Soluble, Functional Apoptotic Protease-Activating Factor-1 Fragments

Web Published:
12/1/2011
Description:

Princeton University Invention # 05-2166

 

           

Apoptosis or programmed cell death is an essential process in the development and homeostasis of humans. Abnormal inhibition of apoptosis is a hallmark of cancer and autoimmune diseases, whereas excessive activation of cell death is implicated in neuro-degenerative disorders such as Alzheimer¿s disease. Apoptosis is executed by the enzymatic activity of a family of special proteases termed ¿caspases¿. Caspases are synthesized as inactive zymogens and must be proteolytically processed to become fully active.

 

The caspase activation cascade downstream of mitochondria is controlled by the apoptotic protease activating factor 1 (Apaf-1), which is responsible for the activation of the initiator caspase-9 and subsequent activation of effector caspases -3 and -7. Apaf-1 plays an essential role in the regulation of programmed cell death in mammalian development and in oncogene and p53-dependent apoptosis. The critical importance of Apaf-1 mediated apoptosis is manifested by the observation that Apaf-1 is frequently inactivated in cancers such as malignant melanoma.

 

Efforts to study Apaf-1 have been hampered by the inability to generate significant quantities of the protein in a form that is sufficiently stable, soluble, and pure to allow such study. For instance, there is no published protocol that allows bacterial expression and purification of a soluble recombinant Apaf-1 fragment longer than 200 amino acids. In addition there is no published protocol that allows the preparation of soluble, stable recombinant Apaf-1 fragments longer than 200 amino acids, except for the full-length Apaf-1 protein in baculovirus-infected insect cells.

 

As a consequence of the lack of availability of reasonable quantities of Apaf-1 protein for research studies, little work has been done to identify compounds which can modulate the activity of Apaf-1. Furthermore, there has been an absence of three-dimensional structure information for any fragment of Apaf-1 other than the soluble N-terminal CARD domain. Knowledge of the physical structure of Apaf-1 protein would significantly aid design and screening of compounds that can modulate the activity of Apaf-1. This invention overcomes these prior limitations by providing a method of producing stable, soluble, pure and active Apaf-1 protein and the determination of the crystal structure of Apaf-1 bound to ADP.

 

Researchers at Princeton have discovered ways to express recombinant Apaf-1 protein in a sufficiently stable, soluble, functional, and pure form. The Princeton team determined the crystal structure of Apaf-1 bound to ADP, which provides the first glimpse of an immensely important protein at atomic resolution (2.2À).  This structure reveals, quite unexpectedly, that the nucleotide-binding pocket of Apaf-1 is much larger that the bound ADP and is lined by featured amino acids that are poised to make specific interactions. These structural observations strongly suggest that bulkier nucleotide analogs can be synthesized to better occupy the nucleotide-binding pocket of Apaf-1.  Biochemical evidence elucidated at Princeton shows that binding to this pocket directly impacts on Apaf-1¿s ability to activate caspase-9. Hence these novel structural features can be used to design nucleotide analogs that either enhance or inhibit Apaf-1¿s ability to activate caspase-9.

 

These novel structural features together with the availability of soluble, functional fragments of human Apaf-1 protein having ADP bound should have profound implications for the screening and rational design of drugs for the therapeutic treatment of cancer and other disease conditions associated with inappropriate regulation of apoptosis.

 

References:

 

Riedl,S. Li,W., Chao,Y., Schwarzenbacher,R., Shi,Y., 14 April 2005, Structure of the Apoptotic protease-activating factor 1 bound to ADP, Nature, Vol. 434, 926-933.

 

Princeton is currently seeking industrial collaboration to commercialize this technology. Patent protection is pending.

 

 

For more information on Princeton University invention # 05-2166 please contact:

 

                        Laurie Tzodikov

                        Office of Technology Licensing and Intellectual Property

                        Princeton University

                        4 New South Building

                        Princeton, NJ 08544-0036

                        (609) 258-7256

                        (609) 258-1159 fax

                        tzodikov@princeton.edu

 

Patent Information:
For Information, Contact:
Laurie Tzodikov
Licensing Associates
Princeton University
tzodikov@Princeton.EDU
Inventors:
Yigong Shi
Keywords: