14. Predicting Protein Interactions

14. Predicting Protein Interactions

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visit MIT OpenCourseWare at ocw.mit.edu. PROFESSOR: OK. So we’ve been talking about
predicting structure proteins. At the end of the
last lecture we started to talk a little bit
about predicting interactions, and that’s going to be the
focus of today’s lecture. And we identified a couple of
different possible prediction challenges. One was quantitative
predictions of what happens when you make specific
mutations in a known protein complex. We talked about
trying to predict the structure of, say,
just a pair of proteins, and then trying to do that
on the global scale for all known proteins. And so last time,
if you recall, we thought that initially maybe
this would be a simple problem. We have proteins of known
structure with a complex. Structure of the
complex is also known. And we want to make predictions
as to the change in affinity when there’s a
specific mutation made. In principle, this
should be easy because we have all those
different formulations for the potential
energy function. And so if we figure out what
the local structural changes are that are due to the insertion
or deletion of some side chain, then we should be
able to predict the change in the
potential energy, and therefore the change in
the energy of the complex. But in fact, it
turned out that it was very, very hard to do that. And so this plot compared–
the black circles were the prediction
algorithms for this problem, compared to just simply
a substitution matrix, the BLOSUM substitution matrix
defined in terms of the area under the curve for
beneficial mutations and deleterious mutations. And you can see that very,
very few of the black dots get far away from what is the
really simple default model. A lot of them do worse. So OK, well maybe that’s
not such a simple problem because it requires a highly
quantitative prediction. Maybe we’ll do
better just trying to predict which
proteins interact at all. And so that’s going to be
the focus of today’s lecture. Now, that also had
a problem, right? Because even if I know the
structure of two proteins, I don’t know necessarily
what surfaces of those proteins interact. And so I have to
figure out this docking problem of which part
of protein A interacts with which part of protein B. That’s the beginning
of my problem, and then I have to make a
series of subsequent decisions. So I’m going to
have to figure out for any potential
partner of my protein, I need to figure out
the docking problem, the relative
position orientation. Now, in this little
cartoon, it’s shown as a completely
static protein that approaches another
static protein. The only thing that’s changing
is the relative coordinates. But of course, there
will be local changes in confirmation, perhaps
even global ones. And so we need to be able
to make some estimates as to what those structural
rearrangements will be when the two
proteins interact. And then after we’ve
come up with our best estimate of the
structural rearrangements, only then can we come up with
an estimate of the energy interaction and
decide whether it’s better than some threshold. OK. So one of the problems that’s
pretty obvious from this is that this kind of
approach in principle, if we do it rigorously
through all the steps, would be extremely slow. Now, another part that’s perhaps
a little bit less obvious is that it’s going to be very
prone to false positives. And why do you
think that might be? What am I not taking
into account here? AUDIENCE: Are you
not taking into account the desolvation
answer is I’m not taking account of
the desolvation, but in fact, I can do that. Right? So some of the potential
energy functions we looked at, the
statistician’s version rather than the physicist’s
makes it pretty easy to incorporate the desolvation. Any other thoughts as to what
I’m not taking into account? What other protein
should I be considering when I’m considering
an interaction problem? So I’ve isolated, in
this case, two proteins. I’m saying, in a universe
where these are the only two proteins that exist, will
they have a favorable energy interaction? What I really need to know is
whether that energy interaction is more favorable than all
the competing interactions that they could have. So even if I find something
that’s potentially a good interaction, it may
not be the best possible interaction. And if I consider then the
concentration of this protein and the concentration of
all the other molecules out there that have
a higher affinity, then it could turn out
that this is actually a rather poor substrate
for my protein, a rather poor interaction partner. So we have that false
positive problem. OK. But let’s focus on the
computational efficiency problem, because
that’s at least one that we can come up with
some nice algorithms to try to solve. So what we want to do is try
to limit our search space. If I want to figure out–
I have a query protein and I want to ask, what
does it interact with, instead of trying to do
the pairwise comparison of this protein with every
other protein in the database, and doing very precise
structural calculations on all of those, maybe
there’s some way that I can prefilter the
set of proteins that it might interact with. And that’s what we’re
going to look at. So we’re going to try
to officially choose potential partners
before we’re doing any structural comparison. And then once we
have those partners, we’re going to try
to avoid having to do detailed calculations
until we have a relatively high degree of confidence that
these proteins could interact by other criteria. And we’re going to look at two
papers that describe algorithms for solving this
problem, and they’re both uploaded to the website. The first thing
that we’ll look at is called PRISM that actually
uses structural calculations. And then we’ll look
at PrePPI, which deals with everything
purely at– without actually explicitly calculating
the structures. OK. So what does PRISM do? Well, it’s based on
the notion that there are a limited number
of architectures that we could look at for
which proteins can interact. And so if we can identify
those architectures, then we can try to figure
out whether a protein is a potential partner
of another one before we do the detailed,
costly calculations. In addition, in
those architectures, not all amino acids
are going to be equal, but there are going
to be some that contribute more to the
energy than others. And so by identifying
those critical residues, we can once again focus
our computational energy on those complexes that are
most likely to be important. OK. So it has these two components–
a rigid-body structural comparison. So that’s that two
proteins are not changing their own
coordinates, they’re just being brought together
in different conformations. And then once the proteins
have passed a series of checks, then we allow for
flexible refinement using the kinds of energies we looked
at in the previous lectures to decide how high affinity
this complex could be. And the critical
thing is that we’re going to make some of
these early decisions after the rigid-body comparison
using structural similarity, evolutionary conservation,
and particularly looking at these regions
that are called hotspots. These are sites where most of
the free energy of interaction occurs during an interface. So it’s not, as I said,
uniformly distributed. So I showed you this
slide last time. It shows chymotrypsin in a
light gray and its interaction with some protein partners. These two share some global
similarity to each other, whereas this partner is
quite different from either of these two globally. But you can see that
at the interface, it’s actually quite similar. And so this gives you hope that
even if you can’t find a direct homologue– so if you
were trying to figure out, what does this protein
in yellow interact with, and you searched the database
and you couldn’t find anything that was its
structural homologue, but if you could figure
out to look for homologues of the lower regions
that interact, you might be able to figure out
that it interacts with the same protein as this
one and this one. OK. So what about this
idea of hotspots? And this was an
idea that was first developed in 1995 by this
paper, Clackson and Wells, where they were looking at the
interaction of a cell surface receptor with its
ligand approaching. And they did
systematic mutagenesis across the surface
of the interface to see when I mutate any
single amino acid to alanine, how much it affects the
energy of interaction. What they found was things
were highly non-uniform. So this lower curve shows
the change in free energy when you mutate particular
individual amino acids to alanine. And you can see there are
big losses of free energy at some places, and
