Globular Protein – Caspase-9 Enzyme

Category: Biology
Date added
2021/04/15
Pages:  6
Words:  1667
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The globular protein caspase-9 initially many not seem very important or significant; it sounds like the ninth of many other caspase proteins. However, this protein plays a vital role in not only the cell’s life cycle, but the life of humans and other mammals as well. Caspase-9 is an enzyme that’s 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 to 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. This protein, once activated, is able to activate other caspases (these 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, the most commonly associated being cancer. Caspase-9 mutation can also lead to ischemia, inflammatory bowel disease, dystrophinopathies, and cerebral hypoxia (GeneCards, 2019). Luckily, 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 as well, all four making up 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, some tissues more so than others, but has a decreased presence in fetal tissues (GeneCards, 2019). This is likely due to development, and decreasing the rate of cell death to allow for the development. At the cellular level, caspase-9 is found mainly in the cytosol, but can also be detected in the mitochondria and the nucleus as well. The location of this protein is key to be able to activate the effector caspases in the pathway, which are also located in the cytosol to be able to cleave portions of the cell to induced cell death.

On a tissue level, the caspase protein is expressed the most in the heart, specifically myocytes, and reasonably expressed in skeletal muscles, liver and pancreas, and less expressed in other tissues (UniProt, 2019). The secondary structure is made up of both helices and sheets in different positions of the strand, as well as turns and bends (Figure 3). Further, Figure 4 is able to show these sheets and helices as a three dimensional, or tertiary structure. Although the individual characteristics and structure of this protein is incredibly interesting, it is the quaternary structure, or complex, that is formed between caspase-9, cytochrome c, and Apaf-1 that is by far the most fascinating. Together these proteins form a heptameric wheel shaped apoptosome, nicknamed the wheel of death (Figure 5). The tertiary structure is important to ensure that the protein is able to bind properly to Apaf-1 and properly provide apoptosis. During binding though, one hypothesis is the “induced conformation model”, where Apaf-1 binds and causes a conformational change to caspase-9 which in turn activates the protein (Li, P., et. al, 2017). This change to caspase-9 is important because it basically acts as the on/off switch to the protein on whether or not to activate the effector caspases. The caspase is activated by phosphorylation at Tyrosine-153. Mutation at this site, turns the Tyrosine into Phenylalanine, thus preventing the phosphorylation and reducing the formation of the caspase 9 subunit (UniProt, 2019). This mutation interferes with cells’ ability to induce apoptosis regularly, and can ultimately lead to different degenerative or developmental disorders, most commonly leading to cancer. The importance of the normal functioning of the apoptosome can also be assumed because of how highly regulated it is.

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

In the alignment, five different species were used; humans, chimpanzee, orangutan, horse, 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 was fairly similar as well to the chimpanzee and human. The horse had a lower similarity, about 79%, and the pufferfish even lower at about 48%. Although there was variance in all of the sequences for the protein, all five species conserved the main function of the caspase. Some residues were consistently conserved in each of the species. For example, two active sites as well as surrounding residues were kept the same, a Histidine at amino acid 237, and a Cysteine at amino acid 287. The Tyrosine at 153 that becomes phosphorylated to activate the protein was also conserved in each of the species (Clustal Omega, 2019). All of these portions of the protein are key in maintaining the function of the protein.

Overall, caspase-9 has a very specific function but 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 goes 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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Globular Protein – Caspase-9 Enzyme. (2021, Apr 15). Retrieved from https://papersowl.com/examples/globular-protein-caspase-9-enzyme/

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