Transcript the ubiquitin system and a putative stimulatory role

The ubiquitin system and a putative
stimulatory role for activator
turnover in gene expression
Chao Wang
Nov 16, 2005
The Ubiquitin System
Ubiquitin (Ub) is a small protein that is composed of 76 amino acids.
Found only in eukaryotic organisms and is not found in either eubacteria or
archaebacteria.
Among eukaryotes, ubiquitin is highly conserved, meaning that the amino acid
sequence does not differ much when very different organisms are compared.
Ub is a heat-stable protein that folds up into a compact globular structure.
It is found throughout the cell and can exist either in free form or as part of a
complex with other proteins. In the latter case, Ub is attached (conjugated) to
proteins through a covalent bond between the glycine at the C-terminal end of
Ub and the side chains of lysine on the proteins.
Ub is involved in many cell processes. Regulating the cell cycle, DNA repair,
embryogenesis, the regulation of transcription, and apoptosis.
Ub is encoded by a family of genes whose translation products
are fusion proteins. The Ub genes typically exist in two states :
A. The ubiquitin gene (red) can
be fused to a ribosomal
protein gene (blue) giving
rise to a translation product
that is a Ub-ribosomal fusion
protein;
B. Ub genes can exist as a linear
repeat, meaning the
translation product is
comprised of a linear chain
of Ub-molecules fused
together (a polyubiquitin
molecule).
Ubiquitin Function
Proteins exist as a linear chain of amino acids. This chain can
degrade over time as such a reaction is thermodynamically favorable
in an aqueous environment. When proteins degrade over time, this is
called protein-turnover. It is the balance between a protein's
degradation and its synthesis that determines the concentration of
that protein inside the cell.
Studies of protein turnover rates have shown that some proteins are
short-lived while others are long-lived. Long-lived proteins
constitute the majority of proteins in the cell. Short-lived proteins are
typically key regulatory proteins and abnormal proteins.
Ub functions to regulate protein turnover in a cell by closely
regulating the degradation of specific proteins. Such a regulatory
role is very important.
Ub functions in an ATP-dependent fashion. But why is this? We
don't need energy to hydrolyze proteins. The reason ATP is
required is because machinery is needed to specifically target the
proteins that need to be degraded. Ub itself does not degrade
proteins. It serves only as a tag that marks proteins for
degradation. The degradation itself is carried out by the 26S
proteasome. In short, proteins that are to be degraded are first
tagged by conjugating them with Ub and these tagged proteins are
then recognized and shuttled to the proteasome for degradation.
The Ubiquitin-Proteasome Pathway
Something else is needed to attach Ub to a protein.
Three types of enzymes are needed in most cases.
1. E1 enzymes known as Ub-activating enzymes. These enzymes
modify Ub so that it is in a reactive state (making it likely that
the C-terminal glycine on Ub will react with the lysine sidechains on the substrate protein).
2. E2 enzymes known as Ub-conjugating enzymes. These
enzymes actually catalyze the attachment of Ub to the
substrate protein.
3. E3 enzymes known as Ub-ligases. E3's usually function in
concert with E2 enzymes, but they are thought to play a role
in recognizing the subtrate protein.
In yeast, there are many types of E1, E2, and E3 enzymes. For
example, 13 different E2 enzymes have been found. While they all
carry out the conjugation reaction, they are apparently tailored for
specific functions.
The general reaction pathway is shown in the figure below. First,
Ub is activated by E1 in an ATP-dependent fashion. E2 and E3 then
work together to recognize the substrate protein and conjugate Ub
to it. Ub can be attached as a monomer or as a previously
synthesized chain (as shown). From this point, the ubiquinated
protein is shuttled to the proteasome for degradation.
What determines if a protein gets tagged by Ub and thus marked
for degradation? Let's consider some of signals thus far discovered.
1.
The N-degron. In 1986, Varshavsky carried out an elegant set of experiments
(Science 234, 179-186) that showed a correlation between the half-life of a
protein and its N-terminal residue. This observation gave rise to the so-called
N-end rule where one could generally predict the lifespan of a protein by its
N-terminal amino acid.
2.
Certain amino acid sequences appear to be signals for degradation. One such
sequence is known as the PEST sequence because short stretch of about eight
amino acids is enriched with proline, glutamic acid, serine, and threonine.
3.
Signals may also be buried in the hydrophobic core. This is why partially
folded or abnormal, mutant proteins may be prone to degradation. When such
proteins exist in their native state, the signals are hidden and the protein is thus
long-lived. But in a partially unfolded state, the signals may be seen by the Ub
machinery caused the protein to become tagged by Ub. This reaction appears
to be hindered by chaperone activity.
Ubiquitination lead to protein degradation
It is not entirely accurate to think of Ub as a simple tag, as Ub does
appear to be involved in degradation.
The proteasome is the structure that actually does the degrading.
Ubiquiton's degradation role may simply be to decrease the rate of
dissociation between proteasomes and interacting substrate proteins.
That is, without Ub, proteins may interact with the proteasome, but
quickly dissociate. Ub slows down this dissociation. A substrate
protein that is conjugated with Ub-chains is thought to interact with
a proteasome for a longer period of time, thus increasing the
likelihood that the proteasome will degrade it. In fact, Ub could
actually function to tether the substrate protein to the proteasome.
Lipford JR, Smith GT, Chi Y, Deshaies RJ. A putative
stimulatory role for activator turnover in gene expression.
Nature. 2005 Nov 3;438(7064):113-6.
California Institute of Technology
The ubiquitin–proteasome system (UPS) promotes the destruction of target
proteins by attaching to them a ubiquitin chain that is recognized by the 26S
proteasome1.
The UPS influences most cellular processes, and its targets include transcriptional
activators that are primary determinants of gene expression. Emerging evidence
indicates that non-proteolytic functions of the UPS might stimulate
transcriptional activity.
The proteolysis of some transcriptional activators by the UPS can stimulate their
function. Authors focused on the role of UPS-dependent proteolysis in the
function of inducible transcriptional activators in yeast, and found that inhibition
of the proteasome reduced transcription of the targets of the activators Gcn4,
Gal4 and Ino2/4. In addition, mutations in SCF-Cdc4, the ubiquitin ligase for
Gcn4, or mutations in ubiquitin that prevent degradation, also impaired the
transcription of Gcn4 targets.
These transcriptional defects were manifested despite the
enhanced abundance of Gcn4 on cognate promoters. Proteasome
inhibition also decreased the association of RNA polymerase II
with Gcn4, Gal4 and Ino2/4 targets, as did mutations in SCF-Cdc4
for Gcn4 targets. Expression of a stable phospho-site mutant of
Gcn4 or disruption of the kinases that target Gcn4 for
turnover alleviated the sensitivity of Gcn4 activity to defects in
the UPS.
This mechanism places Gcn4, Gal4 and Ino2/4 into a class of regulatory
Factors whose activity is required early in a process but whose
subsequent turnover or removal promotes completion of the process or
subsequent reaction cycles.
We call this phenomenon ‘activation by destruction’ and believe that it
might represent a regulatory mechanism for a large class of factors
and might be an important determinant of infection and disease.
The End