[ Silence ]
>> I can't believe
it's gone so quickly.
So, it's a little bittersweet.
I love teaching this
class so it's hard for me
to give this very last lecture.
Okay, so today we're
going to pick
up where we left off last time.
Small molecule control
over signaling pathways.
We already talked about
this last lecture.
Today proposals are due, make
sure you don't leave the class
without handing them in.
If you miss handing it
in for whatever reason
then just bring it
by my office as soon
as possible.
You have until noon,
it's a grace period.
This also applies if you
need to get an abstract in
or something like that.
I'd like you to attach
your abstract
to the proposal --
your graded abstract.
If you don't have your graded
abstract with you today,
run home, get it, bring
it back either to me
or to the T.A.'s,
Kritika or Mariam.
Do that ASAP, there's a
little bit of a grace period
but you don't want
to extend it too far.
I understand there's some
issues at turnitin.com,
did anyone have issues
with that?
All right.
Let's try to get those in the
next 24 hours uploaded, okay?
Sometimes it just jams with --
this is kind of the end of
the quarter so there's a lot
of assignments that
are coming in.
Hopefully turnitin.com will
get their act together.
If it persists as a problem
then I'll make arrangements,
but for now just keep
trying and then get
that turned in in 24 hours.
I do have a quick question
for you for next year.
Did you like having
this due today
or would you rather
have it due Monday?
So, everyone in favor of
today raise your hand.
Okay. Two, three, four.
>> Like, Monday as
in this past Monday?
>> No, no, no.
Monday as in the
upcoming Monday,
like you know, a
few days from now.
Okay, so today, all
in favor of today?
Two, three, four, okay.
And Monday?
Wow, it is almost
completely, evenly divided.
All right.
Well, in that case we'll
probably stick with today then.
The reason is, this gives me
more time to read the proposals.
It actually takes a
lot of time for me
to read 120 10-page papers
and provide comments
on half of them.
So, because of that I
need the extra days.
And also, I feel like you guys
need a little bit of break
to study for your
other final exams.
If you had this hanging over
you, you would imagine just,
you know, working on
it, and working on it,
and working on it, until
the very last minute.
So, okay, well I'm looking
forward to reading them.
So, you can now relax now that
you're here and just make sure
that I get them at the end of
today on this table over here.
All right.
I do have office hours today
immediately after class.
I imagine that you don't have
too many important things
to talk to me about.
I will have office hours next
quarter if you want to stop by.
You're always cordially
welcome to my office, okay?
So you're always
welcome to stop by.
Usually office hours are
the best way to get ahold
of me, or send me an email.
You know, if you need advice
about anything, about careers,
about finding a job,
or whatever,
don't hesitate to call on me.
Okay, so you've been in
my class, you worked hard
for me this quarter, I'll work
hard for you in the future.
All right.
Here's what we talked
about on Thursday.
So, Thursday we wrapped up
our discussion of terpenes
and then we talked
about cell signaling.
And one thing I wanted to press
upon you is that this concept
of using small molecules
to control cells
and to coordinate responses
in cells is really a
universal phenomenon.
This is carried out by the most
simple organisms on the planet
and the most complicated
organisms.
It's carried out by humans,
it's done by humans,
it's done by insects.
It is truly universal.
It's one of those aspects
of life that we find.
Small molecules are
also used extensively
by chemical biologists
as power tools
for dissecting biological
phenomena.
And I showed you examples
of this using both reverse
and forward chemical genetics.
The example of the forward
chemical genetics were those
massive screens to
identify compounds
that could interfere
with mitosis.
Okay? And we saw this during
the cell division, the DNA,
instead of lining up on
the scrimmage line neatly
in the center, was getting, you
know, pushed to the peripheries
and it looked like the complete
reverse of what should happen.
So, forward chemical
genetics is a powerful way
to identify unusual
phenotypes in the cell.
Then using reverse
chemical genetics,
you can use that small molecule
as a powerful tool to figure
out exactly what's happening.
That compound monastrol emerged
as a really interesting
inhibitor of kinesin,
and using that inhibitor
scientists were able to --
chemical biologists,
more specifically,
were able to define a particular
kinesin variant that's critical
to shoving apart the
chromosomes and pushing them
into place during cell division.
And that's absolutely
fascinating.
Okay, so small molecules
offer temporal control,
meaning time control.
They also offer localization,
you can dose them in
a particular spot.
And we even talked about
how they can be used
to investigate phenotypes
that would otherwise be
lethal to the organism.
So all of those aspects are
what make small molecules
such an integral part
of chemical biology
in the modern era.
Okay now, because we often
use these small molecules,
kind of like toxicology,
to investigate cell phenomena
we oftentimes find ourselves
investigating cell
signaling pathways.
And my message to you
there is don't panic.
Okay, so if you open an issue of
Cell or something like that,
and you look at the last couple
of pages they always have
signal transduction diagrams.
And every month to every week --
or every couple weeks they
investigate a different
signaling pathway, and these are
daunting in their complexity,
they look like the Milky
Way in terms of the number
of elements that are present.
And it just -- they
look completely nuts.
Okay? But the message there is
don't panic, instead of looking
at such crazy complexity, we
as chemists can simplify things
down to seven major pathways.
And I contend that if you learn
these seven pathways you'll be
equipped then to understand
anything no matter how complex
it is.
All of that complexity
can be reduced
down to just seven elements
-- or seven different types.
And if we learn those seven
types we'll be in good shape.
Okay, we looked at the
very first of these seven,
which were the nuclear receptor
base pathways, and we looked
at how steroids have potent
activity through binding
to these nuclear receptors.
And then we saw that these
have two different modes,
that there were two modes of
the nuclear receptor pathways,
hetero-dimerization
and homo-dimerization.
Any questions?
All right.
Well let's return to our
seven canonical pathways.
I'll remind you again that we
looked at the steroids binding
to their nuclear receptors
and the key element
here was the notion
that these nuclear receptors
bind directly to the steroids,
and then these complexes
make their way all the way
to the nucleus where DNA
is turned on or turned off
and transcription takes place.
That's a very powerful paradigm
for effecting cell division,
cell growth, and this is why
these steroids like testosterone
and estrogen have such
potent cell-based effects.
Today I want to pick up
our discussion and discuss
with you the other six pathways
found in a cell, and again,
I'm going to be emphasizing
what's common
and what's simple
about these pathways.
We could get crazy complex
but it's just not --
it's not pedagogically useful.
Okay, so I'll show you kind
of the stripped down version
and if you start,
like, you know,
launching a research
project on one
of these pathways
you'll find quickly
that things get a
little more complex,
but not much more complex.
Okay? All right, so
let's get started
with the two-component pathways.
These are pathways in which a
ligand binds with the outside
of the cell up here and then
dimerizes two receptors.
Oh, and by the way, just as a
quick preview, note that many
of the upcoming pathways
are going to feature ligands
that don't even get
inside the cell.
Okay? Like these guys over here.