other places there’s almost no change in the
free energy binding. In a few places you
actually get a benefit from mutating a side
chain to alanine. So in this particular
case, and it’s held up over many,
many cases then, the free energy of binding is
not uniform across the surface, but it’s distributed in what
has been called hotspots. So here is a structure
of the human growth hormone and its receptor. And in red are the
few amino acids that contribute very, very large
amounts– more than one and a half kcals per mole–
to the energy of interaction. And it doesn’t correspond
with any simple structural parameter. So it’s not the amino acids
that have the biggest surface area, for example, or
anything like that. So it’s not trivial to figure
out what these regions are, although there are some
prediction algorithms. So there are studies,
and subsequent ones have indicated that roughly
10% of the amino acids at the interface
are the ones that have the biggest contribution. There are some trends, but
none of these are hard rules. These tend to be rich in
these three amino acids– tryptophan, arginine,
and tyrosine. If you might imagine,
these are regions of the protein that are
highly complimentary. So there’ll be a patch
on one side that’s a hotspot matching
up with another patch on the other protein
that’s also a hotspot. And it’s kind of
an interesting note that around these regions
where the hotspots occur, there are other amino
acids that exclude solvent from the interface. And they call that an o-ring. So these are some
of the features that tend to occur with
protein interfaces. So in this PRISM algorithm,
what they do is the following. They start off with a
template– two proteins that are known to interact– and
they define the interface simply by close approach of
amino acids in one chain to amino
acids in the other. So in this case,
shown in these balls are regions of the
proteins that interact. And then they isolate
the interfacial residues. Ignore the rest of the
protein, because we said that the parts that
interact in different proteins could be homologous even
if the global structures of the proteins are not, right? So we’re going to do our
structural similarity calculations purely on
the interface residues and not on the entire structure. So then with that
template, you can then look at lots of proteins
and see whether they have any structural match
to pieces that interact. So here they’ve identified
this protein, ASPP2, which has structural homology
to I kappa b at the interface. Although globally
it’s quite different. And now, once they have this
potential partner for NF kappa b, this ASPP2,
they’re going to test whether there’s a
good structural match, whether specifically
in the regions that are hotspots– they have
an algorithm for predicting hotspots– whether the
match is good, whether it’s sequence conservation
at those hotspots. And only then do they
do the refinement to do the flexible
refinement of the type that we looked at in the
previous lecture, energy minimization, and
other approaches to figure out what the
best possible structure of this complex
would be, and then what it’s free energy would be. So here’s their
description of the problem. They have template
proteins and targets. They do a structure alignment. They asked whether it
passes some thresholds. These are very, very
fast calculations to do. And only if they pass
these fast calculations do you do more
detailed calculations. And finally, only
if it passes this do you do the very
computationally expensive refinement. And then one critical thing to
remember from this algorithm is that it doesn’t require
the template and its query to be perfectly
matched in structure. In fact, the elements of the
structure at the interface could come from different
parts of the chain. So they don’t take into
account the chain order. So if I had a beta sheet
structure in one protein that looks like this, in my
query these two proteins could be very
indirectly connected. I don’t care that there’s a
huge gap in the insertion. I just care that locally
at the interface, one protein looks a
lot like the other. There was a question
in the back. AUDIENCE: How do you search
a database for 3d structures? Are you just looking
at all the [INAUDIBLE]? PROFESSOR: That’s right. So the question was,
how do you search a database for 3D structure? You do structural
similarity comparisons that are based on
the 3D coordinates. The simplest way to do it,
but not the most efficient, is to find the rigid-body
superpositions that minimize the root mean
squared deviation, which was a metric we gave in one
of the previous lectures. There are faster things
you can do as well. You could imagine that you could
look at certain global features of elements of secondary
structure and so on. And there’s been a lot of work
making those algorithms very fast. Other questions? Good question. So they give an
example in their papers that starting off with this
known structural complex, cyclin-dependent kinase, the
cyclin, and p27, the inhibitor. And then looking for
structural matches. So we can identify this
potential structure match. You refined it, get an
energy of interaction. Try another one that has no
global structural similarity. Again, once it passes
all the checks, you compute the
refinement and the energy. And similarly with this side. And so from this
initial complex, where we had these
two proteins which were known to interact in the
PDP they can make predictions that these other proteins are
likely to interact even though, again, at the global
level, there’s very little sequence similarity. Is that clear? OK. So the advantage of this
is that it eventually does do these structural
refinements that allow us to figure out
the best match between two potential interacting proteins. But that’s also its
weakness because that takes a lot of
computational time. So this other approach called
PrePPI never actually does those structural refinements
of the type we talked about in the previous lecture. So if so, how does it
figure out whether the two proteins are likely to interact? So this is their schematic,
and we’ll go through the steps. So you start off with
two query proteins that you want to know
if they interact. And you do sequence
similarity to a database of known structures. So you find sequence
homologues to those proteins. And so they call
those homology models. MA and MB. And now they look through the
database for all the structural homologues, not
sequence homologues, but structural
homologues of MA and MB. So they get a
series of neighbors that they call NA 1
through n and NB 1 to n. So these are the neighbors
of these homologues. And they asked whether
any of these neighbors, anything in this row,
anything in this row, are known to interact. And that potential
interaction then could be a model for the
interaction of the query, right? So far so good. Then they do a
sequence alignment. They sequence
alignment of MA and MB, which are the known structural
homologues of the queries, and the two proteins that
are known to interact. And so now they’ve got
this potential model for the interaction of the
queries made up of two proteins of known structure that have
homologues that are known to interact. OK? So it’s two steps removed
from the actual interaction. Now, while their
figure says that they do a structural
superposition, that’s not, in fact, what they do. If you look at it carefully,
it’s a sequence analysis. And I’ll take you through
the steps in a second. So they mean structured
in a rather loose way. So they’re only doing
sequence comparisons here. They’re never actually
building a homology model for the queries. OK So this figure comes
from the supplement where, for some
mysterious reason, they’ve changed all
the nomenclature. So things that previously
were called NA and NB have now been called TA and TB. Take what you get. So this is a pair of
interacting proteins where the structure of
the interaction is known. And they’re structural
neighbors of NA and NB, which you don’t know whether
they interact or not. They identify interacting
residues in this structure. That’s why it’s represented by
these black lines connecting blue dots. So these are
interacting residues from the two template proteins
and neighbors NA and NB. And they asked whether the
amino acids in MA and MB also are good matches
for this interface. And they have a number of
criteria for doing that. So they come up
with five measures. The first of those measures
is a structural similarity between these MA proteins and
the MA and MB and NA and NB. Then similarity– OK, similarity
is the structural similarity. Then they asked, how many of the
amino acids at this interface, and what fraction of the
amino acids at the interface can be aligned? So this is a sequence-based
alignment of MA and– well, it’s here called TA,
