CYNTHIA
WOLBERGER'S research is
focused on the molecular mechanisms by which genes are silenced
in eukaryotic
cells. She also studies the assembly
of polyubiquitin chains that play a nondegradative role in the
cell.
A
genome encodes the information needed to produce all of the proteins
and nucleic acids that carry out a cell's essential functions. The
cell utilizes the information by first transcribing an RNA copy of
each gene. The information in the RNA is then translated by the ribosome
to produce a protein; for some genes, the RNA itself is the end product.
Since only a fraction of the encoded protein and RNA molecules is needed
by the cell at any given time, all organisms have elaborate mechanisms
for controlling whether a gene is transcribed. Our research is aimed
at understanding at a molecular level how eukaryotic cells control
gene expression. We use x-ray crystallography to determine the atomic
structures of the protein and nucleic acid complexes that participate
in gene regulation. Together with biochemical and genetic studies,
these structures provide mechanistic insights into the fundamental
processes that govern transcription regulation.
Our research is also focused on the assembly of Lys63-linked polyubiquitin.
These chains of ubiquitin proteins are attached to a variety of cellular
targets, where they play important roles in transcriptional activation
in the inflammatory response, as well as in the repair of DNA damage.
Here, too, the tools of structural biology are revealing how the cell
assembles these chains with the correct linkages and attaches them to
other protein complexes in the cell.
Transcriptional Silencing
The DNA in eukaryotic
cells is packaged by histone proteins, giving rise to a nucleoprotein
complex called chromatin. Some regions of chromatin are relatively
accessible to the transcriptional machinery and contain actively transcribed
genes; other regions are silenced and are organized in a way that prevents
transcription. Transcriptional silencing is brought about by protein
complexes that chemically modify histone proteins, either by introducing
post-translational modifications or by removing them. We study the
enzymes and protein complexes that establish and maintain transcriptional
silencing. One example is Sir2, an enzyme that deacetylates lysine
side chains in an unusual reaction that requires NAD+. Enzymes similar
to Sir2 are found in virtually all organisms, where they are involved
in a variety of critical biological processes, including transcriptional
silencing, DNA repair, chromosome stability, and enzyme regulation.
Sir2 proteins play an intriguing role in life span, with high levels
of Sir2 activity extending life span and low levels of Sir2 activity
leading to shortening of life span in yeast, worms, and flies. Members
of the Sir2 enzyme family also regulate important pathways in humans
that are involved in protection of neurons from degeneration, fat mobilization,
and cell death. Sir2 activity is linked to cellular concentrations of
NAD+, a substrate of Sir2, as well as to levels of nicotinamide, a reaction
product that inhibits Sir2 activity. Some Sir2 enzymes catalyze a somewhat
different reaction in which NAD+ is cleaved to produce ADP ribose, which
is then attached to a substrate in a reaction known as mono-ADP ribosylation.
Understanding the chemistry and regulation of Sir2 proteins, as well
as the basis for the dual enzymatic activity, is central to unraveling
how these enzymes help to regulate different cellular pathways.
Our structural studies of Sir2 enzymes bound to a variety of substrates,
products, and inhibitors have revealed how these proteins carry out their
unique chemical reaction in the cell. The structure of Sir2Af2 bound
to an acetylated peptide corresponding to the carboxyl terminus of p53
showed that peptides bind in a cleft between the two domains of the enzyme,
closing the intervening gap and inserting the acetyl-lysine side chain
deep within the enzyme active site. Structural and biochemical studies
of a bacterial sirtuin, Sir2Tm, bound to a variety of peptide substrates
have shown that interactions between the enzyme and the substrate residues
flanking the acetyl-lysine influence the peptide substrate specificity
of the enzyme. We have also determined structures of Sir2 enzymes bound
simultaneously to both peptide and NAD+ before catalysis, as well as
after the enzymatic reaction has occurred in the crystal.