So here's a ligand,
it's binding up here.
Here's another ligand in
purple binding up here.
Another ligand binding up here.
None of these ligands get
through the plasma membrane,
instead they're going to affect
the conformation of proteins
that are found on
the cell surface
and that conformational change
will transduce the signal
from outside the cell
to inside the cell.
Okay? So that's going to
be our paradigm for today.
All right, let me
skip on, one moment.
Let me see if I can
find where we left off.
Okay, we talked about quite
a bit last time, didn't we?
Okay. Here we are,
number two in our seven.
In this case I want
to talk to you
about controlling cell
differentiation through a class
of molecules called cytokines,
also known as interleukins,
variously called interleukins.
This is one of the annoying
aspects about biology,
is that they don't have a
uniform nomenclature the way we
chemists do.
So, the periodic table is the
same here as it is in Russia,
or Sudan, or anywhere
in the world,
but biologists don't have IUPAC
so they have a little bit
more difficulties getting a
standardized nomenclature.
Okay, so be that as it may,
cytokines are an important class
of molecules for causing
cells to differentiate
and the differentiation
I want to talk to you
about today is the
differentiation
into various blood cells.
Okay? So this includes red
blood cells, erythrocytes.
It also includes B-cells, and
T-cells, which are crucial
to the immune response,
to responding to attack.
But it also includes a number
of other different variants,
like platelets, which we've
already seen as being crucial
to blood clotting, et cetera.
Okay, so all of these signaling
ligands highlighted in bold
and blue are examples of
cytokines that all more
or less have the same
sort of signaling pathway.
So rather than us saying, "All
right, let's learn about IL-4 --
all right, now forget everything
I told you about IL-4,
we're going to talk
about IL-2 next."
Rather than doing that I could
just tell you about one example
and then from that one
example you can figure
out everything else.
Okay? So, let's dive right in.
I'm going to be talking to
you about a favorite of ours,
one that we've seen before.
This is growth hormone.
Human growth hormone
causes acromegaly.
This is characterized by
abnormal growth features
in the face, the face
looks a little distorted,
hands and feet, just abnormally
sized large hands and feet.
I mean, really abnormal.
Okay? So, I'm realizing
this slide is out of place.
All right, we'll get
back to growth hormone.
Let's get back to
-- let's focus in --
the example I'm going to show
you will be erythropoietin,
right here.
And again it's generalizable
to a bunch of other molecules.
Okay, so this is a
very typical mode
for these two-component
pathways.
Two, meaning that they are going
to cause dimerization
on the cell surface.
And so here's the molecule
EPO up here, erythropoietin.
Erythropoietin stimulates the
formation of red blood vessels,
of red blood cells, and the
way it works is EPO, up here,
dimerizes an EPO receptor
on the cell surface
and that allows an
associated kinase, JAK2 kinase,
to phosphorylate each other.
Okay? So this is an example
of transphosphorylation.
This guy phosphorylizes
this guy,
which phosphorylizes this guy.
That in turn allows
phosphorylation of STAT5,
which dimerizes and then STAT5
actually gets into the nucleus
and causes transcription
down here.
Now, a closer look at the
phosphorylation is in order
because this is going
to be a major mode
for relay of the signal.
Okay, so you know like
in track and field
when you have a relay
there's a baton and you kind
of pass the baton,
hand over hand,
this is a similar
type of phenomena.
In this case the baton is going
to be a phosphorylated
amino acid side chain
on the surface of the protein.
And that phosphorylated amino
acid side chain is going
to create a new surface that
then allows something to bind
that otherwise would
not be allowed to bind,
or it could change the
conformation of the protein
after it's phosphorylated.
Okay, so what that means is
STAT5 has one conformation
up here, it gets phosphorylated
and then it dimerizes
and that [inaudible]
phosphorylation,
it's not dimerized, it's
a monomeric protein.
Upon dimerization it has
a different conformation,
one that can bind DNA, one that
can turn on transcription, oh,
and one that can get
through the nuclear pore
to get into the nucleus.
Okay, so this is also
termed a Jak-STAT pathway
because JAK kinase and
STAT5 -- or STAT proteins,
are playing a key role
in this cell signaling.
And note too that
it's fairly direct.
There's only a small number of
proteins between signal up here
and then effect down
here in the nucleus.
I'll show you examples that
aren't nearly as direct.
This is a good place
to start, though.
All right, looks zoom
in and take a look
at exactly what's happening.
So, here is erythropoietin
binding
to its EPO receptor
on the cell surface.
These are also called
Janus-faced kinases
because this is Janus the
doorway god, you know,
that also has two faces,
two homodimerized faces
looking both ways.
In any case, EPO is up here,
so even though EPO is not a
perfectly symmetrical protein it
results in a pseudo symmetrical
dimer of these EPO receptors.
Okay? So it's not --
nothing is exactly symmetric
but it's pretty close
to symmetric.
Okay, and again, that's
the characteristic
of these two-component
signaling pathways.
Now, earlier I showed you
that the STAT5 molecules upon
dimerization combine to DNA.
Here's the mode of
binding to DNA
where the DNA is
coming up towards us.
So imagine this long, you know,
B-strand or B-helix DNA coming
out towards us and
extending way out in space.
And you can see that
the dimer has the DNA
in kind of a vise grip.
It's clamped over the DNA
and its looking actively
for a correct DNA sequence
that it can then grab onto
and signal for transcription.
And again, the transcription
is going
to involve the usual
players, RNA polymerase
and other transcription factors
that jump on and get involved.
Okay, so if all we have to
do is dimerize something,
grab two things together
on the cell surface,
then we chemists can probably
do something like that.
And so, one way to do this
would be to identify a molecule
that binds to one half of the
dimer, like this guy over here
that binds to GyrB, and then
simply form a pseudo symmetric
dimer of it.
It's pseudo symmetric
because notice this stuff
in pink is not exactly symmetric
so this is not C2 symmetric,
meaning you can't just
flip it over like that.
But it doesn't really
matter because as long
as you get these guys close
enough for the two JAK kinases
to auto phosphorylate each other
then the signal transduction
takes place and then the
STAT5 gets phosphorylated
and then it dimerizes and then
it goes on to transcription.
Okay? So, again, all we have to
do is identify a binder to one
of these receptors and then
by simply forming that monomer
into a dimer, boom, we get
something that could turn
on signal transduction.
And anyone have an idea why
you might want to turn on EPO,
or why you might want to
provide erythropoietin
to patients, for example?
What circumstances would
you want red blood cells
being produced?
>> After surgery.
>> After surgery.
Yeah, so after surgery the
patient is low on blood,
lost a lot of blood, and
blood all over the place
so you routinely give EPO as
a way of stimulating blood --
you know, blood -- red
blood cell production.
Of course this is
wildly abused by athletes
who also value red blood cells
for their oxygen
carrying capacity,
and up until recently there
were no effective tests
for erythropoietin abuse.