but was previously called MA. Just to make life complicated. So this is the
sequence-based alignment. These are they interacting
residues, all the blue ones in the structure of
TA and TB interacting. And they asked, what
fraction and what number of these amino acids are aligned
in this sequence alignment? So here they come
up with a number. In this case, I guess, it’s
four amino acids in this– four pairs, I should say, of the
amino acids– one, two, three, and four, indicated
by these four lines– are both interacting in the
structure of the complex and can be aligned to
sequences in MA and MB. And then they use
these other algorithms that are based primarily
on machine learning looking at protein
interfaces to decide whether the sequence of the
amino acids that are going to sit at those places
in the interface are likely to be residues that
typically occur at interfaces. So this is the
kind of statistics that I showed you before
from those old papers that said 10% of the amino acids
are in these hotspots. Certain kinds of amino
acids are predominant there. So the number of algorithms,
and they list a bunch, that they use to
come up with a score to decide whether these
residues, in fact, are statistically likely
to be good matches. So they have these criteria
and they decide then that some fraction of the amino
acids at this interface in MA and MB are likely to
be reasonable ones to be at the interface. So with all that
done, they then use all of these different scores
with a Bayesian classifier, and we’ll talk a
little bit later in this lecture and
probably the next lecture as well as to what a
Bayesian classifier is. But they plug all
those scores in that they’ve derived
from these proteins to decide whether
these two proteins are likely to interact. So the advantage
of this approach is it’s extremely fast. Everything we’ve talked
about are very, very quick calculations. Even the structural
alignments are fast. The sequence alignments,
of course, are. So we get through the whole
database very quickly. So they’ve actually computed the
potential attraction partners of every pair of proteins in
various genomes based solely on these alignments. The disadvantage– so what’s
the disadvantage of this method? AUDIENCE: Can’t get a
de novo interaction? PROFESSOR: We can’t get
any de novo interaction, so if there’s no neighboring
structures that interact, they’ll never come up with it. So that’s an important point. And then the other problem
is, because it doesn’t have the structural
refinement, it’s given up on that
slow calculation, so also loses a lot of
potential specificity. All the conformational
changes that can occur will be lost to an
algorithm like this. So we have these two
competing approaches. Yes, questions in the back. AUDIENCE: Couldn’t this method
actually be used as an input to, say, a refinement
step, for example? PROFESSOR: The
question was, could you use this kind of approach as an
input to the refinement step? And absolutely one could. Is there another
question back there? Other questions? All right. So we’re going to take a slight
turn here in the course lecture and move away from a purely
computational approach and actually look at how
interaction measurements are made. One of the big changes
of the last decade or so is that we’ve gone from an era
when interactions were measured pairwise to interactions
being measured in bulk. So through high
throughput measurements. And we’ll see that that leads
us to some statistical problems which eventually bring us back
to some computational issues as well. So if you want to measure
all the proteins that interact in an
organism, turns out to be, obviously,
very difficult. One big advance that’s
helped with this is the idea of tagging proteins
and using mass spectrometry to figure out what
they interact with. So in these two sets
of papers, which were some of the early
ones being done in yeast, they took one protein at a
time and attached a tag to it. And I’ll talk about exactly
what those tags are, but those are labels
that allow you to attach it to a solid support. And then by attaching
to a solid support, you could then
purify any proteins that stuck to protein one here. And then after you purify them,
you can run them out on a gel, cut them out, and
figure out what the identity of those
interacting proteins were by mass spec. So this sounds very
labor intensive, but it’s still a lot faster than
anything that came before it. And with this
approach, they were able to go through
entire genomes, proteomes I should
say, and figure out all the interacting partners
for very, very large fractions of all the proteins there. So with this approach,
what kinds of proteins do you think are likely
to be false positives? Any thoughts? Yes. AUDIENCE: Proteins
stuck on the column that has nothing to do with
interaction [INAUDIBLE]. PROFESSOR: Exactly. So one thing that can
be quite problematic are proteins that
stick to the column regardless of which
protein you put there. And we’ll see an approach
to getting rid of that. Other kinds of problems? A variant of that. Thoughts? What about proteins
that tend to stick to other proteins
non-specifically, right? Those are going to be
quite problematic too. And what are the
likely false negatives in an approach like this? The proteins that really do
interact with the blue one but aren’t picked up. Yes. AUDIENCE: Weak interaction
partners [INAUDIBLE] PROFESSOR: Weak interaction
partners, things, particularly with
short half lives. Because you do a lot
of washing, so it’s going to be dependent
on half-life. Very good. What else? Yeah. AUDIENCE: Maybe something
that interacts in tag region? PROFESSOR: Something interacts
in the tag region, right. So something
interacts right around here would be lost because this
would sterically interfere. Very good. Anything else? What about the
concentration of proteins. How does that influence
whether they show up here? All right. So if I have a very high
concentration protein, it may interact even though
naturally it doesn’t. They never see each other. They’re in different
compartments. But when [INAUDIBLE]
and do this. But low abundance
proteins are going to be quite problematic because
there’ll be very little of them in these complexes compared to
the high abundance proteins. It won’t be detected
by this method. They will never get to
the mass spec, and so on. So we’ve got both false
positives and false negatives in these approaches. Now, one of the
things that came up was proteins that stick
non-specifically to the column. And there was a
clever approach in one of these early papers that
got picked up to avoid that. And this is called tandem
affinity purification, or TAP-tags. And the idea is the following. We have some gene. And we use homologous
recombination– this was done in
yeast where this is easy– to insert
this sequence, which codes for the following. A piece of protein of
no particular function, as far as anyone
knows, a spacer, followed by this
calmodulin-binding protein, followed by a protease
recognition site, and then by protein A. So once this protein
gets expressed– and it gets expressed
in it’s native levels because you’re inserting
this into the genome. So it’s not on an
exogenous promoter. It’s in its normal position. Whatever that protein
was, then has it as C terminus all these pieces. So how does that help? In the purification, we start
with something, IgG IGG, that binds to protein
A. So now that’s what attaches us to
the solid support. And attached to
the solid support will be all those things
that are nonspecific binders. And so if I have some
nonspecific binder that just likes my solid
support, it’ll be here. Nonspecific. And if I just acid washed
everything off the column and ran my gels with that,
or boiled it off in SDS, I would get the
nonspecific protein too. But what they do instead
is they instead cleave here with a very specific protease
that recognizes this site. It’s called a tobacco
etch virus protease. It has a very long
recognition sequence. You can make sure it doesn’t cut
anywhere in any other protein. And so now, instead of
alluding non-specifically with acid or detergent, you
allude specifically with TEV, and then this part of the
protein will fall off. And then you do a
second purification that relies on this
piece of the protein. So you pull out only
the things that you want that have the CBP,
the calmodulin binding protein, by having different
kind of solid support that has calmodulin
attached to it. And so through this
process, you can get rid of a lot of nonspecific binders. It doesn’t help you with
the false negatives, right? You’ve made the wash
conditions even harsher so you’re going to
lose more proteins. But you’ll pick up
fewer false positives. And then finally, the last
purification procedure actually uses EGTA, which is
