25. Cholesterol Homeostasis 5 & Metal Ion Homeostasis 1

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visit MIT OpenCourseWare at ocw.mit.edu. JOANNE STUBBE: In
the last lecture, we were focused
on the proteasome. And we were focusing
on how you targeted the protein of interest
for degradation by attachment of ubiquitin. So here’s the model
that I presented at the very beginning. And right now, we’re focused
on how do you attach ubiquitins to a protein of interest
that’s going to be degraded. OK, and so here’s the
protein of interest the last time we talked
about all the linkages. We’re going to be
isopeptides where you have lysine on the surface,
or a lysine that’s accessible, that can then get attached
to ubiquitin, which at a C terminal end has glycine. So that’s glycine 76– that’s the linkage
common to everything. And then this ubiquitin, and
you can see from the structure, has a number of lysines
attached depending on what the function
is of the ubiquitin. It can be attached
almost anywhere. For the proteasome, we’re
focused on lysine 48. OK, which again, makes an
isopeptide linkage here. And you need multiple
ubiquitins to be able to get your protein
of interest degraded. OK, so what I want to do now is
talk about the equipment that’s required to attach
this ubiquitin molecule through the glycine
to the protein of interest. And so what we’re focused
on is the three enzymes– E1, E2, E3. So we want to look
at the attachment. And we’re going to
look at E1, E2, E3, and what their
function is in general. And so E1– and it turns out
in human systems there are only two of these proteins– by, again, by homology. And this is going
to be what they call the activating enzyme. OK, and so E1 is
going to be– so since we have thousands
of proteins that are going to be degraded
and only one of these E1s, it’s going to play a role
in many, many reactions. It’s sort of the lynchpin. Over there there’s a
cartoon of what I’m going to write on the board. But E1– and when we
look at the chemistry, a key player in the chemistry
are cysteines and covalent catalysis by forming thioesters. OK, you’ve now seen
this many times in the polyketide synthases. You’ve seen it in
fatty acids synthases. You’ve seen it in
cysteine proteases. This is the motif that nature
uses over and over again. And what she does is takes ATP
and then she takes ubiquitin. And I’m just going to put
G 76 at the C terminal end. So this is the C
terminal carboxylate. And what she does then is uses
ATP to activate the carboxylate so that it can be attached
to the cysteine, which then forms the thioester. OK, and sort of the
strategy is the same. I’m not going to write out
the details of this strategy. But there are two
ways ATP can be used. What does ATP use
to activate this into a good dehydrating agent? What does nature do? What are the two options? AUDIENCE: [INAUDIBLE] JOANNE STUBBE: So adenylate or
phosphorylate, alpha or gamma, you see this over, and
over, and over again. You have seen this used many,
many times in the first half of this course. We have talked about the tRNA
synthetases, the activating domains of the non-ribosomal
peptide synthetases. And so what you have
done in this reaction is activated the carboxylate. And just remember
this so I don’t have to keep writing this down. The G is there. It’s always attached to the
C-terminus of ubiquitin. And now what happens– so this is activated. And so you have a base. You never do any
chemistry with a thiol. And so one then forms that
a thioester by nucleophilic attack on the carbonyl. So what one ends up with
then in the first step is ubiquitin attached
covalently to E1. OK, so let me put that up. Again, all of this is written
down in your hand outs. And so what we have
now is E1, where we have S attached to ubiquitin. OK, so that’s the
activating step. The second step is– involves an E2. And that’s called the
ubiquitin conjugating enzyme. So this is going to react with
E2, the ubiquitin conjugating enzyme. What we see in humans again,
that there are about 40 of these proteins. And we still have
thousands of proteins that are going to be
targeted for degradation. So that would imply
that these E2s can be used in
multiple processes and during the targeting
process for degradation. So we’re going to see that E2– and so there are 40 of these. And we’ll see that E2 also
has a sulfhydryl group that is going to play a key
role in this reaction. So what we’re going
to do now is a simple thiotransesterification. So we’re going to transfer
the ubiquitin to E2 by thiotransesterification. So again, you have base. And you liberate E1. And now what you have is E2
with the attached ubiquitin. OK, so there were
40 of these things. And what does that
have to tell you– what does that tell
you about E1 and E2? I haven’t given you any
information about structure, but there are 40 E2’s,