There are now really good
tests and this is one
of the reasons why you're
seeing all these scandals come
out as athletes are
getting caught.
In fact, there was one in
the news in the last week
where athletes that had competed
eight years ago were caught
because their samples
were still refrigerated
and you can actually detect the
artificial EPO in those samples.
So from eight years ago.
And I think that's going
to happen more and more,
that we chemists are going
to get better and better
at analyzing samples and
finding tiny little quantities
of unnatural compounds
and, you know,
and nailing athletes
who are abusing them.
And I think this is
a good trend, okay,
because as a sports watcher I
want to watch real athletics
and not chemical athletics.
So, okay. A little
off the topic.
Okay, so you now know that you
can readily make a chemical
version of EPO using
a strategy like this.
Okay? So, all right.
Now, another strategy
would be a lot cruder.
Okay, so the thing about
this that's lovely is
that this compound over here
is specific just for GyrB.
Okay, but a much more crude
way to do this would be
to simply get in and
form a covalent dimer.
Right? This is a non-covalent
dimer and it has some chance
to be specific, but a much less
specific way to do this would be
to simply add a chemical
cross-linker.
And I'm showing you this
because it illustrates a really
important reaction and one
that's used quite a bit
in chemical biology
and biochemistry labs.
Okay, so in this reaction on
the interferon-Beta receptor-2,
there are going to be lysines.
Okay, it's almost
unavoidable to have lysines
on the surface of protein.
Lysine is this amino acid side
chain that's frequently charged,
typically positively charged,
and it's very solubilizing.
So the outer surfaces
of proteins are almost always
studded with lysine residues.
And so for this reason you can
react these lysine amine side
chains, shown here in
their neutral state,
with an activated carboxylate.
Okay? So this is like a
carboxylate except instead
of an OH here -- instead of an
O minus, if it's carboxylate
or OH, if it's carboxylate acid,
instead this has an
N-hydroxysuccinimide ester.
Okay? And this NHS ester is
a really good [inaudible].
And so what that means then is
that this can very
readily form an amide bond
and give you a covalent bond.
This is completely analogous
to the O-acylisourea activated
carboxylate acid that we saw
when we talked about
the amide bond formation
by DCC many weeks ago.
Okay? So four weeks ago
or something like that
when we talked about
how to make amide bonds,
we talked about how you have to
activate the carboxylate acid.
In this case we're starting
with something that's already
activated as NHS ester,
and what's extraordinary
about its reactivity is
that it reacts specifically
with amines and not all
of those OH groups
that are present
at 55 molar concentration
in water.
Right? Water is a whole
bunch of OH groups.
And so this kind of
chemical specificity,
or chemo specificity,
allows this to pick
out these amine functionalities
and then if there are two amines
from neighboring interferon-Beta
receptors these two amines can
be dragged together to
form a covalent bond,
where they're linked
together covalently.
And again, once you've
covalently cross-linked things
you've brought these
guys together
and you can start signaling
through the Jak-STAT pathway.
Okay? Now, I want
to take a moment
to show you how this is useful,
how this reaction is useful.
This thing, not so useful.
Okay, yeah, you can do this
experiment, it will work,
but honestly you're not going
to be giving your patients this
compound as a pharmaceutical
because it will react with every
other lysine that it finds.
It has zero specificity, zip.
Okay? And who knows what kind
of allergic reaction the
patient will come down with
if you inject that into them.
Okay, but a much
better way to do this,
or a much more useful aspect
of this reaction is to use it
to modify proteins to
improve their solubility.
So, for example, interferon
alpha 2B, this compound here,
it's a protein therapeutic,
is typically pegylated,
meaning there is this
polyethylene glycol tail that's
appended to the protein.
And so the way this
reaction works is you start
with an NHS ester of peg.
Okay, so again, this
is polyethylene glycol,
this is a polymer that
you've seen before,
maybe not in this
class but certainly
in sophomore organic chemistry.
And because it has an activated
carboxylate it will come along
and modify all these lysines and
even these histidine residents.
And what's remarkable about
this is if you control the pH
and the buffer conditions
you can set this up so
that there is only one position
in this complicated protein
that actually gets modified.
And when we look closely at this
what we find is not the lysines
at pH 6.5 that get modified,
at pH 6.5 all these lysines
are protonated, and again,
if they're protonated --
if these guys are protonated
there's no way they can react
with the activated carboxylate.
Instead at pH 6.5 the only thing
that's available to react is one
of these histidines, I
believe it's this one.
And so, the PEG gets appended,
gets conjugated specifically
just to one of these histidines
over here, and nowhere else
in this complicated protein.
And that's really exquisite
reactivity and selectivity.
And so for this reason this
is used very extensively
and these types of things are
used extensively in therapeutics
and protein therapeutics.
Okay, why would you
want to do this?
Why would anyone want to
put PEG on your protein?
It's a little bit of a trick
question, but come on you guys.
[Inaudible] you seem to be
thinking about something.
>> [Inaudible] increasing
the solubility?
>> Yes. All right, well done.
So, what [inaudible]
suggested was that this PEG
over here is going to be
highly water-soluble, right?
It's going to have a lot
of hydrogen bond acceptors,
and so you could take a protein
that would be relatively
insoluble
and make it more soluble.
In addition, you're
increasing its molecular weight.
N over here can be really big.
It can be hundreds to thousands,
and so you can take a
relatively small protein
that would get excreted
very readily by the kidneys,
increase its molecular weight
and then it gets recycled back
and prevented from
being excreted.
So there's some huge
advantages to solubilization
and even doing things
like decreasing the
protein's antigenicity.
Okay? So decreasing
how recognized it is
by the immune system as
being a foreign invader.
Okay, all those are
really important properties
and so this reaction is
used very extensively.
Okay, any questions about this?
All right, notice that the
HPLC over here indicates
that you're getting one
and only one protein out.
If this was getting
pegylated in a whole bunch
of places you would see
lots and lots of peaks.
Okay? So, the dominant
peak here is number two
and these guys are
relatively small.
All right, I'm going to move on.
I want to talk to you
next about growth hormone
and growth hormone pathways.
And earlier I showed you
this idea of acromegaly,
which is too much growth hormone
leading to gigantism, you know,
excessively large
hands, feet, and chin.
So, this is our third example
of a signaling pathway.
This is a classic, this
is used extensively
by many different signaling
ligands including TGF-Beta
up here.
And in general -- so this
very common, very typical,
this is a biggie -- in
general all of these work
by the same mechanism.
The mechanism is dimerize
the receptors up here
and then autophosphorylate,
meaning this kinase
over here phosphorylates
the one next to it,
and then this guy phosphorylates
the one next to this,
and then that leads
to some sort of --
and then that allows a kinase
to phosphorylate a nearby
other protein that then goes on
and eventually get down
to the DNA down here.
Okay? But the important
thing is dimerization
then autophosporylation.
And we're just going to see that
again, and again, and again.
Okay? So, here, for example is
growth factor receptor up here.
