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Int J Mol Sci
2022 Mar 10;236:. doi: 10.3390/ijms23063008.
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Apoptosis-Inducing Factor 2 (AIF-2) Mediates a Caspase-Independent Apoptotic Pathway in the Tropical Sea Cucumber (Holothuria leucospilota).
Li X
,
Chen T
,
Wu X
,
Jiang X
,
Luo P
,
E Z
,
Hu C
,
Ren C
.
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Apoptosis, also known as programmed cell death, is a biological process that is critical for embryonic development, organic differentiation, and tissue homeostasis of organisms. As an essential mitochondrial flavoprotein, the apoptosis-inducing factor (AIF) can directly mediate the caspase-independent mitochondrial apoptotic pathway. In this study, we identified and characterized a novel AIF-2 (HlAIF-2) from the tropical sea cucumber Holothuria leucospilota. HlAIF-2 contains a conserved Pyr_redox_2 domain and a putative C-terminal nuclear localization sequence (NLS) but lacks an N-terminal mitochondrial localization sequence (MLS). In addition, both NADH- and FAD-binding domains for oxidoreductase function are conserved in HlAIF-2. HlAIF-2 mRNA was ubiquitously detected in all tissues and increased significantly during larval development. The transcript expression of HlAIF-2 was significantly upregulated after treatment with CdCl2, but not the pathogen-associated molecular patterns (PAMPs) in primary coelomocytes. In HEK293T cells, HlAIF-2 protein was located in the cytoplasm and nucleus, and tended to transfer into the nucleus by CdCl2 incubation. Moreover, there was an overexpression of HlAIF-2-induced apoptosis in HEK293T cells. As a whole, this study provides the first evidence for heavy metal-induced apoptosis mediated by AIF-2 in sea cucumbers, and it may contribute to increasing the basic knowledge of the caspase-independent apoptotic pathway in ancient echinoderm species.
2018YFD0901605 Ministry of Science and Technology of the People's Republic of China, 2020YFD0901104 Ministry of Science and Technology of the People's Republic of China, GML2019ZD0402 Southern Marine Science and Engineering Guangdong Laboratory, 42176132 National Natural Science Foundation of China, 41906101 National Natural Science Foundation of China, COMS2020Q03 Chinese academy of sciences
Figure 1. Functional domain and phylogenetic tree of HlAIF-2. (A) Structural domain and active sites of HlAIF-2 predicted using the SMART and ScanProsite programs. (B) Phylogenetic analysis of AIFs among various species using the neighbor-joining method with a bootstrap value of 1000.
Figure 2. Sequence alignment and three-dimensional (3-D) structure of AIF-2 in different species. (A) A.a. sequence alignment of AIF-2 in nine Deuterostomia species. The conserved a.a. residues of H. leucospilota are presented in WebLogo format, and the conserved and similar a.a. residues between different species are labeled in dark blue and light blue, respectively. Residues that interact with FAD or NAD (in H. leucospilota) are marked as “F” or “N”, respectively. Two core consensus sequences of the typical motif “GXGXXG” are boxed in red lines. (B–D) Comparison of the 3-D protein of AIF-2 among human H. sapiens and the sea cucumber A. japonicus and H. leucospilota. Space-filling symbols indicate the most conserved binding sites of FAD (orange) and NAD (blue).
Figure 3. Expression patterns of HlAIF-2. (A) Expression profile of HlAIF-2 mRNA in different tissues. (B) Expression profiles of HlAIF-2 mRNA during embryonic and larval stages. Data are presented as the mean ± SE (n = 3).
Figure 4. Expression analysis of HlAIF-2 after treatment of different exogenous stimulants. (A–C) Temporal expression pattern of HlAIF-2 after treatment of CdCl2 (20 μM), LPS (10 μg/mL) smf poly (I:C) (10 μg/mL). Data are presented as mean ± SE (n = 3), and significant differences are analyzed using one-way ANOVA, and shown as n.s. p > 0.05 and *** p < 0.001.
Figure 5. Subcellular localization of HlAIF-2 in HEK293T cells. “DAPI” represents the DAPI-stained cell nuclei; “EGFP-AIF” represents the EGFP-labeled HlAIF-2 protein; “Merge” represents the combination of cell nuclei and HlAIF-2 protein. Cd2+ treatment (12 h) could trigger HlAIF-2 nuclear translocation, compared with the “Control” group. The arrows indicate the typical cells located in cytoplasm or translocated into nuclei.
Figure 6. Apoptotic property of HEK293T cells transfected with pcDNA3.1/HA/HlAIF-2 recombinant plasmid. (A) Detection of apoptosis by TUNEL assay. “DAPI” represents the DAPI-stained cell nuclei; “FITC” represents the FITC-stained fractured DNA fragments (marker for apoptosis); “Merged” represents the combination of cell nuclei and fractured DNA fragments. (B) Comparison of apoptosis rates for HEK293T cells in different groups. “Blank” represents the blank group (untransfected HEK293T cells); “Control” represents the control group (HEK293T cells transfected with pcDNA3.1/HA); “AIF” represents the experimental group (HEK293T cells transfected with pcDNA3.1/HA/HlAIF-2). The values are expressed as mean ± SE (n = 3), and significant difference was analyzed by the Student’s t-test and shown as *** p < 0.001.
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