a chelating agent. So this interaction
between CBP and calmodulin depends on calcium. EGTA sucks the calcium
out of that interaction. And so it’s, again, a very
specific way of alluding rather nonspecific one, like heat,
salt, acid, or detergent. So this has been one technology,
affinity purification followed by mass spec, that’s
given us a lot of information on protein-protein interactions. And a computing
technology that’s also contributed quite a lot
is called yeast two-hybrid. So in this approach,
you have a reporter gene that normally is not
going to be transcribed. It has at a design DNA binding
site, a DNA binding protein, and your bait protein. And you want to figure
out every protein that can interact with this prey. So the prey now is attached
to an activation domain. If these two proteins
don’t interact, the activation domain never
gets recruited to this reporter, there’s no transcription. But if the green protein and
the blue protein interact, then the activation
domain is going to be recruited to
this promoter and it’s going to turn on transcription,
and then you’ll get a signal. So what are some of the
advantages of this approach? It doesn’t require you
to purify anything. So it should be
much more sensitive to low abundance proteins. So that’s definitely
an advantage. It’ll pick up a lot of those
transient interactions. You may not get
continuous activation, but you’ll get
transient activation. And if you’ve set the
conditions up properly, you can pick up the
transient activation. But it has its own biases,
so none of these techniques are going to be perfect. It’s going to be
biased against proteins that don’t express well. This is, as the name implies,
typically done in yeast. So if you have human
proteins and you express them in yeast, or plant proteins
that you express in yeast, there could be some proteins
that just will not express well in that organism. What else can be a problem? Some proteins don’t do
well in the nucleus, right? So if you’re interested
in interactions with membrane
proteins, it’s going to be very hard to get them
to express in the nucleus, and therefore, you’ll never
pick up those interactions. OK. So we’ve got these two
different technologies– the affinity capture mass
spec and the two-hybrid. Questions on those technologies? Yes. AUDIENCE: Could
another control be for the mass spec
purification just to subtract out everything
that alludes non-specifically. PROFESSOR: The question was,
could you subtract out anything that’s nonspecific. And yes, if you’ve
got what you might call frequent flyers,
proteins that show up in every single
purification, then you can simply ignore those. And that is often done. So that’ll help you
with things that are very nonspecific
for the surface. What’s more of a
problem are proteins that have some affinity
for your protein x but are not really
highly specific for it. So they tend to bind in
certain kinds of patches. Those would be
harder to figure out because they won’t
stick to everything. Good question. Other questions? All right. So we’ve got these
different technologies. What we’d really
like to be able do is we know that there are
problems in each approach. We’d like to be able to compute
the probability that two proteins interact
based on the data. So now we’re turning back to the
more mathematical computational approaches. So if we just consider
one experiment– and we’re going to talk about
gold standard. So what’s a gold standard? It’s a set of proteins that we
have extremely high confidence interact because it was analyzed
by some other technology. Not two-hybrid, non-affinity
capture mass spec, but much, much more direct interactions. By physical measurements,
maybe the structural work. So the number of
criteria that go into it. So we have this
gold standard data set where we know the proteins
definitely interact, and we have our experiment. So clearly anything
in the overlap, we can count as true
positives, right? We detected it. It’s in the database
of gold standards. And things that are in the
gold standard that we missed are obviously false negatives. We report them as
non-interacting, but in fact they do. The question is, how much
of this is true positive? Everything that’s detected
in the experiment but we have no information
for it in the database. So that could be for one
of two reasons, right? That could be that they
really don’t interact. Or it could be that
no one’s measured it. The whole point
of this experiment is to find new things. So is there any way to estimate
what fraction of all the things that are unique to this
experiment are true positives, and what fraction
are false positives? Those we’d like to
try to figure out. Now, if we just
had one experiment, that would be very challenging. But what happens when
we’ve got two experiments? So we have these two affinity
capture mass spec experiments, or maybe affinity capture
mass spec and a two-hybrid. So now let’s think about
the overlap of those two experiments with
the gold standard. So I’ve got this region of
overlap between experiment 1 and experiment 2,
and then this region that’s overlapping
between all three things. Experiment 1, experiment
2, and the gold standard. So these clearly are
two positives, right? They’re high confidence
because I picked them up in both experiments, and
they’re in the gold standard. What about all these things
in what I’ve labeled here region 2? Well, if we believe that
these two experiments are independent of each
other in a rigorous way– so let’s say one’s a
two-hybrid and one’s an affinity capture mass spec,
there’s no particular reason that the false
positives for one would be false positives in the other. In that case, I can
call this region 2 my consensus true positives. I have a very high
confidence that these are true interactors. Everyone buy that? Seem reasonable? OK. So here’s where
the trick comes in. What fraction of all these
consensus true positives are picked up in
the gold standard? This ratio, right? Region 1 over region 2. OK. So now I’ve got this region
of things that are picked up– the true positives from
this experiment, then the gold standard. And then I’ve got this region
that’s unique to experiment 2 and it’s going to be some
mix of true positives and false positives. And the authors of this
paper that are cited here make the following argument. We’re going to assume
that the ratio of I to II is the same as the
ratio of III to IV. So the fraction of
consensus true positives that are picked– these are
independent experiments. So the fraction
of true positives that are picked up
in the gold standard is going to be constant,
whether they’re in the consensus or not. So the fraction at
ratio of I to II is going to be the same
as the ratio of III to IV. So by that then, I can figure
out how much of this region consists of true
positives and how much consists of false positives. Everyone buy that? Yeah. AUDIENCE: Can I check–
are we not saying that the gold standard
represents all true positives? PROFESSOR: Correct. Well, we’re saying that the
gold standard consists of things that we know to interact– AUDIENCE: But there may be more. PROFESSOR: But
there may be more. And the goal of our experiment
is to find those other ones. All right. So if you accept that premise,
which seems plausible, then you can compute what
fraction of all the things that are picked up in
each of these experiments are likely to be true positives. So drum roll please. It turns out that the
number’s not that high. So the fraction of
things in the consensus was 347 out of almost 2000. And if you do the math
then, what you end up with is that the true
fraction in this region, for which we have no
data, is 1,123 out of– and the false piece in this
is going to be almost 15,000. And they went ahead and
did this for a number of different
experiments and computed the fraction of derived false
positives for these data– might be a little bit hard
to see on this screen. But the numbers range
from 50% false positives to, in some cases, over
90% false positives. That’s a little
disturbing, right? So these technologies are good
at picking up interactions, but there’s reason
to be very skeptical. OK. So now we’ve got
a serious problem, because how are we
going to figure out which of these interactions
to trust when we know that a very, very large fraction
of them are false positives? So what could you do? Well, you could take only
the little bit of overlap. You could say, I have that Venn
diagram– method 1, method 2. They did agree on
a bunch of things. So I could take only those. That obviously
throws away a lot. Someone else suggested we could
throw away the sticky proteins, right? So maybe there are
nonspecific proteins that don’t show up
in every experiment, but they show up in a
very, very large fraction of all experiments. Maybe I toss those out. That’s another possibility. But what we really
