1. Discovery

The CED-9 gene was discovered in 1992 while searching the genome of C. elegans for mutations affecting cell death.^[1] The first mutation identified was a dominant gain of function mutation referred to as n1950 that allowed cells to survive when they were fated to die.^[1] The observed phenotype was similar to that observed in CED-3 and CED-4 loss of function mutants (known proteins from the apoptotic pathway).^[1] It was also observed that loss of function mutations in CED-3 and CED-4 were able to rescue cells with a CED-9 loss of function mutation.^[2] These observations suggested that CED-9 functioned upstream of the CED-3 and CED-4 proteins in the same pathway.^[2]

2. Structure

2.1. Gene

The CED-9 gene is located on chromosome 3 of the C. elegans genome.^[3] CED-9 is transcribed from a polycistronic locus that also contains genes required for the mitochondrial Oxidative phosphorylation.^[4] The CED-9 gene has been identified in two distinct transcripts, both transcribed from this locus.^[4] The first was identified as a 1.3 kb transcript encoding only the CED-9 sequence. The second being a rare 2.1 kb bicistronic transcript containing the 1.3 kb transcript and an additional 0.75 kb transcript from an upstream gene found in the gene locus.^[4] this 0.75 kb transcript corresponds to the cytochrome protein cyt-1 that functions in the electron transport chain within the mitochondria.^[4] The bicistronic transcript is then spliced giving rise to the two distinct mature messenger RNA (mRNA) for both genes.^[4] The most prevalent transcript however, is the 1299-nucleotide (~1.3kb) long transcript that encodes an 843-nucleotide mRNA containing 4 exons.^[5]

Structure of the CED-9 protein from two different angles. PBD ID: 1OHU. https://handwiki.org/wiki/index.php?curid=1231441

2.2. Protein

CED-9 encodes the apoptosis regulator CED-9 protein which is an important negative regulator protein in the apoptosis pathway of C. elegans.^[6] The protein consists of 280 amino acids and has a molecular weight of 31824.42 Da.^[7] The structure of this protein has been solved using X-ray diffraction revealing 9 Helices, 2 Beta strands, and 2 turn motifs.^[8] The CED-9 protein belongs to the Bcl-2-like protein family. This refers to the homology between the ced-9 protein and the B-cell lymphoma proteins (Bcl) found in humans, specifically the Bcl-2 protein.^[4] CED-9 contains a BCL domain homologous to Bcl-2 domains BH1, BH2, and part of BH3 as well as a separate domain homologous to BH4 located near the N-terminus.^[9] CED-9 also includes a transmembrane domain on the C-terminal end of the structure that anchors the protein to the mitochondrial membrane.^[10] However, research shown that the C-terminal domain is not necessary for the protein's main function as an inhibitor of the CED-4 protein found in the same apoptosis signalling pathway.^[10]

3. Function

Cell death, or apoptosis during early development is crucial for the correct morphology and refractivity of adult C. elegans.^[11] This process involves a signal and interaction cascade of proteins leading to the engulfment and death of the targeted cell. Proteins in this cascade can be categorized into two groups; pro-apoptotic and anti-apoptotic.^[11] Pro-apoptotic proteins activate the apoptosis pathway while anti-apoptotic proteins suppress the pathway.^[12] CED-9 is classified as an anti-apoptotic protein.^[13]

Simplified outline of the apoptosis pathway observed in C. elegans. https://handwiki.org/wiki/index.php?curid=1302806

Apoptosis in C. elegans is often simplified to interactions between four major proteins in the pathway; EGL-1, CED-9, CED-4, and CED-3. CED-3 is the final protease in the interaction network and is responsible for activating the proteins involved in cell disassembly.^[11] CED-9 is said to protect cells from the apoptosis pathway.^[2] Under normal conditions, in a cell not experiencing apoptotic signalling, CED-9 forms a complex with CED-4 at the membrane of the mitochondria.^[12] This interaction sequesters the pro-apoptotic signalling of CED-4. CED-4 consists of an asymmetric dimer of CED-4a and CED-4b proteins in which CED-9 can specifically recognize and bind CED-4a.^[12] This interaction is a highly specific recognition and binding interaction between the N-terminal tails of both proteins.^[12] When the cell receives an apoptotic signal via a receptor commonly referred to as a "death receptor", the protein EGL-1 is activated.^[14] The active EGL-1 binds CED-9 causing a Conformational change that interrupts and inhibits the CED-9 - CED-4 interaction.^[12] CED-4 is free to dissociate and activate the CED-3 protease effectively triggering the final stages for apoptosis.^[14]

3.1. Mutations

The cells developed during embryogenesis and early life in C. elegans have one of two fates, to live and differentiate or apoptose.^[15] Apoptosis during development is highly regulated and only occurs in specific cells at specific times.^[15] Every cell division and cell death in the development of C. elegans from embryo to adult has been studied and documented to reveal a fixed pattern between individual organisms.^[15] Apoptosis during development is important for the proper morphology and refractivity of C. elegans, but it is not always essential for survival.^[15] Thus, over 100 mutations have been observed and documented as affecting the apoptotic pathway of C. elegans.^[2] Many proteins involved in the interaction cascade were discovered because of these mutations and their resulting phenotype. CED-9 mutants are among the mutations that affect this pathway. CED-9 gain of function mutations are unresponsive to apoptosis signalling and allow cells fated to die, to survive.^[2] A notable example of a CED-9 dominant gain of function mutation would be the n1950 mutation which was the first mutation documented for CED-9 and responsible for the gene's discovery.^[1] Loss of function mutations cause inappropriate cell death in the absence of apoptosis stimuli.^[2] Mutations in CED-9 also reveal its maternal effect; where the genotype of the mother determines the phenotype of the progeny.^[2] Homozygous, loss of function mutants from a heterozygous mother experience some unpredictable cell death, however, give rise to unviable progeny themselves.^[2]

4. Significance

The apoptotic pathway has been conserved in evolutionary history and is vital for the maintenance of multicellular organisms such as humans.^[16] A parallel pathway to the one found in C. elegans is also observed in mammals involving a number of homologous proteins. Disruptions to this pathway often lead to diseases that, in humans, include various cancers, autoimmune diseases, and neurodegenerative disease.^[16] Bcl-2 in particular is often found mutated in many human cancers.^[17] Due to the conserved nature of the apoptotic pathway and the extensive knowledge and understanding available for C. elegans, the organisms apoptotic pathway can be used as a proxy for the human equivalent.^[16] CED-9 is the homologue of Bcl-2 which can provide researchers with information including the pathways the protein is involved in and the consequences of mutation that may parallel pathways or abnormalities in humans.^[17]

5. Interactions

• CED-9: CED-4^[5][18]
• EGL-1: CED-9^[5][18]
• DRE-1: CED-9^[18]