These structures have shed light on the unique mechanism by which Sir2
enzymes catalyze the cleavage of NAD+ and the transfer of an acetyl group
from the peptide lysine to ADP ribose. They have shown us that acetyl-lysine
induces binding of NAD+ in a strained conformation that favors cleavage
of the nicotinamide ring from NAD+. This cleavage sets up a cascade of
events leading to the eventual deacetylation of lysine and the release
of the reaction products. To determine structures of a series of trapped
reaction intermediates, we will address how subsequent steps in the reaction
are catalyzed by the enzyme. These studies may also explain why some
members of the Sir2 family catalyze the transfer of the ADP-ribose portion
of NAD+ to a protein substrate, a reaction that is known as ADP ribosylation.
The activity of Sir2 enzymes can be controlled by small molecules that
bind within the enzyme active site. We have determined how nicotinamide,
a product of the NAD+-dependent deacetylation reaction, binds in the
enzyme active site and interferes with the deacetylation reaction by
promoting a back reaction known as base exchange. We have shown that
Sir2 enzymes contain a single site that is involved in both NAD+ binding
and nicotinamide inhibition. We are studying how other small molecules
either repress or stimulate the enzymatic activity of Sir2 enzymes.
In some organisms, the transcription of genes that lie near the ends
of chromosomes—known as telomeres—is subject to silencing
mediated by Sir2 enzymes. This type of silencing is found in budding
yeast, as well as in a pathogenic yeast, Candida glabrata, and in the
malaria parasite. This transcriptional repression, known as telomeric
silencing, requires additional proteins that help recruit Sir2 enzymes.
Structural studies of the telomere-binding protein Rap1 and of the silencing
proteins recruited by Rap1 are shedding light on how telomeric silencing
is established and maintained.
The nucleus contains a set of enzymes that synthesize NAD+ from a variety
of molecular precursors. The relative activity of these enzymes controls
Sir2 activity by affecting the nuclear concentrations of NAD+ and nicotinamide.
We have determined the structure of a mammalian NAD+ biosynthetic enzyme,
nicotinamide phosphoribosyltransferase (Nampt), bound to reaction products.
Nampt catalyzes the synthesis of nicotinamide mononucleotide from nicotinamide
and phosphoribosylpyrophosphate in a reaction that is enhanced when the
enzyme becomes autophosphorylated. Ongoing structural studies, as well
as biochemical studies of mutant and wild-type enzymes, are being carried
out to elucidate the reaction mechanism and determine the role that autophosphorylation
plays in the catalytic mechanism.
Assembly of Lys63-Linked Polyubiquitin Chains
Ubiquitin is a small protein that serves as the building block for chains
of polyubiquitin, which are attached to other proteins and thereby mediate
a variety of biological processes. Polyubiquitin is formed when the C
terminus of one ubiquitin is joined to one of the seven surface lysines
of ubiquitin. Polyubiquitin chains with different types of lysine linkages
play distinct biological roles. For example, Lys48-linked polyubiquitin
chains target proteins for destruction by the proteasome, whereas Lys63-linked
polyubiquitin chains are nondegradative signals that play a role in DNA
damage tolerance and NF?B activation.
We study the molecular basis for Lys63-linked polyubiquitin chain assembly.
Lys63-linked chains are assembled by a specialized heterodimer that helps
to catalyze formation of a covalent bond between Lys63 of one ubiquitin
and the C terminus of the next. We determined the structure of the yeast
enzyme Mms2/Ubc13, as well as a complex containing ubiquitin covalently
joined to the active-site residue of Ubc13. In the structure, the unexpected
binding of a donor ubiquitin of one Ub~Ubc13/Mms2 complex to the acceptor
binding site of Mms2/Ubc13 in an adjacent complex allowed us to visualize
the molecular determinants of acceptor ubiquitin binding and explain
how Mms2 helps to orient ubiquitin to give rise to a Lys63-linked polyubiquitin
chain. We continue to study polyubiquitin chain assembly by Mms2/Ubc13,
as well as how it cooperates with the ubiquitin ligase, Rad5, to modify
substrates with Lys63-linked polyubiquitin chains.
A portion of this work has been supported by the National Science Foundation
and the National Institutes of Health.
Last
updated: May 2, 2007. |