IEF step, the strips were incubated in caspase-3 activation buffer with or without 25 μg of recombinant human caspase-3 (Cas-3) followed by SDS-PAGE on an 8–18% gradient gel. The gels were stained with 0.1% Coomassie Brilliant Blue G-250 and analyzed by the ProteomWeaver system. Among several identified protein spots listed in Table 1, the arrows on the control gels indicate 12 putative caspase-3 substrates that were significantly decreased after incubation with caspase-3. hnRNP, heterogeneous nuclear ribonucleoprotein; SPR, sepiapterin reductase; APRT, adenine phosphoribosyltransferase; TSase, thymidylate synthase.
FIGURE 4.
FIGURE 4.
In vitro caspase-3 cleavage assay for the identified substrates. pcDNA3.1/V5-His vectors encoding calponin-3 (A), emerin (B), thymidylate synthase (TSase) (C), and TDP-43 (D) cDNAs were transcribed and translated in the presence of 35S-labeled methionine. To confirm whether these identified proteins are indeed cleaved by exogenously added caspase-3, each reactant was incubated for 1.5 h at 37 °C with or without 50 ng of recombinant human caspase-3 (Cas-3) in the presence or absence of 100 μm Z-VAD-fmk, a pan-caspase inhibitor. After incubation, the reactants were separated by SDS-PAGE followed by autoradiography. Arrowheads indicates the full-length proteins, whereas arrows indicate potentially caspase-3-cleaved fragments that do not appear or exist in low levels in the control and in lanes treated with caspase-3 plus Z-VAD-fmk.
FIGURE 5.
FIGURE 5.
Confirmation of anamorsin as a novel caspase-3 substrate. A, MN9D cellular lysates (1.5 mg) were processed for two-dimensional electrophoresis following incubation with or without 25 μg of recombinant human caspase-3 (Cas-3). Arrows indicate the Coomassie Brilliant Blue G-250-stained protein spot, the level of which decreased in the presence of casapse-3 compared with untreated control. Mass spectrometry indicates that this protein spot is anamorsin. B, MN9D cells were treated with or without a prototypic apoptotic inducer, 100 μm 6-hydroxydopamine (6-OHDA) for the indicated time periods. Total cellular lysates were processed for two-dimensional electrophoresis, and the separated protein spots on the gel were stained with 0.1% Coomassie Brilliant Blue G-250. A close-up view of the same gel position as in A is demonstrated for each time period. Arrows indicate the expression level of anamorsin following 6-hydroxydopamine treatment or sham treatment (CTL). C, in vitro caspase-3 cleavage assay confirmed that the metabolically labeled mouse (mAM) and human anamorsin (hAM) were cleaved by 50 ng of caspase-3. Caspase-3-meidated cleavage of full-length anamorsin (AM-FL) resulted in the generation of two cryptic fragments with molecular sizes of 25 (AM-25 kDa) and 10 kDa (AM-10 kDa). This cleavage was blocked in the presence of 100 μm Z-VAD-fmk. D, specificity of caspase-3-mediated cleavage of anamorsin was confirmed by an in vitro caspase cleavage assay. Fifty nanograms of recombinant human caspase-2, -3, -6, -7, -8, and -9 were used for the reaction.
FIGURE 6.
FIGURE 6.
Determination of the anamorsin cleavage site by caspase-3. A, amino acid sequences of mouse anamorsin demonstrate four putative caspase-3 cleavage sites containing tetrapeptide motif DXXD (marked in bold and underlined). B, in vitro caspase-3 (Cas-3) cleavage assay was performed using 35S-labeled WT and each of four anamorsin DXXD mutants. Each reactant was incubated for 1.5 h at 37 °C with or without 50 ng of caspase-3. Note that the cleavage sequence of mouse anamorsin was mapped at DSVD209↓L. AM-NT and AM-CT represent 25- and 10-kDa anamorsin, respectively. C, MN9D cells overexpressing C-terminally V5-His-tagged WT anamorsin (AM-V5-His) or one of four anamorsin DXXD mutants were treated for 24 h with 1 μm staurosporine (STS). Following drug treatment, cellular lysates were processed for an immunoblot analysis using anti-V5 antibody that recognizes the C-terminally V5-tagged fragment of anamorsin and anti-cleaved caspase-3 antibody. Note that DSVD209↓L was also confirmed as a caspase-3-mediated cleavage site of anamorsin. AM-FL, full-length anamorsin.
FIGURE 7.
FIGURE 7.
