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Unlike most HATs, which regulate the expression of only a few genes,
p300/CBP is involved in the activation of a wide variety of genes. In
addition, aberrant p300/CBP activity contributes to pancreatic, colon,
and lung cancer � among the deadliest cancers in humans � as well as
gastric and thyroid cancer and some leukemias. In addition to acting
as an oncoprotein by promoting tumors, p300/CBP also can suppress
tumors.
These unusual properties have made p300/CBP one of the most studied
enzymes in the HAT family, and a target for developing new anti-cancer
drugs, says Ronen Marmorstein, Ph.D., a professor in the Gene
Expression and Regulation Program at Wistar and a senior author and
corresponding author on the study. Philip A. Cole at Johns Hopkins is
also a senior author and corresponding author.
�It�s unusual to have a HAT that�s so implicated in cancer, and even
more unusual to have one that has both tumor suppressor and
oncoprotein activities,� Marmorstein says.
In a report published in the Feb. 14 issue of the journal Nature,
Marmorstein, Cole and their colleagues detail their elucidation of the
three-dimensional structure of a p300/CBP HAT domain, or segment,
bound to a small molecule that inhibits its activity. The study also
reveals how the binding site and chemical mechanism of the enzyme
enable it to regulate a wide variety of genes.
p300/CBP has long been recognized for its ability to involve other
transcription factors in regulating gene expression. About 10 years
ago, when scientists discovered that p300/CBP also had histone
acetyltransferase activity, Marmorstein and his group began work to
determine its three-dimensional structure.
But solving that structure was no easy feat. The researchers attempted
to create crystals of p300/CBP to analyze using x-ray crystallography,
a widely used analytical technique in which x-rays are beamed at
crystals containing the protein of interest. The three-dimensional
structure of the crystallized protein is deduced by analyzing the
pattern of x-ray diffraction caused by the arrangement of atoms in the
protein crystal.
Marmorstein and his colleagues spent years attempting to crystallize
p300/CBP � a process made excruciatingly difficult by the protein�s
tendency to lose its functional form upon isolation for
crystallization.
In 2004 a breakthrough was made when Paul Thompson, a postdoctoral
fellow in the Cole laboratory at the time and coauthor of the current
study, discovered why the protein performed so poorly: p300/CBP not
only works to acetylate histones but also acetylates itself.
Thompson found a way to prevent this self-acetylation using a
�chemical trick� to produce the protein in a form that contained no
acetyl groups. By employing a few additional tricks to counteract the
enzyme�s flexible and �floppy� nature, the scientists were then able
to crystallize it.
Studies of the structure show that p300/CBP contains a binding pocket
that is suitable for associating with a wide range of substrates � the
molecules it binds with � and makes p300/CBP more �promiscuous� than
other HATs, Marmorstein says.
In addition, p300/CBP uses a novel �hit-and-run� chemical mechanism to
convert its substrates to the resulting protein products. The chemical
mechanism differs from those employed by other HATs in that the
histone substrate binds only transiently, leaving after a very brief
encounter.
This hit-and-run mechanism is consistent with the enzyme�s ability to
acetylate a variety of substrates because they don�t have to bind in a
very stable fashion, Marmorstein says.
The chemical mechanism employed by p300/CBP also bodes well for
designing cancer drugs capable of pinpointing p300/CBP without
affecting other enzymes � and causing unwanted side effects. �Because
of p300/CBP�s chemical mechanism, which differs from that of other
HATs, an inhibitor that works against this family of enzymes likely
won�t work against the other ones,� Marmorstein explains.
The scientists are now working to further elucidate the functions of
p300/CBP and to solve larger structures of the protein. They plan to
use the information they have already gained to develop inhibitors of
p300/CBP activity � research that will pave the way for the
development of new anti-cancer therapies, Marmorstein says.
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