so they obviously all have different structures. But these things have got
to be really flexible. And so we have a few
structures, but I don’t think it even
comes close to allowing us to understand really
sort of the specificity of these processes. And this is a major
focus of many people now. They’ve been discovered
for a long time, but people are still
messing around. And I think part
of the complexity relates to the
flexibility, which makes them harder to crystallize. OK, and so E3 are the
ubiquitin ligases. And the latest paper I read– they keep finding new ones–
but there are greater than 600 of these guys. OK, so even there,
since more proteins are targeted for
degradation than 600, E3s are going to be
used multiple times. And E3s are able to
form protein complexes. And again, I’m giving you
sort of a generic overview. But you’ll see in
the next module, one of the key proteins
involved in iron homeostasis gets targeted for
degradation by a ubiquitin ligase that exists in a
complicated protein complex. So that and then adds
to the complexity you need to target
all of your proteins specifically for
degradation in some fashion. OK, so what does E3 do? So we have an E2. And we have an E2 with
ubiquitin attached. And E2 needs to
interact with E3. And this, again, is a complex. It is not a single, necessarily
a single polypeptide. And E3 interacts–
ultimately what we want to do is attach ubiquitin into
our protein of interest. So E3 interacts with
the protein of interest. And as I alluded to
before, but I’m not going to talk about
in any detail, what is the basis of the
interaction of a protein? It’s going around. It’s doing its function. How do you target
it for degradation? In general, you
target for degradation by the N-terminal
modification or by post-translational
modification. And every protein is different. So you might phosphorylate it. You might hydroxylate it. In fact, I told you and
Lizzie told you in the section on ClipX and ClipP, that
there was this thing called the N-end rule. OK, so one of the
things you can do is you can attach different
amino acids to the N-terminus. And in fact, tRNAs are
actually used to do that. And we’re not going to talk
about the details of that. But these things, you know,
you have so many proteins that are floating around. How you’re going
to subtly control when you’re going to
degrade it is not trivial. And that’s a focus of
many people’s attention. So the model here is that–
remember, everything we’re doing is isopeptide linkages. OK so, this needs to be set up. This complex needs to be set
up so the lysine to which the ubiquitin is
going to be attached needs to be adjacent
to the ubiquitin. So you have– a lysine
needs to be activated for nucleophilic attack. And now you’ve attached
your ubiquitin. So this is a direct transfer. Again, you’re forming
the isopeptides. And remember that we said
in the very beginning, you don’t just
have one ubiquitin. You have many ubiquitins. Somebody asked me
after class, how do you get the many
ubiquitins attached? It turns out that
people have started to study this in some detail. And in many cases,
the E2s and E3s interact in a processive way
to attach multiple ubiquitins. I gave you a
reference if anybody wants to read about this. It was published
a couple of years ago about how you control
polyubiquitination. And one of the handouts,
they had an E4. There could be E4s that also
act process to attach ubiquitin. So you can attach more than
one to give you, basically, the protein of interest with the
ubiquitins actually attached. OK, so that is the machinery. We know what a bit
about this machinery, but that’s all I’m going to
actually say in this class. But every system– there are
a lot of people studying this. And there are very
few of these that are understood in really
sort of molecular detail. So here again is, again,
that you have these kinds of isopeptide linkages. Here is that ubiquitin
with the carboxylate. And here is the ubiquitin
with a lysine 48 which you can attach
additional ubiquitins to. And here is another cartoon
of this overall process. So what I didn’t
tell you in general is that E3s come in flavors. So they have little
domains in them. They have HECT– is
that what it’s called? I can’t remember the
names of these domains. I haven’t– yeah, HECT,
H-E-C-T domains, HECT domains, RING domains, U domains, all
of which are distinct and play a role in the details of how
this process actually works. So again, this is
something I don’t expect you to remember
the details from, but this is what you
can imagine happening. So in the cartoon
over here, this is what I just
described, that we have an E2 that has– this
little ball is ubiquitin. Here is our protein of interest,
S. And so what happens is, E2 is attaching ubiquitin
to the protein of interest. OK, so that’s one possibility. And in this case, the
