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JOANNE STUBBE: What I want to do today

is finish up module 7 on reactive oxygen species

and then move on into the last module, which we are obviously

not going to get completely through.

We're going to be focused mostly on purines

and maybe some pyrimidines.

And I'll give you a big overview of what

I think the things are you need to think

about in nucleotide and deoxynucleotide metabolism

as a starting point.

OK.

So we've been talking about module 7 and, in this section,

how you control reactive oxygen species for signaling.

We were going through the generic overview.

And at the end of the last lecture,

this is the system we were talking

about using epidermal growth factor

receptor, which we've now looked at quite a bit as an example.

But what I wanted to point out is

that it's not limited to epidermal growth factor

receptor.

So you have insulin growth factor receptor,

nerve growth factor signaling, VEGF, IL-1, IL-4, et cetera.

And all of these things are all distinct.

They all have different signaling cascades.

But the generic approach that we've

been looking at in the Kate Carroll paper is also,

I think, applicable to these other systems.

And so what I wanted to do was just

make one more point with this, and then what I'm going to do

is summarize the general principles

of post-translational modification

by anything-- we're using post-translational modification

by sulfenylation and then briefly come back

to the methods used.

But we spent a lot of time in recitations in 11 and 12

focused on methods, so I'm not going

to spend very much time on that.

It's also in your PowerPoint handouts.

So the key thing here is the general--

is we have EGF, OK, so that's Epidermal Growth Factor

in the membrane.

We have epidermal growth factor receptor, which you all

know has to dimerize and you all know, at this stage,

is a tyrosine kinase.

And the key thing we're going to be focused on

is if we modify these proteins, what is

the biological consequence, OK?

Do you have any biological consequence?

And if you don't, it's probably just an artifact of the fact

that cysteines react rapidly with hydrogen-- not rapidly,

but they react with hydrogen peroxide

at some level to give you modification.

So this is all, I'm just going to say,

tyrosine kinase activity.

We've already gone through that.

And what happens is you activate the NOX proteins.

And in this case, it's the NOX2 isozymes.

And this is outside, and this is inside the cell.

And NOX2 can generate superoxide--

OK, so let's just put this in parentheses--

which can rapidly generate hydrogen peroxide.

And so the issue is that the superoxide and all

of the hydrogen peroxide needs to come

from the outside of the cell to the inside of the cell.

OK.

So we have hydrogen peroxide.

And what is hydrogen peroxide doing?

So the model is--

and this is what we've been focusing on--

that the hydrogen peroxide can modify the cysteine

by sulfenylation, OK?

So we can go from SH to SOH.

And in the case of the tyrosine kinase

and in the paper you had to read,

it turns out that tyrosine kinase by activity assays

was more active.

So it's phosphorylated.

It's sulfenylated.

That leads to higher activity.

That means it's potentially biologically interesting.

And we also, in the Kate Carroll paper,

looked not only at the activity, but we looked downstream

at the signaling pathways, and we

saw signaling as defined by phosphorylation events.

We saw more signaling.

So those are the kinds of peak criteria

people are looking at for being biologically interesting.

Now, what we also have is a key control,

and, in these cascades, like over there, we also have PTP.

And that's Protein Tyrosine Phosphatase.

And these proteins all have a cysteine at the active site.

We talked about this before.

And the cysteine at the active site, what can it do?

It can really sort of dephosphorylate

the tyrosine kinase.

And if you remove the phosphate, the activity is lowered.

OK.

So again, you have something that activates,

something that removes it.

But what we also know-- so this is the active form,

and this is the key in all these signaling events.

And so what we also have-- so let me

go over here, since I didn't leave quite enough room.

So we have PTP that can also react with hydrogen peroxide

to become sulfenylated.

That's the inactive form.

So when it's in this state, basically, you put a roadblock

in this pathway.

So this is inactive.

OK.

And the Carroll paper spent a lot of time trying to define--

there are lots of protein tyrosine phosphatases

inside the cell-- not anywhere near as many as kinases.

So one protein tyrosine phosphatase

services many proteins.

But both of these guys are regulated by sulfenylation.

