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From CellBiology

Apoptotic Protease Activating Factor 1 (APAF-1)

Apoptotic Protease Activating Factor 1 (Apaf-1) is a protein found in every living cell of the human body. With the aid of mitochondrial cytochrome c, Apaf-1 has the ability to cleave procaspase-9 and activate the apoptotic pathway. Extensive research continues to be done on this protein, especially in regards to cancer therapy. Specifically, researchers are looking for ways to increase activation of this protein in cancer cells so as to induce apoptosis and death in them.

Key Words

Apoptosis, Apoptosome, Caspase, Cytochrome c, Oncogenesis, S Phase.

General Information

The apoptosome and its components.

Function

As discussed in the group project, Apaf-1 is an intrinsic part of the complex activating procaspase-9 to give caspase-9. The steps in this process are as follows:

  1. Apoptotic signals and effector proteins act on the mitochondria to increase its membrane permeability[1].
  2. The disruption of the membrane causes release of mitochondrial cytochrome c through permeability transition pores[2].
  3. Cytochrome c binds to a binding site on Apaf-1 in the cytoplasm.
  4. Apaf-1, using ATP, can then cleave procaspase-9 and release caspase-9.

Active caspase-9 leads to caspase cascade activation and, ultimately, cell death by apoptosis. As caspase-9 activity occurs, Apaf-1 still remains important as it regulates caspase-9’s function within the cell[3]. Note that Apaf-1 requires dATP to replace bound dADP to become active.

Crystal Structure of Apaf-1 with binding domains.



Structure

Apaf-1 can be structurally split into 5 parts: CARD (N-terminal Caspase Recruitment Domain), Helical Domain I, Helical Domain II, α/β Domain and Winged-Helix Domain[4].

If we split up Apaf-1 functionally, we have a CARD (which binds caspase-9), nucleotide-binding oligomerisation domain (NOD; located at the centre of Apaf-1 and binds ADP/ATP) and numerous WD40 repeats (required for cytochrome C binding).

Location in the Cell

Apaf-1 is found in the cytoplasm of cells, often bound to procaspase-9. Once the mitochondria release cytochrome C from their inner membrane, Apaf-1 can bind it and, with the aid of ATP, can become active.

Uses and Recent Research

Histological pictures showing Apaf-1 presence in healthy and cancerous tissue.

Apaf-1 in Metastatic Cancer and use in its prognosis

Since Apaf-1 is so essential to the execution of apoptosis in a cell, it is of no surprise that metastatic cancer cells do not eagerly express it.

Metastatic melanomas have been shown to lose Apaf-1 function which give them properties of longevity and non-responsiveness to apoptotic signals[5]. This poses problems when administering chemotherapies to a cancer patient because apoptotic-activating drugs will not have much efficacy if Apaf-1 is poorly expressed. Gene transfer to upregulate Apaf-1 expression has been shown to be an effective way to counteract this, greatly increasing patient sensitivity to chemotherapies. Hence, we can deduce that Apaf-1 expression in metastatic cancer is an important feature when seeking to obtain a good prognosis.

Increased Apaf-1 expression in neural cancer cells.

Breakthrough Research in treatment of neural cancers

Although many cancers are resistant to chemotherapies inducing Apaf-1 activity, neural cancers have been found to be hypersensitive to cytoplasmic cytochrome C levels[6].

For an unknown reason, neural cancer cells express high Apaf-1 levels in their cytoplasms. This increased expression leads to increased sensitivity of the neural oncogenic cells to high cytochrome C levels within their cytoplasms since the cytochrome C has an increased chance of binding to Apaf-1 to induce apoptosis. The chemotherapy using this principle and delivering high levels of cytochrome C to neural cancer cells has been shown to be efficient in eradicating them. Encouraging the development of this type of chemotherapy further is the fact that neurons and brain parenchyma seem to be particularly resistant to high cytochrome C levels. Further research into this treatment may lead to the discovery of a cytochrome C level that both optimises neural cancer cell death and normal neural tissue survival.

Apaf-1 helps to arrest the cell cycle in the S phase (coloured in blue).

Alternate Role of Apaf-1: Cell cycle regulation

In stark contrast to its role in promoting cell death, Apaf-1 seems to have a very important role in the regulation of the cell cycle[7].

The S phase is an important part of the cell cycle and is where DNA replication occurs. Abnormal replication of DNA can lead to oncogenesis and, hence, regulators are particularly needed in this phase of the cell cycle. Checkpoint kinase 1 (Chk1) in its phosphorylated state is able to arrest cell cycle progression at the S phase in response to the presence of abnormal DNA within the cell.

Research has revealed that Apaf-1 movement to the nucleus is associated with increased phosphorylation of Chk1 and that Apaf-1 depletion leads to reduced activity of Chk1. As a result of such findings, we can infer that Apaf-1 has another important role within the cell; maintaining cellular integrity and suppressing tumour formation as a result.

Summary

  • Apaf-1 is a cytoplasmic protein.
  • It functions in apoptosis and cell cycle regulation.
  • Its expression is extremely important when considering cancer patient prognosis.
  • Apaf-1 expression is important for chemotherapies designed to cause apoptosis in oncogenic cells.

The moral of the story is: Don't mess with Apaf-1 because it can either save or kill your cells!

References

  1. Dash, Philip. Picture of mitochondria in apoptosis. Retrieved May 23, 2009, from Reproductive and Cardiovascular Disease Research Group Web site: http://www.sgul.ac.uk/depts/immunology/~dash/apoptosis/mito.htm
  2. Hill, Mark (2009). 2009 Lecture 18 - Cellbiology. Retrieved May 23, 2009, from Cell Biology Wiki Web site: http://cellbiology.med.unsw.edu.au/cellbiology/index.php?title=2009_Lecture_18#Mitochondrial_Pathway
  3. Online Mendelian Inheritance in Man, OMIM (TM). Johns Hopkins University, Baltimore, MD. MIM Number: 602233 APOPTOTIC PROTEASE ACTIVATING FACTOR 1; APAF1
  4. Structure of the apoptotic protease-activating factor 1 bound to ADP. Riedl SJ, Li W, Chao Y, Schwarzenbacher R, Shi Y. Nature. 2005 Apr 14;434(7035):926-33. PMID:15829969
  5. Inactivation of the apoptosis effector Apaf-1 in malignant melanoma. Soengas MS, Capodieci P, Polsky D, Mora J, Esteller M, Opitz-Araya X, McCombie R, Herman JG, Gerald WL, Lazebnik YA, Cordón-Cardó C, Lowe SW. Nature. 2001 Jan 11;409(6817):207-11. PMID:11196646
  6. Differential Apaf-1 levels allow cytochrome c to induce apoptosis in brain tumors but not in normal neural tissues. Johnson CE, Huang YY, Parrish AB, Smith MI, Vaughn AE, Zhang Q, Wright KM, Van Dyke T, Wechsler-Reya RJ, Kornbluth S, Deshmukh M. Proc Natl Acad Sci U S A. 2007 Dec 26;104(52):20820-5. Epub 2007 Dec 18. PMID:18093951
  7. Nonapoptotic role for Apaf-1 in the DNA damage checkpoint. Zermati Y, Mouhamad S, Stergiou L, Besse B, Galluzzi L, Boehrer S, Pauleau AL, Rosselli F, D'Amelio M, Amendola R, Castedo M, Hengartner M, Soria JC, Cecconi F, Kroemer G. Mol Cell. 2007 Nov 30;28(4):624-37. PMID:18042457