transfer is directly from E2. And that’s what the ones
that have been studied, the RING-finger-containing
domains, do. Alternatively, you
can imagine that E2 could transfer ubiquitin to E3. And once it attaches
it to E3, E3 could attach it to the
protein of interest. So all of those are possible. And there has been one case
where that’s been studied, where E3 attaches to ubiquitin. So I think you’re
going to find actually many variations of this theme. This is an old paper, I think. And I think the more
people study it, probably the more complex
it will end up getting. And so that’s basically
sort of the machinery– with the major
consideration, which I think is actually quite
interesting from a biochemical point of view, is
the N-end rule, how do you modify the N to
target it for degradation. Does it have a half-life
of two minutes? Does it have a
half-life of two hours? And what governs all of that? And you can imagine that
post-translational modification also governs the half-life–
so both of those possible. And those of you interested
in polyubiquitination can look at that reference. And in fact, that paper
uses methodologies we’ve talked about in
class and recitation too. They use rapid chemical
quench technology to measure the rate
constants for putting on multiple ubiquitins. So this rapid chemical
quenched technology continues to appear over and
over again when you want to look at
more details about how these systems actually work. OK, so that is allowing
us to get to the stage where the ubiquitin is attached
to the protein of interest. So and that is via the chamber
of doom, the 20s proteosome. But now what we also would
like to look at a little bit– and this is a very
active area of research– is the lid. OK, and you saw ClipX in
addition to ClipP before. And so I just want
to spend a minute on the 19s lid of
the proteosome. And this lid has proteins
coming and going. And when you isolate
it, you probably lose proteins that
are loosely bound. So this is, again, a complex– you can tell that from
this cartoon over here– a machine of 15 to 20 proteins. OK, and if you look
at this machine, there has been a lot
of people– actually, one of Bob Sauer’s
students at Berkeley has spent a lot of time
studying this human counterpart and has done a lot of really
beautiful cryoEM on this. So again, this methodology
we’ve been talking about has been used. Why are they using cryoEM? Because you can imagine,
this is really hard to get a picture of because
it’s moving around a lot. So if you look
over here, what you will see is that you have
a species called Rp2– Rpt. And there are six of these. So they’re all
slightly different. And this is part of
an AAA ATPase system. So you have the Rpt
equivalent 1 through 6. And this is an ATPase. And it sits on top
of the proteosome. OK, so its function is exactly
like what you guys learned about with ClipX and ClipP. What does it do? It’s going to pull to try
to unfold the protein. And it’s going to use ATP
hydrolysis to then try to thread the protein
into the chamber of doom. So the model, which hasn’t been
anywhere near as well-studied– the best-studied system is
the one that Liz talked about. That’s why we chose
to look at this. It’s sort of doing
the same thing. It’s just there is orders
of level more complexity associated with this. You can imagine how
complicated this is in terms of thinking about– from the Saunders
single-molecule talk, you can imagine this is
even more complicated. So what do you have here that’s
also similar to the ClipX-ClipP system? Here is a hexamer. And remember that we
looked at beta and alpha, they were sevenmers. So again, just like with ClipX
and ClipP, you have a mismatch. So we have a
hexamer-heptamer mismatch just like you did before. And why nature has chosen
to do this, I don’t know. But remember, even the beta– the alpha subunits are inactive. The beta subunits are–
only three out of the seven are active. So it’s just really
quite complex. Now, what are the other things
that could be involved– these other proteins
could be involved in? Well now, what’s
distinct from the two you had in the
ClipX-P system, you had something that
recognized the ssRNA tag. So you had adapter proteins. So here what we
need is something that recognizes the ubiquitins. And these could be– and in the
handouts that I’ve given you, they tell you which
one of these is which. I’m not going to talk
about that detail. But you have Rpn proteins
that recognize ubiquitins. OK, and I have another– people are starting to get
cryoEM pictures of all of this. This is a paper in 2012. Here is the protein of interest. Here is the AAA-type
ATPase system that needs to unfold
the protein of interest and thread it into
the chamber of doom. And you have binding sites
for the polyubiquitin tail. OK, so that’s one thing you need
