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