want to do is actually come up with a
probability estimate. To not have to make
a hard decision, but come up with an
estimate of the probability that things interact
based on all the data. So how do we go
about doing that? So first of all, what happens
if you just require a consensus? So this plot shows
accuracy and coverage of the gold standard for
individual experiments with different thresholds for
deciding what’s interacting, different cutoffs and things. So the individual
experiments are shown here. And then if you
acquire two methods to pick something up, or three
methods to pick something up, you can get better and
better in your accuracy. This is a log-log plot. So if you require
three methods to agree before you call something
a true positive, you can get up to– I’m not
sure exactly what this is, but 80%, 90% possibly. Right? But look at where
you at the y-axis. You’d only get
about less than 1% coverage of the gold standard. So that’s not a great approach. So what we really
want to do, as I said, is to try to estimate the
probability that proteins interact given all of
our available data. And the data could be
specific experiments. Say the two different
mass spec experiments we just referred to. Or as we’ll see a
little bit later in this lecture and possibly
the next one, other kinds of extraneous data that are not
direct physical measurements of interaction, but
might give us confidence that things interact based
on similarity in annotation, or similarity in gene
expression, and so on. And we’ll get into
details of that. OK. So to do this, we need
to have a little bit of a refresher on
Bayesian statistics. So I want to measure
the probability that an interaction is true
given the available data. Right? And I can estimate that based
on the probability of observing the data for things
that I know to be true and these prior estimates. So what’s the prior probability
that an interaction is true and the prior probability of
observing a particular data set. Now, this by itself isn’t
really that helpful. I haven’t told you yet how
to calculate any of the terms on the right. But bear with me. If I want to decide
the likelihood that a protein interacts–
how likely is it? Is it more likely that
it interacts or not? I can compute this ratio. The probability
that the interaction is true given the data
over the probability an interaction is
false given the data. That’s the likelihood ratio. So by this formula, I then
cancel out this probability of the data, the prior
probability of the data. And if I had a way
of calculating this, and we’ll get to it in
a second, then if it’s more likely than not to
be a true interaction, I can call it an interaction,
right, if it’s less likely. So if this ratio
is greater than 1, I accept it as a
true interaction. If this ratio is less
than 1, then I reject it. OK. So now our challenge
is to figure out how to compute these terms. One more thing to note
is if all I want to do is be able to rank every
interaction by this likelihood ratio, rather than coming
up with a hard threshold, then I actually don’t
need all these terms. So this is the likelihood ratio. I can convert it to a log space. So it’s going to be the
sum of these two terms. And if I’m simply
ranking everything by this log likelihood
ratio, this term is the same for
every interaction. It’s just composed of
prior probabilities. So it’s not going to
affect the ranking at all. Any questions on that? Is that clear? Good. So if I just want to come
up with a ranking function, all I need to do–
all– I need to do is to be able to estimate
the probability of observing data for true interactions and
the probability of observing that set of data for
false interactions. Everybody buy that? Yes, please. AUDIENCE: When you say
that prior probability is the same for all
interactions, we’re saying we’re assuming the same
prior probability for all, or is this [INAUDIBLE]? PROFESSOR: That’s
its definition. We mean, what is the prior
probability that proteins interact versus the
prior probability? So it’s independent of the
proteins that we’re looking at. Other questions? All right. So we need a way of
computing this piece of all the things
we’ve looked at before. So how do we get an estimate
of the probability observing a particular
configuration of the data? Meaning, I detect
it in experiment 1 and not in experiment
2, but in experiment 3. What’s the probability of that
given it’s a true interaction? So that’s what we’re going
to dive into right now. OK. So one thing we could
do to make life simpler, and then we’ll remove
this simplification later, but let’s, for the time being,
assume that all of my data are independent. So the two-hybrid is going
to have completely different mistakes than the affinity
capture mass spec. So those two data
sets are going to be completely independent
of each other. So I can write this as a product
of a particular observation– a particular mass
spec experiment and a particular two-hybrid
experiment for true attractions and false interactions. So it’s the product
of the probability that a particular experiment
would detect an interaction if the interaction is
true over the probability that that particular
experiment would detect it if there was no interaction. I’m just going to multiply
all of those probabilities. Yes. AUDIENCE: [INAUDIBLE]. This is one interaction pair? PROFESSOR: That’s right. AUDIENCE: And you
take the product over all the interaction
pairs within one run of the experiment. Is that correct? PROFESSOR: If I
want to determine whether a particular
interaction pair– I want to compute
this log likelihood ratio, or this,
actually, ranking ratio, because I’ve thrown
away the priors. I want to compute this ranking
ratio for a particular pair. So I’ve got protein
A and protein B. And I want to determine
whether I believe it to be more likely
to interact or not, and rank it with all
the others, right? So I’m doing this for
a pair of proteins now. So far so good? Now, for that pair
of proteins, I have a series of observations,
or lack of observations, right? I have a whole bunch
of experiments. This experiment detected
it, that experiment didn’t detect it, this one did. So what’s the probability
of these proteins– these A and B really interact
given that yes, no, yes in my experiments? And then for new protein,
it might be no, no, yes, and what I want to figure out
the probability for this pair. AUDIENCE: So is the scale
of the big letter M, is it on the order of like 10
experiments, 100 experiments, or thousands of experiments? PROFESSOR: Ah. So the question is,
what’s the scale of this. So obviously, that’s going to
depend on what kind of data I bring in, but in
these cases, it’s small. So we have a handful of these
high throughput experiments over entire genomes
and proteomes. So there’s not to be a lot. So in some of
these early papers, there were four
interaction experiments that they were looking at. Now the numbers might
be a little bit bigger, but not significantly greater. All right. So now to compute this, we
need a set of gold standards. But now we don’t just need gold
standard positive interactions, proteins that we know
really do interact. We also need proteins that we
know really don’t interact. Because I want to compute the
probability of an observation given that some interaction
is definitely wrong. So precisely how I
compute these terms is going to depend
on the kinds of data. The experiments I’ve
just been talking about, these high throughput
mass spec, which were the ones which we looked
at the ratio of the consensus, true positives, and estimated
that 96% of all the data were possibly in error. The details of how to do
those calculations are here. I leave you to look that
up if you’re interested. But now what we’re
going to do is we’re going to see how, if
we were to rank interactions based on this term,
we can avoid having to throw out most of our data. So we said if we require all
the experiments to agree, we’re going to have
very, very low coverage. Now we’re instead going
to rank everything based on this likelihood
ratio, or something derived from the
likelihood ratio. So in this paper
where they were simply looking at the
protein-protein interaction data sets to compute
these interactions, they ranked everything based on
that ranking function we just described. And then as you
vary your threshold, you can figure out how many
true positives you have and how many false positives
you have in the gold standard. True interactors and
false interactors. And you can compute
this curve, right? For any particular value
of that ranking ratio, what’s my sensitivity and
what’s my specificity? Are you clear what
this plot means? And here they’ve
plotted the values for individual experiments. And this is the value for