So, sorry, this is growth
factor receptor in purple.
These are also called
receptor tyrosine kinases
because they're acting
as kinases
to phosphorylate tyrosines on
each other, and then you end
up with a whole series
of different phosphorylated
tyrosines.
This thing basically
becomes loaded
up with all these
phosphorylated tyrosines.
Okay, everyone still with me
on the cell surface stuff?
Dimerize, autophosphorylate.
All right, here's where
it gets complicated.
Once you get the dimerization
and the phosphorylation a total
of four pathways
are then activated.
This thing goes on
and, for example,
it engages the STAT
subpathway that we saw earlier
when we talked about
the two-component
signaling pathways.
It always gets into the
MAP kinase subpathway,
which I'll describe next to you.
But it also starts
to effect PI3 kinase
and PLC-Gamma pathways, as well.
These are pathways
associated with fatty acids,
which we briefly alluded
to earlier, last --
or later on Thursday of
last week when we looked
at fatty acid synthesis
and signaling in the cell.
And I'm going to show you
-- I'll be able to pick up
and show you exactly
what's going on when we talk
about that kind of signaling.
All right, before we do
let's take a closer look.
This protein at the
top is growth factor,
human growth hormone,
or growth factor.
It's a ligand and it's going
to dimerize the receptor
tyrosine kinase,
RTK, at the cell surface.
And good news, we've
already seen this protein,
this is a familiar
protein to us.
This is human growth hormone
over here, and it's going
to initiate this dimerization
through having two
binding sites.
And earlier in this
quarter we talked about,
in fact this is one of our
very earliest lectures --
this might have been like
the third week or something.
We talked about one
of these binding sites
and how it had a hot
spot of binding energy
for binding two the
receptor tyrosine kinase,
human growth hormone binding
hormone, on the cell surface.
There is a second binding site
that looks completely different
than this one on the other side
that allows the dimerization
of the receptor tyrosine
kinase to take place.
Okay, so this is our model for
receptor-ligand interactions,
which is why we seen it before.
But you don't necessarily have
to have large proteins
doing this dimerization.
In fact, small molecules can
be very potent at doing this.
And so, for example,
this PD compound
over here has great specificity
for particular growth factor
receptors on the cell surface.
And these numbers over here
are IC50's and nanomolar,
and recall that smaller number
indicates higher potency, right?
Because that means a lower KD
which means greater affinity.
And notice that these compounds
are selective for the FGF,
the fetal growth factor
receptors, FGFR1, 2, and 3,
versus VEGF, this is a vascular
endothelial growth factor
receptor, which stimulates
angiogenesis,
the growth of blood vessels
that we talked about earlier
when we talked about proteases
and defining amino [inaudible].
Okay, so sort of specificity
for one class of these receptors
over other receptors in the
same class is [inaudible]
and medicinal chemists will
spend a lot of time trying
to optimize the structure
of a compound like this one
such that it will be specific
for one of these receptors
or maybe for a whole class of
these receptors, but then avoid,
say, EGFR endothelial
growth factor receptor.
Or, you know, something
like SRC,
which is really broad activity.
Okay, and what this means
then is then you can have ways
of targeting tumors selectively,
and what we're showing
here are tumor treated --
are tumors dissected out of an
animal that had been treated
with the compound on the
left versus controls.
And check at how much
smaller these guys are.
Okay? So that's a good sign.
So, if we had ways of
targeting, blocking,
inhibiting growth factor
receptors we'd have really
potent anti-cancer compounds,
and this is one of the frontiers
in medicinal chemistry
and chemical biology.
Many of these get their
specificity through binding,
not to the ligand binding
site that was highlighted
by this ligand over here where
the ligand likes to bind.
Rather, they seem to prefer to
bind to the ATP binding site,
and that actually seems
to be a surprising area
for gaining specificity.
You wouldn't expect this, right?
Because you'd expect
every kinase that uses --
all kinases use ATP, that's
their source of phosphate --
but you want it -- and you'd
expect them all to bind ATP
in the same sort of way.
But what we're finding
through efforts
of medicinal chemistry is
that actually there's
enough variation
in the ATP binding site that
you can alter the structures
of these compounds and
gain specificity for, say,
FGFR versus all of these
other receptors down here.
Okay? And so what I mean by
this, by varying structural --
varying the structure
would be, for example,
changing the structure of
this urea functionality.
I'm showing with a T-butyl urea.
You might want to
try a phenyl urea.
You might want to
try, I don't know,
a straight butyl
urea, et cetera.
So you could imagine making
lots and lots of little changes
over here, over here, over
here, and doing this to dial
in the specificity for
a particular receptor.
This is a property that's
also known as selectivity.
Okay? Make sense?
All right, let's take a closer
look then at phosphorylation.
Phosphorylation is not just
random, so these guys are going
to be autophosphorylating
each other, you know,
punching Judy or something.
But they're going to do it in a
very specific sequence of blows.
In other words, they're going
to have a very defined pathway
of one phosphorylation allows
the next phosphorylation,
which allows the
next phosphorylation.
And that's illustrated by
these numbers over here
which detail this guy
gets phosphorylated first,
this guy number two, second,
three, four, and five,
and so on and so forth.
And so the longer
this dimer hangs
around the more likely it is
to actually allow signaling
into those various pathways.
And remember those four pathways
that are going to be implicated
by this one receptor up here.
Okay? So the receptor
tyrosine kinase goes to town
but it doesn't start
going wild and randomly,
instead it is very specific and
in that specificity it's going
to be turning on
specific pathways.
Let's take a look at
one of these pathways.
This is the MAP kinase
pathway, this is one of the four
that the protein is
going to signal through.
So, after this phosphorylation
takes place a protein
with an SH2 domain, called SHK1
can grab onto this phosphate
and then get phosphorylated
itself, allowing Grb2 to bind.
Grb2 also has an SH2
domain, SH2 domains bind
to phosphorylated
tyrosine, in fact,
I think we saw them
earlier in the quarter,
but Grb2 has a polyproline
helix that can be bound
by an SH3 domain, allowing
binding to [inaudible],
allowing binding to [inaudible],
and then in the end MEK
can phosphorylate ERK,
which then gets into
the nucleus.
And remember earlier I
told you how there was
alternative nomenclature?
So, MEK can also be
called MAP kinase kinase,
and then it phosphorylates
MAP kinase.
I've even seen MAP
kinase kinase kinase.
So, there's a bunch of other
kinases in this pathway
that I'm sort of not showing you
and I'm also simplifying
the nomenclature.
I'm using the simplest
nomenclature as well.
Okay, the important part here is
that there's kind of a hand off
of signal that involves not just
phosphorylation but interaction
through an SH2 domain,
and we've seen
that earlier in the quarter.
Earlier in the quarter I showed
you how polyproline sequences
will bind into SH3 domains.
In fact, here's the
structure that I showed you.
Here's an SH3 domain in green,
here's the polyproline helix,
and you remember this is the
helix, this is the 310 helix
that if you look down this axis
you see how it looks triangular?