to do with your lid proteins. A second thing you need
to do is that nature recycles the ubiquitins. So what you have is enzymes that
are called deubiquitin enzymes. I think that’s whatever
they label down here. R11 in this molecule,
Up6 are deubiquitin– sorry. [INAUDIBLE] DUBs–
OK, yeah, so they are. Both of these guys that are
involved in clipping off the ubiquitins and recycling. So you have another set of
proteins, deubiquitinating enzymes. And you have an isopeptide
linkage, remember. And what kind of
an enzyme might you expect a deubiquitinating
enzyme to be? What kind of activity
would it have? You want to cut
these things off. What are you– AUDIENCE: Protease. JOANNE STUBBE: –going to do? Protease, OK. And it turns out almost
all of them, there– again, we’re identifying
them continually. They’re not so
sequence-identifiable by looking at bioinformatics. The ones that have been looked
at or all cysteine proteases. So the ones that have
been studied in detail are cysteine proteases. But remember,
they’re recognizing isopeptide linkages,
not peptide linkages. And as in the case of
cysteine proteases, what do they involve? They involve covalent catalysis. So again, here is
another example of stuff you learned in the
first part of the course that you’re going
to– you see over, and over, and over
again in nature. And hopefully, this is
now becoming second nature to you guys, that these kinds
of processes actually happen. OK, so this is the lid. I’m not going to say
anymore about that. You see the equipment. You see how complicated it is. And every system you
study in biology, and if you care
about the regulation, you’re probably kind of have
to think about degradation. And you’re going to have
to individually look at the proteins of
interest and figure out what the E2s and
the E3s are and what the signals are that control
this overall process. So this was just taken
out of some recent review, but it just gives you
an idea of where– you see this is a couple
of years old now– but, where you see this
kind of machinery. We’re going to see it
in the next section. We’re going to see a key
player in sensing iron is degraded by ubiquitination. In addition, you can imagine
progression through the cell cycle, apoptosis,
immune surveillance, they’re all regulated by
protein-mediated degradation. So this is a fundamental
mechanism of regulation. And so having, I think, a
cartoon overview that I’ve given you in class
is really important to have in the back
of your mind when you’re thinking about the system
that you might be working on. And this was a paper that
was very recently published. And so we’ve been focusing
on cholesterol homeostasis. And remember, when I
introduced this topic, we were talking about Insig
and HMG-CoA Reductase. And HMG-CoA Reductase is
targeted for degradation by Insig. That’s why we made
this digression. And if you go back now and look
at what people have pulled out of the literature–
we’re going to look today very briefly at gp78 in your
problem set due this week. We’ll see that gp78,
which people thought was the whole story,
is not the whole story, that there is another E3. Hopefully you will get
that out of the data that I’ve given you
in problem set three. And there is yet
another system involved in cholesterol degradation
of HMG-CoA Reductase, but it’s not limited
to HMG-CoA Reductase. One also has degradation of the
transcription factors SREBP. We’ve talked about those. They use different
targeting enzymes. And furthermore, a
lot of people have been studying the enzymes
involved in cholesterol efflux. And again, these
enzymes here are also targeted for degradation. So the timing of all this
and what’s recognized is central to think– people
thinking about regulation, not only in systems in general,
but cholesterol, specifically. OK, so that’s a summary
of everything we’ve said. And finally, what
I want to do now is just come back to where
we started in this section to finish up. And where we
started was, we were looking at the second
mechanism of regulation and the key role of Insig,
that you’ve already seen, plays a key role
in SREBP control, keeping it in the
endoplasmic reticulation. So now we’re coming
back and looking at HMG Reductase and
the role of Insig in targeting its degradation. And so we’ve seen
these players now over, and over, and over again. So I’m not going to keep
drawing the structures out on the board. But remember, if you have
high cholesterol, what do you want to do with
HMG-CoA Reductase, if you have high cholesterol? Do what? AUDIENCE: Inhibit it. JOANNE STUBBE: Yeah,
you want to inhibit it. And so the way you
inhibit it is you target it to remain in the ER. And so the question
then is then, how does Insig and HMGR in
the presence of cholesterol– and it turns out, the signal
is not cholesterol itself, but the signal is lanosterol. And we talked about that very
