Globular Protein – Caspase-9 Enzyme

The globular protein caspase-9 may initially not seem very important or significant, as it sounds like the ninth of many other caspase proteins. However, this protein plays a vital role, not only in the cell’s life cycle but also in the life of humans and other mammals. Caspase-9 is an enzyme whose function ultimately leads to cell degradation and apoptosis. This protein is a member of the peptidase family C14, and is encoded by the CASP9 gene located on the short (p) arm of the first chromosome (UniProt, 2019).

There are three classes of caspases: initiator caspases, effector caspases, and inflammatory caspases. Caspase-9 belongs to the initiator class and, as this class name suggests, is responsible for initiating the apoptotic signal in the intrinsic or mitochondrial pathway. Once activated, this protein can activate other caspases, those belonging to the effector class, which in turn begin the apoptotic process by cleaving vital cellular constituents (McIlwain, D., Berger, T., & Mak, T., 2013).

Although this is not the only pathway for cells to undergo apoptosis, it aids in regulating the survival of only healthy and fully functioning cells. This is extremely important because the decreased or inability to undergo programmed cell death can allow a collection of mutated cells to continuously divide and multiply without regulation, leading to disease, particularly cancer. Caspase-9 mutation can also lead to ischemia, inflammatory bowel disease, dystrophinopathies, and cerebral hypoxia (GeneCards, 2019). Fortunately, drugs have been created to target the caspase cascade, specifically caspase-9, to either induce or inhibit apoptosis to treat these various diseases. The most common one is the anti-cancer chemotherapy drug Cisplatin, which aids in activating subsequent effector caspases to stimulate apoptosis (GeneCards, 2019). Caspase-9 is an average-sized protein, its main isoform being 416 amino acids in length (see Figure 1 for 1° sequence). Its composition contains all twenty amino acids, but some are expressed more abundantly than others (Figure 2). Leucine makes up almost 12% of the protein, with serine, glycine, and arginine making up 7% to over 8% each, accounting for over a third of the entire sequence. Casp-9 has a molecular weight of approximately 46280 kDa, and a theoretical inflection point of 5.73 (SIB, 2019). It is found ubiquitously in cells of all tissues, with varying abundance in certain tissues, but a decreased presence in fetal tissues (GeneCards, 2019). This likely relates to development, reducing the rate of cell death to allow for growth. At the cellular level, caspase-9 is found mainly in the cytosol but can also be detected in the mitochondria and the nucleus. The location of this protein is key for its ability to activate the effector caspases in the pathway, which also reside in the cytosol, enabling them to cleave portions of the cell and induce death.

On a tissue level, the caspase protein is expressed the most in the heart, specifically myocytes, and is reasonably expressed in skeletal muscles, liver, and pancreas. This protein is less expressed in other tissues (UniProt, 2019). The secondary structure is composed of both helices and sheets in various positions of the strand, in addition to turns and bends (Figure 3). Moreover, Figure 4 depicts these sheets and helices in a three-dimensional or tertiary structure. While the individual characteristics and structure of this protein are incredibly interesting, it is the quaternary structure or complex, formed between caspase-9, cytochrome c, and Apaf-1, which is by far the most fascinating. Together, these proteins forge a heptameric wheel-shaped apoptosome, aptly nicknamed the 'wheel of death' (Figure 5). The tertiary structure is crucial to ensure the proper binding of the protein to Apaf-1, which subsequently facilitates apoptosis. During binding, however, one hypothesis is the "induced conformation model". This model posits that the binding of Apaf-1 induces a conformational shift in caspase-9, thereby activating the protein (Li, P., et al., 2017). The modification in caspase-9 is significant as it essentially controls whether or not the effector caspases are activated. The caspase is activated by phosphorylation at Tyrosine-153. A mutation at this site transforms the Tyrosine into Phenylalanine, inhibiting phosphorylation thus reducing the formation of the caspase 9 subunit (UniProt, 2019). This mutation affects the cells' ability to regulate apoptosis, potentially leading to various degenerative or developmental disorders, most commonly cancer. The normal functioning of the apoptosome's importance is underscored by its high level of regulation.