It looks triangular here,
this is what the
polyproline looks like.
So this is a familiar
concept to us.
This is something that
we've seen before,
we've also seen SH2
domains as well.
Okay? So Grb2 has an SH2 domain
to bind to the phosphotyrosine
on SHC1, but it also has an
SH3 domain that binds to Son
of Sevenless, SOS over here.
Okay? And then SOS binds to Ras,
which binds to Raf, et cetera.
Okay, so everyone still with me?
All right, there's
one other element
that this pathway illustrates
that's also prototypical.
This is one of those things
that you see quite often.
On the cell surface -- cell
surface is not a, you know,
sterile, neatly,
diluted environment.
Rather, the cell
surface is complicated.
It has a lot of shrubbery
on it and in fact,
amongst the shrubbery
is long chains
of this stuff called
heparan sulfate.
This can then attract FGF,
it can attract the ligand
that these growth factor
receptors like to bind
and increase their -- enhance
their apparent affinity
for binding to the receptor
simply by a proximity effect.
Okay? So, the shrubbery up here
the the heparan sulfate grabs
onto any stuff that happens
to be passing by and then
because now it's localized,
up close to the receptor,
the receptor can then more
likely, more easily find it,
and that in turn enhances
the signaling abilities.
Okay? Sound good?
Okay, so these are common modes.
We're seeing the common
mode that I want you
to learn is this
idea that heparan is
out here to enhance binding.
Dimerization takes place.
Autophospherization takes place.
There's binding through
SH2 and SH3 domains,
and then a relay
of phosphorylation.
Phosphorylation,
and then finally
into the nucleus
for transcription.
Okay? It's like Yogi Berra said,
baseball, it's just running,
throwing, catching, hitting,
what could be more
complicated than that?
Okay? So there's a few
elements here but when you get
into this area you just see
these same elements repeated
over and over again.
Learn these elements,
you'll be able
to understand everything else.
Any questions so far?
We good? All right,
let's talk a little bit
about the other pathways
that these receptor tyrosine
kinases like to stimulate.
Another pathway are
these pathways
that are fatty acid
signaling pathways.
These include
phosphoinositol-3-kinase.
This is phosphonoinositol-3.
There are kinases that
phosphorylate this, so PI3K,
phosphoinositol-3-kinase,
I'll just call it PI3K,
phosphorylates the third
hydroxyl of this glycan.
Okay? So this glycan is
appended to a fatty acid chain
and upon phosphorylation
this gives you something --
it gives you a different
structure.
Right? We now have something
that has three phosphate
groups attached to this glycan.
And this then can allow binding,
this is called phosphoinositol-3
or PIP3.
PIP3 can then be bound by
these PIP3 binding sites.
Okay? The effect here --
so this is a protein called
protein-kinase-C, or PKC.
PKC is floating around the cell.
Okay, it's happy as a bee,
you know, it's banging
into flowers looking
for nectar --
whatever it is it does all day.
Okay, until it finds PIP3.
Once it finds PIP3
it gets ensnared
like a fish getting
stuck onto a fishhook.
At that point PKC is
stuck at the cell membrane
and this localizes PKC
at the cell membrane
where it can start doing
all kinds of things.
Okay? So again, this is that
principle of localization.
Okay? Localization is
what allows these things
to then get turned on and then
in turn affect the pathways.
It's kind of like that
old adage in real estate.
Location, location, location.
Okay? And so in the same way
that heparan is confining these
ligands to particular spots
in the cell this PIP3
is going to confine PKC
to particular spots at the
plasma membrane and that
in turn is going to allow the
pathway to get kicked off.
Okay? So it's not simply
activity it's also localization,
localization, localization that
gets things into the right spot
where they can do
the most signaling.
Okay, make sense?
Okay, now this is a
grisly simplified portrait.
When you start looking at this
in more detail you start finding
that there are other ways
of shutting this off.
So for example you can free
up the glycan from the rest
of the [inaudible] tail by a
hydrolase PLC-Gamma over here
and things get more
complicated as well.
Okay. In addition to
the heparan sulfate
that I've already told you
about and the various IP3's
and PIP3's, there's also a
curious role played by calcium
and the release of calcium.
So, IP3, the release of the IP3
from the PIP3 has the effect
of releasing calcium
into the cell and this
in turn unhinges a binding
site to allow binding to stop
down here at the
plasma membrane.
Okay, so all of this
stuff is working together.
Okay, so you have a really
complicated dance where one
of the major goals of the dance
is simply to get the players
into the right space
at the right time.
Okay? And calcium is great
because it diffuses very readily
through the cell and it
works really well for this.
Okay, any questions about
receptor tyrosine kinases,
growth hormone, et cetera?
Okay. This is 90%
of cell signaling,
really is what I just told you.
Okay? I'm now going to go
on and I'll talk to you
about the other -- oh, sorry.
I didn't mean to say 90%.
30%, 40% of cell signaling that
you read about in Science,
Nature, and Cell deals
with these pathways.
I want to talk to you
about another 30% or so.
This is a really important one.
G protein-coupled
receptors are some
of the most common proteins
in the human protium.
These really are extremely
abundant to us humans
and in fact, these
are the proteins
that make life worth living.
These are the things that
allow us to see like Rhodopsin
over here, that allow
us to smell,
like the olfactory
things, allow us to taste,
here's some tasty
ones, et cetera.
All of these smell molecules,
taste molecules work by binding
to G protein-coupled
receptors, which look like this.
This is a trans-membrane
protein,
where the membrane is
going through here.
Okay? So the membrane cuts
through this over here
and the ligand is
very deeply buried.
It gets snuggled down
deep in this GPCR,
and inside the GPCR it changes
the conformation of the GPCR
such that the stuff in here
that's going to be signaling
on the inside, in the cytoplasm
of the protein has
a new conformation.
Okay, so these bind to a
very wide range of ligands.
In fact, many of these are
promiscuous in their activity,
meaning that they'll bind
to a bunch of tasty things,
and once that happens that
allows the conformational change
that communicates from what's
happening outside the cell
to the inside of the cell.
Okay? So that's kind of
at the very cell surface,
let's take a closer look.
Okay? I have to tell you a
little bit about how these work.
I first want to tell
you about dynamic range.
Okay? So I'm going to
digress for a moment
and then we're going to
take a closer look at GPCR.
Okay, so if for example
you had some receptor
that simply was turned
on and off depending
on if you had binding
by a ligand,
you'd have a kind
of a lousy sensor.
Okay, so these have
to act as sensors.
Okay, if you're going to be able
to, say, sense light changing,
you know, for you know,
moving towards light
if you're some organism or, you
know, being able to smell stuff
so that it determines whether
or not you eat this food
that's going to kill you
or not kill you or make it
possible for you to live,
there's an incredible
premium on being able
to sense tiny little quantities
and at the same time being able
to sense really big quantities.
Okay? And that's a property
called dynamic range.