briefly a couple of times. Where do you see lenosterol? If you go back to the
biosynthetic pathway, it’s sort of in the middle. So you have acetyl-CoA. You have lanosterol. And you have another 19 steps
before you get to cholesterol. And somehow, this
senses lanosterol. And people are trying to
understand the details of that. How do you really
know that’s true? That’s not such an
easy thing, as we’ve talked about in recitation. So what we want to
do then is retain HMG-CoA Reductase in the ER. So these are both ER-bound. And in the presence
of lanosterol, we want to target HMG-CoA
Reductase for degradation. That’s the goal. The question is, is how did
people go about studying that? OK, and so it
turns out that they have discovered three
proteins, at least in one of these systems. And the protein that I’m
going to talk about for a very brief period of time is gp78. That was glycoprotein78,
tells you something about its
molecular weight. Again, I don’t expect you
to remember the details. But gp78 interacts with Insig. OK and if you go
back and you look at the little cartoons
I’ve given you, Insig, again, has lots
of transmembrane helices and is stuck in the ER. So what do we know about gp78? And again, you see these
cartoons that Liz has used and I’ve been using,
since we really know nothing about the detailed
structures of these systems. What we know is,
at the N-terminus, we have an Insig binding site. And so people had to study that. And how did they study that? Probably by mechanisms
similar to what you had to– what you thought about
looking at problem set seven. It turns out that gp78
is a ubiquitin ligase, so it’s an E3. So this is an E3
ubiquitin ligase. So this is– gp78 is
an E3 ubiquitin ligase. It has a RING domain. Remember we said there
were little domains that alter the way you
stick the ubiquitin on. Again, we don’t know
the details about this. It has another little
domain called Ubc7. We’re really into
acronym worlds. But what you need to know is
that this is an E2-conjugating enzyme. So what you have now is an
E3, they can bind an E2. That’s the cartoons we just went
through over here, E3 binding to E2. E3 is the gp78. E2 is this little
protein domain. And I think what’s
really interesting about this protein is it has
another little domain called VPC. And this is an ATPase. And if you think about this,
if you want to target something for degradation, where
is the proteosome located that we’ve been talking about? Where is it located in the cell? Actually, there are
multiple proteosomes, but the ones we’ve been focused
on, where is it located? AUDIENCE: Cytosol. JOANNE STUBBE: Yeah, cytosol,
so this is a membrane protein. So how do you get this membrane
protein into the proteosome? OK, that’s not trivial. And this protein,
this VPC domain, uses energy somehow to pull
this out of the membrane so it can get degraded
in the proteosome. So the VPC domain, well,
pulls HMGR out of membrane. And so it gets degraded in
the cytosol by the proteosome, complicated, actually
quite interesting– yeah? AUDIENCE: Is it at all
understood how that pulling out happens? JOANNE STUBBE: I don’t– you
know, maybe, I don’t know how. I haven’t found
anything, but I haven’t looked through the literature
of any of this, the details. My guess is the answer is no,
but you can go look it up. And one of the
questions you can ask is how frequent
does that happen? How often do you
want to degrade– do you have this domain, and
how often is that domain used? And what are the
characteristics of that domain? Probably a lot more is known. I don’t really know
off the top of my head. So this is a cartoon model. And so I’m not going
to draw the model out. So I’ll say the model, you
can just see your PowerPoint. OK, and so this is the
same kind of cartoon we’ve been using
over and over again. So Insig is the center guy. Insig interrupts with
SCAP and cholesterol to keep SREBP in the ER
membrane so you don’t activate transcription of cholesterol
biosynthesis or the LDL receptor. We spent a lot of time on this. So here, Insig is here again. And it interacts
with gp78, which interacts with these
other two proteins, the E2 and whatever this protein
is that helps extract it from the membrane. A key player in all
of this is lanosterol. You have lanosterol
in the membranes. So you could do, potentially,
a similar study that we talked about in recitation this
past week to look at do you see a switch with
lanosterol, what are the lanosterol concentrations? What are the concentrations
of lanosterol? And this is a
cartoon showing this. I have no idea about the
details of this cartoon, but what you’re
going to do then is attach the ubiquitin
using this E2-E3 machinery onto HMG-CoA Reductase. And remember, that protein– we’ve looked at that
now a number of times– has a steroid– sterol-sensor
