1

THE MECHANISM AND REGULATION OF APOPTOSIS

By: Danielle Schleig English 202C 3/17/14 AUDIENCE AND SCOPE The purpose of this document is to assist students in the Penn State course VB SC 231- Introduction to Cancer Research and Medicine. Because of this specific audience, the only source used for creating this document, including both words and images, is a lecture given in the class on February 27th, 2014. The rest of the information comes from common knowledge from other biology classes that a Penn State student taking VB SC 231 should be familiar with. The technical document is meant to act as a review of the complex mechanism of apoptosis for a better understanding of the process and as a study guide for future exams. Apoptosis also appears in other biology classes so any biology students at Penn State may also benefit from reading this paper. INTRODUCTION Apoptosis, or programmed cell death, is the method in which the body removes old or damaged cells so they can be replaced by new cells. Apoptosis is important in embryo development, maintaining tissue health, and allows the body to help survive changing conditions. The process only removes those cells that are unnecessary to the body, damaged beyond repair, or are in some way unhealthy and should not be replicated. There are many factors that can potentially induce apoptosis, including: viral infections hypoxia - low oxygen in the blood buildup of toxins release of certain hormones or cytokines depletion of nutrients Cells can undergo apoptosis through two different pathways; a pathway triggered by intrinsic stimuli and a pathway triggered by extrinsic stimuli. INTRINSIC PATHWAY In the intrinsic pathway, the main regulator is a protein called Cytochrome C. Cytochrome C is stored in the space between mitochondrial membranes. When a cell starts to trigger apoptosis, the permeability of the outer mitochondrial membrane is targeted, producing Mitochondrial Apoptosis-induced Channels (MACs). Once these channels are formed, Cytochrome C can leak out of the mitochondria into the cytoplasm and heptamerize with Apoptotic Protease Activating Factor -1 (APAF-1). The heptamer also binds ATP and pro-caspase-9 to create an apoptosome. CASPASE CASCADE In the intrinsic pathway, the apoptosome cleaves pro-caspase-9 to activate it, initiating a caspase cascade. Cysteine Aspartate-specific Proteases, or caspases, are a group of cysteine proteases that specifically cleave at the amino acid aspartate. Prior to apoptosis induction, the proteases are inactive zymogens, meaning that they must be activated by cutting the protein at a specific point in the amino

2 acid chain. Once the chain is cleaved, large and small subunits change conformation to create a mature enzyme that can recognize specific sequences. These caspases have two different roles in apoptosis, either as initiator caspases or executioner caspases. Initiator pro-caspases include caspases 2, 8, 9, and 10. In addition to being activated by Cytochrome C, these caspases can also be activated through death receptors on the cell surface. These will be explored in more detail when discussing the extrinsic pathway. Once the initiator caspases are activated, they can in turn activate the executioner caspases 3, 6, and 7. Executioner caspases target key proteins including actin, Inhibitor of Caspase Activated DNAse (ICAD), and lamin, all required for cell survival. By destroying these targets, the cell can no longer function and will result in cell death. Once the cascade is triggered, the process is irreversible. INTRINSIC PATHWAY REGULATION While the cascade cannot be reversed, the process can be regulated through inhibition. Inhibitor of Apoptosis Proteins (IAPs) suppress executioner caspases from destroying target proteins. However, Second Mitochondria-derived Activators of Caspases (SMACs) can sequester IAPs and prevent them from inhibiting programmed cell death. There are also pro-apoptotic and anti-apoptotic signals that can regulate apoptosis by regulating the MACs that allow cytochrome C to traverse the plasma membrane and start the cascade. One important regulator is Bcl2 and the rest of its protein family members. Proteins like Bax and Bak are pro-apoptotic, and help to form the channels. Bcl2, Bclxl and Mcl1 prevent MAC formation, helping the cell survive. The full cascade including these different types of regulation is shown in Figure 1.

Figure 1. Intrinsic Pathway Caspase Cascade

CONSEQUENCES OF UNREGULATED PATHWAYS The regulation of apoptosis is essential to maintain homeostasis in the body. These collective proteins help ensure that cells participate in programmed cell death when necessary while allowing healthy cells to continue to thrive. An imbalance in the system inevitably results in disease, one of the most prevalent of which is cancer. For example, Bcl2 is considered to be a proto-oncogene; while it does not cause cancer on its own, a mutation in the Bcl2 gene may prevent the blocking of apoptosis and result in uncontrolled growth in the form of a tumor. The gene was the second gene discovered to be upregulated in B cell lymphoma patients, resulting in the corresponding name Bcl2. EXTRINSIC PATHWAY While the intrinsic and extrinsic pathways have different triggers, they both eventually result in the initiation of the caspase cascade. Extrinsic inducers come from outside the cell instead of from within it, and either cross the cell membrane themselves or bind to a receptor on the exterior of the cell that can continue the signal to the interior. There are five categories of death receptors that can be present on the cell surface and use extrinsic inducers to begin apoptosis. Two of the main extrinsic pathways include the TNF pathway and the FAS pathway. TNF PATHWAY Tumor Necrosis Factor (TNF) is the major extrinsic factor and can induce apoptosis by binding to its corresponding receptors in the cell. TNF is a small cell-signaling molecule referred to as a cytokine that is produced by activated macrophages. While macrophages have many functions, one of their main purposes is to digest cellular debris during apoptosis and break it down for recycling by the cell. This process is extremely important and unique to apoptosis. Without the phagocytotic clearance that the macrophages provide, the cell debris would build up and cause inflammation in the body. When the macrophages release TNF, the molecules bind to the TNFR1 receptor and trigger the pathway that leads to the caspase cascade as in the intrinsic pathway. Before reaching the caspases however, the intermediates TNF Receptor-Associated Death Domain (TRADD) and Fas-Associated Death Domain (FADD) must be activated by the receptor. FAS PATHWAY The FAS pathway in many ways is similar to the TNF pathway after activating their corresponding receptors. The FAS receptor (also called APO1, CD95, or TNFRSf6) is a member of the TNFR family. The FAS receptor binds the FAS ligand (FASL) which causes the receptor to trimerize and helps to bind FADD like in the TNF pathway. From here, both the TNF pathway and the FAS pathway use FADD proteins to form larger molecules that recruit pro-caspases that eventually form the Death-Inducing Signaling Complex (DISC) where the same initiator pro-caspases that are part of the intrinsic pathway can be activated. From there the signaling cascade can work to stimulate apoptosis. A diagram of the extrinsic pathway is included in Figure 2.

Figure 2. Extrinsic Pathway for Apoptosis CONCLUSION Apoptosis is a complex mechanism in which many proteins interact to initiate programmed cell death. Without these methods of organized destruction, the human body would not recycle the necessary materials to create new cells and old cells that malfunction would not be destroyed, leading to a buildup of debris and inflammation. Learning about apoptosis is important to understand biology and also to understand how an imbalance in the process can cause diseases such as cancer. Through the intrinsic and extrinsic pathways, various methods exist for triggering one of cell biologys most important processes, allowing organisms to adapt to changing conditions and develop fully.

WORKS CITED Kanjilal, Sagarika. "Apoptosis." VBSC 231. 107 Wartik, University Park. 27 Feb. 2014. Lecture.