And there's one third thing, and so this is just

giving us the big picture now.

If you have hydrogen peroxide in the cell,

I've already told you that there are enzymes that

can degrade hydrogen peroxide--

peroxiredoxins.

And so that removes the hydrogen peroxide,

which then prevents these things from happening.

So you have peroxiredoxins, which I already talked about.

And so the hydrogen peroxide concentration goes down.

So that's another mechanism of control.

OK.

So the take-home message is shown in this slide.

It's shown in the papers you had to read.

And there are many proteins that have

some variation on this theme, and this

is a really active area of research

to look at this in more detail.

OK.

Yeah?

AUDIENCE: The tyrosine kinase activity, [INAUDIBLE] 160%

or something.

I was just wondering how they actually

classified that as [INAUDIBLE].

JOANNE STUBBE: Active?

So, I mean, in biology, that's a huge effect.

AUDIENCE: OK.

JOANNE STUBBE: So, I mean, to somebody

that's doing something in the test tube, a factor of two

is nothing.

In biology, that's all it takes.

So the question is, is it enough?

And you should always ask that question.

And then you've got to look at the consequences,

and you do more experiments.

If you hadn't seen any effect, well, maybe you

didn't have the right proteins in there,

and you need five more proteins to assay, which

would give you a bigger effect.

OK.

So that's the issue with all of these problems.

That particular experiment, if you go back and look at it,

was done in crude extracts, OK.

And the activity is extremely low.

They had to use a luciferase assay

to be able to measure this and amplify the signal, OK,

which probably has a lot of issues with--

can have a lot of issues.

So if you're not happy with that,

then you're going to have trouble in biology.

So the question is, what is the baseline?

How much slop do you have?

And then you have to do the experiments many, many times.

It's all a question of statistics.

And then do you believe it?

And does the rest of the community believe it?

So that's a good question.

But if that's what they saw, that's what they saw.

And their interpretation was, based on this and other--

they did a lot of experiments in the paper,

and that's why we chose that paper--

suggested this is a good working hypothesis.

So I'm one of these people, you always

start out with the simplest working hypothesis.

You do experiments.

It always gets more complicated, always.

And then you expand it, or you change it.

There's nothing wrong with that.

That's what science is all about.

So NOX.

We've been talking about NOX.

We talked about it for the last couple lectures.

And I had already told you that there

were seven isozymes of NOX.

OK.

We had focused on this guy in the phagosome.

We now are focusing on NOX2 again.

And this guy is also important, but this guy is not.

That's the phagasome oxidase.

So you're changing the factors inside the cell

that govern what happens.

And so each one of these guys--

you Google it, you find another 100 papers on this.

People are trying to understand the details of what's

going on with these systems.

OK.

So that sort of just shows you, again, with generic,

it affects a lot of growth factors, or a lot of cytokines

can use these signaling pathways that NOX is important in.

Many of them, in the model, sulfenylation

is also important.

But in many cases, that remains to be established.

OK.

So what I want to briefly do, then,

is look at the general principles of regulation.

OK.

And I'm just going to briefly outline these.

And we've gone through each one of these examples in the two

recitation sections.

So I'm just sort of reviewing this and making a point.

So is post-translational modification important?

OK.

And I think your generation needs to think about this,

because as the methods become more and more sophisticated,

OK, we've got really amazing mass spec methods if you

can figure out how to do them correctly.

Everything, almost any metabolite in the cells,

can modify a protein.

Acetyl-CoA, it acetylates things.

S-Adenosyl methionine, the universal methylating agent,

methylates things.

OK.

So you have hundreds of modifications on your protein,

OK, and it is, in fact, related to,

in part, I think, the metabolites interacting

with the proteins not enzymatically.

The question is, is it interesting?

So I want you to think about that.

So that's why the general principle is,

what are you going to use as a control?

You can see it.

Are you going to spend five years of your life

chasing this?

Or is it not interesting?

So you need to think about that question.

It's not an easy question.

And that's what everybody is into now.

That's the future for the next five years.

So one of the things we see is that--

and I told you this 20 times--

it needs to be reversible.

OK.