domain, which is lanosterol. And it also has a
cytoplasmic face. That’s the HMG-CoA Reductase. You can cut this off. It’s also active. And we’ve talked about
that a number of times. And so what they have here is
just a cartoon of attaching ubiquitin, which then,
in the end, magic, you end up with degradation
of your membrane-bound system. So this is a major mechanism
of regulation involving cholesterol homeostasis. But what you see when you
look at the problem set that I’ve given you is that
it’s more complicated than that. So you can knock out genes
and you still get it degraded. What is the timescale? How do you do the experiments? And I think that’s
what people are seeing with all of these things. And in part, it
becomes complicated because, if these proteins need
to be modified in some way, it’s not so easy to tell
whether they’ve been modified, and what it is that is
recognized by the E3 ligase. OK, so I think this is sort
of an exciting and interesting area. And we need some
new breakthroughs so that we can better
understand how these degradation systems are integrated
into regulation in general. So that’s just a summary
of the role of Insig, in the presence of cholesterol– or lanosterol, in keeping the
levels of cholesterol low. OK, so we finished the
section on cholesterol. I think I’ve
introduced you to a lot of different kinds of concepts. I’ve told you how important it
is in terms of therapeutics. People are continually
studying this, as you saw by the
news article that Liz had given me last time. We have this PCSK9 that’s
in clinical trials, in addition to the statins. And I think it’s going to
be on people’s radar screens for some time to come. So I think cholesterol
is cool because of the spectacular discoveries
of receptor-mediated endocytosis of
transcription factors that are found in
the ER as opposed to being found in the nucleus. And we’ve also introduced you
to another generic mechanism of control, that by
protein-mediated degradation. So that’s the end of Module 5. And what I’m going to do now– and we’ve posted
this information. Again, the information
will always be posted ahead of class
so that you can actually have the PowerPoints out there. Some things, I’m not
going to write down. In this section, there is
a lot more phenomenology. What I’ll try to do is
give you an overview of why I’ve picked
this phenomenology, but I’m not going to write
down– it takes a long time to write down all
of the phenomenology on the blackboard. And I’m not going to do that. So integrating your
notes of the things I’m going to write down with
your PowerPoint, I think, is really important
for you to do. And I would suggest that
you bring the PowerPoint so you can see
what’s written down and where you might want to
stick in a piece of paper where I expand on something
or really tell you something in much more
detail than what’s written in the PowerPoint. So Module 6, so as I just told
you at the very beginning, these modules are not
really linked together except through thinking
about homeostasis. Everything in the
cell is homeostasis. In the first
lecture, we’re going to be talking about metals and
metal homeostasis in general using the periodic table. OK, but then what I’m going to
do is focus on a single metal. And the single metal I’m going
to be focused on is iron. And so the reading
is also posted. And there are three
things for you to read. One is to think about iron
in the geochemical world. You know, why is
iron so important? If you look at the
periodic table, why aren’t we using aluminum? It’s the most abundant
in the earth’s crust. OK well using iron
and not aluminum? Well, as chemists, we ought to
be able to think about that. Silicon is the
other thing that’s one of the most abundant
things in the earth’s crust. Why aren’t we using silica
and aluminum as life– as the basis for life? And this article,
I think it’s very interesting from a
chemical perspective telling you about how to think
about these kinds of things. Why is it true? And I’ll give you a little
bit of background on that. And then you can do
as much or as little thinking about it as you choose. So the first one, I’m
just going to give you an overview of why metals
are so darn important and try to convince
you that you should all know a lot more about metals
than probably most of you have thought about from
an introductory course. Then in Lecture 2, we’re going
to talk about metal homeostasis in general. And that’s going to be– that could be applied
to any of the metals I’m going to show you
in the periodic table, but I’m going to focus on iron. And then in the
second lecture, we’re going to focus on iron
homeostasis in humans. And we’re going to
look at iron transport from the diet, where
we heard this from. How does it get
taken into the cell? It can get taken into the cell–
