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Apoptosis-Inducing Factor 2 (AIF-2) Mediates a Caspase-Independent Apoptotic Pathway in the Tropical Sea Cucumber (Holothuria leucospilota).

ABSTRACT: 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.

SUBMITTER: Li X

PROVIDER: S-EPMC8954137 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Apoptosis-Inducing Factor 2 (AIF-2) Mediates a Caspase-Independent Apoptotic Pathway in the Tropical Sea Cucumber (Holothuria leucospilota).

Li Xiaomin X Chen Ting T Wu Xiaofen X Jiang Xiao X Luo Peng P E Zixuan Z Hu Chaoqun C Ren Chunhua C

International journal of molecular sciences 20220310 6

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 ...[more]

PMID: 35328428

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Project description:BackgroundThe sea cucumber Holothuria leucospilota belongs to echinoderm, which is evolutionally the most primitive group of deuterostomes. Sea cucumber has a cavity between its digestive tract and the body wall that is filled with fluid and suspended coelomic cells similar to blood cells. The humoral immune response of the sea cucumber is based on the secretion of various immune factors from coelomocytes into the coelomic cavity. The aim of this study is to lay out a foundation for the immune mechanisms in echinoderms and their origins in chordates by using RNA-seq.ResultsSea cucumber primary coelomocytes were isolated from healthy H. leucospilota and incubated with lipopolysaccharide (LPS, 10 μg/ml), polyinosinic-polycytidylic acid [Poly (I:C), 10 μg/ml] and heat-inactived Vibrio harveyi (107 cell/ml) for 24 h, respectively. After high-throughput mRNA sequencing on an Illumina HiSeq2500, a de novo transcriptome was assembled and the Unigenes were annotated. Thirteen differentially expressed genes (DEGs) were selected randomly from our data and subsequently verified by using RT-qPCR. The results of RT-qPCR were consistent with those of the RNA-seq (R2 = 0.61). The top 10 significantly enriched signaling pathways and immune-related pathways of the common and unique DEGs were screened from the transcriptome data. Twenty-one cytokine candidate DEGs were identified, which belong to 4 cytokine families, namely, BCL/CLL, EPRF1, IL-17 and TSP/TPO. Gene expression in response to LPS dose-increased treatment (0, 10, 20 and 50 μg/ml) showed that IL-17 family cytokines were significantly upregulated after 10 μg/ml LPS challenge for 24 h.ConclusionA de novo transcriptome was sequenced and assembled to generate the gene expression profiling across the sea cucumber coelomocytes treated with LPS, Poly (I:C) and V. harveyi. The cytokine genes identified in DEGs could be classified into 4 cytokine families, in which the expression of IL-17 family cytokines was most significantly induced after 10 μg/ml LPS challenge for 24 h. Our findings have laid the foundation not only for the research of molecular mechanisms related to the immune response in echinoderms but also for their origins in chordates, particularly in higher vertebrates.

Project description:Holothuria leucospilota (Echinodermata: Holothuroidea) is a widespread tropical sea cucumber with strong value for the ecological restoration of coral reefs. Therefore, some studies regarding the artificial breeding and cultivation of H. leucospilota have been undertaken recently. However, the biological functions of the digestive system of this species have not been elucidated. In this study, a cDNA coding for α-amylase, an indicator of digestive maturity in animals, was identified from H. leucospilota and designated Hl-Amy. The full-length cDNA of the Hl-Amy gene, which is 1734 bp in length with an open reading frame (ORF) of 1578 bp, encodes a 525 amino acid (a.a.) protein with a deduced molecular weight of 59.34 kDa. According to the CaZy database annotation, Hl-Amy belongs to the class of GH-H with the official nomenclature of α-amylase (EC 3.2.1.1) or 4-α-D-glucan glucanohydrolase. The Hl-Amy protein contains a signal peptide at the N-terminal followed by a functional amylase domain, which includes the catalytic activity site. The mRNA expression of Hl-Amy was abundantly exhibited in the intestine, followed by the transverse vessel with a low level, but was hardly detected in other selected tissues. During embryonic and larval development, Hl-Amy was constitutively expressed in all stages, and the highest expression level was observed in the blastula. By in situ hybridization (ISH), positive Hl-Amy signals were observed in different parts of the three different intestinal segments (foregut, midgut and hindgut). The Hl-Amy recombinant protein was generated in an E. coli system with codon optimization, which is necessary for Hl-Amy successfully expressed in this heterogenous system. The Hl-Amy recombinant protein was purified by immobilized metal ion affinity chromatography (IMAC), and its activity of starch hydrolysis was further detected. The optimal temperatures and pH for Hl-Amy recombinant protein were 55°C and 6.0, respectively, with an activity of 62.2 U/mg. In summary, this current study has filled a knowledge gap on the biological function and expression profiles of an essential digestive enzyme in sea cucumber, which may encourage future investigation toward rationalized diets for H. leucospilota in artificial cultivation, and optimized heterogenous prokaryotic systems for producing recombinant enzymes of marine origins.

Dataset Information

Apoptosis-Inducing Factor 2 (AIF-2) Mediates a Caspase-Independent Apoptotic Pathway in the Tropical Sea Cucumber (Holothuria leucospilota).

Publications

Apoptosis-Inducing Factor 2 (AIF-2) Mediates a Caspase-Independent Apoptotic Pathway in the Tropical Sea Cucumber (<i>Holothuria leucospilota</i>).

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