If the sensor simply operates
on the principle of bound
or unbound your dynamic range
is going to be very limited.
Okay? And let me remind you
that this principle of bound
versus unbound is governed
by an on-rate and an off-rate
that together have a
dissociation constant associated
with it.
The truth is this offers
a limited dynamic range
as epitomized by this
percent activity plot
that we showed earlier
in the class.
Recall all of these
have the sigmoidal plot.
Okay? The sigmoidal
type of relationship
between concentration of ligand,
and activity on the Y-axis.
The problem with this
is that this type
of receptor would be
sensitive only in these three
or four orders of magnitude,
in concentrations from 10
to the minus seven, to
10 to the minus four.
Okay, so like submillimolar
to submicromolar.
And that might be okay, but
it's not so okay if you have
to smell, I don't know, tiny
quantities of some pheromone
to know whether or not,
you know, you're ever going
to find a mate in your lifetime.
Okay? That could be
kind of important
if you're an organism, right?
And so this business
about sensing only
in one particular range is
pretty much worthless, okay,
for really good sensing and
devising really good sensors.
So instead all of the GPR's
rely on a different principle.
Before I get to the
different principle,
does anyone have
questions about this one?
Does it make sense to
you that this is kind
of limited in its utility?
Okay. I always get nervous
when there's no questions.
Take your word for it.
Instead the G protein-coupled
receptors signal
through a G protein.
They all will bind directly to
something called the G protein,
and the G protein is a catalyst
that catalyzes hydrolysis GTP,
this guy up here, magnesium
GTP complex, to magnesium GDP.
So it's simply hydrolysis
of the Gamma phosphate
of GPP to give us GDP.
Okay, straightforward reaction,
a reaction we've seen
before, an easy one.
Right? An easy -- reaction
we can derive in our sleep,
we can draw a mechanism
in our sleep.
Here's the important part.
By coupling binding
to the activity
of an enzyme you can very
sensitively tune the activity
and you can tune the sensor.
Okay? So it becomes a
really exquisite sensor
for low concentrations
and high concentrations.
And I realize it's not
clear at this moment,
I'm going to show you
on the next slide.
Before I do, though,
everyone with me at this idea,
GPCR's bind to G proteins,
G proteins are catalysts?
We're good with that one?
Okay. So check this out.
Here's what's really going on.
Okay? So, the G protein in its
GDP bound state says signal off.
Okay? So GPCR is up here, if
the G protein however is bound
to GDP, signal gets shut off.
On the other hand if it's bound
to GTP the signal
gets turned on.
Okay? And what's important is
that there are two intermediary
enzymes that are going
to catalyze and help
push this equilibrium
between GDP bound and GTP bound.
These two are a guanine
nucleotide exchange factor,
a GEF, that helps push out a
GDP to allow binding by GTP,
and then in the opposite
direction a GTPase-activating
protein, or GAP, that helps
drive the phosphorylation
of GTP to GDP.
Okay, now here's
the way this works.
Your cells can carefully
control the ratio of GEF
to GAP, or GAP to GEF.
And by doing that they
can make the signal,
the sensor more sensitive,
or less sensitive.
Okay? And if you want it
to be more sensitive
you basically set it
up so it's always signal off.
Right? So then you'd have
lots of GAP's around,
the GAP's keep it in this off
state so if anything turns it
on over here then boom,
everything goes on.
On the other hand,
if you want it
to be less sensitive you keep
it in the signal on state
so it really takes quite a
bit to get it shut off again.
Make sense?
Okay, this is a really
powerful principle, and it's one
that I really have not
seen in too many areas
of electronics or chemistry.
And it's one that I think
really could be exploited
for interesting uses.
[Inaudible]?
>> There is only
water [inaudible]
so how can you actually
turn it off?
>> Okay, so you mean
how does this happen?
>> Actually, I think
this only happened
because there is
only water inside --
>> [Inaudible] all of this
is taking place in water.
So it's 55 molar water.
But the GTP over here, bound
by the G protein is getting
neutralized at a very slow rate.
The catalyst here is not
such a great catalyst.
And GTP in water is completely
stable, it's not going
to get hydrolyzed, it needs
an enzyme to hydrolyze it
on the time scale that's
relevant for biology.
Okay? Any a question
over here, Sergio?
[ Inaudible Speaker ]
Okay, so this is happening right
up close to the plasma membrane,
right up close to
the cell surface.
Okay, so the GPCR is right
there at the cell surface,
the G protein is right
below it, grabbing on.
Holding on for dear life.
And then these GEF's and
GAP's are forming, you know,
another piece of this sandwich,
so then you get these
monster complexes.
Okay, yeah, and over here.
>> So these seven [inaudible].
>> Yes.
>> [Inaudible] have
ligand and receptor
and then just [inaudible]?
>> You wouldn't get
the same dynamic range.
Okay? So if you address
the concentration of ligand
down here you're only going
over [inaudible]
magnitude of dynamic range.
Okay? But if you have this GEF
and GAP system then you
can tune this to make --
to kind of shove this over
so that it's now responsive
to ligand over here
or ligand over here.
This gives you responsiveness
over all
of these -- this dynamic range.
Okay? And this is what you need.
You know, let me explain.
Okay, so this is the same
set of receptors that's going
to allow you to see and,
you know, there's times
when you need to be able
to see in dark caves
to be able to survive.
Maybe not you, but certainly
your primeval ancestors.
Right? They would need to be
able to survive in dark caves
where humans can
sense one photon.
Okay? We can sense
just a few photons.
That kind of low
light sensitivity,
incredibly important.
On the other hand, eventually
you get out of the cave
and you're wandering around
in the bright sunlight
and if all you can
see are, you know,
if you can see each photon
individually you're going
to get totally overwhelmed
and you're not going
to be able to see anything.
Right? So you need a system
that allows you to be able
to sense things way down
here but then also be able
to shut off so that it doesn't
get overwhelmed way up here.
Okay, and that's really
the key to this activity.
So affecting the GEF and GAP
ratio controls that sensitivity.
Okay. Question in the middle?
Yeah?
>> Is that why our
eyes -- pupils dilate?
>> That's also part
of it as well.
Okay, but, you know, the
Rhodopsin that sensing photons,
that has to have some way of
being less sensitive at times
and more sensitive
at other times.
But certainly yes, you're right,
dilation of the pupils will
have an effect as well.
And over here?
>> Is there any way
that we can modify like,
the ligand to be more
sensitive because --
>> Okay, but if you
modify the ligand
and you make it more
sensitive --
let's say you're doing
taste or something.
You'd make it more sweet, right?
But eventually, you
know, you're over here.
So you're going to be
like hammering on it,
so now it binds really
well over here.
Eventually it's just
going to get overwhelmed.
It gets saturated.
Okay. Right, and so
everything tastes sweet.
That's not so pleasant either.
Okay. So, what happens
after the G proteins?
So, we're up here,
we have binding
to the G protein-coupled
receptors.