we’ll see a number of ways. But receptor-mediated
endocytosis, and they told us where
have we seen that? There is a protein that allows
iron to be transferred around. Just like with
cholesterol, you had to figure out how to keep
this insoluble thing soluble with– we’re going to see there
is a lot of problems with iron, so we need to figure out how
to control iron’s chemical reactivity. So we use a protein to do that. There is a transferrin. It’s a little protein
called transferrin. There is a transferrin receptor. We’ll talk about that. And then there are many levels
at which iron is regulated. Probably the most
important regulation is a peptide hormone that
I’ll briefly mention, but that’s not what
I’m going to focus on. What I’m going to
focus on is a new kind of regulation
based on regulation of the translational process
and proteins binding to RNA. And right now, that’s a very
active area of research here. It doesn’t have to be a
protein binding to RNA, but small molecules
binding to RNA. Riboswitches are being
found all over the place. And so I’m going to introduce
you to translational control by proteins binding to RNA. And then the third
and fourth lectures are going to be focused
on more on bacteria. We know a lot about
bacterial systems. Almost all bacteria
require iron to survive. And Liz is the
expert, so she can correct anything
I say incorrectly during this lecture. Where did bacteria
get their ion from? Some bacteria get
their iron from rocks. How the heck do you
get iron out of a rock? OK, well, bacteria
have figured that out. We on the other hand
are way up here. We can eat bacteria. We can eat plants. They’ve already figured
out how to get the iron out of the rocks. And so our problem
is much easier. But so bacteria are
amazingly creative. And I’ve just chosen
one of the creative ways to look at how iron is obtained. So we’re going to
talk a little bit about the host-pathogen battle. And I’m going to
use specifically Staphylococcus aureus
as an example because of the resistance problems we
currently have in the clinic. You can get an
iron in many forms. We’re going to focus
on getting iron in the form of heme, which
is a major source of iron for this organism. OK, so that’s where we’re going. Will we get finished
in four lectures? Probably not. Anyhow, so what I’m
going to do today is the first five or six
slides of PowerPoint. And it’s more phenomenology. And then we’ll get into
it, the more details, in the next lecture. So here is the bottom
bottle that– do any of you take Flintstone vitamins? Anyway, I’m not
supposed to digress. I can’t swallow vitamins,
though I like them because they taste good. AUDIENCE: [INAUDIBLE] JOANNE STUBBE: Huh? AUDIENCE: When we were little. JOANNE STUBBE: Do you
remember– does anybody remember who this guy is? No, OK– AUDIENCE: [INAUDIBLE] JOANNE STUBBE: Oh yeah, all
right, so [INAUDIBLE] Fred. OK, well you know, I always
have this generation– I’m much older than you. So anyhow, I mean,
what you learned about in the introductory
course 5.07 is, you learned a lot about
the vitamin bottle, really, how the vitamins that
you have, vitamin A, vitamin C, vitamin, all the vitamin
Bs, et cetera, what they do is greatly expand the
repertoire of reactions that enzymes can catalyze in
all your metabolic pathways. What you don’t learn about
in most introductory courses is the minerals. OK, so they’re on
the bottle too, but you sort of ignore
all of that stuff. And you know, you need iron. You need copper. You need calcium. You need zinc, et cetera. And so what I want
to do is to try to give you very briefly
an overview of why these metals are so important. And again, the focus
is going to be on iron. OK so here is our
periodic table. And these are the metals
that are found inside of us– yeah, I guess maybe. We don’t have tungsten. Liz, do we have tungsten? We don’t have tungsten. I don’t think we
have tungsten in us. So these are found
in bacteria and us. And so if you look at this,
all of these guys over here, where have we seen
magnesium before? I’ve been talking about
that over and over again. Magnesium is bound
to all nucleotides. We’re going to see
this again and again. We’re going to talk about– a
little bit about the proper use of magnesium which makes it
function in that capacity to neutralize the
charge on nucleotides. Sodium, and potassium, and
iron, conduction– calcium is involved in signaling. But what we’re going
to be focusing on are the transition metals. OK, and specifically
within the transition metals, what we’re going
to be focusing on is iron. And this is– it’s
hard to measure the concentrations in their
localizations within the cell. But you can measure
the total concentration by just taking
your cell and then submitting it to some kind
of mass spec analysis. We can see iron
versus manganese. And we’re going to,