an independent database of gold standard interactions. And so now, where
do they come up with their true positives
and their false positives? A lot of this is going to depend
on how representative those are. And all these numbers
are subject to revision if you decide that the true
positives and false positives that people are using
are not accurate enough. So they used two well annotated
databases of interactions. One from MIPS and one from SGD. And you can play those
off against each other as the database
of true positives. In some ways, that’s
the easier thing because people like to report
that proteins interact. They tend not to like to report
the proteins don’t interact. You don’t see a lot of
nature papers saying protein x doesn’t interact
with protein y. So how are you going
to figure out, then, what are your true negatives? So the strategies
that they used– well, one possibility is they’re
annotated to be in complexes, and those complexes are
different from each other. That’s not bad, right? But it’s not a guarantee either. Or this is a little bit better. They’re annotated to be in
different parts of the cell. Of course, if those
annotations aren’t perfect, low concentrations, you
could still be wrong. Or that they have
anti-correlated gene expression. I kind of like this one. So it’s one thing to be not
correlated, but if you’re anti-correlated, seems
pretty suggestive that these two proteins are
never in a complex together. Again, it’s no guarantee
because, as we’ll talk about in some detail later,
RNA levels are not very good predictors
of protein levels. But if you apply enough
of these criteria, you can come up with
a set of proteins that you have fairly
high confidence really don’t interact. You combine that with
the databases of proteins with very high confidence
that they do interact, and you can get the true
positives and false positives that you need for this analysis. all right. So that’s a way of
combining some information. We’re going to see a
generalization of that called Bayesian networks. We’ve mentioned this
already in at least two different
contexts, and it’ll come up again later
in the course as well. So these are very
general methods for reasoning probabilistically. We will see them
in the context here of predicting interactions. We’ll see them later in the
context of gene regulation and signaling as well. What we fundamentally need
to do a Bayesian network is a graphical structure that
represents our understanding what the relationship is
between causes and effects. And a set of
probabilities that allow us to compute things
on this network. We’ll show you examples where
those networks are derived from our prior understanding
of the problem, but also ones where the
structure of the network is learned from the data. And we’re going to see
two primary contexts. First we have this question
of whether proteins interact. That’s what we’ve just
been talking about. So here are four experiments,
the in vitro pulldown experiments and yeast
two-hybrid experiments, that give us relatively
independent information about whether proteins interact. And we’re going to
look at a paper that used those data with
a Bayesian network to compute the probability that
two proteins really do interact based on the combination
of all the data, rather than throwing out
anything that doesn’t fall in the overlap, which could
be a very, very small number. And then later on
we’ll see examples of using Bayesian networks to
understand biological networks. So this might be a set
of transcription factors that are regulating a set of
differentially expressed genes. And the structure of
the graphical network for a Bayesian network
has a lot of similarities to the way we normally
think about transcriptional regulatory networks. So there’s sort of a
natural way of transferring our regulatory problem into
a graphical network problem. But we’re going to focus
on these prediction problems for protein-protein
interactions first. Now, if I just want to compute
the probability of detecting an interaction in various
experiments, given that it’s true or false, I
could explicitly compute that probability. And we saw examples
of that just now. But some of these
Bayesian network problems become much, much
too large to do that. This is a little tiny
piece of a Bayesian network that is supposed to
represent I believe it’s transcriptional
regulatory network. You could never possibly
write down all of the terms in this probability, where every
node could, in principle depend on every other node
in the network. It would just be a
ridiculously large problem. In fact, how large would it be
if I’ve got N binary variables, my gene is on or off, my
interaction is true or false, I have 2 to the N
possible states? Right? And the only constraint
I have, in principle, is that all the probabilities
have to add up to one. So I have 2 to the N minus 1. 2 to the N minus 1 possible
variables that I need to set. So that’s a ridiculously
large number in most contexts. So how do Bayesian networks
help us solve this problem? Well, we represent
our understanding of the problem in a
graphical structure where we have
causes and effects. And there’ll be a direct arrow
from a cause to an effect. I don’t always know the cause. So in our context,
we were trying to figure out whether
two proteins interact. What do we measure? We actually don’t
measure interactions. We measure the result of a
particular experiment, which is a combination of
whether interacted and all sorts of noise
that we’ve just discussed. So the effects that we observe
are detected in experiment one or detected in experiment two. The cause is, did
it interact or not? So the cause is hidden,
the effects are observed. Now, in the case we
were looking at before, we treated all
these probabilities as being independent. But we might know something
about the structure of our experiments, the kinds
of experiments we’re doing, that might lead us to have
a different structure. So we could have an
interaction that gives rise to all different kinds of data. But depending on whether
the protein’s a membrane protein or highly
expressed, it might influence the results
of certain experiments and not influence the
results of others, right? So like a two-hybrid
would be very biased by which one of these? The membrane, right? And then the affinity
capture mass spec could be very
influenced by proteins that are expressed at very
high levels or very low levels. If we assume that all the
interactions are independent, then we multiply probabilities. And we’ll go into
more detail, but this is what we’re looking
at up until now. In cases where we believe that
all the observations are not independent, then
we’re not going to simply multiply things. We’ll see there’s
a more precise way of computing the probabilities. Now in this case, I’ve drawn
the graphical structure because I believe that
I know what’s going on. But in the more general
case that we’ll look at, we’ll actually derive the
structure from the data. One of the nice things
about Bayesian networks is that it removes the
need to have all 2 to the N minus 1 possible parameters,
because it tells us there are certain
independence conditions. So node is independent of its
ancestors given its parents. What does that mean? If I’m trying to reason
about the expression of one of the genes down here, and I
know that this transcription factor is on, I
don’t really care what the probability is
that any particular parent of that transcription
factor is on, right? So I don’t need to know anything
of transcription factor B1 if I know the state of B2. If this is on, then
that’s the only thing that’s going to affect whether
it’s turning on these genes, regardless of what the
activation state of its parent was. Is that clear? Yes. AUDIENCE: The
slide’s saying TF B1. [INAUDIBLE] TF B2? It says TF A1. PROFESSOR: Yeah, sorry. That should say TF B1. Thank you. OK. So we’ll do a little example. It’s admission season
both for graduate school and undergraduate. So let’s do a little
toy example where we’re going to get rid of
the admissions committees and just do
automated admissions. So we’re going to collect
various data about students, and then we’re going to
build a Bayesian network. And that network
is going to decide whether to admit students
into this simplified version. And the only information that
will go into our decision will be the grades on the
transcript and the GREs. Hopefully that’s not the case. And we believe
that certain things influenced your
grades and your GREs. Whether or not the
student is smart certainly should
have some influence, but also the great
inflation at their school will have some influence. So a prediction problem
in a Bayesian network is going from the
causes to the effects. So if I want to predict
whether a student’s admitted, I only need to look upstream. So we want to predict– we