Various protein kinases, like EKT1/2, DYRK1a, and Akt/PkB, as well as the CDK1-CyclinB1 complex, the phosphorylation of Thr125, and even some miRNAs, all work towards regulating caspase, inhibiting its activation when necessary. This protein's suppression is thought to be utilized by cancer cells as a tumor escape mechanism, inhibiting apoptosis and allowing tumors to keep dividing (Li, P., Zhou, L., Zhao, T., Liu, X., Zhang, P., Liu, Y., … Li, Q, 2017). As caspase is so highly regulated, there are many targets available for creating a treatment to fix the protein's faulty expression. This also means that there's a lot of room for error; a small alteration to these pathways can have significant effects on both a cellular and organism level. Caspase-9 is widely researched and experimented on due to its importance and relevance to current efforts in cancer treatment. As this protein is part of the intrinsic pathway toward apoptosis, much research is being done to target this protein, or those upstream in the pathway that lead to caspase-9 activation, in creating drugs that induce apoptosis (Li, P., et. al, 2017). An important experiment to conduct, though, is to create a drug that only targets the protein in tumor cells and not healthy cells after targeting the protein. This could be targeted therapy, or genetic changes made to induce apoptosis and prevent cancer cells from avoiding cell death. After completing a BLAST comparison, we found over 250 results, many of which demonstrated a high percentage of similarity. One species at the top of the list was the Pygmy chimpanzee, which has only two of the 416 amino acids different from the human sequence; amino acid 211 is lysine in humans but glutamic acid in the chimpanzee, and amino acid 221 is glutamine in humans and arginine in chimps (NCBI, 2019). The alterations don’t affect the protein's function, as it still serves as regulation for apoptosis. With the BLAST comparison and the ClustalW homology alignment, we more concisely observed the differences and similarities in the species' sequences (Figure 6).

In the alignment, five different species were used: humans, chimpanzees, orangutans, horses, and Japanese pufferfish. The chimpanzee caspase 9 sequence was very similar to humans', with only two amino acid differences, as seen before with the BLAST. The orangutan's sequence was fairly similar to both the chimpanzee's and human's sequences. The horse's sequence showed a lower similarity, about 79%, and the pufferfish's even lower, at about 48%. Although there was variance in all of the sequences for the protein, all five species conserved the caspase's main function. Some residues were consistently conserved in each of the species. For example, two active sites and surrounding residues were the same: a Histidine at amino acid 237 and a Cysteine at amino acid 287. The Tyrosine at 153, which becomes phosphorylated and activates the protein, was also conserved in each species (Clustal Omega, 2019). All these portions of the protein are key to maintaining its function.

Overall, caspase-9 has a very specific function, but it is one that plays a crucial role in the survival of cells and prevention of life-threatening diseases. Its function, need for high regulation, and the consequences when not properly expressed, go to show how important this protein is to the organism. It was also interesting to see how consistent the function was throughout each of the animals, even when less than half of the protein sequence was conserved. This protein is highly relevant in current medicine and research, and could play a key role in future cancer treatments, which is why I think this protein is so interesting. This caspase may be one of twelve, but with its unique structure and important function, it is definitely one of a kind.

References

1. Chao, Y., Shiozaki, E., Srinivassula, S., Rigotti, D., Fairman, R., & Shi, Y. (2005). Crystal structure of a dimeric caspase-9. doi:10.2210/pdb2ar9/pdb Clustal Omega. (2019). EMBL-EBI. Retrieved from https://www.ebi.ac.uk/Tools/services/web/toolresult.ebi?jobId=clustalo-I20190420- 043501-0920-95162258-p1m&analysis=alignments
2. GeneCards Human Gene Database. (2019). CASP9 Gene (Protein Coding). Retrieved from https://www.genecards.org/cgi-bin/carddisp.pl?gene=CASP9
3. Li, P., Zhou, L., Zhao, T., Liu, X., Zhang, P., Liu, Y., … Li, Q. (2017). Caspase-9: structure, mechanisms and clinical application. Oncotarget, 8(14), 23996–24008. doi:10.18632/oncotarget.15098
4. McIlwain, D., Berger, T., & Mak, T. (2013). Caspase Functions in Cell Death and Disease. Cold Spring Harbor Perspectives In Biology, 5(4), a008656-a008656. doi:10.1101/cshperspect.a008656
5. NCBI National Center for Biotechnology Institute. (2019). Blast Results P55211. Retrieved from https://blast.ncbi.nlm.nih.gov/Blast.cgi SIB Swiss Institute of Bioinformatics. (2019). Casp9 Human. Retrieved from https://web.expasy.org/cgi-bin/protparam/protparam
6. UniProt Consortium. (2019). UniProtKB - P55211 (CASP9_HUMAN). Retreived from https://www.uniprot.org/uniprot/P55211
7. https://www.ebi.ac.uk/Tools/services/web/toolresult.ebi?jobId=clustalo-I20190420-043501-0920-95162258-p1m&analysis=alignments
8. https://www.ebi.ac.uk/Tools/services/web/toolresult.ebi?jobId=clustalo-I20190420-043501-0920-95162258-p1m&analysis=alignments
9. https://www.genecards.org/cgi-bin/carddisp.pl?gene=CASP9 https://blast.ncbi.nlm.nih.gov/Blast.cgi https://web.expasy.org/cgi-bin/protparam/protparam https://www.uniprot.org/uniprot/P55211
10. Chao, Y., Shiozaki, E., Srinivassula, S., Rigotti, D., Fairman, R., & Shi, Y. (2005). Crystal structure of a dimeric caspase-9. doi:10.2210/pdb2ar9/pdb
11. McIlwain, D., Berger, T., & Mak, T. (2013). Caspase Functions in Cell Death and Disease. Cold Spring Harbor Perspectives In Biology, 5(4), a008656-a008656. doi:10.1101/cshperspect.a008656

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