Here are the G proteins.
These have three sub units, and
Alpha, Beta, and Gamma sub unit.
Alpha is going to be
doing the GTPase activity
that I talked about.
This is then going to
lead to adenylate cyclase,
this protein over here.
Which turns over ATP
to give cyclic AMP.
What's neat about
this is that all
of these G protein-coupled
receptors converge
on this adenylate cyclase --
all is a little bit too strong,
but they are largely going
to converge over here
and adenylate cyclase acts
as an integrator of signals,
such as you can get
signals from a couple
of different cell
surface receptors,
turning on adenylate cyclase,
which in turn the
cyclic AMP turns
on protein kinase A. Protein
kinase A we've seen before,
we've talked about this protein.
This is the protein
that does the waltz
as it phosphorylates
different proteins in the cell.
We looked at a single molecule
level earlier in this class
and it's a good friend of --
everyone who's taking Chem 128.
PKA allows CREB, this
transcription factor to turn
on genes, for example, to
turn on genes to do something
about cardiac myocyte
hypertrophy.
Okay. So, it turns out that
the molecules that bind
to these GPCR's are
extremely diverse,
and in fact we humans
have learned to revel
in this chemical diversity.
So, for example, these are
the molecules associated
with the smell of peanuts.
So you know that nice
roasty smell of peanuts?
It's actually -- it's
not one molecule.
Rather, it's a plethora,
a little tiny combinatorial
library of these molecules
over here that are present at
these different concentrations,
that have different
odor thresholds,
and different odor
activity values.
And so the odorant receptors,
which are G protein-coupled
receptors are going to bind
to each of these and then
in turn these are going
to map to specific neurons.
Okay, so lots and lots
of different compounds.
Each one with their own
characteristic smell
but the sort of symphony that we
associate with the roasty smell
of peanuts is a bunch
of these present
at different concentrations.
So we need receptors that
are going to respond to each
of these different
molecules and do it in a way
that then integrates the
signal and gives us something
that reminds us of
roasty peanuts.
Here's the way this works.
These are the smell receptors,
the olfactory receptors
in the nose.
Here is the odorant over here.
Odorant combined to
several different receptors.
Okay? So here's the odorant,
it's going to bind weakly
to this guy, strongly to this
guy, but not at all to this guy,
and maybe to this guy over here.
All of the olfactory cells
that have this receptor
on their surface, all signal
back through the same neuron.
Okay, so if we map
the cells over here
in this olfactory region they're
all going to go back to one
and only one neuron,
all the ones
that have the purple
GPCR in this surface.
All the ones that have
the green GPCR go back
to a different neuron.
This is an astonishing
result that came up --
that was discovered in the
last two decades or so.
But in short what this means
is that everything that binds
to this receptor
signals in the same way.
You get the same neurons firing.
And in the end, using that GAP
to GEF ratio that I discussed
with you this can
allow signaling
that tells the neurons whether
or not you have a strong
binder or a weak binder.
So over here you have a strong
binder, you get more signal out,
over here you have
a weaker binder,
so you have a weaker
signal coming back out.
And again you can
control the dynamic range
such that you don't get
totally overwhelmed if you go
into a peanut factory,
so that you know,
you're smelling peanuts
for the next week
or something like that.
Okay? All right.
Let's talk about vision.
I talked to you -- I
mentioned this earlier,
this is one of those amazing
chemical reactions that all
of us are reliant upon.
We humans are especially
reliant upon vision
as our primary sensing organ.
And what's remarkable about
this is the goal here is
to capture a photon.
And your expectation is you
can't bind to a photon, right?
How do you capture a photon?
So, instead the GPCR associated
with vision, called Rhodopsin --
Rhodopsin over here,
covalently links
to a cofactor called Retinal.
Okay, so this is a structure
Retinal, it forms a shift base
with a lysine in the GPCR.
This is normally
found in a cis-olefin.
Okay, so this 11 12 carbon
carbon double bond cis.
Upon being irradiated by
a photon, that's H new,
the cis-olefin isomerizes
to give a trans-olefin.
And the net effect here is
a flipping of the retinal.
Okay? So we go from
cis to trans,
and this whole cyclohexane has
flipped from being over here
to being now extended.
That has the effect of changing
the conformation of the GPCR
and allowing you to see.
This is how you are
seeing me right now.
This is how it really works.
Okay? Now, in recent years
chemists have started
to get creative and we've
been devising retinals
that are responsive to
different wavelengths.
And some of these might
be really interesting
for allowing us to see
at different wavelengths.
For example, to be able to see
in the infrared, to see in dark,
and so on and so forth.
You can imagine a lot of
interest in this by people
who like to see in the dark.
All right.
Let's take a closer look at
the structure of Rhodopsin.
So, again it's a GPCR,
notice that it's structural,
it's very similar to the GPCR
I showed on the first slide.
All of these GPCR's have
a common structural motif
consisting of seven
alpha helices
that circumvent the membrane
that are trans-membranes.
Here in purple is the retinal,
again, forming a shift base
at one end to a lysine
residue on this alpha helix.
And the protein undergoes a
dramatic conformation change
in response to that
cis-trans isomerization.
Cis-trans isomerization is huge.
Up here, this little change up
here, photon comes splashing
in over here, boom, you get
isomerization, and boom,
the whole protein changes
conformation changing --
effecting its binding to the
G protein, in turn signaling
through that G protein cascade
that we talked about earlier,
in turn leading to
stimulation of neurons
which again is what
you're doing when you see.
Okay, that's kind
of extraordinary.
All right.
Any questions about GPCR's?
Moving on.
Oh, Carl?
>> So does it just
flip back and forth?
>> Yeah, so it's constantly
flipping back and forth
between cis and trans,
and that's what you're
interpreting as vision.
All right.
Ion signaling.
So this is number five
in our discussion.
So, these arrows are
going the wrong way.
Sodium is constantly being
pumped out of the cell,
calcium is being
controlled carefully,
chloride is being
pumped out of the cell,
potassium is constantly
being pumped into the cell.
The cell maintains
an equilibrium
of specific concentrations
of its ions.
Those ions provide a way
of signaling very quickly
in the cell, in fact, most of
the cells fastest responses,
such as vision and stuff
like that are probably
going through ion channels.
Okay? So, the way this works is
that these ion channels can open
up pores that allow ions
to flow in and this leads
to an electrical signal that
can propagate along a neuron,
for example.
Okay so, it's carefully
controlled through transporters
and it provides a
really fast response.
Okay, this is the kind of
response where, you know,
if I suddenly make a loud noise
then you jump or something
like that, that's
the ion response.
You hearing that,
ion channel response.
Seeing, ion channel response.
Okay, so here's what
these channels look like.
This is a calcium
activated potassium channel
that allows potassium
to flow in.
Calcium binds down here and
changes the conformation
and then allows calcium
to flow out of the cell.
You can imagine that because
these channels allow a really
fast response such
as a muscle response,
toxins would very potently
target these types of channels.