again, be focused on iron, which accounts for about 8%. And it’s been estimated in this
article that approximately 50% of all the proteins have
some kind of metal bound. OK, it might involved
in catalysis. It might not. In fact, the metals more likely
are not involved in catalysis. And we’ll look at
that distribution. So we’ll come back to
this a couple of times, but we’re going to be
focusing over here. And what are the
properties of metals that make them so
special to increase the repertoire of
reactions that can be catalyzed inside our bodies? OK, so these guys
are unique from a lot of the reactions
you’ve already studied in your introductory
biochemistry course. And so what I want to do
is sort of just give you a general overview
of where you see metals involved in catalysis. And then we’re going
to focus on iron only. OK, so where do
we see catalysis? We see iron transport. We need to get iron, potassium,
and sodium in the right places, or we’re in trouble. Signaling– signal transduction,
we use calcium all the time. There is huge numbers of people
studying calcium signaling. Where have you seen
oxygen transport? In us– we’re in serious
trouble if oxygen can’t be carried by our
hemoglobin to our tissues. And I’ll show you a
little bit about that. So oxygen transport
is really important. Of central importance
is electron transfer and proton-coupled
electron transfer. Where have you seen that? You’ve seen that in
the respiratory chain. If you go back and you look at
complex I, complex II, complex III, you see all
these metals in there. What are they doing? They’re doing electron
transfer reactions. We’ll talk a little bit,
but not much, about that. So not only is electron transfer
involved in respiration. Electron transfer
plays a central role in all of the
environmental chemistry. And so while, in many
introductory classes, they don’t talk about this– we talk about humans,
because most people are more interested in disease– the coolest chemistry,
without question, hands down, is absolutely associated with
the bacteria and the Archae. OK, they do, like,
amazing things. How do you take nitrogen and
do an eight-electron reduction to ammonia? How do we do that as chemists? 200 atmospheres pressure
in a 400 degrees. This is an incredibly
important reaction. Where does all the nitrogen
from our amino acids come from? What about our nucleic acids? And we skip all this stuff. This is like– I mean, this, to
me, is sort of, like, amazing. Another thing we
skip all the time is where does oxygen,
how does oxygen– how does light take water
and make oxygen gas? Without that, we’d be
in serious trouble. The bacteria would definitely
be taking over the world. And this, I’ll show you, is
sort of an amazing reaction– nucleotide reduction. We may never get there,
but the last module is, I’m going to show you,
you’re making deoxynucleotides. The enzymes can use manganese,
iron, cobalt, and iron sulfur. So they use a wide
range of metals to make the building
blocks required for DNA. OK, signaling, we’ve all– I just talked about calcium
as a signaling agent. But now it’s becoming
clear, because of studies from the lipid
group, and studies from Chris Chang who
is a former lipid group member, signaling of
metals is much more common than we thought. And people are
proposing that, not only is zinc a signaling
agent, but also copper. And there is a lot
of problems in nerve cells with oxidative
damage which we’re going to come back to. So thinking about the levels and
sensing of these levels I think is going to be a future
area that’s going to be very exciting to study. You have to regulate
these metals. Transcriptional,
translational levels, we’re going to talk about. And they’re involved in
many kinds of catalysis. So let me just
close by showing you one last slide, oxygen carriers. You’ve seen this before. That’s hemoglobin. You’ve all studied,
hopefully, hemoglobin and the cooperative binding
of oxygen, how it binds, how it’s released– sort
of an amazing machine. That’s not the only
way that organisms reversibly bind oxygen. This
guy, the horseshoe crab, it uses copper. This guy– these are worms. These are found in– they’re found in the sea, right? So you go to Woods
Hole and they’ll extract these worms for you. Anyhow, what do they have? They have a diiron cluster. And the strategies
of both– they all have to reversibly bind
oxygen. And they’ve all adapted to their
environments to be able to do this in
an efficient way. So what I’ll do next
time is come back and– let me just do one more thing. Anyhow, this is–
think about this. Put it on under pillow. Think about how it works. Look at this. This is the cofactor
of nitrogenase. Not only does it have
iron and molybdenum, but look at that
guy in the center– carbon, carbon 4 minus. Think about that. We’ll come back next time.

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