observe the things on the top. Say, grades and
GREs, and we want to predict whether this student
should be admitted or not. There’s another problem called
an inference problem, which is when we observe
the effect and we want to make inferences
about the causes. So an example of that would
be, you apply for an internship and they say, oh,
she’s a student at MIT. I bet she’s smart. Right? They’re doing an
inference problem. We’ll leave it for you to decide
whether you and your colleagues are as smart as everyone
thinks, but hopefully you are. OK. So we’ve got these two
different kinds of problems. We’ve got prediction
problems from top to bottom, and inference problems
from bottom to top. And we’re going to talk about
conditional probability. So if I’ve got some very
small piece of this network with just two nodes,
I could write out all the possible probabilities
for any pair of those nodes. So the probability that
a student is not smart given that that student has
low grades, the probability that the student is not smart
given that the student has good grades, and so on, for all
possible pairwise comparisons. Or I could write this as a
conditional probability, which tends to be an easier way
to think about the problem. What’s the conditional
probability of a student being smart given that
they’ve got good grades or given that they
have bad grades? They have the same information. For this one, I need
additional information about the total probability of
students being smart or not. And the total number of
variables, as I said, in either case is the same. So these are completely
interchangeable, but it’s a lot easier to reason
with conditional probabilities than with the joint
probability tables. Those we’ll see in a second. So as I’ve said, you don’t
need a full probability table for a Bayesian network. You don’t need two N to
the minus 1 variables. And the fundamental
reason for that is that the joint
probability is only going to depend on the parents. So in this toy example,
the GRE scores over here are not dependent
on grade inflation. Now, that all
hopefully makes sense. Questions? Bayesian networks get
a little murky next, so I’m going to try to
give you into– oh, yes. Question, please. AUDIENCE: You said that
the parents don’t affect their children, but if grade
inflation affects the grades, how does that
influence– will that influence the grade [INAUDIBLE]? PROFESSOR: Sorry, can you
say the question again? AUDIENCE: I guess
I’m just confused by this particular example. What do you mean by
the joint probability? The joint probability of what? PROFESSOR: So if I
want to figure out the probability of some
particular configuration of all the nodes in my network,
I don’t necessarily need to consider
all possibilities. Because for example,
if I want to consider all of the joint
probability samples with settings for the GREs,
whether the student had good GRE scores
or not, that’s not going be influenced by the
student’s school’s grade inflation policies. AUDIENCE: But wouldn’t the
grades be influenced by the– PROFESSOR: But the
grades would be. That’s right. So some of the
variables I can remove and others– some of the
joint probability statements I don’t need to worry
about and others I do. And which ones I
need to consider is determined by
the graph structure. Yes. AUDIENCE: How is the graph
structure determined? PROFESSOR: OK. So how is the graph
structure determined? So it’s determined
in one of two ways. I can draw it in advance because
I believe that I know something about my setting, I believe
that these data are independent. Then it has that
structure like this. Cause and a bunch of
independent effects. Or perhaps I claim to know that
actually two of these things have a common parent as well. In some cases I know. We’ll also talk about how
to learn the structure from the data, which is
the more common setting in regulatory networks. So in these kinds
of problems when trying to decide
how to integrate different proteomic data
sets, typically people make arbitrary decisions
about what the structure is based on their
knowledge of the system. But if you’re trying to figure
out de novo which proteins interact with which, which
proteins regulate which genes, then you have to learn
it from the data. And we’ll talk about how
to do that in a second. Great questions. Any other questions? Anything in the quiet
half of the room? OK. So as I said, this
part of it, I think you can usually
come up with cases that give you fairly
good intuition. One of the things that is true
in these Bayesian networks which most people find a
little bit surprising at first is something called
explaining away. So let’s look at this
Bayesian network. I go outside and I
detect that things are slippery on the grass. So that could be for
a lot of reasons, but one possible reason
is that the grass is wet. OK. What are the causes of
the grass being wet? Well, it could have
rained or the sprinklers might have been on. And depending on this
as an example– so a lot of the Bayesian networks
were developed in Stanford by Judea Pearl and colleagues. And of course, in California
it doesn’t rain that often. So there the season is a strong
determiner of these things. Not so much around here. So in this example
that they like to do, so does the
probability that it’s raining depend on whether
the sprinkler is on or not? Now, the answer
should be no, right? I mean, in reality, when
you think about– there’s no causal relationship
between the sprinkler being on and the rain. But in fact, when we’re
reasoning over these networks, we actually are influenced. In a probabilistic model,
if I know that it’s raining, and I know the grass
is wet, then what do I think about the
sprinkler being on? Do I think it’s just as likely? No, I think it’s
less likely, right? If I go outside and see the
grass is wet, there are clouds, the rain is coming
down, is the sprinkler likely to be on or not? It’s likely to be off, right? So there’s no
causal relationship, but there’s the probabilistic
relationship through the graph structure. And that’s called
explaining away. And you can take a whole
course on how to understand which relationships you
can detect and which not. This is not the place
to try to go into that, but I hope you’ll be
familiar with this problem. And I’ll try to give
you a toy example that makes it a little bit
more obvious in terms of the equations
where this comes from. So imagine this very silly game
where we play, we toss coins. We toss a coin twice. And if it turns up heads
both times, you get a point. If it turns up tails both
times, you get a point. But if one’s a head and one’s a
tail, you don’t get any points. Now, does the probability that I
tossed a head on the first time depend on whether I toss
a tail on the second time? So causally,
obviously not, right? First of all, it
happened earlier in time. And secondly, the coin tosses
are completely independent. But what happens when
I know the outcome? What if I know
what score you got? So if I know your score,
then is the probability that I tossed the
heads on the first time independent of whether I got
a tail on the second time? What do you think? How many people think
it is independent then? How many people think
it’s not independent. Very good. It’s not independent. And obviously, here’s
the math to prove it, but your intuition
does the same thing. So what’s the probability
that I tossed a head on the second time
given that I got a one, I scored, and I tossed a
tail on the first time? Obviously, it’s zero, right? So here’s the
probability of getting a head in the first
time and scoring one, and tails on the second
time is exactly zero. So that’s called
explaining away. You can reduce your
belief in certain parents based on what you know
about the children. Think of this coin toss
example or the rain in California and
the sprinklers. All right. So as this come
up several times, how do we obtain the
Bayesian network structure? There are two problems that
we need to be able to solve. We need to be able to
learn the structure, and we need to be able to
learn these probability tables. If we know structure, how
do we get the probabilities? Well, we need to identify
some objective function we’re going to try to optimize,
and then choose values for all probability
distributions that optimize that
objective function. And that’s the
kind of thing we’ve been doing all along, just
like in the Gibbs sampler. We need some objective
function or protein structure. We need some objective
function that we’re going to try to optimize. So there are two common
ones that are used a lot. There’s maximum likelihood
and the maximum posterior. So maximum likelihood is defined
as the set of param– theta is all the parameters, all
the probability distributions, the probability of getting a
score of one given that you had heads and tails,