And caribatoxin is
one in a large numbers
of different toxins that
target these types of channels.
Many of these toxins cause
paralysis, these are things
that are used by scorpions
and cobras, et cetera.
All right, that's all I have
to say about ion channels.
They're fascinating, ions
flow in, they flow out.
I want to move on.
Death receptors.
So this is a class of compounds
that binds to the cell surface
and instead of causing
dimerization they
cause trimerization.
Okay, so you get
this active trimer.
In fact, dimerization
is associated
with shutting off the pathway.
And so, for example, this
compound over here allows --
encourages formation
of an inactive dimer
that shuts off the pathway.
I'm getting a little
ahead of myself.
Here's what it looks like.
Ligand is up here,
ligand is a trimer,
pon binding the receptor
forms a trimer,
and then you get a series
of kinases [inaudible] --
you get a series of kinases
passing off a phosphorylated
response and then in the end,
NF-Kappa B gets into the cell
and causes transcription
and inflammation.
This is a highly regulated
process that's used extensively
in the immune system.
This includes things like TNF,
this includes interferon,
et cetera.
These are really important
pathways for turning
on inflammation, turning
on the immune system.
Okay. Last pathway,
number seven.
Diffusible gas molecules.
It turns out that we humans
are susceptible to signaling
by tiny little gas molecules.
Things like oxygen.
In fact, all life is going
to be very dependent upon
being able to sense oxygen.
You can imagine that if
you cannot sense low oxygen
conditions you're going to die.
Right? Because you need
to be able to learn
to move towards areas
where you can breathe.
Right? That instinct
to breathe when you're
under water is fundamental to
life and it's really fundamental
to all organisms that
depend on oxygen.
Okay? And so the way this
works is oxygen sensing
by the cell works
by taking advantage
of the chemical properties
of oxygen as an oxidant.
So, there is a concentration
of Hif-1Alpha in the cell.
This Hif-1Alpha is
constantly being synthesized
and constantly being degraded.
And this Hif-1Alpha upon
encountering oxygen oxidizes,
gets oxidized to introduce an
OH group that in turn allows it
to be degraded and then in turn
will prevent these transcription
pathways from taking place.
Okay, so when Hif-1Alpha
is present nuclear --
this gets in, causes
transcription,
which in turn causes -- turns
on anaerobic respiration.
Right? That tells the
cell we don't have a lot
of oxygen present,
we better kick
into gear the anaerobic
respiration aspects
of metabolism.
On the other hand
if oxygen's present
that oxidation introduces a
hydroxyl that in turn leads
to degradation of Hif-1Alpha.
So the key concept here is that
you have this concentration
of the Hif-1A that's
constantly being formed,
constantly being degraded,
and that gives you
a very rapid sense
of how much oxygen is present.
It's kind of like
this Hif-1Alpha is
like a thermostat
for oxygen levels.
It's constantly monitoring how
much oxygen do the cells have,
if the cell doesn't have
enough oxygen it's going to die
if it doesn't switch on these
anaerobic respiration pathways.
Nitric oxide is also another
very common cell signaling
molecule, and this
one works by binding
to iron porphyrin cofactors
of guanylate cyclase,
and in turn causing
blood vessels to relax.
So this is important for the
regulation of blood pressure,
and a number of compounds were
invented to affect this pathway.
And what was astonishing
is actually they weren't --
compounds like Viagra were
found to be less effective
as blood pressure regulators
and more interesting
for their side effects.
Okay? And here are their
structures over here.
These are structures
of Cialis and Viagra,
compounds in the
little blue pill.
Here's the way this works.
Okay, so this is nitric oxide
over here, it's going to bind
to a guanylate cyclase
and in turn that's going
to effect GTP being
converted to cyclic GMP.
These compounds inhibit
guanylate cyclase and in turn
that causes blood
vessel relaxation.
Okay? This is one pathway that's
very dependent upon calcium
concentration, et cetera.
Oh, sorry, actually -- sorry,
the compounds are going
to be targeting PDB5,
this compound over here,
which is effected by
cyclic GMP levels.
Okay. Any questions about any
of the seven signal
transduction pathways?
All right.
In that case that brings us to
my wrap up and the conclusion.
What I've been talking to you
about all quarter is a new way
of thinking about biology.
Thinking about it at the
level of atoms and bonds
where we can actually
make changes,
where we can think creatively.
And I hope for example the
proposal assignment has shown
you what it's like to
be at the frontiers
of this really exciting field.
So, these are processes
that you've probably seen
in other classes, you
know, certainly DNA --
DNA base pairing, et cetera.
But I hope by thinking about
these in terms of chemicals,
in terms of being
compounds that have atoms
and bonds we've introduced
into your thinking new ways
of thinking about
their reactivity.
For example, forming
cross-links,
depurination, et cetera.
Chemical biology lets us
start to address who are we.
It really gets us to answer
really big questions in biology,
and all of the structures that
I'm showing up here are things
that we've seen this quarter,
either we've discussed
in lecture or they're
in the textbook.
These include things
like [inaudible] toxin,
that targets ion channels
and causes paralysis.
So these are organisms on the
planet and we've seen lots
and lots of their
bioactivities in this class.
It also lets us address
where do we come from?
Right? When we talked
about prebiotic synthesis
it was very abstract.
Right? We were using it as
an example of aero pushing,
but chemical biology gives
us a foundation to start
to address really
philosophical questions.
Things that humans
have been grappling
with for a very long
time, and finally we get
to address them really at
the level of atoms and bonds.
And then chemical biology gives
us the control of our destiny.
It gives us the tools that
we need to devise compounds
that allow us to
overcome our being human.
That allow us to address things
like disease, starvation,
nutritional deficit, et cetera.
And we've been talking
about this all quarter.
Here, for example, is
the PEG interon molecule
that I showed earlier
in today's lecture.
This is the PEG, this
big blob next to it,
and then that's the protein
that I showed earlier.
These are really
important molecules.
These are molecules
that affect the world
and that allow us
to address diseases.
And in the end chemical biology
ends up changing society.
The compounds we talk about
have a big impact on our lives.
They have an impact that goes
beyond just medical health,
they have impacts
on, for example,
affecting athletes, for example.
Okay? So I want to
encourage you to join us,
join us at this frontier
of chemical biology.
I hope to see you in the
laboratories here at UC Irvine
and I'm really looking forward
to reading your proposals.
So I want to thank Natalie
for being our projectionist --
or for doing all the
videotaping all quarter.
[ Applause ]
And I have to thank Kritika
up here and Mariam down here
for being really
outstanding T.A.'s.
[ Applause ]
And I want to thank all of
you for a really fun quarter.
I've really enjoyed teaching
you and if you're joining us
on YouTube I hope to see some
comments from you on the class
and whether you enjoyed
it as well.
But I've really enjoyed teaching
this class and so I thank you
for a really great quarter.
Thanks a lot everyone.
Thank you.
[ Applause ]
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