Cleavage of anamorsin in caspase-3-dependent apoptotic death. A and B, MN9D cells were treated for the indicated time periods with a prototypic apoptotic stimulus, 100 μm 6-hydroxydopamine (6-OHDA) (A) or 1 μm staurosporine (STS) (B), in the presence or absence of 100 μm Z-VAD-fmk. Following drug treatment, cellular lysates were processed for immunoblot analyses using anti-anamorsin that recognizes the N-terminal anamorsin fragment (KM3052) and anti-cleaved caspase-3 antibody. GAPDH and actin antibodies were used as loading controls. Note that the cleaved form of anamorsin (AM) was apparent only in activated caspase-3 (cas-3) lanes. C, MN9D cells were treated with 50 μm MPP+ for the indicated time periods. Lysates obtained from staurosporine-treated MN9D cells were used as a positive control for caspase-3-mediated anamorsin cleavage. An immunoblot analysis demonstrates no cleavage of anamorsin in MPP+-treated cells in which no sign of caspase-3 activation was found. D, U-2 OS cells were treated with 50 μm etoposide for 24 h in the presence or absence of 100 μm Z-VAD-fmk. An immunoblot blot analysis indicates that etoposide-induced cleavage of anamorsin was blocked in the presence of Z-VAD-fmk. CTL, control.
FIGURE 8.
FIGURE 8.
Appearance of the cleaved anamorsin fragment in the post-mortem brains of patients with AD or PD. Tissue lysates from the cortices of post-mortem brains from AD patients (A) or the substantia nigra region (SN) of post-mortem brains of patients with PD (B) were subjected to immunoblot analyses using antibodies against anamorsin (KM3052). The identity of two bands marked by asterisks was not determined. Equivalent tissues lysates from age-matched control brains were run in parallel. The intensity of the cleaved form of anamorsin (AM-NT) was plotted for both post-mortem brains (closed squares) and controls (CTL) brains (closed circles) for comparison. Error bars indicate ±S.D. on either side of the mean. *, p < 0.05. The information for AD and PD patients is provided in Tables 2 and 3, respectively. AU, arbitrary units.
FIGURE 9.
FIGURE 9.
Evaluation of antiapoptotic function of anamorsin. A, MN9D control cells (shCon) and anamorsin-knockdown MN9D cells (shAM) were treated with 1 μm staurosporine (STS) for 18 h. The rate of viability was determined by MTT reduction assays. Values are expressed as a percentage of the untreated controls (100%). Bars represents the mean ± S.D. from three independent experiments done in triplicate. *, p < 0.05. Expression levels of anamorsin in cells transfected with anamorsin shRNA or scrambled sequences were determined by immunoblot analysis using anti-anamorsin antibody. B, FW3-1 (anamorsin WT) and FK3-1 (anamorsin KO) cells were treated for 12 h with the indicated concentrations of staurosporine. Cellular lysates were processed for immunoblot analyses using antibody recognizing anamorsin or cleaved caspase-3. The intensity of the cleaved caspase-3 in FK3-1 cells at 1 μm staurosporine was quantitated by densitometry and is expressed as a ratio over the staurosporine-treated FW3–1 cells after normalization against GAPDH. Bars represent the mean ± S.D. from three independent experiments. ***, p < 0.001. C, FK3-1 cells were transiently transfected with the vector alone (pCI-Neo; CTL) or vectors containing FLAG-tagged anamorsin wild type (AM). Twenty-four hours after transfection, cells were treated with 1 μm staurosporine for 12 h. Following drug treatment, cellular lysates were processed for immunoblot analyses using antibodies against anamorsin and cleaved caspase-3 (cas-3). Levels of the cleaved caspase-3 are expressed as a ratio over the staurosporine-treated control cells (1.0) after normalization against GAPDH. Bars represent the mean ± S.D. from three independent experiments. **, p < 0.01. All error bars represent S.D. AU, arbitrary units.
FIGURE 10.
FIGURE 10.
Evaluation of anti-cell death function of anamorsin in primary cultures of cortical neurons. Primary cultures of cortical neurons were prepared using ED14.5 mouse cortices as described under “Experimental Procedures.” At 7 days in vitro, cultures were exposed to lentiviral particles containing one of the two shRNA anamorsin sequences (shAM 1 and shAM 2) or control shRNA sequences (shCon) for 4 days. Cultures were treated with 200 nm staurosporine (STS) for 24 h and subjected to immunoblot analyses using anti-anamorsin and anti-cleaved caspase-3 (A) or MTT reduction assay (B). A, relative intensity of cleaved caspase-3 (cas-3) is expressed as a ratio over the control cultures (1.0) after normalization against GAPDH. Bars represent the mean ± S.D. from three independent experiments. **, p < 0.01; ***, p < 0.001. B, viability was determined by MTT reduction assays. Values are expressed as a percentage of the untreated controls (100%). Bars represent the mean ± S.D. from three independent experiments. ***, p < 0.001. All error bars represent S.D. AU, arbitrary units.

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