whatever it may be. The probability of
getting admitted given that you had certain
GREs and certain grades. So we want to find
the set of parameters, all those probability
distributions, that maximize this. The probability of the
data, our training data, given those parameters. That’s a pretty obvious one. And the maximum posterior
includes some of our beliefs about the prior
probability of the data and the prior probability
of the parameters. This is a little
bit less intuitive because you have
to ask, well, where do those numbers come from? And that, again, is a
whole course unto itself. OK. Now, how do you find
these parameters? Again, it’s the kinds
of search problems that we’ve looked at before,
various kinds of hill climbing. So gradient descent,
expectation maximization, Gibbs sampling, which
you’ve looked at explicitly. And again, the full
details of how to do that are outside of our scope today. OK. So in our example of
this coin toss game, we would use one of
these two functions to try to decide what’s
the probability of getting heads or tails for
any given score. That’s what the kinds
of parameters are. Now, the structure
problem actually turns out to be
really, really hard, because there are a very
exponentially large number of potential structures
to draw from. And unless you’ve got
some prior knowledge, it can be impossible, depending
on how much data you have, to actually build
this structure. So there are many algorithms
that have been proposed. And a lot of our
settings, we’re going to use some kind
of prior knowledge to reduce the search space. So if we’re trying to talk
about transcriptional regulatory networks, it’s very common
to assume that there are only some kinds of nodes that can be
causes and other kinds of nodes that can be effects, right? So in gene expression
it would be effect, and then you would
limit your causes to only be
transcription factors. It would generally be signaling
molecules or something like that, and not allow all
20,000 genes to be causes and all 20,000
genes to be effects. So there are lot of
resources to learn more about Bayesian networks. As I said, you can have
whole courses on this. I think there are a lot of
good tutorials at this website. I’ve also put in the notes
a little toy example for you to work through all the
probabilities, which I think, in the interest of time, we
won’t go through in detail. All right. So to motivate what we’re going
to do in the next lecture, I just want to talk
about other kinds of data that you could bring to bear
on this problem of predicting which proteins interact. We’ll see, then,
how that gets fed into an interaction
Bayesian network to make the predictions. So we’ve talked about affinity
capture and two-hybrid, but what other
kinds of data could we use to predict the
probability interaction? Well, one thing you could use
would be gene expression data. And the idea is that if
two proteins interact, they should be present in the
cell at the same time, right? So we talked about
this a little bit. If they’re
anti-correlated, it seems very unlikely they interact. What about if they’re
correlated, but not perfectly correlated? So here’s a plot that shows
a histogram of proteins that are known to interact, proteins
that are known not to interact. So empty circles are known
interacting proteins, the dark circles are
non-interacting proteins, and the other ones are based
on the experimental data. And the distance here
is the difference between expression profiles. And we’ll talk in coming lecture
about exactly how to compute distance between
expression profiles. But the further to the right
it is, the less similar the expression profiles
are across large data sets. So what you see
is the interacting proteins tend to be shifted
more to the left, more similar expression profiles
than the non-interacting ones. But what do you
notice about this? There’s no way to
draw a line and say, everything to the right
of this is in one class and everything to the
left is another, right? So by itself, it’s not
going to get us very far. There are plenty of
non-interacting proteins that have very highly correlated
gene expression and plenty of interacting proteins
that have poorly correlated gene expression. So it’s a trend, not a rule. Now, what about evolution? So if I look over many, many
organisms, I might expect what? The proteins that
interact with each other are going to appear in
the same species, right? So let’s look at
these two cases. We’ve got a bunch of–
eight different genomes. And I’ve got gene 1 and gene 2,
which I suspect might interact, and gene 3 and gene 4, which
I suspect might interact. Now, looking at
these two patterns of evolution, which one
do we have more confidence in that it interacts? The red one or the green one? So what do we notice about
the difference between them? What’s true of the red one
compared to the green one? Yeah. AUDIENCE: The red one is only
in one branch of the tree. PROFESSOR: The red one is
only one branch in the tree and the green one
is scattered across. So let’s take a vote. Do we believe that
the red one is better evidence of
interaction or the green one is better evidence
of interaction? Red? Green? Can I have an advocate of green. Someone explain their rationale? Anyone in the quiet
side of the room? All right, Ed. AUDIENCE: Because red is only
on one branch of the tree, I’d expect that
they’re naturally more correlated with each other. They have less–
they appear together in [INAUDIBLE] so I’d
expect [INAUDIBLE]. PROFESSOR: OK. So the argument is that red only
occurs in one part of the tree. And so there could be a
very simple explanation for all the reds being in one
part of the tree and one not, which would be a single
loss and gain event. Right? Somewhere early
on, perhaps here, I gain those two proteins. And then they’re inherited
throughout the genome, like most of genes get
inherited throughout the genome. Whereas here, we’ve
got independent events of gain and loss. And at each one of these
independent events, we’re getting them
moving jointly, either in or out of the genome. So there’s more
evidence for green to be interacting than red. Everyone buy that? Even some of the
advocates of red? Questions? Yes. AUDIENCE: Could there be a
way of either objectively or mathematically
[INAUDIBLE] that way, or is it just the
reasoning [INAUDIBLE]? PROFESSOR: One can do
the statistics on it with known ones, right? I think that’s
probably the best way. And we’ll actually see that
in one of these papers that uses– well,
actually, now I don’t recall whether they
use this co-evolution. But yeah, there are
plenty of papers that actually have done
the statistics on that. So it is supported. And a related kind
of question is what’s called the
Rosetta Stone approach. Unfortunately, of
the term Rosetta gets used far too much
in computational biology. So this has nothing to
do with the other Rosetta that we’ve been talking about. And this has to do
with how often you find the same pair of genes in
the same genome versus split up in different genomes. OK. So what we’re going to
look at next time then is an approach that
combines these kinds of data with the protein interaction
physical measurements through the two-hybrid and the
affinity capture mass spec that actually uses the Bayesian
networks we talked about this time to predict
whether two proteins are likely to interact based on
all of the available data. These evolutionary arguments,
the [? sentiality ?] arguments, and then the interaction data. Any final questions? OK, see you next time.

4 Replies to “14. Predicting Protein Interactions”

  1. Thanks for sharing this. Large scale identification of PPIs generated hundreds of thousands interactions, which were collected together in specialized biological databases that are continuously updated in order to provide complete interactomes. The first of these databases was the Database of Interacting Proteins (DIP).

  2. 13:58 // Pg.23
    How can we determine the delta G value? Could you give us a link or so? I assume we are not literally calculating the value based on thermodynamic data but there should be a software that does that. Could you help me how to figure del G value? Thanks

  3. 40:09 // pg. 60
    How does that prior probability is the same for all interactions?? I thought differently. If P(true_PPI) is 0.6, wouldn't that P(false_PPI) is 0.4? Whatever probability we have for the true_PPI, wouldn't the false probability be 1-P(true)? Could anyone help me out? Thanks

  4. Hey, team, I have just started a youtube channel about my PhD. I have just started so the idea of the channel is for you guys to follow along with me as I complete my PhD. My latest video is about how having dyslexia and how it affecting my grades and academia!

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