Protein-Protein
Interactions
These
pages have been contributed by Cosmin
Saveanu (Institut Pasteur, France)
Cells
need interactions
Imagine a cell in which, suddenly, the specific interactions
between proteins would disappear. This unfortunate cell would
become deaf and blind, paralytic and finally would disintegrate,
because specific interactions are involved in almost any physiological
process. Sensing extracellular signals is a matter of receptor
to adaptor interactions and the shape of the cell is maintained
by an intricate network of structural protein interactions.
Finding interactions between proteins involved in common cellular
functions is a way to get a broader view of how they work
cooperatively in a cell. In the following paragraphs, for
reasons of brevity, I will call protein-protein interactions
any type of meaningful interaction that proteins are capable
of (protein-RNA, receptor-ligand, etc.)
Functional genomics "puzzle"
Even
if I feel tempted to place the techniques that identify protein-protein
interactions at the heart of the functional genomics puzzle,
a balanced view is more accurate. Bioinformatics helps scientists
to handle the vast amount of data generated by different genome-wide
approaches. Interesting conclusions about genome evolution and
the relationships between genes and function may be drawn by
comparative genomics. Predictions of function, based
on genome sequence analysis, will become more and more accurate
as the number and diversity of sequenced genomes steadily increases.
A recent review of computational methods for function hunting
is offered by Pellegrini
(2001). Systematic deletions (physical or by RNA
interference analysis, see for example Kamath
et al., (2000) and systematic localization studies
are first steps in functional characterization of genes and
may be of great help in improving the quality of protein interaction
maps. For example, a two-hybrid interaction between a nuclear
and a mitochondrial protein is not likely to be biologically
significant (even if it could be, but that's another story).
Clustering of genes by mRNA expression profiles (a comprehensive
study of expression profiles in yeast under a variety of conditions
was described by Hughes
et al., (2000)) seem to be
an effective way to predict protein function. Protein quantitation
and differences in protein expression under different experimental
conditions may be analysed by 2D-electrophoresis but the ICAT
technique that makes extensive use of mass spectrometry might
be more effective in proteome-wide projects (Gygi
et al., 1999). I should also mention here, the equivalent
of DNA chips in the protein world and the projects of constructing
libraries of specific antibodies raised against every single
protein of a cell (for
a review see Holt et al., 2000). Biochemical
genomics, where the entire ORFeome (see Reboul
et al., 2001 for the term ORFeome) of an organism is cloned,
expressed and individually purified, allow the discovery of
proteins when a biochemical assay for their function has already
been developed (for a review see Grayhack
& Phizicky, 2001). By a different approach, structural
genomics is trying to decipher biochemical protein function
by structural similarity to known proteins, however this method
is limited to predicting the cellular function of a given protein.
Not mentioned in the previous picture but important as always
are the tools of genetics, and genetic screening is a
continuous source of marvelous discoveries.
What
are we going to study today?
In the pre-genomic era it was quite difficult to ask questions
like: "I have this gene, hmmm...it looks nice, has a zinc-finger
motif, let's see what the function is...". However this
type of "fishing expedition" (quote from Marc
Vidal's review in Cell, 2001) may be quite succesful even
if, as one of my colleagues told me: "this is not like
science...you are not asking a functional question...".
In fact, the hunting for function may reveal interesting, and
sometimes unexpected results. Additionally, by the development
of automated, large scale projects an important amount of functional
data may accumulate to be used by the scientific community.
Alternative ways of studying protein function
One,
two, three...infinity
I have the sequence of a predicted protein and I would like
to know if it collaborates with other proteins in a functional
pathway (and I hope that additionally my protein is THE essential
one in this pathway). One possibility is to test whether my
protein:
1. Has a reasonable level of expression.
2. Is associated to a complex in which at least one of the
components has a known function.
For
these purposes, I will isolate the protein fused with an affinity
tag, either by fine-tuning the conditions of isolation such
that the protein is highly enriched in a single affinity purification
step or by using a "generic" method such as that
described by Rigaut
et al., 1999 under the name of TAP (Tandem Affinity
Purification). The figure below shows different kinds
of results when yeast proteins of unknown function were tested
by TAP isolation. Two successive affinity purifications are
done using two tags fused to the protein of interest; the
first step involves binding of the protein A tag to an IgG
column. Under mild conditions the putative complex is released
from the column by a specific viral protease and rebound to
a second column, where calcium dependent interaction of calmodulin
with a calmodulin binding peptide (CBP) will allow the enrichment
of the complex. Recently, a similar technique was used in
a multiple affinity purification (MAFT) using three
different tags in the succesion: calmodulin binding peptide,
histidines, hemagglutinin (Honey
et al., 2001).
Number
of proteins isolated by using the TAP strategy in yeast with
different unknown proteins as "baits"
It
is a good idea to test whether the depletion of the studied
protein gives rise to a particular phenotype. If the tagged
protein used for complex purification is functional, this
phenotype should not be observed. That's why it is an advantage
to study one particular protein by the affinity purification
of the associated complex in a yeast strain where the functional
protein is essential. This way, we can be sure that the tag
did not interfere with the protein function and that the purified
complex might be the 'physiological one'.
Two
hybrids are better than one
A long time ago, in a galaxy far, far away... the two-hybrid
system was described in yeast (Fields
and Song, 1989). The original two-hybrid underwent many
technical modifications but the original idea of reconstructing
a functional factor (transcriptional, enzymatic, signal transduction)
from two pieces that come close together, being in "hybrids"
with proteins that interact specifically, is always present
(see figure below).
If
an interaction is established between the two hybrid proteins
the transcription of a reporter gene is activated and one
can select the cells by testing the activity of the reporter
protein
Reconstituting
a functional transcription factor requires that the hybrids
are transported into the nucleus and it was only ten years
after the original description of the two-hybrid system that
a nuclear localization signal was uncovered in the sequence
of the bacterial LexA, the bait DNA binding domain (Rhee
et al., 2000). Moreover, a one hybrid system with a mutated
LexA sequence was developed. In this system a protein of interest
is fused with both LexA and Gal4 activator domain reconstituting
a stable transcriptional activator. However this activator
is not functional if it is not transported through the nuclear
pore complex at the site of transcription. If the protein
of interest has a nuclear localization signal (NLS) it will
be transported and the activation of transcription will take
place.
(page
last updated: August 15th, 2001)
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